Antigen binding molecules that promote antigen elimination
阅读说明：本技术 促进抗原消除的抗原结合分子 (Antigen binding molecules that promote antigen elimination ) 是由 井川智之 前田敦彦 原谷健太 岩柳有起 橘达彦 于 2012-09-28 设计创作，主要内容包括：本发明人创作了抗原结合分子,所述抗原结合分子含有：在pH酸性范围条件下具有人FcRn结合活性,且抗原结合分子的抗原结合活性随离子浓度条件而变化的抗原结合结构域；以及,在pH中性范围条件下Fcγ受体结合活性与EU编号297位连接的糖链为含岩藻糖糖链的天然型人IgG的Fc区的Fcγ受体结合活性相比高的Fcγ受体结合结构域。(the present inventors have created an antigen binding molecule comprising: an antigen binding domain which has human FcRn binding activity in the pH acidic range and the antigen binding activity of the antigen binding molecule varies depending on the ion concentration condition; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of the Fc region of natural human IgG having fucose-containing sugar chains as the sugar chain linked at EU position 297.)
1. A pharmaceutical composition comprising an antigen binding molecule, wherein said antigen binding molecule comprises: an antigen binding domain which has human FcRn binding activity in the acidic pH range and whose binding activity to an antigen varies depending on the ion concentration; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of the Fc region of natural human IgG having fucose-containing sugar chains as the sugar chain linked at EU position 297.
2. The pharmaceutical composition of claim 1, wherein said antigen is a soluble antigen.
3. The pharmaceutical composition according to claim 1 or2, wherein said ion concentration is a calcium ion concentration.
4. the pharmaceutical composition of claim 3, wherein said antigen binding domain is an antigen binding domain having an antigen binding activity at a high calcium ion concentration that is higher than the binding activity to said antigen at a low calcium ion concentration.
5. The pharmaceutical composition according to claim 1 or 2, wherein the ion concentration condition is a pH condition.
6. The pharmaceutical composition of claim 5, wherein said antigen binding domain is an antigen binding domain having an antigen binding activity in the neutral pH range that is higher than the binding activity to said antigen in the acidic pH range.
7. the pharmaceutical composition according to any one of claims 1 to 6, wherein the antigen-binding molecule is an antigen-binding molecule having a neutralizing activity against the antigen.
8. the pharmaceutical composition of any one of claims 1 to 7, wherein said Fc γ receptor binding domain comprises an Fc region of an antibody.
9. The pharmaceutical composition according to claim 8, wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of the following amino acids are different from the amino acids at the corresponding positions in the natural-type Fc region at the positions in the Fc region represented by the EU numbering: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
10. The pharmaceutical composition according to claim 9, wherein the Fc region contains at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
The amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 225 is any one of Glu, Lys, or Trp;
The amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
the amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
The amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
the amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
the amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
The amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
The amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
the amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
the amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
the amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
The amino acid at position 263 is any one of Ala, Ile, Met or Thr;
the 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
the amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 273 is any one of Phe or Ile;
the amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 275 is any one of Leu or Trp;
the amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
Amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
The amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
The amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
The amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
The amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
The amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
the amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
The amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
The amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
the 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
The amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
the amino acid at the 303 th position is Asp, Gly or Tyr;
The amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
the amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
the amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
the amino acid at position 313 is Phe;
The amino acid at position 315 is Leu;
The amino acid at position 317 is Glu or Gln;
The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
The amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
the amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
The amino acid at position 377 is Gly or Lys;
Asp for the amino acid in position 378;
The amino acid at position 379 is Asn;
the amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
The amino acid at position 421 is Lys;
The amino acid at position 427 is Asn;
The amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
the amino acid at position 434 is Trp;
The amino acid at position 436 is Ile; and
the amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the sugar chain linked at position 297 in the EU numbering system is a fucose-containing sugar chain in the Fc region of a natural human IgG comprising: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
12. The pharmaceutical composition of any one of claims 1-11, wherein said human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
13. The pharmaceutical composition of any one of claims 1 to 11, wherein said human Fc γ receptor is Fc γ RIIb.
14. the pharmaceutical composition according to any one of claims 8 to 13, wherein the Fc region is an Fc region comprising at least one amino acid selected from the group consisting of:
The amino acid at position 238 is Asp, or
The amino acid at position 328 is Glu.
15. a method according to any one of (i) to (vi) below, comprising the step of contacting a cell expressing an Fc γ receptor with a cell in or outside the body, wherein the antigen binding molecule comprises: an antigen binding domain which has human FcRn binding activity in the acidic pH range and whose binding activity to an antigen varies depending on the ion concentration; and an Fc gamma receptor binding domain having Fc gamma receptor binding activity in the neutral pH range which is higher than that of the Fc region of a natural human IgG having fucose-containing sugar chains as the sugar chains attached at position 297 in the EU numbering system,
(i) A method of increasing the number of antigens to which a molecule of antigen binding molecule can bind;
(ii) a method for eliminating an antigen in plasma;
(iii) a method of improving the pharmacokinetics of an antigen binding molecule;
(iv) A method of promoting intracellular dissociation of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule;
(v) a method for promoting the extracellular release of an antigen-binding molecule in a state in which it is not bound to an antigen; or
(vi) a method of reducing the total antigen concentration or the free antigen concentration in plasma.
16. the method of claim 15, wherein said antigen is a soluble antigen.
17. The method of claim 15 or 16, wherein said ion concentration is a calcium ion concentration.
18. The method of claim 17, wherein said antigen binding domain is an antigen binding domain having an antigen binding activity at a high calcium ion concentration that is higher than the binding activity to said antigen at a low calcium ion concentration.
19. The method according to claim 15 or 16, wherein the ion concentration condition is a pH condition.
20. the method of claim 19, wherein said antigen binding domain is an antigen binding domain having an antigen binding activity in the neutral pH range that is greater than the binding activity to said antigen in the acidic pH range.
21. The method according to any one of claims 15 to 20, wherein the antigen-binding molecule is an antigen-binding molecule having a neutralizing activity against the antigen.
22. the method of any one of claims 15 to 21, wherein said fey receptor binding domain comprises an Fc region of an antibody.
23. the method according to claim 22, wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
24. The method according to claim 23, wherein the Fc region comprises at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
The amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 225 is any one of Glu, Lys, or Trp;
The amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
The amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
The amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
The amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
the amino acid at position 244 is His;
the amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
the amino acid at position 249 is any one of Glu, His, Gln or Tyr;
the amino acid at position 250 is either Glu or Gln;
the amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
The amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
the amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
The amino acid at position 263 is any one of Ala, Ile, Met or Thr;
The 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 266 is any one of Ala, Ile, Met or Thr;
the amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 273 is any one of Phe or Ile;
The amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 275 is any one of Leu or Trp;
the amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
The amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
the amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
The amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
the amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
the amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
The amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
the amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
the amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
The amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
The amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
the 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
The amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
the amino acid at the 303 th position is Asp, Gly or Tyr;
the amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
The amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
the amino acid at position 313 is Phe;
the amino acid at position 315 is Leu;
The amino acid at position 317 is Glu or Gln;
the amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
The amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
The amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
The amino acid at position 336 is any one of Glu, Lys or Tyr;
the amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
amino acid 376 is Ala or Val;
The amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
The amino acid at position 379 is Asn;
the amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
Amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
the amino acid at position 427 is Asn;
The amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
The amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
25. The method according to any one of claims 15 to 24, wherein the Fc region of a natural human IgG having a fucose-containing sugar chain as the sugar chain linked at position 297 in the EU numbering system is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
26. the method of any one of claims 15-25, wherein the human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
27. the method of any one of claims 15-25, wherein said human Fc γ receptor is Fc γ RIIb.
28. the method according to any one of claims 22 to 27, wherein the Fc region comprises at least one of the following amino acids at the positions in the Fc region represented by EU numbering:
The amino acid at position 238 is Asp, or
the amino acid at position 328 is Glu.
29. The method according to any one of the following (i) to (vii), comprising: a step of enhancing the Fc γ receptor binding activity under the pH neutral range condition of an Fc γ receptor binding domain of an antigen binding molecule comprising an antigen binding domain and an Fc γ receptor binding domain, which have human FcRn binding activity under the pH acidic range condition and the binding activity to an antigen varies depending on the ion concentration condition, as compared with the Fc region of a natural human IgG having fucose-containing sugar chains as the sugar chains linked at the EU number 297,
(i) a method for modifying an antigen-binding molecule which promotes the uptake of a bound antigen into cells;
(ii) A method of increasing the number of antigens to which a molecule of antigen binding molecule can bind;
(iii) a method for increasing the antigen-eliminating ability in the plasma of an antigen-binding molecule;
(iv) a method of improving the pharmacokinetics of an antigen binding molecule;
(v) a method of promoting intracellular dissociation of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule;
(vi) A method for promoting the release of an antigen-binding molecule, which is taken up into a cell while being bound to an antigen, to the outside of the cell while being unbound to the antigen; or
(vii) Methods of altering antigen binding molecules that reduce the concentration of total or free antigen in plasma.
30. The method of claim 29, wherein said antigen is a soluble antigen.
31. The method of claim 29 or 30, wherein said ion concentration is a calcium ion concentration.
32. The method of claim 31, wherein said antigen binding domain is an antigen binding domain having an antigen binding activity at a high calcium ion concentration that is greater than the binding activity to said antigen at a low calcium ion concentration.
33. The method of claim 29 or 30, wherein the ion concentration condition is a pH condition.
34. The method of claim 33, wherein said antigen binding domain is an antigen binding domain having an antigen binding activity in the neutral pH range that is greater than the binding activity to said antigen in the acidic pH range.
35. the method of any one of claims 29 to 34, wherein said antigen binding molecule is an antigen binding molecule having neutralizing activity against said antigen.
36. the method of any one of claims 29 to 35, wherein said fey receptor binding domain comprises an Fc region of an antibody.
37. The method according to claim 36, wherein the Fc region is an Fc region in which at least one amino acid selected from the group consisting of the following amino acids at the positions indicated by EU numbering is different from the amino acid at the corresponding position in the natural Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
38. The method according to claim 37, wherein the Fc region comprises at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
The amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 225 is any one of Glu, Lys, or Trp;
the amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
The amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
the amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
the amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
The amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
The 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
The amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
The amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
the amino acid at position 256 is any one of Ala, Met or Pro;
the amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
The amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
The amino acid at position 263 is any one of Ala, Ile, Met or Thr;
The 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 273 is any one of Phe or Ile;
the amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 275 is any one of Leu or Trp;
The amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
Amino acid 279 is Ala;
the 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
the amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
the amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
the amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
The amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
The amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
The amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
the amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
The amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
the amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
the amino acid at position 301 is any one of Asp, Glu, His or Tyr;
The amino acid at position 302 is Ile;
The amino acid at the 303 th position is Asp, Gly or Tyr;
The amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
the amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
The amino acid at position 313 is Phe;
the amino acid at position 315 is Leu;
the amino acid at position 317 is Glu or Gln;
the amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
The amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
the amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
The amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
the amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
the amino acid at position 377 is Gly or Lys;
Asp for the amino acid in position 378;
The amino acid at position 379 is Asn;
the amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
the amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
The amino acid at position 427 is Asn;
The amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
the amino acid at position 434 is Trp;
The amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
39. the method according to any one of claims 29 to 38, wherein the Fc region of a natural human IgG having a fucose-containing sugar chain as the sugar chain linked at position 297 in the EU numbering system is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
40. the method of any one of claims 29-39, wherein said human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
41. The method of any one of claims 29 to 39, wherein said human Fc γ receptor is Fc γ RIIb.
42. The method according to any one of claims 36 to 41, wherein the Fc region comprises at least one of the following amino acids at the positions in the Fc region represented by the EU numbering:
the amino acid at position 238 is Asp, or
the amino acid at position 328 is Glu.
43. a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) A step of obtaining the binding activity of the antigen-binding domain to an antigen under a high calcium ion concentration condition;
(b) a step of obtaining the antigen-binding activity of the antigen-binding domain under the condition of low calcium ion concentration;
(c) A step of selecting an antigen-binding domain having an antigen-binding activity obtained in (a) higher than that obtained in (b);
(d) a step of linking the polynucleotide encoding the antigen binding domain selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to the EU-numbering position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
44. a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) A step of obtaining the binding activity of the antibody to the antigen under the condition of high calcium ion concentration;
(b) a step of obtaining the antigen binding activity of the antibody under the condition of low calcium ion concentration;
(c) A step of selecting an antibody having an antigen binding activity obtained in (a) higher than that obtained in (b);
(d) A step of linking a polynucleotide encoding the antigen binding domain of the antibody selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range, to which the EU-position 297 is linked, is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
45. A method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) a step of obtaining the binding activity of the antigen-binding domain to an antigen under a condition of a neutral range of pH;
(b) a step of obtaining an antigen binding activity of the antigen binding domain under a pH acidic range condition;
(c) a step of selecting an antigen-binding domain having an antigen-binding activity obtained in (a) higher than that obtained in (b);
(d) A step of linking the polynucleotide encoding the antigen binding domain selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to the EU-numbering position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
46. a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) A step of obtaining the binding activity of an antibody to an antigen in a neutral pH range;
(b) A step of obtaining an antigen binding activity of an antibody to an antigen in a pH acidic range;
(c) A step of selecting an antibody having an antigen binding activity obtained in (a) higher than that obtained in (b);
(d) A step of linking a polynucleotide encoding the antigen binding domain of the antibody selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to the EU-numbering position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
47. the method according to any one of claims 43 to 46, wherein the antigen is a soluble antigen.
48. The method of any one of claims 43 to 47, wherein the Fcyreceptor binding domain comprises an Fc region of an antibody.
49. The production method according to claim 48, wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of the following amino acids at positions in the EU numbering system of the Fc region are different from the amino acids at corresponding positions in the natural-type Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
50. The production method according to claim 49, wherein the Fc region contains at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
The amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
The amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 225 is any one of Glu, Lys, or Trp;
the amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
the amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
the amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
The amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
the amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
the amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
the amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
The amino acid at position 255 is any one of Glu, Leu or Tyr;
The amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
the amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
the amino acid at position 263 is any one of Ala, Ile, Met or Thr;
The 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 273 is any one of Phe or Ile;
the amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 275 is any one of Leu or Trp;
The amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
Amino acid 279 is Ala;
the 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
The amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
The 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
The amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
the amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
The amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
the amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
the amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
the amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
the amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
the 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
the amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
the amino acid at the 303 th position is Asp, Gly or Tyr;
the amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
the amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
The amino acid at position 313 is Phe;
the amino acid at position 315 is Leu;
the amino acid at position 317 is Glu or Gln;
the amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
The amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 323 is Ile;
the amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
The amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
the amino acid at position 336 is any one of Glu, Lys or Tyr;
the amino acid at position 337 is any one of Glu, His, or Asn;
the amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
the amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
the amino acid at position 379 is Asn;
the amino acid at position 380 is any one of Ala, Asn or Ser;
amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
the amino acid at position 427 is Asn;
the amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
The amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
51. the method according to any one of claims 43 to 50, wherein the Fc region of a natural human IgG having a fucose-containing sugar chain as the sugar chain linked at position 297 in the EU numbering system is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
52. the method of any one of claims 43-51, wherein the human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
53. The method of any one of claims 43 to 51, wherein the human Fc γ receptor is Fc γ RIIb.
54. the production method of any one of claims 48 to 53, wherein the Fc region is an Fc region comprising at least one or more of the following amino acids at a position in the Fc region represented by the EU numbering:
The amino acid at position 238 is Asp, or
the amino acid at position 328 is Glu.
Technical Field
The present invention provides: an antigen-binding molecule that promotes the uptake of bound antigen into cells, an antigen-binding molecule that increases the number of antigens that can be bound per molecule, an antigen-binding molecule that improves pharmacokinetics, an antigen-binding molecule that promotes the dissociation of antigen bound extracellularly within cells, an antigen-binding molecule that promotes the release of antigen from cells without binding to antigen, an antigen-binding molecule that has the function of reducing the concentration of total or free antigen in plasma, a pharmaceutical composition containing the antigen-binding molecule, and methods for producing the same.
Background
Antibodies have been drawing attention as pharmaceuticals because of their high stability in plasma and few side effects. Among them, a large number of IgG-type antibody drugs are available on the market, and a large number of antibody drugs are now being developed (non-patent documents 1 and 2). On the other hand, various techniques have been developed as techniques applicable to second-generation antibody drugs, and techniques for improving effector functions, antigen binding ability, pharmacokinetics, and stability, techniques for reducing the risk of immunogenicity, and the like have been reported (non-patent document 3). In general, since the amount of an antibody drug to be administered is very high, it is considered to be difficult to prepare a subcutaneous preparation, and the production cost is high. As a method for reducing the amount of an antibody drug to be administered, a method for improving the pharmacokinetics of an antibody and a method for improving the affinity between an antibody and an antigen can be considered.
as a method for improving the pharmacokinetics of antibodies, artificial amino acid substitutions of the constant region have been reported (non-patent documents 4 and 5). As a technique for enhancing the antigen binding ability and the antigen neutralizing ability, an affinity maturation technique has been reported (non-patent document 6), and it has been reported that the binding activity to an antigen can be enhanced by introducing a mutation into an amino acid in a CDR region of a variable region or the like. By enhancing the antigen-binding ability, the biological activity in vitro can be improved, the dose can be reduced, and the drug efficacy in vivo (in vivo) can be improved (non-patent document 7).
on the other hand, the amount of antigen that can be neutralized per molecule of antibody depends on affinity, and the affinity can be enhanced to neutralize the antigen with a small amount of antibody, and the affinity of the antibody can be enhanced by various methods (non-patent document 6). Furthermore, one molecule of antigen (bivalent antigen) can be neutralized with one molecule of antibody, as long as it can be covalently bound to the antigen and has infinite affinity. However, the conventional methods are limited to the stoichiometric neutralization reaction of one molecule of antibody with one molecule of antigen (in the case of a bivalent antibody, a bivalent antigen), and it is impossible to completely neutralize the antigen with an amount of antibody equal to or less than the amount of antigen. That is, there is a limitation in the effect of enhancing affinity (non-patent document 9). In the case of neutralizing antibodies, in order to maintain their neutralizing effect for a certain period of time, it is necessary to administer an amount of antibody equal to or greater than the amount of antigen produced in the body during that period, and there is a limit in reducing the amount of antibody required to be administered, only by the above-mentioned antibody pharmacokinetics improvement or affinity maturation techniques. Therefore, in order to maintain the neutralizing effect of an antigen for a target period with an amount of antibody equal to or less than the amount of the antigen, it is necessary to neutralize a plurality of antigens with one antibody. As a novel method for achieving this, an antibody that binds to an antigen in a pH-dependent manner has recently been reported (patent document 1). The pH-dependent antigen-binding antibody that strongly binds to an antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in vivo can dissociate from the antigen in vivo. After dissociating the antigen, the pH-dependent antigen-binding antibody can be bound to the antigen again if the antibody is recycled into plasma by FcRn, and thus binding to a plurality of antigens can be repeated with one pH-dependent antigen-binding antibody.
Furthermore, the retention of antigen in plasma is very short compared to antibodies that are recycled by binding to FcRn. When such an antibody having a long plasma retention property binds to its antigen, the antibody-antigen complex has a long plasma retention property as long as that of the antibody. Therefore, the binding of the antigen to the antibody not only increases the retention in plasma but also increases the concentration of the antigen in plasma.
Thus, the pH-dependent antigen-binding antibody can bind to a plurality of antigens with 1 antibody, and can promote the elimination of antigens from plasma as compared with a normal antibody, and thus has an effect that cannot be achieved by a normal antibody. However, no antibody engineering technique has been reported so far which can repeat the effect of binding to an antigen and further improve the effect of promoting elimination of an antigen from plasma of the pH-dependent antigen-binding antibody.
IgG antibodies have long retention in plasma by binding to FcRn. Binding of IgG to FcRn was observed only under acidic conditions (ph6.0), while binding was not substantially observed under neutral conditions (ph 7.4). IgG antibodies are taken up non-specifically into cells, but return to the cell surface by binding to FcRn in vivo under acidic conditions in vivo, and dissociate from FcRn under neutral conditions in plasma. Introduction of a mutation into the Fc region of IgG, which loses binding to FcRn under pH acidic conditions, becomes unable to be recycled from endosomes into plasma, and retention of antibody in plasma is significantly impaired. As a method for improving the retention of IgG antibodies in plasma, a method for increasing the binding to FcRn under pH acidic range conditions has been reported. By introducing amino acid substitutions into the Fc region of IgG antibodies, binding to FcRn under pH acidic range conditions is increased, efficiency of recycling from endosomes into plasma is increased, and retention in plasma is improved as a result.
Many studies have been made to date on antibody-dependent cytotoxic activity (hereinafter referred to as ADCC) and complement-dependent cytotoxic activity (hereinafter referred to as CDC) of effector functions of IgG-based antibodies, and it has been reported that antibodies of IgG1 subclass have the highest ADCC activity and CDC activity among human IgG (non-patent document 13). Further, antibody-dependent cell-mediated phagocytosis (ADCP), which is the phagocytosis of target cells mediated by IgG-based antibodies, is also suggested as one of the effector functions of antibodies (non-patent documents 14 and 15). Antibodies of the IgG1 subclass are likely to exert these effector functions on tumors, and therefore antibodies of the IgG1 subclass are used as most antibody drugs against cancer antigens.
in order for an IgG antibody to mediate ADCC and ADCP activities, it is necessary to bind the Fc region of the IgG antibody to an antibody receptor (hereinafter referred to as Fc γ receptor or Fc γ R) present on the surface of an effector cell such as a killer cell, a natural killer cell, or an activated macrophage. As a protein family of human Fc γ receptors, isoforms of Fc γ RIa, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, and Fc γ RIIIb have been reported, and their allotypes have also been reported (non-patent document 16).
enhancement of cytotoxic effector functions such as ADCC and ADCP has been attracting attention as a promising means for enhancing the antitumor effect of anticancer antibodies. The importance of Fc γ receptor-mediated effector functions for the purpose of anti-tumor effects of antibodies has been reported using mouse models (non-patent documents 17 and 18). Furthermore, a correlation was observed between the clinical effect on humans and the high-affinity polymorphism (V158) and the low-affinity polymorphism (F158) of Fc γ RIIIa (non-patent document 19). According to these reports, it is suggested that: antibodies having an Fc region optimized for binding to a specific fey receptor mediate stronger effector functions, thereby exerting potent antitumor effects. The balance of the affinity of an antibody for each of the activating receptor containing Fc γ RIa, Fc γ RIIa, Fc γ RIIIa, Fc γ RIIIb, and the inhibitory receptor containing Fc γ RIIb is an important factor in optimizing the effector function of an antibody. Since an antibody can be endowed with a property of mediating a stronger effector function by enhancing affinity for an activated receptor (non-patent document 20), various reports have been made so far as an antibody engineering method for enhancing or improving the antitumor activity of an antibody drug against an anticancer antigen.
It has been shown that: in connection with the binding of the Fc region to the Fc γ receptor, a sugar chain attached to the hinge region of the antibody, several amino acid residues in the CH2 domain, and Asn at EU position 297 bound to the CH2 domain are important (non-patent document 13, non-patent document 21, and non-patent document 22). Around this binding site, various Fc region mutants having Fc γ receptor binding properties have been studied so far, and Fc region mutants having higher affinity for activated Fc γ receptors have been obtained (patent documents 2 and 3). For example, Lazar et al succeeded in increasing the binding of human IgG1 to human Fc γ RIIIa (V158) by about 370-fold by substituting Ser at position 239, Ala at position 330, and Ile at position 332 of human IgG1, which are expressed by EU numbering, with Asp, Leu, and Glu, respectively (non-patent documents 23 and 3). The ratio of binding of Fc γ RIIIa to Fc γ RIIb (a/I ratio) for this variant compared to the native form was about 9-fold. Furthermore, Shinkawa et al succeeded in increasing the binding to Fc γ RIIIa by about 100-fold by deleting fucose from the sugar chain attached to Asn at position 297 indicated by EU numbering (non-patent document 24). By these methods, the ADCC activity of human IgG1 can be greatly improved as compared with that of natural human IgG 1.
as described above, in an antibody targeting a membrane-type antigen, Fc γ receptor binding activity plays an important role in cytotoxic activity, and therefore, a technique of enhancing cytotoxic activity by enhancing Fc γ receptor binding activity using an isotype of human IgG1 having high Fc γ R binding activity is widely used when the cytotoxic activity is required. On the other hand, in antibodies targeting soluble antigens, the role played by the Fc γ receptor binding activity is still unknown, and it is considered that: there was no difference in effect between human IgG1 having high Fc γ receptor binding activity and human IgG2 or human IgG4 having low Fc γ R binding activity. Therefore, no attempt has been made to enhance the Fc γ receptor binding activity of antibodies targeting soluble antigens, and no report has been made on the effect.
Disclosure of Invention
technical problem to be solved by the invention
the present invention has been made in view of the above circumstances, and an object thereof is to provide: an antigen-binding molecule that promotes the uptake of bound antigen into cells, an antigen-binding molecule that increases the number of antigens that can be bound per molecule, an antigen-binding molecule that improves pharmacokinetics, an antigen-binding molecule that promotes the dissociation of antigen bound extracellularly within cells, an antigen-binding molecule that promotes the release of antigen from cells without binding to antigen, an antigen-binding molecule that has the function of reducing the concentration of total or free antigen in plasma, a pharmaceutical composition containing the antigen-binding molecule, and methods for producing the same.
means for solving the problems
the present inventors have conducted intensive studies to achieve the above object, and have created an antigen-binding molecule comprising: an antigen binding domain which has human FcRn binding activity in the pH acidic range and in which the antigen binding activity of the antigen binding molecule varies depending on the ion concentration condition; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range, which is higher than that of an Fc γ receptor binding domain of an Fc region of a natural human IgG having fucose-containing sugar chains, linked to the sugar chain at EU-position 297. Furthermore, the present inventors have found a method of promoting the uptake of bound antigen into cells, a method of increasing the number of antigens to which each molecule of antigen-binding molecule can bind, a method of improving the pharmacokinetics of antigen-binding molecules, a method of promoting the dissociation of antigen bound to antigen-binding molecules outside cells from antigen-binding molecules inside cells, a method of promoting the release outside cells in a state of not binding to antigen, and a method of reducing the total antigen concentration or free antigen concentration in plasma, which comprise a step of bringing the above-mentioned antigen-binding molecules into cell contact with Fc γ receptor-expressing cells inside or outside the body. Further, the present inventors have found a method for producing an antigen-binding molecule having the above properties and, at the same time, found the usefulness of a pharmaceutical composition containing such an antigen-binding molecule or an antigen-binding molecule produced by the production method of the present invention as an active ingredient, thereby completing the present invention.
More specifically, the present invention provides the following [1] to [46 ].
[1] A pharmaceutical composition comprising an antigen binding molecule, wherein the antigen binding molecule comprises: an antigen binding domain which has human FcRn binding activity in the acidic pH range and whose binding activity to an antigen varies depending on the ion concentration; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of the Fc region of natural human IgG having fucose-containing sugar chains as the sugar chain linked at EU position 297.
[2] [1] the pharmaceutical composition according to any one of the above aspects, wherein the antigen is a soluble antigen.
[3] The pharmaceutical composition according to [1] or [2], wherein the ion concentration is a calcium ion concentration.
[4] [3] the pharmaceutical composition according to any one of the above aspects, wherein the antigen-binding domain has an antigen-binding activity at a high calcium ion concentration that is higher than the antigen-binding activity at a low calcium ion concentration.
[5] The pharmaceutical composition according to [1] or [2], wherein the ion concentration condition is a pH condition.
[6] [5] the pharmaceutical composition according to any of the above aspects, wherein the antigen-binding domain has an antigen-binding activity in a neutral pH range that is higher than an antigen-binding activity in an acidic pH range.
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the antigen-binding molecule has a neutralizing activity against the antigen.
[8] the pharmaceutical composition according to any one of [1] to [7], wherein the Fc γ receptor binding domain comprises an Fc region of an antibody.
[9] [8] the pharmaceutical composition according to any one of the above Fc regions, wherein the Fc region has at least one amino acid selected from the group consisting of the following amino acids at the positions indicated by the EU numbering in the Fc region that is different from the amino acid at the corresponding position in the natural-type Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
[10] [9] the pharmaceutical composition according to any one of the above Fc regions, wherein the Fc region contains at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
the amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 225 is any one of Glu, Lys, or Trp;
The amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
The amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
The amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 240 is any one of Ala, Ile, Met or Thr;
The amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
the amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
the amino acid at position 244 is His;
the amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
the amino acid at position 249 is any one of Glu, His, Gln or Tyr;
the amino acid at position 250 is either Glu or Gln;
the amino acid at position 251 is Phe;
The amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
The amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
The amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
The amino acid at position 263 is any one of Ala, Ile, Met or Thr;
The 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 273 is any one of Phe or Ile;
the amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 275 is any one of Leu or Trp;
the amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
Amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
the amino acid at position 281 is Asp, Lys, Pro or Tyr;
the amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
The 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
the amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
the amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
The amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
the amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
the amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
the amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
The amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
the 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
the 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
the amino acid at position 301 is any one of Asp, Glu, His or Tyr;
The amino acid at position 302 is Ile;
The amino acid at the 303 th position is Asp, Gly or Tyr;
the amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
the amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
The amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
the amino acid at position 313 is Phe;
The amino acid at position 315 is Leu;
The amino acid at position 317 is Glu or Gln;
The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 323 is Ile;
the amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
the amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
The amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
the amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
The amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
the amino acid at position 379 is Asn;
The amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
the amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
The amino acid at position 421 is Lys;
the amino acid at position 427 is Asn;
the amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
The amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
[11] the pharmaceutical composition according to any one of [1] to [10], wherein the sugar chain linked at position 297 in the EU numbering system is an Fc region of a natural human IgG having fucose-containing sugar chains, and the Fc region is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
[12] The pharmaceutical composition according to any one of [1] to [11], wherein the human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
[13] The pharmaceutical composition according to any one of [1] to [11], wherein the human Fc γ receptor is Fc γ RIIb.
[14] The pharmaceutical composition according to any one of [8] to [13], wherein the Fc region contains at least one of the following amino acids at the position indicated by the EU number:
The amino acid at position 238 is Asp, or
the amino acid at position 328 is Glu.
[15] a method according to any one of the following (i) to (vi), the method comprising the step of contacting a cell expressing an Fc γ receptor with a cell in or outside the body, the antigen binding molecule comprising: an antigen binding domain which has human FcRn binding activity in the acidic pH range and whose binding activity to an antigen varies depending on the ion concentration; and an Fc gamma receptor binding domain having Fc gamma receptor binding activity in the neutral pH range which is higher than that of the Fc region of a natural human IgG having fucose-containing sugar chains as the sugar chains attached at position 297 in the EU numbering system,
(i) a method of increasing the number of antigens to which a molecule of antigen binding molecule can bind;
(ii) a method for eliminating an antigen in plasma;
(iii) A method of improving the pharmacokinetics of an antigen binding molecule;
(iv) a method of promoting intracellular dissociation of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule;
(v) A method for promoting the extracellular release of an antigen-binding molecule in a state in which it is not bound to an antigen; or (vi) a method of reducing the total antigen concentration or the free antigen concentration in plasma.
[16] the method of [15], wherein the antigen is a soluble antigen.
[17] the method according to [15] or [16], wherein the ion concentration is a calcium ion concentration.
[18] [17] the method according to any one of the above methods, wherein the antigen-binding domain has an antigen-binding activity at a high calcium ion concentration that is higher than an antigen-binding activity at a low calcium ion concentration.
[19] The method according to [15] or [16], wherein the ion concentration condition is a pH condition.
[20] [19] the method according to any one of the above methods, wherein the antigen-binding domain has an antigen-binding activity in a neutral pH range that is higher than an antigen-binding activity in an acidic pH range.
[21] The method according to any one of [15] to [20], wherein the antigen-binding molecule has a neutralizing activity against the antigen.
[22] the method according to any one of [15] to [21], wherein the Fc γ receptor binding domain comprises an Fc region of an antibody.
[23] [22] the method according to [1], wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of the following amino acids are different from the amino acids at the corresponding positions in the natural-type Fc region at the positions indicated by the EU numbering in the Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
[24] [23] the method according to [1], wherein the Fc region contains at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
the amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 225 is any one of Glu, Lys, or Trp;
the amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
The amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
the amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
The amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
The amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
The 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
the amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
The amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
The amino acid at position 255 is any one of Glu, Leu or Tyr;
The amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
the amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
The amino acid at position 263 is any one of Ala, Ile, Met or Thr;
the 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 273 is any one of Phe or Ile;
the amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 275 is any one of Leu or Trp;
the amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
the amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
The 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
the amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
the amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
the amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
The amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
the amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
the amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
the amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
the amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
the amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
the 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
The amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
the amino acid at the 303 th position is Asp, Gly or Tyr;
the amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
The amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
the amino acid at position 313 is Phe;
The amino acid at position 315 is Leu;
the amino acid at position 317 is Glu or Gln;
The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
The amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
The amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
amino acid 376 is Ala or Val;
the amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
the amino acid at position 379 is Asn;
The amino acid at position 380 is any one of Ala, Asn or Ser;
amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
the amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
the amino acid at position 427 is Asn;
the amino acid at position 428 is any one of Phe or Leu;
The amino acid at position 429 is Met;
The amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
[25] The method according to any one of [15] to [24], wherein the Fc region of a natural human IgG having a fucose-containing sugar chain as the sugar chain linked at position 297 in the EU numbering system is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
[26] the method according to any one of [15] to [25], wherein the human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
[27] The method according to any one of [15] to [25], wherein the human Fc γ receptor is Fc γ RIIb.
[28] the method according to any one of [22] to [27], wherein the Fc region contains at least one of the following amino acids at a position in the Fc region represented by the EU number:
the amino acid at position 238 is Asp, or
the amino acid at position 328 is Glu.
[29] The method according to any one of the following (i) to (vii), comprising: a step of enhancing the Fc γ receptor binding activity under the pH neutral range condition of an Fc γ receptor binding domain of an antigen binding molecule comprising an antigen binding domain and an Fc γ receptor binding domain, which have human FcRn binding activity under the pH acidic range condition and the binding activity to an antigen varies depending on the ion concentration condition, as compared with the Fc region of a natural human IgG having fucose-containing sugar chains as the sugar chains linked at the EU number 297,
(i) a method for modifying an antigen-binding molecule which promotes the uptake of a bound antigen into cells;
(ii) a method of increasing the number of antigens to which a molecule of antigen binding molecule can bind;
(iii) A method for increasing the antigen-eliminating ability in the plasma of an antigen-binding molecule;
(iv) A method of improving the pharmacokinetics of an antigen binding molecule;
(v) a method of promoting intracellular dissociation of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule;
(vi) a method for promoting the release of an antigen-binding molecule, which is taken up into a cell while being bound to an antigen, to the outside of the cell while being unbound to the antigen; or
(vii) methods of altering antigen binding molecules that reduce the concentration of total or free antigen in plasma.
[30] [29] the method according to any one of the above methods, wherein the antigen is a soluble antigen.
[31] the method according to [29] or [30], wherein the ion concentration is a calcium ion concentration.
[32] [31] the method according to any one of the above methods, wherein the antigen-binding domain has an antigen-binding activity at a high calcium ion concentration that is higher than an antigen-binding activity at a low calcium ion concentration.
[33] the method according to [29] or [30], wherein the ion concentration condition is a pH condition.
[34] [33] the method according to any one of the above methods, wherein the antigen-binding domain has an antigen-binding activity in a neutral pH range that is higher than an antigen-binding activity in an acidic pH range.
[35] The method according to any one of [29] to [34], wherein the antigen-binding molecule has a neutralizing activity against the antigen.
[36] The method according to any one of [29] to [35], wherein the Fc γ receptor binding domain comprises an Fc region of an antibody.
[37] [36] the method according to [36], wherein the Fc region is an Fc region in which at least one amino acid selected from the following amino acids at the positions indicated by the EU numbering in the Fc region is different from the amino acid at the corresponding position in the natural-type Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 25294 bit, 295 bit, 298 bit, 297 bit, 296 bit, 299 bit, 326 bit, 300 bit, 301 bit, 302 bit, 305 bit, 303 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 325 bit, 323 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
[38] [33] the method according to [ 3], wherein the Fc region contains at least one amino acid selected from the group consisting of:
the amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
the amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 225 is any one of Glu, Lys, or Trp;
The amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
the amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
the amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
The amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
The amino acid at position 245 is Ala;
the amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
The amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
the amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
The amino acid at position 255 is any one of Glu, Leu or Tyr;
The amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
the amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
The amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
the amino acid at position 263 is any one of Ala, Ile, Met or Thr;
the 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 273 is any one of Phe or Ile;
the amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 275 is any one of Leu or Trp;
the amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
Amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
the amino acid at position 281 is Asp, Lys, Pro or Tyr;
The amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
the amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
The amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
The amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
the amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
The amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
the amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
the amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
the amino acid at position 301 is any one of Asp, Glu, His or Tyr;
The amino acid at position 302 is Ile;
the amino acid at the 303 th position is Asp, Gly or Tyr;
the amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
the amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
the amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
the amino acid at position 313 is Phe;
The amino acid at position 315 is Leu;
the amino acid at position 317 is Glu or Gln;
The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
the amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
The amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
the amino acid at position 377 is Gly or Lys;
Asp for the amino acid in position 378;
the amino acid at position 379 is Asn;
the amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
the amino acid at position 396 is Leu;
The amino acid at position 421 is Lys;
the amino acid at position 427 is Asn;
The amino acid at position 428 is any one of Phe or Leu;
The amino acid at position 429 is Met;
the amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
[39] the method according to any one of [29] to [38], wherein the Fc region of a natural human IgG having a fucose-containing sugar chain as the sugar chain linked at position 297 in the EU numbering system is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
[40] The method according to any one of [29] to [39], wherein the human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
[41] the method according to any one of [29] to [39], wherein the human Fc γ receptor is Fc γ RIIb.
[42] the method according to any one of [36] to [41], wherein the Fc region contains at least one of the following amino acids at a position in the Fc region represented by the EU number:
The amino acid at position 238 is Asp, or
the amino acid at position 328 is Glu.
[43] a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) a step of obtaining the binding activity of the antigen-binding domain to an antigen under a high calcium ion concentration condition;
(b) A step of obtaining the antigen-binding activity of the antigen-binding domain under the condition of low calcium ion concentration;
(c) A step of selecting an antigen-binding domain having an antigen-binding activity obtained in (a) higher than that obtained in (b);
(d) a step of linking the polynucleotide encoding the antigen binding domain selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to the EU-numbering position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
[44] A method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) A step of obtaining the binding activity of the antibody to the antigen under the condition of high calcium ion concentration;
(b) a step of obtaining the antigen binding activity of the antibody under the condition of low calcium ion concentration;
(c) a step of selecting an antibody having an antigen binding activity obtained in (a) higher than that obtained in (b);
(d) A step of linking a polynucleotide encoding the antigen binding domain of the antibody selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range, to which the EU-position 297 is linked, is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) A step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
[45] a method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) A step of obtaining the binding activity of the antigen-binding domain to an antigen under a condition of a neutral range of pH;
(b) A step of obtaining an antigen binding activity of the antigen binding domain under a pH acidic range condition;
(c) a step of selecting an antigen-binding domain having an antigen-binding activity obtained in (a) higher than that obtained in (b);
(d) a step of linking the polynucleotide encoding the antigen binding domain selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to the EU-numbering position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
[46] A method for producing an antigen-binding molecule, which comprises the following steps (a) to (f):
(a) a step of obtaining the binding activity of an antibody to an antigen in a neutral pH range;
(b) A step of obtaining an antigen binding activity of an antibody to an antigen in a pH acidic range;
(c) a step of selecting an antibody having an antigen binding activity obtained in (a) higher than that obtained in (b);
(d) a step of linking a polynucleotide encoding the antigen binding domain of the antibody selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to the EU-numbering position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of the Fc region of a native human IgG containing fucose sugar chains;
(e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
[47] The production method according to any one of [43] to [46], wherein the antigen is a soluble antigen.
[48] the production method according to any one of [43] to [47], wherein the Fc γ receptor binding domain comprises an Fc region of an antibody.
[49] [48] the production method according to, wherein the Fc region is an Fc region in which at least one or more amino acids selected from the group consisting of the following amino acids are different from the amino acids at the corresponding positions in the natural-type Fc region at the positions indicated by the EU numbering in the Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits.
[50] [49] the production method according to, wherein the Fc region contains at least one amino acid selected from the group consisting of:
the amino acid at position 221 is any one of Lys or Tyr;
the amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
the amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 225 is any one of Glu, Lys, or Trp;
The amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
the amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
The amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 240 is any one of Ala, Ile, Met or Thr;
the amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
The amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
the amino acid at position 245 is Ala;
the amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
The amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
The amino acid at position 251 is Phe;
The amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
the amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
The amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
the amino acid at position 263 is any one of Ala, Ile, Met or Thr;
The 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 266 is any one of Ala, Ile, Met or Thr;
The amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 273 is any one of Phe or Ile;
The amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 275 is any one of Leu or Trp;
The amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
amino acid 279 is Ala;
the 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
The amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
The amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
the amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
The amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
the amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
The amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
the amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
The amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
The amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
the amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
The amino acid at the 303 th position is Asp, Gly or Tyr;
The amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
The amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
The amino acid at position 313 is Phe;
The amino acid at position 315 is Leu;
The amino acid at position 317 is Glu or Gln;
the amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 323 is Ile;
the amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
The amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
The amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
the amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
The amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
The amino acid at position 379 is Asn;
The amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
the amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
The amino acid at position 427 is Asn;
The amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
the amino acid at position 434 is Trp;
The amino acid at position 436 is Ile; and
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
[51] the production method according to any one of [43] to [50], wherein the Fc γ receptor binding domain is: the sugar chain linked at position 297 in the EU numbering system is an Fc region of any one of natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4 which contains fucose sugar chains.
[52] The production method according to any one of [43] to [51], wherein the human Fc γ receptor is Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), or Fc γ RIIIa (F).
[53] The production method according to any one of [43] to [51], wherein the human Fc γ receptor is Fc γ RIIb.
[54] The production method according to any one of [48] to [53], wherein the Fc region contains at least one of the following amino acids at a position in the Fc region represented by the EU number:
the amino acid at position 238 is Asp, or
The amino acid at position 328 is Glu.
drawings
FIG. 1 is a diagram showing an unlimited mechanism of action of eliminating soluble antigen from plasma by administering an antibody that binds to antigen with ion concentration dependence, which enhances Fc γ receptor binding at neutral pH, as compared with a conventional neutralizing antibody.
FIG. 2 is a graph showing the change in the concentration of human IL-6 receptor in the plasma of a human FcRn transgenic mouse to which H54/L28-IgG1 or Fv4-IgG1 which binds to the human IL-6 receptor pH-dependently was administered.
FIG. 3 is a graph showing changes in the concentration of human IL-6 receptor in the plasma of a human FcRn transgenic mouse when administered with Fv4-IgG1 which binds pH-dependently to the human IL-6 receptor, an altered form of Fv4-IgG1 which binds no mouse FcGamma R, i.e., Fv4-IgG1-F760, and an altered form of Fv4-IgG1 which enhances the binding of mouse FcGamma R, i.e., Fv4-IgG1-F1022 or a low fucose type antibody of Fv4-IgG1, i.e., Fv4-IgG 1-Fuc.
FIG. 4 is a graph showing changes in the concentration of human IL-6 receptor in the plasma of human FcRn transgenic mice when FcRn transgenic mice are administered with Fv4-IgG1-F1093, which is an FcRn binding protein having an increased pH acidic range and contains modifications of Fv4-IgG1, Fv4-IgG1-F1022 and Fv4-IgG1-F1022, as an antigen-binding molecule for the heavy chain.
FIG. 5 is a graph showing the change in concentration of antigen-binding molecules administered in the plasma of human FcRn transgenic mice when FcRn 4-IgG1-F1093, which is an FcRn binding protein having an increased pH range and is an altered form of Fv4-IgG1, Fv4-IgG1-F1022 and Fv4-IgG1-F1022, is administered as an antigen-binding molecule for the heavy chain to the mice.
FIG. 6 is a graph showing the change in the concentration of human IL-6 receptor in the plasma of human FcRn transgenic mice when administered with Fv4-IgG1, an altered form of Fv4-IgG1, i.e., Fv4-IgG1-F1087, which enhances mouse Fc γ R binding (particularly, enhanced binding to mouse Fc γ RIIb, mouse Fc γ RIII), and an altered form of Fv4-IgG1, i.e., Fv4-IgG1-F1182, which enhances mouse Fc γ R binding (particularly, enhanced binding to mouse Fc γ RI, mouse Fc γ RIV).
FIG. 7 is a graph showing the change in concentration of antigen-binding molecules administered in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1087, and the altered forms of Fv4-IgG1-F1087 that bind FcRn with an increased pH range, i.e., Fv4-IgG1-F1180, Fv4-IgG1-F1412, are administered to the mice.
FIG. 8 is a graph showing the change in concentration of antigen-binding molecules administered in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1182, and an altered form of Fv4-IgG1-F1182 that binds to FcRn with an increased pH range, i.e., Fv4-IgG1-F1181, are administered to the mice.
FIG. 9 is a graph showing the change in the concentration of human IL-6 receptor in the plasma of human FcRn transgenic mice when administered with Fv4-IgG1, Fv4-IgG1-F1087, and the modifications of Fv4-IgG1-F1087 that bind FcRn with an increased pH range, i.e., Fv4-IgG1-F1180, Fv4-IgG 1-F1412.
FIG. 10 is a graph showing the change in the concentration of human IL-6 receptor in the plasma of human FcRn transgenic mice when administered to the mice with Fv4-IgG1, Fv4-IgG1-F1182, and an altered form of Fv4-IgG1-F1182 that binds to FcRn with an increased pH range, i.e., Fv4-IgG 1-F1181.
FIG. 11 shows the results of concentration changes of Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 in the plasma of a human FcRn transgenic mouse when Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 is administered to the mouse.
FIG. 12 is a graph showing the results of changes in the concentration of soluble human IL-6 receptor in the plasma of human FcRn transgenic mice when Fv4-IgG1, Fv4-IgG1-F1782, or Fv4-IgG1-F1087 was administered to the mice.
FIG. 13 is a graph showing changes in the concentration of human IL-6 receptor in the plasma of a mouse when administered to normal mice with Fv4-mIgG1, an altered form of Fv4-mIgG1, i.e., Fv4-mIgG1-mF44, in which the binding to mouse Fc γ RIIb and mouse Fc γ RIII is enhanced, and an altered form of Fv4-mIgG1, i.e., Fv4-mIgG1-mF46, in which the binding to mouse Fc γ RIIb and mouse Fc γ RIII is further enhanced.
FIG. 14 is a graph showing changes in the concentration of human IL-6 receptor in the plasma of an Fc γ RIII-deficient mouse when administered with Fv4-mIgG1, an altered form of Fv4-mIgG1 in which binding to mouse Fc γ RIIb and mouse Fc γ RIII is enhanced, i.e., Fv4-mIgG1-mF44, and an altered form of Fv4-mIgG1 in which binding to mouse Fc γ RIIb and mouse Fc γ RIII is further enhanced, i.e., Fv4-mIgG1-mF 46.
FIG. 15 is a graph showing the change in the concentration of human IL-6 receptor in the plasma of an Fc receptor gamma chain-deficient mouse when administered to an Fc receptor gamma chain-deficient mouse with Fv4-mIgG1, an altered form of Fv4-mIgG1, i.e., Fv4-mIgG1-mF44, in which the binding to mouse Fc gamma RIIb, mouse Fc gamma RIII is enhanced, and an altered form of Fv4-mIgG1, i.e., Fv4-mIgG1-mF46, in which the binding to mouse Fc gamma RIIb, mouse Fc gamma RIII is further enhanced.
FIG. 16 is a graph showing the change in the concentration of human IL-6 receptor in the plasma of mice deficient in Fc γ RIIb when administered with Fv4-mIgG1, an altered form of Fv4-mIgG1, i.e., Fv4-mIgG1-mF44, in which the binding to mouse Fc γ RIIb and mouse Fc γ RIII is enhanced, and an altered form of Fv4-mIgG1, i.e., Fv4-mIgG1-mF46, in which the binding to mouse Fc γ RIIb and mouse Fc γ RIII is further enhanced.
FIG. 17 is a graph showing the results of evaluating the platelet aggregation ability of omalizumab-G1 d-v3/IgE immune complexes in a platelet aggregation assay using donor-derived platelets having Fc γ RIIa polymorphism (R/H).
FIG. 18 is a graph showing the results of evaluating the platelet aggregation ability of omalizumab-G1 d-v3/IgE immune complexes in a platelet aggregation assay using donor-derived platelets having Fc γ RIIa polymorphism (H/H).
FIG. 19 is a graph showing the results of evaluating the expression of CD62p on the membrane surface of washed platelets. The graph coated in black shows the results when the cells were stimulated by adding ADP after reacting with PBS, and the uncoated side in the center of the graph shows the results when the cells were stimulated with ADP after reacting with immunocomplexes.
FIG. 20 is a graph showing the results of evaluating the expression of active integrin on the membrane surface of washed platelets. The graph coated in black shows the results when the cells were stimulated by adding ADP after reacting with PBS, and the uncoated side in the center of the graph shows the results when the cells were stimulated with ADP after reacting with immunocomplexes.
[ FIG. 21] is a graph showing the results of evaluating the platelet aggregation ability of omalizumab-G1 d-v3/IgE immune complex and omalizumab-BP 230/IgE immune complex in a platelet aggregation assay using donor-derived platelets having Fc γ RIIa polymorphism (R/H).
FIG. 22 is a graph showing the results of evaluating the expression of CD62p on the membrane surface of washed platelets. The graph coated in gray shows the results when the cells were stimulated by adding ADP after reacting with PBS, the solid line shows the results when they were stimulated with omalizumab-G1 d-v3/IgE immunocomplexes, and the dotted line shows the results when they were stimulated with ADP after reacting with omalizumab-BP 230/IgE immunocomplexes.
FIG. 23 is a graph showing the results of evaluating the expression of active integrin on the membrane surface of washed platelets. The graph coated in gray shows the results when the cells were stimulated by adding ADP after reacting with PBS, the solid line shows the results when they were stimulated with omalizumab-G1 d-v3/IgE immunocomplexes, and the dotted line shows the results when they were stimulated with ADP after reacting with omalizumab-BP 230/IgE immunocomplexes.
[ FIG. 24] the horizontal axis represents the value of the relative binding activity of each PD variant to Fc γ RIIb, and the vertical axis represents the value of the relative binding activity of each PD variant to Fc γ RIIa R type. The value of the amount of binding of each PD variant to each Fc γ R was divided by the value of the amount of binding of the antibody IL6R-F652/IL6R-L (IL6R-F652 is an Fc-modified antibody heavy chain as defined in SEQ ID NO:142, containing a substitution of Pro at position 238 with Asp as indicated by the EU numbering) to each Fc γ R before the introduction of modification as a control, and the resultant value was multiplied by 100 times to obtain the value of the relative binding activity of each PD variant to each Fc γ R. The F652 dot plot in the figure shows the values for IL6R-F652/IL 6R-L.
FIG. 25 shows the values of the relative binding activity of the modified antibodies to Fc γ RIIb, which were obtained by introducing each modification into GpH7-B3(SEQ ID NO:159)/GpL16-k0(SEQ ID NO:160) without P238D modification, on the vertical axis, and the values of the relative binding activity of the modified antibodies to Fc γ RIIb, which were obtained by introducing each modification into IL6R-F652(SEQ ID NO:142)/IL6R-L with P238D modification, on the horizontal axis. The value of the amount of Fc γ RIIb bound by each of the modified antibodies was divided by the value of the amount of Fc γ RIIb bound by the antibody before the modification was introduced, and the resultant value was multiplied by 100 times to obtain the value of the relative binding activity. Here, the modification that the binding enhancing effect on Fc γ RIIb is exerted in both cases of introduction into GpH7-B3/GpL16-k0 without P238D and introduction into IL6R-F652/IL6R-L with P238D are contained in region A; the binding-enhancing effect on FcyRIIb is exhibited when the protein is introduced into GpH7-B3/GpL16-k0 without P238D, but the modification that the binding-enhancing effect on FcyRIIb is not exhibited when the protein is introduced into IL6R-F652/IL6R-L with P238D is contained in region B.
FIG. 26 shows the crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex.
FIG. 27 is a diagram showing the superposition of the crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex and the model structure of the Fc (WT)/Fc γ RIIb extracellular region complex by the least squares method based on the C.alpha.atom distance between the Fc γ RIIb extracellular region and Fc CH2 domain A.
FIG. 28 is a graph showing a crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex and a model structure of the Fc (WT)/Fc γ RIIb extracellular region complex, in which an Fc CH2 domain A and an Fc CH2 domain B were superimposed by the least squares method based on the C.alpha.atomic distance, respectively, and detailed structures in the vicinity of P238D were compared.
FIG. 29 is a diagram showing that, in the crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex, a hydrogen bond is observed between the backbone of Gly at position 237 expressed by EU numbering in Fc CH2 domain A and Tyr at position 160 in Fc γ RIIb.
FIG. 30 is a diagram showing that in the crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex, electrostatic interaction is observed between Asp at position 270 of Fc CH2 domain B, as indicated by EU numbering, and Arg at position 131 of Fc γ RIIb.
[ FIG. 31] the horizontal axis represents the relative binding activity of each 2B variant to Fc γ RIIb, and the vertical axis represents the relative binding activity of each 2B variant to Fc γ RIIa R-type. The value of the amount of binding of each 2B variant to each Fc γ R was divided by the value of the amount of binding of the antibody (altered Fc in which Pro at position 238 was substituted with Asp, as indicated by EU numbering) to each Fc γ R before the introduction of the alteration as a control, and the resultant value was multiplied by 100 times to obtain the value of the relative binding activity of each 2B variant to each Fc γ R.
FIG. 32 shows Glu at position 233 in the EU numbering of Fc chain A and residues around the Fc γ RIIb extracellular region in the crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex.
FIG. 33 shows Ala at position 330 of Fc chain A in the EU numbering system and residues around the Fc γ RIIb extracellular region in the crystal structure of the Fc (P238D)/Fc γ RIIb extracellular region complex.
FIG. 34 shows the structure of Pro at position 271 in the EU numbering of Fc chain B, which is obtained by superimposing the crystal structures of the Fc (P238D)/Fc γ RIIb extracellular region complex and the Fc (WT)/Fc γ RIIIa extracellular region complex by the least squares method based on the C.alpha.atomic distance with respect to Fc chain B.
FIG. 35 is a diagram of the Fc (P208)/Fc γ RIIb extracellular region complex determined by X-ray crystal structure analysis. The CH2 domain and the CH3 domain of the Fc portion are each domain a on the left side and domain B on the right side.
FIG. 36 is a graph in which the structure of the Fc (P208)/Fc γ RIIb extracellular region complex determined by X-ray crystal structure analysis and the structure of the Fc (WT)/Fc γ RIIa extracellular region complex (PDB code: 3RY6) were superimposed in the Fc portion CH2 domain A by the least squares method based on the C.alpha.atomic distance, and compared. In the figure, the portion drawn with thick lines is the structure of the Fc (P208)/Fc γ RIIb extracellular region complex, and the portion drawn with thin lines is the structure of the Fc (WT)/Fc γ RIIa extracellular region complex. In the structure of the Fc (WT)/Fc γ RIIa extracellular domain complex, only domain A of CH2 of the Fc portion is depicted.
FIG. 37 is a diagram showing the detailed structure of the Fc portion CH2 domain A having hydrogen bonds with Tyr at position 160 of Fc γ RIIb in the main chain portion in the vicinity of Asp at position 237 indicated by EU numbering in the X-ray crystal structure of the Fc (P208)/Fc γ RIIb extracellular region complex.
FIG. 38 is a diagram showing the structure of amino acid residues around the Asp side chain at position 237 in the EU numbering of domain A of Fc portion CH2, which is hydrogen-bonded to Tyr at position 160 of Fc γ RIIb in the main chain portion, in the X-ray crystal structure of the Fc (P208)/Fc γ RIIb extracellular region complex.
FIG. 39 is a graph showing the comparison of the X-ray crystal structure of Fc (P238D)/Fc γ RIIb extracellular region complex and the X-ray crystal structure of Fc (P208)/Fc γ RIIb extracellular region complex shown in example 10, which are superimposed on each other in domain B of Fc portion CH2 by the least squares method based on the C.alpha.atomic distance, and the circumferences of the rings from position 266 to position 271 shown by the EU numbers. In this loop, Fc (P208) has H268D changes at position 268, as indicated by EU numbering, and P271G changes at position 271, as indicated by EU numbering, compared to Fc (P238D).
FIG. 40 is a graph showing the structure around Ser239 in domain B of CH2 in the Fc portion, together with the electron density as a 2Fo-Fc coefficient obtained by X-ray crystal structure analysis, in the X-ray crystal structure of the Fc (P208)/Fc γ RIIb extracellular region complex.
FIG. 41 is a graph in which the three-dimensional structure of the Fc (P208)/Fc γ RIIaR extracellular region complex determined by X-ray crystal structure analysis and the three-dimensional structure of the Fc (P208)/Fc γ RIIb extracellular region complex were superimposed by the least squares method based on the C.alpha.atomic distance, and compared.
FIG. 42 is a graph comparing the X-ray crystal structure of the Fc (P208)/Fc γ RIIaR extracellular region complex with the X-ray crystal structure of the Fc (P208)/Fc γ RIIb extracellular region complex, in the vicinity of Asp at position 237 indicated by the EU number in domain A of the Fc portion CH2, with the electron density as a 2Fo-Fc coefficient obtained by X-ray crystal structure analysis.
FIG. 43 is a graph comparing the X-ray crystal structure of the Fc (P208)/Fc γ RIIaR extracellular region complex with the X-ray crystal structure of the Fc (P208)/Fc γ RIIb extracellular region complex at about Asp at position 237 in the EU number of domain B of the Fc portion CH2 with the electron density as a 2Fo-Fc coefficient obtained by X-ray crystal structure analysis.
FIG. 44 is a graph comparing the constant region sequences of G1d and G4 d. In the figure, amino acids enclosed in a thick frame are shown at positions which are different amino acid residues in G1d and G4 d.
FIG. 45 is a graph showing the change in antibody concentration in plasma of GA2-IgG1 and GA2-F1087 in normal mice.
FIG. 46 is a graph showing the change in plasma hIgA concentration in normal mice administered with GA2-IgG1 and GA 2-F1087.
FIG. 47 is a graph showing the change in antibody concentration in plasma of 278-IgG1 and 278-F1087 in C57BL/6J mice.
FIG. 48 is a graph showing the change in plasma hIgE (Asp6) concentration in C57BL/6J mice administered with 278-IgG1 and 278-F1087.
FIG. 49 is a diagram showing the structure of the heavy chain CDR3 of the Fab fragment of the 6RL #9 antibody, as determined by X-ray crystal structure analysis. (i) Heavy chain CDR3 showing the crystal structure obtained under crystallization conditions in the presence of calcium ions. (ii) Heavy chain CDR3 showing the crystal structure obtained under crystallization conditions in the absence of calcium ions.
FIG. 50 is a graph showing the change in the concentration of each antibody in the plasma of normal mice administered with H54/L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL #9-IgG1 antibody.
FIG. 51 is a graph showing the change in the concentration of soluble human IL-6 receptor (hsIL-6R) in the plasma of normal mice administered with H54/L28-IgG1 antibody, FH4-IgG1 antibody, and 6RL #9-IgG1 antibody.
FIG. 52 is a view showing an ion exchange chromatogram of an antibody containing the sequence of human Vk5-2 and an antibody containing the sequence of hVk5-2_ L65 in which the sequence of the sugar chain addition in the sequence of human Vk5-2 is changed. The solid line represents the chromatogram of an antibody having the sequence of human Vk5-2 (heavy chain: CIM _ H, SEQ ID NO:67, and light chain: hVk5-2, SEQ ID NO:4), and the dotted line represents the chromatogram of an antibody having the sequence of hVk5-2_ L65 (heavy chain: CIM _ H (SEQ ID NO:67), light chain: hVk5-2_ L65(SEQ ID NO: 70)).
FIG. 53A is an ion exchange chromatogram of an antibody having the sequence of LfVk1_ Ca (heavy chain: GC _ H, SEQ ID NO:51, and light chain: LfVk1_ Ca, SEQ ID NO:83) and an antibody having a sequence in which the Asp (D) residue in the sequence of LfVk1_ Ca is changed to an Ala (A) residue after storage at 5 ℃ (solid line) or after storage at 50 ℃ (dotted line). The peak of each ion exchange chromatogram after storage at 5 ℃ was defined as the main peak and normalized on the y-axis using the main peak. Is a figure showing a chromatogram of an antibody containing LfVk1_ Ca (SEQ ID NO:83) as a light chain.
FIG. 53B is a diagram showing a chromatogram of an antibody containing LfVk1_ Ca1(SEQ ID NO:85) as a light chain.
FIG. 53C is a diagram showing a chromatogram of an antibody containing LfVk1_ Ca2(SEQ ID NO:86) as a light chain.
FIG. 53D is a diagram showing a chromatogram of an antibody containing LfVk1_ Ca3(SEQ ID NO:87) as a light chain.
FIG. 54A is an ion exchange chromatogram of an antibody comprising the sequence of LfVk1_ Ca (heavy chain: GC _ H, SEQ ID NO:51, and light chain: LfVk1_ Ca, SEQ ID NO:83) and an antibody comprising the sequence of LfVk1_ Ca6 with the Asp (D) residue at position 30 (Kabat numbering) in the sequence of LfVk1_ Ca changed to a Ser (S) residue (heavy chain: GC _ H, SEQ ID NO:51, and light chain: LfVk1_ Ca6, SEQ ID NO:88) after storage at 5 ℃ (solid line) or after storage at 50 ℃ (dashed line). The peak of each ion exchange chromatogram after storage at 5 ℃ was defined as the main peak and normalized on the y-axis using the main peak. Is a figure showing a chromatogram of an antibody containing LfVk1_ Ca (SEQ ID NO:83) as a light chain.
FIG. 54B is a diagram showing a chromatogram of an antibody containing LfVk1_ Ca6(SEQ ID NO:88) as a light chain.
FIG. 55 is a graph showing the relationship between the amino acid profile (denoted as Library) of 290 clones isolated from E.coli into which a gene Library of antibodies that bind to antigens in a Ca-dependent manner was introduced, and the designed amino acid profile (denoted as Design). The horizontal axis indicates the position of the amino acid indicated by Kabat numbering. The vertical axis represents the amino acid distribution ratio.
FIG. 56 is a graph showing sensorgrams of an anti-IL-6R antibody (toclizumab), a 6RC1IgG _010 antibody, a 6RC1IgG _012 antibody, and a 6RC1IgG _019 antibody under a high calcium ion concentration condition (1.2 mM). The horizontal axis shows time and the vertical axis shows RU value.
FIG. 57 is a graph showing sensorgrams of an anti-IL-6R antibody (toclizumab), a 6RC1IgG _010 antibody, a 6RC1IgG _012 antibody, and a 6RC1IgG _019 antibody under a low calcium ion concentration condition (3. mu.M). The horizontal axis shows time and the vertical axis shows RU value.
FIG. 58 is a graph showing the relationship between the amino acid profile (denoted as Library) of 132 clones isolated from E.coli into which a gene Library of antibodies that bind to antigens in a pH-dependent manner was introduced and the designed amino acid profile (denoted as Design). The horizontal axis indicates the position of the amino acid indicated by Kabat numbering. The vertical axis represents the amino acid distribution ratio.
FIG. 59 is a diagram showing sensorgrams of an anti-IL-6R antibody (tollizumab), a 6RPH #01 antibody, a 6RPH #02 antibody and a 6RPH #03 antibody at pH 7.4. The horizontal axis shows time and the vertical axis shows RU value.
FIG. 60 is a diagram showing sensorgrams of an anti-IL-6R antibody (tollizumab), a 6RPH #01 antibody, a 6RPH #02 antibody and a 6RPH #03 antibody at pH 6.0. The horizontal axis shows time and the vertical axis shows RU value.
FIG. 61A is a graph showing ECL responses to native Fc and modified Fc, sera isolated from 15 or 30 independent rheumatic patients. Graphs respectively show graphs of ECL responses to native Fc (fig. 61A) Fv4-YTE (fig. 61B), Fv4-F1166(═ YTE + Q438R/S440E) (fig. 61C), Fv4-F1167(═ YTE + S424N) (fig. 61D), Fv4-LS (fig. 61E), Fv4-F1170(═ LS + Q438R/S440E) (fig. 61F), Fv4-F1171(═ LS + S424N) (fig. 61G), Fv4-N434H (fig. 61H), Fv4-F1172(═ N434H + Q438R/S440E) (fig. 61I), Fv4-F1173(═ N434H + S424N) (fig. 61J).
FIG. 61B is a continuation of FIG. 61A.
FIG. 61C is a continuation of FIG. 61B.
FIG. 61D is a continuation of FIG. 61C.
FIG. 61E is a continuation of FIG. 61D.
FIG. 61F is a continuation of FIG. 61E.
FIG. 61G is a continuation of FIG. 61F.
FIG. 61H is a continuation of FIG. 61G.
FIG. 61I is a continuation of FIG. 61H.
FIG. 61J is a continuation of FIG. 61I.
FIG. 62A is a graph showing ECL reactions to serum isolated from 30 independent rheumatic patients with altered Fc. The graphs show the ECL reactions to Fv4-LS (FIG. 62A), Fv4-F1380 (FIG. 62B), Fv4-F1384 (FIG. 62C), Fv4-F1385 (FIG. 62D), Fv4-F1386 (FIG. 62E), Fv4-F1388 (FIG. 62F), and Fv4-F1389 (FIG. 62G), respectively.
FIG. 62B is a continuation of FIG. 62A.
FIG. 62C is a continuation of FIG. 62B.
FIG. 62D is a continuation of FIG. 62C.
FIG. 62E is a continuation of FIG. 62D.
FIG. 62F is a continuation of FIG. 62E.
FIG. 62G is a continuation of FIG. 62F.
Detailed Description
the following definitions and detailed description are provided to facilitate understanding of the invention described in this specification.
Amino acids
In the present specification, for example, amino acids are labeled with a single-letter code or a three-letter code or both as represented by Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F, Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G, Trp/W, His/H, Tyr/Y, Ile/I, Val/V.
amino acid changes
for changing the amino acid in the amino acid sequence of the antigen-binding molecule, a known method such as the site-specific mutagenesis method (Kunkel et al (Proc. Natl. Acad. Sci. USA (1985)82,488-492)) or overlap extension PCR can be suitably used. The addition, deletion and/or substitution of amino acids can be carried out by any of these known methods as appropriate. The substitution of an amino acid residue means that the substitution is carried out for the purpose of changing, for example, the following (a) to (c) by substitution into another amino acid residue:
(a) a backbone structure of the polypeptide in a region of a fold structure or a helix structure;
(b) Charge or hydrophobicity in the target site; or
(c) The size of the side chain.
amino acid residues are classified into the following groups according to the characteristics of the side chains they contain in their structure:
(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;
(3) acidity: asp and Glu;
(4) Alkalinity: his, Lys, Arg;
(5) Residues that influence chain orientation: gly, Pro; and
(6) aromatic property: trp, Tyr, Phe.
such substitutions of amino acid residues within each group are referred to as conservative substitutions, while substitutions of amino acid residues between different groups are referred to as non-conservative substitutions. The substitution in the present invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution. As a method for changing an amino acid by substitution with an amino acid other than a natural amino acid, various known methods (annu.rev.biophysis.biomol.struct. (2006)35,225-249, proc.natl.acad.sci.u.s.a. (2003)100(11),6353-6357) can be used. It is also possible to suitably use, for example: and a cell-free translation system (Protein Express) in which a tRNA obtained by linking an unnatural amino acid to a complementary amber suppressor tRNA containing a UAG codon (amber codon) that is one of the stop codons is used.
Further, as a expression indicating an amino acid change, it is possible to suitably adopt: the expression of the one-letter code of the amino acid before and after the change is used before and after the number indicating the specific position. For example, when an amino acid substitution is added to the Fc region contained in the antibody constant region, the change in P238D used indicates a substitution of Pro at position 238 to Asp as indicated by EU numbering. That is, the numbers indicate the positions of amino acids represented by EU numbering, the one-letter codes of the amino acids described before indicate the amino acids before substitution, and the one-letter codes of the amino acids described after indicate the amino acids after substitution.
And/or
in the present specification, the meaning of the term "and/or" means a combination of the preceding and following terms of the idiom "and/or" and includes all combinations of "and" with "or" as appropriate. Specifically, for example, "the amino acids at position 326, 328 and/or 428 are substituted" includes the following altered variations of the amino acids:
(a)326 bits, (b)328 bits, (c)428 bits, (d)326 bits and 328 bits, (e)326 bits and 428 bits, (f)328 bits and 428 bits, and (g)326 bits, 328 bits and 428 bits.
antigens
in the present specification, the structure of the "antigen" is not limited to a specific structure as long as it contains an epitope bound by the antigen binding domain. In other words, the antigen may be an inorganic substance or an organic substance, and is preferably a soluble antigen that exists in body fluid of the body in a state that the antigen-binding molecule of the present invention can bind thereto. Examples of the antigen include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF 1a, 8-iso-PGF 2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE-2, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, activin RIBALK-4, activin RIIA, activin RIIB, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS4, ADAMTS5, addressen (addressen), aFGF, ALCAM, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonists, ANG, Ang, APAF-1, ARC, APJ, APART, APL, ARL, ARALV-1, ASI3842, ASIb-M, ASIcB-1/TAB, ADA-2, ADAM-III, ADAM-I, ADAM-II, ADAM, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE-1, Bad, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, B-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP-2a, BMP-3, osteogenic protein (Osteogenin), BMP-4, BMP-2B, BMP-5, BMP-6, Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, BMRPK-1, BMPR-II (BRK-3), BMP, B-NGF, BOK, derived neurotrophic factor (ByS), bone-stimulating factor (ByS), BACE-1, BAD, BIM-BLC, BIM, BLC, BMC, BM, BPDE, BPDE-DNA, BTC, complement factor 3 (C), C3, C5, C, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK, CCL/10, CCR, CD11, CD, CCL, CC, CD11, CD27, CD30, CD (p protein), CD40, CD49, CD66, CD (B-1), CD123, CD137, CD138, CD140, CD146, CD147, CD148, CD152, CD164, CEACAM, CFTR, cGMP, CINC, botulinum toxin, Clostridium Perfringens (Clostridia Perfringens) toxin, CKb-1, CLC, CMV, CMVUL, COXF, CNTN-1, CXCR, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL, CXCR 3CR, CXCL, CXCR, CXCL, CXCR, CXCL, CXCR, CXCL, CXCR, CXCL, DC-SIGN, complement inhibitory factor (decay promoting factor), des (1-3) -IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, neprilysin, eOS, Eot, Eotaxin 1, CAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, Fibroblast Activation Protein (FAP), Fcs, Fas, 1, Flt-1, ferritin, FECX-19, FGF-2, FGF3, FGF-8, FGFR-3, fibrin, FL, FLIP-3, FLIP-I, chemotactic factor eXC-4, Flt-C, Flt-A, factor II, EMR-A, EMR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF-1, GDF-3(Vgr-2), GDF-5(BMP-14, CDMP-1), GDF-6(BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8(Myostatin ), GDF-9, GDF-15(MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Gl 4, glycoprotein b/IIIa (GPICSF/NIIb), GM-46Ib, gp72, GRO-P, half growth factor, HCP-release hormone, HCP, or EGF-c, HCMV gH envelope glycoprotein, HCMV UL, Hematopoietic Growth Factor (HGF), Hep Bgp120, heparanase, Her2, Her2/neu (ErbB-2), Her3(ErbB-3), Her4(ErbB-4), Herpes Simplex Virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120V 3 loop, HLA-DR, HM1.24, HMFG, HRG, Hrk, human cardiac myosin, Human Cytomegalovirus (HCMV), Human Growth Hormone (HGH), HVEM, I-309, IAP, ICAM-1, ICAM-3, IgE, ICOS, IFNg, IgA, Ig receptor, IGF, Ig, IGF binding protein, IGF-IGF 1, FBR, IGF-1, IGF-IL-R, IGF-52, IL-R-1, IGF-I I, IGF, IGF-IL-II, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18R, IL-23, Interferon (INF) - α, INF- β, INF- γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α 2, integrin α 3, integrin α 4/β 1, integrin α 4/β 7, integrin α 5(α V), integrin α 5/β 1, integrin α 5/β 3, integrin α 6, integrin β 1, integrin β 2, Interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP (TGF-1), latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Ltn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, surface hormone, luteinizing hormone, lymphotropic receptor 1-1, lymphotropic receptor 1-Ltn, Lptn, LfT, LtA-Y antigen, and so, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP-1-alpha, MK, MMAC1, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, mullerian inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N-C adhesion factor, NCA90, NCAM, enkephalinase, neurotrophin-3, neurotrophin-4 or neurotrophin-6, Neurturin, NGF-1-alpha, NGF, MMP-3, MPIF, MMP-11, MMP-6, and NGF, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, P150, P95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulosin, relaxin, protein C, PS, PSCA, Prostate Specific Membrane Antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, syncytial virus (RSV), RSV 483F, RSV-5, RSV-S, RSV-1, RSV-S, RSV-5, RSV-S, RSV-1, RSV-S, RSV-1, RSV-S, RSV-1, RSV, PSK, SPARC, Stat, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF-alpha, TGF-beta Pan specificity (TGF-beta Pan specificity), TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, EFTMF 2, Tmpo, TMPRSS2, TNF, TNFa-alpha, TNFa-beta, TNF-C, TNF-beta-2, TNFa-beta, TNF-gamma, TNF-, TNF-RII, TNFRSF10 (TRAIL Apo-2, DR), TNFRSF10 (TRAIL DR, KILLER, TRICK-2-B), TNFRSF10 (TRAIL DcR, LIT, TRID), TNFRSF10 (TRAIL DcR, TRUNDD), TNFRSF11 (RANK R, TRANCE R), TNFRSF11 (OPGOCIF, TR), TNFRSF (TWEAK R FN), TNFRSF13 (TACI), TNFRSF13 (BABARR), TNFRSF (HVATAR, HveA, LIGHT R, TR), TNFRSF (NGFR 75NTR), TNFRSF (BCMA), TNFRSF (GITR AITRR), TNFRSF (TNFROY TAJ, TRADE), TNFRSF19 (RELT), TNFRSF1 (TNFRRI CD120, p-60), TNFRSF1 (FasriF 120, TNFRSR-80, TNFRSF (ApoRSP), TNFRSF (TNFRSF) 1 (TNFRSF 4, TNFRSF (TNFRSF) 1, TNFRSF (TNFRSF-III), TNFRSF (TNFRSF) and TNFRSF (TNFRSF 4, TNFRSF) as, ILA), TNFRSF21(DR6), TNFRSF6 (DcTRAIL R6 TNFRH 6), TNFRST 6 (DcTRAIL R1TNFRH 6), TNFRSF6 (DR6 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFRSF6 (TRAIL Apo-2 ligand, TL6), TNFRSF6 (TRANCE/RANK ligand ODF, OPG ligand), TNFRSF6 (TWEAK Apo-3 ligand, DR6 ligand), TNFRSF6 (6), TNFRSF13 6 (BAFF, TALL 6, THANK, TNFRSF 6), TNFRSF6 (6 ligand, 6), TNFRSF6 (TL1 6/VEGI ligand), TNFRSF6 (GITR ligand, TNFRSF TFTL 36TL 6), TNFRSF1 FasF 6 (TNFSF-a), TNFRSF6 (CONSF, DIF 6), TNFRSF6 (TNFRSF) ligand, TNFRSF7, TNFSF6 (TNFSF 6) ligand, TNFSK 6, TNFSK ligand, TNFSF6 (TNFSF-6) ligand, TNFSK ligand, TNFSF6 (TNFSK ligand, TNFSK 6) ligand, TNFSF6 (TNFSK 6 (TNFSF 6) ligand, TNFSF6 (TNFSF 6, TNFSK ligand, TNFSK 6 (TNFSK ligand, TNFSF, TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing virus Y-associated carbohydrate, TWEAK, TXB2, Ung, uPAR-1, urokinase, VCAM-1, VECAD, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGFR-3(flt-4), VEGI, VIM, viral antigen, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2B/13, WNT3, WNT3A, XCT 4, XCT 595, XCT 599, WNT 869, 3B, 368672, 3627, 3646 XT-B, 3646T-B, 3646 XT-B, 3627, B, 3627, 3695 XT-B, 3627, B, 3627, 3695, 369, WNT-X-3, WNT-3, and, XIAP, XPD, HMGB1, IgA, Abeta, CD81, CD97, CD98, DDR 98, DKK 98, EREG, Hsp 98, IL-17/IL-17 98-20/IL-20R, oxidized LDL, PCSK 98, prokininogen, RON, TMEM16 98, SOD 98, chromogranin A, chromogranin B, tau, VAP 98, polymeric kininogen, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, VIIC 98, C1 98, C72, C2 98, C3, C72, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, acetylcholine receptor, AdipoR1, AdipoR2, ADPribosyl cyclase-1, alpha-4/beta-7 integrin, alpha-5/beta-1 integrin, alpha-v/beta-6 integrin, alpha/beta 1 integrin, angiopoietin ligand-2, Angptl2, anthrax, cadherin, carbonic anhydrase-IX, CD105, CD155, CD158a, CD37, CD 3849 49b, CD51, CD70, CD 387, CD2, anthrax, cadherin, CD105, CD155, CD a, TFPI-3, TPI, TPM, EPCR, PPI, TPA, PPI, TP, CD72, Claudin 18, Clostridium difficile toxin, CS1, delta-like protein ligand 4, DHICA oxidase, Dickkopf-1 ligand, dipeptidyl peptidase IV, EPOR, RSV F protein, factor Ia, FasL, folate receptor alpha, glucagon receptor, glucagon-like peptide 1 receptor, glutamate carboxypeptidase II, GMCSFR, hepatitis C virus E2 glycoprotein, Hepcidin (Hepcidin), IL-17 receptor, IL-22 receptor, IL-23 receptor, IL-3 receptor, Kit tyrosine kinase, leucine rich a-2 glycoprotein 1 (63G 1), lysosphingolipid receptor, membrane glycoprotein OX2, Mesothelin (Mesothelin), MET, MICA, MUC-16, myelin-related glycoprotein LR, neuropilin-1, neuropilin-2, Nogo receptor, PLXNA 23, PLXNA2, PLXNA3, PLXNA 634, PLXNB 5394, PLXNB 464, PLXNB 4662, PLXNB 3884, PLXNB 4642, PLXNB3, PLXNB3, PLXNB3, PLXNB3, PLXN 3, and PLXN 3, The proprotein convertase PC9, P-selectin glycoprotein ligand-1, RAGE, Reticulon 4, RF, RON-8, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SEMA4A, SEMA4B, SEMA4C, SEMA4D, SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, SEMA 6957 2, Shiga-like toxin II, 1-phosphosphingosine receptor-1, ST2, staphylococcal lipoteichoic acid, tenascin, TG2, thymic stromal lymphopoietin receptor, TNF transmembrane glycoprotein family receptor 12A, TSNMB-1, TREM-862, TREM-receptor, TREB 2, and a trophoblast 2, which are soluble in and are used to anchor soluble receptors in body fluids. Among receptors, for example, soluble antigens which are present in body fluids of the body due to any mechanism including digestion with protease, such as receptors expressed on the cell surface, are also suitable as the soluble antigens of the present invention. Examples of such molecules include soluble IL-6R molecules described in the present specification (J.Immunol. (1994)152,4958-4968), CD20, CD52(Br.J.Haematol. (2003)123(5),850-857), and the like. In addition, soluble antigens that exist in body fluids of the body using not only molecules expressed inherently in the body but also infectious organisms such as viruses, antigens presented by these organisms, or infectious molecules belonging to prion proteins and the like can be exemplified as the soluble antigens of the present invention. The body fluid may suitably include: and body fluids such as blood, plasma, serum, urine, lymph, saliva, and tears.
epitope
by epitope, which denotes an antigenic determinant present in an antigen, is meant a site on an antigen to which an antigen binding domain in an antigen binding molecule disclosed in the present specification binds. Thus, for example, an epitope can be defined by its structure. Furthermore, the epitope may also be defined with respect to the antigen binding activity in an antigen binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope may be defined by amino acid residues constituting the epitope. When the epitope is a sugar chain, the epitope may be defined by a specific sugar chain structure.
a linear epitope is an epitope that contains an epitope that is recognized by the primary sequence of amino acids. Linear epitopes typically contain at least 3 amino acids in the native sequence, and most commonly at least 5, e.g., about 8 to about 10, 6 to 20 amino acids.
a stereostructural epitope is an epitope in which the primary sequence of amino acids comprising the epitope is not a single specified component of the epitope being recognized, as opposed to a linear epitope (e.g., the primary sequence of amino acids need not be recognized by an antibody to the specified epitope). The stereostructural epitope may contain an increased number of amino acids relative to the linear epitope. With respect to recognition of a steric epitope, an antibody recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule is folded to form a three-dimensional structure, certain amino acids and/or polypeptide backbones that form the epitope of the three-dimensional structure become juxtaposed so that the epitope can be recognized by an antibody. Methods of determining the stereo structure of an epitope include, for example: x-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electron paramagnetic resonance spectroscopy, but are not limited thereto. For example, see Epitope Mapping Protocols in methods in Molecular Biology (1996), Vol.66, Morris (eds.).
binding Activity
The following examples show the method of confirming the binding to an epitope by a test antigen-binding molecule containing an antigen-binding domain to IL-6R, but the method of confirming the binding to an epitope by a test antigen-binding molecule containing an antigen-binding domain to an antigen other than IL-6R can also be suitably carried out based on the following examples.
for example, containing the IL-6R antigen binding domain of the test antigen binding molecules in the presence of IL-6R molecules in the linear epitope recognition, can be achieved by the following operation to confirm. For the above purpose, a linear peptide containing an amino acid sequence constituting the extracellular domain of IL-6R was synthesized. The peptide may be chemically synthesized. Alternatively, the cDNA for IL-6R can be obtained by genetic engineering techniques using a region encoding an amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide comprising the amino acid sequence constituting the extracellular domain and the antigen-binding molecule to be tested comprising the antigen-binding domain against IL-6R was evaluated. For example, the binding activity of the antigen-binding molecule to an immobilized linear peptide can be evaluated by ELISA using the peptide as an antigen. Alternatively, the binding activity to a linear peptide can be clarified based on the level of inhibition of the linear peptide in the binding of the antigen-binding molecule to an IL-6R-expressing cell. Through these tests, the binding activity of the antigen-binding molecule to a linear peptide can be clarified.
Furthermore, the antigen binding molecules to be tested, which contain an antigen binding domain for IL-6R, recognize a steric epitope, which can be confirmed as follows. For the above purpose, IL-6R-expressing cells were prepared. Examples thereof include: and a case where the antigen-binding molecule to be tested, which contains an antigen-binding domain for IL-6R, strongly binds to an IL-6R-expressing cell when the cell is contacted with the antigen-binding molecule, but the antigen-binding molecule does not substantially bind to an immobilized linear peptide containing an amino acid sequence constituting the extracellular domain of IL-6R. Here, the term "substantially non-binding" means a binding activity of 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less, of the binding activity on human IL-6R-expressing cells.
Examples of a method for measuring the binding activity to IL-6R-expressing cells containing a test antigen-binding molecule directed against the antigen-binding domain of IL-6R include: a method described in Antibodies A Laboratory Manual (EdHarlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, the evaluation can be performed by ELISA or FACS (fluorescence activated cell sorting) using IL-6R-expressing cells as antigens.
In the ELISA format, the binding activity of IL-6R-expressing cells containing a test antigen-binding molecule directed against the antigen-binding domain of IL-6R was quantitatively evaluated by comparing the level of signal generated by the enzyme reaction. That is, the antigen-binding molecule to be detected is added to an ELISA plate on which IL-6R-expressing cells are immobilized, and the antigen-binding molecule to be detected that binds to the cells is detected using an enzyme-labeled antibody that recognizes the antigen-binding molecule to be detected. Alternatively, in FACS, dilution series of the antigen-binding molecule to be tested are prepared, and the binding potency (titer) of the antibody to IL-6R-expressing cells is determined, whereby the binding activity of the antigen-binding molecule to be tested on IL-6R-expressing cells can be compared.
The binding of the antigen-binding molecule to be detected to the antigen expressed on the surface of the cell suspended in a buffer or the like can be detected by flow cytometry. As a flow cytometer, for example, the following devices are known:
FACSCantoTM II
FACSAriaTM
FACSArrayTM
FACSVantageTM SE
FACSCalibur (both trade names of BD Biosciences)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
cell Lab Quanta/Cell Lab Quanta SC (both trade names of Beckman Coulter).
For example, the following methods can be mentioned as examples of preferred methods for measuring the antigen binding activity of a test antigen-binding molecule comprising an antigen binding domain to IL-6R. First, IL-6R-expressing cells are reacted with the antigen-binding molecule to be detected and stained with FITC-labeled secondary antibody that recognizes the antigen-binding molecule to be detected. The antigen binding molecule to be tested is diluted with a suitable, preferably buffered solution, whereby the antigen binding molecule is prepared to the desired concentration for use. For example, it can be used at any concentration between 10. mu.g/ml and 10 ng/ml. Subsequently, the fluorescence intensity and the cell number were measured by FACSCalibur (BD Co.). The amount of antibody bound to the CELLs was reflected in the geometric mean value of fluorescence intensity analyzed using CELL QUEST Software (BD). That is, by obtaining the geometric mean, the binding activity of the antigen-binding molecule to be detected, which is represented by the amount of binding of the antigen-binding molecule to be detected, can be measured.
The antigen binding molecule to be tested, which contains an antigen binding domain for IL-6R, shares an epitope with an antigen binding molecule, as can be confirmed by competition of the two for the same epitope. Competition between antigen-binding molecules is detected by a cross-blocking assay or the like. For example, a competitive ELISA assay is a preferred cross-blocking assay.
specifically, in a cross-blocking assay, the IL-6R protein coated onto the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen binding molecule, and the antigen binding molecule to be tested is added. The amount of the antigen binding molecule to be tested that binds to the IL-6R protein in the well is indirectly related to the binding ability of the candidate competing antigen binding molecule that competes for binding to the same epitope. That is, the greater the affinity of the competing antigen binding molecule for the same epitope, the less active the test antigen binding molecule will bind to a pore coated with the IL-6R protein.
the amount of the antigen binding molecule to be detected bound to the pore via the IL-6R protein can be readily determined by pre-labelling the antigen binding molecule. For example, biotin-labeled antigen-binding molecules are assayed by using an avidin peroxidase conjugate and a suitable substrate. Cross-blocking assays using enzymatic labels such as peroxidase are particularly known as competitive ELISA assays. The antigen binding molecules can be labeled with other labeling substances that can be detected or measured. Specifically, a radioactive label, a fluorescent label, or the like is known.
the competing antigen binding molecule is an antigen binding molecule that binds to substantially the same epitope as the competing antigen binding molecule or that competes for binding to the same epitope as the competing antigen binding molecule if it blocks binding of at least 20%, preferably at least 20-50%, more preferably at least 50%, of the antigen binding molecule to be tested that comprises an antigen binding domain for IL-6R, as compared to the binding activity obtained in a control assay performed in the absence of the candidate competing antigen binding molecule.
in identifying the structure of an epitope bound by a test antigen-binding molecule containing an antigen-binding domain against IL-6R, the test antigen-binding molecule shares an epitope with a control antigen-binding molecule, and the binding activity of the antigen-binding molecules to a peptide obtained by introducing an amino acid mutation into a peptide constituting the epitope can be evaluated by comparing the binding activities of the two antigen-binding molecules.
as a method for measuring such a binding activity, for example, in the ELISA format described above, the binding activity of the antigen-binding molecule to be measured and the binding activity of the control antigen-binding molecule to the linear peptide into which a mutation has been introduced are compared. In a method other than ELISA, the binding activity to the mutant peptide bound to the column can be measured by allowing the antigen-binding molecule to be measured and the control antigen-binding molecule to flow through the column and then quantifying the amount of the antigen-binding molecule eluted from the eluate. Methods of adsorbing a mutant peptide to a column as a fusion peptide with GST, for example, are known.
Furthermore, when the epitope identified is a stereo epitope, the antigen binding molecule to be tested shares an epitope with the control antigen binding molecule, which can be evaluated by the following method. First, IL-6R-expressing cells and IL-6R-expressing cells into which mutations have been introduced into epitopes were prepared. These cells are suspended in an appropriate buffer such as PBS, and the antigen-binding molecule to be tested and the control antigen-binding molecule are added to the resulting cell suspension. Next, washing with an appropriate buffer, and adding to the resulting cell suspension an FITC-labeled antibody capable of recognizing the antigen-binding molecule to be detected and the control antigen-binding molecule. The fluorescence intensity and cell number of the cells stained with the labeled antibody were measured by FACSCalibur (BD). The concentrations of the antigen-binding molecule to be tested and the control antigen-binding molecule are appropriately diluted with an appropriate buffer solution, and thus can be prepared to the desired concentrations for use. For example, it can be used at any concentration between 10. mu.g/ml and 10 ng/ml. The amount of labeled antibody bound to the CELLs was reflected in the geometric mean value of the fluorescence intensity analyzed by CELL QUEST Software (BD). That is, by obtaining the geometric mean, the binding activity of the antigen-binding molecule to be measured and the antigen-binding molecule to be controlled, which are represented by the amount of binding of the labeled antibody, can be measured.
in the present method, for example, "substantially not binding to the mutant IL-6R-expressing cells" can be determined by the following method. First, the test antigen-binding molecule bound to cells expressing the mutant IL-6R and the control antigen-binding molecule are stained with a labeled antibody. Subsequently, the fluorescence intensity of the cells is detected. When FACSCalibur is used as a flow cytometer for fluorescence detection, the obtained fluorescence intensity can be analyzed by CELL QUEST Software. The ratio of increase in fluorescence intensity due to binding of the antigen-binding molecule can be determined by calculating the comparison value (. DELTA.geo-Mean) from the geometric Mean values in the presence and absence of the antigen-binding molecule according to the following calculation formula.
Δ Geo-Mean (in the presence of an antigen binding molecule)/Geo-Mean (in the absence of an antigen binding molecule)
The geometric Mean comparison value (mutant IL-6R molecule. DELTA. Geo-Mean value) obtained by the analysis, which reflects the amount of binding of the antigen-binding molecule to the mutant IL-6R-expressing cells, was compared with the. DELTA. Geo-Mean comparison value, which reflects the amount of binding of the antigen-binding molecule to the IL-6R-expressing cells. In this case, the concentrations of the antigen-binding molecules to be tested used for calculating the Δ Geo-Mean comparison values with respect to the mutant IL-6R-expressing cells and the IL-6R-expressing cells are particularly preferably prepared to be the same or substantially the same concentrations as each other. Antigen binding molecules that are pre-determined to recognize epitopes in IL-6R are used as control antigen binding molecules.
a test antigen-binding molecule is considered to be "substantially not bound to a mutant IL-6R-expressing cell" as long as the comparison value for Δ Geo-Mean of the test antigen-binding molecule with respect to the mutant IL-6R-expressing cell is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the comparison value for Δ Geo-Mean of the test antigen-binding molecule with respect to the IL-6R-expressing cell. The formula for determining the Geo-Mean (geometric Mean) is described in CELL QUEST Software User's Guide (BD biosciences). By comparing the comparison values, it can be evaluated that the epitope of the antigen-binding molecule to be tested is the same as that of the control antigen-binding molecule, as long as the comparison values are substantially the same.
Antigen binding domains
In the present specification, the "antigen binding domain" may be any domain as long as it binds to a target antigen. Preferred examples of such domains include, for example: variable regions of heavy and light chains of an antibody, a module called an A domain of about 35 amino acids contained in a cell membrane protein Avimer existing in the body (WO2004/044011, WO2005/040229), an Adnectin containing a 10Fn3 domain which is a protein binding domain in glycoprotein fibronectin expressed in cell membranes (WO2002/032925), an Affibody (WO1995/001937) having as a scaffold an IgG binding domain of 3 helix bundles (bundles) containing 58 amino acids constituting protein A, a DARPins (derived anti proteins) having a structure in which a turn containing 33 amino acid residues and 2 antiparallel helices (AR) overlap with a subunit Repeat of a loop, a conserved reverse barrel lipid structure (such as a truncated antipyrin protein) (WO2002/020565) supporting neutrophil gelatinase-binding lipid-carrying protein (neutrophil gelatinase-associated protein) in the direction of a central barrel lipid conserved lipid in the order (NGAL 8) molecule And a depressed region of a parallel sheet structure inside a horseshoe-shaped structure formed by overlapping repeated leucine-rich-repeat (LRR) elements of a Variable Lymphocyte Receptor (VLR) which is a jawbreaker that acquires an immune system and does not have an immunoglobulin structure, which is a jawbreaker such as lamprey or haganus, and which is a unipod-free loop region of (4) single-sided loop regions of (WO2003/029462) (WO 2008/016854). Preferred examples of the antigen-binding domain of the present invention include antigen-binding domains comprising the variable regions of the heavy and light chains of an antibody. Examples of such an antigen-binding domain preferably include "scFv (single-chain Fv)", "single-chain antibody", "Fv", "scFv 2 (single-chain Fv 2)", "Fab" and "F (ab') 2".
The antigen binding domains in the antigen binding molecules of the invention may bind to the same epitope. Here, the same epitope may be present in, for example, a protein having the amino acid sequence shown in SEQ ID NO. 1. Furthermore, it can be present in a protein comprising the amino acids 20 to 365 of the amino acid sequence shown in SEQ ID NO. 1. Alternatively, the antigen binding domains in the antigen binding molecules of the invention may bind to epitopes that are different from each other. Here, different epitopes may be present in, for example, a protein comprising the amino acid sequence shown in SEQ ID NO. 1. Furthermore, it can be present in a protein comprising amino acids 20 to 365 of the amino acid sequence shown in SEQ ID NO. 1.
specificity of
Specificity refers to a state in which one of the specifically bound molecules does not exhibit any significant binding to a molecule other than one or more of the binding partner molecules. Furthermore, the present invention can also be applied to a case where the antigen-binding domain is specific to a specific epitope among a plurality of epitopes contained in a certain antigen. In addition, when the epitope bound by the antigen binding domain is contained in a plurality of different antigens, the antigen binding molecule having the antigen binding domain can bind to the various antigens containing the epitope.
antibodies
in the present specification, an antibody refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. The antibody may be isolated from a natural source such as plasma or serum in which the antibody naturally exists or from a culture supernatant of a hybridoma cell producing the antibody, or may be partially or completely synthesized by using a method such as gene recombination. Examples of the antibody include: isotypes of immunoglobulins and subclasses of these isotypes. As human immunoglobulins, 9 types (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM are known. The antibody of the present invention may contain IgG1, IgG2, IgG3, and IgG4 among these isotypes. The IgG constant region also contains naturally occurring mutants thereof. A plurality of allotypic Sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242, as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and the present invention may be any of these Sequences. In particular, the amino acid sequence at positions 356 to 358 in EU numbering is DEL or EEM, which is the sequence of human IgG 1.
Methods for preparing antibodies with desired binding activity are well known to those skilled in the art. The following is an example of a method for producing an antibody that binds to IL-6R (anti-IL-6R antibody). Antibodies that bind to antigens other than IL-6R can also be prepared as appropriate according to the following examples.
The anti-IL-6R antibody can be obtained in the form of a polyclonal or monoclonal antibody using a known method. As the anti-IL-6R antibody, a monoclonal antibody derived from a mammal can be preferably produced. Monoclonal antibodies derived from mammals contain: monoclonal antibodies produced by hybridomas, monoclonal antibodies produced by host cells transformed with expression vectors containing antibody genes using genetic engineering techniques, and the like. The monoclonal antibody of the present invention includes "humanized antibody" and "chimeric antibody".
Hybridomas that produce monoclonal antibodies can be produced by using known techniques, for example, the following methods. That is, mammals are immunized by a general immunization method using the IL-6R protein as a sensitizing antigen. The obtained immune cells are fused with known parental cells by a usual cell fusion method. Next, the monoclonal antibody-producing cells are screened by a usual screening method, whereby hybridomas producing an anti-IL-6R antibody can be selected.
Specifically, the monoclonal antibody is produced, for example, as follows. First, the IL-6R gene whose nucleotide sequence is disclosed in SEQ ID NO. 2 is expressed, whereby the IL-6R protein represented by SEQ ID NO. 1, which is used as a sensitizing antigen for antibody preparation, can be obtained. That is, a gene sequence encoding IL-6R is inserted into a known expression vector, thereby transforming an appropriate host cell. The desired human IL-6R protein is purified from the host cells or culture supernatant by known methods. In order to obtain a soluble form of IL-6R from the culture supernatant, instead of the IL-6R protein represented by SEQ ID NO:1, for example, a soluble form of IL-6R as described by Mullberg et al (J.Immunol. (1994)152(10),4958-4968), i.e., a protein containing amino acids 1 to 357 in the IL-6R polypeptide sequence represented by SEQ ID NO:1, was expressed. In addition, purified native IL-6R protein can also be used as a sensitizing antigen.
As a sensitizing antigen for immunizing mammals, the purified IL-6R protein can be used. In addition, partial peptides of IL-6R may also be used as sensitizing antigens. In this case, the partial peptide can also be obtained by chemical synthesis based on the amino acid sequence of human IL-6R. Alternatively, the expression vector may be obtained by integrating a part of the IL-6R gene into an expression vector and expressing the integrated gene. Further, it can also be obtained by decomposing IL-6R protein with a protease, and the region and size of the IL-6R peptide used as a partial peptide are not particularly limited to a specific embodiment. As a preferred region, any sequence can be selected from the amino acid sequences corresponding to amino acids 20 to 357 in the amino acid sequence of SEQ ID NO. 1. The number of amino acids constituting the peptide as the sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50, preferably 10 to 30 residues can be used as the sensitizing antigen.
Furthermore, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide may be used as the sensitizing antigen. For preparing a fusion protein used as a sensitizing antigen, for example, an Fc fragment of an antibody, a peptide tag, or the like can be preferably used. The vector for expressing the fusion protein can be prepared as follows: the genes encoding the desired two or more polypeptide fragments are fused in frame, and the fused genes are inserted into an expression vector as described above. A method for producing a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed.,9.47-9.58(1989) Cold Spring Harbor Lab. press). Methods for obtaining IL-6R used as a sensitizing antigen and immunization methods using the same are also specifically described in WO2003/000883, WO2004/022754, WO2006/006693, and the like.
The mammal immunized with the sensitizing antigen is not limited to a specific animal, and is preferably selected in consideration of compatibility with a parent cell used for cell fusion. Usually, rodent animals such as mice, rats, hamsters or rabbits, monkeys, etc. are suitable.
the above animals are immunized with the sensitizing antigen according to a known method. For example, as a general method, immunization is carried out by administering a sensitizing antigen into the abdominal cavity or subcutaneously of a mammal by injection. Specifically, the sensitizing antigen diluted with PBS (Phosphate-Buffered Saline) or physiological Saline at an appropriate dilution ratio is mixed with a usual adjuvant, for example, freund's complete adjuvant, as necessary, and the sensitizing antigen is administered to the mammal several times every 4 to 21 days after emulsification. In addition to this, the present invention is,for immunization with the sensitizing antigen, an appropriate carrier may be used. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, albumin or keyhole limpet may be preferably usedthe sensitizing antigen peptide bound to a carrier protein such as hemocyanin is used for immunization.
furthermore, hybridomas producing desired antibodies can also be produced by using DNA immunization as follows. DNA immunization refers to the following immunization methods: in an immunized animal to which a vector DNA constructed so that a gene encoding an antigen protein can be expressed in the immunized animal is administered, a sensitizing antigen is expressed in the body of the immunized animal, thereby imparting immunostimulation. Compared with a conventional immunization method in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages:
capable of maintaining the structure of membrane proteins such as IL-6R to confer immunostimulation
Without purification of the immunizing antigen.
in order to obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing IL-6R protein is administered to the immunized animal. The DNA encoding IL-6R can be synthesized by a known method such as PCR. The resulting DNA is inserted into an appropriate expression vector and administered to an immunized animal. As the expression vector, a commercially available expression vector such as pcDNA3.1 can be preferably used. As a method for administering the vector to the body, a method generally used can be employed. For example, DNA immunization is carried out by introducing gold particles having an expression vector adsorbed thereto into cells of an individual immunized animal using a gene gun. Furthermore, an antibody recognizing IL-6R can be prepared by the method described in International publication WO 2003/104453.
after the mammal was immunized in this manner and the increase in the antibody titer binding to IL-6R in the serum was confirmed, immune cells were collected from the mammal and cell fusion was performed. As a preferred immune cell, spleen cells can be used in particular.
as the cell to be fused with the above immune cell, a mammalian myeloma cell can be used. Myeloma cells are preferably provided with an appropriate selection marker for selection. Selectable marker refers to a property that confers to a cell the ability to survive (or die) under specific culture conditions. Among the selection markers, hypoxanthine-guanine-phosphoribosyltransferase deletion (hereinafter abbreviated as HGPRT deletion) and thymidine kinase deletion (hereinafter abbreviated as TK deletion) are known. Cells with HGPRT and TK deletions have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter referred to as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die, but when fused with normal cells, continue DNA synthesis using salvage pathways of normal cells, and therefore grow in HAT selection medium.
HGPRT-deleted, TK-deleted cells can each be selected by a medium containing 6-thioguanine, 8-azaguanine (hereinafter, referred to as 8AG) or 5' -bromodeoxyuridine. Normal cells with these pyrimidine analogs incorporated into the DNA die. Cells lacking the enzyme that are unable to integrate the pyrimidine analogs can survive in the selection medium. In addition, a selection marker known as G418 resistance can confer resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) through the neomycin resistance gene. Various myeloma cells preferred in cell fusion are known.
As such myeloma cells, P3(P3x63Ag8.653) (J.Immunol. (1979)123(4),1548-1550), P3x63Ag8U.1(Current Topics in Microbiology and Immunology (1978)81,1-7), NS-1(C.Eur.J.Immunol. (1976)6(7),511-519), MPC-11(Cell (1976)8(3),405-415), SP2/0(Nature (1978)276(5685),269-270), FO (J.Immunol. methods (1980)35(1-2),1-21), S194/5.XX0.BU.1(J.Exp.Med. (1978)148(1),313-323), R (210 (1979)277, 133-56133), etc. can be suitably used.
Basically, the aforementioned cell fusion of the immunocytes and myeloma cells is carried out according to a known method, for example, the method of Kohler and Milstein et al (Methods Enzymol. (1981)73,3-46), etc.
more specifically, for example, the cell fusion can be carried out in a normal nutrient medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG), sendai virus (HVJ), etc. are used as fusion promoters, and further, in order to improve the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide is added, if desired.
The ratio of the immune cells to the myeloma cells to be used can be arbitrarily set. For example, it is preferable to make the immune cells 1 to 10-fold relative to myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium and MEM culture medium suitable for the growth of the myeloma cell line, and a normal culture medium used for the cell culture are used, and a serum replacement solution such as Fetal Calf Serum (FCS) may be preferably added.
for cell fusion, predetermined amounts of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution (for example, having an average molecular weight of about 1000 to 6000) heated to about 37 ℃ in advance is usually added at a concentration of 30 to 60% (w/v). The mixture is slowly mixed to form the desired fused cells (hybridomas). Then, by adding appropriate culture media listed above in order and repeating the operation of removing the supernatant by centrifugation, cell fusion agents that are unfavorable for the growth of hybridomas can be removed.
the hybridoma thus obtained can be selected by culturing in a normal selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). The culture using the HAT medium described above may be continued for a time sufficient for the desired cells other than hybridomas (non-fused cells) to die (generally, the sufficient time is several days to several weeks). Next, screening and monoclonality of hybridomas producing desired antibodies are performed by a usual limiting dilution method.
the hybridoma thus obtained can be selected by using a selection medium corresponding to a selection marker possessed by myeloma used for cell fusion. For example, cells with HGPRT or TK deletions can be selected by culturing with HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells successfully fused with normal cells can selectively grow in HAT culture medium. The culture using the HAT medium can be continued for a time sufficient for the desired cells other than hybridomas (non-fused cells) to die. Specifically, the desired hybridoma can be selected usually by culturing for several days to several weeks. Second, screening and monoclonality of hybridomas producing desired antibodies can be performed by a usual limiting dilution method.
The screening and monoclonality of the desired antibody can be preferably carried out by a known screening method based on an antigen-antibody reaction. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the surface of cells. Such monoclonal antibodies can be screened by, for example, FACS (fluorescence activated cell sorting). FACS is a system that can measure the binding of antibodies to the cell surface by analyzing cells contacted with fluorescent antibodies with a laser and measuring the fluorescence emitted by each cell.
in order to screen hybridomas producing the monoclonal antibody of the present invention by FACS, IL-6R-expressing cells are first prepared. Preferably, the cells used for screening are mammalian cells that are forced to express IL-6R. As a control, by using an untransformed mammalian cell used as a host cell, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting a hybridoma that produces an antibody that does not bind to a host cell but binds to an IL-6R-forced expression cell, a hybridoma that produces an IL-6R monoclonal antibody can be obtained.
Alternatively, the binding activity of the antibody to immobilized IL-6R-expressing cells can be assessed based on the principle of ELISA. For example, IL-6R-expressing cells are immobilized in wells of an ELISA plate. The culture supernatant of the hybridoma is brought into contact with the immobilized cells in the well, and the antibody bound to the immobilized cells is detected. Where the monoclonal antibody is derived from a mouse, the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce a desired antibody having antigen-binding ability selected by these screening can be cloned by a limiting dilution method or the like.
the monoclonal antibody-producing hybridoma thus produced can be subcultured in a normal culture medium. In addition, the hybridomas can be stored in liquid nitrogen for a long period of time.
The desired monoclonal antibody can be obtained from the culture supernatant of the hybridoma by culturing the hybridoma according to a usual method. Alternatively, the hybridoma can be administered to a mammal compatible therewith and grown to obtain the monoclonal antibody from ascites thereof. The former method is suitable for obtaining an antibody of high purity.
An antibody encoded by an antibody gene cloned from an antibody-producing cell such as the hybridoma can also be used as appropriate. The cloned antibody gene is integrated into an appropriate vector and introduced into a host, whereby the antibody encoded by the gene is expressed. Methods for the isolation of antibody genes, introduction into vectors, and transformation of host cells have been established, for example, by Vandamm et al (Eur.J.biochem. (1990)192(3), 767-775). Further, a method for producing a recombinant antibody as described below is also known.
for example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells producing the anti-IL-6R antibody. For this purpose, total RNA is usually first extracted from the hybridoma. As a method for extracting mRNA from cells, for example, the following methods can be used:
guanidine ultracentrifugation (Biochemistry (1979)18(24),5294-5299)
AGPC method (anal. biochem. (1987)162(1), 156-159).
The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GE Healthcare Bioscience) or the like. Alternatively, a Kit for directly extracting total mRNA from cells is also commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Bioscience). Using this kit, mRNA can be obtained from hybridomas. A cDNA encoding the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized by using AMV reverse transcription First-strand cDNA Synthesis Kit (manufactured by Biochemical industries, Ltd.). For the synthesis and amplification of cDNA, 5' -RACE method using SMART RACE cDNA amplification kit (Clontech) and PCR (Proc. Natl. Acad. Sci. USA (1988)85(23),8998-9002, Nucleic Acids Res (1989)17(8),2919-2932) can be suitably used. Furthermore, suitable restriction enzyme sites to be described later can be introduced into both ends of the cDNA during the synthesis of the cDNA.
The target cDNA fragment was purified from the resulting PCR product, followed by ligation with vector DNA. After the recombinant vector is prepared and introduced into Escherichia coli or the like in this manner, and the selected colony is selected, a desired recombinant vector can be prepared from the Escherichia coli forming the colony. Whether or not the recombinant vector has the base sequence of the target cDNA can be confirmed by a known method such as the dideoxynucleotide chain termination method.
In order to obtain a gene encoding a variable region, it is convenient to use the 5' -RACE method in which a primer for variable region gene amplification is used. First, cDNA was synthesized using RNA extracted from hybridoma cells as a template to obtain a 5' -RACE cDNA library. A commercially available kit such as SMART RACE cDNA amplification kit can be suitably used for the synthesis of the 5' -RACE cDNA library.
the obtained 5' -RACE cDNA library was used as a template to amplify an antibody gene by a PCR method. Based on the known antibody gene sequence, primers for amplifying the mouse antibody gene can be designed. These primers have different base sequences depending on the subclass of immunoglobulin. Therefore, it is desirable to use a commercially available kit such as the Iso Strip mouse monoclonal antibody isotype typing kit (Roche Diagnostics) to determine the subclasses in advance.
specifically, for example, in order to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ 1, γ 2a, γ 2b, and γ 3 as heavy chains, and a κ chain and a λ chain as light chains can be used. For amplifying variable region genes of IgG, primers that anneal to a portion corresponding to a constant region near the variable region are generally used as primers on the 3' -side. The 5 '-side primer used was a primer attached to the 5' -RACE cDNA library preparation kit.
using the PCR product thus amplified, immunoglobulins composed of a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin against IL-6R is used as an index, and a desired antibody can be screened. For example, in order to obtain antibodies against IL-6R, it is further preferred that the antibodies are specific for binding to IL-6R. Antibodies that bind to IL-6R can be screened, for example, as follows:
(1) a step of contacting an antibody containing a V region encoded by cDNA obtained from the hybridoma with an IL-6R-expressing cell;
(2) a step of detecting binding of the IL-6R-expressing cell to the antibody; and
(3) A step of selecting an antibody that binds to the IL-6R-expressing cell.
methods for detecting binding of an antibody to IL-6R-expressing cells are well known. Specifically, the binding of the antibody to the IL-6R-expressing cells can be detected by FACS or the like as described above. For the evaluation of the binding activity of the antibody, a fixed sample of IL-6R-expressing cells can be suitably used.
As a method for screening an antibody using a binding activity as an index, a panning method using a phage vector can be suitably used. When the antibody gene is obtained from a polyclonal antibody-expressing cell population in the form of a library of subclasses of heavy and light chains, it is advantageous to use a screening method using a phage vector. The genes encoding the variable regions of the heavy and light chains may form a single chain Fv (scFv) by ligation using appropriate linker sequences. By inserting a gene encoding scFv into a phage vector, a phage expressing scFv on the surface can be obtained. After the phage is contacted with the desired antigen, the phage that binds to the antigen is recovered, and DNA encoding scFv having the target binding activity can be recovered. By repeating this operation as necessary, the scFv having the desired binding activity can be concentrated.
after obtaining a target cDNA encoding the V region of the anti-IL-6R antibody, the cDNA is digested by recognizing restriction enzymes inserted into restriction enzyme sites at both ends of the cDNA. Preferred restriction enzymes recognize and digest a base sequence that appears less frequently in the base sequences constituting the antibody gene. Furthermore, in order to insert 1 copy of the digested fragment into the vector in the correct orientation, it is preferable to insert a restriction enzyme that provides a cohesive end. The antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by in-frame fusion of a gene encoding the constant region (C region) of an antibody and a gene encoding the V region. Here, the term "chimeric antibody" means that the constant region and the variable region are derived from different sources. Therefore, in addition to mouse-human xenochimeric antibodies, human-human allochimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector having a constant region in advance. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme digesting the V region gene can be appropriately placed on the 5' side of an expression vector holding a DNA encoding a constant region (C region) of a desired antibody. The two digested with the same combination of restriction enzymes were fused in-frame, thereby constructing a chimeric antibody expression vector.
for the preparation of anti-IL-6R monoclonal antibodies, the antibody genes are integrated into expression vectors in such a way that expression takes place under the control of expression regulatory regions. The expression control region for expressing the antibody contains, for example, an enhancer and a promoter. In addition, a suitable signal sequence may be added at the amino terminus to allow secretion of the expressed antibody outside the cell. In the examples described later, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO:3) was used as the signal sequence, and an appropriate signal sequence may be added thereto. The expressed polypeptide is cleaved at the carboxy-terminal portion of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, a suitable host cell is transformed with the expression vector, whereby a recombinant cell expressing a DNA encoding an anti-IL-6R antibody can be obtained.
To express the antibody gene, DNAs encoding the heavy chain (H chain) and light chain (L chain) of the antibody are integrated into different expression vectors, respectively. Antibody molecules having both H and L chains can be expressed by co-transforming (co-transfect) the same host cell with a vector incorporating both H and L chains. Alternatively, the DNA encoding the H chain and the L chain may be integrated into a single expression vector and used to transform a host cell (see International publication WO 1994/011523).
many combinations of host cells and expression vectors for producing antibodies by introducing isolated antibody genes into appropriate hosts are known. These expression systems can be used to isolate the antigen binding domains of the invention. When eukaryotic cells are used as host cells, animal cells, plant cells or fungal cells can be suitably used. Specifically, the following cells can be exemplified as the animal cells.
(1) mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, HEK (human embryonic kidney) 293, FreestyleTM293 etc
(2) an amphibian cell: xenopus oocytes and the like
(3) insect cells: sf9, sf21, Tn5, and the like.
Alternatively, as plant cells, there are known antibody gene expression systems based on cells derived from a tobacco (Nicotiana) genus such as tobacco (Nicotiana tabacum). For transformation of plant cells, cells cultured from callus may be used as appropriate.
Further, as the fungal cell, the following cells can be used:
-yeast: genus Saccharomyces such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) and genus Pichia such as Pichia pastoris (Pichia pastoris)
-filamentous fungi: aspergillus (Aspergillus) such as Aspergillus niger.
furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be suitably used. An expression vector containing a gene for the target antibody is introduced into these cells by transformation. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells.
In addition to the above host cells, transgenic animals can also be used for the production of recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding a desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by in-frame insertion into the interior of a gene encoding a protein inherently produced in milk. As the protein secreted into milk, for example, goat β casein or the like can be used. The DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a ewe. The desired antibody can be obtained as a fusion protein with a milk protein in the milk produced by a transgenic goat born from a goat receiving the embryo (or its offspring). In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be administered to the transgenic goat (Bio/Technology (1994),12(7), 699-702).
when the antigen-binding molecule described in the present specification is administered to a human, an antigen-binding domain derived from a genetically recombinant antibody artificially modified for the purpose of reducing heterologous antigenicity to a human can be suitably used as an antigen-binding domain in the molecule. Recombinant antibodies include, for example, Humanized antibodies and the like. These altered antibodies can be suitably prepared using known methods.
The antibody variable region used to prepare the antigen binding domain of the antigen binding molecule described in the present specification is generally composed of 3 complementarity-determining regions (CDRs) held by 4 Framework Regions (FRs). CDRs are regions that substantially determine the binding specificity of an antibody. The amino acid sequences of the CDRs are rich in diversity. On the other hand, the amino acid sequences constituting the FRs often show high identity even among antibodies having different binding specificities. Thus, it is generally considered that the binding specificity of an antibody can be grafted to another antibody by grafting of CDRs.
humanized antibodies are also known as reshaped (reshaped) human antibodies. Specifically, humanized antibodies and the like in which CDRs of an animal other than a human, for example, a mouse antibody are grafted onto a human antibody are known. General genetic recombination methods for obtaining humanized antibodies are also known. Specifically, as a method for grafting CDRs of a mouse antibody into human FRs, for example, overlap extension PCR is known. In the overlap extension PCR, a base sequence encoding the CDR of the mouse antibody to be grafted is added to a primer for synthesizing the FR of the human antibody. Primers were prepared for each of the 4 FRs. In general, it is considered advantageous to graft mouse CDRs onto human FRs, and to select human FRs having high identity with mouse FRs, in order to maintain the CDR functions. That is, it is generally preferable to use a human FR that contains an amino acid sequence having high identity with the amino acid sequence of the adjacent FR of the mouse CDR to be grafted.
in addition, the linked base sequences are designed to be linked in frame with each other. Human FRs were synthesized by each primer alone. As a result, a product obtained by adding DNA encoding mouse CDR to each FR was obtained. The base sequences of each product encoding the mouse CDR were designed to overlap each other. Subsequently, the overlapping CDR portions of the product synthesized using the human antibody gene as a template are annealed to each other, and a complementary strand synthesis reaction is performed. By this reaction, human FRs are linked by mouse CDR sequences.
The final V region gene, which is obtained by ligating 3 CDRs and 4 FRs, is amplified over its entire length by primers which anneal to the 5 'and 3' ends thereof and have an appropriate restriction enzyme recognition sequence added thereto. A vector for expression of a human antibody can be prepared by inserting the DNA obtained as described above and a DNA encoding a human antibody C region into an expression vector so as to perform in-frame fusion. After the integration vector is introduced into a host to establish a recombinant cell, the recombinant cell is cultured to express a DNA encoding the humanized antibody, thereby producing the humanized antibody in a culture of the cultured cell (see European patent publication EP239400, International publication WO 1996/002576).
by qualitatively or quantitatively measuring and evaluating the antigen binding activity of the humanized antibody produced as described above, the human antibody FR can be appropriately selected so that the CDR forms a good antigen binding site when it is ligated by CDR. If necessary, the amino acid residues of the FR may also be substituted in such a manner that the CDRs of the human antibody are reconstructed to form a suitable antigen-binding site. For example, mutations of amino acid sequences can be introduced into FRs by PCR method used for grafting mouse CDRs into human FRs. Specifically, a mutation of a partial base sequence may be introduced into a primer that anneals to an FR. The FR synthesized by such a primer has a mutation in its base sequence introduced therein. By measuring and evaluating the antigen binding activity of the amino acid-substituted mutant antibody by the above-described method, a mutant FR sequence having desired properties can be selected (cancer Res., (1993)53, 851-856).
Furthermore, a transgenic animal having the entire composition of human antibody genes (see International publications WO1993/012227, WO1992/003918, WO1994/002602, WO1994/025585, WO1996/034096, WO1996/033735) is used as an immunized animal, and a desired human antibody can be obtained by DNA immunization.
Further, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed on the surface of a phage by phage display in the form of a single chain antibody (scFv). Phages expressing scFv that bind to the antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After the DNA sequence of the scFv that binds to the antigen is determined, the V region sequence is fused in frame with the desired human antibody C region sequence, and then inserted into an appropriate expression vector, thereby producing an expression vector. The human antibody can be obtained by introducing the expression vector into the above-listed suitable expression cells and expressing the gene encoding the human antibody. These methods are already known (see International publications WO1992/001047, WO1992/020791, WO1993/006213, WO1993/011236, WO1993/019172, WO1995/001438, WO 1995/015388).
in addition to the above, methods for obtaining antibody genes, such as Bernasconi and the like (Science (2002)298,2199-2202) or B cell cloning described in WO2008/081008 (identification and cloning of coding sequences of each antibody, isolation thereof, and use for constructing expression vectors for producing each antibody (in particular, IgG1, IgG2, IgG3, or IgG4) can be suitably used.
EU and Kabat numbering
according to the method used in the present invention, the amino acid positions of the CDRs and FRs assigned to an antibody are in accordance with Kabat's regulations (Sequences of Proteins of Immunological Interest (National Institute of health, Bethesda, Md.,1987 and 1991).
condition of ion concentration
Condition of metal ion concentration
In one embodiment of the present invention, the ion concentration refers to a metal ion concentration. The "metal ion" refers to an ion belonging to group I such as an alkali metal other than hydrogen and a copper group, group II such as an alkaline earth metal and a zinc group, group III other than boron, group IV other than carbon and silicon, group VIII such as an iron group and a platinum group, and each of subgroup a of groups V, VI and VII, and a metal element such as antimony, bismuth, polonium. The metal atom has the property of releasing an electron of atomic valence to form a cation, which is called ionization tendency. It is considered that metals having a large ionization tendency are chemically active.
examples of the metal ion preferable in the present invention include calcium ion. Calcium ions are involved in the regulation of many life phenomena, and calcium ions are involved in the contraction of muscles such as skeletal muscle, smooth muscle and cardiac muscle, the activation of leukocyte motility and phagocytosis, the activation of platelet deformation and secretion, etc., the activation of lymphocytes, the activation of mast cells such as histamine secretion, etc., the cellular response mediated by catecholamine α receptors or acetylcholine receptors, exocytosis, the release of transmitters from neuronal terminals, the axial plasma flow of neurons, etc. As intracellular calcium ion receptors, troponin C, calmodulin, microalbumin, myosin light chain, and the like, which have a plurality of calcium ion binding sites and are considered to be derived from common origins in molecular evolution, and the binding motifs thereof are also known in large numbers. For example, the cadherin domain, the EF hand contained in calmodulin, the C2 domain contained in protein kinase C, the Gla domain contained in coagulation protein factor IX, the C-type lectin contained in asialoglycoprotein receptor or mannose binding receptor, the a domain contained in LDL receptor, annexin, thrombospondin type 3 domain and EGF-like domain are also well known.
In the present invention, when the metal ion is a calcium ion, the calcium ion concentration conditions include a low calcium ion concentration condition and a high calcium ion concentration condition. The binding activity varies depending on the calcium ion concentration condition, which means that the antigen binding activity of the antigen-binding molecule varies depending on the conditions of low calcium ion concentration and high calcium ion concentration. For example, the antigen binding activity of the antigen-binding molecule under the condition of high calcium ion concentration is higher than the antigen binding activity of the antigen-binding molecule under the condition of low calcium ion concentration. In addition, the antigen binding activity of the antigen-binding molecule under the condition of a low calcium ion concentration is higher than the antigen binding activity of the antigen-binding molecule under the condition of a high calcium ion concentration.
In the present specification, the high calcium ion concentration is not particularly limited to a uniform value, and may be a concentration preferably selected from between 100. mu.M and 10 mM. Furthermore, in other embodiments, it may be a concentration selected from between 200. mu.M and 5 mM. Furthermore, in a different embodiment, a concentration of between 500. mu.M and 2.5mM is possible, and in other embodiments a concentration of between 200. mu.M and 2mM is also possible. Furthermore, a concentration between 400. mu.M and 1.5mM is also possible. Particularly preferably, the concentration is selected from the range of 500. mu.M to 2.5mM which is close to the calcium ion concentration in plasma (blood) in the body.
in the present specification, the low calcium ion concentration is not particularly limited to a uniform value, and may be a concentration preferably selected from between 0.1. mu.M and 30. mu.M. In addition, in other embodiments, it may be a concentration selected from between 0.2. mu.M and 20. mu.M. Furthermore, in various embodiments, the concentration may be selected from a range of 0.5. mu.M to 10. mu.M, and in other embodiments, the concentration may be selected from a range of 1. mu.M to 5. mu.M. Furthermore, the concentration may be selected from the range of 2. mu.M to 4. mu.M. Particularly preferably, the concentration is selected from the range of 1. mu.M to 5. mu.M which is close to the ionized calcium concentration in the early endosome in the living body.
in the present invention, the antigen-binding activity under the condition of low calcium ion concentration is lower than that under the condition of high calcium ion concentration means that the antigen-binding activity of the antigen-binding molecule at a calcium ion concentration selected from between 0.1. mu.M and 30. mu.M is weaker than that at a calcium ion concentration selected from between 100. mu.M and 10 mM. Preferably means that the antigen binding activity of the antigen binding molecule at a calcium ion concentration selected between 0.5 μ M and 10 μ M is weaker than the antigen binding activity at a calcium ion concentration selected between 200 μ M and 5mM, particularly preferably means that the antigen binding activity at a calcium ion concentration in early in vivo is weaker than the antigen binding activity at a calcium ion concentration in plasma in vivo, in particular means that the antigen binding activity of the antigen binding molecule at a calcium ion concentration selected between 1 μ M and 5 μ M is weaker than the antigen binding activity at a calcium ion concentration selected between 500 μ M and 2.5 mM.
Whether the antigen-binding activity of the antigen-binding molecule changes depending on the metal ion concentration condition can be determined by using, for example, a known measurement method described in the above-mentioned item of binding activity. For example, in order to confirm that the antigen-binding activity of the antigen-binding molecule becomes higher under the condition of low calcium ion concentration than under the condition of high calcium ion concentration, the antigen-binding activities of the antigen-binding molecule under the conditions of low calcium ion concentration and high calcium ion concentration are compared.
in the present invention, the expression "the antigen-binding activity under the condition of a low calcium ion concentration is lower than the antigen-binding activity under the condition of a high calcium ion concentration" may be expressed such that the antigen-binding activity of the antigen-binding molecule under the condition of a high calcium ion concentration is higher than the antigen-binding activity under the condition of a low calcium ion concentration. In the present invention, "the antigen binding activity under the condition of low calcium ion concentration is lower than that under the condition of high calcium ion concentration" may be described as "the antigen binding ability under the condition of low calcium ion concentration is weaker than that under the condition of high calcium ion concentration", and "the antigen binding activity under the condition of low calcium ion concentration is lower than that under the condition of high calcium ion concentration" may be described as "the antigen binding ability under the condition of low calcium ion concentration is weaker than that under the condition of high calcium ion concentration".
the conditions other than the calcium ion concentration for measuring the antigen-binding activity can be appropriately selected by those skilled in the art, and are not particularly limited. For example, the measurement can be carried out in HEPES buffer at 37 ℃. For example, measurement can be performed using Biacore (GE Healthcare) or the like. In the measurement of the binding activity between the antigen-binding molecule and the antigen, when the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through the chip on which the antigen-binding molecule is immobilized, and when the antigen is a membrane antigen, the binding activity to the membrane antigen can be evaluated by passing the antigen-binding molecule as an analyte through the chip on which the antigen is immobilized.
In the antigen-binding molecule of the present invention, as long as the antigen-binding activity under the condition of low calcium ion concentration is weaker than the antigen-binding activity under the condition of high calcium ion concentration, the ratio of the antigen-binding activity under the condition of low calcium ion concentration to the antigen-binding activity under the condition of high calcium ion concentration is not particularly limited, but the ratio of KD (Ca3 μ M)/KD (Ca2mM) between KD (Dissociation constant: Dissociation constant) under the condition of low calcium ion concentration and KD under the condition of high calcium ion concentration to an antigen is preferably 2 or more, more preferably the value of KD (Ca3 μ M)/KD (Ca2mM) is 10 or more, and still more preferably the value of KD (Ca3 μ M)/KD (Ca2mM) is 40 or more. The upper limit of the KD (Ca 3. mu.M)/KD (Ca2mM) value is not particularly limited, and may be any value such as 400, 1000, 10000, etc., as long as the technique of those skilled in the art can be made. In addition, the KD (Ca 3. mu.M)/KD (Ca 1.2mM) value can also be specified. That is, the KD (Ca 3. mu.M)/KD (Ca 1.2mM) value is 2 or more, more preferably the KD (Ca 3. mu.M)/KD (Ca 1.2mM) value is 10 or more, and still more preferably the KD (Ca 3. mu.M)/KD (Ca 1.2mM) value is 40 or more. The upper limit of the value of KD (Ca 3. mu.M)/KD (Ca 1.2mM) is not particularly limited, and may be any value such as 400, 1000, 10000, etc., as long as the technique of those skilled in the art can be made.
As the value of the antigen binding activity, KD (dissociation constant) can be used when the antigen is a soluble antigen, and Apparent KD (Apparent dissociation constant) can be used when the antigen is a membrane-type antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, and for example, Biacore (GE healthcare), Scatchard plot (Scatchard plot), flow cytometry, or the like can be used.
in addition, as another index showing the ratio of the antigen-binding activity under the condition of a low calcium concentration to the antigen-binding activity under the condition of a high calcium concentration of the antigen-binding molecule of the present invention, kd (Dissociation rate constant), which is an Dissociation rate constant, can also be suitably used, for example. When KD (dissociation rate constant) is used as an index showing the ratio of binding activities in place of KD (dissociation constant), the value of KD (low calcium concentration condition)/KD (high calcium concentration condition), which is the ratio of KD (dissociation rate constant) under the low calcium concentration condition to KD (dissociation rate constant) under the high calcium concentration condition with respect to the antigen, is preferably 2 or more, more preferably 5 or more, further preferably 10 or more, and more preferably 30 or more. The upper limit of kd (low calcium concentration condition)/kd (high calcium concentration condition) is not particularly limited, and may be any value such as 50, 100, 200, etc. as long as it can be made by the technical common knowledge of those skilled in the art.
As the value of the antigen binding activity, kd (dissociation rate constant) may be used when the antigen is a soluble antigen, and Apparent kd (Apparent dissociation rate constant) may be used when the antigen is a membrane antigen. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, and for example, Biacore (GE healthcare), flow cytometry, or the like can be used. In the present invention, when the antigen binding activity of the antigen binding molecule is measured at different calcium ion concentrations, the conditions other than the calcium concentration are preferably the same.
For example, an antigen-binding domain or antibody having an antigen-binding activity under a low calcium ion concentration condition that is one embodiment of the present invention is lower than that under a high calcium ion concentration condition can be obtained by screening an antigen-binding domain or antibody including the following steps (a) to (c):
(a) A step of obtaining the antigen binding activity of the antigen binding domain or antibody under the condition of low calcium concentration;
(b) A step of obtaining the antigen binding activity of the antigen binding domain or antibody under a high calcium concentration condition; and
(c) A step of selecting an antigen-binding domain or antibody having an antigen-binding activity under a low calcium concentration condition that is lower than that under a high calcium concentration condition.
furthermore, an antigen-binding domain or antibody having an antigen-binding activity under a low calcium ion concentration condition that is one embodiment of the present invention is lower than that under a high calcium ion concentration condition can be obtained by screening an antigen-binding domain or antibody or a library thereof including the following steps (a) to (c):
(a) a step of contacting the antigen binding domain or antibody or library thereof with an antigen under high calcium concentration conditions;
(b) A step of subjecting the antigen-binding domain or antibody that binds to the antigen in the aforementioned step (a) to a low calcium concentration condition; and
(c) A step of isolating the antigen binding domain or antibody that is dissociated in the aforementioned step (b).
In addition, as the invention provides a scheme of low calcium ion concentration conditions of antigen binding activity is lower than high calcium ion concentration conditions of antigen binding activity of antigen binding domain or antibody can be included by the following steps (a) - (d) of antigen binding domain or antibody or its library screening to obtain:
(a) a step of contacting a library of antigen binding domains or antibodies with an antigen under low calcium concentration conditions;
(b) a step of selecting an antigen-binding domain or an antibody that does not bind to the antigen in the aforementioned step (a);
(c) A step of allowing the antigen-binding domain or antibody selected in the aforementioned step (b) to bind to an antigen under a high calcium concentration condition; and
(d) A step of isolating the antigen binding domain or antibody that binds to the antigen in the aforementioned step (c).
furthermore, an antigen-binding domain or antibody having an antigen-binding activity under a low calcium ion concentration condition that is an embodiment of the present invention is lower than an antigen-binding activity under a high calcium ion concentration condition can be obtained by a screening method comprising the following steps (a) to (c):
(a) A step of contacting a library of antigen-binding domains or antibodies with a column immobilized with an antigen under a high calcium concentration condition;
(b) A step of eluting the antigen-binding domain or antibody bound to the column in the aforementioned step (a) from the column under a condition of low calcium concentration; and
(c) a step of separating the antigen binding domain or antibody eluted in the aforementioned step (b).
Furthermore, an antigen-binding domain or antibody having an antigen-binding activity under a low calcium ion concentration condition that is an embodiment of the present invention is lower than an antigen-binding activity under a high calcium ion concentration condition can be obtained by a screening method comprising the following steps (a) to (d):
(a) a step of passing the library of antigen-binding domains or antibodies through a column immobilized with an antigen under a low calcium concentration condition;
(b) A step of recovering the antigen-binding domain or antibody eluted without binding to the column in the step (a);
(c) a step of allowing the antigen-binding domain or antibody recovered in the aforementioned step (b) to bind to an antigen under a high calcium concentration condition; and
(d) A step of isolating the antigen binding domain or antibody that binds to the antigen in the aforementioned step (c).
furthermore, an antigen-binding domain or antibody having an antigen-binding activity under a low calcium ion concentration condition that is an embodiment of the present invention is lower than an antigen-binding activity under a high calcium ion concentration condition can be obtained by a screening method comprising the following steps (a) to (d):
(a) a step of contacting a library of antigen binding domains or antibodies with an antigen under high calcium concentration conditions;
(b) a step of obtaining an antigen binding domain or antibody that binds to the antigen in the aforementioned step (a);
(c) A step of subjecting the antigen-binding domain or antibody obtained in the aforementioned step (b) to a low calcium concentration condition; and
(d) a step of isolating an antigen-binding domain or antibody having an antigen-binding activity weaker than that of the standard selected in the step (b) in the step (c).
The foregoing steps may be repeated 2 or more times. Therefore, according to the present invention, there is provided an antigen-binding domain or antibody having an antigen-binding activity under a low calcium ion concentration condition lower than that under a high calcium ion concentration condition, which is obtained by a screening method further comprising repeating the steps (a) to (c) or (a) to (d) 2 or more times. (a) The number of repetitions of the steps (a) to (c) or (a) to (d) is not particularly limited, and is usually 10 or less.
in the screening method of the present invention, the antigen binding activity of the antigen binding domain or antibody under low calcium concentration conditions is not particularly limited as long as the antigen binding activity is between 0.1. mu.M and 30. mu.M of ionized calcium concentration, and the preferred ionized calcium concentration includes the antigen binding activity between 0.5. mu.M and 10. mu.M. More preferable ionized calcium concentration includes ionized calcium concentration in the early in vivo, specifically, antigen binding activity at 1. mu.M to 5. mu.M. The antigen binding activity of the antigen binding domain or antibody under high calcium concentration conditions is not particularly limited as long as the antigen binding activity is between 100. mu.M and 10mM of ionized calcium concentration, and a preferred ionized calcium concentration includes an antigen binding activity between 200. mu.M and 5 mM. More preferred ionized calcium concentration includes ionized calcium concentration in plasma in vivo, and specifically, antigen binding activity at 0.5mM to 2.5 mM.
the antigen binding activity of the antigen binding domain or antibody can be determined by methods known to those skilled in the art, and can be determined appropriately by those skilled in the art for conditions other than ionized calcium concentration. The antigen binding activity of an antigen binding domain or an antibody can be evaluated as KD (Dissociation constant), Apparent KD (Apparent Dissociation constant), Dissociation rate KD (Dissociation rate constant), Apparent KD (Apparent Dissociation rate constant), or the like. They can be measured by methods known to those skilled in the art, and for example, Biacore (GE healthcare), scatchard plot, FACS, or the like can be used.
In the present invention, the step of selecting an antigen-binding domain or antibody having an antigen-binding activity at a high calcium concentration higher than that at a low calcium concentration is the same as the step of selecting an antigen-binding domain or antibody having an antigen-binding activity at a low calcium concentration lower than that at a high calcium concentration.
when the antigen-binding activity under the high calcium concentration condition is higher than the antigen-binding activity under the low calcium concentration condition, the difference between the antigen-binding activity under the high calcium concentration condition and the antigen-binding activity under the low calcium concentration condition is not particularly limited, but the antigen-binding activity under the high calcium concentration condition is preferably 2 times or more, more preferably 10 times or more, and still more preferably 40 times or more the antigen-binding activity under the low calcium concentration condition.
the antigen binding domain or antibody of the present invention obtained by the screening method may be any antigen binding domain or antibody, and for example, the antigen binding domain or antibody may be screened. For example, an antigen binding domain or antibody having a native sequence can be selected, and an antigen binding domain or antibody having a substituted amino acid sequence can be selected.
Libraries
according to one aspect, the antigen binding domain or antibody of the present invention may be obtained from a library formed mainly of a plurality of antigen binding molecules whose sequences are different from each other and whose antigen binding domain contains at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule depending on the ion concentration condition. Examples of the ion concentration include a metal ion concentration and a hydrogen ion concentration.
in the present specification, a "library" refers to nucleic acids and polynucleotides that encode a plurality of antigen binding molecules or a plurality of fusion polypeptides comprising antigen binding molecules, or sequences encoding the same. The sequences of the plurality of antigen-binding molecules or the plurality of fusion polypeptides containing the antigen-binding molecules contained in the library are not a single sequence, but antigen-binding molecules or fusion polypeptides containing the antigen-binding molecules whose sequences are different from each other.
in the present specification, the term "different sequences" in the description of a plurality of antigen-binding molecules having different sequences means that the sequences of the antigen-binding molecules in the library are different from each other. That is, the number of mutually different sequences in a library reflects the number of independent clones having different sequences in the library, and may be regarded as "library size". A typical phage display library is 106to 1012The library size can be scaled up to 10 by using known techniques such as ribosome display14. However, the actual number of phage particles used in panning selections of phage libraries is typically 10 to 10,000 times larger than the library size. This excess multiple is also referred to as "library equivalent number", and indicates that 10 to 10,000 clones having the same amino acid sequence can exist. Therefore, the term "different from each other" in the present invention means that the sequences of the respective antigen-binding molecules in the library except the number of equivalents of the library are different from each other, and more specifically means that the antigen-binding molecules having different sequences from each other exist in an amount of 106to 1014molecule, preferably 107To 1012one molecule, more preferably 108To 1011one molecule, particularly preferably 108To 1012And (4) a molecule.
the term "plurality" in the description of the library of the present invention mainly comprising a plurality of antigen-binding molecules generally refers to a collection of 2 or more of the antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors, and viruses of the present invention. For example, if 2 or more substances are different from each other in specific form, it means that 2 or more substances are present. Examples thereof include mutant amino acids observed at a specific amino acid position in the amino acid sequence. For example, when 2 or more antigen-binding molecules of the present invention having substantially the same, preferably the same, sequence exist except for the flexible residues or except for the specific mutant amino acids at the hypervariable amino acid positions exposed on the surface, there are a variety of antigen-binding molecules of the present invention. In other embodiments, when 2 or more polynucleotide molecules of the present invention having substantially the same, preferably the same, sequence other than the base encoding the flexible residue or other than the base encoding the specific mutant amino acid at the hypervariable amino acid position exposed on the surface are present, the polynucleotide molecules of the present invention are present in a plurality of types.
furthermore, the expression "mainly formed" in the description of the library of the present invention mainly formed of a plurality of antigen-binding molecules reflects the number of independent clones having different sequences in the library againstthe amount of antigen binding molecule that varies with the ionic concentration conditions. In particular, it is preferred that antigen binding molecules exhibiting such binding activity be present in the library in at least 104and (4) a molecule. Furthermore, it is more preferred that the antigen binding domain of the present invention may be present by at least 10 from an antigen binding molecule exhibiting such binding activity5Obtained from a library of individual molecules. It is further preferred that the antigen binding domain of the present invention may be present in at least 10% by weight of the antigen binding molecule exhibiting such binding activity6Obtained from a library of individual molecules. It is particularly preferred that the antigen binding domain of the present invention may be present in at least 10% by weight of the antigen binding molecule exhibiting such binding activity7Obtained from a library of individual molecules. It is also preferred that the antigen binding domain of the present invention may be present in at least 10% by weight of the antigen binding molecule exhibiting such binding activity8obtained from a library of individual molecules. In other expressions, it can be appropriately expressed as the ratio of antigen-binding molecules whose antigen-binding activity differs depending on the ion concentration condition among the number of independent clones differing in sequence in the library. Specifically, the antigen binding domain of the present invention can be obtained from a library in which the antigen binding molecule exhibiting such a binding activity accounts for 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, further preferably 2% to 20%, particularly preferably 4% to 10% of the number of independent clones differing in sequence in the library. The fusion polypeptide, polynucleotide molecule or vector is also the same as described above, and can be expressed by the number of molecules or the ratio of all the molecules. In addition, the case of viruses can be expressed by the number of virus individuals or the ratio of the virus to all the individuals, as described above.
amino acids having antigen-binding activity of antigen-binding domain that varies depending on calcium ion concentration conditions
The antigen binding domain or antibody of the present invention screened by the aforementioned screening method can be prepared by any method, and for example, in the case where the metal ion has a calcium ion concentration, a preexisting antibody, a preexisting library (phage library or the like), an antibody or library prepared from a hybridoma obtained by immunizing an animal or a B cell derived from an immunized animal, an antibody or library obtained by introducing a mutation into an amino acid (e.g., aspartic acid or glutamic acid) or an unnatural amino acid capable of chelating calcium into these antibodies or libraries (a library obtained by introducing an amino acid (e.g., aspartic acid or glutamic acid) or an unnatural amino acid capable of chelating calcium into a specific position, or a library obtained by introducing a mutation into an amino acid (e.g., aspartic acid or glutamic acid) or an unnatural amino acid capable of chelating calcium at a specific position), or the like can be used.
As described above, as an example of the amino acid that changes the antigen binding activity of the antigen binding molecule depending on the ion concentration condition, for example, when the metal ion is a calcium ion, any amino acid may be used as long as it forms a calcium binding motif, regardless of the kind thereof. Calcium binding motifs are well known to those skilled in the art and are described in detail (e.g., Springer et al (Cell (2000)102,275-277), Kawasaki and Kretsinger (Protein Prof. (1995)2,305-490), Moncrief et al (J.mol. Evol. (1990)30,522-562), Chauvaux et al (biochem. J. (1990)265,261-265), Bairoch and Cox (FEBS Lett. (1990)269,454-456), Davis (New Biol. (1990)2,410-419), Schaefer et al (Genomics (1995)25,638-643), onomou et al (EMBO J. (1990)9,349-354), Wurzburg et al (Structure. (14, 6, 9-1048)). That is, the antigen-binding molecule of the present invention may contain any known calcium-binding motif such as C-type lectins such as ASGPR, CD23, MBR, DC-SIGN, and the like. As a preferred example of such a calcium binding motif, there may be mentioned, in addition to the above, the calcium binding motif contained in the antigen binding domain of SEQ ID NO: 62.
further, as an example of an amino acid whose antigen-binding activity of the antigen-binding molecule varies depending on the condition of calcium ion concentration, an amino acid having a metal-chelating action can also be suitably used. Preferred examples of the amino acid having a metal chelating action include: serine (Ser (S)), threonine (Thr (T)), asparagine (Asn (N)), glutamine (Gln (Q)), aspartic acid (Asp (D)), glutamic acid (Glu (E)), and the like.
the position of the antigen-binding domain containing the amino acid is not limited to a specific position, and any position of the heavy chain variable region or the light chain variable region forming the antigen-binding domain may be used as long as the antigen-binding activity of the antigen-binding molecule is changed depending on the calcium ion concentration condition. That is, the antigen-binding domain of the present invention can be obtained from a library mainly comprising antigen-binding molecules having different sequences and amino acids in the antigen-binding domain of the heavy chain, the amino acids changing the antigen-binding activity of the antigen-binding molecule according to the calcium ion concentration condition. In addition, in other non-limiting embodiments, the antigen binding domain of the present invention can be obtained from a library formed mainly of antigen binding molecules that contain the amino acid in CDR3 of the heavy chain and that differ from each other in sequence. In other non-limiting embodiments, the antigen binding domain of the present invention may be obtained from a library formed mainly of antigen binding molecules having the amino acid at position 95, position 96, position 100a, and/or position 101 of the CDR3 of the heavy chain, which are represented by Kabat numbering, and having different sequences from each other.
In a non-limiting embodiment of the present invention, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences and amino acids in the antigen-binding domain of the light chain, the amino acids changing the antigen-binding activity of the antigen-binding molecule according to the calcium ion concentration condition. In other embodiments, the antigen binding domain of the present invention may be obtained from a library formed mainly of antigen binding molecules having different sequences, each of which contains the amino acid in CDR1 of the light chain. In other embodiments, the antigen binding domain of the present invention may be obtained from a library formed mainly of antigen binding molecules having the amino acid at position 30, position 31, and/or position 32 of the CDR1 of the light chain, as represented by the Kabat numbering, and having different sequences from each other.
In addition, in other non-limiting embodiments, the antigen binding domain of the present invention can be obtained from a library mainly formed of antigen binding molecules having different sequences from each other, in which the CDR2 of the light chain contains the amino acid residue. In other embodiments, a library is provided that is formed primarily of antigen binding molecules of the CDR2 of the light chain that contain the amino acid residue at position 50, as indicated by Kabat numbering, and that differ from each other in sequence.
In another non-limiting embodiment, the antigen binding domain of the present invention can be obtained from a library mainly composed of antigen binding molecules having different sequences, each of which contains the amino acid residue in CDR3 of the light chain. In other embodiments, the antigen binding domain of the present invention may be obtained from a library formed mainly of antigen binding molecules having the amino acid residue at position 92 of the CDR3 of the light chain, which is represented by Kabat numbering, and having different sequences from each other.
furthermore, the antigen binding domain of the present invention can be obtained as a different embodiment of the present invention from a library formed mainly of antigen binding molecules in which 2 or 3 CDRs selected from the CDR1, CDR2, and CDR3 of the light chain described above contain the amino acid residues and which are different from each other in sequence. Furthermore, the antigen-binding domain of the present invention can be obtained from a library mainly comprising antigen-binding molecules having different sequences, each of which contains the amino acid residue at any one or more of positions 30, 31, 32, 50 and/or 92 of the light chain represented by Kabat numbering.
In particularly preferred embodiments, it is desirable that the framework sequences of the light and/or heavy chain variable regions of the antigen binding molecule have human germline framework sequences. Thus, in one embodiment of the invention, if the framework sequence is entirely human, it is believed that the antigen binding molecules of the invention will not substantially or completely elicit an immunogenic response when administered to a human (e.g., in the treatment of a disease). In the above sense, the term "comprising germline sequence" according to the invention means that a portion of the framework sequence according to the invention is identical to a portion of any human germline framework sequence. For example, the sequence of heavy chain FR2 of the antigen-binding molecule of the present invention is a sequence obtained by combining heavy chain FR2 sequences of a plurality of different human germline framework sequences, and is also an "antigen-binding molecule containing germline sequences" of the present invention.
Preferred examples of the frame include: sequences of fully human framework regions, now known, included in websites such as V-Base (http:// vbase. mrc-cpe. cam. ac. uk /). The sequences of these framework regions can be suitably used as germline sequences contained in the antigen binding molecules of the invention. Germline sequences can be classified based on their similarity (Tomlinson et al (J.mol. biol. (1992)227,776-798) Williams and Winter (Eur.J. Immunol. (1993)23,1456-1461) and Cox et al (nat. genetics (1994)7, 162-168)). An appropriate germline sequence can be appropriately selected from among V κ classified into 7 subclasses, V λ classified into 10 subclasses, and VH classified into 7 subclasses.
the fully human VH sequence is not limited to the following, and suitable examples include: VH1 subclasses (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69), VH2 subclasses (e.g., VH2-5, VH2-26, VH2-70), VH2 subclasses (VH 2-7, VH 2-9, VH 2-11, VH 2-13, VH 2-15, VH 2-16, VH 2-20, VH 2-21, VH 2-23, VH 2-30, VH 2-33, VH 2-35, VH 2-38, VH 2-43, VH 2-48, VH 2-49, VH 2-53, VH 2-64, VH 2-66, 2-72, VH 2-73, VH 2-72, VH 2-72-31, VH 2-31-2, VH 2-34, VH 2-72-34-72, VH 2-72-31, VH2, VH4-59, VH4-61), VH5 subclass (VH5-51), VH6 subclass (VH6-1), VH7 subclass (VH7-4, VH7-81), etc. They are also described in the publicly known literature (Matsuda et al (J.Exp.Med. (1998)188,1973-1975)) and the like, and the skilled person can appropriately design the antigen-binding molecules of the present invention based on their sequence information. It may also be suitable to use fully human frameworks or sub-regions of frameworks other than these.
the fully human VK sequence is not limited to the following, and suitable examples include: a20, a30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, O18 classified as the Vk1 subclass; a1, a2, A3, a5, a7, a17, a18, a19, a23, O1, O11 classified as the Vk2 subclass; a11, a27, L2, L6, L10, L16, L20, L25 classified as the Vk3 subclass; b3 classified as subclass Vk 4; b2 (also referred to herein as Vk5-2) classified as subclass Vk 5; a10, A14, A26, etc., classified as subclasses VK6 (Kawasaki et al (Eur. J. Immunol. (2001)31,1017-1028), Schable and Zachau (biol. chem. hopper Seyler (1993)374,1001-1022) and Brensing-Kuppers et al (Gene (1997)191, 173-181)).
the completely human VL sequence is not limited to the following, and examples thereof include: v1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, V1-22 classified as VL1 subclass; v2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17 and V2-19 which are classified as VL1 subclasses; v3-2, V3-3 and V3-4 which are classified as VL3 subclasses; v4-1, V4-2, V4-3, V4-4 and V4-6 which are classified as VL4 subclasses; v5-1, V5-2, V5-4, V5-6, etc., which are classified as VL5 subclasses (Kawasaki et al (Genome Res. (1997)7, 250-261)).
typically these framework sequences differ from each other by the difference of one or more amino acid residues. These framework sequences can be used with the "at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule according to the conditions of ionic concentration" of the present invention. Examples of the fully human framework used together with the "at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule according to the ion concentration condition" of the present invention are not limited thereto, and in addition: KOL, NEWM, REI, EU, TUR, TEI, LAY, POM, etc. (e.g., Kabat et al (1991) and Wu et al (J.exp. Med. (1970)132,211-250) as described previously).
Without being bound by a particular theory, it is believed that one reason why the use of germline sequences is expected to rule out adverse immune responses in most individuals is as follows. The consequences of the affinity maturation phase that occurs in the usual immune response cause the variable regions of immunoglobulins to frequently mutate somatic cells. These mutations are mainly generated in the vicinity of CDRs whose sequences are hypervariable, and also affect the residues of the framework regions. Mutations of these frameworks are not present in germline genes and are less likely to be immunogenic in patients. It is due to: the majority of the framework sequences expressed by genes of the germ line to which a typical human population is exposed are predicted to be low or non-immunogenic in patients as a result of immune tolerance. To maximize the likelihood of immune tolerance, the genes encoding the variable regions may be selected from a collection of functional germline genes that are normally present.
in order to produce the antigen-binding molecule of the present invention, which contains an amino acid in the framework sequence thereof, which changes the antigen-binding activity of the antigen-binding molecule according to the calcium ion concentration condition, a known method such as the site-specific mutagenesis method (Kunkel et al (Proc. Natl. Acad. Sci.USA (1985)82,488-492)) or overlap extension PCR can be suitably used.
for example, a library containing a plurality of antigen-binding molecules having different sequences of the present invention can be prepared by combining a light chain variable region selected as a framework sequence containing at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule under calcium ion concentration conditions in advance with a heavy chain variable region prepared as a library of random variable region sequences. As such non-limiting examples, when the ion concentration is a calcium ion concentration, there may be mentioned, for example: a library comprising a combination of a light chain variable region sequence belonging to Vk5-2 family represented by the light chain variable region sequence of SEQ ID NO:62(Vk5-2) and a heavy chain variable region prepared as a library of random variable region sequences.
in addition, the light chain variable region sequence selected as the framework sequence containing at least one amino acid residue whose antigen binding activity of the antigen binding molecule varies depending on the calcium ion concentration condition may be designed to contain various amino acids as residues other than the amino acid residue. In the present invention, such residues are also referred to as flexible residues. The number and position of the flexible residues are not limited to a specific embodiment as long as the antigen binding activity of the antigen binding molecule of the present invention varies depending on the ion concentration condition. That is, the CDR sequences and/or FR sequences of the heavy and/or light chains may contain one or more flexible residues. For example, when the ion concentration is calcium ion concentration, non-limiting examples of the flexible residues introduced into the light chain variable region sequence shown in SEQ ID NO:62(Vk5-2) include the amino acid residues shown in Table 1 or Table 2.
[ Table 1]
[ Table 2]
in the present specification, a flexible residue refers to a variation of an amino acid residue present at a position in the light and heavy chain variable regions where the amino acid residues present at that position are hypervariable when compared to the amino acid sequence of a known and/or native antibody or antigen binding domain. The hypervariable positions are generally present in the CDR regions. In one approach, the data provided by Kabat, Sequences of Proteins of immunological Interest (National Institute of Health BethesdaMd.) (1987 and 1991) was valid in determining the hypervariable position of well-known and/or native antibodies. In addition, numerous databases on the Internet (http:// vbase. mrc-cpe. cam. ac. uk/, http:// www.bioinf.org.uk/abs/index. html) are provided with sequences of a large number of human light and heavy chains collected and their configurations, and information on these sequences and their configurations is useful for determining the hypervariable positions in the present invention. According to the invention, a position is said to be hypervariable when the amino acid has a diversity of preferably about 2 to about 20, preferably about 3 to about 19, preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12, possible different amino acid residues at that position. In several embodiments, an amino acid position may have a diversity of preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, preferably about 12, possible different amino acid residues.
further, a library containing a plurality of antigen-binding molecules of the present invention having different sequences can also be prepared by combining the light chain variable region introduced with at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule under ion concentration conditions with the heavy chain variable region prepared as a library of random variable region sequences. As such non-limiting examples, when the ion concentration is a calcium ion concentration, preferable examples include: a library obtained by combining a light chain variable region sequence obtained by substituting a specific residue of a germline such as SEQ ID NO 5(Vk1), SEQ ID NO 6(Vk2), SEQ ID NO 7(Vk3), or SEQ ID NO 8(Vk4) with at least one amino acid residue whose antigen-binding activity of the antigen-binding molecule varies depending on the calcium ion concentration condition, and a heavy chain variable region sequence prepared as a library of random variable region sequences. As a non-limiting example of the amino acid residue, an amino acid residue contained in CDR1 of a light chain is exemplified. Further, as a non-limiting example of the amino acid residue, an amino acid residue contained in light chain CDR2 is exemplified. Further, as other non-limiting examples of the amino acid residue, amino acid residues contained in the light chain CDR3 are exemplified.
as described above, as non-limiting examples of the amino acid residue included in the light chain CDR1, there can be mentioned: an amino acid residue at position 30, 31 and/or 32 in the CDR1 of the light chain variable region by Kabat numbering. Further, as non-limiting examples of the amino acid residue included in the light chain CDR2, there can be mentioned: the amino acid residue at position 50 as represented by Kabat numbering in the CDR2 of the light chain variable region. Further, non-limiting examples of the amino acid residue included in the light chain CDR3 include: the amino acid residue at position 92, as represented by Kabat numbering, in the CDR3 of the light chain variable region. These amino acid residues may be contained alone or in combination of two or more kinds thereof, as long as they can form a calcium binding motif and/or change the antigen binding activity of the antigen-binding molecule depending on the calcium ion concentration condition. In addition, it is known that troponin C, calmodulin, microalbumin, myosin light chain, etc., which have a plurality of calcium ion binding sites and are thought to be derived from a common origin in molecular evolution, and light chain CDR1, CDR2 and/or CDR3 may also be designed in such a manner as to contain the binding motif. For example, for the above purpose, cadherin domain, EF hand contained in calmodulin, C2 domain contained in protein kinase C, Gla domain contained in coagulation protein factor IX, C-type lectin contained in asialoglycoprotein receptor or mannose binding receptor, a domain contained in LDL receptor, annexin, thrombospondin type 3 domain, and EGF-like domain can be suitably used.
in the case of combining the light chain variable region introduced with at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule under ion concentration conditions with the heavy chain variable region prepared as a library of random variable region sequences, the light chain variable region may be designed so that the sequence of the light chain variable region contains a flexible residue, in the same manner as described above. The number and position of the flexible residues are not limited to a specific embodiment as long as the antigen binding activity of the antigen binding molecule of the present invention varies depending on the ion concentration condition. That is, the CDR sequences and/or FR sequences of the heavy and/or light chains may contain one or more flexible residues. For example, when the ion concentration is calcium ion concentration, non-limiting examples of the flexible residues introduced into the light chain variable region sequence include the amino acid residues shown in Table 1 or Table 2.
as an example of the heavy chain variable regions to be combined, a library of random variable regions can be appropriately given. The method of preparing the random variable region library is suitably combined with known methods. In a non-limiting embodiment of the present invention, an immune library constructed based on lymphocyte-derived antibody genes of an animal immunized with a specific antigen, a patient with an infectious disease, or a human immunized with a vaccine to raise the antibody titer in blood, a cancer patient, or an autoimmune disease can be suitably used as the random variable region library.
in a non-limiting embodiment of the present invention, a synthetic library in which CDR sequences of V genes or reconstructed functional V genes in genomic DNA are replaced with a synthetic oligonucleotide set having a sequence encoding a codon set of an appropriate length can also be preferably used as a random variable region library. At this time, since diversity of the gene sequence of CDR3 of the heavy chain was observed, only the sequence of CDR3 could be substituted. The basis for the diversity of amino acids in the variable region of an antigen-binding molecule is the diversity of amino acid residues at the positions where the antigen-binding molecule is exposed to the surface. The exposed position on the surface refers to a position judged to be exposed on the surface and/or contactable with the antigen based on the structure, the structural totality and/or the modeled structure of the antigen-binding molecule, and is usually a CDR thereof. The location of exposure to the surface is preferably determined using coordinates from a three-dimensional model of the antigen binding molecule using a computer program such as the insight ii program (Accelrys). The location of the exposure to the surface can be determined using algorithms known in the art, such as Lee and Richards (j.mol. biol. (1971)55,379-400), Connolly (j.appl. cryst. (1983)16, 548-558). The determination of the location of the exposure to the surface can be performed using three-dimensional structural information derived from software and antibodies suitable for protein modeling. As software that can be used for the above purpose, SYBYL Biopolymer Module software (Tripos Associates) is preferably used. Typically or preferably, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less when the algorithm requires the user to input a size parameter. Further, methods for determining the area and the area exposed to the surface using software for personal computers are described in Pacios (company. chem. (1994)18(4),377-386, and j.mol. model. (1995)1, 46-53).
furthermore, in a non-limiting embodiment of the present invention, a natural library constructed from antibody genes derived from healthy Human lymphocytes and composed of natural sequences that are antibody sequences free of bias can be used as a random variable region library (Gejima et al (Human Antibodies (2002)11,121-129) and Cardoso et al (Scand. J. Immunol. (2000)51, 337-344)). The amino acid sequence containing a natural sequence described in the present invention refers to an amino acid sequence obtained from the natural library.
in one embodiment of the present invention, the antigen-binding domain of the present invention can be obtained from a library containing a plurality of antigen-binding molecules of different sequences of the present invention by combining a heavy chain variable region selected as a framework sequence containing "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule according to ion concentration conditions" and a light chain variable region prepared as a library of random variable region sequences. As such non-limiting examples, when the ion concentration is a calcium ion concentration, there may be mentioned, for example: the library was prepared by combining the heavy chain variable region sequence of SEQ ID NO 9(6RL #9-IgG1) or SEQ ID NO 10(6KC4-1#85-IgG1) with the light chain variable region sequence prepared as a random variable region sequence library. Alternatively, instead of creating light chain variable regions as a library of random variable region sequences, light chain variable regions having germline sequences may be appropriately selected and created. Examples thereof include: a library obtained by combining the heavy chain variable region sequence of SEQ ID NO:9(6RL #9-IgG1) or SEQ ID NO:10(6KC4-1#85-IgG1) with the light chain variable region having a germline sequence.
in addition, the sequence of the heavy chain variable region selected as the framework sequence containing "at least one amino acid residue which changes the antigen binding activity of the antigen binding molecule depending on the ion concentration condition" may be designed to contain a flexible residue. The number and position of the flexible residues are not limited to a specific embodiment as long as the antigen binding activity of the antigen binding molecule of the present invention varies depending on the ion concentration condition. That is, the CDR sequences and/or FR sequences of the heavy and/or light chains may contain one or more flexible residues. For example, non-limiting examples of the flexible residues introduced into the heavy chain variable region sequence of SEQ ID NO:9(6RL #9-IgG1) include amino acid residues of CDR3 other than the 95 th, 96 th and/or 100a th positions of heavy chain CDR3, in addition to all the amino acid residues of heavy chain CDR1 and CDR2, when the ion concentration is calcium ion concentration. Alternatively, non-limiting examples of the flexible residues introduced into the heavy chain variable region sequence of SEQ ID NO 10(6KC4-1#85-IgG1) include amino acid residues of CDR3 other than position 95 and/or position 101 of the heavy chain CDR3, in addition to all of the amino acid residues of the heavy chain CDR1 and CDR 2.
further, by combining the heavy chain variable region into which "at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule under ion concentration conditions" is introduced with a light chain variable region that is a random variable region sequence library or a light chain variable region having a germline sequence, a library containing a plurality of antigen binding molecules having different sequences from each other can also be created. As such non-limiting examples, when the ion concentration is a calcium ion concentration, preferable examples include: a library which is obtained by combining a heavy chain variable region sequence in which a specific residue of a heavy chain variable region is substituted with at least one amino acid residue whose antigen-binding activity of an antigen-binding molecule varies depending on the calcium ion concentration condition, and a light chain variable region which is a library of random variable region sequences or a light chain variable region having a germline sequence. As a non-limiting example of the amino acid residue, an amino acid residue contained in CDR1 of a heavy chain is exemplified. Further, as a non-limiting example of the amino acid residue, an amino acid residue contained in CDR2 of a heavy chain is also exemplified. Further, as other non-limiting examples of the amino acid residue, amino acid residues contained in CDR3 of the heavy chain are also exemplified. Non-limiting examples of the amino acid residue included in the CDR3 of the heavy chain include amino acids at position 95, 96, 100a and/or 101 in the CDR3 of the heavy chain variable region by Kabat numbering. These amino acid residues may be contained alone or in combination of two or more thereof, as long as they form a calcium binding motif and/or the antigen binding activity of the antigen-binding molecule varies depending on the calcium ion concentration condition.
In the case of combining the heavy chain variable region introduced with at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule under ion concentration conditions with the light chain variable region prepared as a library of random variable region sequences or with a germline sequence, the sequence of the heavy chain variable region may be designed to contain a flexible residue in the same manner as described above. The number and position of the flexible residues are not limited to a specific embodiment as long as the antigen binding activity of the antigen binding molecule of the present invention varies depending on the ion concentration condition. That is, the CDR sequences and/or FR sequences of the heavy chain may contain one or more flexible residues. Further, as the amino acid sequence of CDR1, CDR2 and/or CDR3 of the heavy chain variable region other than the amino acid residues which change the antigen binding activity of the antigen binding molecule depending on the ion concentration condition, a random variable region library can also be suitably used. When a germline sequence is used as the light chain variable region, germline sequences such as SEQ ID NO 5(Vk1), SEQ ID NO 6(Vk2), SEQ ID NO 7(Vk3), SEQ ID NO 8(Vk4) and the like are given as non-limiting examples.
As the amino acid whose antigen-binding activity of the antigen-binding molecule changes depending on the calcium ion concentration condition, any amino acid can be suitably used as long as it forms a calcium-binding motif, and specific examples of such amino acids include amino acids having an electron-donating property. Examples of such an amino acid having an electron donating property include preferably serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid.
Condition of hydrogen ion concentration
further, in one aspect of the present invention, the ion concentration condition refers to a hydrogen ion concentration condition or a pH condition. In the present invention, the condition of the concentration of protons, i.e., nuclei of hydrogen atoms, and the condition of the hydrogen index (pH) are regarded as the same meaning. When the active amount of hydrogen ions in the aqueous solution is represented by aH +, the pH is defined as-log 10aH +. Ionic strength in aqueous solution if (e.g. with 10)-3By comparison), aH + is approximately equal in strength to the hydrogen ion. For example, the ion product of water at 25 ℃ under 1 atmosphere is Kw ═ aH + aOH ═ 10-14Thus, for pure water, aH + ═ aOH ═ 10-7. In this case, pH7 is neutral, an aqueous solution having a pH of less than 7 is acidic, and an aqueous solution having a pH of more than 7 is basic.
in the present invention, when a pH condition is used as the ion concentration condition, examples of the pH condition include a pH acidic range condition of high hydrogen ion concentration or low pH and a pH neutral range condition of low hydrogen ion concentration or high pH. The binding activity varies depending on the pH condition, and it means that the antigen binding activity of the antigen-binding molecule varies depending on the conditions of high hydrogen ion concentration or low pH (pH acidic range) and low hydrogen ion concentration or high pH (pH neutral range). For example, the antigen binding activity of the antigen binding molecule under the condition of the neutral pH range is higher than the antigen binding activity of the antigen binding molecule under the condition of the acidic pH range. In addition, the antigen binding activity of the antigen-binding molecule under the pH acidic range is higher than the antigen binding activity of the antigen-binding molecule under the pH neutral range.
In the present specification, the pH neutral range is not particularly limited to a uniform value, and may be preferably selected from pH6.7 to pH 10.0. In addition, in other embodiments, one may select from pH6.7 to pH 9.5. Furthermore, in various embodiments, the pH can be selected from pH7.0 to pH9.0, and in other embodiments, the pH can be selected from pH7.0 to pH 8.0. Particularly preferably, the pH value is 7.4 which is close to the pH value in plasma (blood) in vivo.
In the present specification, the pH acidity range is not particularly limited to a uniform value, and may be preferably selected from pH4.0 to pH 6.5. In addition, in other embodiments, the pH can be selected from pH4.5 to pH 6.5. Furthermore, in various embodiments, one may select from pH5.0 to pH6.5, and in other embodiments, one may select from pH5.5 to pH 6.5. Particularly preferably, the pH is 5.8 which is close to the ionized calcium concentration in the early endosome in the living body.
in the present invention, the antigen-binding activity of the antigen-binding molecule under the condition of high hydrogen ion concentration or low pH (pH acidic range) is lower than the antigen-binding activity under the condition of low hydrogen ion concentration or high pH (pH neutral range), which means that the antigen-binding activity of the antigen-binding molecule under a pH selected from pH4.0 to pH6.5 is weaker than the antigen-binding activity under a pH selected from pH6.7 to pH 10.0. Preferably means that the antigen binding activity of the antigen binding molecule at a pH selected from between pH4.5 and pH6.5 is weaker than the antigen binding activity at a pH selected from between pH6.7 and pH9.5, more preferably means that the antigen binding activity of the antigen binding molecule at a pH selected from between pH5.0 and pH6.5 is weaker than the antigen binding activity at a pH selected from between pH7.0 and pH 9.0. Furthermore, preferably means that the antigen binding activity of the antigen binding molecule at a pH selected from between pH5.5 and pH6.5 is weaker than the antigen binding activity at a pH selected from between pH7.0 and pH 8.0. Particularly preferably, it means that the antigen-binding activity at a pH in vivo in the early stage in vivo is weaker than that at a pH in plasma in vivo, and specifically means that the antigen-binding activity of the antigen-binding molecule at pH5.8 is weaker than that at pH 7.4.
Whether the antigen-binding activity of the antigen-binding molecule changes depending on the pH condition can be determined by using, for example, a known measurement method described in the aforementioned item of binding activity. That is, the binding activity under different pH conditions was measured by this measurement method. For example, in order to confirm that the antigen binding activity of the antigen-binding molecule under the pH acidic range is higher than the antigen binding activity of the antigen-binding molecule under the pH acidic range, the antigen binding activities of the antigen-binding molecule under the pH acidic range and the pH neutral range are compared.
in the present invention, the expression "the antigen-binding activity under the condition of a high hydrogen ion concentration or a low pH, i.e., pH acidic range is lower than the antigen-binding activity under the condition of a low hydrogen ion concentration or a high pH, i.e., pH neutral range" may also be expressed as the antigen-binding activity under the condition of a low hydrogen ion concentration or a high pH, i.e., pH neutral range, of the antigen-binding molecule is higher than the antigen-binding activity under the condition of a high hydrogen ion concentration or a low pH, i.e., pH acidic range. In the present invention, "the antigen binding activity under the condition of a high hydrogen ion concentration or a low pH, i.e., pH acidic range, is lower than the antigen binding activity under the condition of a low hydrogen ion concentration or a high pH, i.e., pH neutral range" may be described as "the antigen binding activity under the condition of a high hydrogen ion concentration or a low pH, i.e., pH acidic range, is lower than the antigen binding activity under the condition of a low hydrogen ion concentration or a high pH, i.e., pH neutral range", and "the antigen binding activity under the condition of a high hydrogen ion concentration or a low pH, i.e., pH acidic range, is lower than the antigen binding activity under the condition of a low hydrogen ion concentration or a high pH, i.e., pH neutral range" may be described as "the antigen binding activity under the condition of a high hydrogen ion concentration or a low pH, i.e., pH acidic range, is lower than the antigen binding.
the conditions other than the hydrogen ion concentration and the pH for measuring the antigen-binding activity can be appropriately selected by those skilled in the art, and are not particularly limited. For example, the measurement can be carried out in HEPES buffer at 37 ℃. For example, measurement can be performed using Biacore (GE Healthcare) or the like. In the measurement of the binding activity between the antigen-binding molecule and the antigen, when the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through the chip on which the antigen-binding molecule is immobilized, and when the antigen is a membrane antigen, the binding activity to the membrane antigen can be evaluated by passing the antigen-binding molecule as an analyte through the chip on which the antigen is immobilized.
In the antigen-binding molecule of the present invention, as long as the antigen-binding activity under the condition of high hydrogen ion concentration or low pH, i.e., pH acidic range, is weaker than the antigen-binding activity under the condition of low hydrogen ion concentration or high pH, i.e., pH neutral range, the ratio of the antigen-binding activity under the condition of high hydrogen ion concentration or low pH, i.e., pH acidic range, to the antigen-binding activity under the condition of low hydrogen ion concentration or high pH, i.e., pH neutral range is not particularly limited, but the ratio KD (pH5.8)/KD (pH7.4) between the ratio KD (Dissociation constant: Dissociation constant) under the condition of high hydrogen ion concentration or low pH, i.e., pH acidic range to the antigen is preferably 2 or more, more preferably 10 or more in terms of KD (pH5.8)/KD (pH7.4), and more preferably 40 or more in terms of KD (pH5.8)/KD (pH 7.4). The upper limit of the value of KD (pH5.8)/KD (pH7.4) is not particularly limited, and may be any value such as 400, 1000, 10000, etc., as long as the technique of those skilled in the art can be made.
As the value of the antigen binding activity, KD (dissociation constant) can be used when the antigen is a soluble antigen, and Apparent KD (Apparent dissociation constant) can be used when the antigen is a membrane-type antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, and for example, Biacore (GE healthcare), scatchard plot, flow cytometry, or the like can be used.
In addition, as another index showing the ratio of the antigen-binding activity of the antigen-binding molecule of the present invention under the condition of high hydrogen ion concentration or low pH, that is, pH acidic range, to the antigen-binding activity under the condition of low hydrogen ion concentration or high pH, that is, pH neutral range, for example, the Dissociation rate constant kd (Dissociation rate constant) can be suitably used. When KD (dissociation rate constant) is used as an index showing the ratio of binding activity instead of KD (dissociation constant), the value of the ratio KD (under pH acidic range)/KD (under pH neutral range) of KD (dissociation rate constant) at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, to KD (dissociation rate constant) at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, to the antigen is preferably 2 or more, more preferably 5 or more, further preferably 10 or more, and further preferably 30 or more. The upper limit of Kd (under the condition of pH in the acidic range)/Kd (under the condition of pH in the neutral range) is not particularly limited, and may be any value such as 50, 100, 200, etc., as long as it can be made by the common technical knowledge of those skilled in the art.
As the value of the antigen binding activity, kd (dissociation rate constant) may be used when the antigen is a soluble antigen, and Apparent kd (Apparent dissociation rate constant) may be used when the antigen is a membrane antigen. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, and for example, Biacore (GE healthcare), flow cytometry, or the like can be used. In the present invention, when the antigen binding activity of the antigen-binding molecule is measured at different hydrogen ion concentrations, i.e., at different pH, it is preferable that the conditions other than the hydrogen ion concentration, i.e., pH, be the same.
For example, an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than that at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, which is one embodiment of the present invention, can be obtained by screening an antigen-binding domain or antibody including the following steps (a) to (c):
(a) A step of obtaining the antigen binding activity of the antigen binding domain or antibody under the condition of pH acidic range;
(b) A step of obtaining the antigen binding activity of the antigen binding domain or antibody under the condition of pH neutral range; and
(c) a step of selecting an antigen-binding domain or antibody having antigen-binding activity in the pH acidic range lower than that in the pH neutral range.
Furthermore, an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than that at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, which is an embodiment of the present invention, can be obtained by screening an antigen-binding domain or antibody or a library thereof including the following steps (a) to (c):
(a) A step of contacting the antigen binding domain or antibody or library thereof with an antigen under conditions of a neutral range of pH;
(b) A step of subjecting the antigen-binding domain or the antibody that binds to the antigen in the aforementioned step (a) to a pH acidic range condition; and
(c) a step of isolating the antigen binding domain or antibody that is dissociated in the aforementioned step (b).
In addition, the present invention provides an embodiment of an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, which is obtained by screening an antigen-binding domain or antibody or a library thereof including the following steps (a) to (d):
(a) a step of contacting a library of antigen binding domains or antibodies with an antigen under conditions of pH in the acidic range;
(b) A step of selecting an antigen-binding domain or an antibody that does not bind to the antigen in the aforementioned step (a);
(c) a step of allowing the antigen-binding domain or antibody selected in the aforementioned step (b) to bind to an antigen under a condition of a neutral range of pH; and
(d) a step of isolating the antigen binding domain or antibody that binds to the antigen in the aforementioned step (c).
furthermore, an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than that at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, which is an embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c):
(a) A step of contacting a library of antigen-binding domains or antibodies with a column immobilized with an antigen under conditions of a neutral range of pH;
(b) A step of eluting the antigen-binding domain or antibody bound to the column in the aforementioned step (a) from the column under a pH acidic range condition; and
(c) a step of separating the antigen binding domain or antibody eluted in the aforementioned step (b).
Furthermore, an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than that at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, which is an embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d):
(a) a step of passing the library of antigen-binding domains or antibodies through a column on which an antigen is immobilized under a pH acidic range;
(b) A step of recovering the antigen-binding domain or antibody eluted without binding to the column in the step (a);
(c) A step of allowing the antigen-binding domain or antibody recovered in the aforementioned step (b) to bind to an antigen under a condition of a neutral pH range; and
(d) a step of isolating the antigen binding domain or antibody that binds to the antigen in the aforementioned step (c).
Furthermore, an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than that at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, which is an embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d):
(a) a step of contacting a library of antigen binding domains or antibodies with an antigen under conditions in the neutral range of pH;
(b) a step of obtaining an antigen binding domain or antibody that binds to the antigen in the aforementioned step (a);
(c) A step of subjecting the antigen-binding domain or antibody obtained in the aforementioned step (b) to a pH acidic range condition; and
(d) A step of isolating the antigen binding domain or antibody having an antigen binding activity weaker than that of the standard selected in the step (b) above in the step (c) above.
The foregoing steps may be repeated 2 or more times. Therefore, according to the present invention, there is provided an antigen-binding domain or antibody having an antigen-binding activity under a pH acidic range condition lower than an antigen-binding activity under a pH neutral range condition, which is obtained by a screening method further comprising the step of repeating the steps (a) to (c) or (a) to (d) 2 or more times. (a) The number of repetitions of the steps (a) to (c) or (a) to (d) is not particularly limited, and is usually 10 or less.
in the screening method of the present invention, the antigen binding activity of the antigen binding domain or antibody under high hydrogen ion concentration conditions or at low pH, i.e., in the acidic pH range, is not particularly limited as long as it is an antigen binding activity between pH 4.0 and 6.5, and a preferred pH includes an antigen binding activity between pH 4.5 and 6.6. The other preferable pH is an antigen binding activity at a pH of 5.0 to 6.5, and further an antigen binding activity at a pH of 5.5 to 6.5. More preferred pH includes early in vivo pH in vivo, and specifically, antigen binding activity at pH 5.8. The antigen binding activity of the antigen binding domain or antibody under the low hydrogen ion concentration condition or at a high pH, i.e., in a pH neutral range is not particularly limited as long as the antigen binding activity is between pH 6.7 and pH 10, and a preferable pH is an antigen binding activity between pH 6.7 and pH 9.5. The other preferable pH is an antigen binding activity at a pH of 7.0 to 9.5, and further an antigen binding activity at a pH of 7.0 to 8.0. More preferred pH includes plasma pH in vivo, and specifically, antigen binding activity at pH 7.4.
the antigen binding activity of the antigen binding domain or antibody can be determined by methods known to those skilled in the art, and can be determined appropriately by those skilled in the art for conditions other than ionized calcium concentration. The antigen binding activity of an antigen binding domain or an antibody can be evaluated as KD (Dissociation constant), Apparent KD (Apparent Dissociation constant), Dissociation rate KD (Dissociation rate constant), Apparent KD (Apparent Dissociation rate constant), or the like. They can be measured by methods known to those skilled in the art, and for example, Biacore (GE healthcare), scatchard plot, FACS, or the like can be used.
in the present invention, the step of selecting an antigen-binding domain or antibody having a higher antigen-binding activity at a low hydrogen ion concentration or a high pH, i.e., pH neutral range, than that at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, is synonymous with the step of selecting an antigen-binding domain or antibody having a lower antigen-binding activity at a high hydrogen ion concentration or a low pH, i.e., pH acidic range, than that at a low hydrogen ion concentration or a high pH, i.e., pH neutral range.
When the antigen binding activity under the condition of low hydrogen ion concentration or high pH, i.e., pH neutral range is higher than the antigen binding activity under the condition of high hydrogen ion concentration or low pH, i.e., pH acidic range, the difference between the antigen binding activity under the condition of low hydrogen ion concentration or high pH, i.e., pH neutral range and the antigen binding activity under the condition of high hydrogen ion concentration or low pH, i.e., pH acidic range is not particularly limited, but the antigen binding activity under the condition of low hydrogen ion concentration or high pH, i.e., pH neutral range is preferably 2 times or more, more preferably 10 times or more, and still more preferably 40 times or more the antigen binding activity under the condition of high hydrogen ion concentration or low pH, i.e., pH acidic range.
The antigen binding domain or antibody of the present invention obtained by the screening method may be any antigen binding domain or antibody, and for example, the antigen binding domain or antibody may be screened. For example, an antigen binding domain or antibody having a native sequence can be selected, and an antigen binding domain or antibody having a substituted amino acid sequence can be selected.
the antigen-binding domain or antibody of the present invention selected by the screening method can be prepared by any method, and for example, a preexisting antibody, a preexisting library (e.g., phage library), an antibody or library prepared from a hybridoma obtained by immunizing an animal or a B cell derived from an immunized animal, an antibody or library obtained by introducing an amino acid (e.g., histidine or glutamic acid) or unnatural amino acid mutation having a side chain with a pKa of 4.0 to 8.0 into the antibody or library (e.g., a library having an increased content of an amino acid (e.g., histidine or glutamic acid) or unnatural amino acid having a side chain with a pKa of 4.0 to 8.0, a library obtained by introducing an amino acid (e.g., histidine or glutamic acid) or unnatural amino acid mutation having a side chain at a specific position, or the like can be used.
As a method for obtaining an antigen-binding domain or antibody having a higher antigen-binding activity in the neutral pH range at a low hydrogen ion concentration or at a high pH, than in the acidic pH range at a low hydrogen ion concentration or at a low pH, from an antigen-binding domain or antibody prepared from a hybridoma obtained by immunizing an animal or a B cell derived from an immunized animal, preferable examples include: an antigen-binding molecule or antibody in which at least one of the amino acids in the antigen-binding domain or antibody described in WO2009/125825 is substituted with an amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid mutation, or an antigen-binding molecule or antibody in which an amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is inserted in the antigen-binding domain or antibody.
The position at which an amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid mutation is introduced is not particularly limited, and any position may be used as long as the antigen-binding activity in the pH acidic range is higher than the antigen-binding activity in the pH neutral range (KD (pH acidic range)/KD (pH neutral range) or KD (pH acidic range)/KD (pH neutral range)) is higher than that before the substitution or insertion. For example, when the antigen binding molecule is an antibody, the variable region, CDR, or the like of the antibody is preferably used. One skilled in the art can suitably determine the number of amino acids substituted with an amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid or the number of inserted amino acids, by substituting 1 amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, by substituting a plurality of amino acids having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, by substituting 2 or more amino acids having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid), or an unnatural amino acid. In addition, other amino acid deletions, additions, insertions, and/or substitutions may be performed simultaneously, in addition to the substitution with an amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, or the insertion of an amino acid having a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid). An antigen-binding molecule in which an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a pKa of 4.0 to 8.0 in a side chain or an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a pKa of 4.0 to 8.0 in an inserted side chain is substituted with an alanine, which is known to those skilled in the art, or a mutant antigen-binding domain or antibody in which a substitution or insertion of an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a pKa of 4.0 to 8.0 in a side chain is randomly introduced may be selected from among those having larger KD (pH acidic range)/KD (pH neutral range) or KD (pH acidic range)/KD (pH neutral range) values than before the mutation.
as described above, preferred examples of the antigen-binding molecule which is mutated to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain with a pKa of 4.0 to 8.0 and has a lower antigen-binding activity in the acidic pH range than in the neutral pH range include, for example: an antigen-binding molecule having an antigen-binding activity in the neutral pH range after mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid whose side chain has a pKa of 4.0 to 8.0, which is equivalent to the antigen-binding activity in the neutral pH range before mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid whose side chain has a pKa of 4.0 to 8.0. In the present invention, the antigen-binding molecule after mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain pKa of 4.0 to 8.0 has an antigen-binding activity equivalent to that of an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid before mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain pKa of 4.0 to 8.0, means that when the antigen-binding activity of the antigen-binding molecule before mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain pKa of 4.0 to 8.0 is taken as 100%, the antigen-binding activity of the antigen-binding molecule after mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid is at least 10%. The antigen-binding activity at pH7.4 after mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid whose side chain has a pKa of 4.0 to 8.0 may be higher than the antigen-binding activity at pH7.4 before mutation to an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid whose side chain has a pKa of 4.0 to 8.0. When the antigen-binding activity of the antigen-binding molecule is lowered by substitution or insertion with an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain with a pKa of 4.0 to 8.0, the antigen-binding activity may be made equivalent to the antigen-binding activity before the substitution or insertion with an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain with a pKa of 4.0 to 8.0 by substitution, deletion, addition, and/or insertion of 1 or more amino acids in the antigen-binding molecule. The present invention also includes antigen-binding molecules in which the above-described amino acid (e.g., histidine or glutamic acid) having a side chain pKa of 4.0 to 8.0 or an unnatural amino acid is substituted or inserted with 1 or more amino acids, and the binding activity is equalized by the substitution, deletion, addition and/or insertion.
Furthermore, when the antigen-binding molecule is a substance containing an antibody constant region, a preferable alternative to the antigen-binding molecule having antigen-binding activity in the acidic pH range which is lower than that in the neutral pH range is a method in which the antibody constant region contained in the antigen-binding molecule is changed. Specific examples of the modified antibody constant region include, for example: 11,12, 13 or 14.
amino acids that change the antigen-binding activity of the antigen-binding domain depending on the hydrogen ion concentration condition
the antigen binding domain or antibody of the present invention screened by the aforementioned screening method can be prepared by any method, for example, when the ion concentration condition is a hydrogen ion concentration condition or a pH condition, a preexisting antibody, a preexisting library (e.g., phage library), an antibody or library prepared from a hybridoma obtained by immunizing an animal or a B cell derived from an immunized animal, an antibody or library obtained by introducing a mutation of an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain with a pKa of 4.0 to 8.0 into the antibody or library (e.g., a library obtained by introducing a mutation of an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain with a pKa of 4.0 to 8.0 into a specific position or a library obtained by introducing a mutation of an amino acid (e.g., histidine or glutamic acid) or an unnatural amino acid having a side chain with a pKa of 4.0 to 8.0 into a specific position) or the like can be used.
In a non-limiting embodiment of the present invention, a library containing a plurality of antigen-binding molecules having different sequences of the present invention can be prepared by combining a light chain variable region into which "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule according to hydrogen ion concentration conditions" is introduced and a heavy chain variable region prepared as a random variable region sequence library.
As a non-limiting example of the amino acid residue, an amino acid residue contained in CDR1 of a light chain is exemplified. Further, as a non-limiting example of the amino acid residue, an amino acid residue contained in light chain CDR2 is exemplified. Further, as other non-limiting examples of the amino acid residue, amino acid residues contained in the light chain CDR3 are exemplified.
As described above, as non-limiting examples of the amino acid residue included in the light chain CDR1, there can be mentioned: an amino acid residue at position 24, 27, 28, 31, 32 and/or 34 in a CDR1 of the light chain variable region by Kabat numbering. Further, as non-limiting examples of the amino acid residue included in the light chain CDR2, there can be mentioned: amino acid residues at positions 50, 51, 52, 53, 54, 55 and/or 56 in the CDR2 of the light chain variable region by Kabat numbering. Further, non-limiting examples of the amino acid residue included in the light chain CDR3 include: an amino acid residue at position 89, 90, 91, 92, 93, 94 and/or 95A in CDR3 of the light chain variable region by Kabat numbering. These amino acid residues may be contained alone or in combination of two or more kinds thereof, as long as the antigen binding activity of the antigen binding molecule can be changed depending on the hydrogen ion concentration condition.
in the case of combining the light chain variable region into which "at least one amino acid residue that changes the antigen binding activity of the antigen binding molecule depending on the hydrogen ion concentration condition" has been introduced and the heavy chain variable region that is a library of random variable region sequences, the light chain variable region may be designed so that the sequence thereof contains a flexible residue, in the same manner as described above. The number and position of the flexible residues are not limited to a specific embodiment as long as the antigen binding activity of the antigen binding molecule of the present invention varies depending on the hydrogen ion concentration condition. That is, the CDR sequences and/or FR sequences of the heavy and/or light chains may contain one or more flexible residues. For example, non-limiting examples of the flexible residues introduced into the light chain variable region sequence include the amino acid residues shown in Table 3 or Table 4. Further, as the amino acid sequence of the light chain variable region other than the amino acid residue and the flexible residue which change the antigen binding activity of the antigen binding molecule depending on the hydrogen ion concentration condition, there can be preferably used, as non-limiting examples: germline sequences such as Vk1(SEQ ID NO:5), Vk2(SEQ ID NO:6), Vk3(SEQ ID NO:7), Vk4(SEQ ID NO:8), and the like.
[ Table 3]
(positions indicate Kabat numbering).
[ Table 4]
(positions indicate Kabat numbering).
As the amino acid residues which change the antigen binding activity of the antigen binding molecule depending on the hydrogen ion concentration condition, any amino acid residues can be suitably used, and specific examples of such amino acid residues include amino acids having a side chain pKa of 4.0 to 8.0. As such an amino acid having an electron donating property, in addition to a natural amino acid such as histidine or glutamic acid, preferably exemplified are histidine analogs (US20090035836) and unnatural amino acids (bioorg. Med. chem. (2003)11(17), 3761-2768) such as m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa7.21) or 3,5-I2-Tyr (pKa 7.38), and further, as a particularly preferable example of the amino acid residue, an amino acid having a side chain pKa of 6.0 to 7.0 can be cited.
For changing the amino acid sequence of the antigen-binding domain, a known method such as the site-specific mutagenesis method (Kunkel et al (Proc. Natl. Acad. Sci. USA (1985)82,488-492)) or overlap extension PCR can be suitably used. In addition, as a method for changing an amino acid by substituting an amino acid other than a natural amino acid, various known methods (annu.rev.biophysis.biomol.struct. (2006)35,225-249, proc.natl.acad.sci.u.s.a. (2003)100(11),6353-6357) can be used. It is also possible to suitably use, for example: and a cell-free translation system (Protein Express) in which a tRNA obtained by linking an unnatural amino acid to a complementary amber suppressor tRNA containing a UAG codon (amber codon) that is one of the stop codons is used.
preferred examples of the heavy chain variable regions to be combined include a random variable region library. The method of preparing the random variable region library is suitably combined with known methods. In a non-limiting embodiment of the present invention, an immune library constructed based on lymphocyte-derived antibody genes of an animal immunized with a specific antigen, a patient with an infectious disease, or a human immunized with a vaccine to raise the antibody titer in blood, a cancer patient, or an autoimmune disease can be preferably used as the random variable region library.
in a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of the V genes or the reconstructed functional V genes in the genomic DNA are replaced with a synthetic oligonucleotide set having a sequence encoding a codon set of an appropriate length can be preferably used as the random variable region library, as described above. At this time, since diversity of the gene sequence of CDR3 of the heavy chain was observed, only the sequence of CDR3 could be substituted. The basis for the diversity of amino acids in the variable region of an antigen-binding molecule is the diversity of amino acid residues at the positions of the antigen-binding molecule exposed to the surface. The position exposed to the surface refers to a position judged to be exposed to the surface and/or contactable with the antigen based on the structure, the structural totality and/or the modeled structure of the antigen-binding molecule, and is usually a CDR thereof. The location of exposure to the surface is preferably determined using coordinates from a three-dimensional model of the antigen binding molecule using a computer program such as the insight ii program (Accelrys). The location of the exposure to the surface can be determined using algorithms known in the art, such as Lee and Richards (j.mol. biol. (1971)55,379-400), Connolly (j.appl. cryst. (1983)16, 548-558). The determination of the location of the exposure to the surface can be performed using three-dimensional structural information derived from software and antibodies suitable for protein modeling. As software that can be used for the above purpose, SYBYL Biopolymer Module software (Tripos Associates) is preferably used. Typically or preferably, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less when the algorithm requires the user to input a size parameter. Further, methods for determining the area and the area exposed to the surface using software for personal computers are described in Pacios (company. chem. (1994)18(4),377-386, and j.mol. model. (1995)1, 46-53).
Furthermore, in a non-limiting embodiment of the present invention, a natural library constructed from antibody genes derived from healthy Human lymphocytes and having all its components composed of natural sequences that are antibody sequences free of bias can be particularly suitably used as a random variable region library (Gejima et al (Human Antibodies (2002)11,121-129) and Cardoso et al (Scand. J. Immunol. (2000)51, 337-344)).
neutralizing Activity
In one non-limiting aspect of the invention, there is provided an antigen binding molecule and a pharmaceutical composition comprising the antigen binding molecule, the antigen binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range, wherein the antigen binding activity varies depending on the ion concentration condition; and an Fc gamma receptor binding domain having an Fc gamma receptor binding activity in a neutral pH range, which is higher than the Fc gamma receptor binding activity of the Fc region of a natural human IgG having fucose-containing sugar chains at position 297 in the EU numbering system, and has a neutralizing activity against an antigen. In general, the neutralizing activity means an activity of inhibiting a biological activity of a ligand having the biological activity on cells, such as a virus or a toxin. That is, the substance having a neutralizing activity is a substance that binds to the ligand or a receptor to which the ligand binds to inhibit the binding of the ligand to the receptor. A receptor in which binding to a ligand is inhibited due to the neutralizing activity cannot exert biological activity via the receptor. When the antigen binding molecule is an antibody, an antibody having such neutralizing activity is generally referred to as a neutralizing antibody. The neutralizing activity of an analyte can be determined by comparing the biological activity of the ligand in the presence or absence of the analyte.
For example, suitable examples of the ligand considered as the main ligand of IL-6R include: IL-6 represented by SEQ ID NO. 15. IL-6R, a type I membrane protein whose amino terminus forms the extracellular domain, forms a heterotetramer with gp130 receptor dimerized by IL-6 induction (HEINRICH et al (biochem. J. (1998)334, 297-314)). Jak associated with gp130 receptor is activated due to the formation of this heterotetramer. Jak autophosphorylates and phosphorylates the receptor. The phosphorylation sites of receptors and jaks function as binding sites for molecules belonging to the Stat family of SH2, Stat3, MAP kinases, PI3/Akt, other SH 2-bearing proteins or adaptors. Subsequently, Stat, which binds to gp130 receptor, is phosphorylated by Jak. Phosphorylated Stat forms a dimer that is transferred into the nucleus, regulating transcription of target genes. Jak or Stat may also be involved in signaling cascades via other classes of receptors. Uncontrolled signaling cascades of IL-6 are observed in the morbid state of autoimmune diseases or in cancers such as inflammation, multiple myeloma or prostate cancer. Stat3, which functions as an oncogene, is activated homeostatically in a variety of cancers. In prostate cancer and multiple myeloma, there is an interaction between the signaling cascade derived from the IL-6R and the signaling cascade derived from a member of the Epithelial Growth Factor Receptor (EGFR) family (Ishikawa et al (j.clin.exp.hematopathol. (2006)46(2), 55-66)).
Since such intracellular signaling cascades differ for each cell, an appropriate target molecule can be set for each target cell to be targeted, and the intracellular signaling cascades are not limited to the factors described above. By measuring the activation of the signal in the body, the neutralizing activity can be evaluated. In addition, the activation of an in vivo signal can also be detected using as an index the transcription induction action on a target gene present downstream of the in vivo signal cascade. The change in the transcription activity of the target gene can be detected by the principle of reporter gene assay. Specifically, a reporter gene such as GFP (green fluorescent protein) or luciferase is placed downstream of a transcription factor or a promoter region of a target gene, and the activity of the reporter gene is measured, whereby the change in transcription activity can be measured as the reporter gene activity. As a kit for measuring the activation of an in vivo signal, a commercially available kit (for example, Mercury Pathway Profile luciferase System (Clontech) and the like) can be suitably used.
Further, as a method for measuring the neutralizing activity of a receptor ligand acting on a receptor such as EGF receptor family that functions in a direction promoting cell growth, the neutralizing activity of a neutralizing antibody can be evaluated by measuring the growth activity of a target cell. For example, as a method for evaluating or measuring the inhibitory effect based on the neutralizing activity of an anti-HB-EGF antibody on the growth of cells whose growth is promoted by a growth factor of the EGF family such as HB-EGF, the following method can be suitably used. As a method for evaluating or measuring the cell growth inhibitory activity in a test tube, a method of measuring uptake of living cells into a medium to which [3H ] is added]a method of using labelled thymidine as an indicator of DNA replication capacity. As a more convenient method, a dye exclusion method or an MTT method, which measures the ability of a dye such as trypan blue to be excluded from cells under a microscope, can be used. The latter makes use of: live cells have formazan which converts the tetrazolium salt MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) to a blue color(formalzan) product capacity. More specifically, a ligand and a test antibody are added to a culture solution of a test cell, and after a lapse of a certain period of time, an MTT solution is added to the culture solution and allowed to stand for a certain period of time, whereby MTT is taken into the cell. As a result, the yellow compound MTT was converted to a blue compound by intracellular mitochondrial succinate dehydrogenase. Dissolving the blue product,after the development, the absorbance was measured and used as an index of the number of living cells. In addition to MTT, commercially available reagents such as (nacalai tesque) MTS, XTT, WST-1 and WST-8 can be suitably used. In the activity measurement, a binding antibody having the same isotype as that of the anti-HB-EGF antibody and having no such cell growth inhibitory activity as a control antibody, and the anti-HB-EGF antibody were used in the same manner, and the anti-HB-EGF antibody showed a stronger cell growth inhibitory activity than that of the control antibody, whereby the activity could be judged.
as cells for evaluating activity, for example, ovarian cancer cell RMG-1 cell line, which is a cell whose growth is promoted by HB-EGF, or mouse Ba/F3 cells transformed with a vector expressing a gene linked to hEGFR/mG-CSFR which is a fusion protein obtained by in-frame fusion of the extracellular domain of human EGFR with the intracellular domain of mouse GCSF receptor, can be suitably used. Thus, one skilled in the art can use the above-described assay for cell growth activity by appropriately selecting cells for evaluation of activity.
The antigen-binding molecules provided by the present invention can eliminate antigens from plasma, and therefore, it is not absolutely necessary that the antigen-binding molecules themselves have neutralizing activity. However, it is more preferable that the function of the antigen present in the plasma is blocked by exerting a neutralizing activity on the antigen until the antigen is taken into the Fc γ receptor-expressing cells together with the antigen-binding molecule by Fc γ receptor-mediated endocytosis.
In addition, the antigen-binding molecule provided by the present invention can promote the dissociation of an antigen bound to the antigen-binding molecule outside the cell from the antigen-binding molecule inside the cell, and therefore the antigen dissociated from the antigen-binding molecule inside the cell is decomposed in lysosomes. Therefore, it is not absolutely necessary that the antigen binding molecule itself have neutralizing activity. However, it is more preferable that the function of the antigen present in the plasma is blocked by exerting a neutralizing activity on the antigen until the antigen is taken into the Fc γ receptor-expressing cell together with the antigen-binding molecule by Fc γ receptor-mediated endocytosis.
furthermore, the antigen-binding molecule provided by the present invention can reduce the total antigen concentration or the free antigen concentration in plasma, and therefore, it is not absolutely necessary that the antigen-binding molecule itself has a neutralizing activity. However, it is more preferable that the function of the antigen present in the plasma is blocked by exerting a neutralizing activity on the antigen until the antigen is taken into the Fc γ receptor-expressing cells together with the antigen-binding molecule by Fc γ receptor-mediated endocytosis.
FcγReceptors
Fc γ receptor (also referred to as Fc γ R) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, and essentially refers to any member of the family of proteins encoded by the Fc γ receptor gene. For humans, this family includes: fc γ RI (CD64) containing isoforms Fc γ RIa, Fc γ RIb and Fc γ RIc; fc γ RII (CD32) comprising isoforms Fc γ RIIa (comprising allotypes H131 and R131), Fc γ RIIb (comprising Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc; and Fc γ RIII (CD16) comprising isoforms Fc γ RIIIa (comprising allotypes V158 and F158) and Fc γ RIIIb (comprising allotypes Fc γ RIIIb-NA1 and Fc γ RIIIb-NA 2); and any undiscovered human fcyr species or fcyr isoform or allotype, but is not limited thereto. The Fc γ R includes Fc γ rs derived from human, mouse, rat, rabbit and monkey, but is not limited thereto and may be derived from any organism. Included in the mouse Fc γ R class are: fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16) and Fc γ RIII-2(Fc γ RIV, CD16-2) and any undiscovered mouse Fc γ R species or Fc γ R isoform or allotype, but is not limited thereto. Preferred examples of such Fc γ receptors include: human Fc γ RI (CD64), Fc γ RIIa (CD32), Fc γ RIIb (CD32), Fc γ RIIIa (CD16) and/or Fc γ RIIIb (CD 16). The polynucleotide sequence and amino acid sequence of human Fc γ RI are set forth in SEQ ID NOs 16 (NM-000566.3) and 17 (NP-000557.1), respectively; the polynucleotide sequence and amino acid sequence of human Fc γ RIIa (allotype H131) are set forth in SEQ ID NOS: 18(BC020823.1) and 19(AAH20823.1), respectively (allotype R131 is the sequence in which amino acid 166 of SEQ ID NO:19 is substituted with Arg); the polynucleotide sequence and amino acid sequence of Fc gamma RIIb are set forth in SEQ ID NOs 20(BC146678.1) and 21(AAI46679.1), respectively; the polynucleotide and amino acid sequences of Fc γ RIIIa are set forth in SEQ ID NO. 22(BC033678.1) and 23(AAH33678.1), respectively; and the polynucleotide and amino acid sequences of Fc γ RIIIb are set forth in SEQ ID NOs: 24(BC128562.1) and 25(AAI28563.1), respectively (RefSeq accession numbers are shown in parentheses). Whether or not the Fc γ receptor has a binding activity to the Fc region of the IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be confirmed by an ALPHA screening (enhanced luminescence Proximity homogeneity Assay (Amplified luminescence Proximity homogeneity analysis)) or a BIACORE method using the Surface Plasmon Resonance (SPR) phenomenon, in addition to the FACS and ELISA formats described above (proc.natl.acad.sci.usa (2006)103(11), 4005-4010).
furthermore, "Fc ligand" or "effector ligand" means a molecule, preferably a polypeptide, of any biological origin that binds to the Fc region of an antibody to form an Fc/Fc ligand complex. Binding of Fc ligands to Fc preferably induces one or more effector functions. Fc ligands include: fc receptors, Fc γ R, Fc α R, Fc ∈ R, FcRn, C1q, C3, mannan-binding lectin, mannose receptors, staphylococcal protein a, staphylococcal protein G, and viral Fc γ R, but are not limited thereto. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al, (2002) Immunological Reviews 190,123-136) or FCRL (Annu Rev Immunol, 2007; 25:525-60) which are a family of Fc receptors homologous to Fc γ R. The Fc ligand may also include undiscovered molecules that bind Fc.
For Fc γ RI (CD64) including Fc γ RIa, Fc γ RIb and Fc γ RIc and Fc γ RIII (CD16) including isoforms Fc γ RIIIa (containing allotypes V158 and F158) and Fc γ RIIIb (containing allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2), the α chain that binds to the Fc portion of IgG associates with the common γ chain of ITAMs that have the ability to transmit activation signals within the cell. On the other hand, Fc γ RII (CD32) itself, including isoforms Fc γ RIIa (containing allotypes H131 and R131) and Fc γ RIIc, contains ITAMs in its cytoplasmic domain. These receptors are expressed in numerous immune cells such as macrophages, mast cells, antigen presenting cells, and the like. Activation signals transmitted by the binding of these receptors to the Fc portion of IgG promote macrophage phagocytic ability, production of inflammatory cytokines, degranulation of mast cells, and hyperfunction of antigen-presenting cells. As mentioned above, Fc γ receptors having the ability to transduce activation signals are also referred to herein as active Fc γ receptors.
On the other hand, Fc γ RIIb (containing Fc γ RIIb-1 and Fc γ RIIb-2) itself contains in the cytoplasmic domain an ITIM that transmits inhibitory signals. In B cells, activation signals from BCR are inhibited by cross-linking Fc γ RIIb and B Cell Receptor (BCR), and as a result, antibody production by BCR is inhibited. In macrophages, the crosslinking of Fc γ RIII with Fc γ RIIb results in inhibition of phagocytic capacity and the ability to produce inflammatory cytokines. As mentioned above, Fc γ receptors that have the ability to transduce inhibitory signals are also referred to herein as inhibitory Fc γ receptors.
Fcγreceptor binding Activity
The binding activity of any one of Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and/or Fc γ RIIIb to the human Fc γ receptor of the Fc γ R binding domain contained in the antigen binding molecule of the present invention can be confirmed by, in addition to the FACS or ELISA formats described above, an ALPHA screening (enhanced luminescence proximity homogenization assay) or a BIACORE method using the Surface Plasmon Resonance (SPR) phenomenon, etc. (proc.natl.acad.sci.usa (2006)103(11), 4005-4010). The extracellular domain of the human Fc γ receptor can be used as a soluble antigen in these assays.
the ALPHA screening was carried out by the ALPHA technique using 2 kinds of beads, donor and acceptor, based on the following principle. The molecules bound to the donor beads and the molecules bound to the acceptor beads interact biologically, and only in the state where 2 beads are close to each other, a luminescence signal is detected. The photosensitizer within the donor bead, which is excited by the laser, converts the surrounding oxygen to singlet oxygen in an excited state. Singlet oxygen diffuses around the donor bead, and when reaching the adjacent acceptor bead, causes a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor beads and the molecules bound to the acceptor beads do not interact, singlet oxygen generated by the donor beads does not reach the acceptor beads, and thus does not cause a chemiluminescent reaction.
for example, an antigen binding molecule containing a biotin-labeled Fc region is bound to a donor bead and an Fc γ receptor tagged with Glutathione S Transferase (GST) is bound to an acceptor bead. Antigen binding molecules having a native Fc region interact with Fc γ receptors to generate a 520-620nm signal in the absence of antigen binding molecules containing competing Fc region alterations. Antigen binding molecules containing unlabeled Fc region variants compete with antigen binding molecules having native Fc regions for interaction with Fc γ receptors. By quantifying the decrease in fluorescence reflecting the competition result, the relative binding affinity can be determined. It is known to biotinylate an antigen-binding molecule such as an antibody using Sulfo-NHS-biotin or the like. As a method for labeling an Fc γ receptor with GST, a method of expressing a polynucleotide encoding an Fc γ receptor and a polynucleotide encoding GST in a cell or the like holding a vector to which a fusion gene fused in-frame is operably linked and purifying the expression product with a glutathione column can be suitably used. The resulting signal can be suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, san diego).
when one of substances (ligands) for observing the interaction is fixed to the gold thin film of the sensor chip and light is irradiated from the back side of the sensor chip to cause total reflection at the interface between the gold thin film and the glass, a part of the reflected light forms a portion (SPR signal) in which the reflection intensity is reduced. When the other of the substances (analyte) for observing the interaction flows on the surface of the sensor chip, the ligand and the analyte bind to each other, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in refractive index, the position of the SPR signal shifts (conversely, when the binding is dissociated, the position of the signal returns). The Biacore system represents the amount of displacement, i.e., the change in mass of the sensor chip surface, as vertical axis and the change in mass with time as measurement data (sensorgram). From the curves of the sensorgrams, kinetic parameters can be determined: the binding rate constant (ka) and the dissociation rate constant (KD) and the affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. Examples of inhibition assays are described in proc.natl.acad.sci.usa (2006)103(11), 4005-4010.
FcγReceptorsbinding domains
the sugar chain having Fc γ receptor binding activity linked to EU-position 297 is an Fc γ receptor binding domain having higher Fc γ receptor binding activity than that of the Fc region of natural human IgG containing fucose sugar chains, and can be prepared by changing the amino acids in the Fc region of natural human IgG. In addition, Fc γ receptor binding domains may also use domains of any of the previously described antigen binding domains that are characterized by binding to Fc γ receptors. In this case, the Fc γ receptor can be prepared without introducing an amino acid change, or the affinity for the Fc γ receptor can be improved by further introducing a change. Examples of such Fc γ receptor binding domains include: described in Protein Eng Des Sel.2009 Mar; 22(3) 175-88, Protein Eng DesSel.2008 Jan; 21(1):1-10. and J Immunol.2002 Jul 1; 169(1) the Fab fragment antibodies binding to Fc γ RIIIa, camelid-derived single domain antibodies and single chain Fv antibodies, and FASEB J.2009 Feb of 137-44; 23(2) 575 to 85, the Fc γ RI described in (1) binds to a cyclic peptide and the like. Whether or not the Fc γ R binding activity of the Fc γ receptor binding domain is higher than the Fc γ R binding activity of the Fc region of natural human IgG having fucose-containing sugar chains as the sugar chains linked at position 297 in the EU numbering system can be suitably carried out by the method described in the above item of binding activity.
in the present invention, as an example of the initial Fc γ receptor binding domain, an Fc region of human IgG can be suitably used. In the present invention, "amino acid change" or "amino acid change" of the Fc region includes changing to an amino acid sequence different from that of the starting Fc region. Modified variants of the starting Fc region are only required to bind to human Fc γ receptors in the neutral pH range, and any Fc region can be used as the starting Fc region. Furthermore, an altered Fc region in which an altered Fc region has been applied as a starting Fc region and further altered may also be suitably used as the altered Fc region of the present invention. The starting Fc region may mean the polypeptide itself, a composition containing the starting Fc region, or an amino acid sequence encoding the starting Fc region. The starting Fc region may contain the Fc region of a well-known IgG antibody produced by recombination as already outlined in the antibody project. The origin of the starting Fc region is not limited, and can be obtained from any organism other than a human animal or a human. Preferably, the arbitrary organism is preferably an organism selected from the group consisting of mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate. In other embodiments, the starting Fc region is also obtained from a cynomolgus monkey, marmoset monkey, rhesus monkey, chimpanzee, or human. The starting Fc region is preferably obtainable from human IgG1, but is not limited to a particular class of IgG. This means that the Fc region of human IgG1, IgG2, IgG3 or IgG4 can be suitably used as the initial Fc receptor binding domain. Likewise, it is also intended in the present specification that an Fc region of any class or subclass of IgG derived from any of the foregoing organisms can be preferably used as the starting Fc region. Examples of variants or modifications of naturally occurring IgG are described in well known literature (curr. opin. biotechnol. (2009)20(6),685-91, curr. opin. immunol. (2008)20(4),460-470, Protein eng.des. sel. (2010)23(4),195-202, WO2009/086320, WO2008/092117, WO2007/041635, and WO2006/105338), but are not limited thereto.
examples of the change include one or more mutations, for example, a mutation by substitution to an amino acid residue different from that of the starting Fc region, or insertion of one or more amino acid residues into or deletion of one or more amino acids from the amino acid of the starting Fc region, and the like. Preferably, the altered Fc region comprises an amino acid sequence comprising at least a portion of a non-naturally occurring Fc region. This variant must have less than 100% sequence identity or similarity to the starting Fc region. In a preferred embodiment, the variant has an amino acid sequence that is from about 75% to less than 100% identical or similar, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100%, and most preferably from about 95% to less than 100% identical or similar to the amino acid sequence of the starting Fc region. In one non-limiting embodiment of the invention, the starting Fc region and the altered Fc region of the invention have at least a1 amino acid difference between them. The difference in amino acids between the starting Fc region and the altered Fc region can also be suitably defined by the difference in amino acids specified, in particular, at the positions of the amino acid residues specified in the aforementioned EU numbering.
For changing the amino acids in the Fc region, a known method such as site-specific mutagenesis (Kunkel et al (Proc. Natl. Acad. Sci. USA (1985)82,488-492)) or overlap extension PCR can be suitably used. As a method for mutating an amino acid by substitution with an amino acid other than a natural amino acid, various known methods (annu.rev.biophysis.biomol.struct. (2006)35,225-249, proc.natl.acad.sci.u.s.a. (2003)100(11),6353-6357) can be used. It is also possible to suitably use, for example: and a cell-free translation system (Protein Express) in which a tRNA obtained by linking an unnatural amino acid to a complementary amber suppressor tRNA containing a UAG codon (amber codon) that is one of the stop codons is used.
The Fc region having Fc γ receptor binding activity in the pH neutral range contained in the antigen binding molecule of the present invention can be obtained by any method, and specifically, the Fc region having Fc γ receptor binding activity in the pH neutral range can be obtained by changing the amino acids of human IgG-type immunoglobulin used as the starting Fc region. Examples of the Fc region of a preferred IgG-type immunoglobulin to be used for modification include Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and modified forms thereof). Naturally occurring mutants thereof and the like are also included in the Fc region of IgG. Most of the allotypic Sequences of gene polymorphisms in the Fc region of human IgG1, human IgG2, human IgG3 and human IgG4 antibodies are described in Sequences of proteins of immunological interest, NIH Publication No.91-324, and any of them may be used in the present invention. In particular, the amino acid sequence at positions 356 to 358 in EU numbering is DEL or EEM, which is the sequence of human IgG 1.
the amino acid may be changed at any position as long as it has Fc γ receptor binding activity in the neutral range of pH or binding activity to Fc γ receptor binding is improved in the neutral range. The antigen binding molecule preferably contains a change that brings about an effect of enhancing binding to Fc γ receptor in a pH neutral range as compared to the binding activity of the initial Fc region of human IgG1 when the Fc region of human IgG1 is contained as the human Fc region. As amino acid changes for enhancing Fc γ receptor binding activity under conditions of pH neutral range, for example, are reported in WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635, WO2008/092117, WO2005/070963, WO2006/020114, WO2006/116260, and WO2006/023403 and the like.
examples of the amino acid capable of effecting the above-mentioned change include at least one amino acid selected from the group consisting of the following amino acids represented by EU numbering: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits. By these amino acid changes, the Fc region of IgG-type immunoglobulins is enhanced in binding to Fc γ receptors under conditions of pH neutral range.
For use in the present invention, particularly preferred alterations include, for example, alterations of at least one amino acid selected from the group consisting of the following, as represented by EU numbering, in the Fc region:
The amino acid at position 221 is any one of Lys or Tyr;
The amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
The amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
The amino acid at position 225 is any one of Glu, Lys, or Trp;
the amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
the amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
the amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 240 is any one of Ala, Ile, Met or Thr;
The amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
the amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
The amino acid at position 244 is His;
the amino acid at position 245 is Ala;
The amino acid at position 246 is any one of Asp, Glu, His or Tyr;
The 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
The amino acid at position 249 is any one of Glu, His, Gln or Tyr;
The amino acid at position 250 is either Glu or Gln;
The amino acid at position 251 is Phe;
the amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
the amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
the amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
the amino acid at position 263 is any one of Ala, Ile, Met or Thr;
The 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 266 is any one of Ala, Ile, Met or Thr;
the amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
the amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 273 is any one of Phe or Ile;
The amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 275 is any one of Leu or Trp;
The amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
the amino acid at position 281 is Asp, Lys, Pro or Tyr;
the amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
The amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
The amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
the amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
The amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
the amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
the amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
the amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
The amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
The amino acid at the 303 th position is Asp, Gly or Tyr;
the amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
the amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
The amino acid at position 313 is Phe;
the amino acid at position 315 is Leu;
The amino acid at position 317 is Glu or Gln;
The amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
The amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
The amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
the amino acid at position 336 is any one of Glu, Lys or Tyr;
The amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
amino acid 376 is Ala or Val;
The amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
the amino acid at position 379 is Asn;
the amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
The amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
the amino acid at position 427 is Asn;
the amino acid at position 428 is any one of Phe or Leu;
The amino acid at position 429 is Met;
The amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; or
The amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr. The number of amino acids to be changed is not particularly limited, and only one amino acid may be changed, or two or more amino acids may be changed. Examples of combinations of changes in amino acids at two or more positions include combinations shown in Table 5 (Table 5-1 to Table 5-3).
[ Table 5-1]
combinations of amino acids
combinations of amino acids
K370E/P396L/D270E
S239Q/I332Q
Q419H/P396L/D270E
S267D/I332E
V240A/P396L/D270E
S267E/I332E
R255L/P396L/D270E
S267L/A327S
R255L/P396L/D270E
S267Q/A327S
R255L/P396L/D270E/R292G
S298A/I332E
R255L/P396L/D270E
S304T/I332E
R255L/P396L/D270E/Y300L
S324G/I332D
F243L/D270E/K392N/P396L
S324G/I332E
F243L/R255L/D270E/P396L
S324I/I332D
F243L/R292P/Y300L/V305I/P396L
S324I/I332E
F243L/R292P/Y300L/P396L
T260H/I332E
F243L/R292P/Y300L
T335D/I332E
F243L/R292P/P396L
V240I/V266I
F243L/R292P/V305I
V264I/I332E
F243L/R292P
D265F/N297E/I332E
S298A/E333A/K334A
D265Y/N297D/I332E
E380A/T307A
F243L/V262I/V264W
K326M/E333S
N297D/A330Y/I332E
K326A/E333A
N297D/T299E/I332E
S317A/K353A
N297D/T299F/I332E
A327D/I332E
N297D/T299H/I332E
A330L/I332E
N297D/T299I/I332E
A330Y/I332E
N297D/T299L/I332E
E258H/I332E
N297D/T299V/I332E
E272H/I332E
P230A/E233D/I332E
E272I/N276D
P244H/P245A/P247V
E272R/I332E
S239D/A330L/I332E
E283H/I332E
S239D/A330Y/I332E
E293R/I332E
S239D/H268E/A330Y
F241L/V262I
S239D/I332E/A327A
F241W/F243W
S239D/I332E/A330I
[ tables 5-2]
F243L/V264I
S239D/N297D/I332E
H268D/A330Y
S239D/S298A/I332E
H268E/A330Y
S239D/V264I/I332E
K246H/I332E
S239E/N297D/I332E
L234D/I332E
S239E/V264I/I332E
L234E/I332E
S239N/A330L/I332E
L234G/I332E
S239N/A330Y/I332E
L234I/I332E
S239N/S298A/I332E
L234I/L235D
S239Q/V264I/I332E
L234Y/I332E
V264E/N297D/I332E
L235D/I332E
V264I/A330L/I332E
L235E/I332E
V264I/A330Y/I332E
L235I/I332E
V264I/S298A/I332E
L235S/I332E
Y296D/N297D/I332E
L328A/I332D
Y296E/N297D/I332E
L328D/I332D
Y296H/N297D/I332E
L328D/I332E
Y296N/N297D/I332E
L328E/I332D
Y296Q/N297D/I332E
L328E/I332E
Y296T/N297D/I332E
L328F/I332D
D265Y/N297D/T299L/I332E
L328F/I332E
F241E/F243Q/V262T/V264E
L328H/I332E
F241E/F243R/V262E/V264R
L328I/I332D
F241E/F243Y/V262T/V264R
L328I/I332E
F241L/F243L/V262I/V264I
L328M/I332D
F241R/F243Q/V262T/V264R
L328M/I332E
F241S/F243H/V262T/V264T
L328N/I332D
F241W/F243W/V262A/V264A
L328N/I332E
F241Y/F243Y/V262T/V264T
L328Q/I332D
I332E/A330Y/H268E/A327A
L328Q/I332E
N297D/I332E/S239D/A330L
L328T/I332D
N297D/S298A/A330Y/I332E
L328T/I332E
S239D/A330Y/I332E/K326E
L328V/I332D
S239D/A330Y/I332E/K326T
L328V/I332E
S239D/A330Y/I332E/L234I
L328Y/I332D
S239D/A330Y/I332E/L235D
[ tables 5 to 3]
L328Y/I332E
S239D/A330Y/I332E/V240I
N297D/I332E
S239D/A330Y/I332E/V264T
N297E/I332E
S239D/A330Y/I332E/V266I
N297S/I332E
S239D/D265F/N297D/I332E
P227G/I332E
S239D/D265H/N297D/I332E
P230A/E233D
S239D/D265I/N297D/I332E
Q295E/I332E
S239D/D265L/N297D/I332E
R255Y/I332E
S239D/D265T/N297D/I332E
S239D/I332D
S239D/D265V/N297D/I332E
S239D/I332E
S239D/D265Y/N297D/I332E
S239D/I332N
S239D/I332E/A330Y/A327A
S239D/I332Q
S239D/I332E/H268E/A327A
S239E/D265G
S239D/I332E/H268E/A330Y
S239E/D265N
S239D/N297D/I332E/A330Y
S239E/D265Q
S239D/N297D/I332E/K326E
S239E/I332D
S239D/N297D/I332E/L235D
S239E/I332E
S239D/V264I/A330L/I332E
S239E/I332N
S239D/V264I/S298A/I332E
S239E/I332Q
S239E/V264I/A330Y/I332E
S239N/I332D
F241E/F243Q/V262T/V264E/I332E
S239N/I332E
F241E/F243R/V262E/V264R/I332E
S239N/I332N
F241E/F243Y/V262T/V264R/I332E
S239N/I332Q
F241R/F243Q/V262T/V264R/I332E
S239Q/I332D
S239D/I332E/H268E/A330Y/A327A
S239Q/I332E
S239E/V264I/S298A/A330Y/I332E
S239Q/I332N
F241Y/F243Y/V262T/V264T/N297D/I332E
S267E/L328F
G236D/S267E
S239D/S267E
。
the pH conditions for measuring the binding activity of the Fc γ receptor binding domain and the Fc γ receptor contained in the antigen-binding molecule of the present invention may suitably be pH acidic range or pH neutral range conditions. The pH neutral range as a condition for determining the binding activity of the Fc γ receptor binding domain and the Fc γ receptor contained in the antigen binding molecule of the present invention generally means pH6.7 to pH 10.0. Preferably, the pH value is in the range of pH7.0 to pH8.0, preferably selected from pH7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, and particularly preferably pH7.4 which is close to the pH in plasma (blood) in the body. In the present invention, the pH acidic range as a condition under which the Fc γ receptor binding domain contained in the antigen binding molecule of the present invention has a binding activity to Fc γ receptor generally means pH4.0 to pH 6.5. Preferably means pH5.5 to pH6.5, and particularly preferably means pH5.8 to pH6.0 which is close to the pH in the early endosome in the body. As the temperature used in the assay conditions, the binding affinity of the Fc γ receptor binding domain to human Fc γ receptor can be evaluated at any temperature from 10 ℃ to 50 ℃. Preferably, to confirm the binding affinity of the human Fc γ receptor binding domain to Fc γ receptor, a temperature of 15 ℃ to 40 ℃ is employed. More preferably, any temperature of 20 ℃ to 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 ℃, is also used to determine the binding affinity of the fey receptor binding domain to the fey receptor. A temperature of 25 ℃ is a non-limiting example of a protocol of the present invention.
In the present specification, that the Fc γ receptor binding activity of the Fc γ receptor binding domain is higher than that of the native Fc region Fc γ receptor binding activity means that the binding activity of the Fc γ receptor binding domain to human Fc γ receptors of any one of Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and/or Fc γ RIIIb is higher than that of the native Fc γ receptor binding domain to these human Fc γ receptors. For example, the term "means that the binding activity of an antigen-binding molecule containing an Fc γ receptor binding domain is 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, particularly preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, compared with the binding activity of an antigen-binding molecule containing an Fc region of a natural human IgG as a control, based on the above-described analytical method, More than 100 times of the binding activity. As the native Fc γ receptor binding domain, either the original Fc γ receptor binding domain or the Fc γ receptor binding domain of a native antibody of the same subclass may be used.
In the present invention, particularly, as the Fc region of the control natural human IgG, the Fc region of natural human IgG having fucose-containing sugar chains as sugar chains linked to the amino acid at position 297 shown in the EU numbering system is preferably used. Whether or not the sugar chain linked to the amino acid at position 297 shown by EU numbering is a fucose-containing sugar chain can be determined by the method described in non-patent document 24. For example, it can be determined whether or not the sugar chain linked to the Fc region of a natural human IgG is a fucose-containing sugar chain by the following method. By reacting N-glycosidase F (Roche diagnostics) with the natural type human IgG to be tested, the sugar chain is isolated upstream from the natural type human IgG to be tested (Weitzhandler et al (J. Pharma. sciences (1994)83,12, 1670-1675.) then, the concentrated dried product of the reaction solution in which ethanol is reacted to remove protein (Schenk et al (J. Clin. investigation (2001)108(11) 1687-1695)) is fluorescently labeled with 2-aminopyridine (Bigge et al (anal. biochem. (1995)230(2) 229-238). the reagent is removed by solid phase extraction using a cellulose column, the fluorescently labeled 2-AB glycosylated sugar chain is analyzed by normal phase chromatography.by observing the peak to be detected, it can be judged whether the sugar chain linked to the Fc region of the natural type human IgG is a fucose-containing sugar chain, as an example of the natural type human IgG-containing sugar chain linked to the amino acid at the EU position 297, examples thereof include: an Fc region contained in an antibody obtained by expressing a gene encoding an antibody containing an Fc region of a natural human IgG in CHO cells such as CHO-K1(American Type Culture Collection, ATCC No. CRL-61) and DXB11(American Type Culture Collection, ATCC No. CRL-11397). By comparing the Fc γ receptor binding activity of the Fc region contained in these antibodies with that of the Fc region of the present invention as a control, it was confirmed whether the Fc γ receptor binding activity of the Fc γ receptor binding domain of the present invention is higher than that of the Fc region of native human IgG having fucose-containing sugar chains as the sugar chain linked to EU position 297. Further, by measuring the fucose content of the sugar chains linked to the Fc region contained in these antibodies and the fucose content of the sugar chains linked to the Fc region of the present invention using the methods described above and the like, it is also possible to compare the fucose contents linked to the sugar chains contained in the Fc region to be compared.
As the antigen-binding molecule containing the Fc region of a natural-type antibody of the same subclass as a control, an antigen-binding molecule having the Fc region of an IgG monoclonal antibody can be suitably used. The structure of this Fc region is described in: SEQ ID NOS.11 (A is added to the N-terminus of RefSeq accession No. AAC 82527.1), 12 (A is added to the N-terminus of RefSeq accession No. AAB 59393.1), 13(RefSeq accession No. CAA27268.1) and 14 (A is added to the N-terminus of RefSeq accession No. AAB 59394.1). In addition, when an antigen-binding molecule containing an Fc region of an antibody of a specific isotype is used as a test substance, the effect of the Fc γ receptor binding activity of the antigen-binding molecule containing an Fc region to be tested can be verified by using an antigen-binding molecule having an Fc region of an IgG monoclonal antibody of the specific isotype as a control. As described above, it is appropriate to select an antigen binding molecule containing an Fc region whose Fc γ receptor binding activity is verified to be high.
Further, as examples of the Fc γ receptor binding domain suitably used in the present invention, there are also suitably mentioned: an Fc γ receptor binding domain having the property of having a binding activity to a specific Fc γ receptor higher than that to other Fc γ receptors (an Fc γ receptor binding domain having a selective Fc γ receptor binding activity). In the case of using an antibody (Fc region as an Fc γ receptor binding domain) as an antigen binding molecule, one molecule of the antibody binds only one molecule of Fc γ receptor, and thus one molecule of the antigen binding molecule cannot bind to other active-type Fc γ R in a state of binding to an inhibitory-type Fc γ receptor, or cannot bind to other active-type Fc γ receptor or an inhibitory-type Fc γ receptor in a state of binding to the active-type Fc γ receptor.
as described above, examples of the active Fc γ receptor include: fc γ RI (CD64) containing Fc γ RIa, Fc γ RIb and Fc γ RIc, and Fc γ RIII (CD16) containing isoforms Fc γ RIIIa (containing allotypes V158 and F158) and Fc γ RIIIb (containing allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA 2). Further, Fc γ RIIb (containing Fc γ RIIb-1 and Fc γ RIIb-2) is given as a suitable example of the inhibitory Fc γ receptor.
For FcγFc with receptor having selective binding activityγr binding domain
whether or not the Fc γ R binding domain of the present invention has selective binding activity can be confirmed by comparing the binding activity to each Fc γ receptor determined by the method described in the above-mentioned item of Fc γ receptor binding activity. As the selective Fc γ R binding domain contained in the antigen binding molecule provided by the present invention, an Fc γ R binding domain having a binding activity to an inhibitory Fc γ receptor higher than that to an active Fc γ receptor can be used. In a non-limiting embodiment, as the selective Fc γ R binding domain contained in the antigen binding molecule provided by the present invention, an Fc γ R binding domain having a binding activity to Fc γ RIIb (containing Fc γ RIIb-1 and Fc γ RIIb-2) higher than that to an active-type Fc γ receptor selected from the group consisting of Fc γ RI (CD64) containing Fc γ RIa, Fc γ RIIb and Fc γ RIc, Fc γ RIII (CD16) containing isoforms Fc γ RIIIa (containing isoforms V158 and F158) and Fc γ RIIIb (containing isoforms Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2), and Fc γ RII (CD32) containing isoforms Fc γ RIIa (containing isoforms H131 and R131) and Fc γ RIIc may be used. In a non-limiting embodiment of the present invention, as the selective Fc γ R binding domain contained in the antigen binding molecule provided by the present invention, an Fc γ R binding domain having a binding activity to Fc γ RIIb-1 and/or Fc γ RIIb-2 higher than that to Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131, and/or Fc γ RIIc can be used. Whether the Fc γ R binding domain of the subject is an Fc γ R binding domain having selective binding activity to an Fc γ receptor can be determined by comparing the following Fc γ R selectivity indices: for example, the Fc γ R selectivity index represented by formula 1 is a value (ratio) obtained by dividing the KD value of the Fc γ R binding domain pair determined by the method described in the above-mentioned Fc γ receptor binding activity item, Fc γ RIa, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131 and/or Fc γ RIIc by the KD value for Fc γ RIIb-1 and/or Fc γ RIIb-2.
[ formula 1]
Fc γ R selectivity index (KD value for active Fc γ R/KD value for inhibitory Fc γ R)
In formula 1 above, the active Fc γ R refers to Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131, and/or Fc γ RIIc, and the inhibitory Fc γ R refers to Fc γ RIIb-1 and/or Fc γ RIIb-2, and the active Fc γ R and inhibitory Fc γ R used for the KD measurement may be selected from any combination, but as a non-limiting embodiment, a value (ratio) obtained by dividing the KD value of Fc γ RIIa containing allotype H131 by the KD value of Fc γ RIIb-1 and/or Fc γ RIIb-2 may be used.
examples of the Fc γ R selectivity index include: 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or 380 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, 520 or more, 540 or more, 560 or more, 580 or more, 600 or more, 620 or more, 640 or more, 660 or more, 680 or more, 700 or more, 720 or more, 740 or more, 760 or more, 780 or more, 800 or more, 820 or more, 840 or more, 860 or more, 880 or more, 900 or more, 920 or more, 940 or more, 960 or more, 980 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 or more, 10000 or more, or 100000.
as a non-limiting embodiment of the selective Fc γ R binding domain contained in the antigen binding molecule of the present invention, there can be exemplified: an Fc region constituting a part of the constant region represented by human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), IgG3(SEQ ID NO:16) or IgG4(SEQ ID NO:17) (the Fc region of the IgG class refers to an Fc region in which the Fc γ R binding domain contained in the constant region is changed, for example, from cysteine at position 226 to the C-terminus or from proline at position 230 to the C-terminus, as expressed by EU numbering, but not limited thereto). Examples of the method for producing the modified Fc region include the methods described in the above-mentioned items for amino acid modification. As examples of such an altered Fc region, there can be exemplified: an Fc region in which the 238 th amino acid in the EU numbering system is Asp or an Fc region in which the 328 th amino acid in the EU numbering system is Glu, of human IgG (IgG1, IgG2, IgG3, IgG 4). Human IgG (IgG1, IgG2, IgG3, IgG4) having an Fc region in which amino acid at position 238 in the EU numbering system is Asp or an Fc region in which amino acid at position 328 in the EU numbering system is Glu, and an antigen-binding molecule comprising the Fc region, has a higher binding activity to Fc γ RIIb-1 and/or Fc γ RIIb-2 than to Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131 and/or Fc γ RIIC.
The constant region comprising a selective Fc γ R binding domain and the antigen binding molecule comprising the constant region comprised in the antigen binding molecule of the invention may also be: and the peptides constituting human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), an Fc region (the Fc region of IgG3(SEQ ID NO:16) or a part of IgG4(SEQ ID NO:17) (the Fc region of IgG class refers to, for example, from cysteine at position 226 to the C-terminus or from proline at position 230 to the C-terminus, as indicated by EU numbering, but is not limited thereto) (hereinafter, collectively referred to as wild-type Fc region) maintains or reduces the binding activity to an active Fc γ R (Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131 and/or Fc γ RIic) and an antigen-binding molecule containing the Fc region, as compared with an antigen-binding molecule containing the wild-type Fc region.
The extent to which the binding activity of the Fc region containing the selective Fc γ R binding domain contained in the antigen binding molecule of the present invention and the antigen binding molecule containing the Fc region to the active Fc γ R is reduced as compared with a wild-type Fc region and an antigen binding molecule containing a wild-type Fc region is, for example, as follows: 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.005% or less.
The Fc region containing a selective Fc γ R binding domain, the constant region containing the Fc region, and the antigen binding molecule containing the constant region contained in the antigen binding molecule of the present invention may also be: an Fc region having enhanced binding activity to inhibitory Fc γ R (Fc γ RIIb-1 and/or Fc γ RIIb-2) and an antigen-binding molecule comprising the same, as compared with an Fc region (the Fc region of IgG class means, for example, a cysteine at position 226 to the C-terminus or a proline at position 230 to the C-terminus, as expressed in EU numbering, but not limited thereto) constituting a part of a constant region expressed by human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), IgG3(SEQ ID NO:16), or IgG4(SEQ ID NO:17) and an antigen-binding molecule comprising the same.
The degree of binding activity of the Fc region containing the selective Fc γ R binding domain and the antigen-binding molecule containing the Fc region, which are contained in the antigen-binding molecule of the present invention, to the inhibitory Fc γ R is enhanced as compared with a wild-type Fc region and an antigen-binding molecule containing a wild-type Fc region, examples of the degree include: 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 112% or more, 114% or more, 116% or more, 118% or more, 120% or more, 122% or more, 124% or more, 126% or more, 128% or more, 130% or more, 132% or more, 134% or more, 136% or more, 138% or more, 140% or more, 142% or more, 144% or more, 146% or more, 148% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, More than 60 times, more than 70 times, more than 80 times, more than 90 times, more than 100 times, more than 200 times, more than 300 times, more than 400 times, more than 500 times, more than 600 times, more than 700 times, more than 800 times, more than 900 times, more than 1000 times, more than 10000 times, more than 100000 times.
Furthermore, the Fc region comprising a selective Fc γ R binding domain and the antigen binding molecule comprising the Fc region comprised in the antigen binding molecule of the invention may also be: compared with an Fc region (the Fc region of IgG class means, for example, a cysteine at position 226 to the C-terminus or a proline at position 230 to the C-terminus, as expressed in EU numbering, as represented by, but not limited to, the Fc region of IgG class (hereinafter, collectively referred to as wild-type Fc region) constituting a part of the constant region represented by human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), IgG3(SEQ ID NO:16) or IgG4(SEQ ID NO: 17)) and an antigen-binding molecule comprising the wild-type Fc region, the binding activity to active Fc γ Rs (Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa comprising allotype V158, Fc γ RIIIa comprising allotype F158, Fc γ RIIIb comprising allotype F1, Fc γ RIIIb comprising allotype Fc γ RIIIb-NA2, Fc γ RIIa comprising allotype H131, Fc γ RIIa comprising allotype RIIa and Fc γ RIIC/RIIC) is maintained or reduced, and an Fc region having an enhanced binding activity to an inhibitory Fc γ R (Fc γ RIIb-1 and/or Fc γ RIIb-2) and an antigen-binding molecule comprising the same, as compared with an Fc region (the Fc region of IgG class means, for example, a cysteine at position 226 to the C-terminus or a proline at position 230 to the C-terminus, as expressed in EU numbering, but not limited thereto) constituting a part of a constant region expressed by human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), IgG3(SEQ ID NO:16) or IgG4(SEQ ID NO:17) and an antigen-binding molecule comprising the same.
furthermore, the Fc region comprising a selective Fc γ R binding domain and the antigen binding molecule comprising the Fc region comprised in the antigen binding molecule of the invention may also be: compared with an Fc region (the Fc region of IgG class means, for example, a cysteine at position 226 to the C-terminus or a proline at position 230 to the C-terminus, as expressed in EU numbering, but not limited thereto) constituting a part of the constant region represented by human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), IgG3(SEQ ID NO:16) or IgG4(SEQ ID NO:17) (hereinafter, collectively referred to as wild-type Fc region) and an antigen-binding molecule containing the wild-type Fc region, the degree of binding activity of an active type Fc γ receptor (Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, or Fc γ RIIa containing allotype RIIIb 131) is enhanced, an Fc region having a high degree of enhancement of the binding activity to an inhibitory Fc gamma receptor (Fc gamma RIIb-1 and/or Fc gamma RIIb-2) and an antigen-binding molecule comprising the same.
in the present invention, at least one additional modification to the Fc region may be added to the Fc region in which the 238 th amino acid represented by the EU numbering system is Asp or the Fc region in which the 328 th amino acid represented by the EU numbering system is Glu, using the protocols described in the above-mentioned items for amino acid modification. Further, in addition to these changes, further added changes may be contained. The added change may be selected from, for example, any one of a substitution, deletion, or modification of an amino acid, or a combination thereof. For example, alterations that enhance binding activity to Fc γ RIIb, and maintain or reduce binding activity to Fc γ RIIa (H type) and Fc γ RIIa (R type) can be added. By adding such a change, the binding selectivity to Fc γ RII is increased compared to Fc γ RIIa.
of these, a change with increased binding selectivity for Fc γ RIIb as compared with Fc γ RIIa (type R) is preferred, and a change with increased binding selectivity for Fc γ RIIb as compared with Fc γ RIIa (type H) is further preferred. Preferred amino acid substitutions for such changes include, for example: change of substitution of Gly to Trp at position 237 in the EU numbering system, change of substitution of Gly to Phe at position 237 in the EU numbering system, change of substitution of Pro to Phe at position 238 in the EU numbering system, change of substitution of Asn to Met at position 325 in the EU numbering system, change of substitution of Ser to Ile at position 267 in the EU numbering system, change of substitution of Leu to Asp at position 328 in the EU numbering system, change of substitution of Ser to Val at position 267 in the EU numbering system, change of substitution of Ser to Gln at position 267 in the EU numbering system, change of substitution of Ser to Met at position 267 in the EU numbering system, change of Gly to Asp at position 236 in the EU numbering system, change of Ala to Asn at position 327 in the EU numbering system, change of Asn to Ser at position 325 in the EU numbering system, change of Leu to Tyr at position 235 in the EU numbering system, change of Val to Met at position 266 in the EU numbering system, Change of substitution of Leu to Tyr as indicated by the EU number 328, change of substitution of Leu to Trp as indicated by the EU number 235, change of substitution of Leu to Phe as indicated by the EU number 235, change of substitution of Ser to Gly as indicated by the EU number 239, change of substitution of Ala to Glu as indicated by the EU number 327, change of substitution of Ala to Gly as indicated by the EU number 327, change of substitution of Pro to Leu as indicated by the EU number 238, change of substitution of Ser to Leu as indicated by the EU number 239, change of substitution of Leu to Thr as indicated by the EU number 328, change of substitution of Leu to Met as indicated by the EU number 328, change of substitution of Pro to Trp as indicated by the EU number 331, change of substitution of Pro to Tyr as indicated by the EU number 331, change of substitution of Pro to Phe as indicated by the EU number 331 to Asp as indicated by the EU number 327, Change of substitution of Leu to Phe at position 328 of the EU number, change of substitution of Pro to Leu at position 271 of the EU number, change of substitution of Ser to Glu at position 267 of the EU number, change of substitution of Leu to Ala at position 328 of the EU number, change of substitution of Leu to Ile at position 328 of the EU number, change of substitution of Leu to Gln at position 328 of the EU number, change of substitution of Leu to Val at position 328 of the EU number, change of substitution of Lys to Trp at position 326 of the EU number, change of substitution of Lys to Arg at position 334 of the EU number, change of substitution of His to Gly at position 268 of the EU number, change of His to Asn at position 268 of the EU number, change of Ser to Val at position 324 of the EU number, change of Val to Leu at position 266 of the EU number, change of Pro to Gly at position 271 of the EU number, change of substitution of Ile to Phe at position 332 of the EU number, Change of substitution of Ser to Ile at position 324 in the EU numbering system, change of substitution of Glu to Pro at position 333 in the EU numbering system, change of substitution of Tyr to Asp at position 300 in the EU numbering system, change of substitution of Ser to Asp at position 337 in the EU numbering system, change of substitution of Tyr to Gln at position 300 in the EU numbering system, change of substitution of Thr to Asp at position 335 in the EU numbering system, change of substitution of Ser to Asn at position 239 in the EU numbering system, change of substitution of Lys to Leu at position 326 in the EU numbering system, change of substitution of Lys to Ile at position 326 in the EU numbering system, change of Ser to Glu at position 239 in the EU numbering system, change of Lys to Phe at position 326 in the EU numbering system, change of Lys to Tyr at position 326 in the EU numbering system, change of Ser to Asp at position 267 in the EU numbering system, change of Lys to Pro at position 326 in the EU numbering system, Change of substitution of Lys to His at position 326 in the EU numbering system, change of substitution of Lys to Ala at position 334 in the EU numbering system, change of substitution of Lys to Trp at position 334 in the EU numbering system, change of substitution of His to Gln at position 268 in the EU numbering system, change of substitution of Lys to Gln at position 326 in the EU numbering system, change of substitution of Lys to Glu at position 326 in the EU numbering system, change of Lys to Met at position 326 in the EU numbering system, change of Val to Ile at position 266 in the EU numbering system, change of Lys to Glu at position 334 in the EU numbering system, change of Tyr to Glu at position 300 in the EU numbering system, change of Lys to Met at position 334 in the EU numbering system, change of Lys to Thr at position 334 in the EU numbering system, change of Lys to Ser at position 334 in the EU numbering system, change of Lys to His at position 334 in the EU numbering system, Substitution of Lys to Phe at position 334 of EU number, substitution of Lys to Gln at position 334 of EU number, substitution of Lys to Pro at position 334 of EU number, substitution of Lys to Tyr at position 334 of EU number, substitution of Lys to Ile at position 334 of EU number, substitution of Gln to Leu at position 295 of EU number, substitution of Lys to Leu at position 334 of EU number, substitution of Lys to Asn at position 334 of EU number, substitution of His to Ala at position 268 of EU number, substitution of Ser to Asp at position 239 of EU number, substitution of Ser to Ala at position 267 of EU number, substitution of Leu to Trp at position 234 of EU number, substitution of Leu to Tyr at position 234 of EU number, substitution of Gly to Ala at position 237 of EU number, substitution of Gly to Asp at position 237 of EU number, Change of substitution of Gly to Glu at position 237 in the EU numbering system, change of substitution of Gly to Leu at position 237 in the EU numbering system, change of substitution of Gly to Met at position 237 in the EU numbering system, change of substitution of Gly to Tyr at position 237 in the EU numbering system, change of substitution of Ala to Lys at position 330 in the EU numbering system, change of substitution of Ala to Arg at position 330 in the EU numbering system, change of substitution of Glu to Asp at position 233 in the EU numbering system, change of substitution of His to Asp at position 268 in the EU numbering system, change of substitution of His to Glu at position 268 in the EU numbering system, change of substitution of Lys to Asp at position 326 in the EU numbering system, change of Lys to Ser at position 326 in the EU numbering system, change of Lys to Thr at position 326 in the EU numbering system, change of substitution of Val to Ile at position 323 in the EU numbering system, change of Val to Met, The substitution of Tyr for Asp at position 296 in the EU numbering system, the substitution of Lys for Ala at position 326 in the EU numbering system, the substitution of Lys for Asn at position 326 in the EU numbering system, and the substitution of Ala for Met at position 330 in the EU numbering system.
among these changes, preferable amino acid substitutions include, for example: change of substitution of Gly to Trp in position 237 in the EU numbering system, change of substitution of Gly to Phe in position 237 in the EU numbering system, change of substitution of Ser to Val in position 267 in the EU numbering system, change of substitution of Ser to Gln in position 267 in the EU numbering system, change of substitution of His to Asn in position 268 in the EU numbering system, change of substitution of Pro to Gly in position 271 in the EU numbering system, change of substitution of Lys to Leu in position 326 in the EU numbering system, change of substitution of Lys to Glu in position 326 in the EU numbering system, change of substitution of Lys to Met in position 326 in the EU numbering system, change of Ser to Asp in position 239 in the EU numbering system, change of Ser to Ala in position 267 in the EU numbering system, change of Leu to Trp in position 234 in the EU numbering system, change of Leu to Tyr in position 234 in the EU numbering system, change of Gly to Ala in position 237 in the EU numbering system, Change of substitution of Gly to Asp in position 237 in the EU numbering system, change of substitution of Gly to Glu in position 237 in the EU numbering system, change of substitution of Gly to Leu in position 237 in the EU numbering system, change of substitution of Gly to Met in position 237 in the EU numbering system, change of substitution of Gly to Tyr in position 237 in the EU numbering system, change of substitution of Ala to Lys in position 330 in the EU numbering system, change of substitution of Ala to Arg in position 330 in the EU numbering system, change of substitution of Glu to Asp in position 233 in the EU numbering system, change of substitution of His to Asp in position 268 in the EU numbering system, change of substitution of His to Glu in position 268 in the EU numbering system, change of Lys to Asp in position 326 in the EU numbering system, change of Lys to Ser in position 326 in the EU numbering system, change of substitution of Lys to Thr in position 326 in the EU numbering system, change of Val to Ile in position 323 in the EU numbering system, change of Val to Leu in position 323 in the EU numbering, A change in substitution of Val to Met at position 323 in the EU numbering system, a change in substitution of Tyr to Asp at position 296 in the EU numbering system, a change in substitution of Lys to Ala at position 326 in the EU numbering system, a change in substitution of Lys to Asn at position 326 in the EU numbering system, and a change in substitution of Ala to Met at position 330 in the EU numbering system.
the above-described change may be one position or a combination of two or more positions. Preferable examples of these modifications include those described in tables 14 to 15, tables 17 to 24, and tables 26 to 28.
As another non-limiting embodiment of the selective Fc γ R binding domain contained in the antigen binding molecule of the present invention, there can be exemplified: an Fc region in which an Fc γ R binding domain contained in an Fc region represented by human IgG1(SEQ ID NO:14), IgG2(SEQ ID NO:15), IgG3(SEQ ID NO:16) or IgG4(SEQ ID NO:17) is altered. Examples of the method for producing the modified Fc region include the methods described in the above-mentioned items for amino acid modification. As examples of such an altered Fc region, there can be exemplified: an Fc region in which the 238 th amino acid in the EU numbering system of human IgG (IgG1, IgG2, IgG3, IgG4) is Asp and the 271 th amino acid in the EU numbering system is Gly. An Fc region in which the 238 th amino acid in the EU numbering is Asp and the 271 th amino acid in the EU numbering is Gly in human iggs (IgG1, IgG2, IgG3, IgG4) and an antigen-binding molecule comprising the Fc region are: has a higher binding activity to Fc γ RIa-1 and/or Fc γ RIIb-2 than Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa with allotype V158, Fc γ RIIIa with allotype F158, Fc γ RIIIb with allotype Fc γ RIIIb-NA1, Fc γ RIIIb with allotype Fc γ RIIIb-NA2, Fc γ RIIa with allotype H131, and Fc γ RIIa and/or Fc γ RIic with allotype R131.
In the present invention, at least one additional change to the Fc region may be added to the Fc region in which the 238 th amino acid represented by the EU numbering system is Asp and the 271 th amino acid represented by the EU numbering system is Gly, using the protocols described in the above-mentioned amino acid changes. Further, in addition to these changes, further added changes may be contained. The added change may be selected from, for example, any one of or a combination of substitution, deletion, or modification of an amino acid. For example, alterations can be added that maintain or reduce binding activity to active Fc γ receptors (Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131). Alterations may be added to enhance binding activity to inhibitory Fc γ receptor (Fc γ RIIb-1 and/or Fc γ RIIb-2) and maintain or reduce binding activity to Fc γ RIIa (H type) and Fc γ RIIa (R type). In addition, it is possible to add: the degree of enhancement of binding activity to the active Fc γ receptor (Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa containing allotype V158, Fc γ RIIIa containing allotype F158, Fc γ RIIIb containing allotype Fc γ RIIIb-NA1, Fc γ RIIIb containing allotype Fc γ RIIIb-NA2, Fc γ RIIa containing allotype H131, Fc γ RIIa containing allotype R131) is higher than the degree of enhancement of binding activity to the inhibitory Fc γ receptor (Fc γ RIIb-1 and/or Fc γ RIIb-2). By adding this alteration, the binding selectivity to Fc γ RIIb is increased compared to Fc γ RIIa.
As examples of the modified Fc region containing the selective Fc γ R binding domain, a modified Fc region in which at least one of amino acid 238 in the EU numbering system is Asp and amino acid 271 in the EU numbering system is Gly of human IgG (IgG1, IgG2, IgG3, IgG4) and at least one of amino acid 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396 in the EU numbering system is substituted may be exemplified as a non-limiting example.
Further, as a non-limiting example of the altered Fc region containing the selective Fc γ R binding domain, an altered Fc region in which the 238 th amino acid of human IgG (IgG1, IgG2, IgG3, and IgG4) is Asp and the 271 th amino acid of human IgG is Gly, which is expressed by EU numbering, may be exemplified, wherein the altered Fc region is one or more of the following expressed by EU numbering:
Asp for the amino acid at position 233;
the amino acid at position 234 is Tyr;
Asp for the amino acid at position 237;
The amino acid at position 264 is Ile;
The amino acid at position 265 is Glu;
the amino acid at position 266 is any one of Phe, Met or Leu;
the amino acid at position 267 is any one of Ala, Glu, Gly, or Gln;
the amino acid at position 268 is any one of Asp or Glu;
asp for the amino acid at position 269;
The amino acid at position 272 is any one of Asp, Phe, Ile, Met, Asn or Gln;
asp for the amino acid at position 296;
The amino acid at position 326 is either Ala or Asp;
The amino acid at position 327 is Gly;
The amino acid at position 330 is any one of Lys or Arg;
the amino acid at position 331 is Ser;
The amino acid at position 332 is Thr;
the amino acid at position 333 is any one of Thr, Lys or Arg;
The amino acid at position 396 is any one of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg or Tyr.
As a non-limiting example of the above-mentioned modified Fc region containing further at least one modification to the Fc region and further added thereto, there can be exemplified the Fc regions described in tables 6-1 to 6-7:
[ Table 6-1]
(Table 6-2 shows continuation of Table 6-1)
[ tables 6-2]
(Table 6-3 shows continuation of Table 6-2)
[ tables 6 to 3]
(Table 6-4 shows continuation of Table 6-3)
[ tables 6 to 4]
(tables 6-5 are continuation tables of tables 6-4)
[ tables 6 to 5]
(tables 6-6 are continuation tables of tables 6-5)
[ tables 6 to 6]
(tables 6-7 are continuation tables of tables 6-6)
[ tables 6 to 7]
for mice, 4 Fc γ rs, Fc γ RI, Fc γ RIIb, Fc γ RIII, and Fc γ RIV, have been found so far. As Fc γ R corresponding to these, Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, Fc γ RIIIb have been found in humans. Of these Fc γ rs, Fc γ RIIb, which is considered to be the only inhibitory type, is conserved in both humans and mice. Other Fc γ rs, in addition to Fc γ RIIIb, transmit activation signals via an immunoreceptor tyrosine-based activating motif (ITAM), while Fc γ RIIb transmits inhibition signals via an immunoreceptor tyrosine-based inhibiting motif (ITIM) that is present within cells (nat. rev. immunol. 2008)8, 34-47).
as splice variants of Fc γ RIIb, Fc γ RIIb1 and Fc γ RIIb2 are reported. In either human or mouse, Fc γ RIIb1 has a long intracellular domain compared to Fc γ RIIb2, confirming that Fc γ RIIb1 is expressed in B cells and Fc γ RIIb2 is expressed in macrophages, mast cells, dendritic cells, basophils, neutrophils, eosinophils (j.clin.immunol. (2005)25(1), 1-18).
to date, it has been reported that Fc γ RIIb is deficient in function and decreased in expression, which is associated with the onset of autoimmune diseases. For example, it is reported that: in SLE patients, binding of transcriptional activator was weakened due to the influence of gene polymorphism located in the promoter region for expression of Fc γ RIIb, and there were examples of decreased expression of Fc γ RIIb (hum. Genet. (2005)117,220-227, J.Immunol. (2004)172,7192-7199, J.Immunol. (2004)172, 7186-7191). In addition, 2 kinds of gene polymorphisms that the amino acid at position 233 of Fc γ RIIb is Ile or Thr in SLE patients are reported. It is reported that: this site is present in the transmembrane region of Fc γ RIIb, and when the amino acid at position 233 is Thr, Fc γ RIIb is less likely to be present on lipid rafts than when he is present, and as a result, the signaling function of Fc γ RIIb is reduced (nat. med. (2005)11,1056-1058, hum. mol. gene., (2005)14, 2881-2892). For mice, it has also been reported that: the knockout mice in which the Fc γ RIIb gene of C57BL/6 mice was disrupted exhibited SLE-like symptoms such as autoantibody production or glomerulonephritis (Immunity 13(2000)277-285, J.Exp.Med. (2002)195, 1167-1174). It has also been reported that the expression level of Fc γ RIIb is also reduced in mice considered to be a natural pathogenesis model of SLE to date, etc. (Immunogenetics (2000)51,429-435, int. Immunol. (1999)11,1685-1691, Curr. biol. (2000)10,227-230, J.Immunol. (2002)169, 4340-4346). According to these reports, it is considered that Fc γ RIIb controls humoral immunity in mice as well as in humans.
among the Fc γ RIIb functions, Fc γ RIIb endocytosis functions are considered to contribute most significantly to the elimination of an antigen via Fc γ RIIb in the Fc-bearing antibody of the present invention. As described above, Fc γ RIIb1 and Fc γ RIIb2 exist as splice variants of Fc γ RIIb, but the latter have been reported to be primarily involved in endocytosis of the immune complex of antibody and antigen (J.Immunol. (1994),152574-585, Science (1992)256,1808-1812, Cell (1989)58, 317-327). To date, it has been reported that Fc γ RIIb2 in mice is taken up into clathrin-coated pits, causing endocytosis (Cell (1989)58, 317-327). In addition, the dual leucine motif (dileucine motif) is reported to be essential in Fc γ RIIb 2-mediated endocytosis, and is conserved in either humans or mice (EMBO J. (1994)13(13), 2963-2969). According to the above report, it is considered that Fc γ RIIb2 has endocytosis ability in humans as in mice.
On the other hand, Fc γ RIIb1 was reported not to cause endocytosis unlike Fc γ RIIb 2. An insertion in an intracellular domain not found in Fc γ RIIb2 was present in Fc γ RIIb 1. This sequence is believed to inhibit Fc γ RIIb1 uptake into clathrin-coated pits, which results in inhibition of endocytosis (j.cell.biol. (1992)116,875-888, j.cell.biol. (1989)109, 3291-3302). In humans as well as in mice, the insertion sequence is present in Fc γ RIIb1 in a similar part to Fc γ RIIb2, thus predicting a difference in endocytosis capacity of Fc γ RIIb1 from Fc γ RIIb2 in a similar mechanism. Furthermore, it has been reported that about 40% of immunocomplexes on the cell surface are taken up into the cell during 20 minutes, both in humans and in mice (mol. immunol. (2011)49,329-337, Science (1992)256, 1808-1812). According to these reports, it can be predicted that Fc γ RIIb2 causes the immune complex to be taken into cells at a similar rate to that of mice in humans as well as in mice.
in the Fc γ R family, Fc γ RIIb is the only one in humans and mice, and it is assumed that it has ITIM in cells and the distribution of expression cells is the same, and therefore, it also has a function in immune control. Further, considering the fact that the immune complex is taken into the cell at the same rate in both human and mouse, it is considered that: by using mice, the antigen elimination effect of Fc γ RIIb-mediated antibodies in humans can be predicted. In fact, in example 5, when the antigen-binding molecules mF44 and mF46, which have the property of binding to a soluble antigen with pH dependency and have enhanced affinity for mouse Fc γ RIIb and Fc γ RIII, were administered to normal mice, increased clearance of the antigen was shown compared to when mIgG1 was administered, as compared to the antigen-binding molecule mIgG1, which has the property of binding to a soluble antigen with pH dependency.
In example 6 described later, the same experiment was performed using Fc receptor γ chain-deficient mice. In the case of mice, it was reported that Fc γ R other than Fc γ RIIb is expressed only in the coexistence of γ chains, and therefore Fc receptor γ chain-deficient mice express only Fc γ RIIb. The effect of enhancing the selective binding of Fc γ RIIb to accelerate antigen elimination can be examined by administering antigen-binding molecules mF44, mF46 having the property of binding to a soluble antigen pH-dependently to Fc receptor γ chain-deficient mice. According to the results of example 6, the antigen-binding molecules mF44 and mF46, which were administered in Fc receptor gamma chain deficient mice and had the property of binding to soluble antigen in a pH-dependent manner, showed increased clearance of antigen compared to the antigen-binding molecule mIgG1, which was administered in the same mice and had the property of binding to soluble antigen in a pH-dependent manner. Furthermore, from the results of example 6, it was confirmed that the cases of administration of mF44 and mF46 to Fc receptor gamma chain-deficient mice eliminated antigens to almost the same extent as the cases of administration to normal mice.
in example 6, the same experiment was performed using Fc γ RIII deficient mice. Since mIgG1 and mF44, mF46 bound only Fc γ RIIb and Fc γ RIII in mFc γ R, the effect of enhancing selective binding of Fc γ RIIb to accelerate antigen elimination was examined by administering these antibodies to Fc γ RIII-deficient mice. According to the results of example 6, mF44 and mF46 administered in Fc γ RIII deficient mice showed increased clearance of antigen compared to mIgG1 administered in the same mice. Furthermore, from the results of example 6, it was confirmed that the cases of administration of mF44 and mF46 to Fc γ RIII-deficient mice, administration to normal mice, and administration to Fc receptor γ chain-deficient mice eliminated antigens to almost the same extent.
from these results, it was clarified that antigen elimination can be accelerated by enhancing only the selective binding to Fc γ RIIb without enhancing the binding to the active form Fc γ R.
in addition to the previous literature reports examined to date, it is believed that, based on the results of the validation using the above-mentioned mice: in the human body, like in the mouse, Fc γ RIIb mediates uptake of immune complexes into cells, and as a result, an antibody having Fc that enhances selective binding to human Fc γ RIIb accelerates elimination of the antigen. Furthermore, as previously examined, since it is considered that Fc γ RIIb-mediated uptake of immune complexes into cells is produced at the same rate in mice and humans, it is considered that: by using Fc whose affinity for human Fc γ RIIb is similarly enhanced, the same effect as that of an antibody having Fc whose affinity for mouse Fc γ RIIb is enhanced in accelerating antigen elimination can be achieved in the human body.
as described in WO2009/125825, it was shown that: the elimination of soluble human IL-6 receptor in mice to which Fv4-IgG1 (obtained by changing the antigen-binding activity of humanized anti-IL-6 receptor antibody H54/L28-IgG1 in the variable region depending on pH conditions (i.e., by binding to the antigen at pH7.4 and dissociating the antigen at pH 5.8)) was administered together was greatly accelerated compared to the elimination of soluble human IL-6 receptor in mice to which H54/L28-IgG1 and the antigen were administered together. In the present specification, the heavy chain H54-IgG1 and the light chain L28-CK contained in H54/L28-IgG1 are represented by SEQ ID NO:36 and SEQ ID NO:37, respectively, and the heavy chain VH3-IgG1 and the light chain VL3-CK contained in Fv4-IgG1 are represented by SEQ ID NO:38 and SEQ ID NO:39, respectively.
the soluble human IL-6 receptor bound by the antibody H54/L28-IgG1, which binds to the soluble human IL-6 receptor, is recirculated to the plasma together with the antibody via FcRn, whereas the soluble human IL-6 receptor bound by the antibody is dissociated under acidic conditions in vivo by the antibody Fv4-IgG1, which binds to the soluble human IL-6 receptor in a pH-dependent manner. The dissociated soluble human IL-6 receptor is decomposed by lysosomes, so that the elimination of the soluble human IL-6 receptor can be greatly accelerated, and the antibody Fv4-IgG1 which binds to the soluble human IL-6 receptor in vivo binds to FcRn and is then recycled to the plasma. The antibody that is recycled can bind to the soluble human IL-6 receptor again, and thus the binding to the antigen (soluble human IL-6 receptor) and the recycling in plasma by FcRn are repeated. As a result, it is considered that one antibody molecule can repeatedly bind to the soluble human IL-6 receptor many times (WO 2009/125825).
On the other hand, as disclosed in the present invention, it was found that: by administering an antigen-binding molecule having enhanced Fc γ R binding activity of an Fc γ receptor-binding domain contained in an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on pH plasma concentration conditions, an FcRn-binding domain having FcRn binding activity in pH acidic range conditions, and an Fc γ receptor-binding domain, the concentration of a soluble antigen in plasma can be greatly reduced.
without being bound by a particular theory, the unexpected decrease in plasma soluble antigen concentration observed by administration of an antigen binding molecule comprising an antigen binding domain with antigen binding activity that varies with pH plasma concentration conditions, and an FcRn binding domain with FcRn binding activity in the pH acidic range, which enhances Fc γ R binding, can also be demonstrated as described below.
as described above, the antigen-binding molecules such as Fv4-IgG1, which contain an antigen-binding domain whose antigen-binding activity varies depending on the ion concentration condition, are thought to be capable of repeatedly binding to an antigen, but are thought to have an effect of dissociating a soluble antigen in vivo and accelerating elimination from plasma, and are thought to depend on the rate at which the complex of the antigen and the antigen-binding molecule is taken into the body. An antigen-binding molecule having an antigen-binding domain whose antigen-binding activity varies depending on ion concentration conditions, which has enhanced binding activity to various Fc γ rs, is actively taken into cells by binding to various Fc γ rs expressed on cell membranes, and can be recycled into plasma through the recycling mediated by the binding of FcRn to FcRn of the FcRn-binding domain having FcRn-binding activity under the pH acidic range conditions contained in the molecule. That is, since the antigen-binding molecule that forms a complex with a soluble antigen in plasma is actively taken into cells via Fc γ R expressed on cell membranes, it is considered that the effect of accelerating elimination of the soluble antigen in plasma is more remarkable than that of an antigen-binding molecule in which the binding activity to various Fc γ rs is not enhanced.
The Fc γ R binding activity of an antibody that binds to a membrane-type antigen plays an important role in the cytotoxic activity of the antibody. Therefore, when an antibody used as a drug requires cytotoxic activity, an isotype of human IgG1 having high Fc γ R binding activity can be used, and a technique of enhancing the cytotoxic activity of the antibody by enhancing the Fc γ R binding activity of the antibody is also widely used. On the other hand, the effect exerted by the Fc γ R binding activity of an antibody that binds to a soluble antigen and is used as a drug has not been known so far, and a difference in Fc γ R binding activity between human IgG1 having high Fc γ R binding activity and human IgG2 or human IgG4 having low Fc γ R binding activity is not sufficiently studied so far, because it imparts a physiological effect to the body to which the antibody is administered. Actually, as will be described later in the present embodiment, it was confirmed that: there was no effect on the change in the concentration of soluble antigen in the plasma of individuals administered antibodies that lost Fc γ R binding activity. On the other hand, in the present invention, it is found that: the concentration of soluble antigen is greatly reduced in the plasma of individuals administered an antigen-binding molecule having enhanced Fc γ R binding activity and containing an antigen-binding domain whose soluble antigen-binding activity varies depending on the ion concentration conditions. That is, it can be said that: the advantage of enhanced binding to Fc γ R is achieved by combining an FcRn binding domain having FcRn binding activity under pH acidic range conditions contained in an antigen binding molecule targeting a soluble antigen with an antigen binding domain whose soluble antigen binding varies with ionic concentration conditions.
Antigen binding molecules
in the present invention, the antigen binding molecule is used in the broadest sense to mean a molecule having human FcRn binding activity in the pH acidic range, containing an antigen binding domain and an Fc γ receptor binding domain, and specifically includes various types of molecules as long as it exhibits antigen binding activity. For example, an antibody can be given as an example of a molecule in which an antigen-binding domain binds to an Fc region. The antibody may include a single monoclonal antibody (containing an agonistic antibody and an antagonistic antibody), a human antibody, a humanized antibody, a chimeric antibody, and the like. When used as an antibody fragment, preferred examples thereof include an antigen-binding domain and an antigen-binding fragment (e.g., Fab, F (ab')2, scFv, and Fv). Scaffold molecules for constructing antigen binding domains by using a steric structure such as a conventional stable α/β -barrel protein structure as a scaffold (scaffold) and by making a library of only a part of the structure thereof may be included in the antigen binding molecule of the present invention.
The antigen binding molecules of the invention may contain at least a portion of an Fc region that mediates binding to FcRn and binding to fey receptors. For example, in one non-limiting embodiment, the antigen binding molecule can be an antibody or an Fc fusion protein. A fusion protein refers to a chimeric polypeptide comprising a polypeptide having a first amino acid sequence linked to a polypeptide having a second amino acid sequence not naturally linked to the polypeptide in nature. For example, a fusion protein can contain: an amino acid sequence encoding at least a portion of an Fc region (e.g., a portion of an Fc region that confers binding to FcRn or a portion of an Fc region that confers binding to an fey receptor), and a non-immunoglobulin polypeptide comprising an amino acid sequence encoding, for example, a ligand binding domain of a receptor or a receptor binding domain of a ligand. The amino acid sequences may be present in the individual proteins that are delivered together to the fusion protein, or they may be present in the same protein in general, participating in a new recombination in the fusion polypeptide. The fusion protein can be produced, for example, by chemical synthesis, or by a gene recombination method in which a polynucleotide in which peptide regions are encoded in a desired relationship is produced and expressed.
The domains of the invention may be linked directly by a polypeptide bond or may be linked by a linker. As the linker, any peptide linker or synthetic compound linker which can be introduced by genetic Engineering (for example, the linkers disclosed in Protein Engineering (1996)9(3),299-305) and the like can be used, and a peptide linker is preferable in the present invention. The length of the peptide linker is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose, and a preferable length is 5 amino acids or more (the upper limit is not particularly limited, and is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids.
For example, preferable examples of the peptide linker include:
Ser
Gly·Ser
Gly·Gly·Ser
Ser·Gly·Gly
Gly·Gly·Gly·Ser(SEQ ID NO:26)
Ser·Gly·Gly·Gly(SEQ ID NO:27)
Gly·Gly·Gly·Gly·Ser(SEQ ID NO:28)
Ser·Gly·Gly·Gly·Gly(SEQ ID NO:29)
Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO:30)
Ser·Gly·Gly·Gly·Gly·Gly(SEQ ID NO:31)
Gly·Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO:32)
Ser·Gly·Gly·Gly·Gly·Gly·Gly(SEQ ID NO:33)
(Gly·Gly·Gly·Gly·Ser(SEQ ID NO:28))n
(Ser·Gly·Gly·Gly·Gly(SEQ ID NO:29))n
[ n is an integer of 1 or more ], and the like. Among them, those skilled in the art can appropriately select the length or sequence of the peptide linker according to the purpose.
Synthetic chemical linkers (chemical crosslinkers) are crosslinkers commonly used in crosslinking of peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl suberate) ester (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimidyoxycarbonyloxy) ethyl ] sulfone (BSOCOES), bis [2- (sulfosuccinimidyloxycarbonyloxy) ethyl ] sulfone (sulfo-BSOCOES), and the like, these crosslinking agents are commercially available.
when a plurality of linkers are used to connect the domains, the same type of linker may be used for all of the linkers, or different types of linkers may be used.
In addition to the linkers exemplified as described above, linkers having peptide tags such as a His tag, an HA tag, a myc tag, and a FLAG tag can be suitably used. Further, the property of being bound to each other by hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions, or a combination thereof can also be suitably utilized. For example, the affinity between CH1 and CL of an antibody can be used, or the Fc region derived from the aforementioned bispecific antibody can be used in association with a heterologous Fc region. Further, disulfide bonds formed between domains can also be suitably utilized.
to link the domains with peptide bonds, the polynucleotides encoding the domains are linked in-frame. Methods for in-frame ligation of polynucleotides are known, such as ligation of restriction fragments, fusion PCR, and overlap PCR, and these methods may be used alone or in combination as appropriate for the preparation of the antigen-binding molecule of the present invention. As used herein, the terms "connected," "fused," "connected," or "fused" are used interchangeably. These terms refer to the joining of two or more polypeptide etc. components or constituents to form a structure by all means including the above-mentioned chemical binding means or recombinant methods. Where two or more components or constituents are polypeptides, in-frame fusion refers to the joining of units of two or more reading frames to form a longer reading frame joined in such a way as to maintain the correct reading frame for the polypeptide. When a two-molecule Fab is used as the antigen binding domain, an antibody as the antigen binding molecule of the present invention in which the antigen binding domain and Fc region are linked in-frame by a peptide bond without a linker can be used as a preferred antigen binding molecule of the present application.
FcRn
unlike Fc gamma receptors belonging to the immunoglobulin superfamily, FcRn, particularly human FcRn, is structurally similar to Major Histocompatibility Complex (MHC) class I polypeptides, having 22 to 29% sequence identity to class I MHC molecules (Ghetie et al, immunol. today (1997)18(12), 592-598). FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β 2 microglobulin). Like MHC, the α chain of FcRn contains 3 extracellular domains (α 1, α 2, α 3), and a short cytoplasmic domain anchors the protein to the cell surface. The α 1 and α 2 domains interact with the FcRn binding domain in the Fc region of antibodies (Raghavan et al (Immunity (1994)1, 303-315).
FcRn is expressed in the maternal placenta or yolk sac of mammals and is involved in the transfer of IgG from the mother to the fetus. In addition, in the small intestine of rodent newborns expressing FcRn, FcRn is involved in the movement of maternal IgG from the ingested colostrum or milk through the brush-like rim epithelium. FcRn is expressed in a large variety of other tissues and various endothelial cell lines. It is also expressed in human adult vascular endothelium, the muscular vasculature and hepatic sinus capillaries. FcRn is thought to bind to IgG, recycling it to the serum, and thus serves to maintain the plasma concentration of IgG. In general, FcRn binding to IgG molecules is strictly pH dependent, with optimal binding observed in the pH acidic range of less than 7.0.
human FcRn, which is a precursor of a polypeptide having a signal sequence shown in SEQ ID NO:34, forms a complex with human β 2-microglobulin in the body (the polypeptide having a signal sequence is shown in SEQ ID NO: 35). As shown in the following reference examples, soluble human FcRn forming a complex with β 2-microglobulin was prepared by using a general recombinant expression method. The binding activity of the FcRn binding domains of the invention to such soluble forms of human FcRn complexed with β 2-microglobulin can be assessed. In the present invention, unless otherwise specified, human FcRn refers to a form capable of binding to the FcRn binding domain of the present invention, and examples thereof include a complex of human FcRn and human β 2-microglobulin.
The antigen binding molecules of the invention have an FcRn binding domain. The FcRn binding domain is not particularly limited as long as the antigen binding molecule has FcRn binding activity in the pH acidic range, and may be a domain having FcRn binding activity directly or indirectly. Examples of such a domain include: fc region of an immunoglobulin of the IgG type having FcRn binding activity directly, albumin domain 3, anti-FcRn antibody, anti-FcRn peptide, anti-FcRn Scaffold (Scaffold) molecule, etc., or a molecule that binds to IgG or albumin having FcRn binding activity indirectly, etc. In the present invention, a domain having FcRn binding activity in the pH acidic range and the pH neutral range is preferable. This domain may be suitably used as it is, if it has FcRn binding activity in the pH acidic range in advance. This domain may confer binding activity to FcRn by altering amino acids in the antigen binding molecule when there is no or weak binding activity to FcRn in the pH acidic range. In addition, the FcRn binding activity can also be improved by changing the amino acid in the domain having FcRn binding activity in the pH acidic range in advance. For amino acid changes in the FcRn binding domain, the change in target can be found by comparing the FcRn binding activity in the pH acidic range before and after the amino acid change.
The FcRn binding domain is preferably a region that directly binds to FcRn. As a preferred example of the FcRn binding domain, an Fc region of an antibody can be cited. However, a region that binds to a polypeptide having an FcRn binding activity such as albumin or IgG may indirectly bind to FcRn via albumin, IgG, or the like. Therefore, as the FcRn binding region in the present invention, a region that binds to a polypeptide having an FcRn binding activity can be suitably used. The Fc region contains the amino acid sequence derived from the constant region of an antibody heavy chain. The Fc region is a portion of the heavy chain constant region of an antibody containing the hinge, CH2 and CH3 domains, starting from the N-terminus of the hinge region at about amino acid 216 as the papain cleavage site, as expressed by EU numbering.
Binding activity of an FcRn binding domain or antigen binding molecule comprising the same to FcRn, in particular human FcRn Property of (2)
The binding activity of the FcRn binding domain of the present invention to FcRn, particularly human FcRn, can be determined by methods well known to those skilled in the art, as described in the aforementioned items of binding activity, and can be determined as appropriate by those skilled in the art for conditions other than pH. The antigen binding activity of an antigen binding molecule and the human FcRn binding activity can be evaluated as KD (Dissociation constant), Apparent KD (Apparent Dissociation constant), Dissociation rate KD (Dissociation rate), Apparent KD (Apparent Dissociation rate), or the like. They can be determined by methods known to those skilled in the art. For example, Biacore (GE healthcare), scatchard plot, flow cytometry, and the like can be used.
Conditions other than pH for determining the binding activity of the FcRn binding domain to FcRn can be appropriately selected by those skilled in the art, and are not particularly limited. For example, the assay can be performed in MES buffer at 37 ℃ as described in WO 2009/125825. The binding activity of the FcRn binding domain of the present invention to FcRn can be measured by a method known to those skilled in the art, and for example, Biacore (GE Healthcare) can be used for the measurement. The binding activity of an FcRn binding domain to FcRn can be evaluated by passing FcRn or an FcRn binding domain or an antigen binding molecule of the present invention containing an FcRn binding domain as an analyte through an FcRn binding domain-immobilized or FcRn binding domain-containing antigen binding molecule of the present invention or an FcRn chip, respectively.
the pH acidic range, which is a condition under which the FcRn binding domain contained in the antigen binding molecule of the present invention has binding activity to FcRn, generally means pH4.0 to pH 6.5. Preferably means pH5.5 to pH6.5, and particularly preferably means pH5.8 to pH6.0 which is close to the pH in the early endosome in the body. The pH neutral range as a condition under which the FcRn binding domain contained in the antigen binding molecule of the present invention has binding activity to FcRn generally means pH6.7 to pH 10.0. The neutral pH range is preferably a range of pH7.0 to pH8.0, preferably selected from pH7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, and particularly preferably pH7.4 close to the pH in plasma (blood) in the living body. Since the human FcRn binding domain or the antigen binding molecule containing the same at ph7.4 has low binding affinity for human FcRn, ph7.0 can be used instead of ph7.4 when it is difficult to evaluate the binding affinity. The binding affinity of the FcRn binding domain to FcRn can be evaluated at any temperature from 10 ℃ to 50 ℃ as the temperature used in the assay conditions. Preferably, a temperature of 15 ℃ to 40 ℃ is used for determining the binding affinity of the FcRn binding domain to human FcRn. More preferably, any temperature of 20 ℃ to 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ℃, is also equally used to determine the binding affinity of the FcRn binding domain to FcRn. A temperature of 25 ℃ is a non-limiting example of a protocol of the present invention.
according to Yeung et al (j. immunol. (2009)182:7663-7671), the binding activity of native human IgG1 to human FcRn was KD 1.7 μ M in the pH acidic range (pH6.0) and substantially no activity was detected in the pH neutral range. Therefore, in a preferred embodiment, the antigen-binding molecule of the present invention having binding activity to human FcRn in the pH acidic range may be used, and includes an antigen-binding molecule having binding activity to human FcRn in the pH acidic range of KD 20 μ M or more and binding activity to human FcRn in the pH neutral range of equivalent to that of natural human IgG. In a more preferred embodiment, the antigen binding molecules of the invention may be used, including those having binding activity for human FcRn of KD 2.0 μ M or stronger in the acidic range of pH. In a further more preferred embodiment, antigen binding molecules with a binding activity for human FcRn of KD0.5 μ M or stronger in the pH acidic range can be used. The KD value is determined by The method described in The Journal of Immunology (2009)182:7663-7671 (in which an antigen-binding molecule is immobilized on a chip and a human FcRn as an analyte is passed).
In the present invention, an Fc region having FcRn binding activity under pH acidic range conditions is preferable. This domain may be used as it is as long as it is an Fc region having FcRn binding activity in advance in the pH acidic range. The domain has no or weak FcRn binding activity under pH acidic range conditions, an Fc region having the desired FcRn binding activity can be obtained by changing the amino acids in the antigen binding molecule, but an Fc region having or enhancing the desired FcRn binding activity under pH acidic range conditions can also be suitably obtained by changing the amino acids in the Fc region. Such amino acid changes in the Fc region that result in the desired binding activity can be found by comparing the FcRn binding activity under pH acidic range conditions before and after the amino acid change. Suitable amino acid changes can be performed by those skilled in the art using well-known methods as those described above for altering Fc γ receptor binding activity.
The Fc region having FcRn binding activity under pH acidic range conditions contained in the antigen binding molecule of the present invention can be obtained by any method, and specifically, an FcRn binding domain having or enhancing FcRn binding activity under pH acidic range conditions can be obtained by changing the amino acids of human IgG-type immunoglobulin used as the starting Fc region. As the Fc region of a preferred IgG-type immunoglobulin for alteration, for example: the Fc region of human IgG (IgG1, IgG2, IgG3 or IgG4 and their variants). For the change to another amino acid, any position of the amino acid may be changed as long as it has an FcRn binding activity under pH acidic range conditions or can enhance human FcRn binding activity under acidic range conditions. When the antigen binding molecule contains the Fc region of human IgG1 as the Fc region, it preferably contains a modification that brings about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG 1. Examples of amino acids that can be modified as described above include: as described in WO2000/042072, amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447, which are represented by EU numbering. Similarly, as amino acids capable of effecting the above-mentioned change, there may be appropriately mentioned, for example: as described in WO2002/060919, the amino acid residues at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and/or 436 in the EU numbering system. Examples of the amino acid capable of effecting the above change include: amino acids at positions 250, 314 and 428 in the EU numbering system as described in WO 2004/092219. Further, as amino acids capable of effecting the above-mentioned change, there may be appropriately mentioned, for example: as described in WO2010/045193, the amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434 and/or 436 represented by EU numbering. By these amino acid changes, the Fc region of an IgG-type immunoglobulin is enhanced in binding to FcRn under pH acidic range conditions.
In the present invention, an Fc region having FcRn binding activity under pH acidic range conditions is preferred. This domain may be used as it is as long as it is an Fc region having FcRn binding activity in advance in the pH acidic range. The domain has no or weak FcRn binding activity under pH acidic range conditions, an Fc region having desired FcRn binding activity under pH acidic range conditions can be obtained by changing amino acids in the antigen binding molecule, but an Fc region having desired FcRn binding activity under pH acidic range conditions can also be suitably obtained by changing amino acids in the Fc region. Such amino acid changes in the Fc region that result in the desired binding activity can be found by comparing the FcRn binding activity under pH acidic range conditions before and after the amino acid change. Suitable amino acid changes can be performed by one skilled in the art using the same well-known methods as those described above for altering Fc γ receptor binding activity.
The Fc region having FcRn binding activity under pH acidic range conditions contained in the antigen binding molecule of the present invention can be obtained by any method, and specifically, an FcRn binding domain having or enhancing FcRn binding activity under pH acidic range conditions can be obtained by changing the amino acids of human IgG-type immunoglobulin used as the starting Fc region. Examples of the Fc region of a preferred IgG-type immunoglobulin to be used for modification include Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and modified forms thereof). For the change to another amino acid, any position of the amino acid may be changed as long as it has an FcRn binding activity under a pH acidic range condition or can enhance a human FcRn binding activity under a pH acidic range condition. When the antigen binding molecule contains the Fc region of human IgG1 as the Fc region, it preferably contains a modification that brings about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG 1. Examples of the amino acid capable of effecting the above-mentioned change include, for example, the amino acids at positions 252, 254, 256, 309, 311, 315, 433 and/or 434 in the EU numbering system and the amino acids at positions 253, 310, 435 and/or 426 in combination of these amino acids, as described in International publication WO 1997/034631. Examples of suitable amino acids include amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447, which are indicated by the EU number as described in International publication WO 2000/042072. Similarly, as an amino acid capable of effecting the above-mentioned change, there may be suitably mentioned, for example, amino acids at position 251, position 252, position 254, position 255, position 256, position 308, position 309, position 311, position 312, position 385, position 386, position 387, position 389, position 428, position 433, position 434 and/or position 436, which are represented by EU numbering as described in International publication WO 2002/060919. Further, examples of the amino acids capable of effecting the above-mentioned change include amino acids at positions 250, 314 and 428 as shown by EU numbering as described in International publication WO 2004/092219. Further, as an amino acid capable of achieving the above change, for example, amino acids at positions 238, 244, 245, 249, 252, 256, 257, 258, 260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316, 317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423, 427, 430, 431, 434, 436, 438, 440 and/or 442 as described in international publication WO2006/020114 are also suitable. Furthermore, as amino acids that can be modified as described above, amino acids at position 251, 252, 307, 308, 378, 428, 430, 434, and/or 436, which are expressed by EU numbering, may be suitably used, for example, as described in international publication WO 2010/045193. By altering these amino acids, the binding of the Fc region of an IgG-type immunoglobulin to FcRn under pH acidic range conditions can be enhanced.
in a non-limiting embodiment in which the Fc region of human IgG1 is contained as the Fc region, the effect of enhancing binding to FcRn under pH acidic range conditions as compared to the binding activity of the initial Fc region of human IgG1 is exerted, and the following are included: (ii) alterations in at least 1 or more amino acids selected from the group consisting of:
the 251 th amino acid is Arg or Leu;
The amino acid at position 252 is any one of Phe, Ser, Thr or Tyr;
The amino acid at position 254 is any one of Ser or Thr;
The amino acid at position 255 is any one of Arg, Gly, Ile or Leu;
The amino acid at position 256 is any one of Ala, Arg, Asn, Asp, Gln, Glu or Thr;
The amino acid at position 308 is any one of Ile or Thr;
Amino acid Pro at position 309;
The amino acid at position 311 is any one of Glu, Leu or Ser;
amino acid position 312 is either Ala or Asp;
amino acid 314 is any one of Ala or Leu;
The amino acid at position 385 is any one of Ala, Arg, Asp, Gly, His, Lys, Ser or Thr;
the amino acid at position 386 is any one of Arg, Asp, Ile, Lys, Met, Pro, Ser or Thr;
Amino acid at position 387 is any one of Ala, Arg, His, Pro, Ser, or Thr;
The amino acid at position 389 is any one of Asn, Pro or Ser;
the amino acid at position 428 is any one of Leu, Met, Phe, Ser or Thr;
the amino acid at position 433 is any one of Arg, Gln, His, Ile, Lys, Pro, or Ser;
The amino acid at position 434 is any one of His, Phe or Tyr; or
the amino acid at position 436 is any one of Arg, Asn, His, Lys, Met or Thr. The number of amino acids to be changed is not particularly limited, and only one amino acid may be changed, or two or more amino acids may be changed.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: wherein the amino acid at position 308 represented by EU numbering is Ile, the amino acid at position 309 is Pro, and/or the amino acid at position 311 is Glu. Moreover, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 308 is Thr, the amino acid at position 309 is Pro, the amino acid at position 311 is Leu, the amino acid at position 312 is Ala, and/or the amino acid at position 314 is Ala. Also, another non-limiting aspect of the modification may be a modification comprising: the 308 th amino acid is Ile or Thr, the 309 th amino acid is Pro, the 311 th amino acid is Glu, Leu or Ser, the 312 th amino acid is Ala, and/or the 314 th amino acid is Ala or Leu. A different non-limiting version of this variation may be a variation comprising: the amino acid at position 308 is Thr, the amino acid at position 309 is Pro, the amino acid at position 311 is Ser, the amino acid at position 312 is Asp, and/or the amino acid at position 314 is Leu.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: the amino acid at position 251 is Leu, the amino acid at position 252 is Tyr, the amino acid at position 254 is Ser or Thr, the amino acid at position 255 is Arg, and/or the amino acid at position 256 is Glu, which are represented by EU numbers.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: the amino acid at position 428 represented by EU numbering is any one of Leu, Met, Phe, Ser or Thr; the amino acid at position 433 is any one of Arg, Gln, His, Ile, Lys, Pro, or Ser; the amino acid at position 434 is any one of His, Phe or Tyr; and/or the amino acid at position 436 is any one of Arg, Asn, His, Lys, Met or Thr. Moreover, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 428 is His or Met, and/or the amino acid at position 434 is His or Met.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: wherein the amino acid at position 385 is Arg, the amino acid at position 386 is Thr, the amino acid at position 387 is Arg, and/or the amino acid at position 389 is Pro, as indicated by EU numbering. Moreover, another non-limiting aspect of the modification may be a modification comprising: asp at amino acid position 385, Pro at amino acid position 386 and/or Ser at amino acid position 389.
In a non-limiting embodiment, in which the Fc region of human IgG1 is contained as the Fc region, the change that brings about an effect of enhancing the binding activity to FcRn under the pH acidic range conditions as compared with the binding activity to the initial Fc region of human IgG1 includes a change in at least 1 or more amino acids selected from the group consisting of:
the amino acid at position 250 is either Gln or Glu; or
the amino acid at position 428 is either Leu or Phe. The number of amino acids to be changed is not particularly limited, and only one or two amino acids may be changed.
In the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: the amino acid at position 250 is Gln and/or the amino acid at position 428 is Leu or Phe, as indicated by EU numbering. Moreover, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 250 is Glu, and/or the amino acid at position 428 is either Leu or Phe.
In a non-limiting embodiment in which the Fc region of human IgG1 is contained as the Fc region, the change that brings about an effect of enhancing binding to FcRn under the pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 includes a change in at least 2 or more amino acids selected from the group consisting of:
The amino acid at position 251 is either Asp or Glu;
The amino acid at position 252 is Tyr;
the amino acid at position 307 is Gln;
amino acid Pro at position 308;
the amino acid at position 378 is Val;
the amino acid at position 380 is Ala;
the amino acid at position 428 is Leu;
the amino acid at position 430 is either Ala or Lys;
the amino acid at position 434 is any one of Ala, His, Ser or Tyr; or
the amino acid at position 436 is Ile. The number of amino acids to be changed is not particularly limited, and only two amino acids may be changed, or three or more amino acids may be changed.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: the amino acid at position 307 is Gln, and the amino acid at position 434 is Ala or Ser, as indicated by EU numbering. Moreover, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 308 is Pro, and the amino acid at position 434 is Ala. Also, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 252 is Tyr, and the amino acid at position 434 is Ala. A different non-limiting version of this variation may be a variation comprising: val for the amino acid at position 378 and Ala for the amino acid at position 434. Another, different non-limiting version of this variation may be a variation comprising: the amino acid at position 428 is Leu, and the amino acid at position 434 is Ala. Also, another different non-limiting one aspect of the modification may be a modification comprising: the amino acid at position 434 is Ala, and the amino acid at position 436 is Ile. Further, another non-limiting one aspect of the modification may be a modification containing: the amino acid at position 308 is Pro, and the amino acid at position 434 is Tyr. Also, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 307 is Gln, and the amino acid at position 436 is Ile.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: any of Gln at amino acid position 307, Ala at amino acid position 380, and Ser at amino acid position 434, which are expressed by EU numbering. Moreover, another non-limiting aspect of the modification may be a modification comprising: gln at amino acid position 307, Ala at amino acid position 380, and Ala at amino acid position 434. Also, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 252 is Tyr, the amino acid at position 308 is Pro, and the amino acid at position 434 is Tyr. A different non-limiting version of this variation may be a variation comprising: asp for the amino acid at position 251, Gln for the amino acid at position 307 and His for the amino acid at position 434.
In a non-limiting embodiment in which the Fc region of human IgG1 is contained as the Fc region, the change that brings about an effect of enhancing binding to FcRn under the pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 includes a change in at least 2 or more amino acids selected from the group consisting of:
The amino acid at position 238 is Leu;
the amino acid at position 244 is Leu;
The amino acid at position 245 is Arg;
Amino acid position 249 is Pro;
The amino acid at position 252 is Tyr;
the amino acid at position 256 is Pro;
the amino acid at position 257 is any one of Ala, Ile, Met, Asn, Ser or Val;
Asp for the amino acid at position 258;
the amino acid at position 260 is Ser;
The amino acid at position 262 is Leu;
The amino acid at position 270 is Lys;
The amino acid at position 272 is any one of Leu or Arg;
the amino acid at position 279 is any one of Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 283 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 285 is Asn;
Phe as amino acid at position 286;
The amino acid at position 288 is any one of Asn or Pro;
the amino acid at position 293 is Val;
The amino acid at position 307 is any one of Ala, Glu or Met;
the amino acid at position 311 is any one of Ala, Ile, Lys, Leu, Met, Val or Trp;
The amino acid at position 312 is Pro;
The amino acid at position 316 is Lys;
the amino acid at position 317 is Pro;
The amino acid at position 318 is any one of Asn or Thr;
the amino acid at position 332 is Phe, His, Lys, Leu, Met, Arg, Ser or Trp;
The amino acid at position 339 is any one of Asn, Thr or Trp;
the amino acid at position 341 is Pro;
The amino acid at position 343 is any one of Glu, His, Lys, Gln, Arg, Thr or Tyr;
the amino acid at position 375 is Arg;
The amino acid at position 376 is any one of Gly, Ile, Met, Pro, Thr or Val;
the amino acid at position 377 is Lys;
the amino acid at position 378 is Asp or Asn;
The amino acid at position 380 is any one of Asn, Ser or Thr;
The amino acid at position 382 is any one of Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 423 is Asn;
the amino acid at position 427 is Asn;
the amino acid at position 430 is any one of Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr;
the amino acid at position 431 is any one of His or Asn;
the amino acid at position 434 is any one of Phe, Gly, His, Trp or Tyr;
The amino acid at position 436 is any one of Ile, Leu or Thr;
The amino acid at position 438 is any one of Lys, Leu, Thr or Trp;
the amino acid at position 440 is Lys; or
the amino acid at position 442 is Lys. The number of amino acids to be changed is not particularly limited, and only two amino acids may be changed, or three or more amino acids may be changed.
in the case of containing the Fc region of human IgG1 as the Fc region, a non-limiting one of the changes that bring about an effect of enhancing binding to FcRn under pH acidic range conditions as compared with the binding activity of the initial Fc region of human IgG1 may be a change that contains: ile at amino acid 257 and Ile at amino acid 311 in the EU numbering system. Moreover, another non-limiting aspect of the modification may be a modification comprising: the amino acid at position 257 is Ile, and the amino acid at position 434 is His. Also, another non-limiting aspect of the modification may be a modification comprising: val for amino acid 376 and His for amino acid 434.
as described later, an Fc region having an FcRn binding activity under a neutral pH range can be suitably used as the FcRn binding domain contained in the antigen binding molecule of the present invention. These Fc regions can also be obtained by any method according to the above-described method for obtaining an Fc region having FcRn binding activity under the pH acidic range, and specifically, an FcRn binding domain having or enhancing FcRn binding activity under the pH neutral range can be obtained by changing the amino acids of human IgG-type immunoglobulin used as the starting Fc region. Examples of the Fc region of a preferred IgG-type immunoglobulin to be used for modification include Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and modified forms thereof). For the change to another amino acid, any position of the amino acid may be changed as long as it has FcRn binding activity under the condition of neutral pH range or can enhance human FcRn binding activity under the condition of acidic range. When the antigen binding molecule contains the Fc region of human IgG1 as the Fc region, it preferably contains a modification that brings about an effect of enhancing binding to FcRn under conditions of pH neutral range as compared with the binding activity of the starting Fc region of human IgG 1. As the Fc region capable of effecting such a change, there can be appropriately mentioned: for example, the human FcRn binding domain is an Fc region in which at least one or more amino acids selected from the group consisting of position 237, position 238, position 239, position 248, position 250, position 252, position 254, position 255, position 256, position 257, position 258, position 265, position 270, position 286, position 289, position 297, position 298, position 303, position 305, position 307, position 308, position 309, position 311, position 312, position 314, position 315, position 317, position 325, position 332, position 334, position 360, position 376, position 380, position 382, position 384, position 385, position 386, position 387, position 389, position 424, position 428, position 433, position 434, and position 436 in the EU-numbering position of the starting Fc region are different from the corresponding amino acids in the natural Fc region.
furthermore, as the Fc region capable of effecting such a change, there may be suitably mentioned, for example, an Fc region containing at least one amino acid selected from the group consisting of the following amino acids represented by EU numbering,
The amino acid at position 237 is Met;
amino acid 238 is Ala;
the amino acid at position 239 is Lys;
the amino acid at position 248 is Ile;
the amino acid at position 250 is any one of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr;
The amino acid at position 252 is any one of Phe, Trp or Tyr;
The amino acid at position 254 is Thr;
the amino acid at position 255 is Glu;
The amino acid at position 256 is any one of Asp, Glu or Gln;
The amino acid at position 257 is any one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val;
The amino acid at position 258 is His;
the amino acid at position 265 is Ala;
The amino acid at position 270 is Phe;
The amino acid at position 286 is either Ala or Glu;
the amino acid at position 289 is His;
The amino acid at position 297 is Ala;
amino acid at position 298 is Gly;
The amino acid at position 303 is Ala;
amino acid at position 305 is Ala;
The amino acid at position 307 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The amino acid at position 308 is any one of Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr;
The amino acid at position 309 is any one of Ala, Asp, Glu, Pro or Arg;
The amino acid at position 311 is any one of Ala, His or Ile;
the amino acid at position 312 is either Ala or His;
the amino acid at position 314 is any one of Lys or Arg;
amino acid 315 is any one of Ala or His;
The amino acid at position 317 is Ala;
The amino acid at position 325 is Gly;
val for the amino acid at position 332;
The amino acid at position 334 is Leu;
the amino acid at position 360 is His;
Amino acid 376 is Ala;
The amino acid at position 380 is Ala;
amino acid 382 is Ala;
the amino acid at position 384 is Ala;
The amino acid at position 385 is any one of Asp or His;
The amino acid at position 386 is Pro;
Amino acid at position 387 is Glu;
The amino acid at position 389 is either Ala or Ser;
Amino acid at position 424 is Ala;
The amino acid at position 428 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 433 is Lys;
the amino acid at position 434 is any one of Ala, Phe, His, Ser, Trp or Tyr; and
The amino acid at position 436 is His.
for example, by using these amino acid changes alone or in combination, the binding of the Fc region of IgG to FcRn in the pH acidic range and/or neutral range can be enhanced, and any amino acid change can be introduced without particular limitation as long as it brings about an effect of improving the retention in plasma.
pharmaceutical composition
When a conventional neutralizing antibody against a soluble antigen is administered, it is predicted that the binding of the antigen to the antibody improves the persistence in plasma. Antibodies typically have long half-lives (1-3 weeks), while antigens typically have short half-lives (1 day or less). Thus, the antigen bound to the antibody in plasma becomes significantly longer in half-life than when the antigen is present alone. As a result, the administration of the existing neutralizing antibody causes an increase in the concentration of the antigen in plasma. Examples of such neutralizing antibodies targeting various soluble antigens include IL-6(J. immunotoxin. (2005)3,131-139), β -amyloid (mAbs (2010)2(5),1-13), MCP-1 (Arthroitis & RHEEMMATISM (2006)54,2387-2392), hepcidin (AAPS J. (2010)4,646-657), sIL-6 receptor (Blood (2008)112(10),3959-64), and the like. It has been reported that the total antigen concentration in plasma increases from baseline to about 10-1000 times (the degree of increase varies depending on the antigen) by administration of conventional neutralizing antibodies. Here, the total antigen concentration in plasma means a concentration which is the total amount of antigen present in plasma, that is, expressed as the sum of antigen concentrations of an antibody-bound type and an antibody-unbound type. For such antibody drugs targeting soluble antigens, it is not preferable to cause an increase in the total antigen concentration in plasma. The reason for this is that in order to neutralize soluble antigens, at least a concentration of antibody in plasma higher than the total antigen concentration in plasma is required. That is, the increase in the total antigen concentration in plasma by 10 to 1000 times, and the antibody concentration in plasma (i.e., the amount of antibody administered) for neutralizing the increase in the total antigen concentration in plasma also means that the increase in the total antigen concentration in plasma by 10 to 1000 times is required. On the other hand, when the total antigen concentration in plasma can be reduced by 10 to 1000 times as compared with the conventional neutralizing antibody, the administration amount of the antibody can be reduced to the same extent. Thus, an antibody that can eliminate soluble antigens from plasma and reduce the total antigen concentration in plasma is significantly more useful than conventional neutralizing antibodies.
the present invention is not limited to a particular theory, and for example, when an antigen-binding molecule containing an FcRn binding domain such as an antigen binding domain having an antigen binding activity that varies depending on an ion concentration condition such that the antigen binding activity in a pH acidic range is lower than the antigen binding activity in a pH neutral range, and an antibody constant region having a human Fc γ receptor binding activity in a pH neutral range is administered to a living body, the uptake into cells in the living body is promoted, so that the number of antigens to which one molecule of the antigen-binding molecule can bind is increased, and the elimination of the antigen concentration in plasma is promoted, for example, as described below.
for example, when an antibody that binds to a membrane antigen is administered into an organism, the antibody is incorporated into an endosome in a cell by internalization together with the antigen in a state of being bound to the antigen after binding to the antigen. Then, the antibody transferred to the lysosome in a state of being bound to the antigen is decomposed by the lysosome together with the antigen. Internalization-mediated elimination in plasma is called antigen-dependent elimination, and has been reported for most antibody molecules (Drug Discov Today (2006)11(1-2), 81-88). When one molecule of IgG antibody binds to an antigen in a bivalent form, one molecule of the antibody is internalized in a state where it binds to two molecules of the antigen, and is directly decomposed in lysosomes. Therefore, in the case of a normal antibody, one molecule of IgG antibody cannot bind to three or more molecules of antigen. For example, in the case of one molecule of IgG antibody having neutralizing activity, more than three molecules of antigen cannot be neutralized.
the reason for the long retention in the plasma (slow elimination) of IgG molecules is that FcRn, which is known as a salvage receptor for IgG molecules, is in play. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in vivo under acidic conditions in vivo. IgG molecules that do not bind to FcRn are then transferred to lysosomes and broken down. On the other hand, IgG molecules bound to FcRn are transferred to the cell surface. Since IgG molecules dissociate from FcRn under neutral conditions in plasma, the IgG molecules are recycled to the plasma.
in addition, when the antigen-binding molecule is an antibody that binds to a soluble antigen, the antibody administered into the body binds to the antigen, and then the antibody is taken into the cell in a state of being bound to the antigen. Most of the antibodies taken into cells are transferred to the cell surface after binding to FcRn in vivo. Since the antibody is dissociated from FcRn under neutral conditions in plasma, it is released to the outside of the cell. However, an antibody containing a normal antigen-binding domain whose antigen-binding activity does not change under the pH plasma concentration condition is released to the outside of the cell in a state of being bound to an antigen, and thus cannot be bound to the antigen again. Therefore, as with the antibody that binds to the membrane antigen, a normal one-molecule IgG antibody, the antigen binding activity of which does not change under the pH plasma concentration condition, cannot bind to more than three molecules of antigen.
An antibody that strongly binds to an antigen under the condition of a neutral pH range in plasma and binds to an antigen in a pH-dependent manner when dissociated from the antigen under the condition of an acidic pH range in vivo (an antibody that binds to an antigen under the condition of a neutral pH range and dissociates under the condition of an acidic pH range), or an antibody that strongly binds to an antigen under the condition of a high calcium ion concentration in plasma and binds to an antigen under the condition of a low calcium ion concentration in vivo (an antibody that binds to an antigen under the condition of a high calcium ion concentration and dissociates under the condition of a low calcium ion concentration) can be dissociated from an antigen in vivo. The antibody that binds to the antigen in a pH-dependent manner or the antibody that binds to the antigen in a calcium ion concentration-dependent manner can bind to the antigen again when the antibody is recycled to plasma through FcRn after dissociation of the antigen. Thus, one molecule of antibody can repeatedly bind to multiple antigen molecules. Furthermore, the antigen bound to the antigen binding molecule is dissociated from the antibody in vivo, and thus is not recycled into plasma but is decomposed in lysosomes. By administering such antigen-binding molecules to the body, the uptake of antigen into cells can be promoted, and the concentration of antigen in plasma can be reduced.
Uptake into cells of the antigen bound by the antigen binding molecule is further facilitated by conferring or enhancing FcRn binding capacity under conditions of pH neutral range (pH7.4) to: an antibody that strongly binds to an antigen under the condition of a neutral pH range in plasma and binds to an antigen in a pH-dependent manner when dissociated from the antigen under the condition of an acidic pH range in vivo (an antibody that binds to an antigen under the condition of a neutral pH range and dissociates under the condition of an acidic pH range), or an antibody that strongly binds to an antigen under the condition of a high calcium ion concentration in plasma and binds to an antigen under the condition of a low calcium ion concentration in vivo (an antibody that binds to an antigen under the condition of a high calcium ion concentration and dissociates under the condition of a low calcium ion concentration). That is, by administering such antigen-binding molecules to the body, elimination of the antigen can be promoted, and the concentration of the antigen in the plasma can be reduced. A general antibody and an antibody-antigen complex thereof having no pH-dependent antigen binding ability or calcium ion concentration-dependent antigen binding ability are taken into cells by nonspecific endocytosis, bind to FcRn under acidic conditions in vivo, are transported to the cell surface, and are recycled into plasma by dissociation from FcRn under neutral conditions on the cell surface. Therefore, when an antibody that binds to an antigen sufficiently in a pH-dependent manner (binds in a pH neutral range, dissociates in a pH acidic range) or sufficiently in a calcium ion concentration-dependent manner (binds in a high calcium ion concentration, dissociates in a low calcium ion concentration) binds to an antigen in plasma and dissociates the bound antigen in vivo, it is considered that the elimination rate of the antigen becomes equal to the rate of uptake of the antibody and its antibody-antigen complex into cells by nonspecific endocytosis. When the pH dependence or calcium ion concentration dependence of the binding between the antibody and the antigen is insufficient, the antigen that is not dissociated from the antibody in vivo is also recycled into the plasma together with the antibody, but when the pH dependence is sufficient, the rate of elimination of the antigen becomes a limit to the rate of uptake into cells by nonspecific endocytosis. In addition, since FcRn transports antibodies from the endosome to the cell surface, a part of FcRn is also considered to be present on the cell surface.
the present inventors considered that an immunoglobulin of IgG type having Fc γ receptor binding activity or having enhanced binding activity under conditions in the neutral pH range could bind to Fc γ receptors present on the cell surface, and that the immunoglobulin of IgG type was taken up into cells dependently by Fc γ receptors by binding to Fc γ receptors present on the cell surface. Fc γ receptor-mediated uptake into cells is faster than uptake into cells using non-specific endocytosis. Therefore, by enhancing the ability to bind to Fc γ receptors under conditions in the neutral range of pH, it is thought that the antigen elimination rate of the antigen-binding molecule can be further accelerated. That is, an antigen-binding molecule having Fc γ receptor binding ability in the neutral pH range delivers antigen into cells more rapidly than normal (native human) IgG-type immunoglobulin, binds to FcRn in vivo to dissociate the antigen, is recirculated into plasma, where it binds to the antigen again, and is taken up into cells via Fc γ receptor. By increasing Fc γ receptor binding capacity in the neutral pH range, the circulation rate of the cycle can be increased, thus increasing the rate of antigen elimination from plasma. Further, by making the antigen-binding activity of the antigen-binding molecule under the pH acidic range lower than that under the pH neutral range, the speed of elimination of the antigen from plasma can be further increased. In addition, since the circulation rate of the circulation is increased to increase the number of the circulation, it is considered that the number of molecules of the antigen to which one molecule of the antigen-binding molecule can bind is increased. The antigen-binding molecule of the present invention contains an antigen-binding domain and an Fc γ receptor-binding domain, and since the Fc γ receptor-binding domain does not affect antigen binding, and according to the above mechanism, it is considered that, independent of the type of antigen, the uptake of antigen into cells by the antigen-binding molecule can be promoted and the elimination rate of antigen can be increased by making the antigen-binding activity (binding ability) of the antigen-binding molecule under the pH acidic range or low calcium ion concentration condition plasma concentration condition lower than the antigen-binding activity (binding ability) under the pH neutral range or high calcium ion concentration condition plasma concentration condition and/or making the Fc γ receptor-binding activity in plasma under the pH increase. Therefore, the antigen-binding molecule of the present invention is considered to exhibit more excellent effects than conventional therapeutic antibodies in terms of reducing side effects caused by antigens, increasing the amount of antibody to be administered, improving the kinetics of the antibody in vivo, and the like.
figure 1 shows the mechanism of elimination of soluble antigen from plasma by administration of an ion concentration-dependent antigen-binding antibody with enhanced binding to Fc γ receptor at neutral pH compared to prior neutralizing antibodies. Hereinafter, an example of an ion concentration-dependent antigen-binding antibody is an antigen-binding antibody having a hydrogen ion concentration (i.e., pH) dependency, but the mechanism is not limited to the hydrogen ion concentration. It is considered that the existing neutralizing antibody having no pH-dependent antigen-binding ability is slowly taken up mainly by nonspecific interaction with cells after binding to a soluble antigen in plasma. The complex of neutralizing antibody and soluble antigen taken into the cell is transferred to the acidic endosome and recycled to the plasma by FcRn. On the other hand, it is considered that, after binding to a soluble antigen in plasma, a pH-dependent antigen-binding antibody that enhances Fc γ receptor binding under neutral conditions is rapidly taken up into cells expressing Fc γ receptors on cell membranes via interaction with Fc γ receptors, in addition to non-specific interaction. Here, the soluble antigen bound to the pH-dependent antigen-binding antibody dissociates from the antibody in acidic endosomes due to the pH-dependent binding ability. Then, the soluble antigen dissociated from the antibody is transferred to lysosomes and is decomposed by the proteolytic activity. On the other hand, the antibody that has dissociated the soluble antigen is bound to FcRn in an acidic endosome, and then is recycled to the cell membrane via FcRn, and is released again into the plasma. The antibody thus freed by recycling can be re-bound to other soluble antigens. By repeating such cycles of Fc γ receptor-mediated uptake into cells, dissociation and decomposition of soluble antigens, recycling of antibodies, and the like, such pH-dependent antigen-binding antibodies that enhance Fc γ receptor binding under neutral conditions can transfer a large amount of soluble antigens to lysosomes, resulting in a reduction in the total antigen concentration in plasma.
That is, the present invention also relates to a pharmaceutical composition comprising the antigen-binding molecule of the present invention, an antigen-binding molecule produced by the modification method of the present invention, or an antigen-binding molecule produced by the production method of the present invention. Administration of the antigen-binding molecule of the present invention or the antigen-binding molecule prepared by the production method of the present invention is useful as a pharmaceutical composition because it has a higher effect of reducing the antigen concentration in plasma than usual antigen-binding molecules, and the immune response of the body to which the antigen-binding molecule is administered and pharmacokinetics in the body are altered. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.
In the present invention, the pharmaceutical composition generally refers to a drug used for the treatment or prevention, or examination/diagnosis of a disease.
the pharmaceutical compositions of the present invention may be formulated using methods well known to those skilled in the art. For example, it may be used parenterally in the form of injections as sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, the preparation can be carried out by mixing in a unit dosage form required for usual approved pharmaceutical practice suitably in combination with a pharmacologically acceptable carrier or vehicle, specifically, suitably in combination with sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, solvent, preservative, binder and the like. The amount of the active ingredient in these preparations is set so that an appropriate capacity of the indicated range can be obtained.
sterile compositions for injection may be formulated using a vehicle such as distilled water for injection in accordance with conventional formulation practice. Examples of the aqueous solution for injection include physiological saline, and isotonic solution containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Can be used in combination with an appropriate solubilizing aid, such as alcohol (ethanol, etc.), polyhydric alcohol (propylene glycol, polyethylene glycol, etc.), nonionic surfactant (polysorbate 80(TM), HCO-50, etc.).
the oily liquid may be sesame oil, soybean oil, or benzyl benzoate and/or benzyl alcohol as dissolution assistant. In addition, it can be compounded with a buffer (e.g., phosphate buffer and sodium acetate buffer), an analgesic (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), an antioxidant. The prepared injection is usually filled in an appropriate ampoule.
The pharmaceutical compositions of the present invention are preferably administered by non-oral administration. Administration is, for example, a composition of an injection type, a nasal administration type, a pulmonary administration type, or a transdermal administration type. Administration can be systemic or local, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.
the administration method may be appropriately selected depending on the age and symptoms of the patient. The amount of the pharmaceutical composition containing the antigen binding molecule administered can be set to: for example, 0.0001mg to 1000mg/kg body weight/time. Alternatively, it is possible to set: for example, the amount is 0.001 to 100000mg per patient, but the present invention is not limited by the above-mentioned value. The administration amount and the administration method vary depending on the body weight, age, symptoms, and the like of the patient, and those skilled in the art can set an appropriate administration amount and administration method in consideration of these conditions.
It should be noted that amino acids contained in the amino acid sequence described in the present invention may be modified post-translationally (for example, modification of glutamine at the N-terminus to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art), and such post-translational modification of amino acids is naturally also included in the amino acid sequence described in the present invention.
methods of using the antigen binding molecules of the invention
The present invention also provides a method according to any one of the following (i) to (vi), which comprises contacting a cell expressing an Fc γ receptor with an antigen-binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity that varies with ion concentration conditions; and an Fc gamma receptor binding domain having an Fc gamma receptor binding activity in a neutral pH range which is higher than that of a native Fc gamma receptor binding domain of fucose-containing sugar chains, the sugar chain linked to position 297 in the EU numbering system,
(i) a method of increasing the number of antigens to which a molecule of antigen binding molecule can bind;
(ii) A method for eliminating an antigen in plasma;
(iii) A method of improving the pharmacokinetics of an antigen binding molecule;
(iv) A method of promoting intracellular dissociation of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule;
(v) A method for promoting the extracellular release of an antigen-binding molecule in a state in which it is not bound to an antigen; or (vi) a method of reducing the total antigen concentration or the free antigen concentration in plasma.
the present invention also provides a method according to any one of the following (i) to (vii), the method comprising: an Fc gamma receptor binding domain in an antigen binding molecule comprising an antigen binding domain having a human FcRn binding activity in the pH acidic range and an Fc gamma receptor binding domain having an antigen binding activity varying depending on the ion concentration condition and an Fc gamma receptor binding domain, has an enhanced Fc gamma receptor binding activity in the pH neutral range as compared with the Fc gamma receptor binding domain of a native Fc gamma receptor binding domain having fucose-containing sugar chains at EU-numbering 297,
(i) A method for modifying an antigen-binding molecule which promotes the uptake of a bound antigen into cells;
(ii) A method of increasing the number of antigens to which a molecule of antigen binding molecule can bind;
(iii) a method for increasing the antigen-eliminating ability in the plasma of an antigen-binding molecule;
(iv) a method of improving the pharmacokinetics of an antigen binding molecule;
(v) A method of promoting intracellular dissociation of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule;
(vi) A method for promoting the release of an antigen-binding molecule, which is taken up into a cell while being bound to an antigen, to the outside of the cell while being unbound to the antigen; or
(vii) Methods of altering antigen binding molecules that reduce the concentration of total or free antigen in plasma.
methods for contacting an antigen-binding molecule with a cell expressing an Fc γ receptor in vivo or in vitro include: for example, (1) a so-called ex vivo (exvivo) method in which plasma containing an antigen-binding molecule and an antigen bound to the antigen-binding molecule is temporarily taken out of the body, then contacted with cells expressing Fc γ receptors, and after a certain period of time, the plasma containing the antigen-binding molecule not bound to the antigen, which is recycled to the outside of the cells (also referred to as re-secretion or recycling), is returned to the body; or (2) methods of administering the antigen binding molecule into the body. In the method (1), the following methods may be used: a method in which plasma containing an antigen bound to an antigen-binding molecule is temporarily taken out of the body, and then the antigen-binding molecule is brought into contact with cells expressing an Fc γ receptor, and the plasma is returned to the body after a lapse of a certain period of time.
In the present invention, any cell may be used as the cell expressing the Fc γ receptor, as long as it expresses the desired Fc γ receptor, and the cell is not limited to a specific cell. In order to specify cells expressing the desired Fc γ receptor, a known database such as the Human Protein expression Atlas (http:// www.proteinatlas.org /) can be used. Furthermore, it can be confirmed whether or not a cell used for contact with the antigen-binding molecule of the present invention expresses a desired Fc γ receptor by a method of confirming the expression of a gene encoding the receptor, and by an immunological method using an antibody that binds to the receptor. Such methods are also known. Contacting a cell expressing an Fc γ receptor with an antigen binding molecule and an antigen that binds to the antigen binding molecule can be performed in vivo, and thus, in the present invention, contacting an antigen binding molecule with a cell expressing an Fc γ receptor comprises administering the antigen binding molecule to the body. The time of contact may be suitably employed, for example, as appropriate during a period of 1 minute to several weeks, 30 minutes to 1 week, 1 hour to 3 days, 2 hours to 1 day, that is, a time required for the antigen-binding molecule or the antigen bound to the antigen-binding molecule to be taken into the cell due to Fc γ receptor-mediated endocytosis. For example, various immune cells can be used as the cells expressing Fc γ receptor.
The method for enhancing the Fc γ receptor binding activity of the Fc γ receptor binding domain in the pH neutral range as compared with the Fc γ receptor binding domain of a native type having fucose-containing sugar chains linked to the sugar chain at EU-position 297, is described in the following item of the method for producing the antigen-binding molecule of the present invention.
Alteration of antigen-binding molecules that promotes intracellular uptake of antigen bound by the antigen-binding molecule method of producing a composite material
the present invention provides a method for modifying an antigen-binding molecule to promote intracellular uptake of an antigen bound to the antigen-binding molecule, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain having a human FcRn binding activity in the pH acidic range and an antigen-binding activity varying depending on the ion concentration condition is increased in the Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains as sugar chains linked at EU number 297 in the pH neutral range.
In the present invention, "uptake of an antigen into a cell" using an antigen-binding molecule means that the antigen is taken into the cell by endocytosis mediated by Fc γ receptor and internalization. Further, in the present invention, "promoting uptake into cells" means that the rate of uptake of antigen-binding molecules that bind to antigens in plasma into cells is promoted and/or the amount of the ingested antigens recycled into plasma is reduced, in addition to the increase in Fc γ receptor binding activity of the antigen-binding molecules in the pH neutral range, the rate of uptake into cells may be accelerated as compared with the antigen-binding molecule before the antigen-binding activity (binding ability) of the antigen-binding molecule is decreased in the pH neutral range or the high calcium ion concentration plasma concentration condition as compared with the antigen-binding activity (binding ability) of the antigen-binding molecule in the pH acidic range or the low calcium ion concentration plasma concentration condition, and preferably accelerated as compared with human IgG of a natural type having fucose-containing sugar chains as the sugar chains linked at position 297 in the EU numbering system, in particular, it is preferable that the activity is promoted compared with any of natural human IgG1, IgG2, IgG3, and IgG 4. Therefore, in the present invention, whether or not the uptake of antigen into cells using the antigen-binding molecule is promoted can be determined by whether or not the rate of uptake of antigen into cells is increased. The rate of uptake of antigen into cells can be calculated, for example, by adding the antigen-binding molecule and the antigen to a culture medium containing Fc γ receptor-expressing cells, and measuring the decrease in the concentration of the antigen in the culture medium over time or the amount of the antigen taken into the Fc γ receptor-expressing cells over time. The method of promoting the rate of antigen uptake into cells using the antigen-binding molecule of the present invention, for example, by administering the antigen-binding molecule, can promote the rate of elimination of the antigen in plasma. Therefore, whether or not the uptake of antigen into cells by the antigen-binding molecule is promoted can also be confirmed by measuring, for example, whether or not the elimination rate of antigen present in plasma is accelerated or the total antigen concentration in plasma is decreased by the administration of the antigen-binding molecule.
in the present invention, "total antigen concentration in plasma" means the total concentration of antigen to which the antigen-binding molecule binds and unbound antigen, or "free antigen concentration in plasma" as the concentration of antigen to which the antigen-binding molecule does not bind. Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are known in the art as described below in the present specification.
in the present invention, "natural type human IgG" means unmodified human IgG and is not limited to a specific class of IgG. In addition, as for "natural human IgG", it is preferable that the sugar chain linked at position 297 in the EU numbering system is a fucose-containing sugar chain. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as "natural human IgG" as long as it can bind to human FcRn in the acidic pH range. Preferably, the "native human IgG" may be human IgG 1.
Method for increasing the number of antigens that a molecule of antigen-binding molecule can bind
The present invention provides a method of increasing the number of antigens to which a molecule of an antigen-binding molecule can bind, the method comprising contacting an antigen-binding molecule with a cell expressing an Fc γ receptor, the antigen-binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity that varies with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in the neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked at EU position 297.
In addition, the present invention provides a method of increasing the number of antigens to which a molecule of an antigen-binding molecule can bind, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain which have human FcRn binding activity in the pH acidic range and have antigen-binding activity varying depending on the ion concentration condition is increased in Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains linked at EU number 297 in the pH neutral range.
the "number of antigens that can be bound by one molecule of antigen-binding molecule" in the present invention means the number of antigens that can be bound until the antigen-binding molecule is decomposed and eliminated. The phrase "increasing the number of antigens to which one molecule of antigen-binding molecule can bind" as used herein means increasing the number of times that an antigen molecule bound to an antigen-binding molecule is dissociated and then bound again to the antigen molecule. The antigenic molecule to which the antigen binding molecule binds may be the same antigenic molecule as that present in the reaction system in which two molecules are present, or may be a different molecule. That is, in other words, the cumulative number of returns of antigen binding molecules bound to antigen in the reaction system. In other words, when one cycle is assumed in which the antigen-binding molecule bound to the antigen is taken into the cell, the antigen is dissociated in the body, and the antigen-binding molecule returns to the outside of the cell, the number of the cycles that can be repeated increases until the antigen-binding molecule is decomposed and eliminated. The antigen binding molecules of the present invention having binding activity to Fc γ receptors in the pH neutral range are taken up into cells of cells expressing the Fc γ receptors due to endocytosis after binding to the Fc γ receptors. The antigen binding molecules of the present invention, which are free from Fc γ receptors in the pH acidic range or low calcium ion concentration plasma concentration conditions, can be recycled outside the cell by binding to FcRn in the pH acidic range. The antigen-binding molecule of the present invention that is circulated to the outside of the cell can be bound to the antigen again after the antigen is dissociated from the antigen-binding molecule under the ionic concentration condition such as pH acidic range or low calcium ion concentration. Therefore, whether or not the number of cycles is increased can be judged by the presence or absence of "promotion of intracellular uptake" as described above or the presence or absence of "improvement of pharmacokinetics" as described below.
Method for eliminating antigen in plasma or method for increasing antigen eliminating ability in plasma of antigen-binding molecule method of
The present invention provides a method of depleting plasma of an antigen comprising contacting a cell expressing an Fc γ receptor with an antigen binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity varying with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in the neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked at EU position 297.
further, the present invention provides a method for increasing the elimination ability of an antigen in plasma of an antigen-binding molecule, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain which have human FcRn binding activity in the pH acidic range and have antigen-binding activity varying depending on the ion concentration condition is increased in Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains linked at EU number 297 in the pH neutral range.
in the present invention, the "method of increasing the ability to eliminate an antigen from plasma" is synonymous with the "method of increasing the ability of an antigen-binding molecule to eliminate an antigen from plasma".
In the present invention, the "antigen-eliminating ability in plasma" refers to an ability to eliminate an antigen present in plasma from plasma when an antigen-binding molecule is administered into the body or when the antigen-binding molecule is secreted in the body. Therefore, in the present invention, the "antigen-eliminating ability in plasma of the antigen-binding molecule is increased", and when the antigen-binding molecule is administered, the rate of antigen elimination from plasma may be increased as compared to when the antigen-binding activity of the antigen-binding molecule in the pH neutral range or the antigen-binding activity in the high calcium ion concentration is decreased as compared to the antigen-binding activity in the pH acidic range or the low calcium ion concentration plasma range. Whether the antigen-eliminating ability in the plasma of the antigen-binding molecule is increased or not can be judged by, for example, administering the soluble antigen and the antigen-binding molecule into the body, and measuring the concentration of the soluble antigen in the plasma after the administration. When the concentration of the soluble antigen in plasma after administration of the soluble antigen and the antigen-binding molecule is decreased, the antigen-eliminating ability in plasma of the antigen-binding molecule can be determined to be increased by increasing the binding activity of the antigen-binding molecule to Fc γ receptor in the pH neutral range, or by decreasing the binding activity of the antigen-binding molecule to the Fc γ receptor in the pH acidic range or in the low calcium ion concentration plasma as compared to the binding activity of the antigen-binding molecule to the antigen in the pH neutral range or in the high calcium ion concentration plasma in addition to the binding activity to the Fc γ receptor. The soluble antigen may be an antigen to which the antigen-binding molecule has bound (an antigen in the state of an antigen-binding molecule complex) or an antigen to which the antigen-binding molecule has not bound, and the concentrations thereof may be determined as "the concentration of the antigen to which the antigen-binding molecule has bound in plasma" and "the concentration of the antigen to which the antigen-binding molecule has not bound in plasma" (the latter is synonymous with "the concentration of free antigen in plasma"), respectively. "Total antigen concentration in plasma" means the total concentration of antigen bound by the antigen binding molecule and antigen unbound by the antigen binding molecule, or "free antigen concentration in plasma" which is the concentration of antigen unbound by the antigen binding molecule, and thus the soluble antigen concentration can be determined as "total antigen concentration in plasma". Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are known in the art as described below in the present specification.
methods of improving the pharmacokinetics of antigen binding molecules
the present invention provides a method of improving the pharmacokinetics of an antigen binding molecule comprising contacting the antigen binding molecule with a cell expressing an Fc γ receptor, in vivo or ex vivo, the antigen binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity varying with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked to position 297 in the EU numbering system.
In addition, the present invention provides a method of improving the pharmacokinetics of an antigen binding molecule, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain which have human FcRn binding activity in the pH acidic range and have antigen-binding activity varying depending on the ion concentration condition is increased in Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains linked at EU number 297 in the pH neutral range.
in the present invention, the terms "improvement in pharmacokinetics", "improvement in pharmacokinetics" and "excellent pharmacokinetics" may mean "improvement in plasma (blood) retention", "excellent plasma (blood) retention" and "prolongation in plasma (blood)" in other words, and these terms are used in the same sense.
The term "improvement of pharmacokinetics" in the present invention includes not only a prolonged period of time from the administration of the antigen-binding molecule to the elimination from plasma (for example, a state in which the antigen-binding molecule is not returned to plasma, such as being degraded in cells) after administration to a human or a non-human animal such as a mouse, a rat, a monkey, a rabbit, or a dog, but also a prolonged period of time during which the antigen-binding molecule is retained in plasma in a state in which it can bind to an antigen (for example, a state in which the antigen-binding molecule is not bound to an antigen) until the antigen is degraded and eliminated after administration. Native human IgG can bind to FcRn derived from non-human animals. For example, native human IgG binds more strongly to mouse FcRn than human FcRn (int. immunol. (2001)13(12),1551-1559), and therefore, in order to confirm the properties of the antigen-binding molecule of the present invention, administration using mouse is preferable. As another example, mice (Methods mol. biol. (2010)602,93-104) in which the original FcRn gene of the mouse is deleted and which have and express a transgene related to the human FcRn gene can also be used as subjects to be administered in order to confirm the properties of the antigen binding molecules of the present invention described below. Specifically, "improvement in pharmacokinetics" also includes a longer time until the antigen-binding molecule not binding to the antigen (antigen-non-binding antigen-binding molecule) is decomposed and eliminated. Even if an antigen-binding molecule is present in plasma, the antigen-binding molecule cannot bind to a new antigen when an antigen is already bound to the antigen-binding molecule. Therefore, the longer the time during which the antigen-binding molecule is not bound to the antigen, the longer the time during which the antigen can be bound to the neoantigen (the more chance that the antigen can be bound to the neoantigen), the shorter the time during which the antigen is not bound to the antigen-binding molecule in the body can be reduced, and the longer the time during which the antigen is bound to the antigen-binding molecule can be increased. As long as the elimination of the antigen from the plasma can be accelerated by administering the antigen-binding molecule, the concentration of the antigen-non-binding antigen-binding molecule in the plasma increases, and further, the time for the antigen to bind to the antigen-binding molecule becomes long. That is, the "improvement in pharmacokinetics of the antigen-binding molecule" in the present invention includes: improvement in any one of the pharmacokinetic parameters of the antigen non-binding antigen binding molecule (any one of increase in half-life in plasma, increase in mean plasma residence time, and decrease in clearance in plasma), or prolongation of the time for which the antigen binds to the antigen binding molecule after administration of the antigen binding molecule, or elimination of the antigen from plasma by the antigen binding molecule is accelerated. Can be judged by measuring any of the half-life in plasma, mean plasma residence time, clearance in plasma, etc., of the antigen-binding molecule or antigen-non-binding antigen-binding molecule (フ ァ ー マ コ キ ネ テ ィ ク ス evolution に よ る understanding (southern mountain hall)). For example, when an antigen-binding molecule is administered to a mouse, rat, monkey, rabbit, dog, human, or the like, the plasma concentration of the antigen-binding molecule or antigen-non-binding antigen-binding molecule is measured, and each parameter is calculated, and the pharmacokinetics of the antigen-binding molecule is improved in cases where the plasma half-life is long or the mean plasma residence time is long. These parameters can be determined by methods known to those skilled in the art, for example, non-compartmental (Noncompartmental) analysis using pharmacokinetic analysis software WinNonlin (Pharsight) according to the attached instructions, whereby appropriate evaluation can be performed. The measurement of the concentration of the antigen-binding molecule not bound to the antigen in plasma can be carried out by a method known to those skilled in the art, and for example, a measurement method in a known method (clin. pharmacol. (2008)48(4),406-417) can be used.
"pharmacokinetic improvement" in the context of the present invention includes an increase in the time that an antigen binds to an antigen binding molecule following administration of the antigen binding molecule. Whether the time for which the antigen binds to the antigen-binding molecule is prolonged after administration of the antigen-binding molecule can be determined by measuring the plasma concentration of the free antigen and determining the time until the plasma concentration of the free antigen or the ratio of the free antigen concentration to the total antigen concentration increases.
the concentration of free antigen not bound to the antigen binding molecule in plasma or the ratio of the concentration of free antigen to the concentration of total antigen can be carried out by methods known to those skilled in the art, and can be determined, for example, by the method described in pharm.res. (2006)23(1), 95-103. When an antigen exhibits a certain function in the body, whether or not the antigen binds to an antigen-binding molecule (antagonist molecule) that neutralizes the function of the antigen can also be determined by evaluating whether or not the function of the antigen is neutralized. The neutralization or lack thereof of the function of the antigen can be evaluated by measuring a certain in vivo marker reflecting the function of the antigen. Whether an antigen binds to an antigen binding molecule (agonist molecule) that activates the function of the antigen can be assessed by measuring certain in vivo markers that reflect the function of the antigen.
The measurement of the concentration of free antigen in plasma, the measurement of the ratio of the amount of free antigen in plasma to the total amount of antigen in plasma, the measurement of the in vivo marker, and the like are not particularly limited, and it is preferable to perform the measurement after a certain time has elapsed from the administration of the antigen-binding molecule. In the present invention, "after a certain period of time has elapsed since the administration of the antigen-binding molecule" is not particularly limited, and can be appropriately determined by one skilled in the art according to the properties of the antigen-binding molecule to be administered, and examples thereof include: for example, 1 day after the administration of the antigen-binding molecule, 3 days after the administration of the antigen-binding molecule, 7 days after the administration of the antigen-binding molecule, 14 days after the administration of the antigen-binding molecule, 28 days after the administration of the antigen-binding molecule, and the like. In the present invention, "concentration of antigen in plasma" means either of the total concentration of antigen bound by the antigen binding molecule and antigen unbound by the antigen binding molecule, i.e., "total antigen concentration in plasma" or the concentration of antigen unbound by the antigen binding molecule, i.e., "concentration of free antigen in plasma".
By administering the antigen-binding molecule of the present invention, the total antigen concentration in plasma can be reduced by 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more compared to the case of administering a reference antigen-binding molecule containing as an Fc γ receptor binding domain a native human IgG Fc region in which the sugar chain linked at position 297 in the EU numbering is a fucose-containing sugar chain, or compared to the case of not administering an antigen-binding molecule containing the antigen-binding domain of the present invention.
The antigen/antigen binding molecule molar ratio can be calculated as follows:
a value is the molar concentration of antigen at each time
b value is the molar concentration of antigen binding molecule at each time
The value of C is the molar concentration of antigen per molar concentration of antigen-binding molecule (molar ratio of antigen/antigen-binding molecule) at each time
C＝A/B。
When the C value is small, the antigen elimination efficiency per antigen-binding molecule is shown to be high, and when the C value is large, the antigen elimination efficiency per antigen-binding molecule is shown to be low.
the antigen/antigen binding molecule molar ratio can be calculated as described above.
by administering the antigen-binding molecule of the invention, the antigen/antigen-binding molecule molar ratio can be reduced by 2, 5, 10, 20, 50, 100, 200, 500, 1000 fold or more compared to the case of administering a reference antigen-binding molecule containing a native human IgG Fc region as a human Fc γ receptor binding domain.
In the present invention, the use of natural-type human IgG1, IgG2, IgG3, or IgG4 as natural-type human IgG for comparison with an antigen-binding molecule in terms of Fc γ receptor binding activity or activity in the body is preferable. Preferably, a reference antigen binding molecule containing the same antigen binding domain as the antigen binding molecule of interest and a native human IgG Fc region as the Fc γ receptor binding domain can be suitably used. More preferably the use of native human IgG1 for comparison with an antigen binding molecule in terms of Fc γ receptor binding activity or activity in vivo with reference native human IgG. In the present invention, as a reference antigen-binding molecule to be compared with the antigen-binding molecule of the present invention, it is also possible to suitably use, depending on the purpose: an antigen binding molecule comprising an antigen binding domain whose antigen binding activity does not vary depending on ion concentration, an antigen binding molecule comprising an FcRn binding domain whose FcRn binding activity under pH acidic range conditions is not enhanced, or an antigen binding molecule comprising an fcgamma receptor binding domain having no selective binding activity to an fcgamma receptor, and the like.
the decrease in total antigen concentration or antigen/antibody molar ratio in plasma can be assessed as described in examples 6, 8 and 13. More specifically, in the absence of cross-reactivity of the antigen binding molecule with the mouse corresponding antigen, evaluation can be performed using either human FcRn transgenic mouse strain 32 or strain 276(Jackson Laboratories, Methods mol. biol. 2010; 602:93-104) by simultaneous administration of antigen antibodies to either the model or the steady state antigen injection model. When an antigen binding molecule cross reacts with a mouse corresponding antigen, it can be assessed by administering only the antigen binding molecule to human FcRn transgenic mouse strain 32 or strain 276(Jackson Laboratories). In the simultaneous administration model, a mixture of antigen binding molecules and antigen is administered to mice. In a steady-state antigen injection model, mice are implanted with an infusion pump filled with an antigen solution to achieve a constant concentration of antigen in plasma, and then are administered with an antigen-binding molecule. The test antigen binding molecules are administered in the same amount. The total antigen concentration in plasma, the free antigen concentration in plasma and the concentration of antigen-binding molecule in plasma are determined at appropriate times using methods well known to those skilled in the art.
In evaluating the effect of an Fc γ receptor binding domain having selective binding activity to an Fc γ receptor, the decrease in the total antigen concentration or the antigen/antibody molar ratio in plasma can be evaluated by either of a simultaneous antigen-antibody injection model or a steady-state antigen injection model using a commonly used C57BL/6J mouse (Charles River Japan) when the antigen-binding molecule does not cross-react with the mouse-corresponding antigen. When the antigen binding molecule cross reacts with the mouse corresponding antigen, evaluation can be performed by injecting only the antigen binding molecule into a commonly used C57BL/6J mouse (Charles River Japan).
The long-term effects of the present invention can be evaluated by measuring the total antigen concentration or free antigen concentration and the antigen/antigen-binding molecule molar ratio in plasma after 2 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after administration. In other words, to evaluate the properties of the antigen binding molecules of the invention, the long term antigen concentration in plasma was determined by measuring the total or free antigen concentration and the antigen/antigen binding molecule molar ratio in plasma 2 days after, 4 days after, 7 days after, 14 days after, 28 days after, 56 days after, or 84 days after the administration of the antigen binding molecule. Whether or not a decrease in the plasma antigen concentration or the antigen/antigen-binding molecule molar ratio is achieved by the antigen-binding molecule described in the present invention can be determined by evaluating the decrease at any 1 or more of the previously described times.
The short-term effects of the present invention can be evaluated by measuring the total antigen concentration or free antigen concentration and the antigen/antigen-binding molecule molar ratio in plasma after 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration. In other words, to evaluate the properties of the antigen binding molecules of the invention, the short term antigen concentration in plasma was determined by measuring the total or free antigen concentration and the antigen/antigen binding molecule molar ratio in plasma 15 minutes after, 1 hour after, 2 hours after, 4 hours after, 8 hours after, 12 hours after, or 24 hours after the administration of the antigen binding molecule.
the route of administration of the antigen binding molecules of the present invention may be selected from intradermal injection, intravenous injection, intravitreal injection, subcutaneous injection, intraperitoneal injection, parenteral injection, and intramuscular injection.
In the present invention, improvement of pharmacokinetics in humans is preferred. When it is difficult to measure the retention in human plasma, the retention in human plasma can be predicted based on the retention in plasma of a mouse (e.g., a normal mouse, a human antigen-expressing transgenic mouse, a human FcRn-expressing transgenic mouse, etc.) or a monkey (e.g., a cynomolgus monkey, etc.).
method for promoting intracellular dissociation of antigen bound to antigen binding molecule outside cell from antigen binding molecule
the present invention provides a method of promoting the intracellular dissociation in a cell from an antigen binding molecule of an antigen bound to the antigen binding molecule extracellularly, the method comprising contacting the antigen binding molecule with a cell expressing an Fc γ receptor, the antigen binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity varying with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked to position 297 in the EU numbering system.
The present invention provides a method of promoting intracellular dissociation in a cell of an antigen bound extracellularly to an antigen binding molecule from the antigen binding molecule, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain which have human FcRn binding activity in the pH acidic range and have antigen-binding activity varying depending on the ion concentration condition is increased in Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains linked at EU number 297 in the pH neutral range.
In the present invention, the site at which the antigen is dissociated from the antigen-binding molecule may be any site as long as it is within the cell, but it is preferably in vivo at an early stage. In the present invention, "the antigen bound to the antigen-binding molecule outside the cell is dissociated from the antigen-binding molecule inside the cell" means that all the antigen taken into the cell without being bound to the antigen-binding molecule is not necessarily dissociated from the antigen-binding molecule inside the cell, and the antigen-binding activity of the antigen-binding molecule in the pH acidic range or the low calcium ion concentration plasma concentration condition is decreased as compared with the antigen-binding activity in the pH neutral range or the high calcium ion concentration plasma concentration condition, and the proportion of the antigen dissociated from the antigen-binding molecule inside the cell is increased as compared with before the Fc γ receptor-binding activity in the pH neutral range is increased. In addition, the method of promoting the intracellular dissociation of an antigen bound to an antigen-binding molecule outside the cell from the antigen-binding molecule can also be said to be a method of imparting a property of facilitating the uptake of the antigen-binding molecule bound to the antigen into the cell and facilitating the intracellular dissociation of the antigen from the antigen-binding molecule to the antigen-binding molecule.
method for promoting extracellular release of antigen-binding molecules in non-antigen-bound state
The present invention provides a method of promoting the release of an antigen binding molecule in a non-antigen bound state to the outside of a cell, the method comprising contacting a cell expressing an Fc γ receptor with a cell in or outside of the body, the antigen binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity varying with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in the neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked at EU position 297.
Further, the present invention provides a method for promoting the release of an antigen-binding molecule, which is taken up into a cell in a state of being bound to an antigen, to the outside of the cell in a state of not being bound to the antigen, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain which have human FcRn binding activity in the pH acidic range and have antigen-binding activity varying depending on the ion concentration condition is increased in Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains linked at EU number 297 in the pH neutral range.
In the present invention, "the antigen-binding molecules taken up into cells in a state of being bound to an antigen are released outside the cells in a state of not being bound to the antigen", it is not necessary that all the antigen-binding molecules taken up into cells in a state of being bound to the antigen are released outside the cells in a state of not being bound to the antigen, and the ratio of the antigen-binding molecules released outside the cells in a state of not being bound to the antigen may be increased as compared with the antigen-binding activity of the antigen-binding molecules under the condition of pH acidity or low calcium ion concentration plasma concentration, as long as the antigen-binding activity of the antigen-binding molecules under the condition of pH neutrality or high calcium ion concentration plasma concentration is decreased, and compared with the ratio before the Fc γ receptor-binding activity under the pH neutrality range is increased. Preferably, the antigen binding molecule that is released outside the cell maintains antigen binding activity. In addition, the method of promoting the release of the antigen-binding molecule incorporated into the cell in the state of being bound to the antigen to the outside of the cell in the state of not being bound to the antigen can also be said to be a method of imparting the antigen-binding molecule with properties of facilitating the incorporation of the antigen-binding molecule bound to the antigen into the cell and facilitating the release of the antigen-binding molecule to the outside of the cell in the state of not being bound to the antigen.
methods of reducing total or free antigen concentration in plasma or reducing total antibodies in plasmaOriginal concentration Methods of altering antigen binding molecules to degree or free antigen concentration
the present invention provides a method of reducing the total or free antigen concentration in plasma, the method comprising contacting cells expressing an Fc γ receptor with an antigen binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity varying with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked to position 297 in the EU numbering system.
in addition, the present invention provides a method of altering an antigen binding molecule that reduces the total antigen concentration or the free antigen concentration in plasma, the method comprising: an Fc gamma receptor binding domain in an antigen-binding molecule comprising an antigen-binding domain and an Fc gamma receptor-binding domain which have human FcRn binding activity in the pH acidic range and have antigen-binding activity varying depending on the ion concentration condition is increased in Fc gamma receptor-binding activity in the pH neutral range as compared with the Fc gamma receptor-binding domain of a native type having fucose-containing sugar chains linked at EU number 297 in the pH neutral range.
methods for evaluating the decrease in total antigen concentration or free antigen concentration in plasma are described in the above-mentioned item of methods for improving the pharmacokinetics of antigen-binding molecules.
ex vivo method for eliminating the antigen from plasma
As a non-limiting example of the use of the antigen-binding molecule provided by the present invention in a method for eliminating the antigen from plasma, the use of the antigen-binding molecule in the following method can also be exemplified: a so-called ex vivo method for eliminating the antigen from plasma, which comprises contacting an immune complex formed by contacting plasma isolated from a subject with an antigen binding molecule of the invention with cells expressing FcRn and Fc γ receptors. Instead of or in combination with the method of administering the antigen-binding molecule into the body, the elimination rate of the antigen in the plasma can also be accelerated by a so-called ex vivo method in which the plasma containing the antigen-binding molecule and the antigen bound to the antigen-binding molecule is temporarily taken out of the body, then contacted with cells expressing FcRn and Fc γ receptors, and after a certain period of time, the plasma containing the antigen-binding molecule not bound to the antigen, which is recirculated to the outside of the cell (also referred to as re-secretion or recirculation), is returned to the body.
In addition, as a non-limiting embodiment of the use of the antigen-binding molecule in the method for eliminating the antigen from plasma provided by the present invention, the use of the antigen-binding molecule in the following method can be exemplified: a so-called ex vivo method for eliminating the antigen from plasma, which method comprises contacting an immune complex present in plasma isolated from a subject to whom the antigen binding molecule of the invention is administered with cells expressing FcRn and Fc γ receptors.
the elimination or absence of the antigen from the plasma can be confirmed as follows: in place of the antigen-binding molecule of the present invention, when an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity does not vary depending on ion concentration, an antigen-binding molecule containing an FcRn-binding domain whose FcRn-binding activity under pH acidic range conditions is not enhanced, or an antigen-binding molecule containing an Fc γ receptor-binding domain having no selective binding activity to an Fc γ receptor are compared as a control, whether the aforementioned elimination rate of an antigen in plasma is accelerated or not is evaluated.
Method for preparing antigen binding molecules
in addition, the present invention provides a method for producing an antigen-binding molecule comprising: an antigen binding domain having human FcRn binding activity in the pH acidic range and antigen binding activity varying with ion concentration conditions; and an Fc γ receptor binding domain having an Fc γ receptor binding activity in a neutral pH range higher than that of a native Fc γ receptor binding domain of fucose-containing sugar chains, the sugar chain linked to position 297 in the EU numbering system.
That is, the present invention provides a method for producing an antigen-binding molecule, comprising the following steps (a) to (f):
(a) a step of obtaining the antigen-binding activity of the antigen-binding domain under the condition of high calcium ion concentration;
(b) a step of obtaining the antigen-binding activity of the antigen-binding domain under the condition of low calcium ion concentration;
(c) a step of selecting an antigen-binding domain having an antigen-binding activity obtained in (a) higher than that obtained in (b);
(d) A step of linking the polynucleotide encoding the antigen binding domain selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity under a pH acidic range condition, and a sugar chain having an Fc γ receptor binding activity under a pH neutral range condition linked to EU position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of a native Fc γ receptor binding domain containing fucose sugar chains;
(e) culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) A step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
further, the present invention provides a method for producing an antigen-binding molecule, comprising the following steps (a) to (f):
(a) a step of obtaining the antigen binding activity of the antibody under the condition of high calcium ion concentration;
(b) A step of obtaining the antigen binding activity of the antibody under the condition of low calcium ion concentration;
(c) a step of selecting an antibody having an antigen binding activity obtained in (a) higher than that obtained in (b);
(d) a step of linking a polynucleotide encoding the antigen binding domain of the antibody selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range and linked to EU position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of a native Fc γ receptor binding domain containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) A step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
also, the present invention provides a method for producing an antigen-binding molecule, comprising the steps of:
(a) A step of obtaining the antigen binding activity of the antigen binding domain under the condition of pH neutral range;
(b) a step of obtaining an antigen binding activity of the antigen binding domain under a pH acidic range condition;
(c) a step of selecting an antigen-binding domain having an antigen-binding activity obtained in (a) higher than that obtained in (b);
(d) A step of linking the polynucleotide encoding the antigen binding domain selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range and linked to EU position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of a native Fc γ receptor binding domain containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
further, the present invention provides a method for producing an antigen-binding molecule, comprising the following steps (a) to (f):
(a) A step of obtaining the antigen binding activity of the antibody under the condition of pH neutral range;
(b) A step of obtaining an antigen binding activity of an antibody to an antigen in a pH acidic range;
(c) a step of selecting an antibody having an antigen binding activity obtained in (a) higher than that obtained in (b);
(d) a step of linking a polynucleotide encoding the antigen binding domain of the antibody selected in (c) to a polynucleotide encoding an Fc γ receptor binding domain which is: a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range and linked to EU position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of a native Fc γ receptor binding domain containing fucose sugar chains;
(e) Culturing a cell into which a vector to which the polynucleotide obtained in (d) is operably linked; and
(f) a step of recovering the antigen-binding molecule from the cell culture broth cultured in (e).
"cell," "cell line," and "cell culture" are used synonymously in this specification, and such designations may include all progeny of a cell or cell line. Thus, the terms such as "transformant" and "transformed cell" are independent of the number of passages, and include primary subject cells and cultures derived therefrom. Furthermore, it is also understood that the content of DNA in all progeny may not be exactly the same due to deliberate or accidental mutation. Progeny of mutants having substantially the same function or biological activity, as screened in the original transformed cell, may also be included. In the case of a description intended to be referred to differently, such an intention should be apparent from the context of the description.
Reference to a control sequence when expressing a coding sequence is a reference to the DNA base sequence required for the expression of the operably linked coding sequence in a particular host organism. For example, suitable regulatory sequences for prokaryotes include promoters, operator sequences as appropriate, ribosome binding sites, and other sequences that may not be well understood. In eukaryotic cells, the use of promoters, polyadenylation signals, and enhancers for expression of a coding sequence is well known.
"operably linked" with respect to a nucleic acid means that the nucleic acid is in a functional relationship with other nucleic acid sequences. For example, a DNA of a presequence (sequence) or secretory leader is operably linked to a DNA of a polypeptide when it is expressed as a preprotein associated with secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence under conditions in which it affects the transcription of the sequence. Alternatively, the ribosome binding site is operably linked to a coding sequence if it is located in a position that facilitates translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secreted leader peptide, contiguous and in reading frame. However, enhancers are not necessarily contiguous. Ligation is achieved by ligation at appropriate restriction sites. In the absence of such sites, synthetic oligonucleotide adaptors or linkers are used according to conventional practice. The ligated nucleic acids can also be prepared by the overlap extension PCR method described above.
"ligation" is a process by which phosphodiester bonds are formed between two nucleic acid fragments. In order to join two fragments, the ends of the fragments must be paired with each other. Optionally, the ends are paired after endonuclease digestion. However, in order to be suitable for ligation, firstly, it is necessary to convert the sticky ends, which are usually formed after endonuclease digestion, into blunt ends. To form blunt ends, the DNA is treated with about 10 units of DNA polymerase I or Klenow fragment of T4 DNA polymerase in a suitable buffer at 15 ℃ for at least 15 minutes in the presence of 4 deoxyribonucleoside triphosphates. Subsequently, the DNA was purified by phenol chloroform extraction and ethanol precipitation, or silicon purification (silica purification). The DNA fragments to be ligated are added to the solution in equimolar amounts. In this solution, ligase such as T4 DNA ligase contained about 10 units per 0.5. mu.g of DNA, in addition to ATP and ligase buffer. When DNA is ligated to a vector, the vector is first linearized by digestion with an appropriate restriction endonuclease. By subsequent treatment of the linearized fragment with bacterial alkaline phosphatase or calf intestinal phosphatase, self-ligation of the fragment during the ligation step is prevented.
in the production method of the present invention, an antigen-binding domain or antibody having a higher antigen-binding activity under a high calcium ion concentration condition than under a low calcium ion concentration condition, which is selected by the method described in the above item of "ion concentration condition", is isolated. Further, an antigen-binding domain or antibody having a higher antigen-binding activity in the pH neutral range than the antigen-binding activity in the pH acidic range, which is selected by the method described in the above item of "ion concentration conditions", is isolated. For example, when an antigen binding domain isolated in this manner is selected from a library, a polynucleotide encoding the antigen binding domain is isolated from a virus such as a phage by ordinary gene amplification, as described in examples described later. When the thus-isolated antigen-binding domain or antibody is selected from a cell culture solution of a hybridoma or the like, an antibody gene or the like is isolated from the cell by ordinary gene amplification as described in the above-mentioned antibody item.
next, a polynucleotide encoding the antigen binding domain isolated as described above is linked in-frame to a polynucleotide encoding an Fc γ receptor binding domain: has a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range linked to the EU position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of a native Fc γ receptor binding domain of fucose-containing sugar chains. As described in the above-mentioned item of Fc γ receptor binding domain, a suitable example of an Fc γ receptor binding domain is an Fc region of an antibody. Further, the Fc γ receptor may be any of Fc γ RIa, Fc γ RIIa (R), Fc γ RIIa (H), Fc γ RIIb, Fc γ RIIIa (V), and Fc γ RIIIa (F).
as the Fc region of the antibody, there may be suitably mentioned an Fc region in which at least one or more amino acids selected from the following amino acids at the positions indicated by EU numbering in the Fc region are different from the amino acids at the corresponding positions in the natural Fc region: 221 bit, 222 bit, 223 bit, 224 bit, 225 bit, 227 bit, 228 bit, 230 bit, 231 bit, 232 bit, 233 bit, 234 bit, 235 bit, 236 bit, 237 bit, 238 bit, 239 bit, 240 bit, 241 bit, 243 bit, 244 bit, 245 bit, 246 bit, 247 bit, 249 bit, 250 bit, 251 bit, 254 bit, 255 bit, 256 bit, 258 bit, 260 bit, 262 bit, 263 bit, 264 bit, 265 bit, 266 bit, 267 bit, 268 bit, 269 bit, 270 bit, 271 bit, 272 bit, 273 bit, 274 bit, 275 bit, 276 bit, 278 bit, 279 bit, 280 bit, 281 bit, 282 bit, 283 bit, 284 bit, 285 bit, 286 bit, 288 bit, 290 bit, 291 bit, 292 bit, 293 bit, 298 bit, 295 bit, 296 bit, 297 bit, 299 bit, 326 bit, 301 bit, 302 bit, 303 bit, 304 bit, 313 bit, 315 bit, 328 bit, 325 bit, 323 bit, 325 bit, 320 bit, 325 bit, 323 bit, 325 bit, 323 bit, 320 bit, 325 bit, 320 bit, 329 bits, 330 bits, 331 bits, 332 bits, 333 bits, 334 bits, 335 bits, 336 bits, 337 bits, 339 bits, 376 bits, 377 bits, 378 bits, 379 bits, 380 bits, 382 bits, 385 bits, 392 bits, 396 bits, 421 bits, 427 bits, 428 bits, 429 bits, 434 bits, 436 bits, and 440 bits. Suitable examples of the natural Fc region include: an Fc region of any one of IgG1, IgG2, IgG3, or IgG 4.
In a non-limiting embodiment, the Fc region of the antibody may suitably include an Fc region containing at least one amino acid selected from the group consisting of:
The amino acid at position 221 is any one of Lys or Tyr;
The amino acid at position 222 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 223 is any one of Phe, Trp, Glu, or Lys;
the amino acid at position 224 is any one of Phe, Trp, Glu or Tyr;
the amino acid at position 225 is any one of Glu, Lys, or Trp;
the amino acid at the 227 position is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 228 is any one of Glu, Gly, Lys or Tyr;
the amino acid at position 230 is any one of Ala, Glu, Gly or Tyr;
the amino acid at position 231 is any one of Glu, Gly, Lys, Pro or Tyr;
The amino acid at position 232 is any one of Glu, Gly, Lys or Tyr;
The amino acid at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 234 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 235 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 240 is any one of Ala, Ile, Met or Thr;
The amino acid at position 241 is any one of Asp, Glu, Leu, Arg, Trp or Tyr;
the amino acid at position 243 is any one of Leu, Glu, Leu, Gln, Arg, Trp or Tyr;
the amino acid at position 244 is His;
the amino acid at position 245 is Ala;
the amino acid at position 246 is any one of Asp, Glu, His or Tyr;
the 247 th amino acid is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val or Tyr;
the amino acid at position 249 is any one of Glu, His, Gln or Tyr;
the amino acid at position 250 is either Glu or Gln;
the amino acid at position 251 is Phe;
The amino acid at position 254 is any one of Phe, Met or Tyr;
the amino acid at position 255 is any one of Glu, Leu or Tyr;
the amino acid at position 256 is any one of Ala, Met or Pro;
The amino acid at position 258 is any one of Asp, Glu, His, Ser or Tyr;
The amino acid at the 260 th position is any one of Asp, Glu, His or Tyr;
the amino acid at position 262 is any one of Ala, Glu, Phe, Ile, or Thr;
The amino acid at position 263 is any one of Ala, Ile, Met or Thr;
the 264 th amino acid is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
The 265 th amino acid is any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 266 is any one of Ala, Ile, Met or Thr;
the amino acid at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 268 is any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val or Trp;
The amino acid at position 269 is Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp or Tyr;
the amino acid at position 271 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the 272 amino acid is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 273 is any one of Phe or Ile;
The amino acid at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 275 is any one of Leu or Trp;
the amino acid at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
amino acid 279 is Ala;
The 280 th amino acid is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp or Tyr;
The amino acid at position 281 is Asp, Lys, Pro or Tyr;
The amino acid at position 282 is any one of Glu, Gly, Lys, Pro or Tyr;
the 283 th amino acid is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg or Tyr;
the amino acid at position 284 is any one of Asp, Glu, Leu, Asn, Thr or Tyr;
the amino acid at position 285 is any one of Asp, Glu, Lys, Gln, Trp or Tyr;
the amino acid at position 286 is any one of Glu, Gly, Pro or Tyr;
The amino acid at position 288 is any one of Asn, Asp, Glu or Tyr;
The amino acid at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp or Tyr;
The amino acid at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln or Thr;
The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr or Tyr;
The amino acid at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 295 is Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val;
The amino acid at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 298 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr;
The 299 th amino acid is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
The 300-position amino acid is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp;
The amino acid at position 301 is any one of Asp, Glu, His or Tyr;
the amino acid at position 302 is Ile;
The amino acid at the 303 th position is Asp, Gly or Tyr;
The amino acid at position 304 is any one of Asp, His, Leu, Asn or Thr;
The amino acid at position 305 is any one of Glu, Ile, Thr or Tyr;
The amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val or Tyr;
the amino acid at position 313 is Phe;
the amino acid at position 315 is Leu;
the amino acid at position 317 is Glu or Gln;
the amino acid at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val or Tyr;
the amino acid at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 323 is Ile;
The amino acid at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp or Tyr;
the amino acid at position 325 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 328 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 329 is Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp or Tyr;
The amino acid at position 332 is any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr;
The amino acid at position 333 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val or Tyr;
the amino acid at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
the amino acid at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp or Tyr;
the amino acid at position 336 is any one of Glu, Lys or Tyr;
the amino acid at position 337 is any one of Glu, His, or Asn;
The amino acid at position 339 is any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser or Thr;
Amino acid 376 is Ala or Val;
the amino acid at position 377 is Gly or Lys;
asp for the amino acid in position 378;
The amino acid at position 379 is Asn;
The amino acid at position 380 is any one of Ala, Asn or Ser;
Amino acid 382 is either Ala or Ile;
amino acid position 385 is Glu;
The amino acid at position 392 is Thr;
the amino acid at position 396 is Leu;
the amino acid at position 421 is Lys;
The amino acid at position 427 is Asn;
the amino acid at position 428 is any one of Phe or Leu;
the amino acid at position 429 is Met;
the amino acid at position 434 is Trp;
the amino acid at position 436 is Ile; and
the amino acid at position 440 is any one of Gly, His, Ile, Leu or Tyr.
In addition, the Fc region of the present invention may also suitably use an Fc region having FcRn binding activity or having enhanced FcRn binding activity under pH acidic range conditions. Examples of such an Fc region include Fc regions of IgG-type immunoglobulins, for example, Fc regions of human IgG (IgG1, IgG2, IgG3, IgG4, and modified forms thereof). In the case of an altered form in which the protein has an FcRn binding activity in the pH acidic range or has an increased human FcRn binding activity in the pH acidic range, an Fc region in which the amino acid at any position is altered may be used, and when the antigen-binding molecule contains an Fc region of human IgG1 as the Fc region, the protein preferably has an altered effect of increasing the binding activity to FcRn in the pH acidic range as compared with the initial Fc region of human IgG 1. Examples of amino acids that can be modified as described above include: as described in WO2000/042072, amino acids at positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and/or 447, which are represented by EU numbering. Similarly, as amino acids capable of effecting the above-mentioned change, there may be appropriately mentioned, for example: as described in WO2002/060919, the amino acid residues at positions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and/or 436 in the EU numbering system. Further, examples of amino acids that can be modified as described above include: amino acids at positions 250, 314 and 428 in the EU numbering system as described in WO 2004/092219. Further, as amino acids capable of effecting the above-mentioned change, there may be appropriately mentioned, for example: as described in WO2010/045193, the amino acids at positions 251, 252, 307, 308, 378, 428, 430, 434 and/or 436 represented by EU numbering. By these amino acid changes, an Fc region in which the binding of the Fc region of an IgG-type immunoglobulin to FcRn is enhanced in the pH acidic range can be used in the production method of the present invention.
as described later, an Fc region having FcRn binding activity in a neutral pH range can be suitably used as the Fc region included in the antigen-binding molecule of the present invention. Based on the above-described method for obtaining an Fc region having FcRn binding activity in the pH acidic range, such an Fc region can be obtained by any method, and specifically, an Fc region containing an FcRn binding domain having FcRn binding activity in the pH neutral range or having enhanced FcRn binding activity can be obtained by changing the amino acids of the Fc region of the human IgG-type immunoglobulin used as the starting Fc region. Examples of the Fc region of a preferred IgG-type immunoglobulin to be used for modification include Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and modified forms thereof). For the change to other amino acids, an Fc region in which the amino acid at any position is changed may be used as long as it has FcRn binding activity in the neutral range of pH or enhances human FcRn binding activity in the neutral range. When the antigen binding molecule contains the Fc region of human IgG1 as the Fc region, it preferably contains a modification that brings about an effect of enhancing binding to FcRn in the pH neutral range as compared with the binding activity of the initial Fc region of human IgG 1. As the Fc region capable of effecting such a change, there may be suitably mentioned, for example: a human Fc region in which at least one or more amino acids selected from the following in positions represented by EU numbering of the starting Fc region differ from the corresponding amino acids of the native Fc region: 237 bit, 238 bit, 239 bit, 248 bit, 250 bit, 252 bit, 254 bit, 255 bit, 256 bit, 257 bit, 258 bit, 265 bit, 270 bit, 286 bit, 289 bit, 297 bit, 298 bit, 303 bit, 305 bit, 307 bit, 308 bit, 309 bit, 311 bit, 312 bit, 314 bit, 315 bit, 317 bit, 325 bit, 332 bit, 334 bit, 360 bit, 376 bit, 380 bit, 382 bit, 384 bit, 385 bit, 386 bit, 387 bit, 389 bit, 424 bit, 428 bit, 433 bit, 434 bit and 436 bit.
In addition, as the Fc region capable of achieving such a change, there may be suitably mentioned, for example, an Fc region containing at least one amino acid selected from the group consisting of the following amino acids represented by EU numbering of the Fc region:
The amino acid at position 237 is Met;
amino acid 238 is Ala;
The amino acid at position 239 is Lys;
the amino acid at position 248 is Ile;
the amino acid at position 250 is any one of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr;
The amino acid at position 252 is any one of Phe, Trp or Tyr;
The amino acid at position 254 is Thr;
The amino acid at position 255 is Glu;
the amino acid at position 256 is any one of Asp, Glu or Gln;
the amino acid at position 257 is any one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val;
the amino acid at position 258 is His;
the amino acid at position 265 is Ala;
the amino acid at position 270 is Phe;
The amino acid at position 286 is either Ala or Glu;
the amino acid at position 289 is His;
The amino acid at position 297 is Ala;
amino acid at position 298 is Gly;
The amino acid at position 303 is Ala;
amino acid at position 305 is Ala;
the amino acid at position 307 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr;
the amino acid at position 308 is any one of Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr;
The amino acid at position 309 is any one of Ala, Asp, Glu, Pro or Arg;
the amino acid at position 311 is any one of Ala, His or Ile;
The amino acid at position 312 is either Ala or His;
The amino acid at position 314 is any one of Lys or Arg;
Amino acid 315 is any one of Ala or His;
the amino acid at position 317 is Ala;
the amino acid at position 325 is Gly;
val for the amino acid at position 332;
The amino acid at position 334 is Leu;
The amino acid at position 360 is His;
amino acid 376 is Ala;
The amino acid at position 380 is Ala;
amino acid 382 is Ala;
the amino acid at position 384 is Ala;
The amino acid at position 385 is any one of Asp or His;
the amino acid at position 386 is Pro;
Amino acid at position 387 is Glu;
the amino acid at position 389 is either Ala or Ser;
amino acid at position 424 is Ala;
The amino acid at position 428 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr;
the amino acid at position 433 is Lys;
the amino acid at position 434 is any one of Ala, Phe, His, Ser, Trp or Tyr;
The amino acid at position 436 is His.
for example, by using these amino acid changes alone or in combination, the binding of the Fc region of IgG to FcRn in the pH acidic range and/or neutral range can be enhanced, and the introduced amino acid changes are not particularly limited, and any amino acid change can be introduced as long as it can bring about an effect of improving the retention in plasma.
The antigen binding molecules of the invention can be isolated from the culture of cells transformed with a desired expression vector to which are operatively linked a polynucleotide encoding an antigen binding domain and a polynucleotide encoding an Fc γ receptor binding domain linked as described above, the Fc γ receptor binding domain being: has a human FcRn binding activity in the pH acidic range, and a sugar chain having an Fc γ receptor binding activity in the pH neutral range linked to the EU position 297 is an Fc γ receptor binding domain having a higher Fc γ receptor binding activity than that of a native Fc γ receptor binding domain of fucose-containing sugar chains. The antigen-binding molecule of the present invention can be produced by a method based on the method for producing an antibody described in the above-mentioned antibody item.
all prior art documents cited in the present specification are incorporated herein by reference.
the present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.