阅读说明：本技术 结合至cd38和cd3的双特异性抗体 (Bispecific antibodies that bind to CD38 and CD3 ) 是由 M.J.伯內特 S.Y.储 G.摩尔 J.德雅莱斯 于 2015-03-30 设计创作，主要内容包括：本发明涉及结合至CD38和CD3的双特异性抗体。本发明提供异二聚体蛋白质,所述异二聚体蛋白质包括结合至CD38和CD3的异二聚体抗体。本发明还涉及核酸组合物,包含所述核酸组合物的宿主细胞,制造所述异二聚体抗体的方法以及通过施用所述异二聚体抗体来治疗有需要的患者的方法。(The present invention relates to bispecific antibodies that bind to CD38 and CD 3. The present invention provides heterodimeric proteins comprising heterodimeric antibodies that bind to CD38 and CD 3. The invention also relates to nucleic acid compositions, host cells comprising the nucleic acid compositions, methods of making the heterodimeric antibodies, and methods of treating a patient in need thereof by administering the heterodimeric antibodies.)

1. A heterodimeric antibody comprising:

a) a first heavy chain comprising:

i) a first variable Fc domain;

ii) a single chain Fv region (scFv) that binds CD 3; and

b) a second heavy chain comprising:

i) a second Fc variable Fc domain; and

ii) a first variable heavy domain; and

c) a first light chain comprising a first variable light chain domain and a first constant light chain domain;

wherein the first variable heavy chain domain and the first variable light chain domain bind to CD 38.

2. A heterodimeric antibody according to claim 1 wherein said heterodimeric antibody is XENP13551 wherein said first heavy chain, said second heavy chain and said first light chain have the sequences depicted in figure 20 or 22.

3. The heterodimeric antibody according to claim 1, wherein the scFv has A sequence comprising vhCDR1 having the sequence T-Y-A-M-XaA1, wherein XaA1 is N, S or H (SEQ ID NO:435), vhCDR2 having the sequence R-I-R-S-K-XaA1-N-XaA2-Y-A-T-XaA 3-Y-A-XaA4-S-V-K-G, wherein XaA1 is Y or A, XaA2 is N or S, XaA3 is Y or A and XaA3 is D or A (SEQ ID NO: 36436) and XaA3 is XaA-V-S-W-F-XaA3-Y, wherein XaA3 is Y or XaA3 is XaA 3-3H-X3 or XaA 3-X3H-3-X3 is V-3 or XaA 3-V-3H-3 and wherein XaA is the sequence V-XaA-3 is V-3 or XaA 3H-3 or XaA 3H-3 is V (SEQ ID) and wherein the sequence XaA-3 is vDR 3-3 is V-3 or XaA-3H-3 is V-3-XaA-3H-3 or XaA-3 is V-3H-3 or XaA-3H-3H-3-XaA-3H-3-XaA-.

4. The composition of any one of claims 1 to 3, wherein the scFv is selected from the group consisting of H1.30_ L1.47, H1.33_ L1.47, and H1.31_ L1.47.47.

5. The composition of claim 1 or 3, wherein the anti-CD 3 variable region has a sequence selected from the group consisting of:

a) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

b) a sequence comprising vhCDR1 having SEQ ID No. 412, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

c) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 414, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

d) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 417, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

e) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 418, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

f) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 421, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

g) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 422, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

h) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 427, and vlCDR3 having SEQ ID No. 430;

i) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 428 and vlCDR3 having SEQ ID No. 430;

j) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 431;

k) a sequence comprising vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

l) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 423, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 432;

m) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 424, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 432;

n) comprises the sequence of vhCDR1 having SEQ ID No. 412, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 417, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

o) comprises the sequence of vhCDR1 having SEQ ID No. 412, vhCDR2 having SEQ ID No. 414, vhCDR3 having SEQ ID No. 419, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

p) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 415, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

q) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 415, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425 and vlCDR3 having SEQ ID No. 430;

r) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 417, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

s) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 419, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 430;

t) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 417, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 433;

u) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 413, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425, and vlCDR3 having SEQ ID No. 433; and

v) comprises the sequence of vhCDR1 having SEQ ID No. 411, vhCDR2 having SEQ ID No. 434, vhCDR3 having SEQ ID No. 416, vlCDR1 having SEQ ID No. 420, vlCDR2 having SEQ ID No. 425 and vlCDR3 having SEQ ID No. 430.

6. A heterodimeric antibody according to any one of claims 1 to 5 wherein said first variable heavy chain domain and said first variable light chain domain are selected from the pair consisting of H1 and L1, H1 and L1.24.24, H1 and L1.96, H1.77 and L1.96, H1.77 and L1.97, H1.72 and L1.97.97, H1.71 and L1.96, and H1.77 and L1.24.

7. A heterodimeric antibody according to any one of claims 1 to 6 wherein said scFv has a charged scFv linker.

8. The composition of any one of claims 1 to 6, wherein the anti-CD 3 variable region comprises a variable heavy chain region and a variable light chain region selected from the group consisting of:

5 and 6 SEQ ID NOs; 9 and 10 for SEQ ID NO; 13 and 14; 17 and 18; 21 and 22; 25 and 26 for SEQ ID NO; 29 and 30; 33 and 34; 37 and 38; 41 and 42; 45 and 46 for SEQ ID NO; 49 and 50 for SEQ ID NO; 53 and 54 SEQ ID NO; 57 and 58; 61 and 62; 65 and 66 for SEQ ID NO; 69 and 70 for SEQ ID NO; 73 and 74; 77 and 78; 81 and 82; 85 and 86 of SEQ ID NO; 89 and 90 for SEQ ID NO; 93 and 94; 97 and 98; 101 and 102; 105 and 106; 109 and 110; 113 and 114; 117 and 118; 121 and 122; 125 and 126; 129 and 130; 133 and 134; 137 and 138 for SEQ ID NO; 141 and 142; 145 and 146; 149 and 150; 153 and 154; 157 and 158; 161 and 162; 165 and 166 SEQ ID NOs; 169 and 170; 173 and 174; 177 and 178; 181 and 182; 185 and 186 SEQ ID NO; 189 and 190; 193 and 194; 197 and 198; 201 and 202; 205 and 206; 209 and 210; 213 and 214; 217 and 218; 221 and 222; 225 and 226 SEQ ID NOs; 229 and 230; 233 and 234; 237 and 238; 241 and 242 for SEQ ID NO; 245 and 246; 249 and 250 in SEQ ID NO; 253 and 254; SEQ ID NOs 257 and 258; 261 and 262; 265 and 266 SEQ ID NOs; 269 and 270; 273 and 274 SEQ ID NO; 277 and 278; 281 and 282; 285 and 286; 289 and 290; 293 and 294 SEQ ID NO; SEQ ID NOs 297 and 298; 301 and 302; 305 and 306; 309 and 310; 313 and 314; 317 and 318 of SEQ ID NO; 321 and 322 SEQ ID NOs; 325 and 326; 329 and 330 SEQ ID NO; 333 and 334; 337 and 338; 341 and 342 in SEQ ID NO; 345 and 346; 349 and 350 SEQ ID NOs; 353 and 354 for SEQ ID NO; 357 and 358; 361 and 362; 365 and 366 SEQ ID NO; 369 and 370; 373 and 374 SEQ ID NOs; 377 and 378; 381 and 382 SEQ ID NO; 385 and 386; 389 and 390; 393 and 394, SEQ ID NOs; 397 and 398; 401 and 402; 405 and 406; SEQ ID NOS: 409 and 410.

9. The composition of claim 7, wherein the charged scFv linker has 3 to 8 positive charges and is selected from the group consisting of SEQ ID NOS 443 to 451.

10. The composition of any one of claims 1 to 9, wherein the scFv has a sequence selected from the group consisting of seq id nos: 4, SEQ ID NO; 8 in SEQ ID NO; 12 is SEQ ID NO; 16 in SEQ ID NO; 20 in SEQ ID NO; 24 is SEQ ID NO; 28 in SEQ ID NO; 32 in SEQ ID NO; 36, SEQ ID NO; 40 in SEQ ID NO; 44 is SEQ ID NO; 48 for SEQ ID NO; 52 in SEQ ID NO; 56 in SEQ ID NO; 60 in SEQ ID NO; 64 is SEQ ID NO; 68 in SEQ ID NO; 72 in SEQ ID NO; 76 in SEQ ID NO; 80 in SEQ ID NO; 84, SEQ ID NO; 88 for SEQ ID NO; 92, SEQ ID NO; 96 in SEQ ID NO; 100 in SEQ ID NO; 104 in SEQ ID NO; 108, SEQ ID NO; 112 in SEQ ID NO; 116 as shown in SEQ ID NO; 120 of SEQ ID NO; 124, SEQ ID NO; 128 for SEQ ID NO; 132 in SEQ ID NO; 136, SEQ ID NO; 140 in SEQ ID NO; 144 in SEQ ID NO; 148, SEQ ID NO; 152, SEQ ID NO; 156 of SEQ ID NO; 160 in SEQ ID NO; 164 in SEQ ID NO; 168, SEQ ID NO; 172 for SEQ ID NO; 176, SEQ ID NO; 180 of SEQ ID NO; 184, SEQ ID NO; 188 SEQ ID NO; 192 of SEQ ID NO; 196 to SEQ ID NO; 200 in SEQ ID NO; 204 in SEQ ID NO; 208 of SEQ ID NO; 212 in SEQ ID NO; 216 SEQ ID NO; 220 in SEQ ID NO; 224 from SEQ ID NO; 228 as shown in SEQ ID NO; 232 in SEQ ID NO; 236, SEQ ID NO; 240 as shown in SEQ ID NO; 244 in SEQ ID NO; 248, SEQ ID NO; 252, SEQ ID NO; 256 of SEQ ID NO; 260 according to SEQ ID NO; 264 in SEQ ID NO; 268, SEQ ID NO; 272, SEQ ID NO; 276 of SEQ ID NO; 280 according to SEQ ID NO; 284, SEQ ID NO; 288 in SEQ ID NO; 292 in SEQ ID NO; 296 as shown in SEQ ID NO; 300 in SEQ ID NO; 304 is SEQ ID NO; 308 is shown in SEQ ID NO; 312 in SEQ ID NO; 316, SEQ ID NO; 320 in SEQ ID NO; 324 of SEQ ID NO; 328, SEQ ID NO; 332, SEQ ID NO; 336 in SEQ ID NO; 340 in SEQ ID NO; 344 as shown in SEQ ID NO; 348, SEQ ID NO; 352 in SEQ ID NO; 356 of SEQ ID NO; 360 of SEQ ID NO; 364 in SEQ ID NO; 368, SEQ ID NO; 372, SEQ ID NO; 376 SEQ ID NO; 380 of SEQ ID NO; 384; 388; 392 of SEQ ID NO; 396 of SEQ ID NO; 400 in SEQ ID NO; 404 of SEQ ID NO; 408 in SEQ ID NO.

11. The composition of claim 2, wherein the scFv has the sequence SEQ ID No. 396.

12. A heterodimeric antibody according to any one of claims 1 to 11, said heterodimeric antibody being selected from the group consisting of: XENP 13243; XENP 13545; XENP 13546; XENP 13547; XENP 13548; XENP 13549; XENP 13550; XENP 13551; XENP 13544; XENP 13752; XENP 13753; XENP 13754; XENP 13756; XENP 13757; and XENP 13694.

13. A heterodimeric antibody according to any of claims 1 to 12 wherein the Fc region of said heavy chain further comprises an FcRn variant.

14. A heterodimeric antibody according to claim 13 wherein said variant is 428L/434S.

15. A nucleic acid composition comprising:

a) a first nucleic acid encoding the first heavy chain of claim 1;

b) a second nucleic acid encoding the second heavy chain of claim 1; and

c) a third nucleic acid encoding the light chain.

16. A nucleic acid composition comprising:

a) a first expression vector comprising a first nucleic acid encoding the first heavy chain of claim 1;

b) a second expression vector comprising a second nucleic acid encoding the second heavy chain of claim 1; and

c) a third expression vector comprising a third nucleic acid encoding the light chain.

17. A host cell comprising the nucleic acid composition of claim 15.

18. A host cell comprising the nucleic acid consisting of claim 16.

19. A method of making a heterodimeric antibody according to any of claims 1 to 14 comprising:

a) providing a first expression vector comprising a first nucleic acid encoding a first heavy chain comprising:

i) a first Fc domain;

ii) a first variable heavy chain; and

b) providing a second expression vector comprising a second nucleic acid encoding a second heavy chain comprising:

i) a first Fc domain; and

ii) a single chain Fv region (scFv) that binds CD 3;

and

c) providing a third expression vector comprising a nucleic acid comprising a light chain;

wherein the variable light chain domains of the first variable heavy chain and the light chain bind CD 38;

d) wherein the first expression vector, the second expression vector, and the third expression vector are transfected into a host cell in a ratio selected from the group consisting of: 1:1.5:1.5, 1:2:1.5, 1:0.667:2, 1:1:2, 1:1.5:2, and 1:2: 2;

e) expressing the first, second and third expression vectors in the host cell, producing a first, second and third amino acid sequence, respectively, whereby the first, second and third amino acid sequences form the heterodimeric antibody.

20. A method of treating a patient in need thereof by administering a heterodimeric antibody according to any one of claims 1 to 14.

Technical Field

The present invention describes novel immunoglobulin compositions for co-conjugating antigens simultaneously, wherein both antigens are monovalent. The novel immunoglobulins described preferably utilize a heterodimeric Fc region. Also described herein are methods of using the novel immunoglobulin compositions, particularly for therapeutic purposes.

Background

Antibody-based therapeutics have been successfully used to treat a variety of diseases, including cancer and autoimmune/inflammatory disorders. However, there is still a need for improvements to such drugs, in particular to enhance their clinical efficacy. One approach being investigated is to engineer additional novel antigen binding sites into antibody-based drugs, thereby allowing a single immunoglobulin molecule to co-engage two different antigens. Such non-native or alternative antibody formats that join two different antigens are often referred to as bispecific antibodies. Because of the considerable diversity of antibody variable regions (Fv) that makes it possible to produce Fv's that recognize virtually any molecule, a typical approach to generating bispecific antibodies is to introduce new variable regions into the antibody.

A variety of alternative antibody formats have been investigated for bispecific antibody targeting (Chames &, 2009, mAbs1[6 ]: 1-9; Holliger & Hudson, 2005, Nature Biotechnology 23[9 ]: 1126-. Initially, bispecific antibodies were prepared by fusing two cell lines each producing a single monoclonal antibody (Milstein et al, 1983, Nature 305: 537-540). Although the resulting hybrid hybridoma or quadroma does produce bispecific antibodies, it is a very small population and requires sufficient purification to isolate the desired antibody. An engineering approach to this is to use antibody fragments to generate bispecific antibodies. Because these fragments lack the complex quaternary structure of the full-length antibody, variable light and heavy chains can be linked in a single genetic construct. Many different forms of antibody fragments have been generated, including bifunctional antibodies, tandem scFv and Fab2 bispecific antibodies (Chames & Baty, 2009, mAbs1[6 ]: 1-9; Holliger & Hudson, 2005, Nature Biotechnology 23[9 ]: 1126-1136; expressly incorporated herein by reference). Although these forms can be expressed at high levels in bacteria and may have beneficial osmotic benefits due to their small size, they are rapidly cleared in vivo and may present manufacturing challenges related to their yield and stability. The main reasons for these disadvantages are that antibody fragments typically lack antibody constant regions and their associated functional properties, including larger size, high stability, and binding to various Fc receptors and ligands to maintain a longer serum half-life (i.e., the neonatal Fc receptor FcRn) or to serve as binding sites for purification (i.e., protein a and protein G).

Recent studies have attempted to address the shortcomings of fragment-based bispecific antibodies by Engineering dual binding into full-length antibody-like forms (Wu et al, 2007, Nature Biotechnology 25[11 ]: 1290-1297; USSN12/477,711; Michaelson et al, 2009, mAbs1[ 2 ]: 128-141; PCT/US 2008/074693; Zuo et al, 2000, Protein Engineering 13[5 ]: 361-367; USSN09/865, 198; Shen et al, 2006, Jbiol Chem 281[16 ]: 10706-10714; L u et al, 2005, J Biol Chem 280[20 ]: 19665-19672; PCT/US 2005/025472; and Kontermann, MAbs 4(2), 182, all expressly incorporated herein) these bispecific antibody-like forms contain a bivalent binding site for the same heavy Fc region, which makes it common for the construction of these bivalent antibody fragments to bind to a bivalent Fc region, which is important as a result of the bivalent binding of these two antigen-like forms.

For many antigens that are of interest as common targets in the therapeutic bispecific antibody format, the desired binding is monovalent rather than bivalent for many immune receptors, cell activation is achieved by cross-linking of monovalent binding interactions the cross-linking mechanism is typically mediated by antibody/antigen immune complexes, or via effector cell-targeted cell conjugation, for example, low affinity Fc γ receptors (Fc γ R), such as Fc γ RIIa, Fc γ RIIb and Fc γ RIIIa binding monovalently to the antibody Fc region monovalent binding fails to activate cells expressing these Fc γ R, however, upon formation of immune complexes or cell-to-cell contact, receptor cross-linking occurs and clustering on the cell surface, causing activation, for receptors responsible for mediating cell killing, such as Fc γ RIIb on Natural Killer (NK) cells, receptor cross-linking and cell activation occur when effector cells engage target cells in a particularly desired manner, such as Fc γ riwsha on Natural Killer (NK) cells, receptor cross-linking occurs when effector cells engage target cells in a particularly desired manner, and cell-receptor binding occurs in a particularly upon a desired manner of effector cells binding to target cells, such as effector cells, receptor binding to antibody receptor antagonist 3, receptor antagonist cells, preferably binding to human cells, receptor binding to antibody receptor antagonist 3, receptor antagonist, receptor binding to human cells, receptor binding to antibody receptor antagonist 3, receptor antagonist, receptor binding to antibody receptor antagonist, receptor binding to human cells, receptor binding to antibody receptor binding to human cells, receptor binding to antibody receptor to human cells, antibody receptor to cell binding to antibody receptor to cell binding to cell type No. 35, antibody receptor to cell type No. 35, antibody to cell-9, receptor to cell binding to antibody receptor to cell binding to antibody receptor to cell binding to antibody receptor to antibody to cell type 35, antibody receptor to antibody receptor to antibody receptor.

CD38, also known as cyclic adenosine diphosphate ribohydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain in hematopoietic cells, a classification of functional roles is thought to be the result of CD 38-mediated signaling, including lymphocyte proliferation, cytokine release, regulation of B-cell and myeloid cell development and survival, and induction of dendritic cell maturation CD38 is dysregulated in many hematopoietic malignancies, including non-Hodgkin's lymphoma, NH L, Burkitt's lymphoma, B L, Multiple Myeloma (MM), B-chronic lymphocytic leukemia (B-C LL), B and T acute peyer's disease leukemia (a LL), T-cell lymphoma (TC 4), acute leukemia (AM L), a form of chronic lymphocytic leukemia (B-C LL), MM and T acute peyer's lymphoma (a LL), T-cell lymphoma (TC 638652), and more recent years of development of hematopoietic stem cell leukemia (CD 639), as well as a prognostic approach, CD 639, CD 6857-expressing a chronic myelogenous leukemia (MM) is a more recently developed as a cancer-resistant to the development of these cancers.

Thus, despite the biophysical and pharmacokinetic hurdles presented by bispecific antibodies produced from antibody fragments, a disadvantage of bispecific antibodies constructed using full length-like antibody formats is that they multivalently engage a common target antigen in the absence of a primary target antigen, thereby causing nonspecific activation and potential toxicity. The present invention solves this problem by introducing a novel form of bi-specific antibody capable of co-conjugating different target antigens.

Summary of The Invention

Accordingly, the present invention provides heterodimeric antibodies that bind to CD3 and CD 38. These heterodimeric antibodies comprise a first heavy chain comprising a first variable Fc domain and a single chain Fv region (scFv) that binds CD 3. These heterodimeric antibodies further comprise a second heavy chain comprising a second Fc variable Fc domain and a first variable heavy chain domain. These heterodimeric antibodies further comprise a first light chain comprising a first variable light chain domain and a second constant light chain domain, wherein the first variable heavy chain domain and the first variable light chain domain bind to CD 38.

In another aspect, the invention provides a heterodimeric antibody selected from the group consisting of: XENP 13243; XENP 13545; XENP 13546; XENP 13547; XENP 13548; XENP 13549; XENP 13550; XENP 13551; XENP 13544; XENP 13752; XENP 13753; XENP 13754; XENP 13756; XENP 13757; and XENP 13694.

In A further aspect, the scFv has A sequence comprising vhCDR1 having the sequence T-Y-A-M-XaA1, wherein XaA1 is N, S or H (SEQ ID NO:435), vhCDR2 having the sequence R-I-R-S-K-XaA1-N-XaA2-Y-A-T-XaA 3-Y-A-XaA4-S-V-K-G, wherein XaA1 is Y or A, XaA2 is N or S, XaA3 is Y or A and XaA3 is D or A (SEQ ID NO:436), or vDR 3 having the sequence H-G-N-F-G-XaA 3-S-Y-S-W-F-XaA3-Y, wherein XaA3 is 3 or XaA 3V-3 is V-3 or XaA 3-3H-XaA 3-X-Y-V-X-V-F-3-X3-Y-X3-Y, wherein XaA is V-XaA3 or XaA 3-3-X3 is V-3 or XaA 3-3H-3 or XaA 3-3H-3-V (SEQ ID, or XaA-3-X-V-3-X-or A-3-V-3-V-X-3-X-.

In another aspect, the scFv is selected from the group consisting of H1.30_ L1.47, H1.33_ L1.47.47, and H1.31_ L1.47.47.

In another aspect, the anti-CD 3 variable region has a sequence selected from the group consisting of seq id no:

a) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

b) comprises a polypeptide having the sequence shown in SEQ ID NO:412, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

c) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:414, having seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

d) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:417 vhCDR3, having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

e) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:418, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

f) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:421 vlCDR1, having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

g) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:422, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

h) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ id NO:427 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

i) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:428 and a vl cdr2 having SEQ ID NO:430, vlCDR 3;

j) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:431 for vlCDR 3;

k) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

1) comprises a polypeptide having the sequence shown in SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:423, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:432 of the vlCDR 3;

m) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:424, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:432 of the vlCDR 3;

n) comprises a polypeptide having the sequence of SEQ ID NO:412, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:417 vhCDR3, having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

o) comprises a polypeptide having the sequence of SEQ ID NO:412, vhCDR1 having the sequence of SEQ ID NO:414, having seq id NO:419 of vhCDR3, having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

p) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:415, a vhCDR2 having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

q) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:415, a vhCDR2 having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

r) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:417 vhCDR3, having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

s) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:419 of vhCDR3, having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, vlCDR 3;

t) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:417 vhCDR3, having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:433, the sequence of vlCDR 3;

u) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:413, having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:433, the sequence of vlCDR 3; and

v) comprises a polypeptide having the sequence of SEQ ID NO:411, vhCDR1 having the sequence of SEQ ID NO:434, a vhCDR2 having the sequence of seq id NO:416, a vhCDR3 having the sequence of SEQ ID NO:420, vl cdr1 having SEQ ID NO:425 and a vl cdr2 having the sequence of SEQ ID NO:430, and a vlCDR 3.

In another aspect, the anti-CD 3 variable region comprises a variable heavy chain region and a variable light chain region selected from the group consisting of:

SEQ ID NO:5 and 6; SEQ ID NO: 9 and 10; SEQ ID NO: 13 and 14; SEQ ID NO: 17 and 18; SEQ ID NO: 21 and 22; SEQ ID NO: 25 and 26; SEQ ID NO: 29 and 30; SEQ ID NO: 33 and 34; SEQ ID NO:37 and 38; SEQ ID NO:41 and 42; SEQ ID NO: 45 and 46; SEQ ID NO: 49 and 50; SEQ ID NO:53 and 54; SEQ ID NO: 57 and 58; SEQ ID NO: 61 and 62; SEQ ID NO: 65 and 66; SEQ ID NO: 69 and 70; SEQ ID NO: 73 and 74; SEQ ID NO: 77 and 78; SEQ ID NO: 81 and 82; SEQ ID NO: 85 and 86; SEQ ID NO: 89 and 90; SEQ ID NO: 93 and 94; SEQ ID NO: 97 and 98; SEQ ID NO: 101 and 102; SEQ ID NO: 105 and 106; SEQ ID NO: 109 and 110; SEQ ID NO: 113 and 114; SEQ ID NO: 117 and 118; SEQ ID NO: 121 and 122; SEQ ID NO: 125 and 126; SEQ ID NO: 129 and 130; SEQ ID NO: 133 and 134; SEQ ID NO: 137 and 138; SEQ ID NO: 141 and 142; SEQ ID NO: 145 and 146; SEQ ID NO:149 and 150; SEQ ID NO: 153 and 154; SEQ ID NO: 157 and 158; SEQ ID NO: 161 and 162; SEQ ID NO: 165 and 166; SEQ ID NO: 169 and 170; SEQ ID NO: 173 and 174; SEQ ID NO: 177 and 178; SEQ ID NO: 181 and 182; SEQ ID NO: 185 and 186; SEQ ID NO: 189 and 190; SEQ ID NO: 193 and 194; SEQ ID NO: 197 and 198; SEQ ID NO: 201 and 202; SEQ ID NO: 205 and 206; SEQ ID NO: 209 and 210; SEQ ID NO: 213 and 214; SEQ ID NO: 217 and 218; SEQ ID NO: 221 and 222; SEQ ID NO:225 and 226; SEQ ID NO: 229 and 230; SEQ ID NO: 233 and 234; SEQ ID NO: 237 and 238; SEQ ID NO: 241 and 242; SEQ ID NO: 245 and 246; SEQ ID NO: 249 and 250; SEQ ID NO: 253 and 254; SEQ ID NO: 257 and 258; SEQ ID NO: 261 and 262; SEQ ID NO: 265 and 266; SEQ ID NO: 269 and 270; SEQ ID NO: 273 and 274; SEQ ID NO: 277 and 278; SEQ ID NO: 281 and 282; SEQ ID NO: 285 and 286; SEQ ID NO: 289 and 290; SEQ ID NO: 293 and 294; SEQ ID NO: 297 and 298; SEQ ID NO:301 and 302; SEQ ID NO: 305 and 306; SEQ ID NO: 309 and 310; SEQ ID NO: 313 and 314; SEQ ID NO: 317 and 318; SEQ ID NO: 321 and 322; SEQ ID NO: 325 and 326; SEQ ID NO: 329 and 330; SEQ ID NO: 333 and 334; SEQ ID NO: 337 and 338; SEQ ID NO:34 l and 342; SEQ ID NO: 345 and 346; SEQ ID NO: 349 and 350; SEQ ID NO: 353 and 354; SEQ ID NO: 357 and 358; SEQ ID NO: 361 and 362; SEQ ID NO: 365 and 366; SEQ ID NO: 369 and 370; SEQ ID NO: 373 and 374; SEQ ID NO:377 and 378; SEQ ID NO: 381 and 382; SEQ ID NO: 385 and 386; SEQ ID NO: 389 and 390; SEQ ID NO: 393 and 394; SEQ ID NO: 397 and 398; SEQ ID NO: 401 and 402; SEQ ID NO: 405 and 406; SEQ ID NO: 409 and 410.

In another aspect, the heterodimeric antibody has a first variable heavy chain domain and a first variable light chain domain selected from the pair consisting of H1 and L1, H1 and L1.24.24, H1 and L1.96.96, H1.77 and L1.96, H1.77 and L1.97, H1.72 and L1.97, H1.71 and L1.96, and H1.77 and L1.24.

In another aspect, the invention provides a heterodimeric antibody as described above, wherein the scFv has a charged scFv linker. The charged scFv linker may have 3 to 8 positive charges and is selected from the group consisting of seq id NO: 443 to 451.

In another aspect, the scFv has a sequence selected from the group consisting of seq id nos: SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO:32, a first step of removing the first layer; SEQ ID NO: 36; SEQ ID NO: 40; SEQ ID NO: 44; SEQ ID NO: 48; SEQ ID NO: 52; SEQ ID NO: 56; SEQ ID NO: 60, adding a solvent to the mixture; SEQ ID NO: 64; SEQ ID NO: 68; SEQ ID NO: 72; SEQ ID NO: 76; SEQ ID NO: 80; SEQ ID NO: 84; SEQ ID NO: 88; SEQ ID NO: 92; SEQ ID NO: 96; SEQ ID NO: 100, respectively; SEQ ID NO: 104; SEQ ID NO: 108; SEQ ID NO: 112, a first electrode; SEQ ID NO: 116; SEQ ID NO: 120 of a solvent; SEQ ID NO: 124; SEQ ID NO: 128; SEQ ID NO: 132; SEQ ID NO: 136; SEQ ID NO: 140 of a solvent; SEQ ID NO: 144, 144; SEQ ID NO: 148; SEQ ID NO: 152; SEQ ID NO: 156; SEQ ID NO: 160; SEQ ID NO: 164; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 176; SEQ ID NO: 180 of the total weight of the composition; SEQ ID NO: 184, a first electrode; SEQ ID NO: 188; SEQ ID NO: 192; SEQ ID NO: 196 parts by weight; SEQ ID NO: 200 of a carrier; SEQ ID NO: 204; SEQ ID NO: 208; SEQ ID NO: 212; SEQ ID NO: 216; SEQ ID NO: 220, 220; SEQ ID NO: 224; SEQ ID NO:228, and (b); SEQ ID NO: 232; SEQ ID NO: 236; SEQ ID NO: 240; SEQ ID NO: 244; SEQ ID NO: 248; SEQ ID NO: 252; SEQ ID NO: 256 of; SEQ ID NO: 260 of a nitrogen atom; SEQ ID NO: 264; SEQ ID NO: 268; SEQ ID NO: 272; SEQ ID NO: 276; SEQ ID NO: 280 parts of; SEQ ID NO: 284; SEQ ID NO: 288; SEQ ID NO: 292; SEQ ID NO: 296; SEQ ID NO: 300, respectively; SEQ ID NO: 304; SEQ ID NO: 308; SEQ ID NO: 312; SEQ ID NO: 316; SEQ ID NO: 320, a first step of mixing; SEQ ID NO: 324, respectively; SEQ ID NO: 328; SEQ ID NO: 332; SEQ ID NO: 336; SEQ ID NO: 340, respectively; SEQ ID NO: 344; SEQ ID NO: 348; SEQ ID NO: 352; SEQ ID NO: 356; SEQ ID NO: 360; SEQ ID NO: 364; SEQ ID NO: 368; SEQ ID NO: 372; SEQ ID NO: 376; SEQ ID NO: 380 of the raw material; SEQ ID NO: 384; SEQ ID NO: 388; SEQ ID NO: 392; SEQ ID NO: 396; SEQ ID NO: 400, respectively; SEQ ID NO: 404; SEQ ID NO: 408.

in another aspect, the heavy chain Fc region further comprises an FcRn variant, including, but not limited to, 428L/434S.

In another aspect, the present invention provides a nucleic acid composition comprising: a first nucleic acid encoding a first heavy chain comprising an Fc region and an scFv that binds to CD 3; a second nucleic acid encoding a second heavy chain comprising a constant heavy chain and a variable heavy chain; and a third nucleic acid encoding a light chain, wherein the Fv regions of the second heavy and light chains bind CD 38. These nucleic acids may be in different expression vectors, or in the same expression vector. The invention provides host cells comprising the nucleic acid compositions.

In another aspect, the invention provides methods of making the heterodimeric antibodies of the invention, the methods comprising: providing a first expression vector comprising a first nucleic acid encoding a first heavy chain comprising a first Fc domain and a first variable heavy chain; providing a second expression vector comprising a second nucleic acid encoding a second heavy chain comprising a first Fc domain and a single chain Fv region (scFv) that binds CD 3; and providing a third expression vector comprising a nucleic acid comprising a light chain; wherein the variable light chain domains of the first variable heavy chain and the light chain bind CD 38. The first expression vector, the second expression vector and the third expression vector are transfected into the host cell at a ratio selected from the group consisting of: 1:1.5, 1:2:1.5, 1:0.667:2, 1:2, 1:1.5:2 and 1: 2. Expressing a first expression vector, a second expression vector, and a third expression vector in a host cell to produce a first amino acid sequence, a second amino acid sequence, and a third amino acid sequence, respectively, whereby the first amino acid sequence, the second amino acid sequence, and the third amino acid sequence form a heterodimeric antibody.

In another aspect, the invention provides a method of treating a patient in need thereof by administering a heterodimeric antibody according to the invention.

Brief description of the drawings

Fig. 1A and 1B depict the sequence of human CD 38. Fig. 1A depicts the full-length sequence, and fig. 1B depicts the extracellular domain.

Figure 2 depicts the human CD3 sequence.

Figures 3A to 3YY depict the amino acid sequences of the stability optimized humanized anti-CD 3 variant scFv, variable heavy chain sequence, and variable light chain sequence. (additionally, it should be noted that for ease of purification, the first sequence is in the form of a histidine tag). The CDRs are underlined. It is understood that the increased stability of the optimized variable and optimized light chains (as well as the scFv chains) can be attributed to the framework regions as well as the CDRs. Thus, it is to be understood that the disclosed complete variable regions include the disclosed framework regions, although the framework regions are not independently numbered. Furthermore, scFv linkers are shown in grey. Each scFv linker can be replaced with a charged scFv linker as depicted in figure 5. That is, any charged scFv linker, whether positively or negatively charged, including those depicted in fig. 5, can be substituted for the regions highlighted in fig. 3A through 3 YY.

Figures 4A to 4I depict the combination of all CD3vhCDR1-3 and vlCDR1-3 sequences with common CDRs useful in the present invention.

Figure 5 depicts suitable positively and negatively charged scFv linkers. Single prior art scFv linkers with a single charge were identified by Whitlow et al, Protein Engineering 6 (8): 989. sup. 995(1993) is referred to as "Whitlow". It should be noted that this linker is used in scFv to reduce aggregation and enhance proteolytic stability.

6A, 6B, 6C and 6D depict novel steric variants it will be appreciated by those skilled in the art that the first column of each table represents the "corresponding" pair of monomers, that is, monomer 1 has 405A and the corresponding steric variant is 394F. it is worth noting that in the case of the asymmetric tri-F form, either monomer may have either variant as long as a certain "chain type" is maintained, that is, the scFv monomer may be monomer 1 or monomer 2. additionally, these sets may optionally and independently be combined with other steric variants including charge pairs, homo-variants, isosteres, pI variants etc. in addition, "monomers" refers to Fc domains, that is, in the tri-F form, one monomer is a scFv construct and the other monomer is a Fab construct, although the fact that in fact there are two amino acid sequences that make up the Fab construct (heavy and light chains) shows many suitable steric variants or "bias (skew)" to be applicable in the present invention, that these steric variants may be used in combination with a number of steric variants (heavy and light chains) such as a6 depicts a number of steric variants that these steric variants would be present if the steric variants were found in combination with a single variant, for example a single variant of a single variant S, i.e.g. a substitution 3626, a substitution of a single variant would be considered in the case where a steric variant would be used in a steric variant, a steric variant would be indicated, a substitution of a substitution 3626, e.7A, a substitution would be considered in a substitution, a substitution of a steric variant, such a substitution of a substitution 3626, such variants would be considered in a substitution, such variants would be expected, such.

Figure 7 depicts a series of engineered heterodimer biased (e.g., "sterically heterodimerized") Fc variants along with heterodimer yield (as determined by HP L C-CIEX) and thermostability (as determined by DSC) — undetermined thermostability is expressed as "n.d.".

Figures 8A to 8c show bispecific immunoglobulins of the "triple F format". FIG. 8A shows scFv-Fc format. Figure 8C depicts a more standard bispecific antibody format, also utilizing pI variants of the invention (and optionally and independently other heterodimerization variants). Figure 8B shows the "tri F" format (also sometimes referred to as the "bottle opener" configuration, (and optionally and independently other heterodimerization variants). many of the embodiments listed herein have the anti-CD 3 component of the bispecific antibody as the scFv, and the anti-CD 38 component as the Fab fragment, although those skilled in the art will appreciate that these can be exchanged where the anti-CD 38 component is the scFv, optionally with a charged linker, and the Fv region of the anti-CD 3 sequence scFv herein is re-engineered into the Fab fragment.

Many, if not all, variants herein are KO variants that can be combined independently and optionally with the set depicted in fig. 9, as well as with any of the heterodimerization variants outlined herein, including steric variants and pI variants E233P/L V/L a/G236del may be combined with any other single or double variant in the list, furthermore, while in some embodiments it is preferred that two monomers contain the same KO variant, it is possible to combine different KO variants on different monomers, and only one monomer comprises the KO variant, see also the legend and the "excise" of USSN 61/913,870, all of which are expressly incorporated by reference in their entirety as if they were referred to as "knockout" or "excise" variants.

Figure 10 shows a series of engineered heterodimers biased towards Fc variants as well as heterodimer yields (as determined by HP L C-CIEX) and thermostability (as determined by DSC) — undetermined thermostability is expressed as "n.d.".

Figure 11. schematic diagram showing the structure of anti-CD 38 x anti-CD 3 bispecific molecule.

Figure 12 Surface Plasmon Resonance (SPR) data for affinity/stability engineered variant anti-CD 38 x anti-CD 3 bispecific molecules.

Figure 13 cytotoxicity (RTCC) assay of fluorescent L DH redirected T cells, showing killing of RPMI8226 multiple myeloma cells by anti-CD 28 x anti-CD 3 bispecific molecules.

Figure 14 RTCC assay (Annexin) V +) showing the killing effect of anti-CD 38 x anti-CD 3 bispecific molecules on RPMI8226 multiple myeloma cells. The ratio of T cells to RPMI8226 cells and the incubation time varied.

Figure 15 table listing the properties of affinity/stability engineered variant anti-CD 38 x anti-CD 3 bispecific molecules. Numbering is according to Kabat.

Figure 16 binding of anti-CD 38 x anti-CD 3 bispecific molecules to cynomolgus monkey CD20+ cells.

Figure 17 anti-CD 38 x anti-CD 3 bispecific molecules killed human plasma cells in SCID mice transplanted with hupmc a significant reduction in human IgG2 and IgE isotype was observed showing the mean ± sem.b L Q on day 22 is < 1 μ g/m L for IgG2 and < 16ng/m L for IgE-data points below B L Q are assigned B L Q values.

Figure 18 anti-CD 38 x anti-CD 3 bispecific molecules killed human plasma cells in SCID mice transplanted with hupmc a significant reduction in human IgM was observed showing mean ± sem at day 22B L Q for IgM was < 0.03 μ g/m L data points below B L Q are designated B L Q values.

Figure 19 anti-CD 38 x anti-CD 3 bispecific antibody cleared CD38+ CD138+ cells in MM PBMCs.

FIGS. 20A through 20Q show the sequences of CD 38X CD3scFv bottle openers of the present invention.

Fig. 21 shows variants of some useful Fc domains of CD 38X CD3 bottle openers.

Figures 22A and 22B depict the amino acid sequences of the anti-CD 38 x anti-CD 3 bispecific antibodies XENP13243 and XENP13551, in which the CDRs are underlined and charged linkers are present (the linkers can be uncharged or substituted with any other positively or negatively charged linker, see figure 7).

FIGS. 23A, 23B, 23C and 23D show DNA sequences encoding anti-CD 38 x anti-CD 3 bispecific antibodies XENP13243 and XENP 13551.

FIG. 24 shows a DNA transfection ratio table for the generation of stable pools of XENP13243 and XENP13551 the relative amounts of DNA transfected by HC-Fab, HC-scFv and L C are listed.

Figures 25A, 25B, 25C and 25D depict cation exchange chromatograms of material purified by protein a from stable pool supernatants collected after 7 days of batch culture of XENP13243 and XENP 13551. The DNA transfection ratios are as listed in figure 24. The integrated peak areas are indicated.

FIG. 26. overview of different protein species generated from the stable pool identified by the cation exchange chromatogram shown in FIG. 25. The DNA transfection rates are listed in FIG. 24.

Figure 27 pharmacokinetics of anti-CD 38 x anti-CD 3 bispecific antibodies XENP13243 and XENP13551 in C57B L/6 mice (n ═ 5 mice/group) half-life values calculated by non-atrioventricular analysis are noted in the legend.

FIG. 28 cytotoxicity of redirected T cells from CD38+ RPMI8226 cells assay consisted of 10,000 RPMI8226 cells incubated with 400,000 purified human T cells at 37 ℃ for 24 hours cytotoxicity was read by lactate dehydrogenase (L DH).

Figure 29 kinetics of binding of human and cynomolgus monkey CD38 and CD3 to XENP13243 and XENP13551 as determined by surface plasmon resonance. The assay was as described.

FIG. 30 XENP13243 (top panel) and XENP13551 (bottom panel) clear CD20-CD38+ cells in cynomolgus monkeys.

Figure 31.XENP13243 (top panel) and XENP13551 (bottom panel) upregulate CD69 in cynomolgus monkey CD8+ T cells.

Figure 32 depicts a list of isosteric variant antibody constant regions and their corresponding substitutions. pI _ (-) indicates a variant with lower pI, and pI _ (+) indicates a variant with higher pI. These variants can be optionally and independently combined with other heterodimerization variants of the invention.

Figure 33 shows some charged linkers and data for a particular anti-CD 3 scFv.

Figure 34 depicts schematics relating to the use of isolated variants (also referred to herein as "pI variants") and combinations with heterodimeric assembly variants (also referred to herein as "biased variants"). These variants can be used in a "plug and play" format, since the role of these variants is easily transferred to different antibodies with different Fv regions and are very stable.

FIG. 35 depicts optimization of the shared anti-CD 3 scFv-Fc. stability was increased in a number of ways, including replacement of rare amino acids, replacement of amino acids with unusual contact residues, linker engineering (for stability and improved purification, e.g., charged scFv linkers), and conversion to the V L-VH orientation.

Figure 36 depicts the use of Fc gene knockouts (or excision variants) that retain wild-type stability but remove all Fc γ R binding.

Fig. 37 depicts in vitro killing and stability data for anti-CD 3X anti-CD 38 bottle opener format.

Figure 38 depicts killing of human myeloma cell lines. XmAb13551 has high affinity for CD3, while XmAb13243 has lower affinity. Daratumumab (Daratumumab) is a bivalent monospecific antibody against CD 38.

Figure 39 shows the long half-life activity of the bottle opener bispecific antibody of the present invention in mice and the corresponding inhibition by human Ig.

Figure 40 shows anti-CD 38X anti-CD 3 function of XmAb13551 and XmAb13243, including clearance of monkey CD38+ cells in blood and bone marrow.

Figure 41 shows that CD38+ cell depletion is associated with T cell redistribution and activation.

Figure 42 shows the development of stable cell lines for the production of XmAb13551 (high CD3 affinity) and its corresponding yields.

FIG. 43 shows some cell culture optimizations performed to improve yield, where titers obtained were ≧ 3 g/L, and no significant difference in heterodimer/homodimer ratios was seen by scale-up.

Figure 44 shows the analysis results from the three-step manufacturing process, which produces very pure heterodimeric bispecific molecules in high yields, over 55%, and effectively removes homodimers, HMW and L MW contaminants, as well as HCP.

FIGS. 45A-45U show a variety of additional heterodimeric formats, any of which may include anti-CD 3 and anti-CD 38 sequences of the invention, FIGS. 45A-45U depict a variety of multispecific (e.g., heterodimeric) formats and combinations of different types of heterodimeric variants that may be used in the invention (these are sometimes referred to herein as "heterodimeric scaffolds"), further, it is noted that all of these formats may include additional variants in the Fc region (as discussed more fully below), including "excision" or "gene knockout" variants (FIG. 7), Fc variants that alter Fc γ R (Fc γ RIIb, Fc γ RIIIa, etc.) binding to Fc receptor, Fc variants that alter binding to Fc receptor, etc. FIG. 45A shows a double scFv-Fc format, identical to all heterodimeric formats herein, which may include heterodimeric variants, as depicted in pI variants, knob-and well as spatial variants or "biased" heavy chain variants ", charge pairs" as depicted in a small set of scFv, heavy chain-heavy chain pairs, and other variants, which may be derived from a single scFv I-scFv 45, scFv-CD-scFv-CD-scFv-CD-scFv-CD-scFv-CD-scFv, which may be used in-CD-scFv-CD-scFv-CD-scFv, which may be used in a single-scFv-CD-scFv-CD-scFv-CD-scFv-CD-scFv-CD-scFv, which may be used in a heavy-CD-scFv-CD-scFv, which may be used in a heavy-scFv, which is depicted in a heavy-scFv, which is depicted in a heavy.

46A, 46B, and 46C depict stability optimized humanized anti-CD 3 variant scFv substitutions are given relative to the H1.1_ L1.4.4 scFv sequence.

FIGS. 47A and 47B variable heavy and variable light chains of the anti-CD 3 sequences used in the present invention, including "stronger" and "weaker" binding sequences. It will be appreciated by those skilled in the art that these may be used in combination with any target tumor antigen binding domain in a Fab or scFv construct.

Figure 48 shows binding affinity in Biacore assay.

FIG. 49 shows heterodimer purity during generation of stable pools using different light chain, Fab-Fc and scFv-Fc ratios.

Figure 50 human IgM and IgG2 clearance of anti-CD 38 x anti-CD 3 bispecific antibodies in a hupmc mouse model.

Fig. 51A and 51B show purification of XENP13243 and XENP13551 designed for CD38 with low and high affinity, respectively.

Fig. 52A, 52B and 52C show binding to human and monkey CD38 and CD3, and Kd.

Fig. 53 shows killing of myeloma cells.

Fig. 54 shows that T cells are catenated killer cells, even when the number of target cells is large.

Figure 55 shows that Fc domains extend half-life.

Figure 56 shows the dose for the experiment of figure 14.

Figure 57 shows higher hlg clearance relative to darunavir.

Figure 58 shows the dose for the experiment of figure 16.

Fig. 59A and 59B bispecific antibodies cleared CD38+ cells in monkey blood and lymphoid organs.

FIGS. 60A, 60B and 60C. Fig. 60A (induced by blood redistribution), fig. 60B (induced by CD 69) and fig. 60C (cytokine release) show that CD38+ cell depletion is associated with T cell redistribution and activation.

Fig. 61 depicts several embodiments of specific applications of the present invention.

Detailed Description

I.Overview

The present invention is directed to novel constructs that provide bispecific antibodies that bind to both CD3 and CD38 antigens. One problem faced by antibody technology is the need for "bispecific" (and/or multispecific) antibodies that bind to two (or more) different antigens simultaneously, thereby generally enabling the different antigens to be accessible and to generate new functions and new therapies. Generally, these antibodies are produced by including genes for each of the heavy and light chains in the host cell. The desired heterodimer (A-B) and two homodimers (A-A and B-B) will thus generally be formed. However, one of the major difficulties in forming multispecific antibodies is that it is difficult to purify and separate heterodimeric antibodies from homodimeric antibodies, and/or heterodimer formation is favored over homodimeric formation.

The present invention is generally directed to the production of heterodimeric proteins, such as antibodies capable of co-engaging an antigen in several ways based on amino acid variants in the constant region that differ in each chain, thereby facilitating heterodimer formation and/or enabling heterodimers to be readily purified relative to homodimers.

Thus, the present invention is directed to novel immunoglobulin compositions that co-engage at least a first antigen and a second antigen. The first and second antigens of the invention are referred to herein as antigen-1 and antigen-2, respectively. One heavy chain of an antibody contains a single chain Fv ("scFv", as defined below), while the other heavy chain is in the form of a "conventional" Fab, comprising a variable heavy chain and a light chain. This structure is sometimes referred to herein as the "tri-F" format (scFv-FAb-Fc) or the "bottle opener" format because it looks substantially similar to a bottle opener (see figures). The two chains are joined together through the use of amino acid variants in the constant region (e.g., Fc domain and/or hinge region) that promote the formation of heterodimeric antibodies, as described more fully below.

The "three F" form of the present invention has several different benefits. As is known in the art, antibody analogs based on two scFv constructs typically present stability and aggregation problems that can be alleviated in the present invention by the addition of "conventional" heavy and light chain pairings. Furthermore, there is no problem of incorrect pairing of heavy and light chains (e.g. heavy chain 1 and light chain 2 are paired equally), as opposed to formats based on two heavy and two light chains.

Various mechanisms can be used to produce the heterodimers of the invention. Furthermore, those skilled in the art will understand and as described more fully below, that these mechanisms can be combined to ensure a high degree of heterodimerization.

One mechanism, generally referred to in the art as "knob and holes" (KIH), or sometimes referred to herein as "biased" variants, can also optionally be engineered with the referenced amino acids to create steric effects favoring heterodimer formation over homodimer formation; this is sometimes referred to as a "button and hole," as USSN 61/596,846; ridgway et al, Protein Engineering 9 (7): 617 (1996); atwell et al, j.mol.biol. 1997270: 26; described in U.S. patent No. 8,216,805, which is incorporated herein by reference in its entirety. The figures identify numerous "button and well" based "monomer a-monomer B" pairs. Furthermore, as in Merchant et al, Nature Biotech.16: 677(1998), these "knob and hole" mutations can be combined with disulfide bonds to bias heterodimerization.

Another mechanism for generating heterodimers is sometimes referred to as "electrostatic ligation" as described by Gunasekaran et al, j.biol.chem.285 (25): 19637(2010) (incorporated herein by reference in its entirety), which is sometimes referred to herein as "charge-pairing". in this embodiment, electrostatic agents are used to bias formation towards heterodimerization.

In the present invention, there are several basic mechanisms that can facilitate the purification of heterodimeric proteins, one based on the use of pI variants such that each monomer has a different pI, thereby allowing isoelectric purification of a-A, A-B and B-B dimer proteins. Alternatively, the "tri F" format also allows for separation based on size. As outlined further below, it is also possible to "bias" the formation of heterodimers relative to homodimers (as outlined generally below). Thus, combinations of spatially heterodimerized variants with pI or charge pair variants are particularly suitable for use in the present invention. In addition, as outlined more fully below, the scFv monomers in the trif format may include charged scFv linkers (either positively or negatively charged), thereby further enhancing the pI for purification purposes. It will be appreciated by those skilled in the art that some tri-F formats may use charged scFv linkers without additional pI adjustments, and that biased variants (and combinations of Fc, FcRn and KO variants) containing charged scFv linkers are indeed used with the present invention.

In the present invention, which utilizes pI as a separation mechanism to produce the heterodimeric trif form, amino acid variants can be introduced into one or both monomeric polypeptides; that is, the pI of one monomer (referred to herein simply as "monomer a") can be engineered differently than monomer B, or both monomer a and monomer B become charged, with the pI of monomer a increasing and the pI of monomer B decreasing. As outlined more fully below, the pI changes for either monomer or both monomers can be made by removing or adding a charged residue (e.g., a neutral amino acid is replaced with a positively or negatively charged amino acid residue, e.g., glycine is replaced with glutamic acid), changing a charged residue from positively or negatively charged to an opposite charge (aspartic acid to lysine), or changing a charged residue to a neutral residue (e.g., losing charge; lysine to serine). Many of these variations are shown in the drawings.

Thus, in this embodiment of the invention, provision is made to cause sufficient pI change in at least one monomer so that heterodimers can be separated from homodimers. It will be understood by those skilled in the art, and as discussed further below, this can be done by using a "wild-type" heavy chain constant region and a variant region engineered to increase or decrease its pI (wt a- + B or wtA-B), or by adding one region and decreasing the other region (a + -B-or a-B +). It should be noted that in this discussion it is not important which monomer comprises an scFv and which monomer comprises a Fab.

Thus, in general, one component of the invention is amino acid variants in the constant region of an antibody, which variants are directed to altering the isoelectric point (pI) of at least one, if not both, monomers of a dimeric protein by incorporating amino acid substitutions ("pI variants" or "pI substitutions") in one or both monomers, thereby forming "pI heterodimers" (when the protein is an antibody, these are referred to as "pI antibodies"). As shown herein, separation of heterodimers from two homodimers can be achieved if the pI of the two monomers differ by as little as 0.1 pH units, and pH unit differences of 0.2, 0.3, 0.4, and 0.5 or higher are all used in the present invention.

It will be appreciated by those skilled in the art that the number of pI variants included on one or both monomers to achieve good separation will depend in part on the starting pI of the scFv and Fab of interest. That is, to determine which monomer is undergoing engineering or in which "direction" (e.g., more positively or more negatively charged), the Fv sequences of the two target antigens are calculated and a decision is made therefrom. As is known in the art, different fvs will have different starting pIs, and this is used in the present invention. Generally, as outlined herein, the pI is engineered such that the total pI difference per monomer is at least about 0.1log, with 0.2 to 0.5 being preferred (as outlined herein).

In addition, it will be understood by those skilled in the art and as outlined herein that heterodimers can be separated from homodimers based on size. For example, as shown in fig. 8, a heterodimer with two scfvs (fig. 8A) can be separated from a heterodimer in the "tri F" form (fig. 8B) and a bispecific mAb (fig. 8C). This can be further used in higher valency states, where additional antigen binding sites are utilized. For example, as additionally shown, one monomer will have two Fab fragments and the other will have one scFv, thereby creating size and molecular weight differences.

Furthermore, it will be understood by those skilled in the art and as outlined herein that the forms outlined herein may also be extended to provide tri-and tetra-specific antibodies. In this embodiment (some of which variations are depicted in the figures), it will be appreciated that some antigens may be bivalent binding (e.g., two antigen binding sites are directed to a single antigen; e.g., A and B may be part of a typical bivalent association and C and D may optionally be present and optionally be the same or different). It is understood that any combination of Fab and scFv can be utilized to achieve the desired results and combinations.

In the case of heterodimerization using pI variants, a modular approach is provided to design and purify multispecific proteins, including antibodies, by using heavy chain constant regions. Thus, in some embodiments, heterodimeric variants (including biased and purified heterodimeric variants) are not included in the variable region, and thus, each individual antibody must be engineered. Furthermore, in some embodiments, the pI is altered by importing pI variants from different IgG isotypes without introducing significant immunogenicity, thereby resulting in a significantly reduced likelihood of immunogenicity arising from the pI variants. Thus, another problem to be solved is to address low pI constant domains with high human sequence content, e.g., minimizing or avoiding non-human residues at any particular position.

In one embodiment, the heterodimeric antibody is monovalent for engagement of one antigen using scFv and monovalent for engagement of another antigen using FAb. This form may also vary, as outlined below; in some embodiments, all three antigens are monovalent engagements, one antigen is a bivalent engagement and the other antigen is a monovalent engagement (e.g., a and C engage the same antigen, and B engages a different antigen), and so forth.

In addition, the side benefits that can be derived from this pI engineering are prolonged serum half-life and increased FcRn binding. That is, as described in USSN 13/194,904 (incorporated in its entirety by reference), decreasing the pI of antibody constant domains, including those found in antibodies and Fc fusions, can prolong serum retention in vivo. These pI variants used to increase serum half-life also help to alter pI to achieve purification.

In addition, additional amino acid variants can be introduced into the bispecific antibodies of the invention to add additional functionality, as outlined herein. For example, amino acid changes within the Fc region can be added (to one monomer or two monomers) to facilitate an increase in ADCC or CDC (e.g., change binding to fey receptors); allowing or increasing the yield of toxin and drug addition (e.g. for ADC); as well as increasing binding to FcRn, and/or increasing the serum half-life of the resulting molecule. As further described herein and as will be understood by one of skill in the art, any and all of the variants outlined herein can be optionally and independently combined with other variants.

Similarly, another class of functional variants is "Fc γ excision variant" or "Fc knock-out (FcKO or KO) variant". In these embodiments, for some therapeutic applications, it is desirable to reduce or eliminate normal binding of the Fc domain to one or more or all of the Fc γ receptors (e.g., Fc γ R1, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, etc.) to avoid additional mechanisms of action. That is, for example, in many embodiments it is generally desirable to cleave Fc γ RIIIa binding to eliminate or significantly reduce ADCC activity.

In addition, the invention provides novel humanized anti-CD 3 sequences, including CDR sets, complete variable light and heavy chains, and linked scfvs, which may optionally include charged scFv linkers. These optimized sequences used may be in other antibody formats.

The invention additionally provides novel anti-CD 38 sequences, including CDR sets, complete variable light and heavy chains, and linked scfvs, which may optionally include charged scFv linkers. These optimized sequences used may be in other antibody formats.

Thus, the present invention provides novel constructs capable of generating bispecific, bivalent antibodies that bind to both CD3 and CD 38.

Thus, in some embodiments, the antibody constructs provided herein comprise an anti-CD 3 antigen-binding domain that binds "strong" or "high affinity" to CD3 (e.g., one example is the heavy and light chain variable domains depicted as H1.30_ L1.47 (optionally including charged linkers, as appropriate)).

II.Definition of

In order to provide a more complete understanding of the present application, several definitions are set forth below. These definitions are intended to cover grammatical equivalents.

"excision" herein means a reduction or removal of activity. Thus, for example, "excise Fc γ R binding" means that the Fc region amino acid variant has less than 50% initial binding, with preferably less than 70%, 80%, 90%, 95%, 98% loss of activity, and generally, activity is below detectable binding levels in a Biacore assay, as compared to an Fc region without the particular variant. When a resection variant (also sometimes referred to herein as an "Fc γ R resection variant," "Fc knockout" ("FcKO") variant, or "knockout" ("KO") variant) is used, variants having particular application are those depicted in fig. 35.

As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" means a cell-mediated reaction that contemplates that nonspecific cytotoxic cells expressing Fc γ R recognize bound antibody on target cells and subsequently lyse the target cells. ADCC is associated with binding to Fc γ RIIIa; increasing binding to Fc γ RIIIa results in an increase in ADCC activity.

As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis means a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc γ R recognize bound antibodies on target cells and subsequently phagocytose the target cells.

"modification" means herein amino acid substitutions, insertions and/or deletions in the polypeptide sequence, or changes to a moiety chemically attached to a protein. For example, the modification may be a change in carbohydrate or PEG structure attached to the protein. "amino acid modification" means herein amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are typically directed to DNA-encoded amino acids, e.g., 20 amino acids with codons in DNA and RNA.

By "amino acid substitution" or "substitution" is meant herein that an amino acid at a particular position in a parent polypeptide sequence is replaced with a different amino acid. In particular, in some embodiments, the substitutions are for non-naturally occurring amino acids at a particular position that are not naturally occurring in an organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide in which the glutamic acid at position 272 is replaced with tyrosine, in this case an Fc variant. For clarity, a protein engineered to alter a nucleic acid coding sequence without altering the starting amino acid (e.g., CGG (encoding arginine) to CGA (still encoding arginine) to increase expression levels by the host organism) is not an "amino acid substitution"; that is, although a new gene encoding the same protein is produced, if the protein has the same amino acid at a specific position from which it is initiated, it is not an amino acid substitution.

As used herein, "amino acid insertion" or "insertion" means the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Additionally, -233ADE or a233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, G236-or G236# or G236del indicates a glycine deletion at position 236. In addition, EDA 233-or EDA233# indicates that the sequence GluAspAla was deleted from position 233. Similarly, some heterodimerization variants include "K447 del," meaning that the lysine at position 447 is deleted.

As used herein, "variant protein" or "protein variant" or "variant" means a protein which differs from the parent protein due to at least one amino acid modification the protein itself, a composition comprising the protein, or an amino acid sequence encoding the same, preferably the protein variant has at least one amino acid modification as compared to the parent protein, e.g., about one to about seventy amino acid modifications as compared to the parent protein, and preferably has about one to about five amino acid modifications, 737 is in some embodiments a human wild-type sequence, e.g., an Fc region from IgGl, IgG2, IgG3 or IgG4, as described below, although a human sequence having a variant may also serve as a "parent polypeptide" -the protein variant sequence herein preferably has at least about 80% identity, and most preferably at least about 90% identity, more preferably at least about 95%, 98%, 99% identity, as compared to the parent protein sequence of the protein sequence of which is referred to herein as the amino acid substitution of the amino acid sequence of the parent protein, which is referred to the amino acid modification of the amino acid sequence of the protein itself, which is referred to the amino acid modification of the amino acid sequence of the amino acid of the parent protein, as described herein, or which is referred to the invention in the invention, e.g. the invention, which is referred to the invention as the invention, the invention is referred to which is referred to the invention, the invention is preferably the invention, the invention is preferably the invention, the invention is characterized by the invention is not the invention is characterized by the invention, the invention is characterized by the invention.

As used herein, "protein" means herein at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides and peptides, peptidyl groups may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., "analogs," such as peptoids (see Simon et al, PNAS USA 89 (20): 9367(1992), incorporated in their entirety by reference.) it will be understood by those skilled in the art that amino acids may be naturally occurring or synthetic (e.g., amino acids not encoded by D NA.) for example, high phenylalanine, citrulline, ornithine and norleucine are considered synthetic amino acids for purposes of the present invention and may utilize amino acids in the D-and L- (R or S) configurations.

As used herein, "residue" means a position in a protein and the identity of its associated amino acid. For example, asparagine 297 (also known as Asn297 or N297) is the residue at position 297 in human antibody IgG 1.

As used herein, "Fab" or "Fab region" means a polypeptide comprising the VH, CH1, V L, and C L immunoglobulin domains Fab may refer to this region isolated, or in the context of a full-length antibody, an antibody fragment, or a Fab fusion protein, "Fv" or "Fv fragment" or "Fv region" as used herein means a polypeptide comprising the V L and VH domains of a single antibody.

As used herein, "IgG subclass modification" or "isotype modification" means an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, since at EU position 296 IgG1 comprises tyrosine and IgG2 comprises phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

As used herein, "non-naturally occurring modification" means an amino acid modification that is not of the same type. For example, since IgG does not contain a serine at position 434, substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or mixtures thereof) is considered to be a non-naturally occurring modification. "isotype" modification refers to the import of one isotype amino acid at one position into the backbone of a different isotype; for example, the IgG1 amino acids were imported into the same position in the IgG2 backbone.

As used herein, "amino acid" and "amino acid identity" mean one of the 20 naturally occurring amino acids encoded by DNA and RNA.

As used herein, "effector function" means a biochemical event caused by the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP and CDC.

As used herein, "IgG Fc ligand" means a molecule, preferably a polypeptide, in any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, Fc γ RI, Fc γ RII, Fc γ RIII, FcRn, C1q, C3, mannose binding lectin, mannose receptor, staphylococcal protein a, streptococcal protein G, and viral Fc γ R. Fc ligands also include Fc receptor homologs (FcRH), which are a group of Fc receptors homologous to Fc γ R (Davis et al, 2002, Immunological Reviews 190: 123-136, incorporated by reference in their entirety). Fc ligands may include molecules that bind Fc not found. Specific IgG Fc ligands are FcRn and Fc γ receptors. As used herein, "Fc ligand" means a molecule, preferably a polypeptide, bound to the Fc region of an antibody in any organism to form an Fc/Fc ligand complex.

As used herein, "Fc γ receptor" or "Fc γ R" means any member of a family of proteins that bind to the Fc region of IgG antibodies and are encoded by Fc γ R genes in humans, this family includes, but is not limited to, Fc γ RI (CD64), including isoforms Fc γ RIa, Fc γ RIb and Fc γ RIc, Fc γ RII (CD32), including isoforms Fc γ RIIa (including isoforms H131 and R131), Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc, and Fc γ RIII (CD16), including isoforms Fc γ RIIIa (including isoforms V158 and F158), and Fc γ RIIIb (including isoforms Fc γ RIIb-NA1 and Fc γ RIIb-NA2) (jefferfferis et al, 2002, Immunol L ett: 57-65, incorporated in their entirety by reference), and any undiscovered human Fc γ R or Fc γ R3525, including mouse Fc γ R358, mouse Fc γ R, rat Fc γ RI, mouse Fc γ RI (CD 358), mouse Fc γ RI (mouse Fc γ RI), and Fc γ RI (CD 16).

As used herein, "FcRn" or "neonatal Fc receptor" means a protein that binds to the Fc region of IgG antibodies and is at least partially encoded by an FcRn gene, FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys as is known in the art, a functional FcRn protein comprises two polypeptides, commonly referred to as heavy and light chains, the light chain is β -2-microglobulin and the heavy chain is encoded by an FcRn gene, FcRn or FcRn protein refers to a complex of the FcRn heavy chain and β -2-microglobulin unless otherwise specified herein FcRn variants are used to increase binding to FcRn receptors and in some cases, to increase serum half-life, Fc variants that increase binding to FcRn receptors and correspondingly increase serum half-life include, but not limited to, fcra, 434S, L, 308F, 428I, 39259/434S, 259I/308F, 436I/428I, or V/46s, 436V/254, Y/252, and Y/428 variants, or three of these may be in the form as shown in the figures, since each of these Fc variants may be three different monomers, or three, e.e.e.e.g., three.

As used herein, "parent polypeptide" means a starting polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered form of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding the same. Thus, as used herein, "parent immunoglobulin" means an unmodified immunoglobulin polypeptide that is modified to produce a variant, and "parent antibody" means an unmodified antibody that is modified to produce a variant antibody, as used herein. It should be noted that "parent antibody" includes known commercially available, recombinantly produced antibodies, as outlined below.

By "Fc fusion protein" or "immunoadhesion" is meant herein a protein comprising an Fc region typically linked (optionally via a linker moiety, as described herein) to a different protein, such as a binding moiety linked to a target protein (as described herein).

As used herein, "position" means the location in a protein sequence. Positions may be numbered sequentially or according to accepted formats, such as the EU index for antibody numbering.

In the context of the monomers of the heterodimeric proteins of the invention, "strand-type" is herein intended to mean that heterodimeric variants are incorporated into each monomer in a similar manner as two DNA strands that "match", thereby retaining the ability to "match" to form heterodimers. For example, if some pI variants are engineered to be monomeric a (e.g., to make the pI higher), then spatial variants ("charge pairs") that can be utilized do not interfere with the pI variants, e.g., the charge variants that make the pI higher are placed on the same "strand" or "monomer" to retain both functions.

As used herein, "target antigen" means a molecule to which the variable region of a given antigen specifically binds. The target antigen may be a protein, carbohydrate, lipid or other compound. Preferred target antigens of the invention are CD3 and CD 38.

As used herein, "target cell" means a cell that expresses a target antigen.

As used herein, "variable region" means a region of an immunoglobulin that comprises one or more Ig domains encoded substantially by vk, V λ and/or VH genes that constitute the kappa, λ and heavy chain immunoglobulin loci, respectively.

"wild-type or WT" as used herein means an amino acid sequence or a nucleotide sequence found in nature, including allelic variants. The WT protein has an amino acid sequence or a nucleotide sequence that is not intentionally modified.

As is well understood in the art, "single chain variable fragment," "scFv," or "single chain Fv" is herein intended to refer to a fusion protein of an antibody variable heavy and light chains, typically connected by a linker peptide. Typical scFv linkers are well known in the art, are generally 10 to 25 amino acids long and include glycine and serine.

By "charged scFv linker" is meant herein a scFv linker that utilizes charged amino acids to produce and purify heterodimeric antibodies comprising at least one scFv. Suitable charged scFv linkers are shown in the figures, although other linkers may be used. In general, compared to standard uncharged scFv linkers, as commonly used (GGGGS)_3-5The charged scFv linker used in the present invention has 3 to 8 charge changes (negative or positive) of 3 to 8(3, 4,5, 6,7 or 8 are all possible). Those skilled in the art will appreciate that heterodimeric antibodies utilizing two scfvs may have one charged linker and one neutral linker (e.g., a positively or negatively charged scFv linker) or two oppositely charged scFv linkers (one positively and one negatively charged).

Heterodimeric proteins

The present invention is directed to multispecific antibodies that produce multispecific, particularly bispecific binding proteins, and specifically, one monomer comprising an scFv and another monomer comprising an Fv. As discussed herein, many of the disclosed embodiments utilize scFv that bind CD3 and Fv (or Fab) that bind CD 38. Alternatively, vh and vl anti-CD 38 sequences herein may be used in scFv constructs wherein the anti-CD 3 monomer is a Fab.

Antibodies

Antibodies useful in the present invention can take a variety of forms as described herein, including conventional antibodies as well as antibody derivatives, fragments, and mimetics described below.

Conventional antibody building blocks typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light chain" (typically having a molecular weight of about 25 kDa) and one "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention is directed to the IgG class, which has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG 4. Thus, as used herein, "isotype" means any subclass of immunoglobulin defined according to the chemical and antigenic characteristics of the constant region. It is understood that the therapeutic antibody may also comprise a mixture of isotypes and/or subclasses. For example, as shown in U.S. publication 2009/0163699 (incorporated by reference), the present invention encompasses pI engineering of IgG1/G2 mixtures.

The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art as the "Fv domain" or "Fv region". In the variable region, the V domains of the heavy and light chains each aggregate into three loops, forming an antigen binding site. Each loop is called a complementarity determining region (hereinafter referred to as "CDR"), in which changes in amino acid sequence are most pronounced. "variable" refers to the fact that the sequences of certain segments of the variable region differ significantly between antibodies. The variations within the variable region are not evenly distributed. In contrast, the V region consists of relatively invariant elongated regions called Framework Regions (FR) of 15 to 30 amino acids separated by vastly varying shorter regions called "hypervariable regions" which are each 9 to 15 amino acids long or longer.

Each VH and V L is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, FR1-CDR1-FR2-CD R2-FR3-CDR3-FR4, arranged in that order from amino terminus to carboxy terminus.

Hypervariable regions generally encompass amino acid residues from about amino acid residues 24-34 (L CDR 1; "L" represents the light chain), 50-56 (L CDR2) and 89-97 (L CDR3) in the light chain variable region and about 31-35B (HCDR 1; "H" represents the heavy chain), 50-65(HCDR2) and 95-102(HCDR 3); Kabat et al, SEQ OF PROTEINS IMMUNO 6 OGICA L INTEREST, 5 th edition, Public Health Service, National Institutes OF Health, Bethesda, Md. (1991); and/or those residues that form hypervariable loops (e.g., residues 26-32 (L CDR1), 50-52 (L CDR2) and 91-96 (L CDR3), and those residues in the heavy chain variable region (e.g., CDR 26-32, 24-4653, 24 DR 4653, 24-55, 24H.; and 51-55H.; Bioshift loop-53 (Bioshift), described in the invention).

Throughout this specification, when referring to residues in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), the Kabat numbering system is generally used (e.g., Kabat et al, supra (1991)).

The CDRs help to form the antigen binding site, or more precisely, the epitope binding site, of the antibody. An "epitope" refers to a determinant that interacts with a particular antigen binding site in the variable region of an antibody molecule, referred to as an antigenic determinant. Epitopes are groups of molecules that generally have specific structural characteristics as well as specific charge characteristics, such as amino acid or sugar side chains, etc. A single antigen may have more than one epitope.

Epitopes can comprise amino acid residues that are directly involved in binding (also referred to as immunodominant components of the epitope) as well as other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a particular antigen binding peptide; in other words, the amino acid residues are within the scope of a particular antigen binding peptide.

The epitope can be a conformational epitope or a linear epitope. Conformational epitopes are created by spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. Conformational epitopes are distinguished from non-conformational epitopes in that binding to the former is lost in the presence of denaturing solvents, whereas binding to the latter is not.

Epitopes typically comprise at least 3 and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies recognizing the same epitope can be validated in a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to the target antigen, e.g., "binning".

Based on the degree OF conservation OF these SEQUENCES, it separates individual primary SEQUENCES into CDRs and frameworks, and lists them (see seq OF immu no L ogic L intest, 5 th edition, NIHpublication, No.91-3242, e.a. Kabat et al, incorporated by reference in their entirety).

In the IgG subclass of immunoglobulins, several immunoglobulin domains are present in the heavy chain. By "immunoglobulin (Ig) domain" is meant herein an immunoglobulin region having a distinct quaternary structure. Of interest in the present invention are heavy chain domains, including constant heavy Chain (CH) domains and hinge domains. In the case of IgG antibodies, the IgG isotype has three CH regions each. Thus, for IgG, the "CH" domains are as follows: "CH 1" refers to position 118 as in Kabat, indexed according to EU 220. "CH 2" refers to position 237 as in Kabat, according to the EU index and "CH 3" refers to position 341 as in Kabat, according to the EU index and 447. As shown herein and described below, pI variants can be in one or more CH regions, as well as hinge regions (as discussed below).

It should be noted that the sequences depicted herein begin with the CH1 region at position 118; variable regions are not included unless indicated. For example, according to EU numbering, SEQ ID NO:2, designated as position "1" in the sequence listing, corresponds to position 118 of the CH1 region.

Another class of Ig heavy chain domains is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" is meant herein a flexible polypeptide comprising amino acids between a first constant domain and a second constant domain of an antibody. The IgG CH1 domain structurally terminates at EU position 220 and the IgG CH2 domain begins at residue EU position 237. Thus, for IgG, an antibody hinge is defined herein to include positions 221 (D221 in IgG 1) to 236 (G236 in IgG 1), wherein the numbering is according to the EU index in Kabat. In some embodiments, for example in the case of an Fc region, a lower hinge region is included, wherein "lower hinge" generally refers to position 226 or 230. pI variants can also be produced in the hinge region, as shown herein.

The light chain typically comprises two domains, a variable light chain domain (containing the light chain CDRs and forming, with the variable heavy chain domain, the Fv region) and a constant light chain region (commonly referred to as C L or ck).

Another region of interest for additional substitutions outlined below is the Fc region. As used herein, "Fc" or "Fc region" or "Fc domain" means a polypeptide that comprises an antibody constant region but does not include a first constant region immunoglobulin domain and in some cases does not include a partial hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-termini of these domains. For IgA and IgM, Fc may comprise J chains. For IgG, the Fc domain comprises the immunoglobulin domains C γ 2 and C γ 3(C γ 2 and C γ 3) and a lower hinge region between C γ 1 (C γ 1) and C γ 2(C γ 2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined to include residues C226 or P230 to its carboxy terminus, where numbering is according to the Eu index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, for example to alter binding to one or more fcyr receptors or to FcRn receptors, as described more fully below.

In some embodiments, the antibodies are full-length. By "full-length antibody" is meant herein the structure that constitutes the native biological form of the antibody, including the variable and constant regions, including one or more modifications as outlined herein.

Alternatively, an antibody may comprise a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively.

Other antibody fragments that may be used include fragments comprising one or more of the CH1, CH2, CH3, hinge and C L domains of the invention that are engineered to produce a heterodimer, for example, an Fc fusion is one in which the Fc region (CH2 and CH3, optionally with a hinge region) is fused to another protein.

As used herein, "Fab" or "Fab region" means a polypeptide comprising VH, CH1, V L, and C L immunoglobulin domains.Fab may refer to this region isolated, or in the context of a full-length antibody, an antibody fragment, or a Fab fusion protein.

Chimeric and humanized antibodies

In some embodiments, the Antibodies may be a mixture of different species from, for example, chimeric and humanized Antibodies in general, "chimeric Antibodies" and "humanized Antibodies" refer to Antibodies that combine regions from more than one species in general, "humanized Antibodies" conventionally comprise variable regions from mouse (or in some cases rat) and constant regions from human beings in general "humanized Antibodies" refer to non-human Antibodies that are exchanged between variable domain framework regions and sequences found in human Antibodies in general in human Antibodies in which the entire Antibodies except for the variable domain framework regions are encoded by human-derived polynucleotides or are internal and external to the CDRs thereof in general in the human Antibodies in which a portion or the entire of these CDRs are encoded by nucleic acids derived from non-human organisms and are transplanted into the β -fold-laid-in framework of the human antibody variable regions to generate Antibodies whose specificity is determined by the generation of such Antibodies described in, for example, WO 92/11018, Jones, 1986, Nature 321: 522: 525, 19810, 19835, 19810, 19835, 19810, 10, 19810, 10, 19810, 10, 35, 92, 35, 92, 35, 92, 35, 92, 35, 92, 35, 92, 35, 92, 35.

Multispecific antibody constructs

It will be appreciated by those skilled in the art and as discussed more fully below, that the heterodimeric fusion proteins of the invention are in a variety of configurations, with the preferred embodiment shown in figure B being a "tri F" construct.

Heterodimeric heavy chain constant region

Thus, the present invention is based on the use of a monomer containing a variant heavy chain constant region as the first domain to provide a heterodimeric protein. "monomer" means herein one half of a heterodimeric protein. It should be noted that traditional antibodies are in fact tetramers (two heavy chains and two light chains). In the context of the present invention, a heavy chain-light chain pair (if applicable, e.g. if the monomer comprises a Fab) is considered to be a "monomer". Similarly, the heavy chain region comprising an scFv is considered a monomer. Essentially, each monomer contains enough heavy chain constant regions to allow heterodimerization engineering, whether for the entire constant region, e.g., Ch 1-hinge-Ch 2-Ch 3; an Fc region (CH2-CH 3); or just the CH3 domain.

The variant heavy chain constant region may comprise all or a portion of the heavy chain constant region, including the full length construct, CH 1-hinge-CH 2-CH3, or a portion thereof, including, for example, CH2-CH3 or CH3 alone. Furthermore, the heavy chain region of each monomer may be the same backbone (CH 1-hinge-CH 2-CH3 or CH2-CH3) or different backbones. Also included within this definition are N-terminal and C-terminal truncations and additions; for example, some pI variants include the addition of a charged amino acid at the C-terminus of the heavy chain domain.

Thus, in general, one monomer of the "tri F" constructs of the invention is (scFv region-hinge-Fc domain) and the other is (VH-CH 1-hinge-CH 2-CH3 plus the linked light chain), and heterodimerization variants, including steric and pI variants, Fc and FcRn variants, and additional antigen binding domains (optionally with linkers) included in these regions.

In addition to the heterodimerization variants outlined herein (e.g., steric variants and pI variants), the heavy chain region may contain additional amino acid substitutions, including changes that alter Fc γ R and FcRn binding as discussed below.

In addition, some monomers may utilize linkers for the scFv portion of the "bottle opener". In the trif format, one charged scFv linker is used. As described herein, a charged scFv linker can be either positively or negatively charged, depending on the intrinsic pI of an scFv for a target antigen and the intrinsic pI of a Fab for another target antigen. In the double scFv format, a single charged scFv linker is used on one monomer (positively or negatively charged) or on both monomers (one positively and one negatively charged). In this embodiment, the charges of each of the two linkers need not be the same (e.g., one is +3, and the other is-4, etc.).

In one embodiment, the anti-CD 3 antigen-binding site is an scFv, and includes a positively charged scFv linker. Alternatively, the anti-CD 38 antigen binding site may be an scFv of an "decapping" construct.

Heterodimerization variants include a variety of different types of variants, including, but not limited to, spatial variants (including charged variants) and pI variants, which can optionally and independently be combined with any other variant. In these embodiments, it is important that "monomer A" matches "monomer B"; that is, if the heterodimeric protein is based on steric and pI variants, then these need to be correctly matched to each monomer: for example, a working set of spatial variants (1 set on monomer a, 1 set on monomer B) is combined with a set of pI variants (1 set on monomer a, 1 set on monomer B), thereby allowing variants on each monomer to be designed to achieve the desired function. In case for example the steric variation can also change the charge, the correct set needs to match the correct monomer.

Notably, the heterodimerization variants outlined herein (e.g., including but not limited to those shown in the figures) can be optionally and independently combined with any other variant and on any other monomer. Thus, for example, pI variants from monomer 1 of one figure can be added to other heterodimerization variants of monomer 1 or from monomer 2 in a different figure. That is, it is important for heterodimerization that there are variant "sets", one set for one monomer and one set for another monomer. It does not matter whether these sets are from a combination of fig. 1 and 1 (e.g., monomer 1 list can match) or a transformation (pI variant of monomer 1 and spatial variant of monomer 2). However, as described herein, when combined as outlined above, the "chain type" should be preserved, thereby favoring heterodimerization; for example, a charged variant with increased pI should be used with an increased pI variant and/or scFv linker with increased pI, etc. In addition, for additional Fc variants (e.g., for fcyr binding, FcRn binding, cleavage variants, etc.), either monomer or both monomers may optionally and independently include any of the listed variants. In some cases, both monomers have additional variants and in some cases, only one monomer has additional variants, or these variants can be combined.

Heterodimerization variants

The present invention provides multispecific antibody formats, for example, on a "three F" or "bottle opener" scaffold as depicted in fig. 8B.

Space variant

In some embodiments, heterodimer formation may be facilitated by the addition of a steric variant. That is, by altering the amino acids in each heavy chain, the probability of different heavy chains associating to form a heterodimeric structure is higher than that of forming a homodimer with the same Fc amino acid sequence. Suitable spatial variants are shown in the figures.

One mechanism, generally referred to in the art as "knob and hole", can also optionally be engineered with the amino acids mentioned to produce steric effects favoring heterodimer formation over homodimer formation; this is sometimes referred to as a "button and hole," as USSN 61/596,846; ridgway et al, Protein Engineering 9 (7): 617 (1996); atwell et al, j.mol.biol. 1997270: 26; described in U.S. patent No. 8,216,805, which is incorporated herein by reference in its entirety. Figures 4 and 5 (described further below) identify numerous "button and well" based "monomer a-monomer B" pairs. Furthermore, as in Merchant et al, Nature Biotech.16: 677(1998), these "knob and hole" mutations can be combined with disulfide bonds to bias heterodimerization.

Another mechanism for generating heterodimers is sometimes referred to as electrostatic veneering, as described by Gunasekaran et al, j.biol.chem.285 (25): 19637(2010) (incorporated herein by reference in its entirety), which is sometimes referred to herein as "charge pair". in this embodiment, electrostatic agents are used to bias formation towards heterodimerization.

Additional monomer a and monomer B variants can optionally and independently be combined in any amount with other variants, such as pI variants outlined herein, or other spatial variants shown in fig. 37 of US 2012/0149876, the figures and legends of which are expressly incorporated herein by reference.

In some embodiments, the spatial variants outlined herein may optionally and independently be incorporated into one or both monomers with any heterodimerization variant including pI variants (or other variants, such as Fc variants, FcRn variants, excision variants, etc.).

pI (isoelectric point) variants of heterodimers

In general, it will be appreciated by those skilled in the art that there are two general classes of pI variants: variants that increase the pI of the protein (alkaline changes) and variants that decrease the pI of the protein (acidic changes). As described herein, all combinations of these variants can be performed: one monomer may be wild type or a variant with a pI that is not significantly different from wild type, and the other monomer may be more basic or more acidic. Alternatively, each monomer is varied, one being more basic and one more acidic.

Preferred pI variant combinations are shown in the figures.

Heavy chain acidic pI changes

Thus, when one monomer comprising a variant heavy chain constant domain is made more positive (e.g., the pI is reduced), one or more of the following substitutions may be made: S119E, K133E, K133Q, T164E, K205E, K205Q, N208D, K210E, K210Q, K274E, K320E, K322E, K326E, K334E, R355E, K392E, K447 deletion, addition of peptide DEDE at the c-terminus, G137E, N203D, K274Q, R355Q, K392N, and Q419E. As outlined herein and shown in the figures, these changes are shown with respect to IgG1, but all isotypes and mixtures of isotypes can be altered in this manner.

In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q may also be used.

Basic pI Change

Thus, when one monomer comprising a variant heavy chain constant domain is made more negative (e.g., increasing pI), one or more of the following substitutions may be made: Q196K, P217R, P228R, N276K and H435R. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes and mixtures of isotypes can be changed in this manner.

Antibody heterodimeric light chain variants

In the case of antibody-based heterodimers, pI variants may also be produced in the light chain, for example where at least one monomer comprises a light chain in addition to the heavy chain domain. Amino acid substitutions that reduce the pI of the light chain include, but are not limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E and the addition of peptide DEDE at the c-terminus of the light chain. Constant lambda light chain-based changes in this class include one or more substitutions in R108Q, Q124E, K126Q, N138D, K145T, and Q199E. In addition, the pI of the light chain can be increased.

Homotypic variants

In addition, many embodiments of the invention are based on the "import" of pI amino acids at specific positions in one IgG isotype into another isotype, thereby reducing or eliminating the possibility of unwanted immunogenicity being introduced into variants. That is, IgG1 is a common isotype of therapeutic antibody for a variety of reasons, including highly potent effector function. However, the pI of the heavy chain constant region of IgG1 was higher than that of IgG2(8.10 vs 7.31). By introducing residues of IgG2 at specific positions into the IgG1 backbone, the pI of the resulting monomer is reduced (or increased) and additionally exhibits a longer serum half-life. For example, IgG1 has glycine (pI 5.97) at position 137, whereas IgG2 has glutamic acid (pI 3.22); the input of glutamate will affect the pI of the resulting protein. As described below, multiple amino acid substitutions are typically required to significantly affect the pI of a variant antibody. However, it should be noted that even changes in the IgG2 molecule can increase serum half-life, as discussed below.

In other embodiments, non-homotypic amino acid changes are made to reduce the overall charge state of the resulting protein (e.g., by changing an amino acid with a higher pI to an amino acid with a lower pI), or to achieve structural adaptations for stability, etc., as described further below.

In addition, significant changes in each monomer of the heterodimer can be seen by pI engineering of the heavy and light chain constant domains. As discussed herein, the pI phase difference of at least 0.5 for the two monomers can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point.

Equal row variant

Furthermore, as shown in fig. 47, isosteric pI variants, e.g., charge variants approximately the same size as the parent amino acid, can be prepared.

Calculate pI

The pI of each monomer can depend on the pI of the variant heavy chain constant domain and the pI of the total monomer including the variant heavy chain constant domain and the fusion partner. Thus, in some embodiments, the change in pI is calculated based on the variant heavy chain constant domain using the table in the figures. Alternatively, the pI of each monomer can be compared. Similarly, the pI of the "starting" variable region (e.g., scFv or Fab) is calculated to understand which monomer will be engineered in which direction.

pI variants additionally conferring better FcRn binding in vivo

Where the pI variant reduces the pI of the monomer, it may have the additional benefit of improving serum retention in vivo.

Although still to be tested, it is believed that the Fc region has a longer half-life in vivo, since binding to FcRn in the endosome at pH6 sequesters Fc (Ghetie and Ward, 1997 immunological today.18 (12): 592-598, incorporated by reference in its entirety). The nuclear endosome compartment then recirculates Fc to the cell surface. Once this compartment is open to the extracellular space, the higher pH, about 7.4, induces Fc release back into the blood. In mice, Dall 'Acqua et al showed that Fc mutants with increased FcRn binding at pH6 and pH7.4 actually have reduced serum concentrations and the same half-life as wild-type Fc (Dall' Acqua et al, 2002, j.immunol.169: 5171-5180, incorporated by reference in its entirety). The increased affinity of Fc for FcRn at ph7.4 is believed to inhibit Fc release back into the blood. Thus, Fc mutations that increase Fc half-life in vivo would ideally increase FcRn binding at lower pH values while still allowing Fc release at higher pH values. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, His residues were found to be unexpected at important positions in the Fc/FcRn complex.

Recently, it has been suggested that antibodies with variable regions having lower isoelectric points may also have longer serum half-lives (Igawa et al, 2010PEDS.23 (5): 385-392, incorporated by reference in its entirety). However, this mechanism is still to be understood. In addition, the variable regions vary from antibody to antibody. Constant region variants with lower pI and longer half-life will provide a more modular approach to improving the pharmacokinetic properties of antibodies, as discussed herein.

The pI variants used in this embodiment and their use in purification optimization are disclosed in the accompanying figures.

Combinations of variants

It will be understood by those skilled in the art that all of the heterodimerization variants set forth can be optionally and independently combined in any manner so long as they retain their "chain type" or "monomer differentiation". Furthermore, all of these variants can be combined in any heterodimeric form.

In the case of pI variants, although a particularly suitable embodiment is shown in the figures, other embodiments can also be produced following the basic principle of varying the pI difference between two monomers to facilitate purification.

The antibodies of the invention are typically isolated or recombinant. "isolated" when used to describe various polypeptides disclosed herein means that the polypeptide is identified and isolated and/or recovered from a cell or cell culture expressing the polypeptide. Typically, the isolated polypeptide is prepared by at least one purification step. By "isolated antibody" is meant an antibody that is substantially free of other antibodies having different antigenic specificities.

"specific binding" or "specifically binds to" or is "specific for" a particular antigen or epitope means binding that is distinctly different from the non-specific interaction. Specific binding can be measured, for example, by determining the difference in binding of one molecule from the binding of a control molecule, which is generally a molecule of similar structure but with no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Specific binding to a particular antigen or epitope can be exhibited, for example, by a KD for the antibody for the antigen or epitope of at least about 10-4M, at least about 10-5M, at least about 10-6M, at least about 10-7M, at least about 10-8M, at least about 10-9M, or at least about 10-10M, at least about 10-11M, at least about 10-12M, or higher, wherein KD refers to the off-rate of the particular antibody-antigen interaction. Typically, the antibody that specifically binds to an antigen has a KD that is 20, 50, 100, 500, 1000, 5,000, 10,000 or more fold greater than the KD for the control molecule relative to the antigen or epitope.

In addition, specific binding to a particular antigen or epitope can be exhibited, for example, by the antibody's KA or KA to the antigen or epitope being at least 20, 50, 100, 500, 1000, 5,000, 10,000, or more fold higher than the epitope's KA or KA relative to a control, where KA or KA refers to the association rate of a particular antibody-antigen interaction.

Modified antibodies

For example, the VH and V L domains can be stabilized by incorporating disulfide bridges to link the molecules (Reiter et al, 1996, Nature Biotech.14: 1239-1245, incorporated by reference in its entirety).

Covalent modification of antibodies is included within the scope of the invention and is typically (but not always) performed post-translationally. For example, several types of covalent modifications of antibodies are introduced into molecules by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues.

The cysteinyl residue is most often reacted with α -haloacetate (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxamidomethyl derivatives the cysteinyl residue can also be derivatized by reaction with bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylk-butene diimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, mercury p-chlorobenzoate, 2-chloromercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1, 3-diazole, and the like.

Furthermore, modifications at cysteines are particularly useful for antibody-drug conjugate (ADC) applications (described further below). In some embodiments, the antibody constant region may be engineered to contain one or more cysteines that are particularly "thiol-reactive," thereby making the drug moiety more specific and controllable for placement. See, e.g., U.S. patent No. 7,521,541, incorporated herein by reference in its entirety.

Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this reagent has relatively high specificity for histidyl side chains. Also useful for bromobenzoyl methyl bromide; the reaction is preferably carried out at pH6.0 in 0.1M sodium cacodylate.

Other suitable reagents for derivatizing the α -amino-containing residue include imide esters such as methyl picoliniminate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2, 4-pentanedione, and transaminases catalyzed reaction with glyoxylic acid.

Arginyl residues are modified by reaction with one or more conventional reagents, among which are phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because of the higher pKa of the guanidine functional group. In addition, these reagents can react with the groups of lysine as well as arginine-amino groups.

Specific modifications can be made to tyrosyl residues, where it is of particular interest to introduce spectroscopic tags into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. The chloramine-T method described above is suitable for use in radioimmunoassays, using 125I or 131I to iodinate tyrosyl residues to produce labeled proteins.

Pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R ' -N ═ C ═ N — R '), where R and R ' are optionally different alkyl groups, such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-aza-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl groups are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional reagents is suitable for crosslinking the antibody to a water-insoluble carrier matrix or surface for use in a variety of methods, including the methods described below. Common crosslinking agents include, for example, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as the ester formed with 4-azidosalicylic acid, homobifunctional imide esters, including disuccinimidyl esters, such as 3, 3' -dithiobis (succinimidyl propionate), and bifunctional maleimides, such as bis-N-maleimidyl-1, 8-octane. Derivatizing agents such as methyl-3- [ (p-azidophenyl) dithio ] imine propionate give photoactivatable intermediates capable of forming crosslinks in the presence of light. Alternatively, protein immobilization is carried out using water-insoluble reactive matrices, such as the cynomolgusgen bromide activated carbohydrates and the reactive matrices described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537 and 4,330,440 (all incorporated by reference in their entirety).

Glutaminyl and asparaginyl residues are often deamidated to give the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under slightly acidic conditions. Any form of these residues is within the scope of the present invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α -amino group of lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp 79-86 [1983], incorporated by reference in its entirety), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Furthermore, it will be understood by those skilled in the art that labels, including fluorescent labels, enzymatic labels, magnetic labels, radioactive labels, etc., may be added to the antibodies (as well as other compositions of the invention).

Glycosylation

Another type of covalent modification is an alteration in glycosylation. In another embodiment, an antibody disclosed herein can be modified to include one or more engineered glycoforms. As used herein, "engineered glycoform" means a carbohydrate composition covalently attached to an antibody, wherein the carbohydrate composition is chemically distinct from a parent antibody. The engineered glycoforms can be used for a variety of purposes, including, but not limited to, enhancing or reducing effector function. One preferred form of engineered glycoform is defucosylation, which has been shown to correlate with increased ADCC function, possibly by binding more tightly to the Fc γ RIIIa receptor. In this context, "defucosylated" means that the majority of the antibodies produced in the host cell are substantially free of fucose, e.g., 90%, 95%, 98% of the antibodies produced do not have significant fucose as a component of the carbohydrate portion of the antibody (typically attached at position N297 in the Fc region). Functionally defined, defucosylated antibodies generally exhibit an affinity for the Fc γ RIIIa receptor of at least 50% or higher.

Engineered glycoform can beProduced by various methods known in the art: (Et al, 1999, NatBiotechnol 17: 176-180; davies et al, 2001, Biotechnol Bioeng 74: 288-; shields et al, 2002, J Biol Chem 277: 26733. about.26740; shinkawa et al, 2003, J Biol Chem 278: 3466-; US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739a 1; PCT WO01/29246A 1; PCT WO 02/31140a 1; PCT WO 02/30954a1, all incorporated by reference in their entirety; (Technique [ Biowa, Inc., Princeton, NJ ]]；Glycosylation engineering technology [ Glycart Biotechnology AG, Z ü rich, Switzerland]) Many of these techniques are based, for example, on the regulation of enzymes involved in the glycosylation pathway (e.g., FUT8[ α 1, 6-fucosyltransferase) by expressing IgG in various organisms or cell lines (e.g., L ec-13 CHO cells or rat hybridoma YB2/0 cells) that are engineered or otherwise treated]And/or (β 1-4-N-acetylglucosaminyltransferase III [ GnTIll ]]) Or by modifying the carbohydrate after expression of IgG to control the degree of fucosylation and/or to bisect oligosaccharides covalently attached to the Fc region. For example, the "glycoengineered antibodies" or "SEA technology" of Seattle Genetics works by adding modified sugars that inhibit fucosylation during manufacture; see, e.g., 20090317869, incorporated herein by reference in its entirety. Engineered glycoforms typically refer to different carbohydrates or oligosaccharides; thus, the antibody may comprise an engineered glycoform.

Alternatively, an engineered glycoform may refer to an IgG variant comprising different carbohydrates or oligosaccharides. As known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, as discussed below), or the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites in antibodies is typically achieved by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations (for O-linked glycosylation sites) may also be made by the addition or substitution of one or more serine or threonine residues in the starting sequence. For convenience, it is preferred to alter the antibody amino acid sequence by variation at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases, thereby generating codons that will translate into the desired amino acids.

Another way to increase the number of carbohydrate moieties on an antibody is chemical or enzymatic coupling of glycosides to proteins. These procedures have the advantage that they do not require the production of proteins in host cells that have the capacity to carry out N-linked and O-linked glycosylation. Depending on the coupling mode used, the sugar may be attached to (a) arginine and histidine; (b) a free carboxyl group; (c) a free sulfhydryl group, such as the sulfhydryl group of cysteine; (d) free hydroxyl groups, such as the hydroxyl groups of serine, threonine, or hydroxyproline; (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan; or (f) an amide group of glutamine. These methods are described in WO 87/05330 and Aplin and Wriston, 1981, crccrit. rev. biochem., page 259-306, both incorporated by reference in their entirety.

Removal of the carbohydrate moiety present on the starting antibody (e.g. post-translationally) may be achieved chemically or enzymatically. Chemical deglycosylation requires exposing the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment allows most or all of the sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) to be cleaved, while the polypeptide remains intact. Chemical deglycosylation is described in hakimudin et al, 1987, arch.biochem.biophysis.259: 52 and Edge et al, 1981, anal. biochem.118: 131 (all incorporated by reference in their entirety). This can be done by using Thotakura et al, 1987, meth. enzymol. 138: 350 (incorporated by reference in their entirety) to enzymatically cleave the carbohydrate moiety on the polypeptide. As described by Duskin et al, 1982, J.biol.chem.257: 3105 (incorporated by reference in its entirety), the use of the compound tunicamycin may prevent glycosylation at potential glycosylation sites. Tunicamycin prevents the formation of protein-N-glycoside linkages.

Another class of covalent modifications of antibodies includes those described by, for example, 2005-2006 PEG Catalog from Nektar therapeutics (available at the Nektar website); antibodies are attached to various non-protein polymers, including but not limited to various polyols, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner set forth in U.S. Pat. nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337 (all incorporated by reference in their entirety). In addition, amino acid substitutions may be made at various positions within the antibody to facilitate addition of polymers such as PEG, as is known in the art. See, e.g., U.S. publication No. 2005/0114037a1, incorporated by reference in its entirety.

Additional Fc variants for additional functions

In addition to pI amino acid variants, numerous useful Fc amino acid modifications can be made for a variety of reasons, including, but not limited to, altering binding to one or more Fc γ R receptors, altering binding to FcRn receptors, and the like.

Thus, proteins of the invention may include amino acid modifications, including heterodimerization variants outlined herein, including pI variants.

Fc gamma R variants

Thus, numerous useful Fc substitutions may be made to alter binding to one or more fcyr receptors, substitutions that increase binding and decrease binding may be useful, for example, it is known that increasing binding to fcriiia generally results in increased ADCC (antibody-dependent cell-mediated cytotoxicity; in such cell-mediated reactions, nonspecific cytotoxic cells expressing fcyr recognize bound antibody on target cells followed by lysis of target cells.) similarly, in some cases, decreasing binding to fcyriib (inhibitory receptor) is also useful.

Furthermore, as specifically disclosed in USSN 12/341,769 (incorporated herein by reference in its entirety), there are additional Fc substitutions that can be used to increase binding to FcRn receptors and increase serum half-life, including, but not limited to, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, and 259I/308F/428L.

Fc excision variants

Additional variants for use in the present invention are variants that cleave (e.g., reduce or eliminate) binding to Fc γ receptors. This would be desirable to reduce the potential mechanism of action (e.g., reduce ADCC activity) of the heterodimeric antibodies of the invention. Numerous suitable Fc-excision variants are depicted in fig. 35, and may or may not optionally and independently include combination with any other heterodimerization variants (including pI variants and steric variants).

The variants have the scFv-121/121-fused-scFv-121-fused-scFv-121-fused-P-121-fused-scFv-121-fused-P-121-fused-scFv-dockerin-121-fused-P-121-fused-dockerin-121-fused-P-121-fused-scFv-121-fused-scFv-121-fusion variants (the variants have the scFv-dockerin-No-F-No-F-No-F-No-F-No-F-No-F-No-F-No-F-No-F-No-F-No-F-No-F-FcNo-F-FcNo-F-FcNo-F-FcNo-F-FcNo-F-Fc-F-FcNo-F-FcNo-F-No-F-No-F-FcNo-No-F-No-F-Fc-F-No-F-No-F-No-F.

Connector

If desired, the invention also optionally provides linkers, e.g., to add additional antigen binding sites, as depicted, e.g., in fig. 11, 12 and 13, wherein the "other end" of the molecule contains additional antigen binding components. In addition, as outlined below, linkers are also optionally used in Antibody Drug Conjugate (ADC) systems. When used to join components of a central mAb-Fv construct, a linker is generally a polypeptide comprising two or more amino acid residues joined by peptide bonds and is used to join one or more components of the invention. Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-. A variety of linkers may be used in some embodiments described herein. It will be appreciated by those skilled in the art that at least three different connector types are used in the present invention.

"linker" is also referred to herein as "linker sequence", "spacer", "tethering sequence" or grammatical equivalents thereof. Homobifunctional or heterobifunctional linkers are well known (see 1994 Pierce Chemical company for crosslinker technical section, page 155-200, incorporated by reference in its entirety). (Note the distinction between "linker" and "scFv linker" and "charged scFv linker"). Various strategies can be used to covalently link the molecules together. These include, but are not limited to, polypeptide linkages between the N-and C-termini of proteins or protein domains, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant techniques or peptide synthesis. The linker peptide may comprise mainly the following amino acid residues: gly, Ser, Ala or Thr. The length of the linker peptide should be suitable to link two molecules in such a way that the molecules assume the correct conformation with respect to each other, whereby they retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used. Useful linkers include glycine-serine polymers including, for example, (GS) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least one; glycine-alanine polymers; alanine-serine polymers; and other flexible connectors. Alternatively, a variety of non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol and polypropylene glycol, may be used as the linker, that is, may be used as the linker.

Other linker sequences may include any sequence of any length of the C L/CH 1 domain, but not all residues of the C L/CH 1 domain, such as the first 5-12 amino acid residues of the C L/CH 1 domain.

Antibody-drug conjugates

In some embodiments, the multispecific antibodies of the present invention are conjugated to a drug to form antibody-drug conjugates (ADCs). In general, ADCs are used in oncology applications where the local delivery of cytotoxic or cytostatic agents using antibody-drug conjugates allows for the targeted delivery of the drug moiety to the tumor, thereby achieving higher efficacy, lower toxicity, etc. A review of this technology is provided in Ducry et al, Bioconjugate chem, 21: 5-13 (2010); carter et al, Cancer J.14 (3): 154 (2008); and sensor, Current opin. chem.biol. 13: 235-244(2009), are all incorporated herein by reference in their entirety.

In general, conjugation is by covalent attachment to an antibody (as described further below), and generally relies on a linker, often a peptide linkage (which may be designed to be sensitive or insensitive to cleavage by a protease at a target site, as described below). further, as described above, linkage of linker-drug units (L U-D) may be achieved by attachment to cysteine within the antibody.

Accordingly, the present invention provides conjugates of multispecific antibodies and drugs. As described below, the drug in the ADC can be a variety of agents, including, but not limited to, cytotoxic agents, such as chemotherapeutic agents, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes (i.e., radioconjugates). In other embodiments, the invention additionally provides methods of using ADCs.

Drugs for use in the present invention include cytotoxic drugs, particularly those used in cancer therapy. Such agents generally include DNA damaging agents, antimetabolites, natural products, and analogs thereof. Exemplary classes of cytotoxic agents include enzyme inhibitors, such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors; a DNA intercalator; a DNA lysing agent; a topoisomerase inhibitor; anthracycline family drugs; vinblastine drugs; mitomycins; bleomycin; a cytotoxic nucleoside; pteridine family drugs; enedialkynes; podophyllotoxins; dolastatins (dolastatins); maytansinoids; a differentiation-inducing agent; and the taxoids.

Members of these classes include, for example, methotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside (cytosine arabinoside), melphalan, leuconostin (leuosine), lurasidone (1eurosideine), actinomycin, daunomycin, doxorubicin, mitomycin C, mitomycin A, carminomycin (caminomycin), aminopterin, tylosin (tallysomycin), podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere (taxotere), retinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin (calicheamicin), eposalamycin (esperamicin), ene-diyne, duocarmycin A, calicheamicin (calicheamicin), Camptothecin, maytansinoids (including DM1), monomethyl auristatin E (MMAE), monomethyl auristatin f (mmaf), and maytansinoids (DM4), and analogs thereof.

The toxins used may be in the form of antibody-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.92 (19): 1573-1581; Mandler et al (2000) Bioorganic & Med. chem. L ets 10: 1025 1028; Mandler et al (2002) Bioconjugate chem.13: 786-791), maytansinoids (EP 1391213; L iu et al (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623) and calicheamicin (L ode et al (1998) Cancer Res.58: 2928; Hinman et al (1993) Cancer Res. 3353: 36-3342) toxins may inhibit cellular cytotoxicity including binding of proteins, DNA binding and microtubule-isomerase binding mechanisms.

Conjugates of multispecific antibodies and one or more small molecule toxins such as maytansinoids, dolastatins, auristatins, trichothecenes, calicheamicins, and CC1065, as well as toxin-active derivatives of these toxins are contemplated.

Maytansinoids

Maytansinoid compounds suitable for use as maytansinoid drug moieties are well known in the art and may be isolated from natural sources according to known methods, prepared using genetic engineering techniques (see Yu et al (2002) PNAS 99: 7968-. As described below, drugs can be modified by incorporating functional active groups, such as thiol or amine groups, for conjugation to antibodies.

Exemplary maytansinoid drug moieties include those having modified aromatic rings, such as C-19-dechlorination (U.S. Pat. No. 4,256,746) (prepared by reduction of ansamitocin P2 with lithium aluminum hydride), C-20-hydroxy (or C-20-demethyl) +/-C-19-dechlorination (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using streptococci or actinomycetes, or dechlorination using L AH), and C-20-demethoxy, C-20-acyloxy (- -OCOR) +/-dechlorination (U.S. Pat. No. 4,294,757) (prepared by acylation using acid chlorides), as well as those having modifications at other positions.

Exemplary maytansinoid drug moieties also include those having modifications such as C-9-SH (U.S. Pat. No. 4,424,219) (prepared by reacting maytansinol with H2S or P2S 5), C-14-alkoxymethyl (demethoxy/CH 2OR) (U.S. Pat. No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No. 4,450,254) (prepared by Nocardia), C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by converting maytansinol with streptococci), C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from the peach tree (Trewianudlflora)), C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylating maytansinol with streptococci), and 4, 5-deoxy (U.S. Pat. No. 4,371,533) (prepared by reducing maytansinol with titanium trichloride/L).

Particularly useful are DM1 (disclosed in U.S. patent No. 5,208,020, incorporated by reference) and DM4 (disclosed in U.S. patent No. 7,276,497, incorporated by reference). See also 5,416,064, WO/01/24763, 7,303,749, 7,601,354, USSN 12/631,508, WO02/098883, 6,441,163, 7,368,565, WO02/16368 and WO04/1033272 (all expressly incorporated by reference in their entirety) for other maytansinoid derivatives and methods.

ADCs containing maytansinoids, methods for their preparation and their therapeutic use are disclosed, for example, in U.S. patent nos. 5,208,020, 5,416,064, 6,441,163 and european patent EP 0425235B 1, the disclosures of which are expressly incorporated herein by reference L iu et al, proc. natl. acad. sci. usa 93: 8618-8623(1996) describe ADCs comprising a maytansinoid, designated DM1, linked to monoclonal antibody C242 directed against human colorectal cancer, which conjugates have been found to be highly cytotoxic against cultured colon cancer cells and to exhibit anti-tumor activity in an in vivo tumor growth assay.

Chari et al, Cancer Research 52: 127-.

Auristatin and dolastatin

In some embodiments, the ADC comprises a conjugate of a multispecific antibody and dolastatin or dolastatin peptide analogs and derivatives auristatin (U.S. Pat. nos. 5,635,483, 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother.45 (12): 3580-3584) and to have anti-cancer (U.S. Pat. No. 5,663,149) and anti-fungal activity (Pettit et al (1998) Antimicrob. Agents Chemother.42: 2961-2965). Dolastatin or auristatin drug moieties can be attached to an antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminally linked monomethylauristatin drug moieties DE and DF as disclosed in the American Association for Cancer Research, volume 45, Abstract Number 623 and described in U.S. patent publication No. 2005/0238648 (the disclosure of which is expressly incorporated by reference in its entirety), filed by sender et al, 3/month 28, 2004.

An exemplary auristatin embodiment is MMAE (see U.S. patent No. 6,884,869, expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649, 5,767,237, and 6,124,431, expressly incorporated by reference in their entirety).

Other exemplary embodiments comprising MMAE or MMAF and various linker components (as further described herein) have the following structures and abbreviations (wherein Ab refers to an antibody and p is 1 to about 8):

typically, peptide-based Drug moieties may be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments such peptide bonds may be prepared, for example, according to liquid phase Synthesis methods well known in The art of peptide chemistry (see E.Schroder and K. L ubke, "The Peptides", Vol.1, pp.76-136, 1965, Academic Press.) The auristatin/dolastatin Drug moieties may be prepared according to The methods of U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J.am.Chem.111: 5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13: 243-, Pettit, G.R. et al, Synthesis, 1996, Pettit et al (J.chem. c. 7815: 719; Natrons.2003: 778; Biotechn 777: 778; Biotec et al (1996) J.chem. TM.111: 635,635,635,635,24).

Calicheamicin

In other embodiments, the ADC comprises a conjugate of an antibody of the invention with one or more calicheamicin molecules Mylotarg is, for example, the first commercially available ADC drug and utilizes calicheamicin γ 1 as the payload (see U.S. Pat. No. 4,970,198, incorporated by reference in its entirety). other calicheamicin derivatives are described in U.S. Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001, 5,767,285 and 5,877,296, all expressly incorporated by reference. the antibiotic calicheamicin family is capable of generating double-stranded DNA breaks at subpicomolar concentrations. for the preparation of calicheamicin family conjugates, see U.S. Pat. Nos. 5,712,374, 5,714,5,739,116, 5,767,285, 5,701, 5,770, 5,710,773,001, 5,877,296 (all belonging to the species of calicheamicin Cyaner family), and thus the anti-tumour cell line antibody can be taken up easily by the internalizing antibody molecule, such as opposed to the anticancer agent (see, as Adenoid, 2, Fancoal, Falcan et al; see U.3,3375, Fancoal, Falcan et al).

Duocarmycin

CC-1065 (see 4,169,888, incorporated by reference) and duocarmycin are members of the family of antitumor antibiotics utilized in ADCs. These antibiotics appear to act by sequence-selective alkylation at adenine N3 in the minor groove of DNA, thus initiating a series of events that cause apoptosis.

Important members of duocarmycins include duocarmycin a (U.S. Pat. No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat. No. 5,101,038, incorporated by reference), as well as large-scale analogs as described in U.S. Pat. nos. 7,517,903, 7,691,962, 5,101,038, 5,641,780, 5,187,186, 5,070,092, 5,070,092, 5,641,780, 5,101,038, 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149, WO2009/017394a1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302, and 7,507,420 (all expressly incorporated by reference).

Other cytotoxic agents

Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin (streptozoicin), vincristine and 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, madecasin A chain, α -sarcin (alpha-sarcin), Aleurites fordii (Aleurites fordii) protein, dianthin (dianthin) protein, Phytolacca americana (Phytolacca americana) protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curculin (curcin), croton toxin (crotin), Saponaria saponaria (saponaria) inhibitor, gelonin (gelonin), mitogenin (mitogellin), restrictocin (ristocetin), phenomycin (phyromycin), enomycin (enomycin) and theimomycin (see, for example, WO 93/21232, 1993).

The invention further encompasses ADCs formed between an antibody and a compound having nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; a DNase).

Examples include radioisotopes of At211, I131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and L u.

The radiolabel or other label may be incorporated into the conjugate by known means. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels, such as Tc99m or I123, Re186, Re188 and In111, may be attached via a cysteine residue In the peptide. Yttrium-90 may be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) biochem. Biophys. Res. Commun.80: 49-57) can be used to incorporate iodine-123. Other methods are described in detail by "monoclonal antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

For compositions comprising multiple antibodies, the drug loading is represented by p, which is the average number of drug molecules per antibody.drug loading can range from 1 to 20 drugs per antibody (D). the average number of drugs per antibody in the preparation of the conjugation reaction can be characterized by conventional means, such as mass spectrometry, E L ISA assays, and HP L C.

In some cases, the isolation, purification, and characterization of homogeneous antibody-drug conjugates (where p is a value) from antibody-drug conjugates with other drug loadings can be achieved by means such as reverse phase HP L C or electrophoresis.

The antibody-drug conjugate complex may be produced by any technique known to the skilled artisan. Briefly, an antibody-drug conjugate complex may include as antibody units a multispecific antibody, a drug, and optionally a linker to join the drug to the binding agent.

A variety of different reactions can be used to covalently attach drugs and/or linkers to binding agents. This can be achieved by reaction of amino acid residues of the binding agent (e.g., antibody molecule), including the amine group of lysine, the free carboxylic acid groups of glutamic and aspartic acids, the sulfhydryl group of cysteine, and various portions of aromatic amino acids. A commonly used non-specific covalent attachment method is a carbodiimide reaction that links the carboxyl (or amino) group of a compound to the amino (or carboxyl) group of an antibody. In addition, bifunctional reagents such as dialdehydes or imidoesters are used to link the amino group of the compound to the amino group of the antibody molecule.

Further, it is a schiff base reaction that can be used to attach drugs to the binding agents. This process involves the periodate oxidation of a drug containing a diol or hydroxyl group, thereby forming an aldehyde, which is then reacted with a binding agent. Attachment occurs via the formation of a schiff base with the amino group of the binding agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binders. Other techniques are known to the skilled artisan and are within the scope of the invention.

In some embodiments, the intermediate, i.e., linker precursor, is reacted with the drug under appropriate conditions. In other embodiments, reactive groups on the drug and/or intermediate are used. The reaction product between the drug and the intermediate, or derivatized drug, is then reacted with the multispecific antibodies of the present invention under appropriate conditions.

It will be appreciated that for the purposes of preparing the conjugates of the invention, the desired compound may also be chemically modified in order to make the compound more susceptible to reaction. For example, functional groups, such as amine, hydroxyl, or sulfhydryl groups, may be appended to a drug at locations that have a minimal or acceptable effect on the activity or other properties of the drug.

Connector sub-unit

Typically, the antibody-drug conjugate complex comprises a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker in an appropriate environment releases the drug unit from the antibody. For example, solid tumors that secrete certain proteases can serve as targets for cleavable linkers; in other embodiments, intracellular proteases are utilized. In yet other embodiments, the linker unit is non-cleavable and the drug is released, for example, by degradation of the antibody in lysosomes.

In some embodiments, the linker can be cleaved by a cleaving agent present in the intracellular environment (e.g., within the lysosome or endosome or caveolae). The linker may, for example, be a peptidyl linker that is cleaved by intracellular peptidases or proteases, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long or longer.

Lytic agents may include, but are not limited to, cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in release of the active drug inside the target cell (see, e.g., Dubowchik and Walker, 1999, pharm. therapeutics 83: 67-123.) peptidyl linkers that can be cleaved by enzymes are present in CD38 expressing cells.

In some embodiments, the peptidyl linker that can be cleaved by an intracellular protease is a Val-Cit linker or a Phe-L ys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin in the presence of a Val-Cit linker).

In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis performed at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) may be used. (see, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, pharm. therapeutics 83: 67-123; Neville et al, 1989, biol. chem.264: 14653-14661.) such linkers are relatively stable under neutral pH conditions (such as those in blood), but are unstable below pH5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to the therapeutic agent via an acylhydrazone bond; see, e.g., U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker may be cleaved under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known In the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene), SPDB, and SMPT (see, e.g., Thorpe et al, 1987, Cancer Res.47: 5924-

In other embodiments, the linker is a malonate linker (Johnson et al, 1995, anticancer Res.15: 1387-93), a maleimidoylbenzoyl linker (L au et al, 1995, Bioorg-Med-chem.3 (10): 1299-1304), or a 3' -N-amide analog (L au et al, 1995, Bioorg-Med-chem.3 (10): 1305-12).

In yet other embodiments, the linker unit is not cleavable, and the drug is released by antibody degradation. (see U.S. publication No. 2005/0238649, incorporated herein by reference in its entirety and for all purposes).

In many embodiments, the linker is self-degrading. As used herein, the term "self-degrading spacer" refers to a bifunctional chemical moiety capable of covalently linking two spaced chemical moieties together into a tripartite stable molecule. If its bond to the first moiety is cleaved, it will spontaneously dissociate from the second chemical moiety. See, for example, WO2007059404a2, WO06110476a2, WO05112919a2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431, WO04/043493, and WO02/083180, which are directed to drug-cleavable matrix conjugates in which the drug and the cleavable matrix are optionally linked by a self-degrading linker, and all are expressly incorporated by reference.

Generally, the linker is substantially insensitive to the extracellular environment. As used herein, with respect to a linker, "substantially insensitive to the extracellular environment" means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linker in an antibody-drug conjugate complex sample is cleaved when the antibody-drug conjugate complex is present in the extracellular environment (e.g., plasma).

Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate complex with plasma for a predetermined period of time (e.g., 2, 4,8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes internalization when conjugated to the therapeutic agent (that is, in the context of the linker-therapeutic agent portion of the antibody-drug conjugate complex as described herein). In yet other embodiments, the linker, when conjugated to an auristatin compound and a multispecific antibody of the invention, promotes cell internalization.

Various exemplary linkers that may be used in the compositions and methods of the invention are described in WO 2004-010957, U.S. publication No. 2006/0074008, U.S. publication No. 20050238649, and U.S. publication No. 2006/0024317 (each of which is incorporated herein by reference and for all purposes).

Drug loading

Drug loading is expressed as p and is the average number of drug moieties per antibody in the molecule the drug loading ("p") can be 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more moieties (D) per antibody, although average numbers are often fractional or fractional in general drug loadings of 1 to 4 are often useful, and 1 to 2 are also useful.

In the case of p, the quantitative distribution of the ADC can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs (where p is a certain value) from ADCs with other drug loadings can be achieved by means such as electrophoresis.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, when the attachment site is a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups to which a linker may be attached. In certain embodiments, higher drug loadings, e.g., p > 5, may cause some antibody-drug conjugates to aggregate, insolubilize, poison, or otherwise lose cell permeability. In certain embodiments, the drug loading of the ADCs of the present invention ranges from 1 to about 8; from about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shown that for certain ADCs, the optimal drug moiety ratio per antibody may be below 8, and may be from about 2 to about 5. See US 2005-0238649 a1 (incorporated herein by reference in its entirety).

In certain embodiments, less than the theoretical maximum amount of drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that are not reactive with the drug-linker intermediate or linker reagent (as discussed below). Generally, antibodies comprise a plurality of free reactive cysteine thiol groups, which groups may be attached to a drug moiety; in fact, the majority of cysteine thiol residues in antibodies are present in the form of disulfide bridges. In certain embodiments, the antibody can be reduced under partial or total reducing conditions with a reducing agent such as Dithiothreitol (DTT) or Tricarbonylethylphosphine (TCEP) to produce a reactive cysteine thiol group. In certain embodiments, the antibody is subjected to denaturing conditions, thereby exhibiting a reactive nucleophilic group, such as lysine or cysteine.

The loading (drug/antibody ratio) of the ADC can be controlled in different ways, for example: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting conjugation reaction time or temperature; (iii) partial or limiting reduction conditions for cysteine thiol modification; (iv) the amino acid sequence of the antibody is engineered by recombinant techniques, thereby modifying the number and position of cysteine residues to control the number and/or position of linker-drug attachments (a thio Mab or thio Fab prepared as disclosed herein and in WO2006/034488 (herein incorporated by reference in its entirety)).

It will be appreciated that where more than one nucleophilic group is reacted with a drug-linker intermediate or linker reagent followed by a drug moiety reagent, the resulting product is a mixture of ADC complexes distributed with one or more drug moieties attached to the antibody the average number of drugs per antibody can be calculated from the mixture by a dual E L ISA antibody assay specific for the antibody and specific for the drug.

In some embodiments, homogeneous ADCs with a single loading value may be separated from the conjugate mixture by electrophoresis or chromatography.

Method for determining the cytotoxic effect of ADC

Methods are known for determining whether a drug or antibody-drug conjugate exerts cytostatic and/or cytotoxic effects on cells. In general, the cytotoxic or cytostatic activity of an antibody drug conjugate can be measured by: exposing mammalian cells expressing the target protein of the antibody drug conjugate to a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and apoptosis-inducing effects (caspase activation) of the antibody drug conjugates.

To determine whether an antibody drug conjugate exerts cytostatic effects, thymidine incorporation assays may be used. For example, cancer cells expressing a target antigen seeded at a density of 5,000 cells/well in a 96-well plate can be cultured for a period of 72 hours and exposed to 0.5 μ Ci of 3H-thymidine for the last 8 hours of the 72 hour period. Incorporation of 3H-thymidine in cultured cells was measured in the presence and absence of antibody drug conjugates.

To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by an increase in plasma membrane permeability; cell expansion; and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. Any of these assays for the effect of cancer cells indicate that the antibody drug conjugates can be used to treat cancer.

Cell viability can be measured by measuring the uptake of dyes such as neutral red, trypan blue or A L AMARTM blue in cells (see, e.g., Page et al, 1993, Intl.J. Oncology 3: 473-476). in such assays, cells are incubated in dye-containing media, washed, and the residual dye is measured spectrophotometrically, thereby reflecting the cellular uptake of the dye.

Alternatively, tetrazolium salts, such as MTT, are used in quantitative colorimetric assays for survival and proliferation of mammalian cells by detecting viable cells rather than dead cells (see, e.g., Mosmann, 1983, J.Immunol.Methodss 65: 55-63).

Examples of such assays, including TUNE L (for detecting labeled nucleotides incorporated in fragmented DNA) and assays based on the E L ISA, are described in Biochemica, 1999, phase 2, pages 34-37 (RocheMolecular Biochemicals).

Apoptosis can also be determined by measuring changes in cell morphology. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the absorption of certain dyes (e.g., fluorescent dyes, such as, for example, acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described in Duke and Cohen, Current Protocols in immunology (Coligan et al, eds., 1992, pp. 3.17.1-3.17.16). The cells may also be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and observed for chromatin condensation and sidedness and nuclear membranes of the cells. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell contraction.

The presence of apoptotic cells may be measured in the adherent and "floating" portions of the culture. For example, the two fractions can be collected by removing the supernatant, trypsinizing the attached cells, pooling the samples obtained after a centrifugal washing step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation). (see, e.g., Piazza et al, 1995, Cancer Research 55: 3110-16).

In vitro, the effect of therapeutic compositions of multispecific antibodies of the invention can be evaluated in suitable animal models. For example, an allogeneic cancer model can be used in which cancer explants or passaged xenograft tissue are introduced into immunocompromised animals, such as nude or SCID mice (Klein et al, 1997, Nature Medicine 3: 402-408). Efficacy can be measured using assays that measure tumor formation, tumor regression, or metastasis inhibition, among others.

Therapeutic compositions for practicing the foregoing methods may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired method of delivery. Suitable carriers include any material that retains the anti-tumor function of the therapeutic composition when combined with the therapeutic composition and that does not generally react with the immune system of the patient. Examples include, but are not limited to, a variety of standard pharmaceutical carriers such as sterile phosphate buffered saline solution, bacteriostatic water, and the like (see generally, Remington's pharmaceutical sciences 16 th edition, a.osal. eds., 1980).

Antibody compositions for in vitro administration

Formulations of the antibodies used in accordance with the present invention are prepared by mixing antibodies of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. eds. [1980]), either in lyophilized formulations or in aqueous solution, acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphates, citrates and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) proteins such as protein, glycine, mannitol, or other ionic protein chelators such as polyethylene glycol, mannitol, polyethylene glycol, polyethylene.

The formulations herein may also contain more than one active compound, preferably those having complementary activities that do not adversely affect each other, as desired for the particular indication being treated. For example, it may be desirable to provide antibodies with other specificities. Alternatively, or in addition, the composition may comprise a cytotoxic agent, a cytokine, a growth inhibitory agent, and/or a small molecule antagonist. Such molecules are preferably present in combination in amounts effective to achieve the intended purpose.

The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-based microcapsules and poly (methylmethacylate) microcapsules, respectively, embedded in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. eds (1980).

Formulations intended for in vivo administration should be sterile or near sterile. This is easily achieved by filtration through sterile filtration membranes.

Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules, examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), L-glutamic acid copolymer with L-gamma-ethyl glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., L UPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer with leuprolide acetate)), and poly-D- (-) -3-hydroxybutyric acid.polymers, such as ethylene-vinyl acetate and lactic acid-glycolic acid, are capable of releasing the molecule within 100 days, while certain hydrogels release the protein within a shorter period of time.

When the encapsulated antibody is maintained in vivo for a prolonged period of time, it may denature or aggregate due to exposure to moisture at 37 ℃, thereby resulting in a loss of biological activity and possibly a change in immunogenicity. Depending on the mechanisms involved, reasonable strategies can be devised to achieve stability. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation through thiol-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and creating specific polymer matrix compositions.

Application method

The antibodies and chemotherapeutic agents of the invention are administered to a subject according to known methods, such as intravenously, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Preferably, the antibody is administered intravenously or subcutaneously.

Method of treatment

In the methods of the invention, therapy is used to provide a positive therapeutic response to a disease or disorder. "positive therapeutic response" is intended to mean an improvement in a disease or disorder, and/or an improvement in a symptom associated with the disease or disorder. For example, a positive therapeutic response refers to one or more of the following disease improvements: (1) a decrease in the number of neoplastic cells; (2) increased neoplastic cell death; (3) neoplastic cell survival is inhibited; (5) tumor growth inhibition (i.e., slowing to some extent, preferably stopping); (6) increased patient survival; and (7) a degree of reduction in one or more symptoms associated with the disease or disorder.

A positive therapeutic response in any given disease or condition can be determined by a standardized response standard specific to that disease or condition. Tumor response can be assessed by changes in tumor morphology (i.e., total tumor burden, tumor size, etc.) using screening techniques such as Magnetic Resonance Imaging (MRI) scans, x-ray radiographic imaging, Computed Tomography (CT) scans, bone scan imaging, endoscopy, and tumor biopsy sampling, including Bone Marrow Aspiration (BMA) and tumor cell counts in circulation, among others.

In addition to these positive therapeutic responses, subjects undergoing therapy may also experience the beneficial effects of disease-related symptom improvement.

Thus, for B cell tumors, the subject may experience so-called B symptoms, i.e., a reduction in night sweats, fever, weight loss, and/or rubella. For premalignant conditions, therapy with a multispecific therapeutic agent may prevent the development of the associated malignant condition and/or extend the time before the development of the associated malignant condition, e.g., the development of multiple myeloma in a subject suffering from monoclonal agammaglobulinemia of unknown significance (MGUS).

Improvement in disease can be characterized by a complete response. "complete response" is intended to mean the absence of clinically detectable disease and normalization of radiographic studies of any previous abnormalities (in the case of myeloma, bone marrow and cerebrospinal fluid (CSF) or abnormal monoclonal proteins).

Such a response may last at least 4 to 8 weeks, or sometimes 6 to 8 weeks, after treatment according to the methods of the invention. Alternatively, improvement of the disease may be classified as a partial response. By "partial response" is intended a reduction of at least 50% of all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured tumor volume, or the amount of abnormal monoclonal protein) in the absence of new lesions, which response may last for 4 to 8 weeks, or 6 to 8 weeks.

The treatment according to the invention comprises a "therapeutically effective amount" of the agent used. A "therapeutically effective amount" refers to an amount effective, at a desired dosage and for a desired period of time, to achieve the desired therapeutic result.

The therapeutically effective amount may vary depending on a variety of factors, such as the disease state; age, sex, and weight of the individual; and the ability of the drug to elicit a desired response in the individual. A therapeutically effective amount is also an amount by which the therapeutically beneficial effect of the antibody or antibody portion exceeds any toxic or detrimental effect thereof.

A "therapeutically effective amount" of a tumor therapy can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated in an animal model system that predicts efficacy in human tumors.

Alternatively, such a property of the composition can be assessed by examining the ability of the compound to inhibit cell growth or induce apoptosis in vitro using assays known to skilled practitioners. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise improve the symptoms in a subject. One of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered; several divided doses can be administered over time; or the dose may be reduced or increased proportionally as indicated by the urgency of the treatment. The parenteral composition can be formulated in unit dosage form for administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable as a single dose for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

The specification for the unit dosage form of the present invention is dictated by and directly dependent on the following factors: (a) the unique characteristics of the active compounds and the specific therapeutic effect to be achieved, and (b) limitations inherent in the art of compounding such active compounds for the treatment of sensitivity in a subject.

Effective dosages and dosage regimens for the multispecific antibodies used in the present invention depend on the disease or disorder to be treated and may be determined by one of skill in the art.

An exemplary, non-limiting range of therapeutically effective amounts of multispecific antibodies for use in the present invention is from about 0.1 to 100mg/kg, such as from about 0.1 to 50mg/kg, for example from about 0.1 to 20mg/kg, such as from about 0.1 to 10mg/kg, for example about 0.5mg/kg, such as about 0.3mg/kg, about 1mg/kg or about 3 mg/kg. In another embodiment, the antibody dose administered is 1mg/kg or higher, such as a1 to 20mg/kg dose, for example a 5 to 20mg/kg dose, for example an 8mg/kg dose.

A medical practitioner having ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start with a drug in a pharmaceutical composition at a dose that is lower than the dose required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In one embodiment, the multispecific antibody is administered by infusion at a weekly dose of 10 to 500mg/kg, such as 200 to 400 mg/kg. Such administration may be repeated, for example, 1 to 8 times, such as 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours.

In one embodiment, the multispecific antibody is administered by slow continuous infusion over an extended period of time, if necessary over 24 hours, to reduce side effects, including toxicity.

In one embodiment, the multispecific antibody is administered in a weekly dose of 250mg to 2000mg, such as, for example, 300mg, 500mg, 700mg, 1000mg, 1500mg or 2000mg, up to 8 times, such as 4 to 6 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours. This regimen may be repeated one or more times, if necessary, after, for example, 6 months or 12 months. The dosage can be determined or adjusted by measuring the amount of the compound of the invention in the blood after administration, e.g. by taking a biological sample, and using an anti-idiotypic antibody targeting the antigen binding region of the multispecific antibody.

In another embodiment, the multispecific antibody is administered once a week for 2 to 12 weeks, such as 3 to 10 weeks, such as 4 to 8 weeks.

In one embodiment, the multispecific antibody is administered according to maintenance therapy, e.g., once a week for 6 months or more.

In one embodiment, the multispecific antibody is administered according to a protocol comprising infusion of the multispecific antibody once, followed by infusion of a conjugate of the multispecific antibody and a radioisotope. This protocol can be repeated, for example, after 7 to 9 days.

In non-limiting examples, the treatment according to the invention may be performed at least on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39 or day 40, or on day 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 12, week 11, week 12, week 13, week 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23 day, day 24 day, day 25 day 26 day, day 27, day 28 day, day 29 day, day 30 day, week 3, week 4, week 6 week, week 7 week, week 8 week, week, At least one week of week 14, week 15, week 16, week 17, week 18, week 19 or week 20, or any combination thereof, using a single dose, or every 24, 12, 8, 6,4 or 2 hours, or any combination thereof, in divided doses at about 0.1-100mg/kg per day, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg or 100 mg/kg.

In some embodiments, the multispecific antibody molecule is used in combination with one or more additional therapeutic agents, e.g., chemotherapeutic agents. Non-limiting examples of DNA-damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin, and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunomycin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine (decarbazine), methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include: paclitaxel (paclitaxel), docetaxel and related analogs; vincristine, vinblastine and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF- κ B inhibitors, including Iκ B kinase inhibitors; antibodies that bind to proteins that are overexpressed in cancer and thereby down-regulate cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and other inhibitors of proteins or enzymes known to be up-regulated, over-expressed or activated in cancer, the inhibition of which down-regulates cellular replication.

In some embodiments, the antibodies of the invention may be used in(bortezomib) treatment is administered prior to, concurrently with or after treatment.

All cited references are expressly incorporated herein by reference in their entirety.

While specific embodiments of the invention have been described above for purposes of illustration, it will be understood by those skilled in the art that various changes in detail may be made without departing from the invention as described in the following claims.

Examples

The following examples are provided to illustrate the invention. These examples are not intended to limit the invention to any particular application or theory of operation. For all constant regions discussed in the present invention, the numbering is according to the EU index as in Kabat (Kabat et al, 1991, Sequences of Proteins of Immunological Interest, 5 th edition, United State public Health Service, National Institutes of Health, Bethesda, incorporated by reference in their entireties). It will be appreciated by those skilled in the antibody art that this convention consists of non-sequential numbering in specific regions of immunoglobulin sequences, thereby enabling a normalized reference to conserved positions in the immunoglobulin family. Thus, the position of any given immunoglobulin as defined by the EU index does not necessarily correspond to its sequential sequence.