阅读说明：本技术 双特异性重组蛋白及其应用 (Bispecific recombinant protein and application thereof ) 是由 宋利平 崔笑添 王健 吴海祥 贾佳娜 范艺 张甘良 李涛 徐红 佘伊莎 龙凯 于 2018-05-08 设计创作，主要内容包括：本发明公开了一种双特异性重组蛋白,其包含高亲和靶向肿瘤的臂和低亲和阻断CD47与SIRPα相互作用的融合蛋白,其中高亲和靶向肿瘤的臂所对应的抗体不结合CD47,其对肿瘤细胞上靶抗原的结合亲和力是低亲和阻断CD47与SIRPα相互作用的融合蛋白所对应的单体融合蛋白同二聚体对肿瘤细胞上CD47结合亲和力的至少6倍,其中低亲和阻断CD47与SIRPα相互作用的融合蛋白包含SIRPα胞外截短体。还公开了编码重组蛋白的核酸分子及所述重组蛋白和核酸分子在制备治疗肿瘤的药物中的用途。本发明的双特异性重组蛋白显著提高具有调节巨噬细胞功能重组蛋白的肿瘤靶向饱和结合丰度并且降低非肿瘤靶向的副作用,在临床上有很大的应用价值。(The invention discloses a bispecific recombinant protein which comprises a high-affinity tumor-targeting arm and a fusion protein for blocking the interaction of CD47 and SIRPa by low affinity, wherein an antibody corresponding to the high-affinity tumor-targeting arm does not bind to CD47 and has at least 6 times of the binding affinity to a target antigen on a tumor cell compared with the binding affinity of a monomer fusion protein homodimer of a fusion protein for blocking the interaction of CD47 and SIRPa by low affinity to CD47 on the tumor cell, and wherein the fusion protein for blocking the interaction of CD47 and SIRPa by low affinity comprises an SIRPa extracellular truncation. Also discloses a nucleic acid molecule for coding the recombinant protein and application of the recombinant protein and the nucleic acid molecule in preparing a medicament for treating tumors. The bispecific recombinant protein of the invention obviously improves the tumor targeted saturation binding abundance of the recombinant protein with the function of regulating macrophages and reduces the side effect of non-tumor targeting, thereby having great application value in clinic.)

1. A recombinant protein comprising a tumor targeting subunit and a subunit that blocks the interaction of CD47 with sirpa.

2. The recombinant protein according to claim 1, wherein the tumor targeting subunit is a half-antibody.

3. The recombinant protein of claim 1 or 2, wherein the subunit that blocks the interaction of CD47 with sirpa is a single chain protein.

4. The recombinant protein of any one of the preceding claims, wherein the tumor targeting subunit is an IgG1 antibody.

5. The recombinant protein of any one of the preceding claims, wherein the tumor targeting subunit targets a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUDG-CSF, MUSC-72, MUDDN-B, MUDG-B, PSDL-B, CTFP-B, CTCD B, PDGFR-B, CD B, PSDL-B, CD-B, CD B, PSDL-B, CD B, PSDL-36363672, CD B, CD 363672, CD 36III-B, CD 36III-B, PSDL-36III-B, PSDL-36III, PSDL-B, PSDL-36III, PSDL-B, PSDL-36III, PSDL-B, and PSDL-B, binding.

6. The recombinant protein of any one of the preceding claims, wherein the tumor targeting subunit and the subunit that blocks the interaction of CD47 with sirpa are bound by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the foregoing binding means.

7. The recombinant protein of any one of the preceding claims, further comprising an Fc region; preferably, the Fc region comprises an Fc region native sequence or an Fc non-native sequence; more preferably, the Fc region is a human Fc region; even more preferably, wherein the binding of the high affinity tumor targeting subunit to the low affinity subunit that blocks the interaction of CD47 with SIRPa is via a nanobs-int-holes binding.

8. The recombinant protein of any one of the preceding claims, wherein the tumor targeting subunit is a half antibody targeting a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUSC-CSF, MUSC-72, MUDDN-B, MUDG-B, PSMG-B, CTFP-B, CTCD B, CD B, PSMGDF-B, CD B, PSNFR-B, CD 363672, PSNFR-B, CD 36III-B, CD-B, PSNFR-B, CD-B, PSNFR-36III-B, PSNFR-B, CD-36III-B, CD-B, PSDMTF-36III-B, PSNFR-36III-B, PSNFR-36III-B, PSNFR-B, PSTFPI-36III-B, PSNFR-36III-B, PSNFR-B, PSNFL-B, PSNFR-B, PSNFL-B, PSNFR-B, CD B, PSNFR-B, PSNFL-B, PSNFR; half antibodies, preferably IgG1 antibodies, optionally human murine chimeric half antibodies, humanized half antibodies, fully human half antibodies; more preferably a half antibody of humanized or fully human IgG1 antibody.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a recombinant protein and application thereof.

Background

With the research on the tumor treatment field, the research and development and application of tumor molecule targeting treatment drugs are receiving more and more extensive attention. Based on the advantages of strong targeting property, small side effect, obvious curative effect and the like, the antibody medicament quickly becomes the tumor targeted treatment fieldHot spot drugs of the domain. At present, dozens of antibody medicines targeting tumors, such as rituximab, aiming at a CD20 target molecule, are approved for clinical application and achieve remarkable curative effect(Rituximab, the first mab approved for cancer treatment in the United states, originally used to treat non-Hodgkin lymphoma, a product of Roche),(ibritumomab tiuxetan, originally approved by the U.S. FDA for the treatment of poorly differentiated non-Hodgkin's lymphoma resistant to rituximab, available from IDEC Pharmaceuticals.),(tositumomab and iododine I131 tositumomab, GSK output),Herceptin against Her2 target molecule (ofatumumab, product of GSK)(trastuzumab, a well-known drug for the treatment of breast cancer, product of Genentech),(pertuzumab, Roche output),Avastin against VEGF or its receptor target molecule (ado-trastuzumab emtansine, available from Roche)(bevacizumab available from Genentech/Roche),(ramucirumab, product of EliLilly) and the like,erbitux for EGFR target(cetuximab, one of the ten well-marketed anti-cancer drugs worldwide, originally approved for the treatment of rectal cancer, by Eli Lilly); products,(panitumumab, a drug for treating colorectal cancer, produced by Amgen) and the like against PD-L1 target molecule(atezolizumab, available from Roche) et al (Cao Rui et al, research on antibody drugs for tumor-targeted therapy, J.Biol.Pharma, China 2016, 36 (6): 15-18).

The anti-tumor action mechanism of the clinically applied anti-tumor antibody medicament (unconjugated naked antibody) targeting tumor cells is mainly realized by two functions of the antibody. First, the first function of an antibody is affinity, i.e., the antibody exerts its effector function after specifically binding to a tumor surface target antigen, killing tumor cells. The antibody molecule can block the signal path of tumor growth factor, induce apoptosis or inhibit the formation of new blood vessel in the tumor microenvironment by combining with target antigen. Secondly, the second function of antibodies is achieved by means of the immune system, i.e. killing of tumor cells can also be mediated by the immune system to mediate cell death, such as Antibody-constant region mediated Antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and Antibody-dependent phagocytosis (ADCP). Increasing data indicate that antibody-mediated immune killing activity is an important mechanism of action for the antibody to exert anti-tumor efficacy (for review see Barnhart BC, el al. role of Fc-Fc γ R interactions in the antibody activity of therapeutic antibodies, immunology and Cell Biology, 2017, 95: 340-

In recent years, studies have confirmed that tumor cells can continue to divide and grow by means of approaches such as modification of self-surface antigens and change of microenvironment around tumor tissues, so-called tumor immune escape. For example, tumor cells inhibit the immune function of macrophages by means of high expression of CD47 and mutual combination with inhibitory receptor signal regulatory protein alpha (SIRP alpha) on the surfaces of the macrophages, thereby obviously inhibiting the activity of immune cells. Meanwhile, high expression of CD47 by tumor cells inhibits Fc receptor-mediated phagocytosis, ultimately affecting the tumor-targeted therapeutic effect of antibody drugs (Willingham SB, et al. the CD47-signal regulatory protein Alpha (SIRP α) interactive target for human soluble tumors. proceedings of the National Academy of Sciences of the United States of America.2012, 109(17): 6662-6667).

CD47 is a widely expressed membrane glycoprotein, which is a receptor and a ligand for SIRP alpha, and can form a CD47-SIRP alpha signal complex to mediate a series of reactions such as apoptosis, proliferation, immunity and the like. In 2000, Oldenborg et al demonstrated that CD47 is an important signal on the cell surface for regulating macrophage phagocytosis (Oldenborg PA, et al, role of CD47 as a marker of self on red blood cells, science, 2000, 288 (5473): 2051-2054). CD47 can bind to SIRP alpha on the surface of macrophage by releasing the signal of ' eating me ' separately ', phosphorylate its immunoreceptor tyrosine-based inhibition motif (ITIM), and then recruit SH2-containing protein tyrosine phosphatase SHP1(SH 2-conjugating protein tyrosine phosphatase 1) protein, and generate a series of cascade reactions to inhibit phagocytosis of the macrophage. Young erythrocytes express higher CD47 releasing the "self-eat me" signal to macrophages, while aged erythrocytes, CD47, are down-regulated and eventually cleared by macrophages.

Since CD47 is widely expressed on the surface of various cancer cells, the target can be used for treating various cancers, and the mouse allogeneic tumor transplantation model has demonstrated the effectiveness of CD47 blocking, CD47 has become a novel target for cancer immune checkpoint therapy (Vonderheide R H. CD47 block as animal checkpoint therapy for cancer Nature Medicine,2015,21(10): 1122) 1123). The anti-CD47 antibody, SIRP alpha-Fc fusion protein and the like are researched into medicines, and the inhibition effect of CD47 on immune cells is relieved by blocking a CD47-SIRP alpha signal channel, so that certain anti-tumor activity is shown. However, since erythrocytes also express CD47 protein in large quantities, anti-CD47 antibody therapy with high affinity binding to CD47 protein can produce toxic side effects such as hemagglutination, anemia, etc. (McCracken MN, et al molecular Pathways: Activating T Cell Cancer Cell pharmaceuticals from blood blocks of CD47 "Don' T Eat Me" signals. clinical Cancer Research,2015,21(16):3597 + 3601), which makes clinical development of anti-CD47 antibody drugs extremely difficult, and no anti-CD47 antibody drugs have entered phase III clinic so far; the wild type SIRP alpha-Fc fusion protein has low affinity with CD47, so the drug effect is not obvious; researchers also have made high affinity mutations to wild type sirpa and obtained sirpa mutants with a thousand-fold increased affinity and exhibited better antitumor effects (Weiskopf K, et al. engineered sirpa varians as immunothereutical adoptions to anti-cancer agents science, 2013,341(6141):88-91), but whether or not multiple mutations had an effect on the safety, immunogenicity, stability and target specificity of the fusion protein in humans remains to be examined in clinical observation.

On the other hand, studies have shown that anti-CD47 antibody can significantly improve the antitumor effect by combining with other tumor-targeted therapeutic antibodies, such as CD47+ CD20, CD47+ Her2 (Chao MP, et al anti-CD47 antibody conjugates with the use of the combination of therapeutic to promoter and therapeutic non-Hodgkin lymphoma. cell,2010,142(5): 699-713; Zhao XW, et al CD47-personal regulatory protein-alpha (SIRP alpha) interaction for a bar for anti-tumor cell modification of the National Academy of Sciences of the United States 108.2011 18345). However, there is great uncertainty as to whether and when an anti-CD47 antibody can be marketed; the use of tumor-targeting antibody drugs in combination after marketing also makes it difficult for patients to bear their very expensive combination therapy costs.

Therefore, there is a need to develop a new tumor-targeted drug, which can specifically target and treat tumors, activate and enhance the immune function of patients, significantly improve the tumor treatment effect on the premise of ensuring safety, and reduce the drug cost of patients.

In addition, in view of potential safety risks (such as anemia, erythrocyte agglutination, killing of CD47 positive non-tumor target cells, and the like) of monovalent or multivalent antibodies or recombinant proteins targeting CD47, species specificity exists due to the binding of human sirpa and human CD47, and the use of human blood is limited by ethics, genetic resources, and the like, so an early in vivo/in vitro immune safety evaluation method for evaluating the immune safety of antibodies or recombinant proteins targeting CD47 is urgently to be developed.

Disclosure of Invention

The invention provides a bispecific recombinant protein and application thereof, which can remarkably improve the tumor targeted saturation binding abundance of the recombinant protein with the function of regulating macrophages and can remarkably reduce the side effect of non-tumor targeting.

In one aspect, the invention provides a bispecific recombinant protein, wherein the bispecific recombinant protein comprises a high affinity tumor-targeting arm and a low affinity fusion protein that blocks the interaction of CD47 with sirpa.

In one embodiment, the high affinity tumor-targeting arms do not bind CD47, and the binding affinity of the antibody to the target antigen on the tumor cell of the high affinity tumor-targeting arms is at least 6-fold, optionally 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or higher fold value, or any value therebetween, of the binding affinity of the monomeric fusion protein homodimer corresponding to the fusion protein that low affinity blocks CD47 from interacting with sirpa for CD47 on the tumor cell;

the low affinity fusion protein that blocks the interaction of CD47 with sirpa has no higher binding affinity for CD47 than the binding affinity of a monomeric fusion protein homodimer containing a sirpa extracellular truncation for CD47. The fusion protein with low affinity for blocking the interaction of CD47 and SIRPa comprises a SIRPa extracellular truncation.

In one embodiment, the sirpa extracellular truncation comprises part or all of the extracellular amino acid sequence of a human sirpa (wild-type or CD47 non-high affinity mutant).

In yet another embodiment, the sirpa extracellular truncation is a human sirpa extracellular truncation. In a specific embodiment, the sirpa extracellular truncation comprises an amino acid sequence selected from any one of a1) -a4) as follows: a1) SEQ ID No. 30; a2) 31 in SEQ ID No; a3) SEQ ID No. 32; a4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any one of the above amino acid sequences, the monomer of which has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of a1), a2) or a3) monomer for CD47 protein.

In one embodiment, the bispecific recombinant protein has a left and a right arm structure oppositely arranged, wherein the high affinity tumor targeting arm is located at the position of the left arm, and the low affinity fusion protein blocking the interaction of CD47 with sirpa is located at the position of the right arm; preferably, the left arm is a Fab or Fab' form of an immunoglobulin and the right arm is a sirpa extracellular truncation. Wherein, the 'structure with left and right arms oppositely arranged' of the invention is described by referring to the conventional 'Y' -shaped structure of immunoglobulin, in this case, the left and right sides of the dual specificity recombinant protein of the invention have different targeted specificity functional proteins; the structural description is mainly distinguished from that two specific functional proteins can form an upper structure and a lower structure by connecting a C terminal and an N terminal. Therefore, the spatial positions of the left and right arms in the present invention are not specifically limited to the structure of the bispecific recombinant protein, but are only for distinguishing the two arm structures of the bispecific recombinant protein from the upper and lower structures formed by the connection of the C-terminal and N-terminal, when one is located on one side, the other is correspondingly located on the opposite side; obviously, the left and right spatial positions of the two can be interchanged.

In another embodiment, the length of the right arm is adapted to the proximity of the epitope to be bound by the left arm to the surface of the target cell membrane. Preferably, when the arms of the high-affinity targeting tumor need to be combined with an epitope at the membrane proximal end of a target cell, the SIRPa extracellular truncation is selected to contain the amino acid sequences shorter than a1) -a 4).

In one embodiment, the high affinity tumor-targeting arm targets a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUDG-CSF, MUSC-72, MUDDN-B, MUDG-B, PSDL-B, CTFP-B, CTCD B, PDGFR-B, CD B, PSDL-B, CD-B, CD B, PSDL-B, CD B, PSDL-36363672, CD B, CD 363672, CD 36III-B, CD 36III-B, PSDL-36III-B, PSDL-36III, PSDL-B, PSDL-36III, PSDL-B, PSDL-36III, PSDL-B, and PSDL-B, binding.

Preferably, when the target is CD20, EGFR or PD-L1, the sirpa extracellular truncation selects a 1); when the target is HER2, the sirpa extracellular truncation selects either a1) or a 2).

In one embodiment, the high affinity tumor-targeting arms are bound to a fusion protein that blocks the interaction of CD47 with sirpa with low affinity by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the above binding means.

In yet another embodiment, the high affinity tumor targeting arm and the low affinity fusion protein that blocks the interaction of CD47 with sirpa further comprise an Fc region. Typically, the Fc region of the invention comprises the native sequence of the Fc region. However, the Fc region of the present invention may have one or more existing amino acid sequence alterations or modifications in the native sequence of the Fc region, for example, an amino acid sequence alteration or modification in which the C1q binding activity of the Fc region is altered.

In another embodiment, the fusion protein that blocks the interaction of CD47 with sirpa with low affinity is a fusion protein comprising a sirpa extracellular truncation and a binding sequence for binding to the arm, the sirpa extracellular truncation and the binding sequence for binding to the arm optionally being linked by a linker sequence, the binding sequence for binding to the arm optionally comprising an Fc region.

In another embodiment, the Fc region of the present invention is conceptual in that although it may not actually be present, antibody engineering may be performed based on the amino acid sequence of the desired Fc region variant to produce a polypeptide or fusion protein comprising this sequence or DNA encoding the amino acid sequence of the desired Fc region variant.

In another embodiment, the Fc region can be an Fc region variant. As used herein, "Fc region variant" refers to an Fc region obtained by modification of one or more amino acid residues of an Fc native amino acid sequence. Methods of modification are well known to those skilled in the art and include, but are not limited to, site-directed mutagenesis of a DNA sequence encoding an Fc region, for example using PCR mutagenesis and cassette mutagenesis to prepare variants of the Fc region. For example, one or more amino acid residues of the Fc region may be deleted in order to enhance binding to FcR. For example, in one embodiment, amino acid inserted Fc region variants can be made in order to alter the effector function of the Fc region.

In one embodiment, for example, at least one amino acid residue (e.g., 1-2 amino acid residues, typically no more than 10 amino acid residues) can be introduced near one or more sites of the Fc region identified as affecting FcR binding. "nearby" means within 1-2 amino acid residues from the identified Fc region site that affects FcR binding. Such Fc region variants may exhibit enhanced or reduced FcR binding and/or ADCC activity. To prepare such insertional variants, the co-crystal structure of a polypeptide comprising an FcR binding region (e.g., the extracellular domain of a target FcR) with an Fc region into which amino acid residues are to be inserted may be evaluated to relate to variants of the Fc region having, for example, enhanced FcR binding. Such insertions are typically placed in loops in the Fc region.

In one embodiment, Fc region variants can be prepared that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells and/or bind Fc γ receptors (fcyr) with greater affinity than recombinant proteins containing a native Fc region by introducing appropriate amino acid sequence modifications in the native Fc region. The Fc region variants of the invention typically comprise at least one amino acid modification in the Fc region. Preferably, multiple amino acid modifications are combined. For example, Fc region variants may include substitutions of 2, 3, 4, 5 or more amino acid residues, as in the specific FcR binding site identified.

The native Fc region is preferably a human Fc region, for example the native sequence of an Fc region of human IgG1(a or non-a isotype), IgG2, IgG3 or IgG 4.

In another embodiment, the binding of the high affinity tumor-targeting arms to the low affinity fusion protein that blocks the interaction of CD47 with sirpa is via a knob-int-holes binding. For example, the knobs-into-holes are of the "knobs" type highlighted by the T366W mutation, and of the "holes" type dented by the 1 amino acid mutation (Y407V) or the "holes" type dented by the 3 amino acid mutations (T366S, L368A and Y407V). The mutations are shown from left to right as the original amino acid residue, the mutation site and the substituted amino acid residue, respectively, according to the Kabat numbering scheme [ Eu number scheme of Kabat et al (1991) ], e.g. in T366W, T is the original amino acid residue, 366 is the mutation site and W is the amino acid residue of the substitution T.

In one embodiment, the high affinity tumor-targeting arm is a half-antibody targeting a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUSC-CSF, MUSC-72, MUDDN-B, MUDG-B, PSMG-B, CTFP-B, CTCD B, CD B, PSMGDF-B, CD B, PSNFR-B, CD 363672, PSNFR-B, CD 36III-B, CD-B, PSNFR-B, CD-B, PSNFR-36III-B, PSNFR-B, CD-36III-B, CD-B, PSDMTF-36III-B, PSNFR-36III-B, PSNFR-36III-B, PSNFR-B, PSTFPI-36III-B, PSNFR-36III-B, PSNFR-B, PSNFL-B, PSNFR-B, PSNFL-B, PSNFR-B, CD B, PSNFR-B, PSNFL-B, PSNFR; half antibodies, preferably IgG1 antibodies, optionally human murine chimeric half antibodies, humanized half antibodies, fully human half antibodies; more preferably a half antibody of humanized or fully human IgG1 antibody.

In one embodiment, the fusion protein that blocks the interaction of CD47 with sirpa with low affinity comprises an amino acid sequence selected from any one of the following b1) -b 4): b1) 26 is SEQ ID No; b2) 27 in SEQ ID No; b3) 28 in SEQ ID No; b4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any of the above amino acid sequences, wherein a homodimer thereof has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of b1), b2) or b3) homodimer for CD47 protein. The sequence of the signal peptide region, the linker sequence, the hinge region, the Fc region and/or the binding sequence included in the above-mentioned sequences may be arbitrarily replaced according to a manner known to those skilled in the art or a commonly used signal peptide sequence, linker sequence, hinge region, Fc region and/or binding sequence.

In another embodiment, the bispecific recombinant protein comprises the following sequence:

wherein when the high affinity tumor targeting arm targets CD20, the high affinity tumor targeting arm comprises SEQ ID No. 16 and SEQ ID No. 17, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26 or SEQ ID No. 27 or SEQ ID No. 28;

wherein when the high affinity tumor targeting arm targets EGFR, the high affinity tumor targeting arm comprises SEQ ID No. 19 and SEQ ID No. 8, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26 or SEQ ID No. 27 or SEQ ID No. 28;

wherein when the high affinity tumor targeting arm is targeted to Her2, the high affinity tumor targeting arm comprises SEQ ID No. 20 and SEQ ID No. 21, or SEQ ID No. 22 and SEQ ID No. 23, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26 or SEQ ID No. 27 or SEQ ID No. 28; or the like, or, alternatively,

wherein when the high affinity tumor targeting arm is targeted to PD-L1, the high affinity tumor targeting arm comprises SEQ ID No. 24 and SEQ ID No. 13, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26 or SEQ ID No. 27 or SEQ ID No. 28.

In one aspect, the invention provides a nucleic acid molecule encoding a bispecific recombinant protein. Preferably, wherein the nucleic acid molecule encoding the high affinity tumor targeting arm is in the same DNA strand as the nucleic acid encoding the fusion protein of the low affinity block CD47 interacting with sirpa or the nucleic acid molecule encoding the high affinity tumor targeting arm is in a different DNA strand from the nucleic acid encoding the fusion protein of the low affinity block CD47 interacting with sirpa.

In another aspect, the invention provides an expression vector comprising a nucleic acid molecule.

In one aspect, the invention provides a cell comprising an expression vector.

In another aspect, the present invention also provides a method of making a bispecific recombinant protein comprising: 1) providing a high affinity tumor-targeting arm; 2) providing a fusion protein that blocks the interaction of CD47 with sirpa with low affinity; 3) the recombinant protein is formed by contacting the high affinity tumor targeting arms with a low affinity fusion protein that blocks the interaction of CD47 with sirpa.

In one embodiment, a method of making a recombinant protein comprises causing a host cell comprising an expression vector comprising a nucleic acid molecule encoding the recombinant protein to express the recombinant protein.

In another aspect, the invention also provides methods of tumor-targeted therapy.

In one embodiment, the tumor is selected from the group consisting of hematological tumors and solid tumors, such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, fibrosarcoma protrusions of the skin, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.

In yet another aspect, the invention also provides a pharmaceutical composition comprising a recombinant protein or fusion protein of the invention and optionally an adjuvant, excipient or pharmaceutically acceptable carrier. The composition may contain a pharmaceutically acceptable carrier. The compositions may be presented in any form of pharmaceutical formulation including, but not limited to, injections, powders, lyophilized powders, and the like. The pharmaceutical composition in the form of the pharmaceutical preparation can be prepared according to the conventional technology of pharmaceutical formulation, and comprises the steps of fusing the pharmaceutical active ingredient, the recombinant protein or the fusion protein of the invention and a pharmaceutical carrier, and preparing into the required dosage form according to the conventional technology of pharmaceutical formulation.

In one embodiment, the invention also provides a pharmaceutical composition comprising an expression vector encoding a nucleic acid molecule of a recombinant protein or fusion protein of the invention and optionally a pharmaceutically acceptable carrier.

In another aspect, the present invention also provides a method of treating a tumor comprising administering to a patient or subject a therapeutically effective amount of a pharmaceutical composition of the present invention. The tumors express additional target molecules including, but not limited to, 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD37, CD38, CD40, CD52, CD79 52, CD52, CEACAM 52, CLDN18.2, CLDN 52, CS 52, CXCR 52, DLL-4, EGFR, EGP-1, PP 52, EphA 52, ETBR, FGFR 52, FN-alpha, GCC, GD 52, GPC-3, NMB, HER 72, NORD 52, PD-52, HLA-1, CSF-1-CSF-3-MUDAP-52, MUDG 52, PDGFR-52, CTF-52, CTP-52, PDGF-52, CTC, CTFA-52, PDGF-52, CTP-52, PDGF-3, PDGF-52, PDGF-3, PDGF-52, PDGF-3, PDGF-52, PDGF-3, PDGF-52, PDGF-3, PDGF-52, PDGF-3, PDGF-52, PDGF-3, PDGF-52, PDGF-52, PDGF-3, PDGF-52, PDGF-, MSB0010718C, BCMA, CD 138.

In yet another aspect, the invention also provides an in vivo gene therapy comprising introducing into a patient or subject a therapeutically effective amount of a nucleic acid molecule encoding a recombinant protein or fusion protein of the invention, or a derivative thereof.

In another aspect, the present invention also provides a method for assessing the early immunological safety of a recombinant protein/antibody targeting CD47 and having ADCC activity in vitro, the method comprising: a) providing a targeted recombinant protein/antibody (including monovalent or multivalent) having ADCC activity; b) detecting the ADCC activity of the recombinant protein/antibody; c) the recombinant proteins/antibodies (either monovalent or multivalent) are evaluated for early immune safety.

In one embodiment, the present invention provides a method for assessing the early immunological safety of a recombinant protein/antibody targeting CD47 and having ADCC activity in vitro, comprising: a) preparing effector cells, such as, but not limited to, human NK92MI-CD16a effector cells; b) contacting the effector cell with the recombinant protein/antibody; c) detecting the ADCC activity of the recombinant protein/antibody; d) and evaluating the early immune safety of the recombinant protein/antibody according to the ADCC activity result.

In another embodiment, the present invention provides a method for assessing the early immunological safety of a recombinant protein/antibody targeting CD47 and having ADCC activity in vitro, comprising: a) harvesting well-grown human NK92MI-CD16a effector cells, and resuspending the harvested effector cells to a cell density of 1 × 10⁵Cell/ml to 5X 10⁶Individual cells/ml; b) incubating the recombinant protein/antibody diluted in a gradient with the effector cells prepared in a), wherein the incubation time is 0.5-5 h; c) after incubation is finished, measuring LDH activity, and calculating the cell lysis rate; d) the early immunological safety of the recombinant protein/antibody was evaluated based on the cell lysis rate, wherein the immunological safety of the recombinant protein/antibody resulting in a lower cell lysis rate was high.

In yet another aspect, the invention also provides a method for assessing the safety of early immunity of a recombinant protein/antibody (including monovalent or multivalent) targeting CD47 in vivo, comprising: a) providing recombinant proteins/antibodies (including monovalent or multivalent) targeting CD 47; b) providing a Hu-NSG mouse; c) contacting the recombinant protein/antibody with a Hu-NSG mouse; d) evaluating the early immune safety of the recombinant protein/antibody in the Hu-NSG mice.

In one embodiment, the present invention provides a method for assessing the early immune safety of a recombinant protein/antibody (including monovalent or multivalent) targeting CD47 in vivo, comprising: a) providing a Hu-NSG mouse; b) administration of recombinant proteins/antibodies (including monovalent or multivalent); c) after 24-96h of administration, taking venous blood of the mice, cracking red blood cells, incubating the rest cells for 15-60min by using fluorescence labeled anti-human CD45, anti-human CD19 or anti-human CD3 antibodies, and detecting by flow cytometry; d) the early immune safety of the recombinant protein/antibody was evaluated based on the cell clearance rate, wherein the immune safety of the recombinant protein/antibody that resulted in lower clearance of immune cells (except target cells) was high.

The invention has the beneficial effects that:

the invention reduces the combination of the recombinant protein of the invention with CD47 targets on non-tumor cells (such as erythrocytes, NK cells, T cells and the like) through the fusion protein for blocking the interaction between CD47 and SIRP alpha by low affinity, thereby improving the safety of the recombinant protein of the invention (such as avoiding anemia, erythrocyte agglutination and CD47 positive non-tumor target cell killing caused by anti-CD47 antibody/recombinant protein such as Hu5F9-G4, and reducing the risk caused by uncertain factors such as potential safety, immunogenicity and stability caused by high affinity mutated SIRP alpha-Fc fusion protein).

It is unexpectedly found in the present invention that the binding affinity of the recombinant protein of the present invention to tumor cells can be significantly improved by the interaction between the high-affinity targeting tumor cell arms and the low-affinity blocking CD47 and sirpa, so as to mediate the action of killing tumor cells with high efficiency (including but not limited to the effector function exerted by the antibody after the antibody is specifically bound to the tumor surface target antigen, and the targeted phagocytosis action of macrophages).

The recombinant protein of the invention realizes double-target effect through the high-affinity tumor targeting arm (such as a half antibody) and the fusion protein for blocking the interaction between CD47 and SIRPa through low affinity, and the result of the invention shows that the recombinant protein of the invention exerts anti-tumor effect through various mechanisms by means of various effects between the high-affinity tumor targeting arm and the fusion protein for blocking the interaction between CD47 and SIRPa through low affinity, and the drug effect is higher.

Compared with the combination of two separated targeting antibodies, the recombinant protein has the advantages of lower cost, more convenient use and the like, so that the problems of low patient compliance, overhigh treatment cost and the like under the condition of the combination of the antibodies can be solved.

Compared with the classical bispecific antibody (consisting of 2 half antibodies), the molecular weight of the fusion protein for blocking the interaction between CD47 and SIRPa is smaller than that of the single arm (half antibody) of the classical bispecific antibody, so the tissue transmittance of the recombinant protein is better than that of the classical bispecific antibody, and the drug effect is higher.

The fusion protein for blocking the interaction between the CD47 and the SIRPa comprises SIRPa extracellular truncation bodies with different lengths, can be quickly optimized and paired with arms (such as a half antibody) of a high-affinity targeting tumor, avoids the situation that a conventional bifunctional antibody is possibly influenced by the spatial structures of two target antigens when synchronously combining the two target antigens (particularly, when the length difference between antibody recognition sites of the two target antigens and a cell membrane is larger, the synchronous combination of the tumor targeting antigen and the CD47 antigen is difficult), enables the obtained recombinant protein to be better combined with tumor cells, and further plays a better role in killing the tumor.

The invention provides a novel method for evaluating the safety of an immunotherapy medicament targeting CD47 in vitro. Conventional anti-CD47 antibody safety assessments at an early stage are generally evaluated by detecting the effect of the drug on erythrocyte agglutination. However, because of species specificity of the binding of human sirpa to CD47, human blood must be used to evaluate the safety of whether recombinant proteins/antibodies targeting CD47 have hemagglutination in humans, and the use of human blood is limited by ethics, genetic resources, and the like. Furthermore, the experiment of erythrocyte agglutination does not allow for early evaluation of the immunological safety of the drug. The method (early in vitro immune safety evaluation experiment) for detecting ADCC activity, which is optimized, is used for replacing the traditional human erythrocyte agglutination experiment and is used for evaluating the early immune safety of the recombinant protein/antibody (comprising monovalent or multivalent) which targets CD47 and has ADCC activity, and the evaluation method is simple and quick and is not limited by blood sources.

The invention provides a novel method for evaluating the in vivo safety of an immunotherapy medicament targeting CD47. The in vivo immune safety evaluation of the medicine before clinic generally uses non-human primates, the number of required samples is large, and the difficulty and the cost of preparing early samples are increased. Other species have no mature early in vivo immune safety evaluation method, so that great potential safety hazard of the medicine in future clinical research is caused, and waste of research and development investment is brought. The invention provides a Hu-NSG mouse, wherein an early safety evaluation experiment carried out on the Hu-NSG mouse can simulate the safety condition of a medicament in a human immune system, compared with preclinical or clinical research, the Hu-NSG mouse has the advantages of low preparation difficulty and cost, low detection cost, high detection efficiency and the like, and the risk of research and development of tumor immunotherapy medicaments is reduced.

The invention provides the recombinant protein, realizes high affinity and specific targeting on the tumor through a high affinity targeting tumor arm, and realizes the blocking of the interaction of CD 47-SIRPa through a fusion protein for blocking the interaction of CD47 and SIRPa through low affinity.

The recombinant protein of the invention overcomes the technical problems of low yield and difficult purification of the target product of the conventional bifunctional antibody. In one embodiment of the invention, the right arm (Fc knob mutation) of the recombinant protein is a single-chain protein, and when the left arm light chain, the left arm heavy chain (Fc hole mutation) and the right arm (Fc knob mutation) are expressed together in cells, more than 80% of the expression products are target proteins after protein A purification, thereby effectively improving the expression yield. Although a small amount of left arm dimer, right arm dimer or left and right arm monomer exists, the products have obvious difference of molecular weight, charge distribution and the like, and can be easily removed by conventional purification means such as ion exchange chromatography, hydrophobic chromatography, molecular exclusion chromatography, affinity chromatography and salting-out method (such as ammonium sulfate precipitation method), and the method is suitable for industrial scale-up production.

The fusion protein for blocking the interaction between the CD47 and the SIRP alpha by low affinity can form a recombinant protein which can antagonize the immunosuppressive efficacy of the CD47 with arms (such as but not limited to half antibodies) of a high affinity targeting tumor.

The invention overcomes the defect that the prior SIRP alpha-Fc fusion protein in the prior art has unobvious drug effect due to low affinity with CD47, and also avoids a plurality of adverse effects of high immunogenicity, high non-tumor targeting specificity and the like possibly brought by the SIRP alpha high-affinity mutant. Meanwhile, the recombinant protein of the invention has weak binding affinity to CD47 expressed on the surface of normal cells (such as erythrocytes), so that the side effects of erythrocyte agglutination, anemia and the like generated by the conventional anti-CD47 antibody treatment are reduced or avoided.

It will be understood by those skilled in the art that the scope of the present invention is not unduly limited to such specific embodiments of the invention as to achieve one of the benefits of the invention either explicitly or implicitly or not explicitly or any generalisation thereof. It should be understood by those skilled in the art that any product, method or use that achieves any of the benefits or combinations of benefits, expressed or implied by the present invention, within the scope of the present invention, means that the technical problem to be solved by the present invention is solved, and the corresponding technical effects are achieved.

In particular, the present invention relates to the following embodiments:

embodiment 1. a bispecific recombinant protein comprising a tumor-targeting arm and a fusion protein that blocks the interaction of CD47 with sirpa.

Embodiment 2. the bispecific recombinant protein of embodiment 1 wherein the fusion protein that blocks the interaction of CD47 with sirpa comprises a sirpa extracellular truncation (including a human sirpa wild-type extracellular truncation or a human sirpa extracellular truncation of a CD47 non-high affinity mutation).

Embodiment 3 the bispecific recombinant protein of embodiment 2 wherein the sirpa extracellular truncation comprises part or all of an extracellular amino acid sequence of sirpa.

Embodiment 4 the bispecific recombinant protein of any one of embodiments 1-3, wherein the high affinity tumor-targeting arms do not bind CD47 and the binding affinity of the antibody to the target antigen on the tumor cell for the high affinity tumor-targeting arms is at least 6-fold greater than the binding affinity of the homodimer of the monomeric fusion protein to the fusion protein that blocks the interaction of CD47 with sirpa for CD47 on the tumor cell.

Embodiment 5. the bispecific recombinant protein of any one of embodiments 1 to 4, wherein the sirpa extracellular truncation comprises an amino acid sequence selected from any one of the following a1) -a 4):

a1)SEQ ID No:30；

a2)SEQ ID No:31；

a3)SEQ ID No:32；

a4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any one of the above amino acid sequences, the monomer of which has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of a1), a2) or a3) monomer for CD47 protein.

Embodiment 6. the bispecific recombinant protein of any one of embodiments 1 to 5, wherein the bispecific recombinant protein has a left and a right arm structure arranged oppositely, the high affinity tumor targeting arm is located at the left arm position, and the low affinity fusion protein blocking the interaction of CD47 with SIRPa is located at the right arm position.

Embodiment 7. the bispecific recombinant protein of embodiment 6, which has the left arm of the Fab or Fab' form of an immunoglobulin and the right arm of the sirpa extracellular truncation.

Embodiment 8. the bispecific recombinant protein of embodiment 7, wherein the length of the right arm is adapted to the distance of the epitope to be bound by the left arm from the surface of the target cell membrane.

Embodiment 9. the bispecific recombinant protein of embodiment 8, whose high affinity targeting tumor arm needs to bind to the epitope at the membrane proximal end of the target cell, the sirpa extracellular truncation is selected to contain the shorter amino acid sequences of a1) -a 4).

Embodiment 10 the bispecific recombinant protein of any one of embodiments 1 to 9, wherein the tumor-targeting arm targets a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUDG-CSF, MUSC-72, MUDDN-B, MUDG-B, PSDL-B, CTFP-B, CTCD B, PDGFR-B, CD B, PSDL-B, CD-B, CD B, PSDL-B, CD B, PSDL-36363672, CD B, CD 363672, CD 36III-B, CD 36III-B, PSDL-36III-B, PSDL-36III, PSDL-B, PSDL-36III, PSDL-B, PSDL-36III, PSDL-B, and PSDL-B, binding.

Embodiment 11. the bispecific recombinant protein of embodiment 10, wherein when the target is CD20, EGFR or PD-L1, the sirpa extracellular truncation selects a 1); when the target is HER2, the sirpa extracellular truncation selects a1 or a 2).

Embodiment 12. the bispecific recombinant protein of any one of embodiments 1 to 11, which tumor-targeting arms are bound to a fusion protein that blocks the interaction of CD47 with sirpa by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the above binding means.

Embodiment 13 the bispecific recombinant protein of any one of embodiments 1 to 12, wherein the tumor-targeting arm and/or the fusion protein blocking the interaction of CD47 with sirpa further comprises an Fc region.

Embodiment 14 the bispecific recombinant protein of embodiment 13, wherein the Fc region comprises an Fc region native sequence or an Fc non-native sequence.

Embodiment 15 the bispecific recombinant protein of embodiment 14 wherein the Fc region is a human Fc region.

Embodiment 16. the bispecific recombinant protein of embodiment 15 whose high affinity tumor-targeting arms bind to fusion proteins with low affinity that block the interaction of CD47 with sirpa via the knobs-endo-holes.

Embodiment 17 the bispecific recombinant protein of any one of embodiments 1 to 16, wherein the tumor-targeting arm is a half antibody targeting a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUSC-CSF, MUSC-72, MUDDN-B, MUDG-B, PSMG-B, CTFP-B, CTCD B, CD B, PSMGDF-B, CD B, PSNFR-B, CD 363672, PSNFR-B, CD 36III-B, CD-B, PSNFR-B, CD-B, PSNFR-36III-B, PSNFR-B, CD-36III-B, CD-B, PSDMTF-36III-B, PSNFR-36III-B, PSNFR-36III-B, PSNFR-B, PSTFPI-36III-B, PSNFR-36III-B, PSNFR-B, PSNFL-B, PSNFR-B, PSNFL-B, PSNFR-B, CD B, PSNFR-B, PSNFL-B, PSNFR; half antibodies, preferably IgG1 antibodies, optionally human murine chimeric half antibodies, humanized half antibodies, fully human half antibodies; more preferably a half antibody of humanized or fully human IgG1 antibody.

Embodiment 18 the bispecific recombinant protein of any one of embodiments 1 to 17, wherein the fusion protein that blocks the interaction of CD47 with sirpa is a fusion protein comprising a sirpa extracellular truncation and a binding sequence for binding to the arm, which are optionally linked by a linker sequence, said binding sequence for binding to the arm being optionally an Fc region.

Embodiment 19 the bispecific recombinant protein of any one of embodiments 1 to 18, wherein the fusion protein blocking the interaction of CD47 with sirpa comprises an amino acid sequence selected from any one of the following b1) -b 4):

b1)SEQ ID No:26；

b2)SEQ ID No:27；

b3)SEQ ID No:28；

b4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any of the above amino acid sequences, wherein a homodimer thereof has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of b1), b2) or b3) homodimer for CD47 protein.

Embodiment 20 the bispecific recombinant protein of embodiment 19 wherein the high affinity tumor targeting arms comprise SEQ ID No 16 and SEQ ID No 17 when said high affinity tumor targeting arms target CD20, and the fusion protein with low affinity blocking the interaction of CD47 with sirpa comprises SEQ ID No 26; wherein when the high affinity tumor targeting arm targets EGFR, the high affinity tumor targeting arm comprises SEQ ID No. 19 and SEQ ID No. 8, and the low affinity fusion protein that blocks the interaction of CD47 and SIRPa comprises SEQ ID No. 26; wherein when the high affinity tumor targeting arm is targeted to Her2, the high affinity tumor targeting arm comprises SEQ ID No. 20 and SEQ ID No. 21, or SEQ ID No. 22 and SEQ ID No. 23, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26 or SEQ ID No. 27; or wherein the high affinity tumor targeting arm comprises SEQ ID No 24 and SEQ ID No 13 and the low affinity fusion protein that blocks the interaction of CD47 and SIRPa comprises SEQ ID No 26 when the high affinity tumor targeting arm is targeted to PD-L1.

Embodiment 21. nucleic acid molecules encoding the bispecific recombinant protein of any one of embodiments 1 to 20.

Embodiment 22 the nucleic acid molecule of embodiment 21, the nucleic acid molecule encoding the high affinity tumor-targeting arm is in the same DNA strand or in a different DNA strand as the nucleic acid encoding the low affinity fusion protein that blocks the interaction of CD47 with sirpa.

Embodiment 23. expression vectors comprising the nucleic acid molecules of embodiment 21 or 22.

Embodiment 24. a cell comprising the expression vector of embodiment 23.

Embodiment 25. a method of making a bispecific recombinant protein of any one of embodiments 1-20 comprising:

1) providing an arm that targets a tumor;

2) providing a fusion protein that blocks the interaction of CD47 with sirpa;

3) contacting the tumor-targeting arms with a fusion protein that blocks the interaction of CD47 with sirpa to form the recombinant protein.

Embodiment 26 the production method of embodiment 25, wherein the cell of embodiment 24 is allowed to express the recombinant protein.

Embodiment 27 the method of embodiment 25, wherein contacting comprises binding by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the above binding modes.

Embodiment 28. the method of any one of embodiments 25 to 27, wherein contacting comprises combining by the knobs-into-holes technique.

The fusion protein of embodiment 29, wherein the fusion protein comprises a sirpa extracellular truncation and a binding sequence for binding to another polypeptide.

Embodiment 30 the fusion protein of embodiment 29, wherein the binding sequence for binding to another polypeptide is an Fc region; optionally wherein the Fc region comprises a holes mutation and/or a knobs mutation.

Embodiment 31 the fusion protein of embodiment 29 or 30 wherein the sirpa extracellular truncation comprises part or all of the extracellular amino acid sequence of a human sirpa wild-type and non-high affinity mutants thereof.

Embodiment 32 the fusion protein of embodiment 31, wherein the sirpa extracellular truncation comprises an amino acid sequence selected from any one of a1) -a4) as follows:

a1)SEQ ID No:30；

a2)SEQ ID No:31；

a3)SEQ ID No:32；

a4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any one of the above amino acid sequences, the monomer of which has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of a1), a2) or a3) monomer for CD47 protein.

Embodiment 33 the fusion protein of any one of embodiments 29 to 32, comprising an amino acid sequence selected from any one of the following b1) -b 4):

b1)SEQ ID No:26；

b2)SEQ ID No:27；

b3)SEQ ID No:28；

b4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any of the above amino acid sequences, wherein a homodimer thereof has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of b1), b2) or b3) homodimer for CD47 protein.

Embodiment 34 encoding the embodiment of 29-33 any fusion protein nucleic acid molecules.

Embodiment 35. an expression vector comprising the nucleic acid molecule of embodiment 34.

Embodiment 36. a cell comprising the expression vector of embodiment 35.

Embodiment 37. a method of making the fusion protein of any one of embodiments 29-33 comprising:

1) providing a sirpa extracellular truncation;

2) providing a binding sequence for binding to another polypeptide;

3) contacting a sirpa extracellular truncation with a binding sequence for binding to another polypeptide to form the fusion protein.

Embodiment 38. method of making the fusion protein of any one of embodiments 29-33, wherein the cell of embodiment 32 is allowed to express the fusion protein.

Embodiment 39 a pharmaceutical composition comprising a recombinant protein according to any one of embodiments 1-20 or a fusion protein according to any one of embodiments 29-33, and optionally an adjuvant, excipient or pharmaceutically acceptable carrier.

Embodiment 40 the pharmaceutical composition of embodiment 39, which is in the form of an injection or a lyophilized powder.

An embodiment 41. a pharmaceutical composition comprising a nucleic acid molecule encoding a recombinant protein according to any one of embodiments 1-20 or a fusion protein according to any one of embodiments 29-33, and optionally a pharmaceutically acceptable carrier.

Embodiment 42. use of the fusion protein of any one of embodiments 29 to 33 for the preparation of a recombinant protein of any one of embodiments 1 to 20.

Embodiment 43. use of the recombinant protein of any one of embodiments 1 to 20 or the fusion protein of any one of embodiments 29 to 33 for the preparation of a medicament for the treatment of a tumor.

Embodiment 44 the use of embodiment 43, wherein the tumor is selected from the group consisting of hematological and solid tumors such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusions, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

Embodiment 45. use of a nucleic acid molecule encoding the recombinant protein of any one of embodiments 1-20 or the fusion protein of any one of embodiments 29-33, or a derivative thereof, for the manufacture of a medicament for in vivo gene therapy.

Embodiment 46. a method for assessing the safety of early immunity of a recombinant protein/antibody targeting CD47 and having ADCC activity in vitro, the method comprising:

a) providing a targeted recombinant protein/antibody (including monovalent or multivalent) having ADCC activity;

b) detecting the ADCC activity of the recombinant protein/antibody;

c) evaluating the early immune safety of the recombinant protein/antibody.

Embodiment 47 a method for assessing the safety of early immunity of a recombinant protein/antibody targeting CD47 and having ADCC activity in vitro, the method comprising:

a) preparing effector cells, such as, but not limited to, human NK92MI-CD16a effector cells;

b) contacting effector cells with recombinant proteins/antibodies (monovalent or multivalent, inclusive);

c) detecting the ADCC activity of the recombinant protein/antibody;

d) and evaluating the early immune safety of the recombinant protein/antibody according to the ADCC activity result.

Embodiment 48 the method of evaluating embodiment 47, comprising:

a) harvesting well-grown human NK92MI-CD16a effector cells, and resuspending the harvested effector cells to a cell density of 1 × 10⁵Cell/ml to 5X 10⁶Individual cells/ml;

b) incubating the recombinant protein/antibody diluted in a gradient with the effector cells prepared in a), wherein the incubation time is 0.5-5 h;

c) after incubation is finished, measuring LDH activity, and calculating the cell lysis rate;

d) the early immunological safety of the recombinant protein/antibody was evaluated based on the cell lysis rate, wherein the immunological safety of the recombinant protein/antibody resulting in a lower cell lysis rate was high.

Embodiment 49. a method for assessing the safety of early immunity of a recombinant protein/antibody (monovalent or multivalent, inclusive) targeting CD47 in vivo, comprising:

a) providing recombinant proteins/antibodies (including monovalent or multivalent) targeting CD 47;

b) providing a Hu-NSG mouse;

c) contacting the recombinant protein/antibody with a Hu-NSG mouse;

d) evaluating the early immune safety of the recombinant protein/antibody in the Hu-NSG mice.

Embodiment 50. a method for assessing the safety of early immunity of a recombinant protein/antibody (monovalent or multivalent, inclusive) targeting CD47 in vivo, comprising:

a) providing a Hu-NSG mouse;

b) administration of recombinant proteins/antibodies (including monovalent or multivalent);

c) after 24-96h of administration, taking venous blood of the mice, cracking red blood cells, incubating the residual cells for 15-60min by using a fluorescence-labeled anti-human CD45, anti-human CD19 or anti-human CD3 antibody, and detecting by flow cytometry;

d) the early immune safety of the recombinant proteins/antibodies was evaluated based on the cell clearance rate, wherein the immune safety of the recombinant proteins/antibodies that resulted in lower immune cell clearance rate (excluding target cells) was high.

The invention also relates to, but is not limited to, the following items:

item 1. a bispecific recombinant protein, wherein the bispecific recombinant protein comprises a high affinity tumor-targeting arm and a low affinity fusion protein blocking the interaction of CD47 with SIRPa,

the high affinity tumor-targeting arms do not bind CD47, and the high affinity tumor-targeting arms correspond to an antibody that binds to a target antigen on a tumor cell with at least 6-fold greater binding affinity than the monomeric fusion protein homodimer to which the fusion protein that blocks the interaction of CD47 with sirpa corresponds to for CD47 on a tumor cell;

the binding affinity of the fusion protein for blocking the interaction of the CD47 and the SIRPa to the CD47 is not higher than the binding affinity of the homodimer of the monomeric fusion protein containing the SIRPa extracellular truncation to the CD 47; the SIRPa extracellular truncation comprises a human SIRPa wild-type extracellular truncation or a human SIRPa extracellular truncation of a CD47 non-high affinity mutation.

2. The bispecific recombinant protein of item 1, the fusion protein with low affinity for blocking the interaction of CD47 with SIRPa comprises a SIRPa extracellular truncation comprising an amino acid sequence selected from any one of the following a1) -a 4): a1) SEQ ID No. 30; a2) 31 in SEQ ID No; a3) SEQ ID No. 32; a4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any one of the above amino acid sequences, the monomer of which has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of a1), a2) or a3) monomer for CD47 protein.

3. The bispecific recombinant protein of item 1 or 2, which has a left-arm structure and a right-arm structure arranged oppositely, wherein the high-affinity tumor-targeting arm is located at the position of the left arm, and the low-affinity fusion protein for blocking the interaction of CD47 and SIRPa is located at the position of the right arm; preferably, the left arm is a Fab or Fab' form of an immunoglobulin and the right arm is a sirpa extracellular truncation.

4. The bispecific recombinant protein of item 3, wherein the length of the right arm is matched with the distance of the antigen space epitope to be bound by the left arm from the surface of a target cell membrane; preferably, when the arms of the high-affinity targeting tumor need to be combined with an epitope at the membrane proximal end of a target cell, the SIRPa extracellular truncation is selected to contain the amino acid sequences shorter than a1) -a 4).

5. The bispecific recombinant protein according to any one of items 1 to 4, wherein the high affinity tumor-targeting arm targets a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUSC-CSF, MUSC-72, MUDDN-B, MUDG-B, PSMG-B, CTFP-B, CTCD B, CD B, PSMGDF-B, CD B, PSNFR-B, CD 363672, PSNFR-B, CD 36III-B, CD-B, PSNFR-B, CD-B, PSNFR-36III-B, PSNFR-B, CD-36III-B, CD-B, PSDMTF-36III-B, PSNFR-36III-B, PSNFR-36III-B, PSNFR-B, PSTFPI-36III-B, PSNFR-36III-B, PSNFR-B, PSNFL-B, PSNFR-B, PSNFL-B, PSNFR-B, CD B, PSNFR-B, PSNFL-B, PSNFR;

preferably, when the target is CD20, EGFR or PD-L1, the sirpa extracellular truncation selects a 1); when the target is HER2, the sirpa extracellular truncation selects either a1) or a 2).

6. The bispecific recombinant protein of any one of items 1 to 5, wherein the high affinity tumor-targeting arm is bound to a fusion protein with low affinity that blocks the interaction of CD47 with SIRPa by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by salt bonds, or by a combination of two or three of the above binding means.

7. The bispecific recombinant protein of item 6, further comprising an Fc region; preferably, the Fc region comprises an Fc region native sequence or an Fc non-native sequence; more preferably, the Fc region is a human Fc region; even more preferably, wherein the binding of the high affinity tumor targeting arms to the low affinity fusion protein that blocks the interaction of CD47 with SIRPa is via a knob-int-holes binding.

8. The bispecific recombinant protein of item 7, wherein the high affinity tumor-targeting arm is a half-antibody targeting a target selected from the group consisting of: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, HLA-DR, ICAM-1, IGF-B-3, MUSC-CSF, MUSC-72, MUDDN-B, MUDG-B, PSMG-B, CTFP-B, CTCD B, CD B, PSMGDF-B, CD B, PSNFR-B, CD 363672, PSNFR-B, CD 36III-B, CD-B, PSNFR-B, CD-B, PSNFR-36III-B, PSNFR-B, CD-36III-B, CD-B, PSDMTF-36III-B, PSNFR-36III-B, PSNFR-36III-B, PSNFR-B, PSTFPI-36III-B, PSNFR-36III-B, PSNFR-B, PSNFL-B, PSNFR-B, PSNFL-B, PSNFR-B, CD B, PSNFR-B, PSNFL-B, PSNFR; half antibodies, preferably IgG1 antibodies, optionally human murine chimeric half antibodies, humanized half antibodies, fully human half antibodies; more preferably a half antibody of humanized or fully human IgG1 antibody.

9. The bispecific recombinant protein of item 7 or 8, wherein the fusion protein with low affinity that blocks the interaction of CD47 with SIRPa comprises an amino acid sequence selected from any one of the following b1) -b 4): b1) 26 is SEQ ID No; b2) 27 in SEQ ID No; b3) 28 in SEQ ID No; b4) an amino acid sequence obtained by adding, deleting, modifying and/or conservatively substituting at least one amino acid residue, such as 1 to 5 amino acid residues, in any of the above amino acid sequences, wherein a homodimer thereof has an amino acid sequence having a binding affinity for CD47 protein which is not higher than the binding affinity of b1), b2) or b3) homodimer for CD47 protein.

10. The bispecific recombinant protein according to item 9,

wherein when the high affinity tumor targeting arm targets CD20, the high affinity tumor targeting arm comprises SEQ ID No. 16 and SEQ ID No. 17, and the low affinity fusion protein that blocks the interaction of CD47 and SIRPa comprises SEQ ID No. 26;

wherein when the high affinity tumor targeting arm targets EGFR, the high affinity tumor targeting arm comprises SEQ ID No. 19 and SEQ ID No. 8, and the low affinity fusion protein that blocks the interaction of CD47 and SIRPa comprises SEQ ID No. 26;

wherein when the high affinity tumor targeting arm is targeted to Her2, the high affinity tumor targeting arm comprises SEQ ID No. 20 and SEQ ID No. 21, or SEQ ID No. 22 and SEQ ID No. 23, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26 or SEQ ID No. 27; or the like, or, alternatively,

wherein when the high affinity tumor targeting arm is targeted to PD-L1, the high affinity tumor targeting arm comprises SEQ ID No. 24 and SEQ ID No. 13, and the low affinity fusion protein for blocking the interaction of CD47 and SIRPa comprises SEQ ID No. 26.

11. A nucleic acid molecule encoding the bispecific recombinant protein of any one of items 1 to 10; preferably, wherein the nucleic acid molecule encoding the high affinity tumor targeting arm is in the same DNA strand as the nucleic acid encoding the fusion protein of the low affinity block CD47 interacting with sirpa or the nucleic acid molecule encoding the high affinity tumor targeting arm is in a different DNA strand from the nucleic acid encoding the fusion protein of the low affinity block CD47 interacting with sirpa.

12. An expression vector comprising the nucleic acid molecule of item 11.

13. A host cell comprising the expression vector of item 12.

14. A method for producing a bispecific recombinant protein, which comprises expressing the recombinant protein using the cell of item 13.

15. The use of the bispecific recombinant protein of any one of items 1 to 10 in the preparation of a medicament for treating a tumor; preferably, the tumor is selected from the group consisting of hematological tumors and solid tumors, such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, fibrosarcoma protrusion of the skin, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

Drawings

FIG. 1 is a schematic structural diagram of an insert to be inserted into an expression vector according to the present invention.

FIG. 2 is a plasmid map of an exemplary expression vector pCHO-TE2 used in the present invention.

FIG. 3 is a schematic structural view as an exemplary embodiment of the recombinant protein of the present invention.

FIGS. 4A-4B are SDS-PAGE electrophoresis of recombinant protein purified by protein A. Lanes 1-6 of FIG. 4A are reducing samples, respectively: 1: marker, 2: Anti-CD20 mAb (Ofatumumab), 3: Anti-CD47 mAb (Hu5F9-G4), 4: SIRP α D1-Fc, 5: Ofa-Fc1-D1-Fc2, 6: Ofa-Fc1-D1-D2-D3-Fc 2; lanes 7-12 of FIG. 4A are non-reducing samples, which are: 7: marker, 8: Anti-CD20 mAb (Ofatumumab), 9: Anti-CD47 mAb (Hu5F9-G4), 10: SIRP α D1-Fc, 11: Ofa-Fc1-D1-Fc2, 12: Ofa-Fc1-D1-D2-D3-Fc 2. FIG. 4B shows a non-reducing sample Ofa-Fc1-D1-D2-Fc2 in lane 1, a Marker in lane 2, and a reducing sample Ofa-Fc1-D1-D2-Fc2 in lane 3.

FIG. 5 shows the results of ELISA assay for binding affinity (protein level) of the recombinant protein of the present invention to human CD47.

FIG. 6 shows the results of flow cytometry for the binding affinity (cellular level) of the recombinant protein of the present invention to human CD47.

FIG. 7 shows the results of flow cytometry to determine the binding force of the recombinant protein of the present invention to the corresponding left arm target. Hu5F9-G4, Ofa-Fc1-D1-Fc2, SIRP α D1-Fc correspond to the mean fluorescence intensity after binding of the samples Hu5F9-G4, Ofa-Fc1-D1-Fc2, SIRP α D1-Fc to Raji cells not blocked by the anti-CD47 antibody Hu5F9-G4(Fab)2, while Hu5F9-G4(b), Ofa-Fc1-D1-Fc2(b), SIRP α D1-Fc (b) correspond to the mean fluorescence intensity after binding of the samples Hu5F9-G4, Ofa-Fc1-D1-Fc2, SIRP α D1-Fc to Raji cells blocked by the anti-CD47 antibody Hu5F9-G4(Fab) 2.

FIG. 8 shows the results of flow cytometry to determine the binding affinity of the recombinant protein of the present invention to CD47 and left arm target double positive cells. FIG. 8A used Raji cells (CD20+ CD47), FIG. 8B used SKBR-3 cells (Her2+ CD47), FIG. 8C used A431 cells (EGFR + CD47), and FIG. 8D used NCI-H441 cells (PD-L1+ CD 47).

FIG. 9 shows the result of ELISA assay for competitive binding of the recombinant proteins of the present invention to CD47 with SIRPa D1-Fc and anti-CD47 antibody.

FIG. 10 shows the results of flow cytometry to determine the binding affinity of the recombinant protein of the present invention and its CD47 high-affinity mutant, anti-CD47 antibody and anti-CD20 antibody to human CD47.

FIG. 11 shows the results of the in vitro immunological safety evaluation experiment of the recombinant protein of the present invention.

FIG. 12 shows the results of the experimental results of the efficacy of the recombinant protein of the present invention in mouse model NSG subcutaneously transplanted to Raji lymphoma.

FIG. 13 shows the results of the measurement of B cell content 96 hours after Hu-NSG mice were treated with different samples at the same dose. Among them, 0.9% physiological saline was used in FIG. 13A, Hu5F9-G4 (6.7. mu.g/mouse) was used in FIG. 13B, and Ofa-Fc1-D1-Fc2 (5. mu.g/mouse) was used in FIG. 13C.

FIG. 14 is Ofa-Fc1-D1-Fc2 and Ofa-Fc1-D1^mEarly safety evaluation of-Fc 2 in Hu-NSG mice (FACS immunocytotyping assay). Each of FIGS. 14A to D used Ofa-Fc1-D1-Fc2 (1. mu.g/mouse), in which FIGS. 14A and 14B are graphs showing the results of examination before administration and FIGS. 14C and 14D are graphs showing the results of examination after 72 hours of administration. FIGS. 14E-H all used Ofa-Fc1-D1^mFc2 (1. mu.g/mouse), wherein FIGS. 14E and 14F are graphs of the results of the assay before administration, and FIGS. 14G and 14H are graphs of the results of the assay after 72 hours of administration.

FIG. 15 is a 96 hour FACS immunocytotyping assay after treatment of Hu-NSG mice with different samples at high dose. FIGS. 15A-B show the results 96 hours post-dose, where panel A used Hu5F9-G4 (200. mu.g/dose) and FIG. 15B used Ofa-Fc1-D1-Fc2 high dose (150. mu.g/dose). FIGS. 15C-D are results at 14 days post-dose, with FIG. 15C using Hu5F9-G4(200 μ G/dose) and FIG. 15D using Ofa-Fc1-D1-Fc2 high dose (150 μ G/dose).

FIG. 16 shows the binding of the recombinant protein of the invention to dual targets (Her2, CD47) in SKBR-3. FIG. 16A shows the results of testing samples of Anti-Her2(T) -Fc1-D1-Fc2 and Anti-Her2(T) -Fc1-D1-D2-Fc2, and FIG. 16B shows the results of testing samples of Anti-Her2(P) -Fc1-D1-Fc2 and Anti-Her2(P) -Fc1-D1-D2-Fc 2.

FIGS. 17A-E are the corresponding amino acid and DNA sequences of exemplary proteins of the invention.

FIG. 18A is the effect of two doses of Ofa-Fc1-D1-Fc2 on cynomolgus monkey red blood cell numbers, and FIG. 18B is the effect of two doses of Ofa-Fc1-D1-Fc2 on cynomolgus monkey hemoglobin.

FIG. 19 shows the results of the two doses of Ofa-Fc1-D1-Fc2 in detecting the B cell content of cynomolgus monkey.

FIG. 20 shows Ofa-Fc1-D1-Fc2,Effect on growth of human B-cell lymphoma Daudi subcutaneous graft tumor.

FIG. 21 shows Ofa-Fc1-D1-Fc2,Effect on body weight of tumor-bearing mice.

FIG. 22 shows Ofa-Fc1-D1-Fc2,Tumor photograph of therapeutic effect on human B-cell lymphoma Daudi subcutaneous graft tumor.

Detailed Description

For the purposes of promoting an understanding of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It should be understood, however, that these specific embodiments are not intended to limit the scope of the invention. Any alterations and further modifications in the described embodiments, and any further applications of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.

Recombinant proteins

As used herein, the term "recombinant protein" refers to an artificially designed/constructed protein, rather than a naturally occurring protein. The "recombinant" in the "recombinant protein" of the present invention does not represent the manner in which it is produced, and is used merely to indicate that the "recombinant protein" does not naturally occur. The recombinant protein of the present invention may be an expressed protein, and may be an assembled protein.

Optionally, the recombinant proteins of the invention comprise high affinity tumor-targeting arms and low affinity fusion proteins that block the interaction of CD47 with sirpa.

As used herein, "high affinity tumor targeting" means that the recombinant protein of the present invention has a higher or approximately equivalent tumor binding affinity as compared to prior art tumor-binding antibody-based drugs, which typically have EC50 at nM or pM levels. Preferably, in the recombinant proteins of the invention, the binding affinity of the antibody to the target antigen on the tumor cell corresponding to the high affinity tumor-targeting arm is at least 6-fold, optionally 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or higher fold value, or any value therebetween, of the binding affinity of the monomeric fusion protein homodimer corresponding to the fusion protein with low affinity blocking the interaction of CD47 with sirpa on to CD47 on the tumor cell. Optionally, in the recombinant proteins of the invention, the binding affinity of the antibody to the target antigen on the tumor cell that corresponds to the high affinity tumor-targeting arm is at least 6-fold, optionally 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or higher fold value, or any value therebetween, of the binding affinity of the sirpa-Fc homodimer to CD47 on the tumor cell that corresponds to the fusion protein that blocks the interaction of CD47 with sirpa.

As used herein, "low affinity block CD47 from interacting with sirpa" means that the recombinant protein of the invention is capable of blocking CD47 from interacting with sirpa with lower affinity. Preferably, the fusion protein with low affinity that blocks the interaction of CD47 and sirpa in the recombinant protein of the invention has no higher binding affinity for CD47 than the homodimer of the monomeric fusion protein containing a sirpa extracellular truncation for CD47. More preferably, the binding affinity of the human SIRPa-Fc fusion protein to CD47 is not higher.

When the binding affinity of the antibody corresponding to the arm of the bispecific recombinant protein with high affinity targeting tumor to the target antigen on the tumor cell is at least 6 times of that of the monomer fusion protein homodimer corresponding to the arm of the fusion protein for blocking the interaction between CD47 and SIRPa to the CD47 on the tumor cell, the defects of obviously improving the tumor targeting saturated binding abundance of the recombinant protein with macrophage function and reducing the side effect of non-tumor targeting can be realized.

Methods for determining binding affinity as used herein are well known to those skilled in the art and include, for example, but are not limited to, ELISA and/or flow cytometry.

Optionally, the signal-regulating protein α extracellular truncation (including wild-type extracellular truncations and non-high affinity mutants thereof) -Fc fusion proteins and half-antibodies of the present invention may be produced by engineering the heavy chain of an antibody to form heterodimers, in particular, recombinant proteins may be obtained, for example, by Knobs-into-holes linkage and/or interchain disulfide bond and/or salt bond mediation.

Optionally, when the "tumor-targeting arm" or "half-antibody" or "left arm" or "half-antibody structure" or "Ig molecule monomer" is linked to the "CD 47-targeting arm" or "right arm" or "signal-regulatory protein α truncation-Fc fusion protein" or "sirpa-Fc" via the nanobs-endo-holes to give a recombinant protein, the structure of the resulting recombinant protein is shown in fig. 3. Wherein the left arm is a tumor targeting half antibody including, but not limited to, an anti-5T 4 half antibody, an anti-AGS-16 half antibody, an anti-ALK 1 half antibody, an anti-ANG-2 half antibody, an anti-B7-H3 half antibody, an anti-B7-H4 half antibody, an anti-c-fms half antibody, an anti-c-Met half antibody, an anti-CA 6 half antibody, an anti-CD 123 half antibody, an anti-CD 19 half antibody, an anti-CD20 half antibody, an anti-CD 22 half antibody, an anti-EpCAM half antibody, an anti-CD 30 half antibody, an anti-CD 32B half antibody, an anti-CD 37 half antibody, an anti-CD 38 half antibody, an anti-CD 40 half antibody, an anti-CD 52 half antibody, an anti-CD 70 half antibody, an anti-CD 74 half antibody, an anti-CD 79B half antibody, an anti-CD 98 half antibody, an anti-CEA half antibody, an anti-CEA C5 half antibody, an anti-C18.2 half antibody, a CLDN 66 half antibody, an anti-C867 half antibody, an anti-EGCS 867 half antibody, an anti-EGCP 3 half antibody, an anti-EGCP 363672 half antibody, an anti-EGFR half antibody, an anti-EGCP 363672 antibody, an anti-EGFR half antibody, an anti-EGFR antibody, an anti-C3636363636363672 antibody, an anti-C3 half antibody, an anti-C3 antibody, an anti-C3 half antibody, an anti-C3 half antibody, an anti-C3 antibody, an anti-C-III antibody, an anti-C-III antibody, an anti-C-III antibody, an anti-C-III antibody, an anti-C-human antibody, an anti, anti-EphA 3 half-antibody, anti-ETBR half-antibody, anti-FGFR 2 half-antibody, anti-FN half-antibody, anti-FR-alpha half-antibody, anti-GCC half-antibody, anti-GD 2 half-antibody, anti-GPC-3 half-antibody, anti-GPNMB half-antibody, anti-HER2 half-antibody, anti-HER 3 half-antibody, anti-HLA-DR half-antibody, anti-ICAM-1 half-antibody, anti-IGF-1R half-antibody, anti-IL-3R half-antibody, anti-LIV-1 half-antibody, anti-MSLN half-antibody, anti-MUC 16 half-antibody, anti-MUC 1 half-antibody, anti-NaPi 2b half-antibody, anti-lectin-4 half-antibody, anti-Notch 2 half-antibody, anti-Notch 1 half-antibody, anti-PD-L1 half-antibody, anti-PD-L2 half-antibody, anti-PDGFR-alpha half-antibody, anti-PS half-antibody, anti-PSMA half-antibody, anti-SLK 6 half-antibody, anti-STEAP 1 half-VEGFR antibody, anti-CD 73727 anti-CD 6384 half-antibody, anti-HER 1 half-HER antibody, anti-HER-MUC-TNF-11 half-antibody, anti-NAP-TNF-half-antibody, anti-TNF-antibody, anti-TNF-CD-TNF-CD-4 half-CD-antibody, anti-CD-antibody, anti-CD b half-CD-4 half-CD-4 half-CD-antibody, anti-CD-, anti-CSF-1R half antibody, anti-MSB 0010718C half antibody, anti-BCMA half antibody, anti-CD 138 half antibody. The right arm is a fusion protein formed by connecting an extracellular truncation of SIRP alpha (including a human SIRP alpha wild type and a non-high affinity mutant thereof) with a hinge region and an Fc region of an IgG antibody. When the right arm Fc region is a hole mutant, the corresponding left arm Fc region is a knob mutant; when the right arm is a knob mutant, the Fc region corresponding to the left arm is a hole mutant. As known to those skilled in the art, the Fc region may also be mutated simultaneously by multiple holes and/or knobs.

Table 1 below is an exemplary molecular structure of the recombinant protein.

Table 1 molecular Structure example of recombinant protein

Serial number	Left arm	Right arm
			1	Anti-CD20-Fc1	D1-Fc2
2	Anti-CD20-Fc1	D1-D2-Fc2
			3	Anti-CD20-Fc1	D1-D2-D3-Fc2
4	Anti-CD20-Fc1	D1m-Fc2
			5	Anti-CD20-Fc1	D1m-D2-Fc2
6	Anti-PD-L1-Fc1	D1-Fc2
			7	Anti-PD-L1-Fc1	D1-D2-Fc2
8	Anti-PD-L1-Fc1	D1-D2-D3-Fc2
			9	Anti-PD-L1-Fc1	D1m-Fc2
10	Anti-PD-L1-Fc1	D1m-D2-Fc2
			11	Anti-EGFR-Fc1	D1-Fc2
12	Anti-EGFR-Fc1	D1-D2-Fc2
			13	Anti-EGFR-Fc1	D1-D2-D3-Fc2
14	Anti-EGFR-Fc1	D1m-Fc2
			15	Anti-EGFR-Fc1	D1m-D2-Fc2
16	Anti-Her2-Fc1	D1-Fc2
			17	Anti-Her2-Fc1	D1-D2-Fc2
18	Anti-Her2-Fc1	D1-D2-D3-Fc2
			19	Anti-Her2-Fc1	D1m-Fc2
20	Anti-Her2-Fc1	D1m-D2-Fc2

D1^mA high affinity mutant representing sirpa extracellular truncation D1; d1 represents the extracellular D1 domain of human SIRP alpha wild type and its non-high affinity mutants; fc means a wild-type Fc region, Fc1 means an Fc region with hole or holes mutation, Fc2 means an Fc region with knob or knobs mutation

The corresponding amino acid sequence and DNA sequence of the recombinant protein of the invention are shown in FIGS. 17A-E and in the sequence listing file of the invention.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

Tumor-targeting arms

As used herein, the term "tumor-targeting arm" or "half-antibody" or "left arm" or "half-antibody structure" or "Ig molecule monomer" refers to a heterodimeric glycoprotein consisting of one light (L) and one heavy (H) chain of an antibody, which is the basic structure that makes up an immunoglobulin molecule and is used interchangeably in the present invention; the molecular weight corresponds to half the molecular weight of the antibody, about 75000 daltons, with the light chain linked to the heavy chain by a covalent disulfide bond. The heavy and light chains also have regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the terms "tumor-targeting arm", "half-antibody" or "left arm" or "half-antibody structure" or "Ig molecule monomer" can be a variety of tumor-targeting IgG proteins. Tumor target molecules include, but are not limited to, 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32B, CD79B, CD B, CEACAM B, CLDN18.2, CLDN B, CS B, CXCR B, DLL-4, EGFR, EGP-1, ENPP B, EphA B, ETBR, FGFR B, FN, FR-alpha, GCC, GD B, GPC-3, GPNMB, HER B, DR-HLA-1, STEICAM-361, CSF-B, MUFR-72, MUFR-alpha, GCC, GD B, PDG-B, CTPD-B, CTFA-B, CTFP-B, SLRP-B, SLEP-B, SLN-B, SLEP-B, SLTFPD-B, SLPSMC-B, SLRP-B, SLN-B, SLTFPD-B, SLPSGL-B, SLTFPD-B, SLK-B, SLTFPD-B, SLPSD B, SLTFPD-B, SLK-B, SLN-B, SLK-B, SLPSD-B, SLN-B, SLK-B, SLC-B, SLPSD-B, SLTFMC-B, SLTFPD-B, SLTFPI-B, SLN-B, SLK-B, SLC, SLTFPI-B, SLPSD 36138, and PSD 36138.

The Fc sequence of the "tumor-targeting arm", "half-antibody" or "left arm" or "half-antibody structure" or "Ig molecule monomer" may employ hole or holes mutants and/or knob mutants.

Targeting arms of CD47

As used herein, the terms "arm targeting CD 47" or "right arm" or "signal-regulatory protein a truncation-Fc fusion protein" or "sirpa-Fc" or "fusion protein blocking the interaction of CD47 with sirpa" are used interchangeably in the present invention. As known to those skilled in the art, the "arm" or "right arm" targeting CD47 or "signal-modulator-alpha-truncation-Fc fusion protein" or "sirpa-Fc" or "fusion protein blocking the interaction of CD47 with sirpa" molecules can vary in length. Optionally, a variety of molecules of different lengths can be formed by linking the extracellular truncation of sirpa (including human sirpa wild-type and non-high affinity mutants thereof) to the hinge and Fc regions of IgG1 antibody to form "CD 47-targeting arms" or "right arms" or "signal-regulatory protein α truncation-Fc fusion proteins" or "sirpa-Fc" or "fusion proteins that block the interaction of CD47 with sirpa". IgG1 may be human IgG 1.

The Fc of an "arm targeting CD 47" or "right arm" or "signal-regulatory protein α truncation-Fc fusion protein" or "sirpa-Fc" or "fusion protein blocking the interaction of CD47 with sirpa" may employ a knob or knob mutant and/or a hole or holes mutant.

"tumor-targeting arm" or "half-antibody" or "left arm" or "half-antibody structure" or "Ig molecule monomer" and "CD 47-targeting arm" or "right arm" or "signal-regulatory protein α truncation-Fc fusion protein" or "sirpa-Fc" or "fusion protein that blocks the interaction of CD47 with sirpa" as known to those skilled in the art, can be engineered to form heterodimeric recombinant proteins by engineering the Fc fragment (domain). Specifically, the recombinant protein of the present invention can be obtained by intermolecular force, a covalent bond such as interchain disulfide bond, a salt bond-mediated bond, or any combination of two or three of the above-mentioned three techniques. Optionally, the recombinant proteins of the invention are bound by the knobs-into-holes technique.

knobs-intoholes technique

As used herein, the term "knob-to-holes" or "knob-to-hole" or "button" is used to introduce different mutations into the two CH3 domains of a heavy chain to promote heterodimerization of the heavy chains, using genetic Engineering techniques to create a knob (knob) on one heavy chain and a button (hole) on the other heavy chain, which then preferentially snap together to form an asymmetric antibody (Ridgway JB 621. Knobs-to-holes' Engineering of antibodies CH3 domains for fatty chain ligation. protein Engineering,1996,9(7): 617). As known to those skilled in the art, multiple buttons (knobs) and/or buttons (holes) may be made on one heavy chain, and correspondingly, multiple buttons (holes) and/or buttons (knobs) may be made on the other heavy chain.

SIRPα

As used herein, the term "sirpa" is a Signal Regulatory Protein a (Signal Regulatory Protein a), also known as CD172 a. The signal regulatory protein (SIRP) is a transmembrane glycoprotein and comprises three family members, SIRP alpha (CD172a), SIRP beta (CD172b) and SIRP gamma (CD172 g). The three members have similar outer ends of the membrane, but different inner regions of the membrane. The outer ends of the membrane each contain three immunoglobulin (Ig) -like regions, the first of which belongs to the IgV region and the second and third of which belong to the IgC region. The membrane inner region of SIRP alpha (CD172a) contains two inhibitory signal domains, which can transmit inhibitory signals and inhibit the corresponding functions of cells. The membrane interior regions of SIRP beta (CD172b) and SIRP gamma (CD172g) are very short and have no signal transmission region, butSIRP β (CD172b) can transmit an activation signal through an Adaptor protein (such as DAP 12). SIRP protein is mainly expressed in macrophageDendritic Cells (DCs), and neuronal cells. The invention particularly relates to a human SIRP alpha wild type and a CD47 non-high-affinity mutant thereof.

SIRP alpha extracellular truncation

"extracellular truncation" refers to a truncation directed to a protein that functions across a membrane. "SIRPa extracellular truncation", as used herein, refers to a human SIRPa wild-type and its CD47 non-high affinity mutants, selectively truncating all or part of its amino acid sequence located in the outer space of the cell membrane.

As used herein, the terms "D1, D2, D3" refer to the three extracellular Ig-like domains of SIRP α, in the order from the amino terminus of the Protein, the D1 domain (Ig variable region-like domain, IgV region), the D2 domain (Ig constant region-like domain, IgC region) and the D3 domain (Ig constant region-like domain, IgC region) (Lee WY, et al. the Role of cis differentiation of Signal Regulatory Protein α (SIRP α) in Binding CD47.J Biol Chem, 2010, 285 (49): 37953-37963).

SIRP alpha-Fc fusion protein

The term "sirpa-Fc fusion protein" as used herein refers to a fusion protein comprising a sirpa extracellular truncation, a linker sequence, and an Fc region. The linker sequence and/or Fc region included in the above sequences may be optionally substituted according to a manner well known to those skilled in the art or a commonly used linker sequence and/or Fc region.

In order to avoid the effect of glycosylation, D1 was mutated from asparagine to alanine (ref: Lee W Y, et al. novel Structural definitions on SIRP. alpha. that media Binding to CD47.journal of Immunology,2007,179(11): 7741-.

D1, D2 and D3 of the invention also comprise corresponding linker sequences.

Linker sequences

As used herein, the term "linker sequence" refers to an amino acid sequence that links a sirpa extracellular truncation and a binding sequence, optionally, a hinge region of an IgG antibody, optionally, comprising a hinge region and an IgG heavy chain CH1 domain. The linker sequence or the hinge region sequence included in the above sequence may be arbitrarily substituted according to a manner known to those skilled in the art or a commonly used linker sequence or hinge region sequence.

Binding sequences

As used herein, the term "binding sequence" refers to a sequence that binds the arms of a high affinity tumor-targeting to a fusion protein that has a low affinity to block the interaction of CD47 with sirpa, optionally the binding sequence comprises a hinge region and an Fc region, more optionally the Fc region comprises a knob or knob and/or hole or holes mutation. The binding sequence or hinge or Fc region sequence contained in the above sequence may be arbitrarily replaced according to a manner well known to those skilled in the art or a commonly used binding sequence or hinge or Fc region sequence.

CD47

CD47 is a transmembrane glycoprotein belonging to a member of the immunoglobulin superfamily and is expressed on the surface of almost all cells including erythrocytes. Ligands for CD47 include adhesion factor (integrin), thrombospondin 1(thrombospondin-1), and signal-regulating protein (SIRP). CD47 has a variety of biological functions including cell migration, T cells, dendritic cell activation, axonal development, and the like. In addition, CD47 inhibits phagocytosis of macrophages by interacting with sirpa. CD47 transmits a so-called "Don' T eat me" signal in this manner, which protects normal cells such as red blood cells, B cells, T cells, etc. from phagocytosis by macrophages.

Ofa

As used herein, the terms "Ofa", "Ofatumumab", "Anti-CD 20 (Ofatumumab)" are used interchangeably herein to denote the Anti-CD20 antibody Ofatumumab.

Obi

As used herein, the terms "Obi", "Obinutuzumab", "Anti-CD 20 (Obinutuzumab)" are used interchangeably in the present invention to denote the Anti-CD20 antibody Obinutuzumab.

Hu5F9-G4

As used herein, the terms "Anti-CD 47 mAb", "Anti-CD 47 antibody", "Hu 5F 9-G4" are used interchangeably in the present invention to denote the Anti-CD47 antibody Hu5F 9-G4.

Anti-EGFR mAb

As used herein, the terms "Anti-EGFR mAb", "JMT 101" are used interchangeably in the present invention to denote the Anti-EGFR antibody JMT 101. JMT101 is a humanized anti-EGFR monoclonal antibody, see ZL201210406288.3 patent BA 03.

Trastuzumab

As used herein, the terms "Trastuzumab", "Anti-Her 2(T) mAb", "Herceptin" are used interchangeably herein to refer to the Anti-Her2 antibody Trastuzumab.

Pertuzumab

As used herein, the terms "Pertuzumab", "Anti-Her 2(P) mAb", "Perjeta" are used interchangeably herein to denote the Anti-Her2 antibody Pertuzumab.

Atezolizumab

As used herein, the terms "tecentiq", "Atezolizumab" are used interchangeably herein to refer to the anti-PD-L1 antibody Atezolizumab.

SIRPαD1-Fc

As used herein, the terms "sirpa D1-Fc", "D1-Fc" are used interchangeably in the present invention to denote a dimer of the single chain fusion protein sirpa D1-Fc.

Ofa-Fc1

Represents an Ofatumumab half-antibody having an Fc region with hole mutation.

Anti-Her2(T)-Fc1

Represents a Transtuzumab half-antibody with hole mutated Fc region.

Anti-Her2(P)-Fc1

Represents Pertuzumab half antibody with hole mutated Fc region.

Anti-EGFR-Fc1

Represents an anti-EGFR half-antibody having a hole-mutated Fc region.

D1-Fc2

Represents a fusion protein comprising an extracellular truncation of the sirpa D1 domain and an Fc region with a knob mutation.

D1-D2-Fc2

Represents a fusion protein comprising an extracellular truncation of the sirpa D1 and D2 domains and an Fc region with a knob mutation.

D1-D2-D3-Fc2

Represents a fusion protein comprising an extracellular truncation of the sirpa D1, D2 and D3 domains and an Fc region with a knob mutation.

Treatment of

As used herein, the terms "treatment," "therapy," and "treating" are used interchangeably. The term "treating" includes controlling the progression of a disease, disorder, condition, and associated symptoms, preferably reducing the effect of one or more symptoms of the disease, disorder, condition. This term includes curing the disease or eliminating the symptoms altogether. The term includes relief of symptoms. This term also includes, but is not limited to, non-curative palliative treatment. The term "treating" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the invention to prevent or delay, alleviate or ameliorate the progression of a disease, disorder, condition, or the effects of one or more symptoms of a disease, disorder, condition.

Administration of

As used herein, the term "administering" refers to delivering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the invention to a subject. Administration may be systemic or local. Administration may be by an administration device, such as a syringe. Modes of administration include, but are not limited to, embedding, nasal inhalation, spraying, injection, and the like. Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration, and the like.

TABLE 2-1 correspondence of sequence names and sequence numbers

TABLE 2-2 correspondence of recombinant proteins and sequences

EXAMPLE 1 construction of expression vectors

According to the designed molecular structure, the amino acid sequences of all the components are spliced together, an optimal DNA coding sequence is designed according to the preference of Chinese hamster (Cricetulus griseus) to codons, enzyme cutting sites required by gene cloning operation and the like are eliminated, then cloning sites, a Kozak sequence and a signal peptide coding sequence are sequentially added to the 5 'end of the sequence, and a termination codon and a cloning site are sequentially added to the 3' end of the sequence, as shown in figure 1.

And (3) carrying out whole gene synthesis, directionally cloning the whole gene between corresponding cloning sites of an expression vector pCHO-TE2 (purchased from Thermo Fisher) by utilizing cloning sites at a 5 'end and a 3' end, and obtaining an expression plasmid after sequencing verification. The cloning sites used at the 5 'and 3' ends were EcoRV and PacI sites. FIG. 2 is a plasmid map of expression vector pCHO-TE 2.

EXAMPLE 2 preparation of expression plasmid, cell transfection, and expression and purification of target protein

Preparation of expression plasmid

Glycerol bacteria containing expression plasmids (1mL of escherichia coli liquid containing expression plasmids is added with 0.5mL of 60% sterile glycerol solution and mixed well) according to the proportion of 1:1000 in proportion was inoculated into liquid LB medium. The cells were cultured at 37 ℃ and 220rpm for 16 hours by shaking and then centrifuged to collect the cells. The expression plasmid was extracted using endotoxin-free plasmid macroextraction kit (DP117, available from Tiangen Biochemical technology, Inc. (Beijing) according to the standard procedures provided in the kit instructions.

Cell transfection, protein expression

After the resulting expression plasmid was filtered through a 0.22 μm filter, 3mg of the plasmid (where the product was a typical antibody molecule and the ratio of the light chain to heavy chain expression plasmid was 1:1 (molar ratio); and the product was a recombinant protein and the ratio of the light chain to heavy chain to right arm expression plasmid was 1:1:1 (molar ratio); see Table 3 for details) was added to 50mL of Opti MEM I Reduced Serum Medium (available from GIBCO) and mixed well. 6mg of the transfection reagent Polyetherimide (PEI, available from Polysciences, dissolved in sterile ultrapure water at a concentration of 1 mg/mL) was pipetted into 50mL Opti MEM I Reduced Serum Medium and mixed well. The resulting PEI solution was added to an Opti MEM I Reduced Serum Medium solution containing the plasmid and mixed well. After standing at room temperature for 15 minutes, the mixture of plasmid and PEI was slowly and uniformly added to a volume of 1L at a cell density of 3X 10⁶Individual cells/mL of host cell CHO-S (purchased from Thermo Fisher) suspension, incubated at 37 ℃ with 5% CO₂Culturing in an incubator. After 4 hours, a feed medium corresponding to 7% of the initial volume was added (the feed medium formulation was 80g of CD Efficient feed C AGT (from Gibco), 75g of 5X 00483 (from Kerry) dissolved in one liter of water). The culture temperature was lowered to 33 ℃ and harvested after 6 days of culture. The cell suspension was centrifuged at 10000g at 10 ℃ for 30 minutes, and the supernatant obtained by centrifugation, i.e., the cell culture harvest, was used for purification of the target protein.

Protein purification

Protein A affinity capture is exemplified by the following method Ofa-Fc1-D1-Fc 2.

The cell culture harvest is centrifuged for 30min at 10000rpm to remove cells and fragments thereof, and then a protein A affinity column (cargo number: 17-5438-02, GE Healthcare) is loaded for elution to harvest the target protein. SDS-PAGE detects protein purity.

The protein A purification method is a conventional protein purification method well known to those skilled in the art, and the specific test method can refer to the GE Healthcare protein A product instruction and the GE antibody purification manual.

The theoretical molecular weights of four proteins of SIRP alpha D1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2 are respectively as follows: 37.8kD, 110.7kD, 121.7kD and 131.4 kD. The results of SDS-PAGE protein electrophoresis are shown in FIG. 4A and FIG. 4B.

Protein electrophoresis (SDS-PAGE): the results show (FIG. 4A and FIG. 4B) that the target proteins in each lane were efficiently expressed and purified, wherein Ofa-Fc1-D1-Fc2 (FIG. 4A lane 11), Ofa-Fc1-D1-D2-Fc2 (FIG. 4B lane 1), Ofa-Fc1-D1-D2-D3-Fc2 (FIG. 4A lane 12) showed different degrees of left arm dimer (Ofa-Fc1-Ofa-Fc1), right arm dimer (SIRP α -Fc2) and/or multimer.

TABLE 3 expression plasmid ratios

Remarking: d1^mA high affinity mutant representing sirpa extracellular truncation D1; d1 represents the extracellular D1 domain of human SIRP alpha wild type and its non-high affinity mutants; fc is a wild-type Fc region, Fc1 is an Fc region with hole or holes mutation, Fc2 is an Fc region with knob or knobs mutation

Example 3 detection of target affinity, target Competition binding Activity

1. Method for detecting affinity of CD47, CD20, EGFR and Her2 targets

Recombinant proteins Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1^m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1^m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1^mThe binding affinity of-D2-Fc 2, Obi-Fc1-D1-D2-D3-Fc2 to the targets CD47 and CD20 was determined by ELISA and/or flow cytometry. The following method is exemplified by Ofa-Fc1-D1-Fc2, and is applicable to a weight whose left arm is a CD20 targetAnd (4) detecting the histone.

Recombinant proteins Anti-Her2(T) -Fc1-D1-Fc2, Anti-Her2(T) -Fc1-D1^m-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1^m-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1^m-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1^mThe binding affinity of-D2-Fc 2, Anti-Her2(P) -Fc1-D1-D2-D3-Fc2 to the targets CD47 and Her2 was determined by ELISA and/or flow cytometry. The following method takes Anti-Her2(T) -Fc1-D1-Fc2 as an example, and is suitable for detecting the recombinant protein with the left arm as the Her2 target.

Recombinant proteins Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1^m-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1^mThe binding affinity of D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2 to the targets CD47 and EGFR was determined by ELISA and/or flow cytometry. The following method takes Anti-EGFR-Fc1-D1-Fc as an example, and is suitable for detecting recombinant protein with the left arm as an EGFR target.

Recombinant proteins Anti-PD-L1(Ate) -Fc1-D1-Fc2, Anti-PD-L1(Ate) -Fc1-D1^m-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1^m-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1^mThe binding affinity of-D2-Fc 2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 to the targets CD47 and PD-L1 was determined by ELISA and/or assay. The following method takes Anti-PD-L1(Ate) -Fc1-D1-Fc2 as an example, and is suitable for detection by taking a recombinant protein with a PD-L1 target point as an example.

ELISA was used to detect the affinity of Ofa-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-Fc2 to the target CD47：

The microplate (cat # 9018, Corning) was coated with 100. mu.l of 1. mu.g/ml CD47-His (12283-H08H-200, Sino Biological) and 4 ℃ CStanding overnight; after washing with PBST solution (PBS containing Tween20 at a concentration of 0.1%), blocking the ELISA plate with PBS + 1% BSA at room temperature for 2 hours; after washing, the coated plates were added with diluted Ofa-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-Fc2 (starting at 1000ng/ml, 2.5-fold dilution, 11 points in total), 100. mu.l per well, and incubated at 25 ℃ for 1 hour; discarding the sample and washing three times with PBST solution; after adding 100. mu.l of diluted mouse anti-human IgG Fc-HRP (1:10000) (Ab7499, abcam), incubating at 25 ℃ for 1 hour; discarding the solution and washing three times with PBST solution; after addition of TMB (P0209, beyond the range of 20 minutes) and development in the dark, H was added₂SO₄The reaction was stopped and the OD (450-650nm) was read on a microplate reader.

The test results show that the anti-CD47 antibody Hu5F9-G4, SIRP alpha D1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1^m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1^m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1^m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1^m-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1^mD2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2 are each capable of binding to CD 47; Ofa-Fc1-D1-Fc2, 2-Fc 2-D2-D2-D2-Fc 2, 2-Fc 2-D2-D2-D2-Fc 2, Anti-EGFR-Fc 2-D2-Fc 2, Anti-EGFR-Fc 2-D2-D2-D2-Fc 2 have a slightly weaker binding affinity for CD2 than the Anti-CD2 antibodies F2-G2 and/or SIRP α D2-Fc.

The above test data prove that the recombinant protein of the invention can specifically target the CD47 antigen of tumor cells at the protein level, and the binding affinity with CD47 is not higher than that of SIRPa D1-Fc fusion protein to CD47 protein; the recombinant protein can reduce or avoid side effects such as Erythrocyte agglutination, anemia and/or non-tumor target cell killing and the like caused by the treatment of a SIRP alpha high-affinity mutant by the treatment of an anti-CD47 antibody (Petrova PS, et al. TTI-621(SIRP alpha Fc): A CD47-Blocking Innate ImmunoImmunoCheckpoint Inhibitor with Broad antibody Activity and minimum Erythrocyte binding. Clin Cancer Res, 2017, 23(4): 1068-.

For example, as shown in FIG. 5, except that the Anti-CD20 antibody Ofatumumab and the Anti-EGFR antibody JMT101 were not able to bind to CD47, the Anti-CD47 antibodies Hu5F9-G4, SIRP α D1-Fc, Ofa-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-Fc2 all bound to CD47, but their affinities (EC) were due to the fact that Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 only the right arm bound to CD47 antigen (EC 56-Fc 1-D1-Fc 42, Anti-EGFR-Fc1-D1-Fc 2)₅₀＝52.57ng/mL/EC₅₀93.86ng/mL) was weaker than anti-CD47 antibody (EC)₅₀5.439ng/mL) and/SIRPa D1-Fc (EC)₅₀＝6.118ng/mL)。

Flow cytometry detection of Ofa-Fc1-D1-Fc2 affinity for the target CD47：

A431 cells (human epidermal carcinoma cells), well-growing cells were collected and counted, centrifuged and the cells were resuspended to 3X 10 with PBS + 2% FBS (purchased from Gibco)⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged off and 9 dilutions of Ofa-Fc1-D1-Fc2 (starting from 15000ng/ml, 5-fold gradient dilutions, total 9 concentrations) were added separately and the 96-well plates were incubated for 1 hour at 4 ℃ in a refrigerator; after washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

Since A431 cells do not express CD20 antigen and cannot bind to Ofatumumab and Obinuzumab, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1 can be evaluated at the cellular level using A431 cells^m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1^m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1^mBinding affinity of-D2-Fc 2, Obi-Fc1-D1-D2-D3-Fc2 to CD47.

The test results showed that the anti-CD47 antibodies Hu5F9-G4, SIRP alpha D1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1, except for the anti-CD20 antibodies Ofatumumab and Obinutuzumab, which could not bind to A431^m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1^m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1^m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2 can be combined with A431 cells; Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2 have slightly weaker binding affinity for CD47 than the anti-CD47 antibody and/or SIRP α D1-Fc for CD47, consistent with the data trend of ELISA.

The above test data prove that the recombinant protein of the invention can specifically target the CD47 antigen of tumor cells at a cellular level, and the binding affinity with CD47 is not higher than that of SIRPa D1-Fc fusion protein for CD 47; the recombinant protein can reduce or avoid side effects of erythrocyte agglutination, anemia and/or non-tumor target cell killing and the like caused by SIRP alpha high-affinity mutant treatment generated by anti-CD47 antibody treatment.

For example, as shown in FIG. 6, the anti-CD47 antibodies Hu5F9-G4, SIRPa D1-Fc, and Ofa-Fc1-D1-Fc2 all bound to A431 cells. Specifically, Ofa-Fc1-D1-Fc2 had slightly weaker affinity than the anti-CD47 antibody and/or SIRPa D1-Fc, consistent with the trends in the ELISA data.

Flow cytometry detection of Ofa-Fc1-D1-Fc2 affinity for the target CD20：

Raji cells (human B-cell lymphoma) (purchased from Shanghai academy of sciences cell bank), well-growing cells were collected and counted, centrifuged and the cells were resuspended to 3X 10 with PBS + 2% FBS⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged off and 100. mu.L of PBS + 2% FBS (control group) or 1.5. mu.g/mL of Hu5F9-G4(Fab)2 (experimental group) which is an anti-CD47 antibody (Fc was cleaved by pepsin, kit: Thermo Fisher, 44988), incubated at 4 ℃ for 1 hour; after washing with PBS + 2% FBS, 7 dilutions of Ofa-Fc1-D1-Fc2, Hu5F9-G4, SIRP α D1-Fc (starting from 6250ng/ml, 4-fold gradient dilution, total 7 concentrations, FIG. 7 post-transformation molarity) were added, respectively, and incubated at 4 ℃ for 1 hour; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

The test results show that the anti-CD47 antibody Hu5F9-G4(Fab)2 can effectively block the binding of the anti-CD47 antibody Hu5F9-G4 and/or SIRP alpha D1-Fc with CD47 on Raji cells; however, the blocking effect of Hu5F9-G4(Fab)2 on CD47 antigen does not significantly inhibit the binding of Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2 to Raji cells.

The above experimental data demonstrate that in the case that the tumor cell surface CD47 antigen is blocked and the mutual binding of sirpa-CD 47 is blocked, the recombinant protein of the present invention can still rely on the specific binding of its left arm to the corresponding antigen on the tumor cell, and the left arm affinity is not significantly affected by the blocking of the right arm binding.

For example, as shown in fig. 7, the anti-CD47 antibody Hu5F9-G4(Fab)2 can effectively block the binding of the anti-CD47 antibody Hu5F9-G4 and/or sirpa D1-Fc to CD47 on Raji cells, but the blocking effect of Hu5F9-G4(Fab)2 to CD47 antigen does not significantly inhibit the binding of Ofa-Fc1-D1-Fc2 to Raji cells, indicating that Ofa-Fc1-D1-Fc2 can still rely on its left arm (anti-CD 20 half antibody) to bind specifically to CD20 antigen on Raji cells after the sirpa-CD 47 binding is blocked, and the affinity is not significantly affected by the blocking of right arm binding.

Flow cytometry detection of bispecific binding to targets CD20 and CD47：

Raji cells (human B-cell lymphoma) (purchased from Shanghai academy of sciences cell bank), well-growing cells were collected and counted, centrifuged and the cells were resuspended to 3X 10 with PBS + 2% FBS⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 12 dilutions of Ofa-Fc1-D1-Fc2, Ofatumumab, Hu5F9-G4, SIRP α D1-Fc (50000ng/ml, 25000ng/ml, 6250ng/ml, then 4-fold gradient diluted for 12 concentrations, FIG. 8A for post-transformation molarity) were added separately and incubated at 4 ℃ for 1 hour; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

Because the Raji cell surface expresses CD20 and CD47 antigens simultaneously, the anti-CD20 antibody Ofatumumab, Obinutuzumab, the anti-CD47 antibody Hu5F9-G4 and SIRP alpha D1-Fc can be specifically combined with the Raji cell, but the maximum average fluorescence intensity achieved is different.

The test results show that Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1^m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1^m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1^mD2-Fc2, Obi-Fc1-D1-D2-D3-Fc2 also bound Raji cells with a higher maximum mean fluorescence intensity.

The above experimental data prove that the recombinant protein of the invention can be specifically combined with tumor cells and shows a remarkable molecular number advantage compared with anti-CD20 antibody Ofatumumab, Obinutuzumab and/or anti-CD47 antibody Hu5F9-G4 and/or SIRP alpha D1-Fc under the same supersaturated protein sample concentration environment. Preferably, under the same supersaturated protein sample concentration environment, the saturation binding abundance of the recombinant protein of the invention and the tumor cells is greater than the sum of the saturation binding abundances of the anti-CD20 antibody and the SIRPa D1-Fc and the tumor cells.

TABLE 4 maximum mean fluorescence intensity and EC50(nM) for antibody/recombinant protein and Raji cell binding

For example, as shown in fig. 8A and table 4, the anti-CD20 antibody Ofatumumab, the anti-CD47 antibody Hu5F9-G4, and sirpa D1-Fc all bound specifically to Raji cells, but the maximum mean fluorescence intensities achieved were different; meanwhile, Ofa-Fc1-D1-Fc2 can also bind to Raji cells and has higher maximum average fluorescence intensity, which indicates that the molecular number of Ofa-Fc1-D1-Fc2 protein molecules which can be specifically bound by the Raji cells is obviously higher than the molecular number of anti-CD20 antibody Ofatumumab or anti-CD47 antibody Hu5F9-G4 or SIRP alpha D1-Fc, respectively, the molecular number of the anti-CD20 antibody Ofatumumab and the anti-CD47 antibody Hu5F9-G4 and the molecular number of the anti-CD20 antibody Ofatumumab and the SIRP alpha D1-Fc under the same supersaturated protein sample concentration environment.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of simultaneously binding to the tumor-targeting antigen and the CD47 antigen can bind to tumor cells more than the anti-CD47 antibody or sirpa D1-Fc combined with other anti-tumor targeting therapeutic antibodies, thereby achieving more significant anti-tumor efficacy.

Flow cytometry detection of bispecific binding to the targets Her2 and CD47：

SKBR-3 cells (human breast cancer cells) (purchased from Shanghai Zhongji cell Bank), well-grown cells were collected and counted, centrifuged and resuspended to 2X 10 with PBS + 2% FBS⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged off and added separately to 10 dilutions each of Anti-Her2(T) -Fc1-D1-Fc2, Trastuzumab and Hu5F9-G4(433.2nM initial, 4 fold gradient dilution, total 10 points) and incubated for 1 hour at 4 ℃; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

As the cell surface of SKBR-3 expresses Her2 and CD47 antigens simultaneously, the anti-Her2 antibodies Trastuzumab, Pertuzumab, the anti-CD47 antibodies Hu5F9-G4 and SIRP alpha D1-Fc can be specifically combined with the SKBR-3 cells, but the maximum average fluorescence intensity can be different.

The test results show that Anti-Her2(P) -Fc1-D1-Fc2, Anti-Her2(P) -Fc1-D1^m-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1^m-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1^m-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1^m-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1^m-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1^m-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1^m-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1^mD2-Fc2, Anti-Her2(T) -Fc1-D1-D2-D3-Fc2 also bound SKBR-3 cells with a higher maximum mean fluorescence intensity.

The above experimental data prove that, under the same supersaturated protein sample concentration environment, the recombinant protein of the invention can be specifically combined with tumor cells and shows a remarkable molecular number advantage compared with the anti-Her2 antibody Trastuzumab, Pertuzumab and/or anti-CD47 antibody Hu5F9-G4 and/or SIRP alpha D1-Fc. Preferably, under the same supersaturated protein sample concentration environment, the saturation binding abundance of the recombinant protein of the invention and the tumor cells is greater than the sum of the saturation binding abundances of the corresponding anti-Her2 antibody and SIRPa D1-Fc and the tumor cells.

TABLE 5 maximum mean fluorescence intensity and EC50(nM) for binding of antibody/recombinant protein to SKBR-3 cells

For example, as shown in fig. 8B and table 5, both anti-Her2 antibody Trastuzumab and anti-CD47 antibody Hu5F9-G4 were able to specifically bind to SKBR-3 cells, but the maximum mean fluorescence intensities achieved were different; meanwhile, Anti-Her2(T) -Fc1-D1-Fc2 can also be combined with SKBR-3 cells and has higher maximum average fluorescence intensity, which indicates that the number of Anti-Her2(T) -Fc1-D1-Fc2 protein molecules which can be specifically combined by the SKBR-3 cells is respectively and obviously higher than the number of Anti-Her2 antibody Trastuzumab or Anti-CD47 antibody Hu5F9-G4 molecules and higher than the sum of the number of Anti-Her2 antibody Trastuzumab and Anti-CD47 antibody Hu5F9-G4 molecules under the same supersaturated protein sample concentration environment.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of simultaneously binding to the tumor-targeting antigen and the CD47 antigen can bind to tumor cells more than the anti-CD47 antibody or sirpa D1-Fc combined with other anti-tumor targeting therapeutic antibodies, thereby achieving more significant anti-tumor efficacy.

Flow cytometry detection and targetingBispecific binding of EGFR and CD47：

A431 cells (human epidermal carcinoma cells) (purchased from basic medical research institute of Chinese academy of medical sciences), well-grown cells were collected and counted, centrifuged and the cells were resuspended to 2X 10 with PBS + 2% FBS⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged off and 11 dilutions each of Anti-EGFR-Fc1-D1-Fc2, JMT101, SIRPa D1-Fc and Hu5F9-G4(216.6nM initial, 4 fold gradient dilution, 11 concentrations total) were added and incubated for 1 hour at 4 ℃; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

As the cell surface of A431 expresses EGFR and CD47 antigens at the same time, the anti-EGFR antibody JMT101, the anti-CD47 antibodies Hu5F9-G4 and SIRP alpha D1-Fc can be specifically combined with the A431 cells, but the maximum average fluorescence intensity can be different.

The test results show that Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1^m-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1^mD2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2 also bound to A431 cells with higher maximum mean fluorescence intensity.

The above test data prove that the recombinant protein of the invention can be specifically combined with tumor cells and shows a remarkable molecular number advantage compared with anti-EGFR antibody JMT101 and/or anti-CD47 antibody Hu5F9-G4 and/or SIRP alpha D1-Fc under the same supersaturated protein sample concentration environment. Preferably, under the same supersaturated protein sample concentration environment, the saturation binding abundance of the recombinant protein of the invention and the tumor cells is greater than the sum of the saturation binding abundances of the corresponding anti-EGFR antibody and SIRPa D1-Fc and the tumor cells.

TABLE 6 maximum mean fluorescence intensity and EC50(nM) for antibody/recombinant protein and A431 cell binding

For example, as shown in FIG. 8C and Table 6, the anti-EGFR antibody JMT101, the anti-CD47 antibodies Hu5F9-G4 and SIRPa D1-Fc all bound specifically to A431 cells, but the maximum mean fluorescence intensities reached were different, and EC was₅₀Are respectively EC₅₀(JMT101)＝0.598nM、EC₅₀(SIRPαD1-Fc)＝3.677nM、EC₅₀(Hu5F9-G4) ═ 0.865nM, it can be seen that the binding affinity of JMT101 to a431 cells is more than 6 times that of sirpa D1-Fc to a431 cells; meanwhile, Anti-EGFR-Fc1-D1-Fc2 can also be combined with A431 cells and has higher maximum average fluorescence intensity, which indicates that the number of molecules of Anti-EGFR-Fc1-D1-Fc2 protein which can be specifically combined by the A431 cells is respectively and obviously higher than the number of molecules of Anti-EGFR antibody JMT101 or SIRPa D1-Fc or Anti-CD47 antibody Hu5F9-G4 and higher than the sum of the number of molecules of Anti-EGFR antibody JMT101 and SIRPa D1-Fc under the same supersaturated protein sample concentration environment.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of simultaneously binding to the tumor-targeting antigen and the CD47 antigen can bind to tumor cells more than the anti-CD47 antibody or sirpa D1-Fc combined with other anti-tumor targeting therapeutic antibodies, thereby achieving more significant anti-tumor efficacy.

Flow cytometry detection of bispecific binding to targets PD-L1 and CD47：

NCI-H441 cells (human lung adenocarcinoma cells) (purchased from Beijing Beinana institute of Biotechnology) were stimulated with 10ng/ml hIFN-. gamma. (BD, cat # 554616) at 2X 10⁷Cells were harvested for 18h, digested, counted, centrifuged and resuspended to 3X 10 with PBS + 2% FBS⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 12 dilutions of Anti-PD-L1(Ate) -Fc1-D1-Fc2 and Atezolizumab (433.2nM, 216.6nM, then 4-fold gradient dilution, 12 concentrations total) were added separately and incubated for 1 hour at 4 ℃; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; washing with PBS + 2% FBSAfter suspension, fluorescence was detected by flow cytometry (Accuri C6, BD).

Because NCI-H441 cell surface simultaneously expresses PD-L1 and CD47 antigens, anti-PD-L1 antibody Atezolizumab, 13G4, 12A4, anti-CD47 antibody Hu5F9-G4, SIRP alpha D1-Fc can be specifically combined with NCI-H441 cell.

The test results show that Anti-PD-L1(Ate) -Fc1-D1-Fc2, Anti-PD-L1(Ate) -Fc1-D1^m-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1^m-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1^mD2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 were also able to bind to NCI-H441 cells and had higher maximum mean fluorescence intensities.

The test data prove that the recombinant protein can be specifically combined with tumor cells and shows certain molecular number advantage compared with anti-PD-L1 antibodies Atezolizumab, 13G4 and 12A4 under the same supersaturated protein sample concentration environment. For example, under the same supersaturated protein sample concentration environment, the recombinant protein of the invention can bind to tumor cells more than the anti-PD-L1 antibody Atezolizumab, and shows a significant advantage in molecular number.

TABLE 7 maximum mean fluorescence intensity and EC50(nM) for antibody/recombinant protein and NCI-H441 cell binding

For example, as shown in FIG. 8D and Table 7, the anti-PD-L1 antibody Atezolizumab binds specifically to NCI-H441 cells; meanwhile, Anti-PD-L1(Ate) -Fc1-D1-Fc2 can also bind to NCI-H441 cells and has higher maximum average fluorescence intensity, which indicates that the NCI-H441 cells have the same supersaturated protein sample concentrationUnder the environment, the number of molecules of Anti-PD-L1(Ate) -Fc1-D1-Fc2 protein capable of being specifically bound is higher than that of Anti-PD-L1 antibody Atezolizumab. EC (EC)₅₀Are respectively EC₅₀(Atezolizumab)＝0.2006nM、EC₅₀(SIRPαD1-Fc)＝1.643nM、EC₅₀(Hu5F9-G4) ═ 0.3865nM, it was found that the binding affinity of Atezolizumab to NCI-H441 cells was 6 times or more greater than that of sirpa D1-Fc to NCI-H441 cells.

As will be appreciated by those skilled in the art, the above experimental results suggest that more binding to tumor cells can be achieved using the recombinant protein of the present invention capable of simultaneous binding to both the tumor-targeting antigen and the CD47 antigen, and thus more significant anti-tumor efficacy, than using the anti-CD47 antibody in combination with other anti-tumor targeting therapeutic antibodies.

2. Detection of target competitive binding activity

The following methods, exemplified by Ofa-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-Fc2, measure competitive binding activity of the target CD47 to SIRPa by ELISA.

ELISA detection Ofa-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-Fc2 competitive binding Activity：

The microplate (9018, Corning) was coated with 100. mu.l of 1. mu.g/ml CD47-His (12283-H08H-200, Sino Biological) and left overnight at 4 ℃; after PBST is cleaned, the ELISA plate is sealed for 2 hours at room temperature by PBS + 1% BSA; after washing, the coated plates were added with diluted mixtures of Ofa-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-Fc2 and biotinylated SIRP α D1-Fc (biotin labeling kit, 21925, Thermo, loading concentration 100ng/ml) at different concentrations (starting at 1000ng/ml, 3-fold gradient dilution, total 11 concentrations), respectively, and incubated at 25 ℃ for 1 hour at 100 μ l per well; discarding the sample and washing three times with PBST solution; adding 100 μ l diluted streptavidin-HRP (1:10000) (ML-0437P-HRP, ZI501-1 grinding chemical reagent), and incubating at 25 deg.C for 1 hr; discarding the solution and washing three times with PBST solution; after addition of TMB (P0209, beyond the range of 20 minutes) and development in the dark, H was added₂SO₄The reaction was stopped and the OD (450-650nm) was read on a microplate reader.

The test results show that the Anti-CD47 antibodies Hu5F9-G4, SIRP alpha D1-Fc, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc 1-D1-D2-D2-Fc 2, Anti-EGFR-Fc 2-D2-D2-D2-Fc 2, Anti-Her Fc2 (Anti) -Fc 2D 2-Fc 2D 2-Fc2 (Fc 2-Fc 2D-2-Fc 2D-Fc 2, Anti-Her (T) -Fc-D-Fc, Anti-Her (T) -Fc-D-D-Fc, Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-D-Fc, Anti-PD-L (Ate) -Fc-D-Fc, Anti-PD-L (13G) -Fc-D-Fc, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 can competitively bind to CD47 antigen with biotin-labeled SIRP alpha D1-Fc to different degrees and exert competitive binding activity.

For example, as shown in FIG. 9, the Anti-CD47 antibodies Hu5F9-G4, SIRPa D1-Fc, Ofa-Fc1-D1-Fc2 and Anti-EGFR-Fc1-D1-Fc2 all competed with biotin-labeled SIRPa D1-Fc for binding to CD47 antigen to varying degrees, exerting competitive binding activity; Ofa-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-Fc2 bound CD47 competitively less strongly than Anti-CD47 antibodies Hu5F9-G4 or SIRP α D1-Fc, respectively, consistent with the affinity results described above in this example.

Example 4 recombinant protein early in vitro immune safety evaluation experiment

Recombinant protein Ofa-Fc1-D1^m-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Anti-EGFR-Fc1-D1^m-Fc2、Anti-EGFR-Fc1-D1^m-D2-Fc2、Anti-Her2(T)-Fc1-D1^m-Fc2、Anti-Her2(T)-Fc1-D1^m-D2-Fc2、Anti-Her2(P)-Fc1-D1^m-Fc2、Anti-Her2(P)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1^m-Fc2、Anti-PD-L1(Ate)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1^m-Fc2、Anti-PD-L1(12A4)-Fc1-D1^mAn early in vitro immune safety evaluation experiment of D2-Fc2, which was performed as Ofa-Fc1-D1^m-Fc2 is taken as an example,the method is suitable for detecting the recombinant protein of the right arm containing the SIRP alpha extracellular truncation body high-affinity mutant.

Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1-D2-Fc2, Ofa-Fc1-D1-D2-D3-Fc2, Obi-Fc1-D1-Fc2, Obi-Fc1-D1-D2-Fc2, Obi-Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-Her 1 (P) -Fc1-D1-Fc 1, Anti-Her 1 (P) -Fc1 (P) -Her-1-Fc 1 (P) -1-Her-1-Fc 1 (P) -Her-1-Fc 1 (P), Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, An early in vitro immune safety evaluation experiment of Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 is suitable for the detection of recombinant protein containing SIRP alpha extracellular truncation in the right arm by taking Ofa-Fc1-D1-Fc2 as an example.

Flow cytometry detection of specific binding to CD47 before and after double-antibody specific right arm mutation：

NCI-H441 cells (human Lung adenocarcinoma cells) (purchased from Beijing Beiner institute of Biotechnology, Biotech), cells were harvested by digestion and counted, centrifuged and resuspended in PBS + 2% FBS to 3X 10⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and added with 12 dilutions each of Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1^m-Fc2, Hu5F9-G4, Ofatumumab (433.2nM, 216.6nM, then 4-fold gradient dilution, 12 concentrations total), incubation for 1 hour at 4 ℃; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

TABLE 8 flow cytometry detection of specific binding to CD47 before and after double antibody-specific right arm mutation

For example, as shown in FIG. 10 and Table 8, the anti-CD47 antibodies Hu5F9-G4, Ofa-Fc1-D1-Fc2, Ofa-Fc1-D1, except that the anti-CD20 antibody Ofatumumab was unable to bind to CD47^m-Fc2 all bind to CD47, and Ofa-Fc1-D1^m-Fc2(EC₅₀0.529nM) has higher binding affinity to NCI-H441 cells than Ofa-Fc1-D1-Fc2 (EC)₅₀8.68nM), wherein D1^mIs a high affinity mutant of SIRP alpha in the D1 region (i.e., Seq ID No:10 of CN 106519036A).

Recombinant protein early in vitro immune safety evaluation experiment

(1) Preparing human effector cell suspension:

well-grown human NK92MI-CD16a effector cells (purchased from Boehringer) which stably and highly express CD16a were harvested, centrifuged (201g, 5min) to discard the supernatant, resuspended in 5ml of MEM (phenol Red-free) basal medium (purchased from Gibco, 51200-⁶Individual cells/ml, used as human effector cell suspension.

(2) Incubation of effector cells with antibodies:

the MEM (phenol red-free) basic culture medium is divided into 50 mul/hole to the corresponding hole of a 96-hole black bottom-penetrating culture plate, and the diluted Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1 is added^mFc2 bispecific antibody dilution gradients of 25. mu.l/well each and duplicate wells were set. Mu.l (60000 cells/well) of the human effector cell suspension prepared in (1) was added. Evenly mixed, Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1^mthe-Fc 2 bispecific antibody reached a final concentration gradient (433.2nM start, 4-fold gradient dilution, ten concentrations total) and was then reacted at 37 ℃ for 5.5 hours, adding lysis buffer (Promega-derived kit, G7891) to the control group and incubated for 0.5 h.

(3) And (3) detecting ADCC activity:

after incubation, the plate was placed in a safety cabinet, and the plate was cooled naturally to room temperature by opening the lid for about 15 min. LDH substrate reaction solution (kit from Promega, G7891) which is balanced for 30min at room temperature is added into a culture plate, each well is 100 mul, after the solution is gently mixed uniformly and incubated for 15min at room temperature, stop solution (kit from Promega, G7891) is immediately added into each well at 50 mul, and after the solution is mixed uniformly, the fluorescence value is read on a microplate reader.

The test results show that recombinant proteins and/or antibodies targeting CD47 with ADCC activity lead to NK cell killing interactions due to the expression of CD47 antigen on NK cells. Thus, compared to Ofa-Fc1-D1 containing a SIRPa extracellular truncate high affinity mutant^m-Fc2 or Ofa-Fc1-D1^m-D2-Fc2 or Anti-EGFR-Fc1-D1^m-Fc2 or Anti-EGFR-Fc1-D1^m-D2-Fc2 or Anti-Her2(T) -Fc1-D1^m-Fc2 or Anti-Her2(T) -Fc1-D1^m-D2-Fc2 or Anti-Her2(P) -Fc1-D1^m-Fc2 or Anti-Her2(P) -Fc1-D1^m-D2-Fc2 or Anti-PD-L1(Ate) -Fc1-D1^m-Fc2 or Anti-PD-L1(Ate) -Fc1-D1^m-D2-Fc2 or Anti-PD-L1(13G4) -Fc1-D1^m-Fc2 or Anti-PD-L1(13G4) -Fc1-D1^m-D2-Fc2 or Anti-PD-L1(12A4) -Fc1-D1^m-Fc2 or Anti-PD-L1(12A4) -Fc1-D1^m-D2-Fc2, Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1-D2-Fc2 or Ofa-Fc1-D1-D2-D3-Fc2 or Obi-Fc1-D1-Fc2 or Obi-Fc1-D1-D2-Fc2 or Obi-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc 1-D1-D1-Fc 1 or Anti-EGFR-Fc 1-D1-D1-Fc 1 or Anti-Her 1 (P) -Fc1 or Anti-Fc 1 or Anti-Fc 1 (Anti-Fc 1 (or Anti-Fc 1-Fc 1( Fc-D-Fc or Anti-Her (T) -Fc-D-D-Fc or Anti-EGFR-Fc-D-Fc or Anti-PD-L (ace) -Fc-D-D-Fc or Anti-PD-L (13G) -Fc-D-Fc or Anti-PD-L (13G) -Fc-D-Fc the-PD-L1 (12A4) -Fc1-D1-Fc2 or Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2 or Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 obviously reduces the toxic and side effects caused by NK cells and at least 1000 times of the toxic and side effects due to the weak affinity effect of the Anti-PD-L1(12A4) with the CD47 antigen.

For example, as shown in FIG. 11, when the antibody/recombinant protein concentration reached 10^-1At nM, Ofa-Fc1-D1^mFc2 cell lysis had begun, when the antibody/recombinant protein concentration reached 10³At nM, Ofa-Fc1-D1^mFc2 cell lysis rate reached 15.25%, whereas Ofa-Fc1-D1-Fc2 was at 10³no cell lysis was observed with nM.

As known to those skilled in the art, the above test results suggest that the recombinant protein having ADCC activity and targeting the CD47 antigen with low affinity has higher immune safety.

As known to those skilled in the art, the above test results suggest that the method for detecting ADCC activity optimized according to the present invention (i.e. the early in vitro immune safety evaluation experiment) can be used to evaluate the safety of early immunity of recombinant proteins (containing monovalent or multivalent) or antibodies (containing monovalent or multivalent) targeting CD47 and having ADCC activity, and that the evaluation method is simple and fast and is not limited by blood source.

EXAMPLE 5 in vivo inhibition of tumor cell growth by recombinant proteins

Recombinant proteins-Fc-D-Fc, -Fc-D-D-Fc, -Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 in vivo tumor cell growth inhibition experiments, and the following method is exemplified by Ofa-Fc1-D1-Fc2 and is suitable for detecting recombinant proteins containing SIRPa extracellular truncates in the right arm.

Male NSG mice (purchased from Edmol) were inoculated subcutaneously with human B-cell lymphoma Raji cells, and the tumor group was treated after inoculation of tumor cellsWeave to 80mm³-150mm³Divided into the following 2 groups (6 mice per group, each group was intraperitoneally injected according to the indicated amounts): 1) vehicle control group (Tris-citrate, pH 6.5); 2) Ofa-Fc1-D1-Fc2 group (150. mu.g/mouse); the administration was 2 times per week for 2 weeks. Tumor growth was observed before (day 0), on days 3, 5, 7, 10, 12, and 14 after administration, respectively, and the tumor size was measured to evaluate the tumor-suppressing effect of Ofa-Fc1-D1-Fc 2.

The test results show that on the NSG mouse model of Raji lymphoma subcutaneously transplanted, the recombinant proteins-Fc-D-Fc, -Fc-D-D-Fc, Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her (T) -Fc-D-D-Fc, Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-D-Fc, Anti-PD-L (Ate) -Fc-D-Fc, Anti-PD-L (13G) -Fc-D-Fc, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 activate targeted phagocytosis of macrophages and/or macrophage-mediated Antibody-dependent phagocytosis (ADCP) by blocking the CD47-SIRP alpha signaling pathway, thereby exhibiting significant tumor inhibition effects.

For example, as shown in fig. 12, NSG mice transplanted subcutaneously with Raji lymphoma on the abscissa receive time (days) of drug treatment, and tumor volume (mm) on the ordinate³). FIG. 12 shows that after a period of drug Ofa-Fc1-D1-Fc2 treatment, Ofa-Fc1-D1-Fc2 showed significant tumor suppression trend compared to vehicle control group, wherein the tumor suppression rate of Ofa-Fc1-D1-Fc2 group at day 14 could reach 63.14%.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of simultaneously binding to the tumor targeting antigen and the CD47 antigen can obtain significant tumor suppression efficacy in NSG mice transplanted with tumor cells subcutaneously.

Example 6 recombinant protein in vivo early immune safety evaluation experiment

Recombinant protein Ofa-Fc1-D1^m-Fc2、Ofa-Fc1-D1^m-D2-Fc2、Anti-EGFR-Fc1-D1^m-Fc2、Anti-EGFR-Fc1-D1^m-D2-Fc2、Anti-Her2(T)-Fc1-D1^m-Fc2、Anti-Her2(T)-Fc1-D1^m-D2-Fc2、Anti-Her2(P)-Fc1-D1^m-Fc2、Anti-Her2(P)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1^m-Fc2、Anti-PD-L1(Ate)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-Fc2、Anti-PD-L1(13G4)-Fc1-D1^m-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1^m-Fc2、Anti-PD-L1(12A4)-Fc1-D1^mEvaluation of safety of in vivo early immunization with D2-Fc2, the following method is Ofa-Fc1-D1^mThe Fc2 is used as an example, and is suitable for detecting the recombinant protein containing the SIRPa extracellular truncate high-affinity mutant in the right arm.

Recombinant proteins-Fc-D-Fc, -Fc-D-D-Fc, -Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 in vivo early immune safety evaluation, the following method takes Ofa-Fc1-D1-Fc2 as an example, and is suitable for the detection of recombinant protein containing SIRPa extracellular truncation in the right arm.

Since the CD20 antigen is highly expressed on B cells, the experiment represents the killing condition of tumor cells through the content measurement of the B cells. For the safety evaluation of the in vivo early immunity of recombinant proteins targeting other tumor antigens and CD47 antigen, the experimental mice described in this example were implanted with corresponding tumor cells subcutaneously.

Tumor specific targeting:

selecting human CD34⁺HSC transplantation NSG (Hu-NSG) female mice (purchased from aldmo) were divided into the following 3 groups (3 mice per group, each group was injected intravenously according to the indicated amounts): 1) 0.9% saline control group; 2) hu5F9-G4 (6.7. mu.g/mouse); 3) Ofa-Fc1-D1-Fc2(5 μ g/mouse), administered 1 time, 96h after administration, collecting blood from tail vein of mouse 80 μ l in anticoagulation tube containing heparin sodium, and lysing erythrocytes with freshly prepared hemolysate (BD phase lysate)^TMAnd the cargo number: 555899): double distilled water for equal volume mixing preparation), washing the residual cells with PBS + 2% FBS for resuspension, incubating the cells with fluorescent antibody (PE anti-human CD45 (cat # 304039)/FITC anti-human CD19 (cat # 302206)/APC anti-human CD3 (cat # 300312) all from BioLegend) for 30min, washing the cells with PBS + 2% FBS for resuspension, and passing the cells through a flow cytometer (Accuri)^TMC6, BD).

The test results show that after the Hu-NSG mouse model is used for 96 hours by using Ofa-Fc1-D1-Fc2 and an anti-CD47 antibody Hu5F9-G4 at the same dose, Ofa-Fc1-D1-Fc2 preferentially eliminates B cells (namely target cells) expressing CD20 antigen, and the anti-CD47 antibody Hu5F9-G4 preferentially eliminates non-target cells with higher expression abundance of CD47, such as T cells and the like, due to the high affinity action of the anti-CD47 antibody Hu5F9-G4 with the CD47 antigen.

For example, as shown in fig. 13, the anti-CD47 antibody Hu5F9-G4 (fig. 13B) significantly cleared non-target cells (e.g., T cells) with higher abundance of CD47 expression compared to 0.9% saline (fig. 13A) (B cells, i.e., CD20 antigen target cells, account for an increase from 40.9% before drug administration to 73.5% after 96 hours of drug administration) in all cells tested; and compared with 0.9% physiological saline (FIG. 13A), the recombinant protein Ofa-Fc1-D1-Fc2 (FIG. 13C) has significant B cell (namely CD20 antigen target cells) clearance, i.e. Ofa-Fc1-D1-Fc2 has preferential B cell clearance 96 hours after drug administration (B cells account for 3.7% of all cells to be detected after drug administration from 40.9% before drug administration to 96 hours).

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of synchronously binding with the tumor targeting antigen and the CD47 antigen preferentially eliminates the cells and/or tumor cells where the tumor targeting antigen is located under the same dosage condition.

Immune safety at low dose:

selecting human CD34⁺HSC transplantation NSG (Hu-NSG) female mice (purchased from aldmo) were divided into the following 2 groups (3 mice per group, each group was injected intravenously according to the indicated amounts): 1) Ofa-Fc1-D1-Fc2 (1. mu.g/mouse); 2) Ofa-Fc1-D1^mFc2 group (1 μ g/mouse), 1 time of administration, and 72h after administration, 80 μ l of blood was collected from the mouse tail vein in an anticoagulation tube containing heparin sodium, and erythrocytes were lysed with freshly prepared hemolysate (BD phase lysate)^TMAnd the cargo number: 555899): double distilled water for equal volume mixing preparation), residual cells are washed and resuspended by PBS + 2% FBS, fluorescent antibody (PE anti-human CD45 (cat # 304039)/FITC anti-human CD19 (cat # 302206)/APC anti-human CD3 (cat # 300312) and cells are incubated for 30min, after PBS + 2% FBS is washed and resuspended, samples of Ofa-Fc1-D1-Fc2(1 μ g/cell) group are passed through a flow cytometer (Accuri)^TMC6, BD) assay, Ofa-Fc1-D1^mFc2 (1. mu.g/cell) group samples were passed through a flow cytometer (Novocyte)^TM3130, ACEA).

The results of the experiments show that low doses of recombinant protein are used in the Hu-NSG mouse model when-Fc-D-Fc, -Fc-D-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her2(T) -Fc1-D1-Fc2, Anti-Her2(T) -Fc1-D1-D2-Fc2, Anti-Her2(T) -Fc1-D1-D2-D3-Fc2, Anti-EGFR-Fc1-D1-Fc2, Anti-EGFR-Fc1-D1-D2-Fc2, Anti-EGFR-Fc1-D1-D2-D3-Fc2, Anti-PD-L1(Ate) -Fc1-D1-Fc2, Anti-PD-L1(Ate) -Fc1-D1-D2-Fc2, Anti-PD-L1(Ate) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(13G4) -Fc1-D1-Fc2, and Anti-PD-L1 (.13G4) -Fc1-D1-D2-Fc2, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12a4) -Fc1-D1-Fc2, Anti-PD-L1(12a4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12a4) -Fc1-D1-D2-D3-Fc2 are significantly cleared after a single 72 hours of administration, while cells that do not express left arm antigen (e.g., T cells, other immune cells) do not see a significant effect; Ofa-Fc1-D1 containing SIRP alpha extracellular truncation body high-affinity mutant^m-D2-Fc2 or Anti-EGFR-Fc1-D1^m-Fc2 or Anti-EGFR-Fc1-D1^m-D2-Fc2 or Anti-Her2(T) -Fc1-D1^m-Fc2 or Anti-Her2(T) -Fc1-D1^m-D2-Fc2 or Anti-Her2(P) -Fc1-D1^m-Fc2 or Anti-Her2(P) -Fc1-D1^m-D2-Fc2 or Anti-PD-L1(Ate) -Fc1-D1^m-Fc2 or Anti-PD-L1(Ate) -Fc1-D1^m-D2-Fc2 or Anti-PD-L1(13G4) -Fc1-D1^m-Fc2 or Anti-PD-L1(13G4) -Fc1-D1^m-D2-Fc2 or Anti-PD-L1(12A4) -Fc1-D1^m-Fc2 or Anti-PD-L1(12A4) -Fc1-D1^mAfter 72 hours of single administration of D2-Fc2, although target cells (such as tumor cells) expressing the left arm antigen are significantly eliminated, cells (such as T cells and other immune cells) not expressing the left arm antigen are also eliminated to a significant extent.

For example, as shown in fig. 14, Ofa-Fc1-D1-Fc2 was significantly depleted (almost completely depleted) of B cells (CD20 antigen target cells) compared to cells (e.g., T cells, other immune cells) that did not express CD20 antigen 72 hours after a single administration (fig. 14C, 14D) compared to cells before administration (fig. 14A, 14B). Ofa-Fc1-D1^m72 hours after a single administration of Fc2 (FIGS. 14G, 14H) compared to before administration (FIGS. 14E, 14F), cells that do not express CD20 antigen (e.g., T cells, other immune cells) were cleared to a significant extent despite significant clearance of B cells (CD20 antigen target cells).

As known by those skilled in the art, the above test results suggest that the recombinant protein containing the SIRPa extracellular truncation body in the right arm has higher tumor-specific targeting effect and higher immune safety than the recombinant protein containing the SIRPa extracellular truncation body high-affinity mutant in the right arm under the same dosage condition.

Immune recovery at high doseThe function is as follows:

selecting human CD34⁺HSC transplantation NSG (Hu-NSG) female mice (purchased from aldmo) were divided into the following 2 groups (3 mice per group, each group was injected intravenously according to the indicated amounts): 1) hu5F9-G4 (200. mu.g/mouse); 2) Ofa-Fc1-D1-Fc2(150 μ g/mouse), administered 1 time, 4 days after administration and 14 days after administration, blood was collected from the tail vein of mouse in 80 μ l in anticoagulation tube containing heparin sodium, and erythrocytes were lysed with freshly prepared hemoglobin (BD phase lysate)^TMAnd the cargo number: 555899): double distilled water for equal volume mixing preparation), washing the residual cells with PBS + 2% FBS for resuspension, incubating the cells with fluorescent antibody (PE anti-human CD45 (cat # 304039)/FITC anti-human CD19 (cat # 302206)/APC anti-human CD3 (cat # 300312) all from BioLegend) for 30min, washing the cells with PBS + 2% FBS for resuspension, and passing the cells through a flow cytometer (Accuri)^TMC6, BD).

The test results show that high doses of Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1-D2-Fc2 or Ofa-Fc1-D1-D2-D3-Fc2 or Obi-Fc1-D1-Fc2 or Obi-Fc1-D1-D2-Fc2 or Obi-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR 2(P) -Fc 2(P) -Herti-Fc 2D-Fc -Fc or Anti-Her (T) -Fc-D-D-Fc or Anti-EGFR-Fc-D-Fc or Anti-PD-L (Ate) -Fc-D-Fc or Anti-PD-L (13G) -Fc-D-Fc or Anti-PD-L (13G) -Fc-D-Fc -D-D-Fc or Anti-PD-L (12A) -Fc-D-D-Fc and the Anti-CD antibody Hu 5F-G, 96 hours after a single administration, -Fc-D-Fc, -Fc-D-D-Fc, Anti-EGFR-Fc-D-Fc, Anti-PD-Fc-D-Fc, Anti-EGFR-Fc-D-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-EGFR-Fc-D-Fc, Anti-PD-L (Ate) -Fc-D-Fc, Anti-EGFR-Fc-D-Fc, Anti-PD-L (Ate) -Fc-D-Fc, Anti-PD-L (Ate) -Fc-D-Fc, Anti-PD-L (Ate) -Fc-D-D-Fc, Anti-PD-L (13G) -Fc-D-Fc, Anti-PD-L (12A) -Fc-D-Fc, Anti-PD-L (12A) -Fc-D-D-Fc and Anti-CD antibody Hu 5F-G can see that B cells (CD antigen target cells) and non-target cells (such as T cells and other immune cells) are largely protected A clearing phenomenon. After 14 days, Ofa-Fc1-D1-Fc2 or Ofa-Fc1-D1-D2-Fc2 or Ofa-Fc1-D1-D2-D3-Fc2 or Obi-Fc1-D1-Fc2 or Obi-Fc1-D1-D2-Fc2 or Obi-Fc1-D1-D2-D3-Fc2 or Anti-EGFR-Fc1-D1-Fc2 or Anti-EGFR-Fc1-D1-D2-Fc2 or Anti-EGFR-Fc 2-D2-D2-Fc 2 or Anti-Her2(P) -2-Fc 2-D2-Fc 2 or Anti-Fc 2(P) -Fc 2-Fc 2-Fc 2(P) -Her-Fc 2-Fc 2-Fc2 or-Fc 2(P) -Her-Fc 2-Fc 2-Fc 2(P) or-Fc 2-Fc 2-Fc 2(P) or-Fc 2-Fc 2-Fc 2-Fc 2(P) or-Fc 2-Fc 2-Fc 2-Fc D-D-Fc or Anti-Her (T) -Fc-D-D-Fc or Anti-EGFR-Fc-D-Fc or Anti-PD-L (ace) -Fc-D-Fc or Anti-PD-L (13G) -Fc-D-Fc or Anti-PD-D-Fc or Anti-PD- Mice in the group of L1(12A4) -Fc1-D1-Fc2 or Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2 or Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 still have their B cells (CD20 antigen target cells) cleared, while other non-target cells (such as T cells) expressing CD47 are significantly restored; mice in the group of anti-CD47 antibody Hu5F9-G4 did not recover neither B cells (CD20 antigen target cells) nor non-target cells expressing CD47.

For example, as shown in fig. 15, although a large amount of B cells (CD20 antigen target cells) and non-target cells (e.g., T cells) expressing CD47 were eliminated at 96 hours after administration in both the high dose anti-CD47 antibodies Hu5F9-G4 (fig. 15A) and Ofa-Fc1-D1-Fc2 (fig. 15B), mice in the group Ofa-Fc1-D1-Fc2 (fig. 15D) had their B cells (CD20 antigen target cells) cleared, but non-target cells (e.g., T cells) expressing CD47 other than the B cells (CD20 antigen target cells) were significantly restored after 14 days, while the anti-CD47 antibodies Hu5F9-G4 (fig. 15C) had no signs of restoration in both the B cells (CD20 antigen target cells) and the non-target cells (e.g., T cells) expressing CD47.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of synchronously binding with the tumor targeting antigen and the CD47 antigen, which expresses non-target cells (e.g., immune cells such as T cells) of CD47, has recoverability under high dosage administration conditions, and the recombinant protein has higher immune safety.

As known to those skilled in the art, the above test results suggest that the in vivo early immune safety evaluation method of the present invention can be used to evaluate the early immune safety of the target CD47 recombinant protein (containing monovalent or multivalent) or antibody (containing monovalent or multivalent).

Example 7 Effect of different truncations on target affinity binding Activity

Recombinant proteins-Fc-D-Fc, -Fc-D-D-Fc, -Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc 4-D4-D4-Fc 4, Anti-PD-L4 (12A4) -Fc 4-D4-D4-Fc 4 are respectively expressed by Anti-Her 4 (T) -Fc 4-D4-Fc 4, Anti-Her 4 (T) -Fc 4-D4-Fc 72, Anti-Her 4 (P) -Fc 4-D4-Fc 72-Fc 4 and Anti-Her 4 (P) -Fc 4-D4-Fc 72-Fc 4-Fc 72 are respectively expressed by the same length as the same in vitro recombinant protein.

Flow cytometry detection of bispecific binding to the targets Her2 and CD47：

SKBR-3 cells (human breast cancer cells) (purchased from Shanghai Zhongkou)Cell bank), well-growing cells were collected and counted, centrifuged and the cells were resuspended to 2 × 10 with PBS + 2% FBS⁶Concentration of individual cells/ml. The cells were added to a 96-well plate U-plate (cat # 3799, Corning) at 100. mu.l/well and allowed to stand for at least 15 minutes; the supernatant was centrifuged and 11 dilutions of each of Anti-Her2(T) -Fc1-D1-Fc2, Anti-Her2(T) -Fc1-D1-D2-Fc2, Anti-Her2(P) -Fc1-D1-Fc2 and Anti-Her2(P) -Fc1-D1-D2-Fc2(433.2nM initial, 4-fold serial dilutions for 11 concentrations) were added and incubated for 1 hour at 4 ℃; after PBS + 2% FBS wash, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4 ℃ for 1 hour; after washing the resuspended suspension with PBS + 2% FBS, the fluorescence was detected by flow cytometry (Accuri C6, BD).

As Trastuzumab and Pertuzumab act on different epitopes of Her2 antigen respectively, and the distances between 2 epitopes and cell membranes are greatly different, the recombinant proteins Anti-Her2(T) -Fc1-D1-Fc2, Anti-Her2(T) -Fc1-D1-D2-Fc2, Anti-Her2(P) -Fc1-D1-Fc2, Anti-Her2(P) -Fc1-D1-D2-Fc2 can be specifically bound with SKBR-3 cells, but the binding affinities and the maximum average fluorescence intensities which can be achieved are different.

The test results show that the affinity of Transtuzumab 2(T) -Fc1-D1-Fc2 and SKBR-3 cells is better than that of Trans-Her 2(T) -Fc1-D1-D2-Fc2 because the Trastuzumab and Her2 epitope (Pedersen M W, et al. targeting Three Distingct HER2 Domains with a Recombinant Antibody Mixture Trastuzumab response. molecular Cancer Therapeutics,2015,14(3):669 and 680) are closer to the surface of cell membrane. Pertuzumab is far away from the Her2 epitope (Her2 extracellular II domain) from the surface of a cell membrane, so that Anti-Her2(P) -Fc1-D1-Fc2 has the same affinity with SKBR-3 cells as compared with Anti-Her2(P) -Fc1-D1-D2-Fc 2.

The above experimental data prove that the recombinant protein composed of the truncations with different lengths in the right arm can influence the affinity of the recombinant protein and the target cells. For the left arm combined with the near-membrane-end epitope, when the right arm selects a shorter SIRP alpha truncation, the capacity of the recombinant protein to combine double targets can be greatly improved. However, for the left arm binding with the epitope at the far membrane end, the advantage is lost by selecting the shorter SIRPa truncation at the right arm, and the farther the antigen target at the left arm is away from the cell membrane, the longer the SIRPa truncation at the right arm is needed to achieve the optimal matching.

For example, as shown in FIG. 16, Anti-Her2(T) -Fc1-D1-Fc2 (EC)₅₀2.04nM) binds SKBR-3 cells significantly better than Anti-Her2(T) -Fc1-D1-D2-Fc2 (EC)₅₀25.95nM) (fig. 16A). While Anti-Her2(P) -Fc1-D1-Fc2 ability to bind SKBR-3 cells (EC)₅₀15.22nM) with Anti-Her2(P) -Fc1-D1-D2-Fc2 (EC)₅₀11.03nM) equivalent (fig. 16B).

As known to those skilled in the art, the above experimental results suggest that the right arm adaptively selects a human SIRPa extracellular truncation with a suitable length according to the distance between the target antigen space epitope and the surface of the target cell membrane, so as to effectively improve the binding capacity of the recombinant protein and the target cell.

Example 8 acute toxicity test of recombinant proteins

Recombinant proteins-Fc-D-Fc, -Fc-D-D-Fc, -Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, The acute toxicity test of Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2 and Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 is applicable to the detection of recombinant proteins containing SIRPa extracellular truncation in the right arm by taking Ofa-Fc1-D1-Fc2 as an example.

An appropriate amount of Ofa-Fc1-D1-Fc2 solution (4.02mg/mL) was diluted to 0.25 and 2.5mg/mL with Ofa-Fc1-D1-Fc2 buffer for injection (Tris-citrate, pH 6.5) for administration to group 1 and group 2, respectively, of the experimental animals.

4 healthy female cynomolgus monkeys, purchased from Guangxi Guidong Primates development experiment Co., Ltd, aged 4 years, were permitted to produce the experimental animals by the Guangxi Zhuang autonomous region scientific and technological Community, and the production permit of the experimental animals was SCXK Gui 2016 0001. Before administration, detailed clinical observation and weighing are carried out, and no abnormality is found. Body weight ranges from 2.47 to 2.85kg on the day of dosing.

TABLE 9 Experimental design

Administration by intravenous injection

The 4 female cynomolgus monkeys were divided into two groups, two in each group, and administered by intravenous injection at a dose of 0.5mg/kg, 5mg/kg and a volume of 2 mL/kg. The experimental design is shown in table 9 above. The administration was performed once on the day of administration, and the administration was continued for 28 days after completion. The cynomolgus monkeys were housed in stainless steel mobile cages, 1 per cage. Alternating light and dark illumination is provided for approximately 12 hours each day. Experimental monkey compound feeds were purchased from australian cooperative feeds ltd, beijing, with animals fed freely during the experiment except for specific fasts. The feed batch is tested by Shanghai spectral Nib test technology, Inc. (PONY) for specific microorganisms, heavy metals and pesticide residues in the feed. During the test period, all animals had free water through the water bottles, the drinking water used was purified water filtered and sterilized by a reverse osmosis system, and the pH, hardness, heavy metals and microorganisms of the drinking water were detected by a meter.

During the test period, all test animals were observed twice daily (once in the morning and afternoon) under cage observation including but not limited to morbidity, damage, mortality and food and water supply. All animals tested were subjected to 1 detailed clinical observation prior to the trial. Detailed clinical observations were made at least 1 time per day after dosing for all experimental animals during the trial. Observations include, but are not limited to, morbidity, mortality, damage and food and water supply conditions, skin, hair, eyes, ears, nose, mouth, chest, abdomen, external genitalia, limbs, respiratory and circulatory systems, autonomic effects (e.g., salivation), nervous system (e.g., tremor, convulsions, stress and abnormal behavior). Animal body weights were determined for D-1 (pre-dose), D1, D4, D8, D11, D15, D18, D22, D25 and pre-dissection, respectively. The food consumption of the animals within 24h (24h +/-1 h) was determined at D2, D4, D8, D11, D15, D18, D22 and D25, respectively. Electrocardiography was carried out using D-1, D2, D14, and D28, and recording was carried out using standard II leads (8 leads) at a recording speed of 50 mm/sec.

Clinical pathology sample collection and hematology, coagulation, blood biochemistry and lymphocyte typing tests were performed before (D-1), after D2, D7, D14 and D28, respectively; urine samples were collected before dosing, on day 28 post-dosing and subjected to urinalysis.

All animals were fasted overnight (at least 10 hours) before sample collection but not water deprived. A blood sample (4.5-6mL) was taken from the femoral vein, of which approximately 1.8mL of whole blood was used for coagulation analysis in an anticoagulation tube containing sodium citrate; approximately 1mL of whole blood was placed in an anticoagulation tube containing K3-EDTA for hematological analysis; approximately 2mL of whole blood was placed in a separation gel blood collection tube (without anticoagulant) for blood biochemical analysis, requiring centrifugation of the serum according to standard procedures. Meanwhile, the serum samples after D-1 before administration and D2 after administration are separated and subjected to T/B cell typing detection by a flow cytometer.

Animals were euthanized at D29 and heart, liver, spleen, lung and kidney tissues were collected and stored, and liver, lung (including main bronchi), kidney, spleen, heart, adrenal gland, pituitary, thyroid and parathyroid, thymus, ovary, uterus (including cervix), brain, etc. tissues were weighed.

The results show that when Ofa-Fc1-D1-Fc2 is administered by single intravenous injection, the administration dose is 0.5mg/kg and 5mg/kg respectively, no obvious abnormality related to drug action is seen in the animals after 28 days of continuous observation after administration, and the food consumption and the body weight of the animals fluctuate within a normal range during the period; compared with the prior to administration, the related detection data of animal blood coagulation, urine and electrocardiogram has no significant change after administration; after the animal is dissected, all organs are generally observed in a normal range, and the weight, the body coefficient and the heart-brain ratio of the animal organs are also in a normal range.

After administration of D2, the lymphocyte count and lymphocyte percentage of the low and high dose animals were significantly reduced, and the D7 was returned to normal level, which may be related to the drug action.

Recombinant proteins-Fc-D-D-Fc, -Fc-D-D-Fc, Anti-EGFR-Fc-D, Anti-EGFR-Fc-D-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, The results are similar to those of Ofa-Fc1-D1-Fc2 in the same dosage for Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc 2.

TABLE 10 recombinant protein vs. cynomolgus monkey erythrocyte number (10)¹²Individual cells/L) of the cells

As shown in FIG. 18A and Table 10, neither of the Ofa-Fc1-D1-Fc2 doses at 0.5mg/kg nor at 5mg/kg had an effect on cynomolgus monkey red blood cell numbers; as shown in FIG. 18B and Table 10, Ofa-Fc1-D1-Fc2 at both 0.5mg/kg and 5mg/kg did not affect cynomolgus monkey hemoglobin.

TABLE 11 Effect of recombinant proteins on cynomolgus monkey hemoglobin (g/L)

Recombinant proteins-Fc-D-D-Fc, -Fc-D-D-Fc, Anti-EGFR-Fc-D, Anti-EGFR-Fc-D-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, The effects of Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 on the number of cynomolgus monkey erythrocytes and hemoglobin at equal doses were similar to the results described above for Ofa-Fc1-D1-Fc 2.

As shown in FIG. 19, the two doses of Ofa-Fc1-D1-Fc2, both at 0.5mg/kg and 5mg/kg, resulted in significant depletion of animal B cells (CD20 antigen target cells) as measured by T/B cell typing.

As known by those skilled in the art, the above test results suggest that the recombinant protein of the present invention, which can synchronously bind to the tumor-targeting antigen and the CD47 antigen, has no obvious abnormality related to drug action after a single administration, and the food consumption and body weight of the animal fluctuate within the normal range during the period; compared with the prior to administration, the related detection data of animal blood coagulation, urine and electrocardiogram has no significant change after administration; after the animal is dissected, all organs are generally observed in a normal range, and the weight, the body coefficient and the heart-brain ratio of the animal organs are also in a normal range.

As known to those skilled in the art, the above test results suggest that the recombinant protein of the present invention, which can simultaneously bind to the tumor targeting antigen and the CD47 antigen, has no influence on the number of erythrocytes and the number of hemoglobin in animals after a single administration.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of synchronously binding with the tumor targeting antigen and the CD47 antigen preferentially eliminates the cells and/or tumor cells where the tumor targeting antigen is located under the same dosage condition.

Example 9 recombinant protein in vivo tumor growth inhibition assay

Recombinant proteins-Fc-D-Fc, -Fc-D-D-Fc, -Anti-EGFR-Fc-D-Fc, Anti-EGFR-Fc-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 in vivo tumor growth inhibition experiments, the following method is exemplified by Ofa-Fc1-D1-Fc2 and is suitable for the detection of recombinant proteins containing SIRPa extracellular truncation in the right arm.

Ofa-Fc1-D1-Fc2 is colorless clear liquid with concentration of 1.14-4.02mg/ml, and is stored at-80 deg.C;(rituximab injection): a colorless clear liquid, specification 100mg/10ml, batch number H0205, stored at 2-8 ℃ in the dark. Formulation buffer (Tris-citrate, pH 6.5): colorless clear liquid, and storing at 2-8 deg.C. The preparation method comprises the following steps: Ofa-Fc1-D1-Fc2,All are diluted by preparation buffer solution; the formulation buffer is administered directly as a solvent.

Cell: CD20 positive human B-cell lymphoma Daudi cells were purchased from the chinese academy of sciences cell bank. The culture conditions comprise adding 10% fetal calf serum, cyan and streptomycin into RPMI 1640 culture medium, and culturing at 37 deg.C with 5% CO₂Air incubator. And (4) carrying out passage twice a week, collecting cells when the cells are in an exponential growth phase, counting and inoculating.

Experimental animals: female NOD-SCID mice, 6-7 weeks, were purchased from Shanghai Ling Biotech, Inc. Producing license numbers: SCXK (Shanghai) 2013-0018; animal certification numbers 2013001829463, 2013001827545. A breeding environment: SPF grade. The use and welfare of the experimental animals were carried out in compliance with the provisions of the International Commission on evaluation and approval of Experimental animals (AAALAC). The health and death of the animals are monitored daily and routine examinations include observations of the effects of test substances and drugs on the daily performance of the animals, such as behavioral activities, weight changes, physical signs of appearance, etc.

Each mouse was inoculated subcutaneously 1.5X 10⁷Daudi cells, mean tumor volume reached 100-150mm at day 18 post inoculation³At times, the doses were grouped according to tumor volume and administered (D0). Intravenous (IV) drug in mice; the solvent group is injected with the same volume of solvent, and the injection volume is 0.1mL/10g body weight; the dosages and schedules of administration are shown in table 12.

Tumor diameter was measured twice weekly with a vernier caliper and tumor volume (V) was calculated as:

tumor volume (V) was calculated as:

V＝1/2×a×b2

wherein a and b represent length and width, respectively.

T/C(％)＝(T-T0)/(C-C0)×100

Wherein T, C is the tumor volume at the end of the experiment; t0, C0 are tumor volumes at the beginning of the experiment; t is the administration group, C is the control group.

Tumor inhibition rate (TGI) (%) 100-T/C (%).

When tumors regress, tumor inhibition rate (TGI) (%) 100- (T-T0)/T0 × 100

Partial tumor regression (PR) is defined if the tumor shrinks from the starting volume, i.e., T < T0 or C < C0; if the tumor completely disappears, it is defined as complete tumor regression (CR).

After the experiment is finished, or when the tumor volume of the animal reaches 1500mm³At the end of euthanasia, animals were sacrificed under carbon dioxide anesthesia and the tumors were dissected and weighed for photographing.

Comparison between two groups of tumor volumes or tumor weights using the two-tailed Student's t test, P <0.05 was defined as a statistically significant difference.

Ofa-Fc1-D1-Fc2(5mg/kg, IV, 2 times per week, 5 times total) significantly inhibited the growth of Daudi subcutaneous transplanted tumors with a tumor inhibition rate of 80.8% and partial regression of 1/6 mouse tumors;the tumor inhibition rate of (7mg/kg, IV, 2 times per week, 5 times total) against Daudi subcutaneous transplanted tumors was 24.5%. Tumor-bearing mice were generally better tolerated the above drugs (table 12, fig. 20, 21, 22).

TABLE 12Ofa-Fc1-D1-Fc2,Therapeutic effect on human B-cell lymphoma Daudi cell subcutaneous transplantation tumor

Note: randomly grouping, with the first dose time being D0; IV: and (4) performing intravenous injection.

As can be seen, Ofa-Fc1-D1-Fc2 andhas different degrees of inhibition on the growth of subcutaneous transplantation tumor of the Daudi cell lymphoma Daudi cell with CD20 positive, wherein Ofa-Fc1-D1-Fc2 is obviously better than that of subcutaneous transplantation tumor of the Daudi cell lymphoma Daudi cellThe tumor-bearing mice can be better tolerant to the medicines on the whole.

Recombinant proteins-Fc-D-D-Fc, -Fc-D-D-Fc, Anti-EGFR-Fc-D, Anti-EGFR-Fc-D-D-D-Fc, Anti-Her (P) -Fc-D-Fc, Anti-Her (T) -Fc-D-Fc, Anti-Her2(T) -Fc 1-D1-D1-Fc 1, Anti-Her 1 (T) -Fc 1-D1-D1-D1-Fc 1, Anti-EGFR-Fc 1-D1-D1-D1-Fc 1, Anti-PD-L1(Ate) -Fc1-D1-Fc 72, Anti-PD-L1(Ate) -Fc 1-D1-D1-Fc 72-D1-D1-Fc 72-Fc 1-Fc 72, Anti-PD-L1 (3613G) -Fc 72-1-Fc 72 (Ate) -1-Fc 72-1-Fc 72 (Ate) -1-Fc 72-Fc-1 (Ate) -1-Fc 72-1-Fc 72-Fc-1-Fc-1-Fc 72-Fc 72-1-Fc-1-Fc 72-Fc-1 (Ate) -1-Fc 72-Fc-1-Fc-1-Fc 72-Fc-1-Fc-1-Fc-1-Fc 72 (Ate) -1-Fc 72-Fc 72 (Ate) -1-Fc 72-Fc-1-Fc 72-Fc 72-Fc-1-Fc-1-Fc 72-1-Fc-1-Fc-1-Fc-, The antitumor effect of Anti-PD-L1(13G4) -Fc1-D1-D2-D3-Fc2, Anti-PD-L1(12A4) -Fc1-D1-Fc2, Anti-PD-L1(12A4) -Fc1-D1-D2-Fc2, and Anti-PD-L1(12A4) -Fc1-D1-D2-D3-Fc2 on NOD-SCID mice transplanted tumors and the tolerance of mice to recombinant proteins were similar to those of Ofa-Fc1-D1-Fc 2.

As known to those skilled in the art, the above experimental results suggest that the recombinant protein capable of synchronously binding with the tumor targeting antigen and the CD47 antigen has an unexpectedly significant anti-tumor effect in mice compared with the tumor targeting antibody under the same dosage condition.

The use and welfare of the experimental animals were carried out in compliance with the provisions of the International Commission on evaluation and approval of Experimental animals (AAALAC). The health and death of the animals are monitored daily and routine examinations include observations of the effects of test substances and drugs on the daily performance of the animals, such as behavioral activities, weight changes, physical signs of appearance, etc.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, any theory, mechanism, proof, or finding stated herein is meant to further enhance understanding of the present invention, and is not intended to limit the present invention in any way to such theory, mechanism, proof, or finding. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character.

Sequence Listing

<110> Shanghai Jinmant Biotech Co., Ltd

<120> bispecific recombinant protein and application thereof

<130> P21018849CD

<150> CN201710317926.7

<151> 2017-05-08

<150> CN201711269620.5

<151> 2017-12-05

<160> 44

<170> PatentIn version 3.5

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Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu

100 105 110

Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met

115 120 125

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

130 135 140

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

145 150 155 160

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

165 170 175

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

180 185 190

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

195 200 205

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

210 215 220

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

225 230 235 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 2

<211> 234

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<220>

<223> Ofa light chain amino acid sequence

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Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

100 105 110

Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 3

<211> 468

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<213> Artificial Sequence

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<223> Obi heavy chain amino acid sequence

<400> 3

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe

35 40 45

Ser Tyr Ser Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn

65 70 75 80

Gly Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 4

<211> 239

<212> PRT

<213> Artificial Sequence

<220>

<223> Obi/Obi-Fc1 light chain amino acid sequence

<400> 4

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro

20 25 30

Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser

35 40 45

Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys

115 120 125

Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 5

<211> 461

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu5F9-G4 heavy chain amino acid sequence

<400> 5

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

20 25 30

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn

35 40 45

Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp

50 55 60

Met Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys

65 70 75 80

Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala

85 90 95

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

100 105 110

Cys Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

225 230 235 240

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

245 250 255

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

260 265 270

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

275 280 285

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

305 310 315 320

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

325 330 335

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

340 345 350

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

355 360 365

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

370 375 380

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

385 390 395 400

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

405 410 415

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

420 425 430

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

435 440 445

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455 460

<210> 6

<211> 236

<212> PRT

<213> Artificial Sequence

<220>

<223> Hu5F9-G4 light chain amino acid sequence

<400> 6

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

20 25 30

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr

35 40 45

Ser Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln

50 55 60

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

65 70 75 80

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

85 90 95

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

100 105 110

Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 7

<211> 466

<212> PRT

<213> Artificial Sequence

<220>

<223> JMT101 heavy chain amino acid sequence

<400> 7

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

20 25 30

Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn

35 40 45

Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

50 55 60

Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe

65 70 75 80

Thr Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

85 90 95

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

100 105 110

Ala Arg Ala Leu Asp Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 8

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> JMT101/Anti-EGFR-Fc1 light chain amino acid sequence

<400> 8

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro

20 25 30

Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr

35 40 45

Asn Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu

50 55 60

Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser

65 70 75 80

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu

85 90 95

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Glu Trp Pro

100 105 110

Thr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

115 120 125

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

130 135 140

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

145 150 155 160

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

165 170 175

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

180 185 190

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

195 200 205

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

210 215 220

Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 9

<211> 468

<212> PRT

<213> Artificial Sequence

<220>

<223> Trastuzumab heavy chain amino acid sequence

<400> 9

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

20 25 30

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp

35 40 45

Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

50 55 60

Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser

65 70 75 80

Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala

85 90 95

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

100 105 110

Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 10

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> Trastuzumab light chain amino acid sequence

<400> 10

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr

35 40 45

Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

50 55 60

Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser

65 70 75 80

Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

85 90 95

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro

100 105 110

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

115 120 125

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

130 135 140

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

145 150 155 160

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

165 170 175

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

180 185 190

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

195 200 205

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

210 215 220

Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 11

<211> 359

<212> PRT

<213> Artificial Sequence

<220>

<223> SIRP alpha D1-Fc amino acid sequence

<400> 11

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val

20 25 30

Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser

35 40 45

Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala

50 55 60

Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr

65 70 75 80

Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser

85 90 95

Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys

100 105 110

Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr

115 120 125

Glu Leu Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

130 135 140

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

145 150 155 160

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

165 170 175

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

180 185 190

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

195 200 205

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

210 215 220

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

225 230 235 240

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

245 250 255

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

260 265 270

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

275 280 285

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

290 295 300

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

305 310 315 320

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

325 330 335

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

340 345 350

Leu Ser Leu Ser Pro Gly Lys

355

<210> 12

<211> 467

<212> PRT

<213> Artificial Sequence

<220>

<223> Atezolizumab heavy chain amino acid sequence

<400> 12

Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

85 90 95

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210> 13

<211> 233

<212> PRT

<213> Artificial Sequence

<220>

<223> Atezolizumab/Anti-PD-L1(Ate) -Fc1 light chain amino acid sequence

<400> 13

Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

20 25 30

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val

35 40 45

Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

50 55 60

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr

100 105 110

His Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 14

<211> 1092

<212> DNA

<213> Artificial Sequence

<220>

<223> D1-Fc2 DNA sequence

<400> 14

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60

gaggagctgc aggtcatcca gcccgataag agcgtgtccg tggccgcagg agaatctgcc 120

atcctgcatt gcaccgtgac ctctctgatc cccgtgggcc caatccagtg gttcagagga 180

gccggaccag ctagagagct gatctacaac cagaaggagg gccacttccc cagagtgaca 240

accgtgtccg agtctaccaa gcgggagaac atggacttct ccatctccat ctccgccatc 300

acaccagccg acgccggcac ctactattgc gtgaagttcc ggaagggctc cccagatacc 360

gagtttaaga gcggcgccgg aacagagctg agcgtgcggg ctaagcctga caagacccac 420

acctgtcccc cttgtcctgc ccctgaactg ctgggcggac cttccgtgtt cctgttcccc 480

ccaaagccca aggacaccct gatgatctcc cggacccccg aagtgacctg cgtggtggtg 540

gatgtgtccc acgaggaccc tgaagtgaag ttcaattggt acgtggacgg cgtggaagtg 600

cacaacgcca agaccaagcc tagagaggaa cagtacaact ccacctaccg ggtggtgtcc 660

gtgctgaccg tgctgcacca ggattggctg aacggcaaag agtacaagtg caaggtgtcc 720

aacaaggccc tgcctgcccc catcgaaaag accatctcca aggccaaggg ccagccccgg 780

gaaccccagg tgtacacact gccccctagc agggacgagc tgaccaagaa ccaggtgtcc 840

ctgtggtgtc tcgtgaaagg cttctacccc tccgacattg ccgtggaatg ggagtccaac 900

ggccagcctg agaacaacta caagaccacc ccccctgtgc tggactccga cggctcattc 960

ttcctgtaca gcaagctgac agtggacaag tcccggtggc agcagggcaa cgtgttctcc 1020

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gtccctgagc 1080

cccggcaaat ga 1092

<210> 15

<211> 1389

<212> DNA

<213> Artificial Sequence

<220>

<223> D1-D2-Fc2 DNA sequence

<400> 15

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60

gaggagctgc aggtcatcca gcccgataag agcgtgtccg tggccgcagg agaatctgcc 120

atcctgcatt gcaccgtgac ctctctgatc cccgtgggcc caatccagtg gttcagagga 180

gccggaccag ctagagagct gatctacaac cagaaggagg gccacttccc cagagtgaca 240

accgtgtccg agtctaccaa gcgggagaac atggacttct ccatctccat ctccgccatc 300

acaccagccg acgccggcac ctactattgc gtgaagttcc ggaagggctc cccagatacc 360

gagtttaaga gcggcgccgg aacagagctg agcgtgcggg ctaagccttc tgctccagtg 420

gtgtcaggac cagcagctag agctacccct cagcacaccg tgtccttcac ctgcgagtct 480

cacggcttct cccctagaga catcaccctc aagtggttca agaacggcaa cgagctgtcc 540

gacttccaga ccaacgtgga tccagtgggc gagagcgtgt cttactccat ccactccacc 600

gccaaggtgg tgctgacaag ggaggacgtg cactcccagg tcatttgcga ggtggcacac 660

gtgacattgc agggcgaccc cctgagggga accgccaact tgagtgacaa gacccacacc 720

tgtccccctt gtcctgcccc tgaactgctg ggcggacctt ccgtgttcct gttcccccca 780

aagcccaagg acaccctgat gatctcccgg acccccgaag tgacctgcgt ggtggtggat 840

gtgtcccacg aggaccctga agtgaagttc aattggtacg tggacggcgt ggaagtgcac 900

aacgccaaga ccaagcctag agaggaacag tacaactcca cctaccgggt ggtgtccgtg 960

ctgaccgtgc tgcaccagga ttggctgaac ggcaaagagt acaagtgcaa ggtgtccaac 1020

aaggccctgc ctgcccccat cgaaaagacc atctccaagg ccaagggcca gccccgggaa 1080

ccccaggtgt acacactgcc ccctagcagg gacgagctga ccaagaacca ggtgtccctg 1140

tggtgtctcg tgaaaggctt ctacccctcc gacattgccg tggaatggga gtccaacggc 1200

cagcctgaga acaactacaa gaccaccccc cctgtgctgg actccgacgg ctcattcttc 1260

ctgtacagca agctgacagt ggacaagtcc cggtggcagc agggcaacgt gttctcctgc 1320

tccgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgtc cctgagcccc 1380

ggcaaatga 1389

<210> 16

<211> 471

<212> PRT

<213> Artificial Sequence

<220>

<223> Ofa-Fc1 heavy chain amino acid sequence

<400> 16

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu

100 105 110

Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met

115 120 125

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

130 135 140

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

145 150 155 160

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

165 170 175

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

180 185 190

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

195 200 205

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

210 215 220

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

225 230 235 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 17

<211> 234

<212> PRT

<213> Artificial Sequence

<220>

<223> Ofa-Fc1 light chain amino acid sequence

<400> 17

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

100 105 110

Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 18

<211> 468

<212> PRT

<213> Artificial Sequence

<220>

<223> Obi-Fc1 heavy chain amino acid sequence

<400> 18

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Ser Asn Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr

65 70 75 80

Pro Phe Thr Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln

85 90 95

Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Ala Leu Asp Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 19

<211> 468

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-EGFR-Fc1 heavy chain amino acid sequence

<400> 19

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Ser Asn Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr

65 70 75 80

Pro Phe Thr Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln

85 90 95

Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Ala Leu Asp Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 20

<211> 469

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-Her2(T) -Fc1 heavy chain amino acid sequence

<400> 20

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

85 90 95

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

370 375 380

Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly Lys

465

<210> 21

<211> 234

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-Her2(T) -Fc1 light chain amino acid sequence

<400> 21

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp

35 40 45

Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr

100 105 110

Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 22

<211> 468

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-Her2(P) -Fc1 heavy chain amino acid sequence

<400> 22

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn

65 70 75 80

Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 23

<211> 234

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-Her2(P) -Fc1 light chain amino acid sequence

<400> 23

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr

100 105 110

Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 24

<211> 467

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-PD-L1(Ate) -Fc1 heavy chain amino acid sequence

<400> 24

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

85 90 95

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210> 25

<211> 1641

<212> DNA

<213> Artificial Sequence

<220>

<223> D1-D2-D3-Fc2 DNA sequence

<400> 25

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60

gaggagctgc aggtcatcca gcccgataag agcgtgtccg tggccgcagg agaatctgcc 120

atcctgcatt gcaccgtgac ctctctgatc cccgtgggcc caatccagtg gttcagagga 180

gccggaccag ctagagagct gatctacaac cagaaggagg gccacttccc cagagtgaca 240

accgtgtccg agtctaccaa gcgggagaac atggacttct ccatctccat ctccgccatc 300

acaccagccg acgccggcac ctactattgc gtgaagttcc ggaagggctc cccagatacc 360

gagtttaaga gcggcgccgg aacagagctg agcgtgcggg ctaagccttc tgctccagtg 420

gtgtcaggac cagcagctag agctacccct cagcacaccg tgtccttcac ctgcgagtct 480

cacggcttct cccctagaga catcaccctc aagtggttca agaacggcaa cgagctgtcc 540

gacttccaga ccaacgtgga tccagtgggc gagagcgtgt cttactccat ccactccacc 600

gccaaggtgg tgctgacaag ggaggacgtg cactcccagg tcatttgcga ggtggcacac 660

gtgacattgc agggcgaccc cctgagaggc acagcaaact tgagcgagac aattagagtg 720

ccccccaccc tggaagttac acagcagccc gttagagccg agaaccaggt caacgtcacc 780

tgccaggtca gaaagtttta tccacagaga ctgcagctga cctggctcga gaacggaaac 840

gtgagcagaa cagagaccgc cagcaccgtg acagagaaca aggacgggac ctacaactgg 900

atgagttggc tgctggtgaa cgtcagcgcc cacagagacg acgtcaagct gacctgcgac 960

aagacccaca cctgtccccc ttgtcctgcc cctgaactgc tgggcggacc ttccgtgttc 1020

ctgttccccc caaagcccaa ggacaccctg atgatctccc ggacccccga agtgacctgc 1080

gtggtggtgg atgtgtccca cgaggaccct gaagtgaagt tcaattggta cgtggacggc 1140

gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacaactc cacctaccgg 1200

gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1260

aaggtgtcca acaaggccct gcctgccccc atcgaaaaga ccatctccaa ggccaagggc 1320

cagccccggg aaccccaggt gtacacactg ccccctagca gggacgagct gaccaagaac 1380

caggtgtccc tgtggtgtct cgtgaaaggc ttctacccct ccgacattgc cgtggaatgg 1440

gagtccaacg gccagcctga gaacaactac aagaccaccc cccctgtgct ggactccgac 1500

ggctcattct tcctgtacag caagctgaca gtggacaagt cccggtggca gcagggcaac 1560

gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1620

tccctgagcc ccggcaaatg a 1641

<210> 26

<211> 363

<212> PRT

<213> Artificial Sequence

<220>

<223> D1-Fc2 amino acid sequence

<400> 26

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val

20 25 30

Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser

35 40 45

Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala

50 55 60

Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr

65 70 75 80

Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser

85 90 95

Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys

100 105 110

Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr

115 120 125

Glu Leu Ser Val Arg Ala Lys Pro Asp Lys Thr His Thr Cys Pro Pro

130 135 140

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

145 150 155 160

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

165 170 175

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

180 185 190

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

195 200 205

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

210 215 220

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

225 230 235 240

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

245 250 255

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

260 265 270

Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

275 280 285

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

290 295 300

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

305 310 315 320

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

325 330 335

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

340 345 350

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 27

<211> 462

<212> PRT

<213> Artificial Sequence

<220>

<223> D1-D2-Fc2 amino acid sequence

<400> 27

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val

20 25 30

Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser

35 40 45

Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala

50 55 60

Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr

65 70 75 80

Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser

85 90 95

Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys

100 105 110

Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr

115 120 125

Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro

130 135 140

Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser

145 150 155 160

His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly

165 170 175

Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser

180 185 190

Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu

195 200 205

Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln

210 215 220

Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Asp Lys Thr His Thr

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

355 360 365

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460

<210> 28

<211> 546

<212> PRT

<213> Artificial Sequence

<220>

<223> D1-D2-D3-Fc2 amino acid sequence

<400> 28

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val

20 25 30

Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser

35 40 45

Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala

50 55 60

Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr

65 70 75 80

Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser

85 90 95

Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys

100 105 110

Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr

115 120 125

Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro

130 135 140

Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser

145 150 155 160

His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly

165 170 175

Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser

180 185 190

Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu

195 200 205

Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln

210 215 220

Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val

225 230 235 240

Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln

245 250 255

Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln

260 265 270

Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser

275 280 285

Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu

290 295 300

Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Asp

305 310 315 320

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

325 330 335

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

340 345 350

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

355 360 365

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

370 375 380

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

385 390 395 400

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

405 410 415

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

420 425 430

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

435 440 445

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

450 455 460

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

465 470 475 480

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

485 490 495

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

500 505 510

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

515 520 525

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

530 535 540

Gly Lys

545

<210> 29

<211> 369

<212> PRT

<213> Artificial Sequence

<220>

<223> D1m-Fc2 amino acid sequence

<400> 29

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val

20 25 30

Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser

35 40 45

Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala

50 55 60

Arg Val Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr

65 70 75 80

Thr Val Ser Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser

85 90 95

Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys

100 105 110

Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr

115 120 125

Glu Leu Ser Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser Asp Lys

130 135 140

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

145 150 155 160

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

165 170 175

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

180 185 190

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

195 200 205

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

210 215 220

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

225 230 235 240

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

245 250 255

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

260 265 270

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

275 280 285

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

290 295 300

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

305 310 315 320

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

325 330 335

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

340 345 350

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360 365

Lys

<210> 30

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> D1 amino acid sequence

<400> 30

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro

115

<210> 31

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> D1-D2 amino acid sequence

<400> 31

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg

115 120 125

Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe

130 135 140

Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu

145 150 155 160

Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr

165 170 175

Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His

180 185 190

Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro

195 200 205

Leu Arg Gly Thr Ala Asn Leu Ser

210 215

<210> 32

<211> 300

<212> PRT

<213> Artificial Sequence

<220>

<223> D1-D2-D3 amino acid sequence

<400> 32

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg

115 120 125

Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe

130 135 140

Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu

145 150 155 160

Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr

165 170 175

Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His

180 185 190

Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro

195 200 205

Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr

210 215 220

Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val

225 230 235 240

Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp

245 250 255

Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr

260 265 270

Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn

275 280 285

Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys

290 295 300

<210> 33

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> D1m amino acid sequence

<400> 33

Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu

35 40 45

Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser

115 120

<210> 34

<211> 1416

<212> DNA

<213> Artificial Sequence

<220>

<223> Ofa-Fc1 heavy chain DNA sequence

<400> 34

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60

gtgcagctgg tggaatctgg cggcggactg gtgcagcctg gcagatccct gagactgtct 120

tgtgccgcct ccggcttcac cttcaacgac tacgccatgc actgggtgcg acaggcccct 180

ggcaaaggcc tggaatgggt gtccaccatc agctggaact ccggctccat cggctacgcc 240

gactccgtga agggccggtt caccatctcc cgggacaacg ccaagaagtc cctgtacctg 300

cagatgaact ccctgcgggc cgaggacacc gccctgtact actgtgccaa ggacatccag 360

tacggcaact actactacgg catggacgtg tggggccagg gcaccacagt gaccgtgtca 420

tctgcttcta ccaagggccc ctccgtgttt cctctggccc cttccagcaa gtccacctct 480

ggcggaacag ccgctctggg ctgcctcgtg aaggactact tccccgagcc tgtgaccgtg 540

tcctggaact ctggcgctct gacatccggc gtgcacacct tccctgctgt gctgcagtct 600

agcggcctgt actccctgtc ctccgtcgtg accgtgcctt ccagctctct gggcacccag 660

acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggaa 720

cccaagtcct gcgacaagac ccacacctgt cccccttgtc ctgcccctga actgctgggc 780

ggaccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat ctcccggacc 840

cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 900

tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 960

aactccacct accgggtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 1020

aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga aaagaccatc 1080

tccaaggcca agggccagcc ccgggaaccc caggtgtaca cactgccccc tagcagggac 1140

gagctgacca agaaccaggt gtccctgagc tgtgcagtga aaggcttcta cccctccgac 1200

attgccgtgg aatgggagtc caacggccag cctgagaaca actacaagac caccccccct 1260

gtgctggact ccgacggctc attcttcctg gtgagcaagc tgacagtgga caagtcccgg 1320

tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1380

acccagaagt ccctgtccct gagccccggc aaatga 1416

<210> 35

<211> 705

<212> DNA

<213> Artificial Sequence

<220>

<223> Ofa-Fc1 light chain DNA sequence

<400> 35

atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60

gagatcgtgc tgacccagtc tcctgccacc ctgtctctga gccctggcga gagagctacc 120

ctgtcctgca gagcctccca gtccgtgtcc tcttacctgg cctggtatca gcagaagccc 180

ggccaggctc cccggctgct gatctacgat gcctccaata gagccaccgg catccctgcc 240

agattctccg gctctggctc tggcaccgac tttaccctga ccatctccag cctggaaccc 300

gaggacttcg ccgtgtacta ctgccagcag cggtccaact ggcccatcac ctttggccag 360

ggcacccggc tggaaatcaa gagaaccgtg gccgctccct ccgtgttcat cttcccacct 420

tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 480

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 540

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 600

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 660

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga 705

<210> 36

<211> 1407

<212> DNA

<213> Artificial Sequence

<220>

<223> Obi-Fc1 heavy chain DNA sequence

<400> 36

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctcag 60

gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gctcctccgt gaaggtgtcc 120

tgcaaggctt ccggctacgc cttctcctac tcctggatca actgggtgcg acaggcccct 180

ggacagggcc tggaatggat gggcagaatc ttccctggcg acggcgacac cgactacaac 240

ggcaagttca agggcagagt gaccatcacc gccgacaagt ccacctccac cgcctacatg 300

gaactgtcct ccctgcggag cgaggacacc gccgtgtact actgcgcccg gaacgtgttc 360

gacggctact ggctggtgta ttggggccag ggcaccctcg tgaccgtgtc ctctgcttct 420

accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 480

gccgctctgg gctgcctcgt gaaggactac ttccccgagc ctgtgaccgt gtcctggaac 540

tctggcgctc tgacatccgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600

tactccctgt cctccgtcgt gaccgtgcct tccagctctc tgggcaccca gacctacatc 660

tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 720

tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780

gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840

acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 960

taccgggtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 1020

aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080

aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140

aagaaccagg tgtccctgtc ctgtgctgtg aaaggcttct acccctccga cattgccgtg 1200

gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260

tccgacggct cattcttcct ggtgagcaag ctgacagtgg acaagtcccg gtggcagcag 1320

ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380

tccctgtccc tgagccccgg caaatga 1407

<210> 37

<211> 1407

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-EGFR-Fc1 heavy chain DNA sequence

<400> 37

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctcag 60

gtccagctcc aggaaagcgg ccccggcctc gtcaaaccct ccgagacact ctccctcaca 120

tgcacagtct ccggcttctc cctcagcaac tacgacgtcc actgggtcag acaggccccc 180

ggcaaaggac tggaatggct cggcgtcatc tggtccggcg gaaacaccga ctacaacacc 240

ccattcacct ccaggctcac catctccgtg gacacctcca agaaccagtt ctccctcaaa 300

ctgagctccg tgaccgccgc cgacaccgct gtctattatt gcgccagagc cctcgactac 360

tacgactacg aattcgccta ctggggccag ggcaccctgg tgaccgtgtc atctgcttct 420

accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 480

gccgctctgg gctgcctcgt gaaggactac ttccccgagc ctgtgaccgt gtcctggaac 540

tctggcgctc tgacatccgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600

tactccctgt cctccgtcgt gaccgtgcct tccagctctc tgggcaccca gacctacatc 660

tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 720

tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780

gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840

acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 960

taccgggtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 1020

aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080

aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140

aagaaccagg tgtccctgag ctgtgcagtg aaaggcttct acccctccga cattgccgtg 1200

gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260

tccgacggct cattcttcct ggtgagcaag ctgacagtgg acaagtcccg gtggcagcag 1320

ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380

tccctgtccc tgagccccgg caaatga 1407

<210> 38

<211> 1410

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-Her2(T) -Fc1 heavy chain DNA sequence

<400> 38

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgag 60

gtgcagttgg tggagagcgg gggggggctg gtgcagcctg gaggaagttt gaggttgagc 120

tgtgccgcaa gcgggttcaa cattaaggac acatacattc actgggtgag gcaggcaccc 180

ggaaagggac tggagtgggt ggctaggatc taccccacca acggctacac aaggtacgcc 240

gacagtgtga agggccggtt caccatttcc gccgacacct ccaagaacac cgcctacctg 300

cagatgaaca gcctgagggc cgaggacacc gccgtctact actgctccag gtggggagga 360

gacggattct atgctatgga ctactgggga cagggcaccc tggtgaccgt gtcatctgct 420

tctaccaagg gcccctccgt gtttcctctg gccccttcca gcaagtccac ctctggcgga 480

acagccgctc tgggctgcct cgtgaaggac tacttccccg agcctgtgac cgtgtcctgg 540

aactctggcg ctctgacatc cggcgtgcac accttccctg ctgtgctgca gtctagcggc 600

ctgtactccc tgtcctccgt cgtgaccgtg ccttccagct ctctgggcac ccagacctac 660

atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagaaggt ggaacccaag 720

tcctgcgaca agacccacac ctgtccccct tgtcctgccc ctgaactgct gggcggacct 780

tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 840

gtgacctgcg tggtggtgga tgtgtcccac gaggaccctg aagtgaagtt caattggtac 900

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc 960

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag 1020

tacaagtgca aggtgtccaa caaggccctg cctgccccca tcgaaaagac catctccaag 1080

gccaagggcc agccccggga accccaggtg tacacactgc cccctagcag ggacgagctg 1140

accaagaacc aggtgtccct gagctgtgca gtgaaaggct tctacccctc cgacattgcc 1200

gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 1260

gactccgacg gctcattctt cctggtgagc aagctgacag tggacaagtc ccggtggcag 1320

cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380

aagtccctgt ccctgagccc cggcaaatga 1410

<210> 39

<211> 705

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-Her2(T) -Fc1 light chain DNA sequence

<400> 39

atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60

gacattcaga tgacccagag cccctcctcc ctctccgcct ccgtgggaga cagagttacc 120

atcacctgca gggcctccca ggacgtgaac accgccgtgg cctggtacca gcagaaaccc 180

ggcaaagccc ccaaactgct catctactcc gcctcatttc tgtacagcgg cgtgccctcc 240

cgcttctccg gttccagatc cggcaccgac ttcaccctga ctatctcctc cctccagccc 300

gaagacttcg ccacctacta ctgccagcag cactacacca ccccccccac cttcggccag 360

ggcacaaagg tcgaaatcaa gagaaccgtg gccgctccct ccgtgttcat cttcccacct 420

tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 480

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 540

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 600

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 660

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga 705

<210> 40

<211> 1407

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-Her2(P) -Fc1 heavy chain DNA sequence

<400> 40

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgag 60

gtgcagttgg tggagagcgg gggggggctg gtgcagcctg gaggaagttt gaggttgagc 120

tgtgccgcaa gcgggttcac atttacagac tacacaatgg actgggtgag gcaggcaccc 180

ggaaagggac tggagtgggt ggctgatgtg aatcccaata gcggagggag catttacaac 240

cagagattca aggggcggtt caccttgtcc gtggacagga gcaagaacac actgtacctg 300

cagatgaaca gcctgagggc cgaggatacc gccgtctact attgcgccag gaacctcgga 360

ccctccttct attttgacta ctggggccag ggaaccctgg tgaccgtgtc atctgcttct 420

accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 480

gccgctctgg gctgcctcgt gaaggactac ttccccgagc ctgtgaccgt gtcctggaac 540

tctggcgctc tgacatccgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600

tactccctgt cctccgtcgt gaccgtgcct tccagctctc tgggcaccca gacctacatc 660

tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 720

tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780

gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840

acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 960

taccgggtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 1020

aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080

aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140

aagaaccagg tgtccctgag ctgtgcagtg aaaggcttct acccctccga cattgccgtg 1200

gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260

tccgacggct cattcttcct ggtgagcaag ctgacagtgg acaagtcccg gtggcagcag 1320

ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380

tccctgtccc tgagccccgg caaatga 1407

<210> 41

<211> 705

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-Her2(P) -Fc1 light chain DNA sequence

<400> 41

atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60

gacattcaga tgacccagag cccctcctcc ctctccgcct ccgtgggaga cagagttacc 120

atcacctgca aagccagcca ggacgtgagc atcggcgtgg cctggtacca gcagaaaccc 180

ggcaaagccc ccaaactgct catttactcc gcctcatacc gttacaccgg cgttccctcc 240

cgcttcagcg gatccggctc cggaaccgac ttcaccctga ctatctcctc cctccagccc 300

gaagacttcg ccacctacta ctgccagcag tactacattt acccctacac cttcggccag 360

ggcaccaagg tggaaatcaa gagaaccgtg gccgctccct ccgtgttcat cttcccacct 420

tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 480

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 540

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 600

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 660

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga 705

<210> 42

<211> 1404

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-PD-L1(Ate) -Fc1 heavy chain DNA sequence

<400> 42

atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60

gtgcagctgg tggaaagcgg cggcggcctg gtgcagccgg gcggcagcct gcgcctgagc 120

tgcgcggcga gcggctttac ctttagcgat agctggattc attgggtgcg ccaggcgccg 180

ggcaaaggcc tggaatgggt ggcgtggatt agcccgtatg gcggcagcac ctattatgcg 240

gatagcgtga aaggccgctt taccattagc gcggatacca gcaaaaacac cgcgtatctg 300

cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt attgcgcgcg ccgccattgg 360

ccgggcggct ttgattactg gggccagggc accctggtga ccgtgtcatc tgcttctacc 420

aagggcccct ccgtgtttcc tctggcccct tccagcaagt ccacctctgg cggaacagcc 480

gctctgggct gcctcgtgaa ggactacttc cccgagcctg tgaccgtgtc ctggaactct 540

ggcgctctga catccggcgt gcacaccttc cctgctgtgc tgcagtctag cggcctgtac 600

tccctgtcct ccgtcgtgac cgtgccttcc agctctctgg gcacccagac ctacatctgc 660

aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggaacc caagtcctgc 720

gacaagaccc acacctgtcc cccttgtcct gcccctgaac tgctgggcgg accttccgtg 780

ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 840

tgcgtggtgg tggatgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 900

ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa ctccacctac 960

cgggtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag 1020

tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaaa agaccatctc caaggccaag 1080

ggccagcccc gggaacccca ggtgtacaca ctgcccccta gcagggacga gctgaccaag 1140

aaccaggtgt ccctgagctg tgcagtgaaa ggcttctacc cctccgacat tgccgtggaa 1200

tgggagtcca acggccagcc tgagaacaac tacaagacca ccccccctgt gctggactcc 1260

gacggctcat tcttcctggt gagcaagctg acagtggaca agtcccggtg gcagcagggc 1320

aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1380

ctgtccctga gccccggcaa atga 1404

<210> 43

<211> 1110

<212> DNA

<213> Artificial Sequence

<220>

<223> D1m-Fc2 DNA sequence

<400> 43

atggagtgga gctgggtgtt cttgttcttc ttgtccgtga ccaccggggt gcacagcgag 60

gaggagttgc agatcatcca gcctgacaag agcgtgagcg tggccgccgg ggagagcgct 120

attctgcact gtaccatcac ctccctcttc cccgtgggcc ccattcagtg gttcagggga 180

gccgggcccg ccagagttct gatttacaac cagaggcagg gcccctttcc ccgggttacc 240

actgtctctg agaccaccaa gcgggagaac atggatttca gcatctccat cagcaacatt 300

actcccgccg acgccggcac ctactactgc atcaaattca gaaagggctc tcccgacacc 360

gaattcaaaa gcggcgccgg caccgaactg tccgtgcgag ctaagccctc cgagcccaaa 420

tcctcagaca agacccacac ctgtccccct tgtcctgccc ctgaactgct gggcggacct 480

tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 540

gtgacctgcg tggtggtgga tgtgtcccac gaggaccctg aagtgaagtt caattggtac 600

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc 660

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag 720

tacaagtgca aggtgtccaa caaggccctg cctgccccca tcgaaaagac catctccaag 780

gccaagggcc agccccggga accccaggtg tacacactgc cccctagcag ggacgagctg 840

accaagaacc aggtgtccct gtggtgtctc gtgaaaggct tctacccctc cgacattgcc 900

gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 960

gactccgacg gctcattctt cctgtacagc aagctgacag tggacaagtc ccggtggcag 1020

cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1080

aagtccctgt ccctgagccc cggcaaatga 1110

<210> 44

<211> 462

<212> PRT

<213> Artificial Sequence

<220>

<223> D1m-D2-Fc2 amino acid sequence

<400> 44

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val

20 25 30

Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser

35 40 45

Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala

50 55 60

Arg Val Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr

65 70 75 80

Thr Val Ser Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser

85 90 95

Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys

100 105 110

Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr

115 120 125

Glu Leu Ser Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser Gly Pro

130 135 140

Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser

145 150 155 160

His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly

165 170 175

Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser

180 185 190

Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu

195 200 205

Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln

210 215 220

Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Asp Lys Thr His Thr

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

355 360 365

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460