CN112969476B

CN112969476B - Multi-specific protein molecules

Info

Publication number: CN112969476B
Application number: CN201980072293.5A
Authority: CN
Inventors: 应华; 张玲; 杨筱莹; 葛虎; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2018-12-07
Filing date: 2019-12-06
Publication date: 2024-06-14
Anticipated expiration: 2039-12-06
Also published as: WO2020114479A1; TW202033218A; CN112969476A

Abstract

To multi-specific protein molecules. In particular to a multispecific antibody with a novel structural form. The multispecific antibody can be combined with CD3 and other tumor-associated antigens simultaneously, and can combine with tumor-associated antigen expression cells and activate CD3 positive T cells at the same time, so that the T cells can promote the specific killing of tumor cells expressing tumor-associated antigens. Meanwhile, the preparation and the application of the multispecific antibody are also provided.

Description

Multi-specific protein molecules

Technical Field

The present invention relates to multispecific antibodies, such as multispecific antibodies that bind CD3 and tumor-associated antigens.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

CD3 is a T cell co-receptor consisting of four distinct chains (Wucherpfennig, pages (2010)"Structural Biology Of The T cell Receptor：Insights Into Receptor Assembly,Ligand Recognition,And Initiation Of Signaling,"Cold Spring Harb.Perspect.Biol.2(4)：a005140;1-14, K.W., et al; chetty, R.et al (1994)"CD3：Structure,Function,And Role Of Immunostaining In Clinical Practice,"J.Pathol.173(4)：303-307;Guy,C.S. (2009), "Organization Of Proximal Signal Initiation At The TCR:CD3 Complex," Immunol. Rev.232 (1): 7-21).

In mammals, complexes formed by CD3 subunits associate with molecules of T Cell Receptors (TCRs) to generate activation signals in T lymphocytes (Smith-Garvin, j.e. et al (2009) "T Cell Activation," annu.rev. Immunol 27:591-619). In The absence of CD3, TCRs do not assemble and degrade properly (Thomas, S.et al (2010)"Molecular Immunology Lessons From Therapeutic T cell Receptor Gene Transfer,"Immunology 129(2)：170-177). research found that CD3 bound to The membranes of all mature T cells and hardly bound to other cell types (Janeway, C.A.et al (2005): immunobiology: the immunone SYSTEM IN HEALTH AND DISEASE, "sixth edition, GARLAND SCIENCE Publishing, N.Y., pp.214-216; sun, Z.J. et al (2001)"Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε：γHeterodimer,"Cell 105(7)：913-923;Kuhns,M.S., et al) (2006)"Deconstructing The Form And Function Of The TCR/CD3 Complex,"Immunity.2006Feb,24(2)：133-139).

Constant CD3 epsilon signaling components of the T Cell Receptor (TCR) complex on T cells have been used as targets to promote the formation of immunological synapses between T cells and tumor cells. Co-conjugation of CD3 and tumor antigen (co-engagement) activates T cells, causing lysis of tumor cells expressing tumor antigens (Baeuerle et al (2011)"Bispecific T Cell Engager For Cancer Therapy,"In：Bispecific Antibodies,Kontermann,R.E.(Ed.)Springer-Verlag;2011：273-287). this method allows bispecific antibodies to interact fully with T cell compartments (components) with high specificity for tumor cells and is widely applicable to a large number of cell surface tumor antigens.

B7H3 is one of the members of the B7 family, belonging to the class I transmembrane proteins, comprising an amino-terminal signal peptide, an extracellular immunoglobulin-like variable (IgV) and constant (IgC) region, a transmembrane region and a cytoplasmic tail containing 45 amino acids (Tissue antibodies.2007 Aug;70 (2): 96-104). Currently, B7H3 exists mainly in 2 types of sheared bodies, B7H3a and B7H3B. The B7H3a extracellular segment consists of IgV-IgC 2 immunoglobulin domains, also called 2IgB H3, while the B7H3B extracellular segment consists of IgV-IgC-IgV-IgC 4 immunoglobulin domains, also called 4IgB H3.

The B7H3 protein is not expressed or extremely expressed in normal tissues and cells, but is highly expressed in various tumor tissues, and is closely related to the progress of tumors, survival of patients and prognosis. B7H3 has been reported clinically to be overexpressed in many cancer types, particularly in non-small cell Lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer (Lung cancer.2009Nov;66 (2): 245-249;Clin Cancer Res 2008Aug 15;14 (16): 5150-5157). In addition, it has also been reported in the literature that in prostate Cancer, the expression intensity of B7H3 is positively correlated with clinical pathology malignancy such as tumor volume, extra-prostatic invasion or Gleason score, and also with Cancer progression (Cancer Res.2007Aug 15;67 (16): 7893-7900). Similarly, in glioblastoma multiforme, B7H3 expression is inversely related to event-free survival, and in pancreatic cancer, B7H3 expression is related to lymph node metastasis and pathological progression. Thus, B7H3 is considered a new tumor marker and potential therapeutic target.

Some CD3 antibody molecules are disclosed in the prior art, such as OKT3, UCHT-1, SP34 (Silvana Pessano et al. The EMBO journal 1985,4 (2): 337-344), and also such as CN103703024, WO2017055389, CN102171248, etc. However, in the development of drugs, there is still a need to develop CD3 antibody molecules that are safer and more potent.

Disclosure of Invention

In one aspect, the present disclosure provides a multi-specific protein molecule comprising a first polypeptide chain and a second polypeptide chain, wherein:

The first polypeptide chain comprising, in order from amino terminus to carboxy terminus, a first binding region for a first target antigen, a second binding region for a second target antigen, and a first Fc region,

The second polypeptide chain comprising, in order from the amino terminus to the carboxy terminus, a third binding region for a third target antigen and a second Fc region,

The second binding region and/or the third binding region does not comprise a constant region domain of an antibody,

The regions within the first polypeptide chain and the second polypeptide chain are linked by peptide bonds and/or linkers.

In some embodiments, wherein the antigen binding region of the multispecific protein molecule, specifically the first binding region, and/or the second binding region, and/or the third binding region is a single chain antibody (scFv).

In some embodiments, wherein the second target antigen of the multi-specific protein molecule is CD3 and the first target antigen and third target antigen are the same or different tumor-associated antigens (TAAs); or the first target antigen is CD3 and the second and third target antigens are the same or different tumor-associated antigens (TAAs).

In some embodiments, wherein the Tumor Associated Antigen (TAA) of the multi-specific protein molecule is selected from AFP, ALK, B H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, beta-catenin (beta-catenin), brc-ab1, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, caspase -8(caspase-8)、CALR、CCR5、CD19、CD20(MS4A1)、CD22、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin 18.2、 cyclin-B1, CYP1B1, EGFR, EGFRvIII, erbB2/Her2, erbB3, erbB4, ETV6-AML, epCAM, ephA2, fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, globoH, glypican-3 (glypican-3), GM3, gp100, her2, HLA/B-raf, HLA/k-Ras 3, hTERT, IL13R alpha 2, LMP2, th Light, leY, MAGE proteins (e.g., MAGE-1, -2, -3, -RT-12, -RT-1, STERT-12, -RG-1, STEGE proteins, STEP-2, SAGE-3, SAGE-1, SAOGHT-2, SAOGhR-3, SAOGhX-1, -SAOGY, SAOGY-3, SAOGY-1; tn), TRP-1, TRP-2, tyrosinase and urolysin-3 and 5T4 (Trophoblast glycoprotein). Preferably, the Tumor Associated Antigen (TAA) is selected from the group consisting of B7H3, BCMA, CEA, CD, CD20, CD38, CD138, claudin18.2, PSMA and mesothelin. More preferably

In some embodiments, wherein said first polypeptide chain of said multispecific protein molecule has the structure of formula I:

V _a1-L1-V_b1-L2-V_c2-L2-V_d 2-L4-Fc1 of formula I,

The second polypeptide chain has the structure of formula II:

v _e3-L5-V_f 3-L6-Fc2 of formula II,

The V _a1、V_b1、V_c2、V_d2、V_e and V _f 3 are the light chain variable region or the heavy chain variable region of an antibody, and the V _a 1 and V _b 1, the V _c 2 and V _d 2, and the V _e 3 and V _f 3 are not the light chain variable region or the heavy chain variable region, respectively.

In some embodiments, wherein the first polypeptide chain of the multispecific protein molecule has a structure as shown below:

VH_TAA-L1-VL_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，

VH_TAA-L1-VL_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，

VL_TAA-L1-VH_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，

VL_TAA-L1-VH_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，

VH_CD3-L1-VL_CD3-L2-VH_TAA-L3-VL_TAA-L4-Fc1，

VH_CD3-L1-VL_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1，

VL _CD3-L1-VH_CD3-L2-VH_TAA-L3-VL_TAA -L4-Fc1 or

VL_CD3-L1-VH_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1；

And the second polypeptide chain has a structure as shown below:

VL _TAA-L5-VH_TAA-L6-F_C 2 or VH _TAA-L5-VL_TAA-L6-F_C 2.

In some embodiments, wherein the first polypeptide chain of the multispecific protein molecule has the structure of VL _TAA-L1-VH_TAA-L2-VH_CD3-L3-VL_CD3 -L4-Fc1 and the second chain has the structure of VL _TAA-L5-VH_TAA-L6-F_C.

In some embodiments, wherein the first polypeptide chain of the multispecific protein molecule has the structure of VH _CD3-L1-VL_CD3-L2-VL_TAA-L3-VH_TAA -L4-Fc1 and the second chain has the structure of VH _TAA-L5-VL_TAA-L6-F_C.

In some embodiments, wherein the TAA is B7H3.

In some embodiments, wherein the first Fc region and the second Fc region of the multispecific protein molecule are the same Fc or different Fc. Preferably, the first Fc region is a knob-Fc and the second Fc region is a hole-Fc; or the first Fc region is a hole-Fc and the second Fc region is a knob-Fc.

In some embodiments, wherein the carboxy-terminus of the first Fc region of the multispecific protein molecule is linked to a His tag (His tag) or the carboxy-terminus of the second Fc region is linked to a His tag.

In some embodiments, wherein the antigen binding region of the multispecific protein molecule to CD3 comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48. 49 and 50, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from any one of i) to v) below:

i) Respectively as SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 38 and 39;

ii) the sequences as set forth in SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 40 and 41;

iii) Respectively as SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 40 and 42;

iv) the sequences as set forth in SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 40 and 43; or (b)

V) the sequences as set forth in SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 47 and 45.

In some embodiments, wherein the antigen binding region of the multispecific protein molecule for CD3 comprises an amino acid sequence as set forth in SEQ ID NO:36 and/or a light chain variable region selected from the group consisting of SEQ ID NOs: 29. 30, 31, 32 and 35.

In some embodiments, wherein the antigen binding region of the multispecific protein molecule for CD3 comprises an amino acid sequence as set forth in SEQ ID NO: 55. 56, 57, 58, 61, 62, 63, 64, 65 or 68.

In some embodiments, wherein the tumor-associated antigen in the multispecific protein molecule is B7H3, the antigen-binding region for B7H3 comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12. 13 and 14, LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 9. HCDR1, HCDR2 and HCDR3 shown in figures 10 and 11.

In some embodiments, wherein the antigen binding region of the multi-specific protein molecule for B7H3 comprises: as set forth in SEQ ID NO:8 and/or the light chain variable region as set forth in SEQ ID NO: 7; or as set forth in SEQ ID NO:16 and/or the light chain variable region as set forth in SEQ ID NO:15, and a heavy chain variable region shown in seq id no.

In some embodiments, wherein the antigen binding region of the multispecific protein molecule for B7H3 comprises the amino acid sequence set forth in SEQ ID NO: 51. 52, 53 or 54.

In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 72. 73, 74, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86 or 87, and/or said second polypeptide chain is selected from the group consisting of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 71. 88 or 70.

In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 71. 88 or 70, and:

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: indicated at 72;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 73;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 74;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 75;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 76;

The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: indicated at 77;

The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 78;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 79;

The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 80;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:83, shown in the figure;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 84;

the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: indicated at 85;

The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: indicated at 86; or alternatively

The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: shown at 87.

In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain having an amino acid sequence set forth in SEQ ID NO:73 and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: shown at 71.

In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain having an amino acid sequence set forth in SEQ ID NO:78, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: shown at 71.

In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain having an amino acid sequence set forth in SEQ ID NO:76, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: shown at 71.

In another aspect, the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a multi-specific protein molecule according to the foregoing, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. Preferably, the therapeutically effective amount comprises from 0.1 to 3000mg (more preferably from 1 to 1000 mg) of the multi-specific protein molecule as described above in a unit dose of the composition.

In another aspect, the present disclosure relates to an isolated nucleic acid molecule encoding a multi-specific protein molecule as described previously.

In another aspect, the present disclosure relates to a recombinant vector comprising an isolated nucleic acid molecule as described previously.

In another aspect, the present disclosure relates to a host cell selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or insect cells, transformed with a recombinant vector as described above.

In another aspect, the present disclosure relates to a method for producing a multi-specific protein molecule as described above, the method comprising the steps of culturing a host cell as described above in a medium to form and accumulate the multi-specific protein molecule as described above, and recovering the multi-specific protein molecule from the culture.

In another aspect, the present disclosure relates to a multi-specific protein molecule as described above or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above, preferably the drug is a drug that activates T cells, more preferably the drug is a drug that treats cancer, or a drug that treats an autoimmune or inflammatory disease.

In another aspect, the present disclosure relates to the use of a multi-specific protein molecule as described above or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above, in the preparation of a medicament for activating T cells.

In another aspect, the present disclosure relates to the use of a multi-specific protein molecule as described above or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above, in the manufacture of a medicament for the treatment of cancer, or for the treatment of an autoimmune or inflammatory disease.

In another aspect, the present disclosure relates to a method of activating T cells, comprising administering to a subject a therapeutically effective amount of a multi-specific protein molecule as described above or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above.

In another aspect, the present disclosure relates to a method of treating cancer or an autoimmune or inflammatory disease, the method comprising administering to a subject a therapeutically effective amount of a multi-specific protein molecule as described above or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above. Preferably, the method comprises administering to the subject a composition comprising a unit dose of 0.1-3000mg of a multi-specific protein molecule as described above, or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above.

In some embodiments, any of the foregoing cancers is selected from the group consisting of carcinoma, lymphoma, blastoma (blastoma), sarcoma, and leukemia or lymphoid malignancy. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung carcinoma, non-small cell lung carcinoma (NSCLC), head and Neck Squamous Cell Carcinoma (HNSCC), glioma, hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute Lymphoblastic Leukemia (ALL), acute Myeloid Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myeloid Leukemia (CML), primary mediastinum large B-cell lymphoma, mantle Cell Lymphoma (MCL), small Lymphocytic Lymphoma (SLL), T-cell/tissue cell enriched large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (MCL-1) myelodysplastic syndrome (MDS), gastrointestinal (or gastrointestinal) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, ewing's sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell Renal Cell Carcinoma (RCC), head and neck cancer, throat cancer, hepatobiliary carcinoma (hepatobiliary cancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma (lymphoplasmacytic lymphoma), myelodysplastic syndrome, myeloproliferative neoplasms, neuroendocrine neoplasms, merkel cell carcinoma, testicular carcinoma, and skin carcinoma. In some embodiments, wherein the cancer is a B7-H3 positive cell-associated cancer; preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gall bladder cancer, glioblastoma and melanoma.

In some embodiments, any of the foregoing autoimmune or inflammatory diseases is selected from: rheumatoid arthritis, psoriasis, crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, sjogren's syndrome, autoimmune hemolytic anemia after transplant rejection, bullous pemphigoid, graves ' disease, hashimoto thyroiditis, systemic Lupus Erythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia.

Drawings

Fig. 1A and 1B: FIG. 1A is a schematic diagram of a bivalent, bispecific antibody, and FIG. 1B is a schematic diagram of a monovalent, bispecific antibody.

Fig. 2A to 2D: flow cytometry detects the binding activity of antibodies to cells expressing or not expressing the corresponding antigen. FIG. 2A shows the detection of the binding activity of different antibodies to A498 cells expressing human B7H3, FIG. 2B shows the detection of the binding activity of different antibodies to CT26 cells overexpressing human B7H3, FIG. 2C shows the detection of the binding activity of different antibodies to CT26 cells not expressing human B7H3, and FIG. 2D shows the detection of the binding activity of different antibodies to Jurkat recombinant cells expressing CD 3. The ordinate in fig. 2A to 2D represents the geometric mean of the fluorescence signal.

Fig. 3A to 3B: detection of killing activity of A498 by bispecific antibodies containing different CD3 scFv. FIG. 3A is the killing activity of a B7H3 monovalent bispecific antibody. FIG. 3B shows the killing activity of B7H3 bispecific antibodies. In addition to the weaker killing activity of 155, 156, 185 and 186 on a498, the remaining bispecific antibodies, whether monovalent or bivalent, show more pronounced killing activity.

Fig. 4A to 4B: comparison of killing activity of B7H3 single diabodies containing the same CD3 scFv against a 498. FIG. 4A is a comparison of the killing activity of HRH 1-containing B7H3 monovalent (181) and bivalent (131) bispecific antibodies. FIG. 4B is a comparison of the killing activity of HRH 7-containing B7H3 monovalent (187) and bivalent (177) cells. Experimental results show that the B7H3 bi-specific antibodies have obvious A498 killing activity compared with the B7H3 monovalent bi-specific antibodies, and meanwhile, the killing activity of the B7H3 bi-specific antibodies is obviously enhanced compared with that of the B7H3 monovalent bi-specific antibodies.

Fig. 5A to 5C: the killing activity of B7H3 bivalent bispecific antibodies containing the same CD3 heavy chain variable region and different structural sequences on A498 is detected. FIG. 5A is a comparison of killing activity between bispecific antibodies of different arrangement of the first HRH 2-containing polypeptide chains (AFF 1, AFF2, AFF3, AFF 4) B7H 3. FIG. 5B is a comparison of killing activity between (AFF 3, AFF 3-B) B7H3 bispecific antibodies with different sequences of the second HRH 2-containing polypeptide chain. The results show that the B7H3 bivalent bispecific antibodies with the same sequence and different VH and VL arrangement sequences have obvious A498 cell killing activity, and the intermolecular killing activities of different structural sequences are similar. FIG. 5C is a graph showing comparison of killing activity of bispecific antibodies comprising different structures of the same B7H3 scFv and CD3 scFv, wherein the killing activity of bispecific antibody 127 is superior to that of 201 and 202, and each of three bispecific antibodies 127 and 201 and 202 tested can kill A498 tumor cells in vitro.

Fig. 6A to 6B: activation detection of Jurkat recombinant cells by different antibodies. FIG. 6A is an antibody-mediated B7H3 target-specific Jurkat recombinant cell activation in the presence of A498-containing cells; FIG. 6B is a schematic of antibody mediated activation of non-B7H 3 target specific Jurkat recombinant cells in the absence of A498 cells. The antibodies indicated by the legends in fig. 6A and 6B are identical.

Fig. 7A to 7B: activation assay of Jurkat recombinant cells with different valency bispecific antibodies containing the same CD3 scFv. FIG. 7A is an antibody-mediated B7H3 target-specific Jurkat recombinant cell activation of B7H3 mono/diabody-specific antibodies in the presence of A498-containing cells; FIG. 7B is a B7H3 mono/diabody mediated activation of Jurkat recombinant cells that are not B7H3 target specific in the absence of A498 cells.

Fig. 8A to 8C: different antibodies stimulated PBMCs to generate B7H3 target specific cytokine secretion assays in the presence of a498 cells. FIG. 8A is a comparison of IFN gamma secretion levels from different antibody-stimulated PBMC, FIG. 8B is a comparison of TNF alpha secretion levels from different antibody-stimulated PBMC, and FIG. 8C is a comparison of IL-2 secretion levels from different antibody-stimulated PBMC. Figures 8A-8C show that antibodies 118, 127 and 132 significantly stimulated PBMC production of B7H3 target-specific cytokine secretion. The antibodies indicated by the legends in fig. 8A to 8C are identical.

Fig. 9A to 9C: different antibodies stimulated PBMCs to produce non-B7H 3 target specific cytokine secretion assays in the presence of CHOK1 cells (not expressing B7H 3). FIG. 9A is a comparison of IFNγ levels secreted by PBMC stimulated by different antibodies, FIG. 9B is a comparison of TNFα levels secreted by PBMC stimulated by different antibodies, and FIG. 9C is a comparison of IL-2 levels of PBMC secreting cells stimulated by different antibodies. FIGS. 9A-9C show that antibodies 118, 127 and 132 do not stimulate PBMC for non-B7H 3 target specific cytokine secretion, and are highly safe. The antibodies indicated by the legends in fig. 9A to 9C are identical.

Fig. 10A to 10E: anti-tumor efficacy detection of bispecific antibodies in a mouse a498 model of human PBMC reconstitution. Fig. 10A is a tumor-inhibiting activity assay for low doses of B7H3 bispecific antibody, with low doses of 118 and 119 antibodies still showing some tumor-inhibiting activity and showing some dose dependence. FIG. 10B is a tumor inhibiting activity assay of a 0.3mpk and 0.6mpk dose of a B7H3 bispecific antibody, with the 113 antibody exhibiting dose dependent in vivo tumor inhibiting activity. FIG. 10C is a graph of the tumor inhibiting activity of a B7H3 bispecific antibody at doses of 0.12mpk and 0.36mpk, with 118 antibodies showing significant tumor inhibiting activity at both doses. FIG. 10D is a graph showing that the anti-tumor activity of B7H3 bispecific antibody at a dose of 0.36mpk, the 126, 127 and 128 antibodies all showed significant anti-tumor activity. Fig. 10E is the tumor inhibiting activity of 127 antibodies at different doses and at different dosing frequency. In fig. 10A to 10E, veccle represents a negative control group to which PBS was administered.

Fig. 11A to 11B: anti-tumor efficacy of bispecific antibodies in hCD3KI mouse models. FIGS. 11A and 11B show the tumor-inhibiting effect of 118 and 132, respectively, in the hCD3KI mouse model.

Detailed Description

Terminology

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).

The term "multispecific protein molecule" refers to a protein molecule capable of specifically binding to two or more antigens or epitopes of interest. A protein molecule capable of specifically binding to two antigens or epitopes of interest is referred to as a bispecific protein molecule, and a "bispecific protein molecule" comprising an antibody or antigen binding fragment of an antibody (e.g. a single chain antibody) is herein interchangeable with a "bispecific antibody".

The term "binding region" or "binding region" for an antigen refers to a region or portion (part) of a multispecific protein molecule or in an antibody molecule that is capable of specifically binding to an antigen, and the antigen binding region may be a portion of a ligand binding domain that is capable of binding directly to an antigen, or may be a domain comprising an antibody variable region that is capable of binding directly to an antigen.

The term "antibody (Ab)" encompasses any antigen binding molecule or molecular complex that includes at least one Complementarity Determining Region (CDR) that specifically binds or interacts with a particular antigen (e.g., CD 3). The term "antibody" comprises: immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, and multimers thereof (e.g., igM) that are linked to each other by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. This heavy chain constant region comprises three regions (domains), CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a region (domain, CL 1). The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR, also termed framework regions, framework regions). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the disclosure, the FR of the anti-CD 3 antibody (or antigen binding portion thereof), the anti-B7H 3 antibody (or antigen binding portion thereof), or the anti-other antigen of interest may be identical to the human germline sequence, or may be modified naturally or artificially. The antibody may be of a different subclass (subclass), e.g., an IgG (e.g., an IgG1, igG2, igG3, or IgG4 subclass), igA1, igA2, igD, igE, or IgM antibody.

The term "antibody" also encompasses antigen binding fragments of complete antibody molecules. The terms "antigen binding portion", "antigen binding domain", "antigen binding fragment" and the like of an antibody, as used herein, encompass any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen binding fragment of an antibody may be derived, for example, from a whole antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable and (optionally) constant regions of the antibody. Such DNA is known and/or may be readily obtained from, for example, commercial sources, DNA databases (including, for example, phage-antibody databases), or may be synthesized. Such DNA may be sequenced and manipulated chemically or by using molecular biology techniques, such as arranging one or more variable and/or constant regions into a suitable configuration, or introducing codons, producing cysteine residues, modifying, adding or deleting amino acids, and the like.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragments; (v) a single chain Fv (scFv) molecule; (vi) a dAb fragment. Other engineered molecules, such as region-specific antibodies, single domain antibodies, region-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, diabodies, etc.), small modular immune medicine (SMIP), and shark variable IgNAR regions, are also encompassed within the term "antigen-binding fragment" as used herein.

The antigen binding fragment of an antibody will typically comprise at least one variable region. The variable region may be of any size or amino acid composition and will generally comprise CDRs adjacent to or within one or more framework sequences. In antigen-binding fragments having VH and VL regions bound to each other, the VH and VL regions may be located opposite each other in any suitable arrangement. For example, the variable region may be dimerised and contain a VH-VL or VL-VH dimer.

In certain embodiments, the antigen binding fragment of an antibody is in any configuration of variable and constant regions, which may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may be comprised of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids such that it creates flexible and semi-flexible linkages between adjacent variable and/or constant regions in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention may comprise homodimers or heterodimers (or other multimers) configured with any of the variable and constant regions listed above that are non-covalently linked to each other and/or to one or more monomeric VH or VL regions (e.g., with disulfide bonds).

"Murine antibodies" are monoclonal antibodies of mouse origin prepared according to the knowledge and skill in the art in the present disclosure. The preparation is carried out by injecting a test subject with an antigen, and then isolating hybridomas expressing antibodies having a desired sequence or functional property, and when the injected test subject is a mouse, the antibodies produced are murine antibodies.

The "chimeric antibody (chimeric antibody)" is an antibody in which a variable region of a murine antibody and a constant region of a human antibody are fused, and can reduce an immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of a human IgG1, igG2, igG3, igG4 or variant thereof, preferably comprises a human IgG1, igG2 or IgG4 heavy chain constant region, or an IgG1, igG2 or IgG4 heavy chain constant region variant using amino acid mutations (e.g., YTE mutations or back mutations, L234A and/or L235A mutations, or S228P mutations).

The term "humanized antibody (humanized antibody)", including CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting CDR sequences of an animal-derived antibody, e.g., a murine antibody, into the framework regions of the variable regions of a human antibody. The heterologous reaction induced by chimeric antibodies due to the large number of heterologous protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available in the Internet http:// www.vbase2.org/and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition). To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal back or back mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured for CDRs by phage display.

Due to the contact residues of the antigen, grafting of CDRs may result in reduced affinity of the generated antibody or antigen binding fragment thereof for the antigen due to the framework residues that are in contact with the antigen. Such interactions may be the result of a high degree of somatic mutation. Thus, it may still be desirable to graft such donor framework amino acids to the framework of a humanized antibody. Amino acid residues from a non-human antibody or antigen binding fragment thereof involved in antigen binding can be identified by examining the variable region sequence and structure of an animal monoclonal antibody. Residues in the CDR donor framework that differ from the germline can be considered relevant. If the closest germ line cannot be determined, the sequences can be compared to the consensus sequences of the subclasses or the consensus sequences of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be the result of highly mutated somatic cells, thereby playing an important role in binding.

In one embodiment of the present disclosure, the antibody or antigen binding fragment thereof may further comprise a light chain constant region of human or murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of human or murine IgG1, igG2, igG3, igG4 or variant thereof.

"Human antibody" is used interchangeably with "human antibody" and may be an antibody derived from a human or obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigen stimulation and may be produced by any method known in the art. In certain techniques, the elemental elements of the human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines in which the endogenous heavy and light chain loci are disrupted by targeted disruption of the endogenous heavy and light chain loci contained in the cell lines. Transgenic organisms can synthesize human antibodies specific for human antigens, and the organisms can be used to produce human antibody-secreting hybridomas. The human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies may also be constructed by gene or chromosome transfection methods as well as phage display techniques, or from in vitro activated B cells, all of which are known in the art.

"Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope except for possible variant antibodies (e.g., comprising naturally occurring mutations or mutations generated during manufacture of monoclonal antibody preparations, which are typically present in minor amounts). Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (formulation) is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods, and other exemplary methods for preparing monoclonal antibodies, are described herein.

The terms "full length antibody", "whole antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in substantially complete form, as distinguished from antigen binding fragments as defined below. The term particularly refers to antibodies whose heavy chain comprises an Fc region.

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, so that they can produce a single protein chain (known as a single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

Antigen binding fragments can also be incorporated into single chain molecules comprising a pair of tandem Fv fragments (VH-CH 1-VH-CH 1) that, together with a complementary light chain polypeptide, form a pair of antigen binding regions (Zapata et al, 1995Protein Eng.8 (10): 1057-1062; and U.S. Pat. No. 5,262).

Fab is an antibody fragment having a molecular weight of about 50,000da and having antigen binding activity in a fragment obtained by treating an IgG antibody molecule with protease papain (cleavage of amino acid residue 224 of the H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds.

F (ab') 2 is an antibody fragment having a molecular weight of about 100,000Da and having antigen binding activity and comprising two Fab regions linked at hinge positions, obtained by digestion of the lower part of the two disulfide bonds in the IgG hinge region with pepsin.

Fab 'is an antibody fragment having a molecular weight of about 50,000Da and antigen binding activity obtained by cleavage of the disulfide bond of the hinge region of the above F (ab') 2. Fab 'can be produced by treating F (ab') 2 that specifically recognizes and binds to an antigen with a reducing agent such as dithiothreitol.

In addition, the Fab ' may be produced by inserting DNA encoding a Fab ' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryotic organism to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 (including 1,2,3 or 4) repeated variants (Holliger et al (1993), proc NATL ACAD SCI USA.90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al (1995), protein eng.8:725-731, choi et al (2001), eur J Immuno.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J Mol biol.293:41-56 and Roovers et al (2001), cancer Immunol immunother.50:51-59. Described.

By "multispecific antibody" is meant an antibody comprising two or more antigen-binding domains capable of binding two or more different epitopes (e.g., two, three, four, or more different epitopes), which may be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind two different epitopes.

The term "diabody" of a tumor-associated antigen refers to a bispecific antibody having two antigen binding regions for a tumor-associated antigen target, e.g. a B7H3 diabody refers to the bispecific antibody comprising two antigen binding regions for B7H 3. The term "monovalent bispecific antibody" refers to a bispecific antibody having only one antigen binding region for a target, e.g., a B7H3 monovalent bispecific antibody refers to a bispecific antibody comprising one antigen binding region for B7H 3.

"Linker" or "L1" for linking two protein domains refers to a connective polypeptide sequence for linking protein domains, typically with some flexibility, without losing the original function of the protein domain by the use of a Linker.

Diabodies (diabodies) refer to antibody fragments in which scFv is dimerized, and are antibody fragments having bivalent antigen-binding activity. In the divalent antigen binding activity, the two antigens may be the same or different.

DsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine residue via a disulfide bond between cysteine residues. Amino acid residues substituted with cysteine residues may be selected based on predictions of the three-dimensional structure of the antibody according to known methods (Protein engineering.7:697 (1994)).

Antigen binding fragments in some embodiments of the present disclosure may be produced by: obtaining the coding cDNA of the VH and/or VL and other domains required of the monoclonal antibody specifically recognizing and binding to the antigen of the disclosure, constructing DNA encoding the antigen binding fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into the prokaryote or eukaryotic organism to express the antigen binding fragment.

The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The numbering of residues in the Fc region is that of the EU index as in Kabat. Kabat et al Sequences of Proteins of Immunological Interest th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, md.,1991. The Fc region of an immunoglobulin generally has two constant region domains, CH2 and CH3. The "first Fc" is also referred to herein as "Fc1", and the second Fc is also referred to herein as "Fc2".

V _a1-L1-V_b1-L2-V_c2-L2-V_d 2-L4-Fc1 "and V _e3-L5-V_f 3-L6-Fc2" V _a1、V_b1、V_c2、V_d2、V_e and V _f are the light chain variable region or the heavy chain variable region of an antibody, V _a 1 and V _b 1 bind to a first antigen (first target antigen) epitope, V _c 2 and V _d 2 bind to a second antigen (second target antigen) epitope, V _e 3 and V _f 3 bind to a third antigen (third target antigen) epitope, and the first, second and third antigen epitopes may be the same or different.

Similarly in "VH _TAA-L1-VL_TAA-L2-VH_CD3-L3-VL_CD3 -L4-Fc1", VH _TAA and VL _TAA represent epitopes where the antibody variable regions bind to tumor-associated antigens, and VH _CD3 and VL _CD3 represent epitopes where the antibody variable regions bind to CD 3.

The present disclosure "knob-Fc" refers to a point mutation comprising T366W in the Fc region of an antibody to form a knob-like spatial structure. Correspondingly, "hole-Fc" refers to a point mutation comprising T366S, L368A, Y407V in the Fc region of an antibody to form a hole-like spatial structure. Knob-Fc and hole-Fc form heterodimers more easily due to steric hindrance. To further promote heterodimer formation, point mutations at S354C and Y349C can also be introduced at knob-Fc and hole-Fc, respectively, to further promote heterodimer formation via disulfide bonds. Meanwhile, to eliminate or attenuate ADCC effect caused by antibody Fc, 234A and 235A substitution mutations may also be introduced into Fc. For example, preferred knob-Fc and hole-Fc of the present disclosure are set forth in SEQ ID NOs: 69 and 70. In a bispecific antibody, either a knob-Fc or a hole-Fc can be used as the Fc region of the first polypeptide chain or as the Fc region of the second polypeptide chain, and in the same bispecific antibody, the Fc regions of the first and second polypeptide chains are not both knob-Fc or hole-Fc.

The term "amino acid difference" or "amino acid mutation" refers to a change or mutation in an amino acid of a variant protein or polypeptide as compared to the original protein or polypeptide, including the insertion, deletion or substitution of 1 or several amino acids on the basis of the original protein or polypeptide.

The "variable region" of an antibody refers to the variable region of an antibody light chain (VL) or the variable region of an antibody heavy chain (VH), alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of 4 Framework Regions (FR) connected by 3 Complementarity Determining Regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together tightly by the FR and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody. There are at least 2 techniques for determining CDRs: (1) Methods based on cross-species sequence variability (i.e., kabat et al Sequences of Proteins of Immunological Interest, (5 th edition, 1991,National Institutes of Health,Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et Al, J.molecular. Biol.273:927-948 (1997)). As used herein, a CDR may refer to a CDR determined by either method or by a combination of both methods.

The term "antibody framework" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain that does not have CDRs.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat e.a. et al, (1991) Sequences of proteins of immunological INTEREST NIH Publication 91-3242). As used in some embodiments herein, CDRs may define CDR1, CDR2, and CDR3 (LCDR 1, LCDR2, LCDR 3) of the light chain variable domain, and CDR1, CDR2, and CDR3 (HCDR 1, HCDR2, HCDR 3) of the heavy chain variable domain, e.g., as defined for CD3 antibody CDRs in the present disclosure, under the Kabat rules (Kabat et al Sequences of Proteins of Immunological Interest, (5 th edition, 1991,National Institutes of Health,Bethesda MD)). In other embodiments, the definition of the CDRs of an antibody may also be performed using rules such as IMGT, for example, in the definition of the CDRs of a B7H3 antibody, i.e., using the IMGT rules.

"Antibody constant region domain" refers to a domain derived from the constant region of the light and heavy chains of an antibody, including CL and CH1, CH2, CH3 and CH4 domains derived from different classes of antibodies. The hinge region (hinge region) in antibodies used to connect the CH1 and CH2 domains of the heavy chain is not within the scope of the "antibody constant region domain" as defined in the present disclosure.

The term "tumor antigen" refers to a substance, optionally a protein, produced by a tumor cell, including "tumor-associated antigens" or "TAAs" (which refer to proteins produced in a tumor cell and differentially expressed in cancer compared to corresponding normal tissue) and "tumor-specific antigens" or "TSAs" (which refer to tumor antigens produced in a tumor cell and specifically expressed or aberrantly expressed in cancer compared to corresponding normal tissue).

Non-limiting examples of "tumor associated antigens" include, for example, AFP, ALK, B H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, beta-catenin, brc-ab1, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, caspase -8(caspase-8)、CALR、CCR5、CD19、CD20(MS4A1)、CD22、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin 18.2、 cyclin-B1, CYP1B1, EGFR, EGFRvIII, erbB2/Her2, erbB3, erbB4, ETV6-AML, epCAM, ephA, fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, globoH, glypican-3 (glypican-3), GM3, gp100, her2, HLA/B-raf, HLA/k-Ras, HLA/MAGE-A3, hTERT, IL13Rα2, LMP2, kappa-Light, leY, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6 and-12), MART-1, RGS, STEO-5, STEO-35, TRP-35, STEP-35, TRP-3, STEP-35, SAoP-35, and TRP-35.

"CD3" refers to an antigen expressed on T cells as part of a polymolecular T Cell Receptor (TCR) and which consists of a homodimer or heterodimer formed by two of the following four receptor chains: CD3- ε, CD3- δ, CD3- ζ, and CD3- γ. Human CD3- ε (hCD 3 ε) comprises UniProtKB/Swiss-Prot: an amino acid sequence as described in P07766.2. Human CD 3-delta (hCD 3 delta comprises the amino acid sequence described in UniProtKB/Swiss-Prot: P04234.1 thus, unless explicitly stated to be from a non-human species, such as "mouse CD3", "monkey CD3", etc., the term "CD3" refers to human CD3.

An "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes generally comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology, volume 66, g.e.Morris, ed. (1996).

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 ^-8 M, e.g., about less than 10 ^-9M、10^- ¹⁰M、10^-11 M or less.

The term "affinity" refers to the strength of interaction between an antibody and an antigen at a single epitope. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple amino acid sites by weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" of an antibody or antigen binding fragment thereof (e.g., fab fragment) generally refers to an antibody or antigen binding fragment having a K _D of 1E ^-9 M or less (e.g., K _D、1E^-11 M or less, K _D、1E^-12 M or less, K _D、1E^-13 M or less, K _D、1E^-14 M or less, K _D, etc.) of 1E ^-10 M or less.

The term "KD" or "K _D" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibody binds to the antigen with a dissociation equilibrium constant (KD) of less than about 1E ^-8 M, e.g., less than about 1E ^-9M、1E^-10 M or 1E ^-11 M or less, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR) techniques. The smaller the KD value, the greater the affinity.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" means a construct capable of delivering one or more genes or sequences of interest and preferably expressing it in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. For example, the mice may be immunized with an antigen or fragment thereof, the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to a CDR region of non-human origin. Human FR germline sequences can be obtained from the website http by aligning IMGT human antibody variable region germline gene databases with MOE software: i/www.imgt.org/or from the journal of immunoglobulins, 2001ISBN 012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the enterobacteriaceae (enterobacteriaceae), such as strains of escherichia coli (ESCHERICHIA COLI) or Salmonella (Salmonella); the family of Bacillaceae (baciliaceae) such as bacillus subtilis (Bacillus subtilis); pneumococci (Pneumococcus); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line), HEK293 cells (non-limiting examples are HEK293E cells) and NS0 cells.

The engineered antibody or antigen binding fragment can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As an alternative prior art, mammalian expression systems can lead to glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expressing antibodies that specifically bound to the antigen. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using a protein A or protein G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treatment" is meant administration of an internal or external therapeutic agent, such as a composition comprising any of the compounds of the embodiments of the present disclosure, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to a subject patient or population in an amount effective to alleviate one or more symptoms of the disease to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) may not be effective in alleviating each of the symptoms of the target disease, it should be determined according to any statistical test methods known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the symptoms of the target disease should be alleviated in a statistically significant number of patients.

"Amino acid conservative modifications" or "amino acid conservative substitutions" refer to the substitution of an amino acid in a protein or polypeptide with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, rigidity, etc.), such that changes can be made frequently without altering the biological activity or other desired characteristics (e.g., antigen affinity and/or specificity) of the protein or polypeptide. Those skilled in The art recognize that in general, single amino acid substitutions in The non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., watson et al, (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub. Co., page 224 (4 th edition)). Furthermore, substitution of structurally or functionally similar amino acids is less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in the following table "exemplary amino acid conservative substitutions".

Exemplary amino acid conservative substitutions

An "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening the severity, or delaying the onset of a disorder, including biochemical, histological and/or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes that are exhibited during the development of the disorder. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-related disorders of the present disclosure, reducing the dosage of other agents required to treat a disorder, enhancing the efficacy of another agent, and/or slowing the progression of a target antigen-related disorder of the present disclosure in a patient.

"Exogenous" refers to substances produced outside of an organism, cell or human body as the case may be. "endogenous" refers to substances produced in cells, organisms or humans, as the case may be.

"Homology" and "identity" are used interchangeably herein to refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, when sequences are optimally aligned, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches or homologies at 100 positions in the two sequences, then the two sequences are 95% homologous. Typically, the comparison is made when two sequences are aligned to give the greatest percent homology. For example, the comparison may be performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give a maximum match between the respective sequences over the entire length of the respective reference sequences.

The following references relate to BLAST algorithms that are often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): altschul, S.F. et al, (1990) J.mol.biol.215:403-410; gish, W.et al, (1993) Nature Genet.3:266-272; madden, t.l. et al, (1996) meth.enzymol.266:131-141; altschul, S.F. et al, (1997) Nucleic Acids Res.25:3389-3402; zhang, j et al, (1997) Genome res.7:649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also known to those skilled in the art.

"Isolated" refers to a purified state and in this case means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth media. In general, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.

"Pharmaceutical composition" means a mixture comprising one or more compounds of the present disclosure or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivery of an antibody or antigen-binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antimicrobial or antifungal), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyols, sorbitol, and sodium chloride.

The term "cancer," "cancerous," or "malignant" refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma (blastoma), sarcoma, and leukemia or lymphoid malignancy. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung carcinoma, non-small cell lung carcinoma (NSCLC), head and Neck Squamous Cell Carcinoma (HNSCC), glioma, hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute Lymphoblastic Leukemia (ALL), acute Myeloid Leukemia (AML), chronic Lymphoblastic Leukemia (CLL), chronic Myeloid Leukemia (CML), primary mediastinal large B-cell lymphoma, mantle Cell Lymphoma (MCL), small Lymphocytic Lymphoma (SLL), T-cell/tissue cell enriched large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (MCL-1) myelodysplastic syndrome (MDS), gastrointestinal (or gastrointestinal) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, ewing's sarcoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell Renal Cell Carcinoma (RCC), head and neck cancer, throat cancer, hepatobiliary carcinoma (hepatobiliary cancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma (lymphoplasmacytic lymphoma), myelodysplastic syndrome, myeloproliferative neoplasms, neuroendocrine neoplasms, merkel cell carcinoma, testicular carcinoma, and skin carcinoma.

An "inflammatory disorder" refers to any disease, disorder or syndrome in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, damage to host tissue, or loss of tissue function. "inflammatory disease" also refers to a pathological condition mediated by the pooling of chemotaxis of leukocytes or neutrophils.

"Inflammation" refers to a localized protective response caused by injury or destruction of tissue, which serves to destroy, attenuate or eliminate (sequester) harmful substances and injured tissue. Inflammation is significantly associated with a pool of leukocyte or neutrophil chemotaxis. Inflammation may be caused by pathogenic organisms and viruses as well as non-infectious causes such as reperfusion or stroke following trauma or myocardial infarction, immune responses to exogenous antigens, and autoimmune responses.

An "autoimmune disease" refers to any group of diseases in which tissue damage is associated with a humoral or cell-mediated response to a component of the body itself. Non-limiting examples of autoimmune diseases include rheumatoid arthritis, psoriasis, crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, sjogren's syndrome, autoimmune hemolytic anemia following transplant rejection, bullous pemphigoid, grave's disease, hashimoto's thyroiditis, systemic Lupus Erythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia, and the like.

Further, another aspect of the present disclosure relates to a method for immunodetection or assay of an antigen of interest, a reagent for immunodetection or assay of an antigen of interest, a method for immunodetection or assay of a cell expressing an antigen of interest, and a diagnostic agent for diagnosis of a disease associated with a positive cell of an antigen of interest, comprising as an active ingredient a monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes and binds to an antigen of interest.

In the present disclosure, the method for detecting or determining the amount of the antigen of interest may be any known method. For example, it includes immunological detection or assay methods.

The immunodetection or assay method is a method of detecting or assaying the amount of an antibody or an antigen using a labeled antigen or antibody. Examples of immunodetection or assay methods include radio-labeled immune antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent Immunoassays (FIA), luminescent immunoassays, western immunoblotting, physicochemical methods, and the like.

The above-described diseases associated with target antigen-positive cells can be diagnosed by detecting or assaying cells expressing the target antigen with the monoclonal antibodies or antibody fragments of the present disclosure.

For detecting the cells expressing the polypeptide, a known immunodetection method may be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, and the like are preferably used. Furthermore, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, a living sample for detecting or determining an antigen of interest is not particularly limited as long as it has a possibility of containing cells expressing the antigen of interest, for example, tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue juice, or culture solution.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions, depending on the desired diagnostic method. Reagents for performing the antigen-antibody reaction include buffers, salts, and the like. Reagents for detection include reagents commonly used in immunological detection or assay methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, a substrate corresponding to the label, and the like.

The details of one or more embodiments of the invention are set forth in the description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. Other features, objects, and advantages of the invention will be apparent from the description and claims. In the description and claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the invention. These examples should not be construed in any way as limiting the scope of the invention, which is defined by the claims.

Detailed Description

Preparation and screening of antibodies

Methods for preparing monoclonal antibodies are known in the art. One method that may be employed is the method of Kohler, g. ,(1975)"Continuous Cultures Of Fused Cells Secreting Antibody Of PredefinedSpecificity,"Nature256：495-497, et al, or a modification thereof. Typically, monoclonal antibodies are formed in non-human species, such as mice. Typically, mice or rats are used for immunization, but other animals, such as rabbits, alpacas, may also be used. Antibodies are prepared by immunizing mice with an immunogenic amount of cells, cell extracts or protein preparations comprising human CD3 or other antigen of interest (e.g., human B7H 3). The immunogen may be, but is not limited to, a primary cell, a cultured cell line, a cancerous cell, a nucleic acid, or a tissue.

In one embodiment, monoclonal antibodies that bind to the antigen of interest are obtained by using host cells that overexpress the antigen of interest as an immunogen. Such cells include, for example and without limitation, human T cells, cells that overexpress human B7H 3.

To monitor antibody responses, small biological samples (e.g., blood) can be obtained from animals and tested for antibody titer against the immunogen. The spleen and/or some large lymph nodes may be removed and dissociated into single cells. If desired, spleen cells can be screened by applying the cell suspension (after removal of non-specifically adherent cells) to antigen-coated plates or wells. B cells expressing membrane-bound antigen-specific immunoglobulins will bind to the plate and will not be washed out by the remaining suspension. The resulting B cells or all dissociated spleen cells can then be fused with myeloma cells (e.g., X63-ag8.653 and cells from SaIk Institute, cell Distribution Center, san Diego, CA). Polyethylene glycol (PEG) can be used to fuse spleen or lymphocytes with myeloma cells to form hybridomas. The hybridomas are then cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, otherwise known as "HAT medium"). The resulting hybridomas are then plated by limiting dilution and analyzed for the production of antibodies that specifically bind to the immunogen using, for example, FACS (fluorescence activated cell sorting) or Immunohistochemical (IHC) screening. Subsequently, the monoclonal antibody-secreting hybridomas are selected for culture in vitro (e.g., in a tissue culture flask or hollow fiber reactor) or in vivo (e.g., as ascites in mice).

As another alternative to cell fusion techniques, epstein-Barr virus (EBV) immortalized B cells can be used to prepare monoclonal antibodies of the present invention. Hybridomas are propagated and subcloned, as desired, and supernatants are assayed for anti-immunogenic activity by conventional assay methods (e.g., FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, etc.).

In another alternative, monoclonal antibodies against the antigen of interest (e.g., CD3, B7H 3) and any other equivalent antibodies can be sequenced and recombinantly produced by any method known in the art (e.g., humanization, production of fully human antibodies using transgenic mice, phage display technology, etc.). In one embodiment, an anti-antigen of interest (e.g., CD3, B7H 3) monoclonal antibody is sequenced and then the polynucleotide sequence is cloned into a vector for expression or proliferation. The sequences encoding the antibodies of interest may be maintained in a vector in a host cell and the host cell may then be propagated and frozen for later use.

The polynucleotide sequences of anti-CD 3 monoclonal antibodies and any other equivalent antibodies may be used in genetic manipulation to produce "humanized" antibodies to improve the affinity or other characteristics of the antibodies. The general principle of humanized antibodies involves retaining the basic sequence of the antigen-binding portion of the antibody while replacing the non-human remainder of the antibody with human antibody sequences. There are four general steps for humanizing monoclonal antibodies. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains, (2) designing the humanized antibody, i.e., determining which antibody framework regions to use in the humanization process, (3) actual humanization methods/techniques, and (4) transfection and expression of the humanized antibody. See, for example, U.S. patent nos. US 481757, US5807715, US5866692 and US6331415.

Preparation and screening of B7H3 antibodies

B cells were isolated using human PBMC, spleen, lymph node tissue, and RNA was extracted to construct a natural single-chain phage antibody library. And packaging the constructed natural single-chain phage antibody library to form phage particles, then carrying out panning by adopting a liquid phase method, combining phage with biotinylated B7H3 liquid phase, and separating by adopting streptavidin magnetic beads. In order to obtain a positive sequence combined with human B7H3, washing and screening are carried out by adopting biotinylated human B7H3, and a plurality of monoclonal colonies are picked and packaged into phage single-chain antibodies for phage ELISA test. The binding activity of the monoclonal phage to human B7H3 and murine B7H3 was tested separately and the B7H3 antibody was obtained by screening.

The B7H 3-related antigen for detection is as follows:

detection of human B7H3 antigen

Commercial products (SinoBiological cat # 11188-H08H)

The sequence is as follows:

Annotation: the cross line is marked as B7H3 extracellular region; the italic part is His tag (His-tag).

Monkey B7H3 antigen for detection

Commercial products (SinoBiological cat # 90806-C08H)

The sequence is as follows:

annotation: the cross line is marked as B7H3 extracellular region; the italic part is His tag.

Murine B7H3 antigen for detection

Commercial products (SinoBiological cat # 50973-M08H)

The sequence is as follows:

Human B7H3 full-length amino acid sequence

Annotation:

the double transverse line part is signal peptide (SIGNAL PEPTIDE:1-28);

The cross-hatched area is the B7H3 extracellular region (Extracellular domain:29-466), where 29-139 is Ig-like V-type 1 Domain and 145-238 is Ig-like C2-type 1 Domain;243-357 is Ig-like V-type 2 Domain,363-456 is Ig-like C2-type 2 Domain;

The dash-dot line part is a transmembrane region part (Transmembrane domain:467-487);

the italics are the intracellular areas (Cytoplasmic domain:488-534).

Monkey B7H3 full-length amino acid sequence

Annotation:

the double transverse line part is signal peptide (SIGNAL PEPTIDE:1-28);

the italics are the intracellular areas (Cytoplasmic domain:488-534).

Full-length amino acid sequence of murine B7H3

Annotation:

the double transverse line part is signal peptide (SIGNAL PEPTIDE:1-28);

The cross-hatched area is the B7H3 extracellular region (Extracellular domain:29-248);

the dash-dot line part is a transmembrane region part (Transmembrane domain:249-269);

the italics are the intracellular areas (Cytoplasmic domain:270-316).

The sequences of the B7H3 antibody H1702 obtained by screening and the CDR sequences defined by IMGT numbering rules are shown below:

>h1702 VH

>h1702 VL

note that: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italics in sequence is FR sequence and underlined is CDR sequence.

TABLE 1 light chain, heavy chain CDR sequence listing for B7H3 antibody h1702

To further enhance the performance of the bispecific antibody, cysteine substitution mutations were performed in VH and VL of B7H3 antibody H1702, respectively, a G103C (amino acid natural sequence numbering at position 103 of SEQ ID No. 16) mutation was introduced in the light chain variable region, a G44C (amino acid natural sequence numbering at position 44 of SEQ ID No. 15) mutation was introduced in the heavy chain variable region to form a pair of disulfide bonds, and the heavy chain and light chain variable region sequences of the mutated anti-B7H 3 single chain antibody were as follows:

B7H3 VH44C：

B7H3 VL103C：

Preparation and screening of CD3 antibodies

On the basis of the murine CD3 antibody, humanized CD3 antibodies can be obtained through means of mutation, library establishment, humanized transformation, screening and the like.

The CD3 antigen-related sequence information is as follows

Detection of human CD3 antigen

Commercial products (SinoBiological cat # CT 038-H2508H)

The sequence is as follows:

human CD3 epsilon (Human CD3 epsilon)

Annotation:

The cross-hatched area is the CD3 epsilon extracellular region (Extracellular domain:23-126); italics is His tag Human CD3 delta

Annotation:

The cross-hatched area is the CD3 delta extracellular region (Extracellular domain:22-105); the italic part is Flag tag.

Monkey CD3 antigen for detection

Commercial products (Acro biosystem cat # CDD-C52W4-100 ug)

The sequence is as follows:

monkey CD3 epsilon

Annotation:

The cross-hatched area is the CD3 epsilon extracellular region (Extracellular domain:22-117); italic part is His tag

Monkey CD3 delta

Annotation:

the cross-hatched area is the CD3 delta extracellular region (Extracellular domain:22-105); italic part is Flag label

Murine CD3 antigen for detection

The commercial product (SinoBiological cat # CT 033-M2508H) sequences are as follows:

Murine CD3 epsilon

Annotation:

The cross-hatched area is the CD3 epsilon extracellular region (Extracellular domain:22-108); italic part is His tag

Murine CD3 delta

Annotation:

Human CD3 epsilon full-length amino acid sequence

Annotation:

The double transverse line part is signal peptide (SIGNAL PEPTIDE:1-22);

The cross-hatched area is the CD3 epsilon extracellular region (Extracellular Domain:23-126), where 32-112 is Ig-like Domain;

the dash-dot line portion is a transmembrane region portion (Transmembrane domain: 127-152);

The italics are the intracellular areas (Cytoplasmic domain:153-207).

Human CD3 delta full-length amino acid sequence

Annotation:

The double transverse line part is signal peptide (SIGNAL PEPTIDE:1-21);

The cross-hatched area is the CD3 delta extracellular region (Extracellular domain:22-105);

The dash-dot line portion is a transmembrane region portion (Transmembrane domain:106-126);

the italic portion is the intracellular area (Cytoplasmic domain:127-171).

Monkey CD3 epsilon full-length amino acid sequence

Annotation:

The double transverse line part is signal peptide (SIGNAL PEPTIDE:1-21);

the cross-hatched area is the CD3 epsilon extracellular region (Extracellular domain:22-117);

The dash-dot line portion is a transmembrane region portion (Transmembrane domain:118-138);

The italics are the intracellular domains (Cytoplasmic domain:139-198).

Monkey CD3 delta full-length amino acid sequence

Annotation:

The double transverse line part is signal peptide (SIGNAL PEPTIDE:1-21);

the italic portion is the intracellular area (Cytoplasmic domain:127-171).

Murine CD3 epsilon full-length amino acid sequence

Annotation:

The double transverse line part is signal peptide (SIGNAL PEPTIDE:1-21);

The cross-hatched area is the CD3 epsilon extracellular region (Extracellular domain:22-108);

the dash-dot line part is a transmembrane region part (Transmembrane domain:109-134);

the italics are the intracellular areas (Cytoplasmic domain:135-189).

Murine CD3 delta full-length amino acid sequence

Annotation:

The double transverse line part is signal peptide (SIGNAL PEPTIDE:1-21);

The italics are the intracellular areas (Cytoplasmic domain:127-173).

Through repeated analysis and optimization, a series of anti-CD 3 humanized antibody sequences are obtained, and the heavy chain variable region sequences are as follows:

TABLE 2 CD3 humanized antibody heavy chain variable region sequence listing

The light chain variable region sequence is as follows:

>HRL

Note that: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italics in sequence is FR sequence and underlined is CDR sequence. Here and in Table 3 below, the CDR (LCDR 1-LCDR3 and HCDR1-HCDR 3) amino acid residues in the light and heavy chain variable regions of the CD3 humanized antibodies conform in number and position to the known Kabat numbering convention.

TABLE 3 CD3 antibody CDR sequence listing

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Construction and preparation of Single chain antibodies

The light and heavy chain variable region derived from the B7H3 antibody described above and the light and heavy chain variable region derived from the CD3 antibody may be linked to form an scFv against B7H3 and an scFv against CD3, respectively, wherein the linker (linker) may be selected from linker sequences well known in the art, and exemplary linkers may be selected from: (GGGGS) n or (GGGGS) n GGG, wherein n may be 1,2, 3 or 4.

Exemplary scFv against B7H3 are as follows:

TABLE 4 sequence listing of different anti-B7H 3 Single chain antibodies (scFv)

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Exemplary anti-CD 3 scFv are as follows:

TABLE 5 different anti-CD 3 Single chain antibodies (scFv) sequence listing

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Construction and preparation of bispecific antibodies B7H3 bispecific antibodies and B7H3 monovalent bispecific antibodies

In some embodiments of the disclosure, the B7H3 bispecific antibody is structured as shown in fig. 1A, with or without a His tag attached to the C-terminus. The two chains containing Fc are designed asymmetrically, sharing two B7H3 antigen binding domains and one CD3 antigen binding domain, wherein the B7H3 antigen binding domain is in the form of scFv on each of the two chains. The Fc region can maintain the normal half-life and good stability of the antibody, the design of the two chains greatly reduces the probability of mismatch, and the uniformity of the sample and the yield of the target antibody are improved. The molecular structure (Format) of a specific bispecific antibody is shown in table 6 below, and furthermore, the molecular structure of a B7H3 monovalent bispecific antibody used in some embodiments of the present disclosure has only an Fc domain in the second polypeptide chain, does not contain an antigen binding domain, and the structure is shown in fig. 1B.

TABLE 6 schematic structural tables of bispecific antibodies

Note that: the carboxyl terminus of the first polypeptide chain or the second polypeptide chain of this table may or may not be linked to a His tag. L1, L2, L3, L4, L5 and L6 represent linkers for linking each antigen binding domain to the Fc region.

TABLE 7 selection of linker (linker) sequences

Joint	Structure or sequence
		L1	(GGGGS) n or (GGGGS) n GGG
L2	(GGGGS)n
		L3	(GGGGS)n
L4	GGGDKTHTCPPCP(SEQ ID NO：98)
		L5	(GGGGS)n
L6	GGGDKTHTCPPCP(SEQ ID NO：98)

Wherein n is selected from 1,2, 3 or 4; preferably, n in L1 is 2 or 3, more preferably 3; n in L2 is 1 or 2, more preferably 1; n in L3 or L5 is 3. Alternatively, the linker used to attach the antigen binding domain to the Fc region may be selected from any other linker that may be used to attach the functional domain of the antibody, and is not limited to the above sequence.

The Fc1 and Fc2 in Table 6 above may be the same sequence of Fc, or may be a knob-Fc and a hole-Fc or a hole-Fc and a knob-Fc, respectively, and in some embodiments of the present disclosure, the sequences of knob-Fc and hole-Fc are preferably the sequences shown in Table 8:

TABLE 8 different Fc sequence Listing

The light and heavy chain variable region, single chain antibody and bispecific antibody can be obtained by constructing a DNA encoding the polypeptide or antigen binding fragment by encoding cDNA of VH and/or VL and other domains required, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and introducing the expression vector into a prokaryote or eukaryotic organism to express the polypeptide or antigen binding fragment.

Example 1 preparation of bispecific antibody molecules and Positive control molecules, negative control molecules

According to the design method of the bispecific antibody molecule of the present disclosure, specific bispecific antibody molecules are designed and prepared, and exemplary molecular amino acid sequences are shown in table 9 below:

TABLE 9 bispecific antibody sequence listing

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Note that: the second polypeptide chains of the above table B7H3 bispecific antibody molecules 113, 118, 119, 126, 127, 128, 131, 132, 154, 155, 156, 161, 162, 171, 172 and 177 are all SEQ ID NOs: 71 VL _B7H3-L5-VH_B7H3 -L6-hole-Fc; the second polypeptide chains of B7H3 monovalent bispecific antibody molecules 181-187 are all SEQ ID NOs: 70.

The amino acid sequences of the bispecific antibodies of the negative control (NC 1, NC2, NC 3) and the positive control (MGD 009) used in the present disclosure are as follows:

NC1: the B7H3 binding domain was replaced with a non-cognate antibody (anti-fluorescein antibody), but the CD3 binding domain was retained, amino acid sequence reference ：The anti-fluorescein antibody used to form the control DART diabody was antibody 4-4-20(Gruber.M.et al.(1994).

Chain 1 (VH _CD3-VL_CD3-VL_ctrl-VH_ctrl -knob-Fc)

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Chain 2 (VL _ctrl-VH_ctrl -hole-Fc)

NC2: non-relevant antibodies retaining the B7H3 binding domain, except that the CD3 binding domain was replaced with an anti-fluoroscein

Chain 1 (VH _ctrl-VL_ctrl-VL_B7H3-VH_B7H3 -knob-Fc)

Chain 2 (VL _B7H3-linker-VH_B7H3 -linker-Fc)

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Note that: the sequence is VL _B7H3-linker-VH_B7H3 -linker-Fc. The underlined part of the sequence is the B7H3 antibody sequence, and the italic part is the hole-Fc sequence.

NC3

Chain 1 (VL _ctrl-VH_ctrl-VH_CD3-VL_CD3 -knob-Fc-His tag)

Chain 2 (VL _ctrl-VH_ctrl -hole-Fc)

The positive control MGD009 comprises three chains, the amino acid sequence of which is as follows, see published patent application WO2017030926A1 for preparation and amino acid sequence:

Chain 1 (B7H 3VL-CD3 VH-Fc)

Chain 2 (CD 3VL-B7H3 VH)

Chain 3 (Fc)

201 (DART-Fc triple chain Structure) 201 chain 1 (B7H 3VL-CD3 VH-E-Fc)

201 Chain 2 (CD 3VL-B7H3 VH-K)

201 Chain 3

202 (Four-chain structure in which the quantitative ratio of substances of four chains is chain 1: chain 2: chain 3: chain 4=1:2:1:1)

202 Chain 1 (B7H 3VH-CH 1-Fc)

202 Chain 2 (B7H 3 VL-CL)

202 Chain 3 (B7H 3VH-CH1-CD3 VH-CL)

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202 Chain 4 (CD 3VL-CH 1)

Example 2 expression and purification of CD3-B7H3 bispecific antibodies

HEK293E cells were transfected with plasmid expressing bispecific antibody (chain 1: chain 21: 1), and after 6 days, the expression supernatants were collected and centrifuged at high speed to remove impurities. The clarified supernatant was purified using Ni Sepharose excel column (GE HEALTHCARE). The column was washed with PBS until the A280 reading was reduced to baseline, the column was washed with PBS+10mM imidazole, the non-specifically bound impurity proteins were removed, and the effluent was collected, and finally the protein of interest was eluted with a PBS solution containing 300mM imidazole, and the elution peak was collected. After the eluted sample was properly concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with 550 buffer (10 mM acetic acid, pH5.5, 135mM NaCl) to collect the desired peak. Samples were transferred to 559 buffer (10 mM acetic acid, pH5.5,9% sucrose) via desalting column or ultrafiltration centrifuge tube, and stored frozen at-80 ℃.

Test example 1 BIAcore assay for bispecific antibodies for affinity experiments on B7H3 and CD3

The antibodies were detected for B7H3 and CD3 affinity in the form of capture antibodies. BsAb was captured affinity using CM5 biosensing chip (Cat.# BR-1005-30, GE) or Protein A (Cat.# 2927556, GE) coupled with Anti-Human IgG Antibody (Cat.# BR-1008-39, lot.#10260416, GE) and then the reaction signal was detected in real time on the chip surface by the Biacore T200 instrument to obtain binding and dissociation curves. After dissociation was completed for each experimental cycle, the chips were washed and regenerated with regeneration buffer glycine1.5 (cat#br 100354, GE) or 3M MgCl ₂ (from Human antibody capture kit, cat#br 100839, GE). After the end of the experiment, the data were fitted with the (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0.0 software to give affinity values.

The sequence of the different V regions was fixed, and the affinity of the bispecific antibody to CD3 was slightly different when different CD3 antibody VH sequences were used, and the affinity of the antibody to CD3 was the weakest when HRH-6 and HRH-5 sequences were used, and binding to CD3 could not be detected on Biacore.

Table 10 results of Biacore detection of antigen affinity of bispecific antibodies with AFF3 Structure

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Exemplary antibodies comprising HRH3 as the heavy chain variable region of the CD3 antigen binding domain were selected for testing, wherein test antibodies 118, 127, 132 have affinities of 10 ^-9 and 10 ^-8 M levels for human (human) B7H3 and human CD3, respectively, comparable to that of MGD009, and have strong cross-binding activity to both monkey (cyno) B7H3 and human CD 3.

Table 11 results of Biacore assays for antigen affinities of bispecific antibodies comprising different structural sequences of HRH3

Test example 2, determination of antibody binding Capacity at cellular level

The binding capacity of the bispecific antibody to the cell surface antigen was examined by FACS. For the binding of cell surface antigens B7H3 and CD3, A498 (ATCC, HTB-44), CT26/hB7H3 (recombinant cell line overexpressing human B7H3 in murine CT26, built internally, CT26 derived from the department of Chinese cell Bank, TCM 37) and Jurkat recombinant cell line (Jurkat cells derived from ATCC, PTS-TIB-152, recombinant cell line overexpressing luciferase (luciferase) gene on Jurkat cell basis, inserted into the NFAT response element upstream of the gene) were used, respectively.

Cells resuspended in FACS buffer (98% PBS,2% FBS) were added to 96-well U-bottom plates (corning, 3795), and gradient diluted antibodies were added, incubated 1h at 4℃for 2 washes in FACS buffer, then APC anti-human IgG Fc Antibody (biolegend, cat #409306,1:50 dilution) was added to each well, incubated 30min at 4℃for 2 washes, and cells were resuspended in FACS buffer, and finally fluorescent signal values were read with FACS CantoII (BD).

The results show that both the B7H3 bivalent bispecific antibodies 118, 127 and 132 and the negative control antibody NC2 (retaining the B7H3 binding domain, only the CD3 binding domain was replaced with a non-relevant antibody) were able to bind to the a498 cell line that highly expressed B7H3 (see fig. 2A), were gradient dependent, were more binding than MGD009, and were B7H3 target specific, the negative control antibody NC1 (B7H 3 binding domain was replaced with a non-relevant antibody, but retaining the CD3 binding domain) did not bind to a 498. Similarly, bispecific antibodies 118, 127 and 132, mgd009 and NC2 bound strongly to CT26/hB7H3 (see fig. 2B), but not to CT26 cell lines that did not express B7H3 (see fig. 2C), also fully demonstrated that the tested bispecific antibodies specifically bound to cell membrane surface B7H3 targets. The difference in binding capacity of antibodies 118, 127 and 132 to MGD009 was more pronounced on the B7H3 overexpressing cell line CT26/hB7H3 than on the a498 cell line, suggesting that the B7H3 bivalent bispecific antibody was more pronounced and a better safety window on the high expressing B7H3 cells than the B7H3 monovalent bispecific antibody MGD 009.

Bispecific antibodies 118, 127 and 132 and negative control antibody NC1 were all able to bind to the Jurkat recombinant cell line (see FIG. 2D) and had a gradient dependent effect. Wherein 118 and NC1 have binding capacity comparable to MGD009, and 127 and 132 have slightly weaker binding capacity, probably due to the fact that the CD3 binding domain is between B7H3 binding domain and FC, binding to Jurkat recombinant cells is affected by some steric hindrance. Negative control antibody NC2, which does not contain a CD3 binding domain, did not bind to Jurkat recombinant cells, indicating that the binding of the bispecific antibody to Jurkat was CD3 target specific.

Test example 3 in vitro PBMC killing experiments

Bispecific antibody mediated killing of tumor cells by PBMC experiments were performed by quantitative detection of cell proliferation. ATP content in cells is detected by CELL TITER-glo, and ATP is an index of metabolism of living cells and is directly proportional to the number of cells in culture.

Four different target cells (TARGET CELL, T) were used, including three tumor cell lines (A498, U87 (Proc. Natl. Acad. Sci. Cell bank, TCHu, 138), detroit562 (ATCC, CCL-138)) with different B7H3 expression levels, and a negative control cell line CHOK1 (ATCC, CCL-61) that did not express B7H 3. Effector cells (E) are PBMCs from healthy volunteers. Target cells were seeded in 96-well plates, cultured overnight, and the next day, equal amounts of freshly extracted PBMC and gradient diluted test bispecific antibody (300 nM final concentration, 1:3 dilution) or PBS (control), effector and target cells, no antibody were added to each well. A blank (blank, medium only, no cells or antibodies) was set, E: T ratio, 10:1, 5:1 for A498, U87, detroit562 and CHOK1 cells, respectively. After culturing for 48 hours, the signal values were read by using CELL TITER-glo detection (see the description), and finally converted into inhibition ratios by using an ELISA reader, and the data were processed and analyzed by using GRAPHPAD PRISM 5.

Inhibition%inhibition =100% - (signal value _{Sample of} -signal value _{Blank space})/(signal value _Control -signal value _{Blank space})

(Inhibition％＝100％-(Signal_sample-Signal_blank)/(Signal_control-Signal_blank))

3.1 Comparison of antibodies containing different affinity CD3 antigen binding Domains

Different bispecific antibodies constructed using different affinities of CD3 scFv exhibited different in vitro target cell killing efficacy (see fig. 3A and 3B), with the minimal killing efficacy of bispecific antibodies 155, 156, 185, and 186 containing HRH5 and HRH6, respectively, consistent with the detection results of Biacore affinity.

3.2 Comparison of B7H3 monovalent and bivalent bispecific antibodies

For bispecific antibodies constructed with scFvs containing different heavy chain variable regions of anti-CD 3 antibodies, AFF3 (131 and 177 for this exemplary antibody) and AF3 (181 and 187 for this exemplary antibody) were compared (see FIGS. 4A and 4B), the AFF3 structure B7H3 bispecific antibody of CD3-B7H3 significantly enhanced cell killing activity in vitro over the B7H3 monovalent bispecific antibody of AF3 structure. This applies to all bispecific antibodies containing different CD3 VH.

TABLE 12 structural order of antibodies

3.3 Influence of different structure ordering molecular structures of B7H3 bivalent bispecific antibody on tumor killing activity

The tumor cell killing activity of B7H3 bispecific antibody molecules 161, 162, 113 and 126 (see fig. 5A), and 113 and 143 (see fig. 5B), which have different structural sequences of the same antigen binding domain components, were tested in parallel, all using HRH2 as the heavy chain variable region in the CD3 antigen binding domain. The results show that B7H3 bi-valent specific antibody molecules with different structural sequences have remarkable killing effect on A498 cells. Wherein 161, 162, 113 and 126 have a killing activity comparable to or slightly better than that of MGD009. The different structural arrangement sequences have little influence on the tumor cell killing activity of the B7H3 bivalent bispecific antibody.

TABLE 13 structural comparison Table of different test antibodies

3.4 Bispecific antibodies have killing efficacy against tumor cell lines with different B7H3 expression levels

The in vitro killing effect of three bispecific antibodies 118, 127 and 132 to be tested on the a498, U87 and Detroit562 tumor cell lines was tested. Killing efficacy correlated positively with B7H3 expression levels, e.g., EC50 at 118 at a498, U87 and Detroit562 were 0.34, 2.4 and 14.5nM, respectively. Three antibody molecules all showed this trend. All bispecific antibodies did not kill B7H3 negative control cell line CHOK1, and negative control bispecific antibody NC1 did not kill all target cell lines, which together demonstrate that cell killing requires redirecting effector cells to B7H3 positive target cells by bispecific antibodies for target specific killing.

Table 14 bispecific antibody mediated PBMC redirected killing of different target cell lines to be tested

3.5 Comparison of killing of A498 cells by bispecific antibodies of different Structure

The in vitro killing effect of three bispecific antibodies 127 and 201, 202 to be tested on the a498 tumor cell line was tested. The results show (see fig. 5C) that bispecific antibodies of three structures all had tumor killing activity, with the killing activity of bispecific antibody 127 being better than 201 and 202.

Test example 4 in vitro T cell activation experiments

The activation function of the bispecific antibody on T cells was tested by using a Jurkat recombinant cell line to detect NFAT-driven expression of luciferase reporter gene (luciferase) after Jurkat activation in the presence or absence of an a498 tumor cell line.

A498 cells were seeded on 96-well cell culture plates (1X 10 ⁵/ml, 100. Mu.L/well) and incubated at 37℃in a 5% CO ₂ incubator for 20-24h. The following day, after removal of the cell culture supernatant, 90. Mu.l of Jurkat recombinant cell suspension (5.5X10 ⁵/ml) and 10. Mu.l of the test bispecific antibody (final concentration of up to 500nM, 1:3 gradient dilution) were added to each well, and negative controls (control, with A498 and Jurkat recombinant cells, no antibody) and blank controls (blank, medium only, no cells or antibodies) were set up and incubated in a 5% CO ₂ incubator at 37℃for 5-6h. The non-tumor cell specific Jurkat recombinant cell activation is to add Jurkat recombinant cell and antibody to be tested directly into blank 96 well culture plate. After the co-cultivation is finished, 100 μl of Bright-Glo Reagent (Bright-Glo ^TM luciferases ASSAY SYSTEM, promega, cat#: E2620) is added to each well, the wells are placed at room temperature for 5-10min, a multifunctional microplate reader is used for reading chemiluminescent signal values, and the calculation formula of the fluorescence enhancement factor (Fold increase) is as follows: enhancement multiple= ((signal value _{Sample of} -signal value _{Blank space})/(signal value _Control -signal value _{Blank space})

(Fold increase＝(Signal_sample-Signal_blank)/(Signal_control-Signal_blank))

4.1 Different arrangement sequences of B7H3 bivalent molecules can activate T cells effectively

B7H3 bispecific antibodies 118, 127 and 132 were tested for activation of Jurkat recombinant cells in the presence or absence of a498, respectively, to verify the specific and non-specific activation effects of the bispecific antibodies on T cells. The results show that the B7H3 bispecific antibodies 118, 127 and 132 of different sequences are effective in activating the Jurkat recombinant cell line in the presence of tumor cell line a498 (see fig. 6A), significantly inducing luciferase expression, as negative control antibody NC1 is unable to induce luciferase expression, which may prove that activation of the Jurkat recombinant cells is B7H3 target-specific. Activation of Jurkat recombinant cells was achieved by co-recruiting Jurkat recombinant cells expressing CD3 and tumor cells expressing B7H3 by bispecific antibodies, with only Jurkat recombinant cells, but no a498 cells (see fig. 6B), luciferase expression was very low and only weak signals were detected at the highest few antibody concentration points.

TABLE 15 structural order of antibodies

4.2 Comparison of B7H3 monovalent and bivalent bispecific antibodies

The bivalent CD3-B7H3 bispecific antibody significantly enhanced target-specific T cell activation compared to the B7H3 monovalent bispecific antibody, which is consistent with enhanced in vitro tumor killing capacity of the B7H3 bi-price B7H3 monovalent molecule in test example 3. At the same time, non-target specific T cell activation remains unchanged. Thus, B7H3 bivalent molecule (131) has stronger potency than B7H3 monovalent molecule (181) (see fig. 7A), but side effects due to non-specific activation of T cells are not enhanced (see fig. 7B).

TABLE 16 antibody Structure

Antibodies to	First polypeptide chain	Second polypeptide chain
			131	VL_B7H3-L1-VH_B7H3-L2-VH_CD3-L3-VL_CD3-L4-F_C1	VL_B7H3-L5-VH_B7H3-L6-F_C2
181	VL_B7H3-L1-VH_B7H3-L2-VH_CD3-L3-VL_CD3-L4-F_C1	Fc2

Test example 5 in vitro cytokine secretion experiments

Effector cells redirect target cells under the mediation of bispecific antibodies, releasing cytokines while killing target cells. Wherein the cytokine secretion is to quantitatively detect the cytokine content in the cell culture supernatant by ELISA method, including IL2, IFNgamma and TNF alpha.

The experimental design and antibodies used were the same as described in test example 4, and cell culture supernatants were collected at the end of the in vitro killing experiment into 96-well plates (Corning # 3795) and stored at-20 ℃ for later use. In ELISA experiments, frozen culture supernatants were removed, thawed at room temperature, centrifuged at 3500rpm for 10mins, and supernatants were collected for ELISA experiments. The ELISA procedure is described in the specification of the Kit (Human IL-2 ELISA Kit, human IFN-gamma ELISA Kit, human TNF-alpha ELISA Kit, xinbo, cat#EHC003.96, EHC102g.96, EHC103a.96).

The results indicate that the test bispecific antibody was effective in inducing secretion of IL2, ifnγ and tnfα by PBMCs in the co-presence of PBMC and B7H3 positive target cell a498 (see fig. 8A-8C), with the highest level of cytokine secretion induced by MGD009 and 118 followed by 127 and 132, whereas the cytokine secretion induced by the negative control antibody NC1 was not within the detection sensitivity range. In the co-presence of PBMC and B7H3 negative cells CHOK1 (see fig. 9A-9C), MGD009 significantly induced ifnγ and tnfα release at the highest three concentration points, whereas the three bispecific antibodies tested, 118, 127 and 132, failed to induce ifnγ and tnfα release, suggesting that the three bispecific antibodies tested have better safety in non-target specific cytokine secretion compared to MGD 009.

Test example 6, efficacy experiment of mouse A498 model reconstructed from human PBMC

This test example uses the NOG mouse (beijing vernalia laboratory animal inc.) a498 model (ATCC) reconstituted with human PBMC to evaluate the anti-tumor efficacy of the inventive test CD3-B7H3 bispecific antibodies in mice.

A498 cells 5×10 ⁶ cells/mouse/100 μl (containing 50% matrigel) were inoculated subcutaneously in the right rib of NOG mice, mice were randomly grouped when tumor volumes of tumor-bearing mice reached around 130-150mm ³, 5-6 per group, and the day of grouping was defined as day 0 of the experiment. PBMCs of freshly extracted 2 volunteers were mixed at a 1:1 ratio on day 0 or day 1, injected into the abdominal cavity of NOG mice at 5×10 ⁶ cells/100 μl, and each antibody was started to be intraperitoneally injected 2 times per week for 6 total administrations, 2 times per week for tumor volume, animal weight and data were monitored. Vehicle is a negative control group to which the antibody was administered in place of PBS buffer.

Antibodies 118 and 119 have exhibited some tumor-inhibiting efficacy at lower doses (fig. 10A) and are dose-dependent. Wherein the tumor inhibition rates (TGI) of antibody 118 at the end of the experiment (day 20) at doses of 0.01mpk and 0.03mpk were 22.17% and 60.39%, respectively.

Antibody 113 has shown some tumor-inhibiting effect on day 14, with tumor-inhibiting rates of 70.05% (p < 0.05) and 60.78% (p < 0.05) for the 0.6mpk and 0.3mpk dose groups (FIG. 10B), respectively, and continued to increase and be dose-dependent by day 20 with tumor-inhibiting rates greater than 100% (p < 0.001) and 77.92% (p < 0.05), respectively.

At doses of 0.12mpk and 0.36mpk (FIG. 10C), antibody 118 had reached doses of 0.12mpk and 0.36mpk at day 12 of 39.18% and 57.44% (p < 0.001), respectively, and tumor inhibition rates by day 21 increased to 81.72% (p < 0.01) and greater than 100% (p < 0.001), respectively, with complete tumor regression (1/6) for even one mouse at the 0.36mpk dose.

At the 0.36mpk dose (FIG. 10D), antibody 126 reached 47.78% (p < 0.01) tumor suppression at day 21. Antibody 128 has shown significant tumor inhibition (tgi= 56.37%) at Day 19, with tumor inhibition rates rising to 69.28% (p < 0.001) by Day 21. The antibody 127mpk had a tumor inhibition rate of 76.20% (p < 0.001) at day 12, and the tumor inhibition effect continued to be enhanced by day21, with a tumor inhibition rate of greater than 100% (p < 0.001), with 3 tumor volumes in 5 animals retracted compared to the group, and 2 tumors completely regressed.

The antitumor activity of antibody 127 was repeated in another experiment (FIG. 10E), with tumor inhibition rates of 90.6% (p < 0.001) at day 14, and 95.80% (p < 0.001) at day 17. 127 was still effective at lower doses (0.12 mpk) and at lower frequency (1 times per week, 127-0.36 mpk-qw) with tumor suppression rates of 51.37% (p < 0.001) and 96.20% (p < 0.001), respectively, on day 17.

Test example 7, efficacy experiment of hCD3KI mouse model

In the experiment, a Balb/c-hCD3 mouse is subcutaneously inoculated with a CT26-hB7H3 tumor cell line (CT 26 cells are derived from a cell bank of the Chinese sciences, TCM37, and the CT26-hB7H3 cells are obtained through expressing hB7H 3), so that the inhibition effect of the CD3-B7H3 bispecific antibody of the invention on tumor growth in the mouse is evaluated.

Female hCD3E Balb/c transgenic mice were purchased from the Nandina model institute (accession number 201801374/5/6, license SCXK (Su) 2015-0001).

CT26-hB7H3 cells 8X 10 ⁵ cells/mouse/100. Mu.l were inoculated subcutaneously in the right rib of hCD3 mice. Mice were randomly grouped when tumor volumes of tumor-bearing mice reached about 80-120mm ³, 7 per group. The day of the grouping was defined as day 0 of the experiment and intraperitoneal injection of each antibody was started, 2 times per week, 5 times per week, 2 times per week tumor volumes, animal weights were monitored and data recorded. Vehicle is a negative control group to which the antibody was administered in place of PBS buffer.

The results show that antibody 118 showed strong efficacy at 1mpk dose immediately after the initial administration (FIG. 11A), with a tumor suppression rate of 38.34% (p < 0.05) on day 13.

Antibody 132 had a trend to inhibit tumor growth at the 3.6mpk dose (fig. 11B), with a tumor inhibition rate of 26.35% at day 13.

Test example 8, rat in vivo PK experiment

The experiment is to inject CD3-B7H3 bispecific antibody into the tail of SD rat, and detect the antibody concentration in the serum of the rat at different time points, so as to evaluate the metabolism of CD3-B7H3 bispecific antibody in SD rat.

The rat tail is intravenous with 3mg/kg of the test drug and the administration volume is 5mL/kg. Blood was collected at each time point of 5min, 8h, 1d, 2d, 4d, 7d, 10d, 14d, 21d, 28d before and after administration. The concentration of antibodies in serum was detected by ELISA using two different ELISA methods, respectively, B7H3 antigen (1. Mu.g/mL) or CD3 antigen (1. Mu.g/mL) were plated, anti-human Fc-HRP (abcam, ab 98624) as secondary antibodies. The pharmacokinetic parameters of the test drug were calculated using Winnolin software and the principal pharmacokinetic parameters are shown in table 17.

Antibodies 118, 127 and 132 had half-lives in the B7H3 antigen binding region of 4.9-8.1 days, slightly longer than MGD009, reaching normal IgG antibody levels, and the CD3 antigen binding region had half-lives of 3.2-5.6 days. Wherein, the kinetic parameters of the antibody 118 in two different antigen binding regions of B7H3 and CD3 are not greatly different, which indicates that the molecule has better integrity in vivo and half-life of 4.9 days and 4.4 days respectively. Antibody 127 has half-lives of 4.9 and 3.2 days in the two different antigen binding regions of B7H3 and CD3, respectively, and differs significantly in exposure and clearance rates, with the CD3 moiety being worse, since the CD3 moiety is internal to the molecular structure, more likely due to the weakening of the binding function of the CD3 moiety than the molecular cleavage. Antibody 132 is based on the 127 molecular sequence of antibody, and a pair of disulfide bonds are added into B7H3 scFv, so that the modification greatly improves the half life (65-75%) of the molecule and greatly improves the exposure and the clearance rate.

TABLE 17 Primary pharmacokinetic parameter Table in rats

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Claims

1. A multi-specific protein molecule comprising a first polypeptide chain and a second polypeptide chain, wherein:

The second target antigen is CD3 and the first and third target antigens are B7H3, or

The first target antigen is CD3, and the second target antigen and the third target antigen are B7H3,

The first binding region, the second binding region and the third binding region are all single chain antibodies,

2. The multi-specific protein molecule of claim 1, wherein the first polypeptide chain has the structure of formula I:

V _a1-L1-V_b1-L2-V_c2-L2-V_d 2-L4-Fc1 of formula I,

The second polypeptide chain has the structure of formula II:

v _e3-L5-V_f 3-L6-Fc2 of formula II,

3. The multi-specific protein molecule according to any one of claims 1 to 2, said first polypeptide chain having the structure shown below:

VH_TAA-L1-VL_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，

VH_TAA-L1-VL_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，

VL_TAA-L1-VH_TAA-L2-VH_CD3-L3-VL_CD3-L4-Fc1，

VL_TAA-L1-VH_TAA-L2-VL_CD3-L3-VH_CD3-L4-Fc1，

VH_CD3-L1-VL_CD3-L2-VH_TAA-L3-VL_TAA-L4-Fc1，

VH_CD3-L1-VL_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1，

VL _CD3-L1-VH_CD3-L2-VH_TAA-L3-VL_TAA -L4-Fc1 or

VL_CD3-L1-VH_CD3-L2-VL_TAA-L3-VH_TAA-L4-Fc1；

And the second polypeptide chain has a structure as shown below:

VL _TAA-L5-VH_TAA-L6-F_C 2 or VH _TAA-L5-VL_TAA-L6-F_C 2.

4. A multi-specific protein molecule according to any one of claims 1 to 3, wherein the first Fc region and the second Fc region are the same Fc or different Fc.

5. The multi-specific protein molecule according to any one of claims 1 to 4, wherein the first Fc region is a knob-Fc and the second Fc region is a hole-Fc; or the first Fc region is a hole-Fc and the second Fc region is a knob-Fc.

6. The multi-specific protein molecule of any one of claims 1 to 5, wherein the carboxy terminus of the first Fc region is linked to a His tag or the carboxy terminus of the second Fc region is linked to a His tag.

7. The multi-specific protein molecule according to any one of claims 1 to 6, wherein the antigen binding region for CD3 comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48. 49 and 50, LCDR1, LCDR2 and LCDR3, and said heavy chain variable region is a heavy chain variable region selected from any one of i) to v) below:

i) Comprising the amino acid sequences as set forth in SEQ ID NO: 37. heavy chain variable regions of HCDR1, HCDR2 and HCDR3 shown at 38 and 39;

ii) comprises the amino acid sequence set forth in SEQ ID NO: 37. heavy chain variable regions of HCDR1, HCDR2 and HCDR3 shown at 40 and 41;

iii) Comprising the amino acid sequences as set forth in SEQ ID NO: 37. heavy chain variable regions of HCDR1, HCDR2 and HCDR3 shown at 40 and 42;

iv) comprises the amino acid sequence set forth in SEQ ID NO: 37. heavy chain variable regions of HCDR1, HCDR2 and HCDR3 shown in 40 and 43; or (b)

V) comprises the amino acid sequence set forth in SEQ ID NO: 37. heavy chain variable regions of HCDR1, HCDR2 and HCDR3 shown at 47 and 45.

8. The multi-specific protein molecule of claim 7, wherein the antigen binding region for CD3 comprises the amino acid sequence as set forth in SEQ ID NO:36, and/or is selected from the group consisting of the light chain variable region shown in SEQ ID NO: 29. 30, 31, 32 and 35.

9. The multi-specific protein molecule of claim 8, wherein the antigen binding region for CD3 comprises the amino acid sequence set forth in SEQ ID NO: 55. 56, 57, 58, 61, 62, 63, 64, 65 or 68.

10. The multi-specific protein molecule according to any one of claims 1 to 9, wherein the antigen binding region for B7H3 comprises an antibody light chain variable region and a heavy chain variable region, wherein:

The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12. 13 and 14, LCDR1, LCDR2, and LCDR3, and said heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 9. HCDR1, HCDR2 and HCDR3 shown in figures 10 and 11.

11. The multi-specific protein molecule of claim 10, wherein the antigen binding region for B7H3 comprises:

As set forth in SEQ ID NO:8 and/or the light chain variable region as set forth in SEQ ID NO: 7;

Or as set forth in SEQ ID NO:16 and/or the light chain variable region as set forth in SEQ ID NO:15, and a heavy chain variable region shown in seq id no.

12. The multi-specific protein molecule of claim 11, wherein the antigen binding region for B7H3 comprises the amino acid sequence as set forth in SEQ ID NO: 51. 52, 53 or 54.

13. The multi-specific protein molecule according to any one of claims 1 to 12, comprising a first polypeptide chain selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 72. 73, 74, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86 or 87, and/or said second polypeptide chain is selected from the group consisting of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 71. or 88.

14. A multi-specific protein molecule comprising a first polypeptide chain and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 71. or 88, and:

15. A pharmaceutical composition comprising a therapeutically effective amount of a multi-specific protein molecule according to any one of claims 1 to 14, together with one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

16. An isolated nucleic acid molecule encoding the multi-specific protein molecule of any one of claims 1 to 14.

17. A recombinant vector comprising the isolated nucleic acid molecule of claim 16.

18. A host cell transformed with the recombinant vector of claim 17, said host cell selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

19. The host cell of claim 18, which is a eukaryotic cell.

20. The host cell of claim 19, wherein the eukaryotic cell is a mammalian cell or an insect cell.

21. A method for producing a multi-specific protein molecule according to any one of claims 1 to 14, the method comprising the steps of culturing the host cell according to claims 18-20 in a medium to form and accumulate the multi-specific protein molecule according to any one of claims 1 to 14, and recovering the multi-specific protein molecule from the culture.

22. Use of a multi-specific protein molecule according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15, or an isolated nucleic acid molecule according to claim 16, for the preparation of a medicament for the treatment of cancer; the cancer is selected from squamous cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, head and neck squamous cell carcinoma, glioma, acute myeloid leukemia, renal carcinoma, ovarian carcinoma, liver carcinoma, colorectal carcinoma, endometrial carcinoma, prostate carcinoma, thyroid carcinoma, melanoma, neuroblastoma, pancreatic carcinoma, glioblastoma, ewing's sarcoma, gastric carcinoma, bladder carcinoma, breast carcinoma, colon carcinoma, hepatocellular carcinoma, renal cell carcinoma, head and neck carcinoma, and esophageal carcinoma.

23. The use of claim 22, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, non-small cell lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, glioblastoma, and melanoma.