CN112969476A - Multispecific protein molecules - Google Patents

Abstract

Relates to multispecific protein molecules. In particular to a multispecific antibody with a novel structural form. The multispecific antibody can be simultaneously combined with CD3 and other tumor-associated antigens, and can be combined with tumor-associated antigen expression cells and simultaneously combined with and activate CD3 positive T cells, thereby promoting the killing of the T cells on the tumor cell specificity expressing the tumor-associated antigens. Also provides the preparation and application of the multispecific antibody.

Description

Multispecific protein molecules Technical Field

The present invention relates to multispecific antibodies, such as those 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, K.W. et al (2010) "Structural Biology Of The T cell Receptor: instruments Into Receptor Assembly, Ligand Recognition, And identification Of Signal, Cold Spring Harb.Perspectrum. biol.2(4): a 005140; pages 1-14; Chetty, R. et al (1994)" CD3: Structure, Function, And bed Of immunological In Clinical Practice, "J.Pathol.173 (4): 303;" Guy, C.S. et al (2009) "organic Signal analysis In TCR: compact therapy," CD 3.2: "Reg.1-21).

In mammals, complexes formed by the CD3 subunits associate with molecules of the T Cell Receptor (TCR) to produce Activation signals in T lymphocytes (Smith-Garvin, J.E. et al (2009) "T Cell Activation," Annu.Rev.Immunol.27: 591-619). Without CD3, the TCR did not assemble and degrade properly (Thomas, S. et al (2010) "Molecular Immunology threads From Therapeutic T cell Receptor Gene Transfer," Immunology 129(2): 170-. CD3 was found To bind To The membranes Of all mature T cells And hardly bind To other Cell types (Janeway, C.A. et al (2005): immunology: The Immune System In Health And Disease, "sixth edition, Garland Science Publishing, NY, pp.214-216; Sun, Z.J. et al (2001)" Mechanisms binding To T Cell Receptor signalling And derived modified By The CD 3. gamma. heterodorimer, "D.E. 105(7):913, D.S. 923; Kuhns, M.S. et al (2006)" binding for d.T.S. TCR/CD3, D.S. 139. C.S. 139. III et al (133. III, D.S. III) III TCR/CD 3. III).

The constant CD3 epsilon signaling component of the T Cell Receptor (TCR) complex on T cells has been used as a target to promote the formation of immunological synapses between T cells and tumor cells. Co-conjugation (co-gag) of CD3 and a tumor antigen activates T cells, resulting In lysis of tumor cells expressing the tumor antigen (Baeuuerle et al (2011) "Bispecific T Cell engage For Cancer Therapy," In: Bispecific Antibodies, Kontermann, R.E. (Ed.) Springer-Verlag; 2011: 273-287). This approach allows bispecific antibodies to interact comprehensively with a T cell compartment (component) with high specificity for tumor cells and is widely applicable to a large number of cell surface tumor antigens.

B7H3 is one of the B7 family members, belonging to type I transmembrane proteins, comprising an amino-terminal signal peptide, an extracellular immunoglobulin-like variable (IgV) and constant (IgC) regions, a transmembrane region and a cytoplasmic tail of 45 amino acids (Tissue antibodies, 2007 Aug; 70(2): 96-104). Currently, there are mainly 2 shears, B7H3a and B7H3B, for B7H 3. The extracellular segment of B7H3a consists of IgV-IgC 2 immunoglobulin domains, also known as 2IgB7H3, while the extracellular segment of B7H3B consists of IgV-IgC-IgV-IgC 4 immunoglobulin domains, also known as 4IgB7H 3.

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

CD3 antibody molecules such as OKT3, UCHT-1, SP34 (Silvera Pessano et al, the EMBO journal.1985,4(2):337-344) are disclosed in the prior art, while CD3 antibody molecules such as CN103703024, WO2017055389, CN102171248 are disclosed. However, in the development of drugs, there is still a need to develop CD3 antibody molecules with greater safety and efficacy.

Disclosure of Invention

In one aspect, the present disclosure provides a multispecific 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,

said second polypeptide chain comprising, in order from amino-terminus to 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 said multispecific protein molecule, in particular 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 said second target antigen of said multispecific protein molecule is CD3, and said first and third target antigens 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, the Tumor Associated Antigen (TAA) of the multispecific protein molecule is selected from AFP, ALK, B7H3, 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(MS4A 20), CD20, CD123, CD133, CD138, CDK 20, CEA, Claudin18.2, cyclin-B20, CYP1B 20, EGFR, EGFRvIII, 20/Her 20, ErbETETE 72, ErbB 20, ErbB 36V 20-20, Ephra LR-B20, Epigra-B20, Epigy-B20, HLA-20, IgG-e.g 3, IgG-g 3, IgG-72, IgG-g 3, IgG-K, HLA/MAGE-A3, hTERT, IL13R α 2, LMP2, κ -Light, LeY, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6 and-12), MART-1, mesothelin (mesothelin), ML-IAP, MOv- γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16(CA-125), MUM1, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF- β, TMPRSS2, Tang-Nori antigen (Thompson-novell antigen; Tn), TRP-1, TRP-2, tyrosinase and urokinase-3 and 5T4(Trophoblast glycoprotein). Preferably, the Tumor Associated Antigen (TAA) is selected from B7H3, BCMA, CEA, CD19, 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 _d2-L4-Fc1 formula I,

the second polypeptide chain has the structure of formula II:

V _e3-L5-V _f3-L6-Fc2 formula II,

the V is_a1、V _b1、V _c2、V _d2、V _e3 and V _f3 is a light chain variable region or a heavy chain variable region of an antibody, and the V _a1 and V _b1, the V _c2 and V _d2 with said V _e3 and V _f3 are not both light chain variable regions or heavy chain variable regions, respectively.

In some embodiments, the first polypeptide chain of the multispecific protein molecule has 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 said second polypeptide chain has the structure shown below:

VL _TAA-L5-VH _TAA-L6-F _C2 or VH_TAA-L5-VL _TAA-L6-F _C2。

In some embodiments, the first polypeptide chain of the multispecific protein molecule has a VL_TAA-L1-VH _TAA-L2-VH _CD3-L3-VL _CD3-L4-Fc1, and said second chain has VL_TAA-L5-VH _TAA-L6-F _C2.

In some embodiments, the first polypeptide chain of the multispecific protein molecule has a VH_CD3-L1-VL _CD3-L2-VL _TAA-L3-VH _TAA-L4-Fc1, and said second chain has VH_TAA-L5-VL _TAA-L6-F _C2.

In some embodiments, the TAA is B7H 3.

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

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

In some embodiments, the antigen binding region to CD3 in the multispecific protein molecule comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 48. LCDR1, LCDR2 and LCDR3 shown at 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 shown in SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 38 and 39;

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

iii) are as set forth in SEQ ID NO: 37. HCDR1, HCDR2 and HCDR3 shown at 40 and 42;

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

v) are respectively shown in SEQ ID NO: 37. 47 and 45, HCDR1, HCDR2, and HCDR 3.

In some embodiments, the antigen binding region against CD3 in the multispecific protein molecule 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, the antigen binding region against CD3 in the multispecific protein molecule 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 to B7H3 comprises an antibody light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 12. 13 and 14, and the heavy chain variable region comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown in fig. 10 and 11.

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

In some embodiments, the antigen binding region to B7H3 in the multispecific protein molecule comprises an amino acid sequence as 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 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 polypeptides having an amino acid sequence as set forth in SEQ ID NOs: 71. 88 or 70.

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

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 72 is shown;

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 73;

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

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

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

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 77;

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 78, respectively;

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

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 80 is shown in the figure;

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 83 is shown;

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

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 85 is shown;

the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 86, respectively; or

The amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 87, respectively.

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

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

In some embodiments, wherein the multispecific protein molecule comprises a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 76, and the amino acid sequence of said second polypeptide chain is as 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 multispecific protein molecule according to the foregoing, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. Preferably, the therapeutically effective amount is a unit dose of the composition comprising 0.1-3000mg (more preferably 1-1000mg) of the multispecific protein molecule as described above.

In another aspect, the present disclosure relates to an isolated nucleic acid molecule encoding a multispecific protein molecule as described above.

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

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 multispecific protein molecule as described above, comprising the steps of culturing a host cell as described above in a culture medium to form and accumulate a multispecific protein molecule as described above, and recovering the multispecific protein molecule from the culture.

In another aspect, the present disclosure relates to a multispecific protein molecule as hereinbefore described or a pharmaceutical composition as hereinbefore described, or an isolated nucleic acid molecule as hereinbefore described, as a medicament, preferably the medicament is a medicament which activates T cells, more preferably the medicament is 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 the use of a multispecific 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 to activate T cells.

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

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 multispecific 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 containing 0.1-3000mg of the multispecific protein molecule as described above, or the pharmaceutical composition as described above, or the isolated nucleic acid molecule as described above.

In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma (blastoma), sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung carcinoma, non-small-cell lung carcinoma (NSCLC), squamous cell carcinoma of the Head and Neck (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 mediastinal large B-cell lymphoma, Mantle Cell Lymphoma (MCL), Small Lymphocytic Lymphoma (SLL), large B-cell lymphoma enriched with T-cells/histiocytes, multiple myeloma, myeloid cell leukemia-1 protein (Mcl-1), myelodysplasia syndrome (MDS), Gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic 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 cancer (hepatobiliary cancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, meckel cell carcinoma, testicular cancer, and skin cancer. In some embodiments, wherein the cancer is a B7-H3 positive cell-associated cancer; preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

In some embodiments, any of the autoimmune or inflammatory diseases described above is selected from: 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.

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 assays for the binding activity of antibodies to cells expressing or not expressing the corresponding antigen. Fig. 2A is a graph of the binding activity of different antibodies to a498 cells expressing human B7H3, fig. 2B is a graph of the binding activity of different antibodies to CT26 cells overexpressing human B7H3, fig. 2C is a graph of the binding activity of different antibodies to CT26 cells not expressing human B7H3, and the results show that none of the antibodies bind to CT26 cells not expressing human B7H3, and fig. 2D is a graph 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: the killing activity of bispecific antibodies containing different CD3scFv against a498 was tested. Fig. 3A is the killing activity of B7H3 monovalent bispecific antibody. FIG. 3B shows the killing activity of B7H3 bivalent diabody. In addition to the weaker killing activity of 155, 156, 185, and 186 against a498, the remaining bispecific antibodies, whether monovalent or bivalent B7H3, showed more pronounced killing activity.

Fig. 4A to 4B: B7H3 bivalent bispecific antibody containing the same CD3scFv was compared for killing activity against a 498. Fig. 4A compares the killing activity of B7H3 monovalent (181) and bivalent (131) bispecific antibodies containing HRH 1. FIG. 4B compares the killing activity of B7H3 monovalent (187) and bivalent (177) containing HRH 7. The experimental results show that the B7H3 bivalent bispecific antibody has obvious A498 killing activity compared with the B7H3 monovalent bispecific antibody, and meanwhile, the B7H3 bivalent bispecific antibody has obviously enhanced killing activity compared with the B7H3 monovalent bispecific antibody.

Fig. 5A to 5C: the killing activity of B7H3 bivalent bispecific antibody containing the same CD3 heavy chain variable region, but different structural order, against a498 was tested. Fig. 5A is a comparison of killing activity between the first polypeptide chain containing HRH2 with different (AFF1, AFF2, AFF3, AFF4) B7H3 bivalent bispecific antibody. FIG. 5B shows the comparison of the killing activity of the second bivalent bispecific antibody against B7H3 (AFF3, AFF3-B) with HRH 2-containing polypeptide chains in different sequences. The results show that the B7H3 bivalent bispecific antibody with the same sequence and different VH and VL arrangement sequences has obvious A498 cell killing activity, and the intermolecular killing activity of different structural sequences is similar. FIG. 5C is a comparison of the killing activity of bispecific antibodies of different structures comprising the same B7H3scFv and CD3scFv, and all three bispecific antibodies 127, 201 and 202 tested can kill A498 tumor cells in vitro, wherein the killing activity of bispecific antibody 127 is better than that of 201 and 202.

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

Fig. 7A to 7B: activation assay of Jurkat recombinant cells with different valencies of bispecific antibody containing the same CD3 scFv. FIG. 7A is the antibody-mediated activation of Jurkat recombinant cells specific for the B7H3 target in the case of B7H3 mono/bivalent bispecific antibody in A498-containing cells; figure 7B is B7H3 mono/bivalent bispecific antibody mediated activation of non-B7H 3 target-specific Jurkat recombinant cells in the absence of a498 cells.

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

Fig. 9A to 9C: different antibodies stimulated PBMCs to produce cytokine secretion assays non-B7H 3 target specific in the presence of CHOK1 cells (not expressing B7H 3). FIG. 9A is a comparison of IFN γ levels secreted by PBMCs stimulated by different antibodies, FIG. 9B is a comparison of TNF α levels secreted by PBMCs stimulated by different antibodies, and FIG. 9C is a comparison of IL-2 levels of PBMCs-secreting cells stimulated by different antibodies. FIGS. 9A-9C show that the 118, 127 and 132 antibodies were unable to stimulate PBMC for cytokine secretion that is not target-specific for B7H3, and were highly safe. Antibodies indicated by legends in fig. 9A-9C are identical.

Fig. 10A to 10E: anti-tumor efficacy testing of bispecific antibodies in a mouse a498 model reconstituted with human PBMCs. FIG. 10A is a graph showing the tumor suppressor activity assay of the low dose B7H3 bivalent bispecific antibody, and the low dose 118 and 119 antibodies still showed some tumor suppressor activity and showed some dose dependence. Fig. 10B is a graph of the tumor suppressor activity assay for 0.3mpk and 0.6mpk doses of B7H3 bivalent bispecific antibody, 113 antibody showing dose dependent tumor in vivo suppressive activity. Fig. 10C is a graph of the tumor suppressor activity assay for the B7H3 bivalent bispecific antibody at 0.12mpk and 0.36mpk doses, with the 118 antibody showing significant tumor suppressor activity at both doses. Fig. 10D is a measure of the tumor suppressor activity of the B7H3 bivalent bispecific antibody at a dose of 0.36mpk, and all of the 126, 127, and 128 antibodies showed significant tumor suppressor activity. Fig. 10E shows the tumor suppressor activity of 127 antibody at different doses and at different dosing frequencies. In FIGS. 10A to 10E, Vehicle represents a negative control group to which PBS was administered.

Fig. 11A to 11B: antitumor efficacy of bispecific antibodies in hCD3 KI mouse model. Fig. 11A and 11B are the tumor inhibitory effects of 118 and 132, respectively, in the hCD3 KI mouse model.

Detailed Description

Term(s) for

The three letter codes and the one letter codes for amino acids used in this disclosure are as described in j. diol. 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 a target antigen. Protein molecules capable of specifically binding to two antigens or epitopes of a target antigen are referred to as bispecific protein molecules, and "bispecific protein molecules" comprising antibodies or antigen-binding fragments of antibodies (e.g., single chain antibodies) are interchangeable herein with "bispecific antibodies".

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

The term "antibody" encompasses any antibody that includes at least one Complementarity Determining Region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD3)(CDR) of an antigen binding molecule or molecular complex. The term "antibody" comprises: immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM). 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 CH 3. 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 hypervariable regions, known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FRs), also known as 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, FR 4. In various embodiments of the disclosure, the anti-CD 3 antibody (or antigen-binding portion thereof), anti-CD 3 antibody, anti-CD binding portion thereof, anti-CD 3 antibodyB7H3The antibody (or antigen-binding portion thereof) or FR against other antigens of interest may be identical to human germline sequences, or may be modified naturally or artificially. The antibody may be of a different subclass (subclass), for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subclass), IgA1, IgA2, IgD, IgE, or IgM antibody.

The term "antibody" also encompasses antigen-binding fragments of a complete antibody molecule. 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, synthetically or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived, for example, from whole antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable and, optionally, constant regions. Such DNA is known and/or can be readily obtained from, for example, commercially available sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. This DNA may be sequenced and manipulated chemically or by using molecular biology techniques, such as arranging one or more variable and/or constant regions in a suitable configuration, or introducing codons, generating cysteine residues, modifying, adding or deleting amino acids, and the like.

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

An antigen-binding fragment of an antibody will typically comprise at least one variable region. The variable region may be a region 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 a VH region associated with a VL region, the VH and VL regions may be located opposite one another in any suitable arrangement. For example, the variable regions may be dimerized and contain VH-VL or VL-VH dimers.

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 hinge or linker region, in whole or in part. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, allowing for flexible and semi-flexible linkages between adjacent variable and/or constant regions in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of the invention may comprise homodimers or heterodimers (or other multimers) configured with any of the variable and constant regions listed above non-covalently linked to each other and/or to one or more monomeric VH or VL regions (e.g., in disulfide bonds).

The "murine antibody" is in the present disclosure a monoclonal antibody derived from a mouse prepared according to the knowledge and skill in the art. Prepared by injecting a test subject with an antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties, which antibodies are murine when the injected test subject is a mouse.

"chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody and a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into expression vector, and finally expressing chimeric antibody molecule in eukaryotic system or 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 a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprises a heavy chain constant region of human IgG1, IgG2 or IgG4, or a heavy chain constant region variant of IgG1, IgG2 or IgG4 using amino acid mutations (e.g., YTE mutation or back mutation, L234A and/or L235A mutation, or S228P mutation).

The term "humanized antibody", including CDR-grafted antibodies, refers to an antibody produced by grafting CDR sequences of an antibody of animal origin, for example, a murine antibody, into a framework region (or framework region) of a human antibody variable region. Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of heterogenous protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of, for example, human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at the Internet http:// www.vbase2.org /), as well as in Kabat, E.A. et al, 1991 Sequences of Proteins of Immunological Interest, 5 th edition. To avoid the reduction of immunogenicity and the resulting reduction of activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies after further affinity maturation of the CDRs by phage display.

Because of the contacting residues of the antigen, grafting of the CDRs can result in a reduction in the affinity of the resulting antibody or antigen-binding fragment thereof for the antigen due to the framework residues that are contacted with the antigen. Such interactions may be the result of a high degree of somatic mutation. Thus, there may still be a need to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues from the non-human antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by examining the sequence and structure of the variable region of an animal monoclonal antibody. Residues in the CDR donor framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subclass consensus sequence or a consensus sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be likely the result of somatic hypermutation and thus play 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 a kappa, lambda chain of human or murine origin or a variant thereof, or further comprise a heavy chain constant region of IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin.

"human antibody" is used interchangeably with "human antibody", and can be an antibody derived from a human or an antibody obtained from a transgenic organism that is "engineered" to produce specific human antibodies in response to antigenic stimuli and can 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 targeted for disruption, including targeted disruption of the endogenous heavy and light chain loci in the cell lines. The transgenic organisms can synthesize human antibodies specific for human antigens, and the organisms can be used to produce human antibody-secreting hybridomas. A human antibody can 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 can also be constructed by gene or chromosome transfection methods as well as phage display techniques, or by 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 the same epitope, except for possible variant antibodies (e.g., antibodies containing naturally occurring mutations or mutations generated during the manufacture of monoclonal antibody preparations, which variants are typically present in minor amounts). Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the identity of the antibody as 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 for use 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 that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods, as well as other exemplary methods for preparing monoclonal antibodies, are described herein.

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

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, such that it is possible to generate a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science242: 423-. Such single chain antibodies are also intended to be encompassed within 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. Antigen binding portions can 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-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al, 1995 Protein Eng.8(10): 1057-1062; and U.S. Pat. No. 5,5641870).

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

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 the hinge position, 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 having an antigen-binding activity, which is obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F (ab') 2. Fab 'can be produced by treating F (ab')2, which 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 the Fab ' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote 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) joined by a linker. Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g. variants using 1-4 (including 1, 2, 3 or 4) repeats(Holliger et al (1993) Proc Natl Acad Sci USA.90: 6444-. Other linkers useful in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

"multispecific antibody" refers to 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 epitopes may be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind two different epitopes.

The term "bivalent bispecific antibody" to a tumor-associated antigen refers to a bispecific antibody having two antigen-binding regions against a certain tumor-associated antigen target, e.g. a B7H3 bivalent bispecific antibody refers to a bispecific antibody comprising two antigen-binding regions against 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" used to link two protein domains together refers to a linking polypeptide sequence used to link protein domains, usually with some flexibility, such that the use of a Linker does not lose the original function of the protein domains.

Diabodies (diabodies) are 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.

The dsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues. The amino acid residues substituted with cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (Protein engineering.7:697 (1994)).

In some embodiments of the disclosure, an antigen-binding fragment can be produced by: obtaining cDNA encoding VH and/or VL and other desired domains of the monoclonal antibodies of the present disclosure that specifically recognize and bind to an antigen, constructing DNA encoding the antigen-binding fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.

The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of immunoglobulin heavy chains may vary, the human IgG heavy chain Fc region is generally defined as extending from the 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, published Health Service 5 th edition, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically has two constant region domains, CH2 and CH 3. The "first Fc" is also referred to herein as "Fc 1" and the second Fc is also referred to as "Fc 2".

“V _a1-L1-V _b1-L2-V _c2-L2-V _d2-L4-Fc1 ' and ' V '_e3-L5-V _f3-L6-Fc 2' V _a1、V _b1、V _c2、V _d2、V _e3 and V _f3 is the variable region of the light chain or the variable region of the heavy chain of the antibody, V _a1 and V _b1 binds to a first antigen (first target antigen) epitope, V _c2 and V _d2 binds to a second antigen (second target antigen) epitope, V _e3 and V _f3 binds to a third antigen (third target antigen) epitope, and the first, second and third epitopes may be the same or different.

Analogous "VH_TAA-L1-VL _TAA-L2-VH _CD3-L3-VL _CD3in-L4-Fc 1 ", VH_TAAAnd VL_TAARepresenting antibody variableThe region binds to an epitope of a tumor-associated antigen, VH_CD3And VL_CD3Indicating that the antibody variable region binds to an epitope of CD 3.

The present disclosure "knob-Fc" refers to the inclusion of a point mutation of 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 antibody Fc region to form a hole-like spatial structure. Knob-Fc and hole-Fc are more prone to heterodimer formation due to steric hindrance. To further promote the formation of heterodimers, point mutations of S354C and Y349C were also introduced into knob-Fc and hole-Fc, respectively, to further promote the formation of heterodimers via disulfide bonds. Meanwhile, substitution mutations of 234A and 235A may also be introduced into Fc in order to eliminate or reduce ADCC effect by the antibody Fc. For example, preferred knob-Fc and hole-Fc of the present disclosure are shown in SEQ ID NO: 69 and 70. In bispecific antibodies, either knob-Fc or 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 at the same time.

The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide as compared to the original protein or polypeptide, including the occurrence of 1 or several amino acid insertions, deletions or substitutions based on 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 is known in the art, the variable regions of the heavy and light chains each consist of 4 Framework Regions (FRs) connected by 3 Complementarity Determining Regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held tightly together by the FRs 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) a cross-species sequence variability based approach (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.Molec.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 that variable domain. It is essentially a variable domain without CDRs.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes 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 herein in some embodiments, the CDRs may define the CDRs 1, 2 and 3(LCDR1, LCDR2, LCDR3) of the light chain variable domain and the CDRs 1, CDR2 and CDR3(HCDR1, HCDR2, HCDR3) of the heavy chain variable domain in Kabat's rules (Kabat et al Sequences of Proteins of Immunological Interest, (5 th edition, 1991, National Institutes of Health, Bethesda MD)), for example the definition of the CD3 antibody CDRs in this disclosure. In other embodiments, the CDRs of the antibody may be defined using rules such as IMGT, for example, in the definition of the CDRs of the B7H3 antibody, i.e., using the IMGT rules.

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

The term "tumor antigen" refers to a substance produced by a tumor cell, optionally a protein, including a "tumor-associated antigen" or "TAA" (which refers to a protein produced in a tumor cell and differentially expressed in cancer compared to corresponding normal tissue) and a "tumor-specific antigen" or "TSA" (which refers to a tumor antigen 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, B7H3, BAGE proteins, BCMA, BIRC5 (survivin), BIRC7, β -catenin (β -catenin), brc-ab brc, BRCA brc, BORIS, CA brc, CA125, carbonic anhydrase IX, caspase-8 (caspase-8), CALR, CCR brc, CD brc (MS4A brc), CD brc, CD 36123, CD133, CD138, CDK brc, CEA, Claudin18.2, cyclin-B brc, CYP1B brc, EGFR, EGFRvIII, EGGME brc/Her brc, Erb3672, ErbB brc, ETV brc-AML, CAM, FrahA brc, FranLRR-1, EpigLR-gR, EGFRvIII, ErbGE brc, HLA-brc, IgG-3, IgG- α -brc, IgG-3, IgG- α -IgG-3, IgG-3, LMP2, κ -Light, LeY, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6 and-12), MART-1, mesothelin (mesothelin), ML-IAP, MOv- γ, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16(CA-125), MUM1, NA17, NKG2D, NY-BR 9, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC 5, PRLR, PRAME, PSMA (FOLH 5), RAGE proteins, Ras, RGS5, Rho, ROR 5, SART-1, STERT-3 AP, PRLR, PRLH 5, TRTP-5, TROPPSMA (TROPPSK 5), TRTP 5, TROPRON 5, TROPROR 5, TROPRON 5, TROPPSE 5, TROPROPSOTN 5, TROPRON 5, TROPROPSE 5, TROPRON 5, TR.

"CD 3" refers to an antigen expressed on T cells as part of a multi-molecular T Cell Receptor (TCR) and which is composed of homodimers or heterodimers formed by two of the following four receptor chains: CD3- ε, CD3- δ, CD3- ζ and CD3- γ. Human CD3- ε (hCD3 ε) comprises UniProtKB/Swiss-Prot: the amino acid sequence as described in P07766.2. Human CD 3-delta (hCD3 delta comprises the amino acid sequence set forth in UniProtKB/Swiss-Prot: P04234.1. thus, unless specifically indicated to be from a non-human species, e.g., "mouse CD 3", "monkey CD 3", etc., the term "CD 3" refers to human CD 3.

An "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically 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., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996).

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

The term "affinity" refers to the strength of the 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 through weak non-covalent forces; the greater the interaction, the stronger the affinity. The term "high affinity" for an antibody or antigen binding fragment thereof (e.g., Fab fragment), as used herein, generally refers to having 1E^-9K of M or less_D(e.g., 1E)^-10K of M or less_D、1E ^-11K of M or less_D、1E ^-12K of M or less_D、1E ^-13K of M or less_D、1E ^-14K of M or less_DEtc.) or an antigen-binding fragment thereof.

The term "KD" or "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies are administered at less than about 1E^-8M, e.g. less than about 1E^-9M、1E ^-10M or 1E^-11M or less, binds to an antigen, 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, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, 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 Cold spring harbor antibody protocols, chapters 5-8 and 15. For example, a mouse may be immunized with an antigen or fragment thereof, and the resulting antibody can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. Antibodies or antigen-binding fragments of the disclosure are genetically engineered to incorporate one or more human FR regions in a CDR region of non-human origin. Human FR germline sequences can be obtained from the website http:// www.imgt.org/or from the immunoglobulin journal, 2001ISBN012441351 by aligning the IMGT human antibody variable region germline gene database with the MOE software.

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 susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and 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 using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As an alternative prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to the antigen. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using a protein A or protein G Sepharose FF column containing an adjusted buffer. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting the antibody fragment by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. 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 contact of an exogenous drug, therapeutic agent, diagnostic agent, 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. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by a reagent, a diagnostic, a 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 "treating" is meant administering a therapeutic agent, e.g., a composition comprising any of the compounds of the embodiments of the present disclosure, either internally or externally to a patient who has 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 in the 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 development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") 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 commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating each of the symptoms of the target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any of the statistical tests known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative amino acid modification" or "conservative amino acid substitution" refers to the replacement of an amino acid in a protein or polypeptide with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that the alteration can be made without altering the biological activity or other desired characteristics (e.g., antigen affinity and/or specificity) of the protein or polypeptide. One skilled in The art recognizes that, in general, single amino acid substitutions in 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., p. 224 (4 th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to destroy biological activity. Exemplary conservative substitutions are set forth in the following table "exemplary amino acid conservative substitutions".

Exemplary amino acid conservative substitutions

Original residues	Conservative substitutions
Ala(A)	Gly；Ser

Arg(R)	Lys；His
Asn(N)	Gln；His；Asp
Asp(D)	Glu；Asn
Cys(C)	Ser；Ala；Val
Gln(Q)	Asn；Glu
Glu(E)	Asp；Gln
Gly(G)	Ala
His(H)	Asn；Gln
Ile(I)	Leu；Val
Leu(L)	Ile；Val
Lys(K)	Arg；His
Met(M)	Leu；Ile；Tyr
Phe(F)	Tyr；Met；Leu
Pro(P)	Ala
Ser(S)	Thr
Thr(T)	Ser
Trp(W)	Tyr；Phe
Tyr(Y)	Trp；Phe
Val(V)	Ile；Leu

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 severity, or delaying onset of the condition, including biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes exhibited during development of the condition. 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-associated disorders of the disclosure, reducing the dosage of other agents required to treat the disorder, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a target antigen-associated disorder of the disclosure in a patient.

"exogenous" refers to a substance produced outside an organism, cell or human body as the case may be. "endogenous" refers to a substance produced in a cell, organism, or human body as the case may be.

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

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

"isolated" refers to the 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. Generally, 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 antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a mixture containing one or more compounds described in 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 facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

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

The terms "cancer," "cancerous," or "malignant" refer to or describe a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (blastoma), sarcoma, and leukemia or lymphoid malignancies. 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 mediastinal large B-cell lymphoma, Mantle Cell Lymphoma (MCL), Small Lymphocytic Lymphoma (SLL), large B-cell lymphoma rich in T-cells/histiocytes, multiple myeloma, myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), Gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic 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 cancer (hepatobiliary cancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, meckel cell carcinoma, testicular cancer, and skin cancer.

By "inflammatory disorder" is meant any disease, disorder or syndrome in which excessive or unregulated inflammatory responses result in excessive inflammatory symptoms, host tissue damage or loss of tissue function. "inflammatory disease" also refers to a pathological condition mediated by the influx of leukocyte or neutrophil chemotaxis.

"inflammation" refers to a local protective response due to tissue damage or destruction, which serves to destroy, weaken, or eradicate (isolate) harmful substances and injured tissue. Inflammation is significantly associated with a influx of leukocyte or neutrophil chemotaxis. Inflammation can be caused by pathogenic organisms and viruses, as well as non-infectious causes such as reperfusion or stroke following trauma or myocardial infarction, immune and autoimmune responses to exogenous antigens.

"autoimmune disease" refers to any group of diseases in which tissue damage is associated with a humoral or cell-mediated response to the body's own components. 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, blistering pemphigoid, grave's disease, hashimoto's thyroiditis, Systemic Lupus Erythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia, and the like.

Furthermore, another aspect of the present disclosure relates to a method for immunodetection or assay of a target antigen, a reagent for immunodetection or assay of a target antigen, a method for immunodetection or assay of a cell expressing a target antigen, and a diagnostic agent for diagnosis of a disease associated with a target antigen-positive cell, which comprises the monoclonal antibody or antibody fragment specifically recognizing and binding to a target antigen of the present disclosure as an active ingredient.

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

The immunoassay or measuring method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

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

For detecting cells expressing the polypeptide, a known immunoassay method can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, or the like is preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, the living sample for detecting or measuring the target antigen is not particularly limited as long as it has a possibility of containing cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance 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 from the claims. In the specification and claims, the singular forms "a", "an", and "the" 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 herein incorporated by reference. The following examples are set forth in order to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed in any way to limit the scope of the invention, which is defined in the claims.

Detailed Description

Preparation and screening of antibodies

Methods for making monoclonal antibodies are known in the art. One method that may be used is the method Of Kohler, G.et al, (1975) "Continuous Cultures Of Fused Cells calibrating Of predefined specification," Nature256:495-497, or a modification thereof. Typically, monoclonal antibodies are formed in non-human species, such as mice. Usually, mice or rats are used for immunization, but other animals, such as rabbits, alpacas, can also be used. The 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 an 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 overexpressing human B7H 3.

To monitor antibody responses, small biological samples (e.g., blood) can be obtained from the animal and tested for antibody titers against the immunogen. The spleen and/or some large lymph nodes may be removed and dissociated into single cells. Splenocytes can be screened, if desired, by applying a cell suspension (after removal of non-specifically adhered cells) to the antigen-coated plate or well. B cells expressing membrane-bound antigen-specific immunoglobulin will bind to the plate and will not be washed away 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, Calif.). 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 referred to as "HAT medium"). Subsequently, the resulting hybridomas are 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 Immunohistochemistry (IHC) screening. Subsequently, the monoclonal antibody-secreting hybridomas are selected for in vitro (e.g., in tissue culture flasks or hollow fiber reactors) or in vivo (e.g., as ascites fluid in mice) culture.

As another alternative to cell fusion techniques, Epstein-Barr virus (EBV) immortalized B cells can be used to prepare monoclonal antibodies of the invention. If desired, the hybridomas are propagated and subcloned, and supernatants are analyzed for anti-immunogen activity by conventional analytical methods (e.g., FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, etc.).

In another alternative, monoclonal antibodies against the antigen of interest (e.g., CD3, B7H3) 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, monoclonal antibodies against an antigen of interest (e.g., CD3, B7H3) are sequenced and then the polynucleotide sequence is cloned into a vector for expression or propagation. The sequences encoding the antibody 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 the anti-CD 3 monoclonal antibody and any other equivalent antibodies can be used for genetic manipulation to produce "humanized" antibodies to improve the affinity or other characteristics of the antibody. The general principle of humanizing 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) the actual humanization method/technique and, (4) transfection and expression of the humanized antibody. See, for example, U.S. patent nos. US4816567, US5807715, US5866692, and US 6331415.

Preparation and screening of B7H3 antibody

B cells are separated by using human PBMC, spleen and lymph node tissues, RNA is extracted, and a natural single-chain phage antibody library is constructed. And packaging the constructed natural single-chain phage antibody library to form phage particles, performing panning by a liquid phase method, combining the phage with biotinylated B7H3 liquid phase, and separating by streptavidin magnetic beads. To obtain positive sequences for binding to human B7H3, panning was performed using biotinylated human B7H3, and several monoclonal colonies were picked and packaged into phage single chain antibodies for phage ELISA testing. The monoclonal phage were tested for binding activity to human B7H3 and murine B7H3, respectively, and screened to obtain the B7H3 antibody.

The detection uses the B7H3 related antigen as follows:

detection of human B7H3 antigen

Commercial product (SinoBiological cat #11188-H08H)

The sequence is as follows:

note that: the crossline is the extracellular region of B7H 3; the italic part is a His-tag (His-tag).

Monkey B7H3 antigen for detection

Commercial product (SinoBiological cat #90806-C08H)

The sequence is as follows:

note that: the crossline is the extracellular region of B7H 3; the oblique part is a His tag.

Detection mouse B7H3 antigen

Commercial product (SinoBiological cat #50973-M08H)

The sequence is as follows:

note that: the crossline is the extracellular region of B7H 3; the oblique part is a His tag.

Full-length amino acid sequence of human B7H3

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-28);

the cross-line part is B7H3 Extracellular region (Extracellular Domain: 29-466), wherein 29-139 is Ig-like V-type 1 Domain, 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 dot-dashed part is a Transmembrane region part (Transmembrane domain: 467-487);

the oblique portion is divided into intracellular domains (cytotoxic domains: 488-coA 534).

Monkey B7H3 full-length amino acid sequence

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-28);

the cross-line part is B7H3 Extracellular region (Extracellular Domain: 29-466), wherein 29-139 is Ig-like V-type 1 Domain, 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 dot-dashed part is a Transmembrane region part (Transmembrane domain: 467-487);

the oblique portion is divided into intracellular domains (cytotoxic domains: 488-coA 534).

Full-length amino acid sequence of rat B7H3

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-28);

the cross-line segment is B7H3 Extracellular region (Extracellular domain: 29-248);

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

the oblique part is divided into intracellular domains (Cytoplasmic domains: 270-316).

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

>h1702 VH

>h1702 VL

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

TABLE 1 light and heavy chain CDR sequence Listing of antibody H1702 of B7H3

To further improve the performance of bispecific antibodies, cysteine substitution mutations were made in the VH and VL of the H1702 antibody B7H3, respectively, a G103C (amino acid natural sequence numbering, position 103 of SEQ ID NO: 16) mutation was introduced in the light chain variable region, and a G44C (amino acid natural sequence numbering, 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 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 antibody

On the basis of a murine CD3 antibody, a humanized CD3 antibody can be obtained by means of mutation, library construction, humanized modification, screening and the like.

CD3 antigen-related sequence information is as follows

Detection of human CD3 antigen

Commercial product (SinoBiological cat # CT038-H2508H)

The sequence is as follows:

human CD3 epsilon (Human CD3 epsilon)

Note that:

the cross-sectional part is CD3 epsilon Extracellular region (Extracellular domain: 23-126); the oblique part is His label

Human CD3δ

Note that:

the crossline segment is CD3 delta Extracellular region (Extracellular domain: 22-105); the ramp portion is a Flag tag.

Monkey CD3 antigen for detection

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

The sequence is as follows:

monkey CD3 epsilon

Note that:

the cross-line segment is CD3 epsilon Extracellular region (Extracellular domain: 22-117); the oblique part is His label

Monkey CD3 delta

Note that:

the crossline segment is CD3 delta Extracellular region (Extracellular domain: 22-105); the inclined part is Flag label

Mouse CD3 antigen for detection

The commercial product (SinoBiological cat # CT033-M2508H) has the following sequence:

mouse CD3 epsilon

Note that:

the cross-line segment is CD3 epsilon Extracellular region (Extracellular domain: 22-108); the oblique part is His-labeled mouse CD3 delta

Note that:

the crossline segment is CD3 delta Extracellular region (Extracellular domain: 22-105); the inclined part is Flag label

Full-length amino acid sequence of human CD3 epsilon

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-22);

the cross-sectional part is CD3 epsilon Extracellular region (Extracellular Domain: 23-126), wherein 32-112 is Ig-like Domain;

the dot-dashed part is a Transmembrane region part (Transmembrane domain: 127-152);

the oblique portion is divided into intracellular domains (153-207).

Human CD3 delta full-length amino acid sequence

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-21);

the crossline segment is CD3 delta Extracellular region (Extracellular domain: 22-105);

the dot-dash line part is a Transmembrane region part (Transmembrane domain: 106-;

the oblique portion is divided into intracellular domains (127-171).

Monkey CD3 epsilon full-length amino acid sequence

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-21);

the cross-line segment is CD3 epsilon Extracellular region (Extracellular domain: 22-117);

the dot-dash line part is a Transmembrane region part (Transmembrane domain: 118-;

the oblique part is divided into intracellular domains (139-198).

Monkey CD3 delta full-length amino acid sequence

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-21);

the crossline segment is CD3 delta Extracellular region (Extracellular domain: 22-105);

the dot-dash line part is a Transmembrane region part (Transmembrane domain: 106-;

the oblique portion is divided into intracellular domains (127-171).

Full-length amino acid sequence of mouse CD3 epsilon

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-21);

the cross-line segment is CD3 epsilon Extracellular region (Extracellular domain: 22-108);

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

the oblique portion is divided into intracellular domains (135-189).

Murine CD3 delta full-length amino acid sequence

Note that:

the double transverse line part is a Signal peptide (Signal peptide: 1-21);

the crossline segment is CD3 delta Extracellular region (Extracellular domain: 22-105);

the dot-dash line part is a Transmembrane region part (Transmembrane domain: 106-;

the oblique portion is divided into intracellular domains (Cytoplasmic domains: 127-.

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

TABLE 2 sequence listing of the heavy chain variable regions of the humanized antibody of CD3

The light chain variable region sequence is as follows:

>HRL

note: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequences are in italics and the CDR sequences are underlined. The CDR (LCDR1-LCDR3 and HCDR1-HCDR3) amino acid residues in the light and heavy chain variable regions of the humanized antibody of CD3 herein and in Table 3 below are in number and position in accordance with known Kabat numbering convention.

TABLE 3 CDR sequence Listing of the CD3 antibody

Construction and preparation of Single chain antibodies

The light and heavy chain variable regions derived from the above-described B7H3 antibody and the light and heavy chain variable regions derived from the CD3 antibody may be linked to form a scFv against B7H3 and a 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 can be 1, 2, 3, or 4.

Exemplary anti-B7H 3 scfvs are as follows:

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

Exemplary anti-CD 3 scfvs are as follows:

TABLE 5 sequence Listing of the various anti-CD 3 Single chain antibodies (scFv)

Construction and preparation of bispecific antibody B7H3 bivalent bispecific antibody and B7H3 monovalent bispecific antibody Body

The structure of the B7H3 bivalent diabody in some embodiments of the present disclosure is shown in fig. 1A, and its C-terminus may or may not be connected with a His-tag. With the Fc-containing two-chain asymmetric structural design, there are two B7H3 antigen-binding domains and one CD3 antigen-binding domain, one on each chain of the B7H3 antigen-binding domain, both in the form of scFv. The Fc region can maintain the normal half-life period and good stability of the antibody, the mismatching probability is greatly reduced by the design of two chains, and the uniformity of a sample and the yield of a target antibody are improved. The molecular structure (Format) of the specific bispecific antibody is shown in table 6 below, and in addition, the molecular structure of the B7H3 monovalent bispecific antibody used in some embodiments of the present disclosure has only an Fc domain in the second polypeptide chain, and does not comprise an antigen binding domain, the structure schematic is shown in fig. 1B.

TABLE 6 structural schematic of bispecific antibodies

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

TABLE 7 selection of 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 and the Fc region may be selected from any other linker that can be used to attach the antibody functional domain, and is not limited to the linkers defined by the above sequences.

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

TABLE 8 different Fc sequence Listing

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

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

According to the design method of the bispecific antibody molecules 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

Note: the second polypeptide chains of the bivalent bispecific antibody molecules 113, 118, 119, 126, 127, 128, 131, 132, 154, 155, 156, 161, 162, 171, 172 and 177 of table B7H3 above are all SEQ ID NOs: VL shown in 71_B7H3-L5-VH _B7H3-L6-hole-Fc; the second polypeptide chains of the B7H3 monovalent bispecific antibody molecule 181-187 are all SEQ ID NO: 70 as shown in the figure.

The amino acid sequences of the bispecific antibodies of the negative controls (NC1, NC2, NC3) and the positive control (MGD009) used in the present disclosure are as follows:

NC 1: B7H3 binding domain was replaced with a non-relevant antibody (anti-fluorescein antibody, anti-fluorescein), but the CD3 binding domain was retained), the amino acid sequence of which is referenced: the anti-fluorescent antibody used to form The control DART diabody was 4-4-20(Gruber. M.et al (1994)).

Chain 1 (VH)_CD3-VL _CD3-VL _ctrl-VH _ctrl-knob-Fc)

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

NC2 Retention of the B7H3 binding Domain, Only the CD3 binding Domain was replaced with an anti-fluorescein unrelated antibody

Chain 1 (VH)_ctrl-VL _ctrl-VL _B7H3-VH _B7H3-knob-Fc)

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

Note: in the order VL_B7H3-linker-VH _B7H3linker-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 strands, the preparation and amino acid sequence of which is referred to in published patent application WO2017030926A1, the amino acid sequence of which is as follows:

chain 1(B7H3VL-CD3VH-Fc)

Chain 2(CD3VL-B7H3VH)

Chain 3(Fc)

201(DART-Fc three-chain structure) 201 chain 1(B7H3VL-CD3VH-E-Fc)

Chain 2 of 201 (CD3VL-B7H3VH-K)

201 chain 3

202 (four-chain structure, wherein the ratio of the amounts of substances in the four chains is 1:2: 3:4 ═ 1:2:1:1)

Chain 1 202 (B7H3VH-CH1-Fc)

202 chain 2(B7H3VL-CL)

202 chain 3(B7H3VH-CH1-CD3VH-CL)

202 chain 4(CD3VL-CH1)

Example 2 expression and purification of CD3-B7H3 bispecific antibody

Plasmid expressing bispecific antibody (chain 1: chain 2: 1) was transfected into HEK293E cells, expression supernatant was collected after 6 days, and impurities were removed by high speed centrifugation. The clarified supernatant was purified using a Ni Sepharose excel column (GE Healthcare). The column was washed with PBS until the A280 reading dropped to baseline, the column was washed with PBS +10mM imidazole to remove non-specifically bound contaminating proteins and the effluent was collected, and finally the desired protein was eluted with PBS containing 300mM imidazole and the peak was collected. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200(GE) equilibrated with 550 buffer (10mM acetic acid, pH5.5,135mM NaCl), and the desired peak was collected. The sample is transferred to 559 buffer solution (10mM acetic acid, pH5.5, 9% sucrose) by desalting column or ultrafiltration centrifuge tube, subpackaged and frozen at-80 ℃.

Test example 1 BIAcore test for affinity of bispecific antibody for B7H3 and CD3

Antibody pairB7H3 and CD3 affinity assays, in the form of capture antibodies. BsAb was affinity captured by CM5 biosensor chip (Cat. # BR-1005-30, GE) or Protein A (Cat. #29127556, GE) coupled with Anti-Human IgG Antibody (Cat. # BR-1008-39, Lot. #10260416, GE), and then each antigen was passed over the chip surface, and reaction signals were detected in real time by a Biacore T200 instrument to obtain binding and dissociation curves. After dissociation was complete for each experimental cycle, regeneration buffer Glycine1.5(Cat # BR100354, GE) or 3M MgCl was used₂(from Human antibody capture kit, Cat. # BR100839, GE) the chips were washed and regenerated. After the experiment was completed, the data were fitted with (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to obtain affinity values.

The different V regions were ordered by the sequence of the different VH sequences, and the affinity of the bispecific antibody for CD3 was slightly different when different VH sequences of the CD3 antibody were used, and the antibody had the weakest affinity for CD3 and no detectable binding to CD3 on Biacore when HRH-6 and HRH-5 sequences were used.

TABLE 10 antigen affinity Biacore assay of bispecific antibodies of the structure of AFF3

Bispecific antibodies	Comprising CD3VH	BIAcore KD(M)
131	HRH-1	4.07E-08
113	HRH-2	7.72E-08

127	HRH-3	9.72E-08
154	HRH-4	6.97E-08
156	HRH-6	Is not combined with
155	HRH-5	Is not combined with
177	HRH-7	1.62E-07

An exemplary selection of antibodies comprising HRH3 as the heavy chain variable region of the CD3 antigen binding domain was tested, where test antibodies 118, 127, 132 had 10 affinities to human (human) B7H3 and human CD3, respectively^-9And 10^-8M levels, comparable to MGD009, and strong cross-binding activity with both monkey (cyno) B7H3 and human CD 3.

TABLE 11 antigen affinity Biacore assay results for bispecific antibodies comprising different structural sequences of HRH3

Test example 2 measurement of antibody binding Capacity at cellular level

The ability of bispecific antibody to bind to cell surface antigen was tested by FACS. For binding of cell surface antigens B7H3 and CD3, A498(ATCC, HTB-44), CT26/hB7H3 (recombinant cell line overexpressing human B7H3 in murine cells CT26, constructed internally, CT26 from the Chinese academy of cells, TCM37) and Jurkat recombinant cell line (Jurkat cells from ATCC, PTS-TIB-152, recombinant cell line overexpressing luciferase (luciferase) gene upstream of which NFAT response element is inserted) were used, respectively.

Add FACS buffer (buffer) (98% PBS, 2% FBS) resuspended cells to 96-well U-bottom plate (corning,3795) and add gradient diluted antibodies, incubate 1h at 4 ℃ and wash FACS buffer 2 times, then add APC anti-human IgG Fc Antibody (biolegend, Cat #409306,1:50 dilution) per well, incubate 30min at 4 ℃, wash 2 times and then resuspend cells with FACS buffer, finally read fluorescence signal values with FACS CantoII (BD).

The results show that B7H3 bivalent diabodies 118, 127 and 132 and negative control antibody NC2 (retaining B7H3 binding domain, only CD3 binding domain was replaced by irrelevant antibody) all bound to a498 cell line that highly expresses B7H3 (see fig. 2A), with a gradient dependent effect, stronger binding capacity than MGD009, and B7H3 target specific, and negative control antibody NC1(B7H3 binding domain was replaced by irrelevant antibody, but retaining 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 do not express B7H3 (see fig. 2C), well suggesting that the tested bispecific antibodies specifically bound to the cell membrane surface B7H3 target. The difference in binding ability of antibodies 118, 127 and 132 to MGD009 was more evident in the B7H3 overexpressing cell line CT26/hB7H3 than in the a498 cell line, suggesting that the B7H3 bivalent diabody has a more significant binding advantage on the B7H3 overexpressing cells than the B7H3 monovalent diabody MGD009, and a better safety window.

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) with a gradient-dependent effect. Among them, 118 and NC1 were comparable to MGD009 in binding capacity to Jurkat recombinant cells, whereas 127 and 132 were slightly weaker in binding capacity, probably due to the CD3 binding domain being between the B7H3 binding domain and FC, binding to Jurkat recombinant cells was affected by some steric hindrance. Negative control antibody NC2, which did not contain the 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 experiment

The experiment for killing tumor cells by PBMC mediated by bispecific antibody is realized by quantitatively detecting the proliferation condition of cells. Cell Titer-glo was used to detect the amount of ATP in the cells, which is an indicator of viable Cell metabolism and is directly proportional to the number of cells in culture.

Four different target cells (T) were shared, including three tumor cell lines expressing different levels of B7H3 (A498, U87 (Chinese courtyard cell Bank, TCHU138), Detroit562(ATCC, CCL-138)), and a negative control cell line CHOK1(ATCC, CCL-61) not expressing B7H 3. Effector cells (E) were PBMCs from healthy volunteers. Target cells were plated in 96-well plates and cultured overnight, the next day, with an equal amount of freshly extracted PBMC and a gradient of diluted test bispecific antibody (up to a final concentration of 300nM, 1: 3 dilution), or PBS (control), potent effector cells and target cells, no antibody, added to each well. Blank controls (blank, medium only, no cells or antibodies) were set, the ratio of E: T, 10:1, 5:1, 5:1, 5:1 for A498, U87, Detroit562 and CHOK1 cells, respectively. After 48 hours of incubation, the cells were examined by the Cell Titer-glo assay (see the description), the signal values were read by the microplate reader and finally converted to inhibition, and the data were processed and analyzed by Graphpad Prism 5.

Inhibition (% Inhibition) — 100% -, (ii)Signal value_Sample(s)-signal value_{Blank space}) /(Signal value)_Control-signal value_{Air conditioner White colour (Bai)})

(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 CD3scFv of different affinities exhibited different in vitro target cell killing efficacy (see fig. 3A and fig. 3B), in order to contain the bispecific antibodies 155, 156, 185 and 186 of HRH5 and HRH6, respectively, with the weakest killing efficacy, consistent with Biacore affinity assay results.

3.2 comparison of B7H3 monovalent and bivalent bispecific antibodies

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

TABLE 12 antibody structural sequences

3.3 influence of different Structure-ordered molecular Structure of B7H3 bivalent bispecific antibody on tumor killing Activity

Tumor cell killing activity of B7H3 bivalent bispecific antibody molecules 161, 162, 113 and 126 (see fig. 5A), and 113 and 143 (see fig. 5B), having different structural order of the same antigen binding domain module, all using HRH2 as the heavy chain variable region in the CD3 antigen binding domain, was tested in parallel. The results show that the B7H3 bivalent specific antibody molecules with different structural sequences have obvious killing effect on A498 cells. Among them, the killing activity of 161, 162, 113 and 126 is comparable to or slightly superior to that of MGD 009. The different structural arrangement order has little influence on the tumor cell killing activity of the B7H3 bivalent bispecific antibody.

TABLE 13 structural comparison of different test antibodies

3.4 the bispecific antibody has killing effect on tumor cell lines with different B7H3 expression levels

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

TABLE 14 bispecific antibody mediated PBMC directed killing of different target cell lines to be tested

3.5 comparison of killing of A498 cells by bispecific antibodies of different structures

Three bispecific antibodies 127 and 201, 202 to be tested were tested for their killing effect in vitro on a498 tumor cell line. The results showed (see fig. 5C) that the bispecific antibody of three structures all had tumor killing activity, wherein the killing activity of bispecific antibody 127 was superior to 201 and 202.

Test example 4 in vitro T cell activation test

The activation function of bispecific antibodies on T cells was tested by using the Jurkat recombinant cell line to detect the expression of the NFAT-driven luciferase reporter gene (luciferase) after Jurkat activation in the presence or absence of the A498 tumor cell line.

A498 cells were seeded in 96-well cell culture plates (1X 10)⁵100. mu.L/well) at 37 ℃ in 5% CO₂Culturing in an incubator for 20-24 h. The following day, after removing the cell culture supernatant, 90. mu.l of Jurkat recombinant cell suspension (5.5X 10) was added to each well⁵Per ml), and 10 μ l of a gradient dilution of the bispecific antibody to be tested (up to a final concentration of 500nM, 1: 3 gradient dilutions), and negative controls (control, with a498 and Jurkat recombinant cells, no antibody) and blank controls (blank, medium only, no cells or antibodies) were set at 37 ℃, 5% CO₂Culturing in an incubator for 5-6 h. The activation of the Jurkat recombinant cells specific to non-tumor cells is carried out by directly adding the Jurkat recombinant cells and the antibody to be detected into a blank 96-well culture plate. After the completion of the co-culture, 100. mu.l of Bright-Glo Reagent (Bright-Glo)^TMLuciferase Assay System, Promega, Cat #: E2620), placed at room temperature for 5-10min, and read the chemiluminescence signal value using a multifunctional microplate reader, wherein the calculation formula of the fluorescence enhancement factor (Fold increase) is as follows: factor of enhancement ═ signal value_Sample(s)-signal value_{Blank space}) /(Signal value)_Control-signal value_{Blank space})

(Fold increase＝(Signal _sample-Signal _blank)/(Signal _control-Signal _blank))

4.1 different permutations of the bivalent B7H3 molecule were all effective in activating T cells

B7H3 diabodies 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 diabodies on T cells. The results showed that the B7H3 bivalent diabody 118, 127 and 132 in different permutations were able to efficiently activate the Jurkat recombinant cell line in the presence of tumor cell line a498 (see fig. 6A), significantly inducing the expression of luciferase, since the negative control antibody NC1 was not able to induce the expression of luciferase, demonstrating that the activation of Jurkat recombinant cells is B7H3 target specific. Activation of Jurkat recombinant cells was achieved by co-recruiting by bispecific antibodies both Jurkat recombinant cells expressing CD3 and tumor cells expressing B7H3, and in the case of Jurkat recombinant cells alone, without a498 cells (see fig. 6B), luciferase expression was very low, with some weak signals detectable only at the highest few antibody concentration points.

TABLE 15 antibody structural sequences

4.2 comparison of B7H3 monovalent and bivalent bispecific antibodies

Bivalent CD3-B7H3 bispecific antibody significantly enhanced target-specific T cell activation compared to B7H3 monovalent bispecific antibody, consistent with the enhanced in vitro tumor killing ability of B7H3 bivalent versus B7H3 monovalent molecule in test example 3. At the same time, non-target-specific T cell activation remains unchanged. Thus, the B7H3 bivalent molecule (131) had stronger potency than the B7H3 monovalent molecule (181) (see fig. 7A), but the side effects due to T cell non-specific activation were not enhanced (see fig. 7B).

TABLE 16 antibody Structure

Antibodies	A first polypeptide chain	A 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 test

The effector cells redirect the target cells under the mediation of the bispecific antibody, and release cytokines while killing the target cells. The cytokine secretion is measured by ELISA method to quantitatively detect the content of cytokine in cell culture supernatant, including IL2, IFN gamma, 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 ℃ until use. In ELISA experiments, the frozen culture supernatant was removed, thawed at room temperature, at 3500rpm, centrifuged for 10mins, and the supernatant collected for ELISA experiments. The ELISA procedure is detailed in 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 show that the test bispecific antibody can effectively induce the secretion of IL2, IFN gamma and TNF alpha by PBMC in the coexistence of PBMC and target cell A498 positive to B7H3 (see FIGS. 8A-8C), wherein the secretion level of cytokines induced by MGD009 and 118 is highest, and 127 and 132 are second, while the secretion of cytokines induced by negative control antibody NC 1is not in the detection sensitivity range. In the co-presence of PBMC and B7H3 negative cells CHOK1 (see fig. 9A-9C), MGD009 clearly induced the release of IFN γ and TNF α at the top three concentration points, while the three bispecific antibodies tested, 118, 127 and 132, did not, suggesting that the three bispecific antibodies tested had better safety against non-target specific cytokine secretion than MGD 009.

Test example 6 efficacy test of mouse A498 model reconstituted with human PBMC

This test example utilizes a NOG mouse (A498 model (ATCC) reconstituted with human PBMC to evaluate the antitumor efficacy of the CD3-B7H3 bispecific antibody tested in accordance with the present invention in mice.

A498 cells 5X 10⁶The cells/mouse/100. mu.l (containing 50% matrigel) were inoculated subcutaneously in the right flank of NOG mice when the tumor volume of the tumor-bearing mice reached 130-³On the left and right, mice were randomly grouped into 5-6 mice each, and the day of grouping was defined as day 0 of the experiment. PBMCs from 2 freshly extracted volunteers were mixed at a 1:1 ratio on day 0 or day 1 and 5X 10⁶Cells/100 μ l were injected into the abdominal cavity of NOG mice and intraperitoneal injection of each antibody was initiated 2 times per week for 6 total doses, with tumor volume, animal weight monitored 2 times per week and data recorded. Vehicle is a negative control group administered with PBS buffer instead of antibody.

Antibodies 118 and 119 already exhibited some anti-tumor efficacy at lower doses (fig. 10A) and exhibited dose-dependent effects. Among these, antibody 118 had a tumor inhibition rate (TGI) of 22.17% and 60.39% at the end of the experiment (day 20), at 0.01mpk and 0.03mpk doses, respectively.

Antibody 113 has shown some anti-tumor effect at day 14, with tumor inhibition 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, continuing to increase and be dose-dependent by day 20, with tumor inhibition rates of greater than 100% (p <0.001) and 77.92% (p <0.05), respectively.

Under the 0.12mpk and 0.36mpk dose conditions (fig. 10C), antibody 118 at day 12, the 0.12mpk and 0.36mpk doses had reached 39.18% and 57.44%, respectively (p <0.001), and the tumor suppression rates by day21 increased to 81.72% (p <0.01) and greater than 100%, respectively, with complete tumor regression in even one mouse at the 0.36mpk dose (1/6).

At the 0.36mpk dose (fig. 10D), antibody 126 reached 47.78% tumor inhibition (p <0.01) at day 21. Antibody 128 has shown significant tumor suppression effect on Day 19 (TGI 56.37%), with tumor suppression rate rising to 69.28% (p <0.001) by Day 21. Antibody 127mpk, with a tumor suppression rate of 76.20% at day 12 (p <0.001), continued to increase by day21 with a tumor suppression rate greater than 100% (p <0.001), with 3 of 5 animals having tumor volumes that were regressed compared to the cohort, and 2 additional tumors regressing completely.

The anti-tumor activity of antibody 127 was repeated in another experiment (FIG. 10E), with a tumor inhibition rate of 90.6% at day 14 (p <0.001) and an increase of 95.80% by day 17 (p < 0.001). 127 was still effective at lower doses (0.12mpk) and less frequent dosing (1 time per week, 127-0.36mpk-qw), reaching tumor inhibition rates of 51.37% (p <0.001) and 96.20% (p <0.001) on day 17, respectively.

Test example 7, efficacy test of hCD3 KI mouse model

In the experiment, a Balb/c-hCD3 mouse is inoculated with a CT26-hB7H3 tumor cell line (CT26 cells are from a Chinese academy of sciences cell bank, TCM37, and are subjected to hB7H3 expression to obtain CT26-hB7H3 cells) subcutaneously to evaluate the inhibition effect of the CD3-B7H3 bispecific antibody on the tumor growth in the mouse.

Female hCD3E Balb/c transgenic mice, purchased from Nanda institute (license No. 201801374/5/6, permit SCXK (threo) 2015-0001).

CT26-hB7H3 cells 8X 10⁵Cells/mouse/100. mu.l were inoculated subcutaneously in the right flank of hCD3 mice. When carrying a tumorThe tumor volume of the mice reaches about 80-120mm³On the left and right, mice were randomly grouped into 7 mice each. The day of grouping was defined as day 0 of the experiment and intraperitoneal injections of each antibody were started 2 times per week for 5 times, tumor volume, animal weight were monitored 2 times per week and data were recorded. Vehicle is a negative control group administered with PBS buffer instead of antibody.

The results show that antibody 118 showed strong efficacy at 1mpk dose after initial administration (FIG. 11A), with an inhibition of 38.34% at day 13 (p < 0.05).

Antibody 132 had a tendency 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

In the experiment, CD3-B7H3 bispecific antibody is injected into the tail vein of SD rats, and the antibody concentration in the serum of the rats at different time points is detected, so that the metabolism of the CD3-B7H3 bispecific antibody in the SD rats is evaluated.

The tail vein of the rat is injected with 3mg/kg of the test drug, and the administration volume is 5 mL/kg. Blood was collected at each time point before and 5min, 8h, 1d, 2d, 4d, 7d, 10d, 14d, 21d, and 28d after administration. Antibody concentrations in serum were determined by ELISA using two different ELISA methods, respectively, B7H3 antigen (1. mu.g/mL) or CD3 antigen (1. mu.g/mL) plated with anti-human Fc-HRP (abcam, ab98624) as secondary antibody. Pharmacokinetic parameters of the tested drugs were calculated using Winnolin software, and the main pharmacokinetic parameters obtained 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, to achieve normal IgG antibody levels, and the CD3 antigen-binding region of 3.2-5.6 days. Among them, the kinetic parameters of antibody 118 in the two different antigen-binding regions of B7H3 and CD3 were not very different, indicating that the molecule has better integrity in vivo and half-lives of 4.9 and 4.4 days, respectively. The half-lives of antibody 127 in the two different antigen-binding regions of B7H3 and CD3 were 4.9 and 3.2 days, respectively, with the differences in exposure and clearance rates being more pronounced, with the CD3 moiety being poorer, and more likely due to the CD3 moiety being internal to the molecular structure, with the CD3 moiety becoming less functionally bound rather than being broken. The antibody 132 is based on the molecular sequence of the antibody 127, a pair of disulfide bonds are added in the B7H3scFv, the modification greatly improves the half-life period of the molecule (65-75%), and also greatly improves the exposure and the clearance rate.

TABLE 17 Table of major pharmacokinetic parameters in rats

Claims (22)

1. A multispecific 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,

   said second polypeptide chain comprising, in order from amino-terminus to 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.
2. The multispecific protein molecule of claim 1, wherein the first binding region, and/or the second binding region, and/or the third binding region is a single-chain antibody.
3. The multispecific protein molecule of claim 1 or 2, wherein the second target antigen is CD3, and the first and third target antigens are the same or different tumor-associated antigens; or

   The first target antigen is CD3, and the second and third target antigens are the same or different tumor-associated antigens.
4. A multispecific protein molecule according to any one of claims 1 to 3, wherein the tumor-associated antigen is selected from AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5, BIRC7, β -catenin, brc-ab brc, BRCA brc, BORIS, CA brc, CA125, carbonic anhydrase IX, caspase-8, CALR, CCR brc, CD 36123, CD133, CD138, CDK brc, CEA, Claudin18.2, cyclin-B brc, CYP1B brc, EGFR, EGFRvIII, ErbB brc/Her brc, ErbB brc, ge brc, ETV brc-AML, EphA brc, Fra-1, fo3672, gagler, ErbB brc, globinopolysaccharide, gleb brc, glebe brc, HLA-g-3, HLA-g-3, HLA-g, MOv- γ, Muc1, Muc2, Muc3, Muc4, Muc5, CA-125, MUM1, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA, RAGE protein, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF- β, TMPRSS2, Tang-nori antigen, TRP-1, TRP-2, tyrosinase, urolysin-3, and 5T 4.
5. The multispecific protein molecule of any one of claims 1 to 4, wherein the first polypeptide chain has the structure of formula I:

   V _a1-L1-V _b1-L2-V _c2-L2-V _d2-L4-Fc1 formula I,

   the second polypeptide chain has the structure of formula II:

   V _e3-L5-V _f3-L6-Fc2 formula II,

   the V is_a1、V _b1、V _c2、V _d2、V _e3 and V_f3 is a light chain variable region or a heavy chain variable region of an antibody, and the V_a1 and V_b1, the V_c2 and V_d2 with said V_e3 and V_f3 points ofNot both light chain variable regions and not heavy chain variable regions.
6. The multispecific protein molecule of any one of claims 1 to 5, wherein the first polypeptide chain has a structure as shown in:

   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 said second polypeptide chain has the structure shown below:

   VL _TAA-L5-VH _TAA-L6-F _C2 or VH_TAA-L5-VL _TAA-L6-F _C2。
7. The multispecific protein molecule of any one of claims 1 to 6, wherein the first and second Fc regions are the same Fc or different Fc;

   preferably, the first Fc region is knob-Fc and the second Fc region is hole-Fc; or

   The first Fc region is hole-Fc, and the second Fc region is knob-Fc.
8. The multispecific protein molecule of any one of claims 1 to 7, wherein the carboxy-terminal end of the first Fc region is linked to a His-tag or the carboxy-terminal end of the second Fc region is linked to a His-tag.
9. The multispecific protein molecule of any one of claims 1 to 8, wherein the antigen-binding region 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 sequences set forth in SEQ ID NOs: 48. LCDR1, LCDR2 and LCDR3 as shown in 49 and 50, and the heavy chain variable region is a heavy chain variable region selected from any one of i) to v) below:

   i) comprises the sequences respectively shown as SEQ ID NO: 37. 38 and 39, HCDR1, HCDR2, and HCDR 3;

   ii) comprises the amino acid sequences as set forth in SEQ ID NOs: 37. the heavy chain variable regions of HCDR1, HCDR2 and HCDR3 shown at 40 and 41;

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

   iv) comprises the sequences as set forth in SEQ ID NO: 37. 40 and 43, HCDR1, HCDR2 and HCDR 3; or

   v) comprises the sequences as set forth in SEQ ID NO: 37. 47 and 45, HCDR1, HCDR2, and HCDR 3.
10. The multispecific protein molecule of claim 9, wherein the antigen-binding region to 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.
11. The multispecific protein molecule of claim 10, wherein the antigen-binding region to CD3 comprises an amino acid sequence as set forth in SEQ ID NO: 55. 56, 57, 58, 61, 62, 63, 64, 65 or 68.
12. The multispecific protein molecule of any one of claims 1 to 11, wherein the tumor-associated antigen is B7H3, and the antigen-binding region to B7H3 comprises an antibody light chain variable region and a heavy chain variable region, wherein:

    the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 12. 13 and 14, and the heavy chain variable region comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown in fig. 10 and 11.
13. The multispecific protein molecule of claim 12, wherein the antigen-binding region to B7H3 comprises:

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

    or as shown in SEQ ID NO: 16 and/or the light chain variable region as set forth in SEQ ID NO: 15, or a heavy chain variable region as set forth in seq id no.
14. The multispecific protein molecule of claim 13, wherein the antigen-binding region to B7H3 comprises an amino acid sequence as set forth in SEQ ID NO: 51. 52, 53 or 54.
15. The multispecific protein molecule according to any one of claims 1 to 14, comprising a first polypeptide chain selected from the group consisting of 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 polypeptides having an amino acid sequence as set forth in SEQ ID NOs: 71. 88 or 70.
16. A multispecific protein molecule comprising a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the second polypeptide chain being as set forth in SEQ ID NO: 71. 88 or 70, and:

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 72 is shown;

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 73;

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

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

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

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 77;

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 78, respectively;

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

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 80 is shown in the figure;

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 83 is shown;

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

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 85 is shown;

    the amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 86, respectively; or

    The amino acid sequence of the first polypeptide chain is as set forth in SEQ ID NO: 87, respectively.
17. A pharmaceutical composition comprising a therapeutically effective amount of a multispecific protein molecule according to any one of claims 1 to 16, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.
18. An isolated nucleic acid molecule encoding the multispecific protein molecule of any one of claims 1 to 16.
19. A recombinant vector comprising the isolated nucleic acid molecule of claim 18.
20. 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 the recombinant vector of claim 19.
21. A method for producing a multispecific protein molecule according to any one of claims 1 to 16, comprising the steps of culturing a host cell according to claim 20 in a culture medium to form and accumulate the multispecific protein molecule according to any one of claims 1 to 16, and recovering the multispecific protein molecule from the culture.
22. A multispecific protein molecule according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17, or an isolated nucleic acid molecule according to claim 18, as a medicament; preferably the drug is a T cell activating drug, more preferably the drug is a drug for the treatment of cancer, autoimmune disease or inflammatory disease.

Images