ANTI-PD-1-ANTI-VEGFA BISPECIFIC ANTIBODY, PHARMACEUTICAL COMPOSITION AND USE THEREOF

Abstract

Provided are an anti-VEGFA-anti-PD-1 bispecific antibody and a use thereof. Specifically, the anti-VEGFA-anti-PD-1 bispecific antibody comprises: a PD-1-targeted first protein functional region and a VEGFA-targeted second protein functional region. According to an EU numbering system, mutation occurs at two positions of positions 234 and 235 of a heavy chain constant region of the immunoglobulin contained in the bispecific antibody, and after the mutation, an affinity constant of the bispecific antibody with FcγRI, FcγRIIa, FcγRIIIa, and/or C1q is decreased compared to that before the mutation. The bispecific antibody can specifically bind to VEGFA and PD-1, specifically relieve immunosuppression of VEGFA and PD-1 on an organism, and inhibit tumor-induced angiogenesis, and thus has good application prospects.

Claims

1. A bispecific antibody, comprising: a first protein functional region targeting PD-1, and a second protein functional region targeting VEGFA; wherein the first protein functional region is an immunoglobulin, and the second protein functional region is a single chain antibody; or, the first protein functional region is a single chain antibody, and the second protein functional region is an immunoglobulin; wherein, for the immunoglobulin, the heavy chain variable region comprises HCDR1-HCDR3 HCDR3 of amino acid sequences set forth in SEQ ID NOs: 28-30 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 31-33 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 34-36 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 37-39 respectively; or, for the immunoglobulin, the heavy chain variable region comprises HCDR1-HCDR3 HCDR3 of amino acid sequences set forth in SEQ ID NOs: 34-36 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 37-39 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 28-30 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 31-33 respectively; the immunoglobulin is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has mutations at any 2 or 3 of positions 234, 235 and 237, and the affinity constant of the bispecific antibody to FcγRIIIa and/or C1q is reduced after the mutation as compared to that before the mutation; preferably, the affinity constant is measured by a Fortebio Octet system.

2. The bispecific antibody according to claim 1, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A.

3. A bispecific antibody, comprising: a first protein functional region targeting PD-1, and a second protein functional region targeting VEGFA; wherein the first protein functional region is an immunoglobulin, and the second protein functional region is a single chain antibody; or, the first protein functional region is a single chain antibody, and the second protein functional region is an immunoglobulin; wherein, for the immunoglobulin, the heavy chain variable region comprises HCDR1-HCDR3 HCDR3 of amino acid sequences set forth in SEQ ID NOs: 28-30 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 31-33 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 34-36 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 37-39 respectively; or, for the immunoglobulin, the heavy chain variable region comprises HCDR1-HCDR3 HCDR3 of amino acid sequences set forth in SEQ ID NOs: 34-36 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 37-39 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 28-30 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 31-33 respectively; the immunoglobulin is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A.

4. The bispecific antibody according to claim 1, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has one or more mutations selected from: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A.

5. The bispecific antibody according to claim 1, wherein, the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17; or, the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3.

6. The bispecific antibody according to claim 1, wherein the bispecific antibody is selected from any one of the following (1)-(12): (1) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 7; (2) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 11; (3) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 17; (4) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 7; (5) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 11; (6) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 17; (7) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3; (8) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 11; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3; (9) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3; (10) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3; (11) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 11; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3; and (12) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3.

7. The bispecific antibody according to claim 1, wherein, the amino acid sequence of the heavy chain of the immunoglobulin is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain of the immunoglobulin is set forth in SEQ ID NO: 26.

8. The bispecific antibody according to claim 1, wherein the immunoglobulin or an antigen-binding fragment thereof binds to FcγRI with an affinity constant of less than about 10.sup.−6 M, such as less than about 10.sup.−7 M, 10.sup.−8M or 10.sup.−9 M or less; preferably, the affinity constant is measured by a Fortebio Octet system.

9. The bispecific antibody according to claim 1, wherein the immunoglobulin or the antigen-binding fragment thereof binds to C1q with an affinity constant of less than about 10.sup.−9 M, such as less than about 10.sup.−7 M, 10.sup.−8 M or 10.sup.−9 M or less; preferably, the affinity constant is measured by a Fortebio Octet system.

10. The bispecific antibody according to claim 1, wherein the first protein functional region is linked to the second protein functional region either directly or via a linker fragment; and/or the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody either directly or via a linker fragment.

11. The bispecific antibody according to claim 10, wherein the linker fragment is (GGGGS)n, n being a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

12. The bispecific antibody according to claim 1, wherein the numbers of the first protein functional region and second protein functional region are each independently 1, 2 or more.

13. The bispecific antibody according to claim 1, wherein the single chain antibody is linked to a C-terminus of the heavy chain of the immunoglobulin.

14. A bispecific antibody, comprising: a first protein functional region targeting PD-1, and a second protein functional region targeting VEGFA; wherein the number of the first protein functional region is 1, and the number of the second protein functional region is 2; the first protein functional region is an immunoglobulin, and the second protein functional region is a single chain antibody; the amino acid sequence of the heavy chain of the immunoglobulin is set forth in SEQ ID NO: 24, and the amino acid sequence of the light chain of the immunoglobulin is set forth in SEQ ID NO: 26; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 17; the single chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment; and the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; preferably, the amino acid sequences of the first linker fragment and second linker fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19; preferably, the amino acid sequences of the first linker fragment and second linker fragments are set forth in SEQ ID NO: 18.

15. An isolated nucleic acid molecule, encoding the bispecific antibody according to claim 1.

16. A vector, comprising the isolated nucleic acid molecule according to claim 15.

17. A host cell, comprising the isolated nucleic acid molecule according to claim 15.

18. A conjugate, comprising an antibody or an antigen-binding fragment thereof and a conjugated moiety, wherein the immunoglobulin is the bispecific antibody according to claim 1, and the conjugated moiety is a detectable label; preferably, the conjugated moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

19. A kit, comprising the bispecific antibody according to claim 1; wherein preferably, the kit further comprises a second antibody capable of specifically recognizing the immunoglobulin or the antigen binding fragment thereof; optionally, the second antibody further comprises a detectable label, for example, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

20. (canceled)

21. A pharmaceutical composition, comprising the bispecific antibody according to claim 1, wherein, optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable vector and/or excipient.

22. (canceled)

23. A method for treating and/or preventing a malignant tumor, comprising a step of administering to a subject in need thereof an effective amount of the bispecific antibody according to claim 1; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, microsatellite instability-high (MSI-H) and deficient mismatch repair (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, the non-small cell lung cancer is EGFR and/or ALK sensitive mutant non-small cell lung cancer; preferably, the liver cancer is hepatocellular carcinoma; preferably, the renal tumor is renal cell carcinoma; preferably, the breast cancer is triple negative breast cancer; preferably, the urothelial cancer is bladder cancer.

24. (canceled)

25. (canceled)

26. A host cell, comprising the vector according to claim 16.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0259] FIG. 1: Affinity constant assay of VP101(hG1DM) to FcγRI. The antibody concentrations for the curve pairs from top to bottom are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively.

[0260] FIG. 2: Affinity constant assay of bevacizumab to FcγRI. The antibody concentrations for the curve pairs from top to bottom are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively.

[0261] FIG. 3: Affinity constant assay of nivolumab to FcγRI. The antibody concentrations for the curve pairs from top to bottom are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively.

[0262] FIG. 4: Affinity constant assay of VP101(hG1WT) to FcγRI. The antibody concentrations for the curve pairs from top to bottom are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively.

[0263] FIG. 5: Affinity constant assay of VP101(hG4WT) to FcγRI. The antibody concentrations for the curve pairs from top to bottom are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively.

[0264] FIG. 6: Affinity constant assay of VP101(hG1DM) to FcγRIIa_H131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0265] FIG. 7: Affinity constant assay of bevacizumab to FcγRIIa_H131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0266] FIG. 8: Affinity constant assay of nivolumab to FcγRIIa_H131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0267] FIG. 9: Affinity constant assay of VP101(hG1WT) to FcγRIIa_H131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0268] FIG. 10: Affinity constant assay of VP101(hG4WT) to FcγRIIa_H131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0269] FIG. 11: Affinity constant assay of VP101(hG1DM) to FcγRIIa_R131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0270] FIG. 12: Affinity constant assay of bevacizumab to FcγRIIa_R131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0271] FIG. 13: Affinity constant assay of nivolumab to FcγRIIa_R131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0272] FIG. 14: Affinity constant assay of VP101(hG1WT) to FcγRIIa_R131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0273] FIG. 15: Affinity constant assay of VP101(hG4WT) to FcγRIIa_R131. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively.

[0274] FIG. 16: Affinity constant assay of VP101(hG1DM) to FcγRIIIa_V158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0275] FIG. 17: Affinity constant assay of bevacizumab to FcγRIIIa_V158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0276] FIG. 18: Affinity constant assay of nivolumab to FcγRIIIa_V158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0277] FIG. 19: Affinity constant assay of VP101(hG1WT) to FcγRIIIa_V158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0278] FIG. 20: Affinity constant assay of VP101(hG4WT) to FcγRIIIa_V158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0279] FIG. 21: Affinity constant assay of VP101(hG1DM) to FcγRIIIa_F158. The antigen concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0280] FIG. 22: Affinity constant assay of bevacizumab to FcγRIIIa_F158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0281] FIG. 23: Affinity constant assay of nivolumab to FcγRIIIa_F158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0282] FIG. 24: Affinity constant assay of VP101(hG1WT) to FcγRIIIa_F158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0283] FIG. 25: Affinity constant assay of VP101(hG4WT) to FcγRIIa_F158. The antibody concentrations for the curve pairs from top to bottom are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0284] FIG. 26: Affinity constant assay of VP101(hG1DM) to C1q. The antibody concentrations for the curve pairs from top to bottom are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively.

[0285] FIG. 27: Affinity constant assay of bevacizumab to C1q. The antibody concentrations for the curve pairs from top to bottom are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively.

[0286] FIG. 28: Affinity constant assay of nivolumab to C1q. The antibody concentrations for the curve pairs from top to bottom are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively.

[0287] FIG. 29: Affinity constant assay of VP101(hG1WT) to C1q. The antibody concentrations for the curve pairs from top to bottom are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively.

[0288] FIG. 30: Affinity constant assay of VP101(hG4WT) to C1q. The antigen concentrations for the curve pairs from top to bottom are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively.

[0289] FIG. 31: ADCC activity assay of VP101(hG1WT) and VP101(hG1DM) on a CHO-K1-PD1 target cell system expressing PD-1 antigen.

[0290] FIG. 32: CDC activity assay of VP101(hG1WT) and VP101(hG1DM) on a CHO-K1-PD1 target cell system expressing PD-1 antigen.

[0291] FIG. 33: Effects of the antibody VP101(hG1DM) on the secretion of cytokine IFN-γ induced by PBMC and Raji-PDL1 cell mixed culture as detected by ELISA method.

[0292] FIG. 34: Effects of the antibody VP101(hG1DM) on the secretion of cytokine IL-2 induced by PBMC and Raji-PDL1 cell mixed culture as detected by ELISA method.

[0293] FIG. 35: ADCP activity assay of VP101(hG1DM) on a CHO-K1-PD1 target cell system expressing PD-1 antigen.

DETAILED DESCRIPTION

[0294] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be construed as limitations on the scope of the present invention. The cases without the specific descriptions of techniques or conditions were carried out according to the technologies or conditions described in the literature in the art (e.g., see, Guide to Molecular Cloning Experiments, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the product manual. Reagents or instruments used are all commercially available conventional products if the manufacturers thereof are not specified.

[0295] In the following examples of the present invention, the marketed antibody bevacizumab (trade name Avastin®) for the same target was purchased from Roche as a control antibody, or was prepared according to Preparation Example 1.

[0296] In the following examples of the present invention, the marketed antibody nivolumab for the same target (trade name Opdivo®) was purchased from BMS as a control antibody.

[0297] In the following examples of the present invention, the isotype control antibody used is human anti-hen egg lysozyme IgG (anti-HEL, or human IgG, abbreviated as hIgG) whose variable region sequences are derived from the study entitled “Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies”, published by Acierno et al (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). The hIgG1DM and hIgG4WT used in the examples are isotype control antibodies of anti-HEL with hG1DM and hG4WT constant region sequences. The isotype control antibodies were prepared in the laboratory of Akeso Biopharma, Inc.

Preparation Example 1: Preparation of Anti-VEGFA Antibody Bevacizumab

[0298] Chinese Patent Publication CN1259962A is referred to for the amino acid sequences of the heavy chain variable region and the light chain variable region of the marketed anti-VEGFA monoclonal antibody Avastin (bevacizumab). Genscript was entrusted to synthesize nucleotide sequences encoding the heavy chain variable region and the light chain variable region.

[0299] Amino acid sequence of the heavy chain variable region of bevacizumab (bevacizumab-Hv): (123 aa)

TABLE-US-00010 (SEQ ID NO: 1) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAP GKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQ MNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS

[0300] Nucleotide sequence encoding the heavy chain variable region of bevacizumab: (369 bp)

TABLE-US-00011 (SEQ ID NO: 2) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCC AGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACA CCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCA GGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACAC CGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCA CCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAG ATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTG TGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATT TTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC

[0301] Amino acid sequence of the light chain variable region of bevacizumab (bevacizumab-Lv): (107 aa)

TABLE-US-00012 (SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGK APKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGQGTKVEIK

[0302] Nucleotide sequence encoding the light chain variable region of bevacizumab: (321 bp)

TABLE-US-00013 (SEQ ID NO: 4) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTC TGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGG ATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGA AAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCA CTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAA CCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGAT TTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATG GACATTTGGCCAGGGGACTAAAGTCGAGATCAAG

[0303] The heavy chain constant regions were all Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions were all Ig kappa chain C region, ACCESSION: P01834.

[0304] The heavy chain cDNA and the light chain cDNA of bevacizumab were cloned into vector pcDNA3.1, and the recombinant expression plasmid of the antibody bevacizumab was obtained. The recombinant plasmid was transfected into 293F cells. The 293F cell culture medium was purified and then detected.

[0305] The anti-VEGFA monoclonal antibody Avastin (bevacizumab) was thus obtained.

[0306] Preparation Example 2: Sequence Design of Anti-PD-1 Antibody 14C12 and Humanized Antibody 14C12H1L1 thereof and Mutant 14C12H1L1(M)

[0307] The amino acid sequences and encoding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and a humanized antibody 14C12H1L1 thereof are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication No. CN106967172A, respectively.

[0308] Heavy and light chain variable region sequences of 14C12

[0309] Amino acid sequence of the heavy chain variable region of 14C12: (118 aa)

TABLE-US-00014 (SEQ ID NO: 5) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQT PEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLY LQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA

[0310] Nucleotide sequence encoding the heavy chain variable region of 14C12: (354 bp)

TABLE-US-00015 (SEQ ID NO: 6) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGC CCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTT CGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACC CCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAG GCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAG ATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTAT CTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGT ACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTA TTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC

[0311] Amino acid sequence of the light chain variable region of 14C12: (107 aa)

TABLE-US-00016 (SEQ ID NO: 7) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYED MGIYYCLQYDEFPLTFGAGTKLELK

[0312] Nucleotide sequence encoding the light chain variable region of 14C12: (321 bp)

TABLE-US-00017 (SEQ ID NO: 8) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGC CTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGAC ATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAA AGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGAC GGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGAT TACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGC ATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTT GGAGCAGGCACAAAACTGGAACTGAAG

[0313] (2) Heavy and light chain variable region and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1

[0314] Amino acid sequence of the heavy chain variable region of 14C12H1L1: (118 aa)

TABLE-US-00018 (SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPG KGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMN SLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS

[0315] Nucleotide sequence encoding the heavy chain variable region of 14C12H1L1: (354 bp)

TABLE-US-00019 (SEQ ID NO: 10) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCG GCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTT TAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAG GGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACA CCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAG AGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTG AGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACG GGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGAC AGTCTCTAGT

[0316] Amino acid sequence of the light chain variable region of 14C12H1L1: (107 aa)

TABLE-US-00020 (SEQ ID NO: 11) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSW FQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQD YTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLE LK

[0317] Nucleotide sequence encoding the light chain variable region of 14C12H1L1: 321 bp)

TABLE-US-00021 (SEQ ID NO: 12) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTC CGCCTCTGTGGGCGACAGGGTCACCTTCACATGCC GCGCTAGTCAGGATATCAACACCTACCTGAGCTGG TTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACT GATCTACCGGGCTAATAGACTGGTGTCTGGAGTCC CAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGAC TACACTCTGACCATCAGCTCCCTGCAGCCTGAGGA CATGGCAACCTACTATTGCCTGCAGTATGATGAGT TCCCACTGACCTTTGGCGCCGGGACAAAACTGGAG CTGAAG

[0318] Amino acid sequence of the heavy chain (14C12H1) of 14C12H1L1: (448 aa)

TABLE-US-00022 (SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMS WVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTI SRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWF AYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

[0319] Nucleotide sequence encoding the heavy chain (14C12H1) of 14C12H1L1: (1344 bp)

TABLE-US-00023 (SEQ ID NO: 14) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGT GCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAG CTTCCGGATTCGCCTTTAGCTCCTACGACATGTCC TGGGTGCGACAGGCACCAGGAAAGGGACTGGATTG GGTCGCTACTATCTCAGGAGGCGGGAGATACACCT ACTATCCTGACAGCGTCAAGGGCCGGTTCACAATC TCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGT ACTATTGTGCCAACCGCTACGGGGAAGCATGGTTT GCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTC TAGTGCCAGCACCAAAGGACCTAGCGTGTTTCCTC TCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACC GCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCC TGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTC TGACAAGCGGAGTCCATACATTCCCTGCTGTGCTG CAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGT CACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCT ACATCTGTAACGTCAACCACAAGCCCTCCAACACC AAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGA CAAGACACACACCTGTCCCCCCTGTCCTGCTCCCG AACTCCTCGGAGGCCCTAGCGTCTTCCTCTTTCCT CCCAAACCCAAGGACACCCTCATGATCAGCAGAAC CCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCC ATGAGGACCCCGAGGTGAAATTCAACTGGTATGTC GATGGCGTCGAGGTGCACAACGCCAAAACCAAGCC CAGGGAGGAACAGTACAACTCCACCTACAGGGTGG TGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAA GGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCA AGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTAT ACACTGCCTCCCTCCAGGGACGAACTCACCAAGAA CCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTT ATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAAC GGACAGCCCGAGAATAACTACAAGACCACCCCTCC TGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACA GCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAG GGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGC CCTCCACAACCACTACACCCAGAAAAGCCTGTCCC TGAGCCCCGGCAAA

[0320] Amino acid sequence of the light chain (14C12L1) of 14C12H1L1: (214 aa)

TABLE-US-00024 (SEQ ID NO: 15) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSW FQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQD YTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLE LKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

[0321] Nucleotide sequence encoding the light chain (14C12L1) of 14C12H1L1: (642 bp)

TABLE-US-00025 (SEQ ID NO: 16) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTC CGCCTCTGTGGGCGACAGGGTCACCTTCACATGCC GCGCTAGTCAGGATATCAACACCTACCTGAGCTGG TTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACT GATCTACCGGGCTAATAGACTGGTGTCTGGAGTCC CAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGAC TACACTCTGACCATCAGCTCCCTGCAGCCTGAGGA CATGGCAACCTACTATTGCCTGCAGTATGATGAGT TCCCACTGACCTTTGGCGCCGGGACAAAACTGGAG CTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCAT TTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCA CAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTAC CCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAA CGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGA CTGAACAGGACTCAAAAGATAGCACCTATTCCCTG TCTAGTACACTGACTCTGTCCAAGGCTGATTACGA GAAGCACAAAGTGTATGCATGCGAAGTGACACATC AGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAAC CGGGGCGAATGT

[0322] (3) Heavy and light chain variable region sequences of 14C12H1L1(M)

[0323] Individual amino acids in the framework region (light chain) were mutated on the basis of 14C12H1L1 to obtain 14C12H1L1(M).

[0324] Heavy chain variable region 14C12H1(M) of 14C12H1L1(M):

[0325] is identical with the heavy chain variable region 14C12H1 of 14C12H1L1, namely, the amino acid sequence is set forth in SEQ ID NO: 9.

[0326] Light chain variable region 14C12L1(M) of 14C12H1L1(M): (108 aa, mutation positions underlined in the amino acid sequence based on 14C12H1L1)

TABLE-US-00026 (SEQ ID NO: 17) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSW FQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQD YTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLE LKR

Preparation Example 3: Sequence Design of Bispecific Antibodies

[0327] Sequence Design

[0328] The structure of the bispecific antibody of the present invention is in the Morrison form (IgG-scFv), i.e., C-termini of two heavy chains of an IgG antibody are each linked to a scFv fragment of another antibody, and the main composition design of the heavy and light chains is as shown in Table 1 below.

[0329] On the basis of bevacizumab, the VP101 antibody, in which amino acid sequences of the heavy chain variable region and the light chain variable region of 14C12H1L1(M) are taken as a ScFv fragment part, is referred to as VP101(M). Compared to 14C12H1L1, 14C12H1L1(M) effectively optimized the structure of the bispecific antibody and improved the effectiveness.

TABLE-US-00027 TABLE 1 Compositional Design of Heavy and Light Chains of VP101(M) and VP101(G4M) Bispecific Immunoglobulin moiety Linker antibody No. Heavy chain Light chain fragment scFv part VP101(M) Bevacizumab-H Bevacizumab-L Linker1 14C12H1(M).sub.V- Linker1-14C12L1(M).sub.V VP101(G4M) Bevacizumab-G4H Bevacizumab-L Linker1 14C12H1(M).sub.V- Linker1-14C12L1(M).sub.V

[0330] In Table 1 above:

[0331] Those with “V” labeled at lower right corner refer to the variable region of corresponding heavy chain or the variable region of corresponding light chain. For those without “V” label, the corresponding heavy or light chain is the full length comprising the constant region. The corresponding sequences described in the above preparation examples are referred to for the amino acid sequences of these variable regions or the full length and the nucleotide sequences encoding them.

[0332] The amino acid sequence of Linker 1 is GGGGSGGGGSGGGGSG GGGS (SEQ ID NO: 18)

[0333] Optionally, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19) can be used as Linker 2 in place of the aforementioned Linker 1.

[0334] (3) Bevacizumab-H used Ig gamma-1 chain C region (ACCESSION: P01857) as the heavy chain constant region.

[0335] (4) Bevacizumab-G4H used Ig gamma-4 chain C region (ACCESSION: P01861.1) as the heavy chain constant region.

[0336] 2. Expression and Purification of Antibody VP101(M)

[0337] The heavy chain cDNA sequence and the light chain cDNA sequence of VP101 were each cloned into vector pUC57simple (provided by Genscript) to obtain plasmids pUC57simple-VP101H and pUC57simple-VP101L, respectively.

[0338] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were enzyme-digested (HindIII&EcoRI), and heavy and light chains isolated by electrophoresis were subcloned into vector pcDNA3.1, and recombinant plasmids were extracted to co-transfect 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was further eluted in one step with Elution Buffer, and the target sample antibody VP101 was isolated and buffer exchanged into PBS.

[0339] 3. Detection of Antibody VP101(M)

[0340] The purified sample was added to both a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and then boiled for SDS-PAGE electrophoresis detection.

[0341] For differentiation from the mutated antibody of Preparation Example 4, VP101(M) is also referred to as VP101(hG1WT) in the present invention. VP101(M) described above is the “wild-type”, comprising an Ig gamma-1 chain C region (ACCESSION: P01857) as the heavy chain constant region and an Ig kappa chain C region (ACCESSION: P01834) as the light chain constant region.

[0342] Amino acid sequence of the heavy chain of the immunoglobulin moiety in VP101(hG1WT): (453 aa)

TABLE-US-00028 (SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0343] Nucleotide sequence encoding the heavy chain of the immunoglobulin moiety in VP101(hG1WT): (1359 bp)

TABLE-US-00029 (SEQ ID NO: 21) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGT GCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCG CTTCAGGGTACACCTTCACAAACTATGGAATGAAT TGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTG GGTCGGCTGGATCAACACTTACACCGGGGAACCTA CCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTC AGCCTGGATACAAGCAAATCCACTGCCTACCTGCA GATGAACAGCCTGCGAGCTGAGGACACCGCAGTCT ACTATTGTGCTAAATATCCCCACTACTATGGGAGC AGCCATTGGTATTTTGACGTGTGGGGGCAGGGGAC TCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGC CCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGC ACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGT GAAGGACTACTTCCCTGAACCCGTCACCGTGAGCT GGAATAGCGGCGCTCTGACAAGCGGAGTCCATACA TTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTC CCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCC TGGGCACCCAGACCTACATCTGTAACGTCAACCAC AAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGA GCCCAAATCCTGCGACAAGACACACACCTGTCCCC CCTGTCCTGCTCCCGAACTCCTCGGAGGCCCTAGC GTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCT CATGATCAGCAGAACCCCTGAAGTCACCTGTGTCG TCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAA TTCAACTGGTATGTCGATGGCGTCGAGGTGCACAA CGCCAAAACCAAGCCCAGGGAGGAACAGTACAACT CCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTC CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG CAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTG AGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGG GAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGA CGAACTCACCAAGAACCAGGTGTCCCTGACCTGCC TGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTC GAGTGGGAGTCCAACGGACAGCCCGAGAATAACTA CAAGACCACCCCTCCTGTCCTCGACTCCGACGGCT CCTTCTTCCTGTACAGCAAACTGACCGTCGATAAA TCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTC CGTGATGCATGAAGCACTGCACAACCATTATACCC AGAAGTCTCTGAGCCTGTCCCCCGGCAAG

[0344] For differentiation from the mutated antibody of Preparation Example 4, VP101(G4M) is also referred to as VP101(hG4WT) in the present invention. VP101(G4M) described above is the “wild-type”, comprising an Ig gamma-4 chain C region (ACCESSION: P01861.1) as the heavy chain constant region and an Ig kappa chain C region (ACCESSION: P01834) as the light chain constant region.

[0345] Amino acid sequence of the heavy chain of the immunoglobulin moiety in VP101(hG4WT): (450 aa)

TABLE-US-00030 (SEQ ID NO: 22) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0346] Nucleotide sequence encoding the heavy chain of the immunoglobulin moiety in VP101(hG4WT): (1350 bp)

TABLE-US-00031 (SEQ ID NO: 23) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGT GCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCG CTTCAGGGTACACCTTCACAAACTATGGAATGAAT TGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTG GGTCGGCTGGATCAACACTTACACCGGGGAACCTA CCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTC AGCCTGGATACAAGCAAATCCACTGCCTACCTGCA GATGAACAGCCTGCGAGCTGAGGACACCGCAGTCT ACTATTGTGCTAAATATCCCCACTACTATGGGAGC AGCCATTGGTATTTTGACGTGTGGGGGCAGGGGAC TCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGC CCTCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGC ACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCT TGGGCACGAAGACCTACACCTGCAACGTAGATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GTCCAAATATGGTCCCCCATGCCCACCATGCCCAG CACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTG TTCCCCCCAAAACCCAAGGACACTCTCATGATCTC CCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGG TACGTGGATGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACC GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC TGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAG GTGTACACCCTGCCCCCATCCCAGGAGGAGATGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG GCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGG CAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACACAGAAGTCTC TGAGCCTGTCCCTCGGCAAG

[0347] Preparation Example 4: Non-Variable Region Amino Acid Mutation Design Based on Humanized Bispecific Antibody VP101(hG1WT)

[0348] On the basis of VP101(hG1WT) obtained in Preparation Example 3, VP101(hG1DM) was obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A) and a leucine-to-alanine point mutation at position 235 (L235A) in the heavy chain.

[0349] Amino acid sequence of the heavy chain of the immunoglobulin moiety in VP101(hG1DM): (453 aa, mutation positions underlined)

TABLE-US-00032 (SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0350] Nucleotide sequence encoding the heavy chain of the immunoglobulin moiety in VP101(hG1DM): (1359 bp, mutation positions underlined)

TABLE-US-00033 (SEQ ID NO: 25) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGT GCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCG CTTCAGGGTACACCTTCACAAACTATGGAATGAAT TGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTG GGTCGGCTGGATCAACACTTACACCGGGGAACCTA CCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTC AGCCTGGATACAAGCAAATCCACTGCCTACCTGCA GATGAACAGCCTGCGAGCTGAGGACACCGCAGTCT ACTATTGTGCTAAATATCCCCACTACTATGGGAGC AGCCATTGGTATTTTGACGTGTGGGGGCAGGGGAC TCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGC CCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGC ACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGT GAAGGACTACTTCCCTGAACCCGTCACCGTGAGCT GGAATAGCGGCGCTCTGACAAGCGGAGTCCATACA TTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTC CCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCC TGGGCACCCAGACCTACATCTGTAACGTCAACCAC AAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGA GCCCAAATCCTGCGACAAGACACACACCTGTCCCC CCTGTCCTGCTCCCGAAGCTGCTGGAGGCCCTAGC GTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCT CATGATCAGCAGAACCCCTGAAGTCACCTGTGTCG TCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAA TTCAACTGGTATGTCGATGGCGTCGAGGTGCACAA CGCCAAAACCAAGCCCAGGGAGGAACAGTACAACT CCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTC CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG CAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTG AGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGG GAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGA CGAACTCACCAAGAACCAGGTGTCCCTGACCTGCC TGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTC GAGTGGGAGTCCAACGGACAGCCCGAGAATAACTA CAAGACCACCCCTCCTGTCCTCGACTCCGACGGCT CCTTCTTCCTGTACAGCAAACTGACCGTCGATAAA TCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTC CGTGATGCATGAAGCACTGCACAACCATTATACCC AGAAGTCTCTGAGCCTGTCCCCCGGCAAG

[0351] The amino acid sequences and encoding nucleotide sequences of the light chain of the immunoglobulin moiety of VP101(hG1DM), VP101(hG1WT) and VP101(hG4WT) are identical.

[0352] Amino acid sequence of the light chain of the immunoglobulin moiety in VP101(hG1DM): (214 aa)

TABLE-US-00034 (SEQ ID NO: 26) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

[0353] Nucleotide sequence encoding the light chain of the immunoglobulin moiety in VP101(hG1DM): (642 bp)

TABLE-US-00035 (SEQ ID NO: 27) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTC CGCCTCTGTGGGCGACAGGGTCACCATCACATGCA GTGCTTCACAGGATATTTCCAACTACCTGAATTGG TATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCT GATCTACTTCACTAGCTCCCTGCACTCAGGAGTGC CAAGCCGGTTCAGCGGATCCGGATCTGGAACCGAC TTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGA TTTCGCTACATACTATTGCCAGCAGTATTCTACCG TGCCATGGACATTTGGCCAGGGGACTAAAGTCGAG ATCAAGCGGACCGTGGCCGCTCCCAGTGTCTTCAT TTTTCCCCCTAGCGACGAACAGCTGAAATCCGGGA CAGCCTCTGTGGTCTGTCTGCTGAACAACTTCTAC CCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAA CGCCCTGCAGAGTGGCAATTCACAGGAGAGCGTGA CAGAACAGGACTCCAAAGATTCTACTTATAGTCTG TCAAGCACACTGACTCTGAGCAAGGCTGACTACGA AAAGCATAAAGTGTATGCATGTGAGGTCACCCACC AGGGGCTGAGCAGTCCAGTCACCAAGTCATTCAAC AGAGGCGAGTGC

[0354] Example 1: Affinity Constant Assay of FcγRI to VP101(hG1WT) and VP101(hG1DM)

[0355] The Fc receptor FcγRI, also known as CD64, can bind to the Fc fragment of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The binding capacity of a therapeutic monoclonal antibody to Fc receptors will influence the safety and efficacy of the antibody.

[0356] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRI were determined using a Fortebio Octet system in this experiment to evaluate the ADCC activity of each antibody.

[0357] The method for determining the affinity constants of the antibodies to FcγRI using the Fortebio Octet system is briefly described as follows: a sample dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4. 1 μg/mL FcγRla was immobilized on an HIS1K sensor for 50 s. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized CD64 on the sensor to the antibodies at concentrations of 3.12-50 nM (serial two-fold dilution) was determined for 120 s. The antibodies were dissociated in the buffer for 120 s. The sensor was refreshed 4 times in 10 mM glycine pH 1.5, each for 5 s. The detection temperature was 30° C. and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0358] The affinity constants of FcγRI to VP101(hG1WT), VP101(hG4WT) and VP101(hG1DM) as well as control antibodies nivolumab and bevacizumab are shown in Table 1 and FIGS. 1-5.

TABLE-US-00036 TABLE 1 Kinetics for Binding of VP101(hG1WT) and VP101(hG1DM) and Isotypes Thereof to FcγRI Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (1/s) SE (kdis) Rmax (nm) VP101(hG1DM) N/A N/A N/A N/A N/A N/A Bevacizumab 3.68E−09 6.61E+05 2.07E+04 2.44E−03 9.02E−05 0.46-0.49 Nivolumab 6.20E−09 6.98E+05 2.22E+04 4.32E−03 9.88E−05 0.48-0.53 VP101(hG1WT) 3.95E−09 5.67E+05 1.82E+04 2.24E−03 9.02E−05 0.68-0.80 VP101(hG4WT) 8.52E−09 6.28E+05 2.12E+04 5.35E−03 1.07E−04 0.61-0.65 N/A indicates that the antibody had no binding or an extremely weak binding signal to the antigen, and thus the results were not analyzed and no corresponding data was obtained.

[0359] The results show that VP101(hG1WT) can bind to FcγRI with an affinity constant of 3.95E-09M; VP101(hG4WT) can bind to FcγRI with an affinity constant of 8.52E-09M; bevacizumab can bind to FcγRI with an affinity constant of 3.68E-09M; nivolumab can bind to FcγRI with an affinity constant of 6.20E-09M; VP101(hG1DM) had no binding or an extremely weak binding signal to FcγRI, and thus the results were not analyzed and no corresponding data were obtained.

[0360] The results show that the affinity of other antibodies to FcγRI is similar except for no binding of VP101(hG1DM) to FcγRI. The binding activity of VP101(hG1DM) was effectively eliminated.

[0361] Example 2: Affinity Constant Assay of FcγRIIa_H131 to VP101(hG1WT) and VP101(hG1DM)

[0362] The Fc receptor FcγRIIa_H131 (also known as CD32a H131), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0363] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIa_H131 were determined using a Fortebio Octet system in this experiment to evaluate the ADCC activity of each antibody.

[0364] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIa_H131 using a Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, pH 7.4. 5μg/mL FcγRIIa_H131 was immobilized on an NTA sensor for an immobilization time of 60 s. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA (pH 7.4) for 600 s of blocking, and the binding of the immobilized FcγRIIa_H131 on the sensor to the antibodies at concentrations of 12.5-200 nM (serial two-fold dilution) was determined for 60 s. The antibodies were dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine pH 1.7 and 10 nM nickel sulfate. The detection temperature was 30° C. and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0365] The affinity constants of FcγRIIa_H131 to VP101(hG1WT), VP101(hG4WT) and VP101(hG1DM) as well as control antibodies nivolumab and bevacizumab are shown in Table 2 and FIGS. 6-10.

TABLE-US-00037 TABLE 2 Kinetics for Binding of VP101(hG1WT) and VP101(hG1DM) and Isotypes Thereof to FcγRIIa_H131 Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (1/s) SE (kdis) Rmax (nm) VP101(hG1DM) 3.57E−08 1.15E+05 1.01E+04 4.11E−03 3.27E−04 0.41-0.53 Bevacizumab 6.44E−08 2.10E+05 1.78E+04 1.36E−02 5.14E−04 0.49-0.68 Nivolumab N/A N/A N/A N/A N/A N/A VP101(hG1WT) 2.28E−08 2.41E+05 1.48E+04 5.50E−03 3.49E−04 1.40-1.52 VP101(hG4WT) 3.68E−08 2.13E+05 2.43E+04 7.83E−03 6.65E−04 0.41-0.57 N/A indicates that the antibody had no binding or an extremely weak binding signal to the antigen, and thus the results were not analyzed and no corresponding data was obtained.

[0366] The results show that VP101(hG1WT) can bind to FcγRIIa_H131 with an affinity constant of 2.28E-08M; VP101(hG4WT) can bind to FcγRIIa_H131 with an affinity constant of 3.68E-08M; bevacizumab can bind to FcγRIIa_H131 with an affinity constant of 6.44E-08M; VP101(hG1DM) can bind to FcγRIIa_H131 with an affinity constant of 3.57E-08M; while nivolumab had no binding or an extremely weak binding signal to FcγRIIa_H131, and thus the results were not analyzed and no corresponding data were obtained.

[0367] The results show that, except for no binding of nivolumab to FcγRIIa_H131, other antibodies bound to FcγRIIa_H131 with the following strong-to-weak affinities: VP101(hG1WT), VP101(hG1DM), VP101(hG4WT) and bevacizumab.

[0368] Example 3: Affinity Constant Assay of FcγRIIa_R131 to VP101(hG1WT) and VP101(hG1DM)

[0369] The Fc receptor FcγRIIa_R131 (also known as CD32a R131), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0370] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIa_R131 were determined using a Fortebio Octet system in this experiment to evaluate the ADCC activity of each antibody.

[0371] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) using a Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, pH 7.4. 5 μg/mL FcγRIIa_R131 was immobilized on an NTA sensor for an immobilization time of 60 s. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA (pH 7.4) for 600 s of blocking, and the binding of the immobilized FcγRIIa_R131 on the sensor to the antibodies at concentrations of 12.5-200 nM (serial two-fold dilution) was determined for 60 s. The antibodies were dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine pH 1.7 and 10 nM nickel sulfate. The detection temperature was 30° C. and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0372] The affinity constants of FcγRIIa_R131 to VP101(hG1WT), VP101(hG4WT) and VP101(hG1DM) as well as control antibodies nivolumab and bevacizumab are shown in Table 3 and FIGS. 11-15.

TABLE-US-00038 TABLE 3 Kinetics for Binding of VP101(hG1WT) and VP101(hGlDM) and Isotypes Thereof to FcγRIIa_R131 Antibody KD (M) kon (1/Ms) SE (kon) kdis (1/s) SE (kdis) Rmax (nm) VP101(hG1DM) 3.35E−08 1.20E+05 9.72E+03 4.03E−03 3.08E−04 0.57-0.69 Bevacizumab 5.16E−08 2.59E+05 1.72E+04 1.33E−02 4.52E−04 0.42-0.69 Nivolumab 6.93E−08 4.78E+05 1.09E+05 3.31E−02 2.54E−03 0.08-0.16 VP101(hG1WT) 2.42E−08 2.14E+05 1.30E+04 5.17E−03 3.28E−04 1.75-1.92 VP101(hG4WT) 3.57E−08 1.99E+05 1.23E+04 7.09E−03 3.40E−04 0.81-1.05 N/A indicates that the antibody had no binding or an extremely weak binding signal to the antigen, and thus the results were not analyzed and no corresponding data was obtained.

[0373] The results show that VP101(hG1WT) can bind to FcγRIIa_R131 with an affinity constant of 2.42E-08M; VP101(hG4WT) can bind to FcγRIIa_R131 with an affinity constant of 3.57E-08M; bevacizumab can bind to FcγRIIa_R131 with an affinity constant of 5.16E-08M; nivolumab can bind to FcγRIIa R131 with an affinity constant of 6.93E-08M; VP101(hG1DM) can bind to FcγRIIa R131 with an affinity constant of 3.35E-08M.

[0374] The results show that the antibodies bound to FcγRIIa_R131 with the following strong-to-weak affinities: VP101(hG1WT), VP101(hG1DM), VP101(hG4WT), bevacizumab and nivolumab.

[0375] Example 4: Affinity Constant Assay of FcγRIIb to VP101(hG1WT) and VP101(hG1DM)

[0376] The Fc receptor FcγRIIb (also known as CD32b), can bind to the Fc fragment of IgG antibodies and regulate functions of immune cells.

[0377] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIb were determined using a Fortebio Octet system in this experiment to evaluate the binding ability of VP101(hG1WT) and VP101(hG1DM) to the Fc receptor.

[0378] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIb using a Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, pH 7.4. 5 μg/mL hFCGR2B-his was immobilized on an NTA sensor for an immobilization time of 60 s. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA (pH 7.4) for 600 s of blocking, and the binding of the immobilized hFCGR2B-his on the sensor to the antibodies at concentrations of 12.5-200 nM (serial two-fold dilution) was determined for 60 s. The antibodies were dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine pH 1.7 and 10 nM nickel sulfate. The detection temperature was 30° C. and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0379] Example 5: Affinity Constant Assay of FcγRIII_V158 to VP101(hG1WT) and VP101(hG1DM)

[0380] The Fc receptor FcγRIIIa_V158 (also known as CD16a V158), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0381] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIIa_V158 were determined using a Fortebio Octet system in this experiment to evaluate the ADCC activity of each antibody.

[0382] The method for determining the affinity constant of the antibodies to FcγRIIIa_V158 by the Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4. 5 μg/mL FcγRIIIa_V158 was immobilized on an HIS1K sensor for 120 s. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized hFcGR3A(V158)-his on the sensor to the antibodies at concentrations of 31.25-500 nM (serial two-fold dilution) was determined for 60 s. The antibodies were dissociated in the buffer for 60 s. The sensor was refreshed 4 times in 10 mM glycine pH 1.5, each for 5 s. The detection temperature was 30° C. and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0383] The affinity constants of FcγRIIIa_V158 to VP101(hG1WT), VP101(hG4WT) and VP101(hG1DM) as well as control antibodies nivolumab and bevacizumab are shown in Table 4 and FIGS. 16-20.

TABLE-US-00039 TABLE 4 Kinetics for Binding of VP101(hG1WT) and VP101(hG1DM) and Isotypes Thereof to FcγRIIIa_V158 Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (1/s) SE (kdis) Rmax (nm) VP101(hG1DM) 1.34E−07 6.05E+05 2.36E+05 8.11E−02 7.42E−03 0.07-0.21 Bevacizumab 2.76E−08 5.06E+05 1.14E+05 1.39E−02 1.41E−03 0.13-0.51 Nivolumab N/A N/A N/A N/A N/A N/A VP101(hG1WT) 4.35E−08 2.39E+05 3.14E+04 1.04E−02 8.73E−04 0.80-1.22 VP101(hG4WT) N/A N/A N/A N/A N/A N/A N/A indicates that the antibody had no binding or an extremely weak binding signal to the antigen, and thus the results were not analyzed and no corresponding data was obtained.

[0384] The results show that VP101(hG1WT) can bind to FcγRIIIa_V158 with an affinity constant of 4.35E-08M; VP101(hG1DM) can bind to FcγRIIIa_V158 with an affinity constant of 1.34E-07M; bevacizumab can bind to FcγRIIIa_V158 with an affinity constant of 2.76E-08M; while nivolumab and VP101(hG4WT) had no binding or an extremely low binding signal to FcγRIIIa_V158, and thus the results were not analyzed and no corresponding data were obtained.

[0385] The results show that, except for no binding of nivolumab and VP101(hG4WT) to FcγRIIIa_V158, other antibodies bound to FcγRIIIa_V158 with the following strong-to-weak affinities: bevacizumab, VP101(hG1WT) and VP101(hG1DM).

[0386] Example 6: Affinity Constant Assay of FcγRIIIa_F158 to VP101(hG1WT) and VP101(hG1DM)

[0387] The Fc receptor FcγRIIIa_F158 (also known as CD16a F158), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0388] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIIa_F158 were determined using a Fortebio Octet system in this experiment to evaluate the ADCC activity of each antibody.

[0389] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) to FcγRIIIa_F158 using a Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4. 5 μg/mL FcγRIIIa_F158 was immobilized on an HIS1K sensor for 120 s. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized hFcGR3A(F158)-his on the sensor to the antibodies at concentrations of 31.25-500 nM (serial two-fold dilution) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was refreshed 4 times in 10 mM glycine pH 1.5, each for 5 s. The detection temperature was 30° C. and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0390] The affinity constants of FcγRIIIa_F158 to VP101(hG1WT), VP101(hG4WT) and VP101(hG1DM) as well as control antibodies nivolumab and bevacizumab are shown in Table 5 and FIGS. 21-25.

TABLE-US-00040 TABLE 5 Kinetics for Binding of VP101(hG1WT) and VP101(hG1DM) and Isotypes Thereof to FcγRIIIa_F158 Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (1/s) SE (kdis) Rmax (nm) VP101(hG1DM) N/A N/A N/A N/A N/A N/A Bevacizumab 9.32E−08 2.64E+05 7.16E+04 2.46E−02 2.09E−03 0.08-0.20 Nivolumab N/A N/A N/A N/A N/A N/A VP101(hG1WT) 7.41E−08 2.47E+05 5.20E+04 1.83E−02 1.55E−03 0.15-0.48 VP101(hG4WT) N/A N/A N/A N/A N/A N/A N/A indicates that the antibody had no binding or an extremely weak binding signal to the antigen, and thus the results were not analyzed and no corresponding data was obtained.

[0391] The results show that VP101(hG1WT) can bind to FcγRIIIa_F158 with an affinity constant of 7.41E-08M; bevacizumab can bind to FcγRIIIa_F158 with an affinity constant of 9.32E-08M; while nivolumab, VP101(hG4WT) and VP101(hG1DM) had no binding or an extremely low binding signal to FcγRIIIa_F158, and thus the results were not analyzed and no corresponding data were obtained.

[0392] The results show that, except for no binding of nivolumab, VP101(hG4WT) and VP101(hG1DM) to FcγRIIIa_F158, other antibodies bound to FcγRIIIa_F158 with the following strong-to-weak affinities: VP101(hG1WT) and bevacizumab.

[0393] Example 7: Affinity Constant Assay of C1q to VP101(hG1WT) and VP101(hG1DM)

[0394] Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate CDC effects. The binding capacity of a therapeutic monoclonal antibody to C1q will influence the safety and efficacy of the antibody.

[0395] The affinity constants of VP101(hG1WT) and VP101(hG1DM) to C1q were determined using a Fortebio Octet system in this experiment to evaluate the CDC activity of each antibody.

[0396] The method for determining the affinity constant of the antibodies to C1q using a Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4. 50 μg/mL of antibodies were immobilized on an FAB2G sensor at an immobilization height of about 2.0 nm. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized antibodies on the sensor to C1q at concentrations of 0.625-10 nM (serial two-fold dilution) was determined for 60 s. The antigen and the antibodies were dissociated in the buffer for 60 s. The sensor was refreshed 4 times in 10 mM glycine pH 1.7, each for 5 s. The shaking speed of the sample plate was 1000 rpm, the temperature was 30° C. and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0397] The affinity constants of C1q to VP101(hG1WT), VP101(hG4WT) and VP101(hG1DM) as well as control antibodies nivolumab and bevacizumab are shown in Table 6 and FIGS. 26-30.

TABLE-US-00041 TABLE 6 Kinetics for Binding of VP101(hG1WT) and VP101(hG1DM) and Isotypes Thereof to C1q Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (1/s) SE (kdis) Rmax (nm) VP101(hG1DM) N/A N/A N/A N/A N/A N/A Bevacizumab 1.14E−09 6.52E+06 5.64E+05 7.40E−03 5.58E−04 0.51-0.63 Nivolumab N/A N/A N/A N/A N/A N/A VP101(hG1WT) 9.76E−10 5.73E+06 5.49E+05 5.59E−03 6.12E−04 0.32-0.51 VP101(hG4WT) N/A N/A N/A N/A N/A N/A N/A indicates that the antibody had no binding or an extremely weak binding signal to the antigen, and thus the results were not analyzed and no corresponding data was obtained.

[0398] The results show that VP101(hG1WT) can bind to C1q with an affinity constant of 9.76E-10M; bevacizumab can bind to C1q with an affinity constant of 1.14E-09M; while nivolumab, VP101(hG4WT) and VP101(hG1DM) had no binding or an extremely low binding signal to C1q, and thus the results were not analyzed and no corresponding data were obtained.

[0399] The results show that, except for no binding of nivolumab, VP101(hG4WT) and VP101(hG1DM) to FcγRIIIa_F158, other antibodies bound to C1q, and VP101(hG1WT) had a similar affinity to bevacizumab.

[0400] Example 8: ADCC Activity Assay of VP101(hG1WT) and VP101(hG1DM) on CHO-K1-PD1 Cells Expressing PD-1 Antigen

[0401] In order to detect the ADCC effect of the antibodies VP101(hG1WT) and VP101(hG1DM) at the cellular level, the inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen, and established a system of co-culture of normal human PBMC and target cells for detecting the ADCC activity of the antibodies at the cytological level.

[0402] The method for detecting the ADCC activity of VP101(hG1WT) and VP101(hG1DM) on CHO-K1-PD1 cells expressing PD-1 antigen is specified as follows:

[0403] In this experiment, a human PD-1 overexpression vector pCDH-CMV-PD1FL-Puro (pCDH-CMV-Puro purchased from Youbio) was firstly constructed, the expression vector was packaged by virus and then infected CHO-K1 cells, and a CHO-K1-PD1 stable cell line of the drug-resistant stable expression membrane PD-1 protein was obtained after dosing and screening with Puromycin (2 μg/mL). Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation instruction. The isolated PBMCs were resuspended in a 1640 complete medium and stained with trypan blue, cells were counted and the viability of cells was determined. The cells were incubated overnight in a saturated humidity incubator at 37° C. and 5% CO.sub.2. The next day, CHO-K1-PD1 cells and PBMC were collected and centrifuged to remove the supernatant, and then the cell pellets were resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as assay medium), centrifuged and washed 2 times; the cells were counted and the viability of cells was determined, the concentration of the cells was adjusted to a proper range by using the assay medium, and the CHO-K1-PD1 cell suspension (30,000/well) was added into a 96-well plate according to the experimental design; 50 μL of antibody was added, mixed well, and pre-incubated for 1 h at room temperature; after pre-incubation, the cells were added with PBMC (900,000/50 μL/well), mixed well, and incubated for 4 h in a incubator at 37° C. and 5% CO.sub.2. After 4 h, the 96-well plate was removed and centrifuged for 5 min at 250×g; 100 μL of the cell supernatant was carefully transferred to a new 96-well flat-bottom microplate (do not pipette the cell precipitate). 100 μL of freshly prepared reaction solution was added to each well according to the Cytotoxicity Detection Kit instruction. The cells were incubated for 30 min at room temperature in the dark. OD values at 490 nm and 650 nm were measured, wherein the OD values of each group=OD.sub.490nm−OD.sub.650nm. ADCC activity was calculated for each group according to the formula ADCC(%)=(treatment group−negative control group)/(maximum LDH release in target cell−spontaneous LDH release in target cell)× 100%.

[0404] The detection results of the ADCC activity of VP101(hG1WT) and VP101(hG1DM) on CHO-K1-PD1 cells expressing the PD-1 antigen are expressed as ADCC%, and the results are shown in FIG. 31.

[0405] The results show that the positive control 14C12H1L1(G1WT) has significant ADCC activity in the mixed culture system of PBMC and CHO-K1-PD1, indicating that the ADCC system is normal. Compared to the isotype control antibody hIgG1DM, VP101(hG1WT) showed significant ADCC activity and exhibited dose-dependence, whereas VP101(G1DM) did not see ADCC activity. The results show that VP101(hG1DM) produced by mutation based on VP101(hG1WT) has no ADCC activity at the cytological level, and the ADCC effect is eliminated.

[0406] Example 9: CDC Activity Assay of VP101(hG1WT) and VP101(hG1DM) on CHO-K1-PD1 Cells Expressing PD-1 Antigen

[0407] In order to detect the CDC effect of the antibodies VP101(hG1WT) and VP101(hG1DM) at the cytological level, the inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen (see Example 8 for the construction method), and established a system of co-culture of target cells and normal human complement serum for detecting the CDC activity of the antibodies at the cytological level.

[0408] The method for detecting the CDC activity of VP101(hG1WT) and VP101(hG1DM) on CHO-K1-PD1 cells expressing PD-1 antigen is specified as follows:

[0409] On the day of detection, CHO-K1-PD1 cells were collected by trypsinization and centrifuged at 170×g for 5 min; the cell pellets were resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as assay medium), repeatedly centrifuged and washed 2 times; the cells were counted and the viability of cells was determined, the concentration of the cells was adjusted to a proper range by using the assay medium, and the CHO-K1-PD1 cell suspension (30,000/well) was added into a 96-well plate according to the experimental design; 50 μL of antibody was added, mixed well, and pre-incubated for 10 min at room temperature; after pre-incubation, the cells were added with normal human complement serum (final concentration: 2%) at 50 μL/well, mixed well, and incubated for 4 h in an incubator at 37° C. and 5% CO.sub.2. After 4 h, the cells were centrifuged for 5 min at 250×g; 100 μL of the cell supernatant was carefully transferred to a new 96-well flat-bottom plate (do not pipette the cell precipitate). 100 μL of freshly prepared reaction solution was added to each well according to the Cytotoxicity Detection Kit instruction. The cells were incubated for 30 min at room temperature in the dark. OD values at 490 nm and 650 nm were measured, wherein the OD values of each group=OD.sub.490nm−OD.sub.650nm. CDC activity was calculated for each group according to the formula CDC(%)=(treatment group−negative control group)/(maximum LDH release in target cell−spontaneous LDH release in target cell)×100%.

[0410] The detection results of the CDC activity of VP101(hG1WT) and VP101(hG1DM) on CHO-K1-PD1 cells expressing the PD-1 antigen are expressed as CDC%, and the results are shown in FIG. 32.

[0411] The results show that CDC% of the positive control antibody 14C12H1L1(G1WT) had significant difference from that of the isotype control antibody hIgG1DM group in the mixed culture system of normal human complement serum and CHO-K1-PD1, indicating that the CDC detection system is normal. At equivalent dose levels, there was no significant difference in CDC% for both VP101(hG1WT) and VP101(hG1DM) compared to the isotype control.

[0412] Example 10: Pharmacodynamic Activities of VP101(hG1DM) in Mixed Culture System (MLR) of Peripheral Blood Mononuclear Cells and Raji-PDL1 Cells

[0413] In this experiment, a human PD-L1 overexpression vector plenti6.3-PD-L1-BSD (plenti6.3-BSD purchased from invitrogen) was firstly constructed, the expression vector was packaged by virus and then infected Raji cells, and a Raji-PDL1 stable cell line for stably expressing membrane PD-L1 protein was obtained after dosing and screening with BSD (10 μg/mL). Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation instruction, and the isolated PBMCs were resuspended in a 1640 complete medium, counted and frozen. The PBMCs were recovered, added with SEB (Staphylococcus aureus enterotoxin B) and stimulated to culture for two days. Two days later, Raji-PDL1 cells in the logarithmic phase were collected, added with mitomycin C (Sigma, working concentration was 25 μg/mL) and incubated in an incubator for 60 min, and the mitomycin C-treated Raji-PDL1 cells were centrifuged and washed; PBMCs stimulated with SEB for two days were collected and washed; these cells were mixed and cultured according to a proportion of 1:1 of the cell number under the condition of the presence or absence of antibodies. 3 days later, cell supernatant was collected by centrifugation, and IL-2 and IFN-γ concentrations in the supernatant were detected by ELISA.

[0414] The results of secretion of IFN-γ are shown in FIG. 33. The results show that VP101(hG1DM) can effectively promote the secretion of IFN-γ, and its activity is significantly superior to that of nivolumab.

[0415] The results of secretion of IL-2 are shown in FIG. 34. The results show that VP101(hG1DM) can effectively promote the secretion of IL-2 in a dose-dependent relationship, and its activity is significantly superior to that of nivolumab.

[0416] Example 11: No Antibody-Dependent Phagocytic Activity of VP101(hG1DM)on PD-1-Positive Cells

[0417] Antibody dependent cellular phagoxytosis (ADCP) means that the Fc fragment of an antibody bound to a cell surface antigen binds to the Fc receptor of a phagocytically active cell (such as a macrophage), and thus mediates phagocytosis of the antibody-bound cells. For immune checkpoint inhibitor antibodies, such as the PD-1 antibody, the presence of ADCP activity will cause damage to immune cells expressing PD-1 that exert an anti-tumor killing effect, thereby affecting their anti-tumor activity.

[0418] In this experiment, murine macrophages were taken as effector cells, PD-1 overexpression CHO-K1-PD1 cell lines (see Example 8 for the construction method) were taken as target cells. The ADCP effect mediated by the cells was detected. In this experiment, flow cytometry was adopted to detect the ADCP activity of VP101(hG1DM) on cells expressing PD-1, and the results show that VP101(hG1DM) had no ADCP activity, whereas the marketed antibody nivolumab for the same target had significant ADCP activity. The method is specifically as follows:

[0419] The femoral bone marrow of C57 mice (purchased from Guangdong Medical Laboratory Animal Center) was first aseptically collected and lysed by erythrocyte lysis buffer on ice for 5 min. The lysis was terminated with DMEM complete medium (containing 10% FBS), and the lysate was centrifuged at 1000 rpm and washed twice. The cell pellet was resuspended in 10 mL of DMEM complete medium and M-CSF were added at a working concentration of 100 ng/mL. The cells were cultured for 7 days at 37° C. and 5% CO2 in a cell culture chamber for induction. Half of the medium was exchanged and M-CSF was added on days 3 and 5. The induction of cells was completed on day 7. The cells were digested with 0.25% trypsin. Macrophages were collected, and centrifuged at 750×g for 5 min. The supernatant was removed and the cells were suspended in DMEM complete medium (containing 10% FBS) and counted. The cells were adjusted to a proper density and filled into a 96-well conical bottom plate for later use.

[0420] CHO-K1-PD1 cells were collected by conventional methods, centrifuged at 170×g for 5 min, resuspended and counted, and the viability was determined. The cells were washed once with PBS. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM with PBS, and the cells were resuspended with an appropriate amount of diluted CFSE (staining density: 10,000,000 cells/mL) and incubated in an incubator for 20 min. 6 mL of DMEM complete medium (containing 10% FBS) was added to stop staining. The cells were centrifuged at 170×g for 5 min, and the supernatant was removed; 1 mL of DMEM complete medium was added. The cells were incubated in an incubator for 10 min. The antibodies were diluted with DMEM complete medium to 20 μg/mL, 2 μg/mL and 0.2 μg/mL (working concentrations were 10 μg/mL, 1 μg/mL and 0.1 μg/mL), and isotype control antibodies hIgG1DM and hIgG4 were designed. Fresh induced mature macrophages were collected and centrifuged at 750×g for 5 min, and the supernatant was removed; the cells were counted and transferred to a 96-well conical bottom plate, and centrifuged at 1000×g for 5 min, and the supernatant was removed; the cell density of CHO-K1-PD 1-CFSE was adjusted; the diluted antibodies and the target cells were mixed according to the experimental design according to a proportion of 50 μL: 50 μL into the 96-well conical bottom plate containing the macrophages. The cells were resuspended and mixed well, and incubated in an incubator at 37° C. for 2 h. 150 μL of normal-temperature 1% PBSA was added into each well and centrifuged at 1000×g for 5 min, and the supernatant was removed; the cells were washed once with 200 μL of PBSA; APC anti-mouse/human CD11b antibody (500-fold diluted with PBSA) was added to the corresponding samples at 100 μL/sample, mixed well and incubated on ice for 40 min. 150 μL of 1% PBSA was added into each well and centrifuged at 1000×g for 5 min, and the supernatant was removed; each well was washed once with 200 μL of PBSA. 200 μL of 1% PBSA was added into each well for resuspension followed by analysis using a Beckman flow cytometer.

[0421] Macrophages in the system were APC.sup.+ positive, and macrophages involved in phagocytosis were APC and CFSE double positive. The phagocytosis rate was determined as the ratio of the number of double positive cells to the number of APC positive cells, and the antibody-dependent ADCP activity was evaluated. The ADCP activity of each group, represented by P%, was calculated according to the following formulas:

[00001]$P\%=\frac{\mathrm{Number}\mathrm{of}\mathrm{macrophages}\mathrm{involved}\mathrm{in}\mathrm{phagocytosis}}{\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{macrophages}}\times 100\%$

[0422] The results are shown in FIG. 35.

[0423] The results show that nivolumab had a significant ADCP effect in the macrophage +CHO-K1-PD1 system; the phagocytosis rate of VP101(hG1DM) was comparable to that of the isotype control antibody, indicating that VP101(hG1DM) had no ADCP effect. The results indicate that VP101(hG1DM) is likely to have a better anti-tumor effect.

[0424] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions can be made to those details according to all the teachings that have been disclosed, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalent thereof