ANTI-PD-1 ANTIBODY AND MEDICAL USE THEREOF

Abstract

The present invention relates to the field of tumor treatment and molecular immunology, and particularly, to an anti-PD-1 antibody and pharmaceutical use thereof. Specifically, the present invention relates to a monoclonal antibody, wherein a heavy chain variable region of the monoclonal antibody comprises CDRs with amino acid sequences of SEQ ID NOs: 19-21, and/or a light chain variable region of the monoclonal antibody comprises CDRs with amino acid sequences of SEQ ID NOs: 22-24; wherein according to the EU numbering system, a heavy chain constant region of the antibody comprises mutations at any 2 or 3 of positions 234, 235 and 237, and an affinity constant of the antibody to FcγRIIIa and/or C1q is reduced after the mutation as compared to that before the mutation. The monoclonal antibody of the present invention can be well and specifically bind to PD-1, specifically relieve immunosuppression of PD-1 in an organism and activate T lymphocytes.

Claims

1. An antibody, wherein a heavy chain variable region of the antibody comprises HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 19-21, respectively, and a light chain variable region of the antibody comprises LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 22-24, respectively; the antibody is of human IgG1 subtype; wherein, according to the EU numbering system, a heavy chain constant region of the antibody comprises mutations at any 2 or 3 of positions 234,235 and 237, and an affinity constant of the 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 antibody according to claim 1, wherein according to the EU numbering system, the heavy chain constant region of the antibody comprises the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

3. An antibody, wherein a heavy chain variable region of the antibody comprises HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 19-21, respectively, and a light chain variable region of the antibody comprises LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 22-24, respectively; the antibody is of human IgG1 subtype; wherein according to the EU numbering system, a heavy chain constant region of the antibody comprises the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

4. The antibody according to any of claims 1-3, wherein, according to the EU numbering system, the heavy chain constant region of the antibody further comprises one or more mutations selected from: N297A, D265A, D270A, P238D, L328E, E233D, 11268D, 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 antibody according to any of claims 1-4, wherein the heavy chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the light chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 8.

6. The antibody according to any of claims 1-4, wherein the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 4; the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 8; the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 4; or the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 8.

7. The antibody according to any of claims 1-6, wherein the heavy chain is set forth in SEQ ID NO: 16, and the light chain is set forth in SEQ ID NO: 12; or the heavy chain is set forth in SEQ ID NO: 18, and the light chain is set forth in SEQ ID NO: 12.

8. The antibody according to any of claims 1-7, wherein the antibody binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and/or FcγRIIb with an affinity constant greater than about 10.sup.−7 M, for example, greater than about 10.sup.−6 M, 10.sup.−5 M, 10.sup.−4 M, or 10.sup.−3 M or greater; preferably, the affinity constant is measured by a Fortebio Octet system; preferably, the antibody has no binding signal or a binding signal of less than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIay158 and/or FcγRIIb; preferably, the binding signal refers to a response measured by a Fortebio Octet system.

9. The antibody according to any of claims 1-8, wherein the antibody binds to C1q with an affinity constant greater than about 10.sup.−9 M, for example, greater than about 10.sup.−8 M, 10.sup.−7 M, 10.sup.−6 M, or 10.sup.−5 M or greater; preferably, the affinity constant is measured by a Fortebio Octet system; preferably, the antibody has no binding signal or a binding signal of less than 0.1 nm to C1q; preferably, the binding signal refers to a response measured by a Fortebio Octet system.

10. An isolated nucleic acid molecule, encoding the antibody according to any of claims 1-9.

11. A vector, comprising the isolated nucleic acid molecule according to claim 10.

12. A host cell, comprising the isolated nucleic acid molecule according to claim 10 or the vector according to claim 11.

13. A conjugate, comprising the antibody according to any of claims 1-9 and a conjugated moiety, wherein 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.

14. A kit, comprising the antibody according to any of claims 1-9 or the conjugate according to claim 13; wherein preferably, the kit further comprises a second antibody specifically recognizing the antibody; 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.

15. Use of the antibody according to any of claims 1-9 or the conjugate according to claim 13 in preparing a kit for detecting the presence or level of PD-1 in a sample.

16. A pharmaceutical composition, comprising the antibody according to any of claims 1-9 or the conjugate according to claim 13, wherein, optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

17. The pharmaceutical composition according to claim 16, further comprising one or more anti-tumor chemotherapeutics; preferably, the anti-tumor chemotherapeutic is a tyrosine kinase inhibitor; more preferably, the anti-tumor chemotherapeutic is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride salt), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., mesylate salt).

18. The pharmaceutical composition according to claim 16 or 17, wherein the unit dose of the pharmaceutical composition is 100-1000 mg, 200-800 mg, 200-500 mg, 300-600 mg, 400-500 mg, or 450 mg, based on the mass of the antibody.

19. A therapeutic combination, comprising the antibody according to any of claims 1-9, and at least one (e.g., 1, 2 or 3) anti-tumor chemotherapeutic.

20. The therapeutic combination according to claim 19, wherein the anti-tumor chemotherapeutic is a tyrosine kinase inhibitor; preferably, the anti-tumor chemotherapeutic is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride salt), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., mesylate salt).

21. The therapeutic combination according to claim 19 or 20, wherein the unit dose of the antibody is 100-1000 mg, 200-800 mg, 200-500 mg, 300-600 mg, 400-500 mg, or 450 mg.

22. The therapeutic combination according to claim 19 or 20, wherein the unit dose of the anti-tumor chemotherapeutic is 0.1-100 mg, 0.5-50 mg, 1-20 mg, 2-15 mg, 4-12 mg, or 8-12 mg.

23. The therapeutic combination according to any of claims 19-22, wherein the therapeutic combination is a fixed combination, e.g., in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or the therapeutic combination is a non-fixed combination, e.g., the anti-PD-1 antibody and the anti-tumor chemotherapeutic in the non-fixed combination are each in the form of a pharmaceutical composition.

24. A kit product, comprising: the pharmaceutical composition according to any of claims 16-18 or the therapeutic combination according to any of claims 19-23, and a package insert.

25. Use of the antibody according to any of claims 1-9, the conjugate according to claim 13, the pharmaceutical composition according to any of claims 16-18 or the therapeutic combination according to any of claims 19-23 in preparing a medicament for treating and/or preventing a tumor or anemia, or in preparing a medicament for diagnosing a tumor or anemia, wherein preferably the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer and endometrial cancer; preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer; preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; preferably, the tumor is a solid tumor of MSI-H/dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors of MSI-H/dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm.

26. Use of the antibody according to any of claims 1-9, the conjugate according to claim 13, the pharmaceutical composition according to any of claims 16-18 or the therapeutic combination according to any of claims 19-23 in preparing: a medicament for blocking the binding of PD-1 to PD-L1, a medicament for down-regulating the activity or level of PD-1, a medicament for relieving the immunosuppression of PD-1 in an organism, or a medicament for elevating IFN-γ and/or IL-2 expression in T lymphocytes.

27. The antibody according to any of claims 1-9, the conjugate according to claim 13, the pharmaceutical composition according to any of claims 16-18 or the therapeutic combination according to any of claims 19-23 for use in treating and/or preventing a tumor or anemia, or for use in diagnosing a tumor or anemia, wherein preferably the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer and endometrial cancer; preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer; preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; preferably, the tumor is a solid tumor of MSI-H/dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors of MSI-H/dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm.

28. A method for treating and/or preventing a tumor or anemia, or a method of diagnosing a tumor or anemia, comprising: administering to a subject in need an effective amount of the antibody according to any of claims 1-9, the conjugate according to claim 13, the pharmaceutical composition according to any of claims 16-18 or the therapeutic combination according to any of claims 19-23, wherein preferably the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer and endometrial cancer; preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer; preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; preferably, the tumor is a solid tumor of MSI-H/dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors of MSI-H/dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm.

29. The method according to claim 28, wherein the effective amount of the antibody is administered to the subject in need before or after surgical treatment and/or before or after radiotherapy.

30. The method according to claim 28 or 29, wherein the unit dose of the antibody is 0.1-100 mg per kg body weight, preferably 1-10 mg per kg body weight; alternatively, the unit dose of the antibody is 10-1000 mg, preferably 50-500 mg in each subject; preferably, the dose is given once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks; preferably, the route of administration is intravenous drip infusion or intravenous injection.

31. The method according to any of claims 28-30, wherein the administration of the antibody is performed in cycles of 2 or 3 weeks, and preferably, the antibody is administered intravenously on the first day of each cycle; preferably, the antibody is administered once every two or three weeks.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0149] FIG. 1: Affinity constant assay of 14C12H1L1(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.

[0150] FIG. 2: Affinity constant assay of 14C12H1L1(hG4) 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.

[0151] FIG. 3: Affinity constant assay of 14C12H1L1(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.

[0152] FIG. 4: Affinity constant assay of 14C12H1L1(hG1TM) 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.

[0153] FIG. 5: Affinity constant assay of 5C10H2L2-IgG1mt 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.

[0154] FIG. 6: Affinity constant assay of 14C12H1L1(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.

[0155] FIG. 7: Affinity constant assay of 14C12H1L1(hG4) 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.

[0156] FIG. 8: Affinity constant assay of 14C12H1L1(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.

[0157] FIG. 9: Affinity constant assay of 14C12H1L1(hG1TM) 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.

[0158] FIG. 10: Affinity constant assay of 5C10H2L2-IgG1mt 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.

[0159] FIG. 11: Affinity constant assay of 14C12H1L1(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.

[0160] FIG. 12: Affinity constant assay of 14C12H1L1(hG4) 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.

[0161] FIG. 13: Affinity constant assay of 14C12H1L1(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.

[0162] FIG. 14: Affinity constant assay of 14C12H1L1(hG1TM) 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.

[0163] FIG. 15: Affinity constant assay of 5C10H2L2-IgG1mt 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.

[0164] FIG. 16: Affinity constant assay of 14C12H1L1(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.

[0165] FIG. 17: Affinity constant assay of 14C12H1L1(hG4) 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.

[0166] FIG. 18: Affinity constant assay of 14C12H1L1(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.

[0167] FIG. 19: Affinity constant assay of 14C12H1L1(hG1TM) 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.

[0168] FIG. 20: Affinity constant assay of 5C10H2L2-IgG1mt 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.

[0169] FIG. 21: Affinity constant assay of 14C12H1L1(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.

[0170] FIG. 22: Affinity constant assay of 14C12H1L1(hG4) 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.

[0171] FIG. 23: Affinity constant assay of 14C12H1L1(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.

[0172] FIG. 24: Affinity constant assay of 14C12H1L1(hG1TM) 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.

[0173] FIG. 25: Affinity constant assay of 5C10H2L2-IgG1mt 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.

[0174] FIG. 26: Affinity constant assay of 14C12H1L1(hG1DM) to FcγRIIb. 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.

[0175] FIG. 27: Affinity constant assay of 14C12H1L1(hG4) to FcγRIIb. 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.

[0176] FIG. 28: Affinity constant assay of 14C12H1L1(hG1WT) to FcγRIIb. 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.

[0177] FIG. 29: Affinity constant assay of 14C12H1L1(hG1TM) to FcγRIIb. 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.

[0178] FIG. 30: Affinity constant assay of 5C10H2L2-IgG1mt to FcγRIIb. 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.

[0179] FIG. 31: Affinity constant assay of 14C12H1L1(hG1DM) to C1q. The antibody concentrations for the curve pairs from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM and 1.25 nM, respectively.

[0180] FIG. 32: Affinity constant assay of 14C12H1L1(hG4) to C1q. The antibody concentrations for the curve pairs from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM and 1.25 nM, respectively.

[0181] FIG. 33: Affinity constant assay of 14C12H1L1(hG1WT) to C1q. The antibody concentrations for the curve pairs from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM and 1.25 nM, respectively.

[0182] FIG. 34: Affinity constant assay of 14C12H1L1(hG1TM) to C1q. The antibody concentrations for the curve pairs from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM and 1.25 nM, respectively.

[0183] FIG. 35: Affinity constant assay of 5C10H2L2-IgG1mt to C1q. The antigen concentrations for the curve pairs from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM and 1.25 nM, respectively.

[0184] FIG. 36: IFN-γ secretion assay by adding 14C12H1L1 (hG1WT) and 14C12H1L1(hG1TM) to mixed lymphocyte reaction.

[0185] FIG. 37: IL-2 secretion assay by adding 14C12H1L1 (hG1WT) and 14C12H1L1(hG1TM) to mixed lymphocyte reaction.

[0186] FIG. 38: ADCP effect assay of 14C12H1L1(hG1WT), nivolumab, and 14C12H1L1(hG1TM).

[0187] FIG. 39: Killing effect assay of 14C12H1L1(hG1TM)+anlotinib on human non-small cell lung cancer cells.

[0188] FIG. 40: Inhibited proliferation of mouse colorectal cancer MC38 cells by 14C12H1L1(hG1TM).

[0189] FIG. 41: Effectively enhanced immune response of immune cells to human gastric cancer KATO III cells by 14C12H1L1(hG1TM).

[0190] FIG. 42: Effectively enhanced immune response of immune cells to nasopharyngeal cancer CNE-2Z cells by 14C12H1L1(hG1TM).

[0191] FIG. 43: Effectively enhanced immune response of immune cells to mesothelioma NCI-H2452 cells by 14C12H1L1(hG1TM).

[0192] FIG. 44: Effectively enhanced immune response of immune cells to human non-small cell lung cancer NCI-11446 cells by 14C12H1L1(hG1TM).

[0193] FIG. 45: Effectively enhanced immune response of immune cells to nasopharyngeal cancer CNE-2Z cells by 14C12H1L1(hG1TM) in combination with anlotinib hydrochloride.

[0194] FIG. 46: Significantly enhanced immune response of immune cells to MSI-H/dMMR tumor SW48 cells by 14C12H1L1(hG1TM) in combination with anlotinib.

[0195] FIG. 47: Significantly enhanced immune response of immune cells to human colorectal cancer SW837 cells of non-MSI-H/dMMR (i.e., MSS) phenotype by 14C12H1L1(hG1TM).

[0196] FIG. 48: Significantly enhanced immune response of immune cells to human colorectal cancer SW837 cells of non-MSI-H/dMMR (i.e., MSS) phenotype by 14C12H1L1(hG1TM) in combination with anlotinib.

DETAILED DESCRIPTION

[0197] 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 limitation on the scope of the present invention. In the cases where the techniques or conditions are not specified, the examples were implemented according to the techniques or conditions described in the literature in the art (e.g., see, Molecular Cloning: A Laboratory Manual, 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 commercially available conventional products if the manufacturers thereof are not specified.

[0198] In the following experiments of the present invention:

[0199] BALB/c mice were purchased from Guangdong Medical Laboratory Animal Center.

[0200] The anti-PDL1 antibody 5C10H2L2-IgG1mt was prepared by methods described in PCT Publication No. WO2017148424A1.

[0201] The anti-PD-1 antibody nivolumab (trade name: Opdivo) was purchased from the Bristol-Myers Squibb.

[0202] Human peripheral blood mononuclear cells were isolated and prepared in Akeso Biopharma, Inc., with informed consent of the donor.

[0203] Raji-PDL1 is a cell expressing human PD-L1 constructed by Akeso Biopharma on the basis of human B cells Raji via transfection.

[0204] Ficoll-Paque™ PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare.

[0205] Human IL-2 ELISA kit was purchased from Dakewe Biotech Co., Ltd.

[0206] RPMI 1640 medium, DMEM medium, Trypsin-EDTA (0.25%) phenol red and Blastidin were all purchased from Gibco.

[0207] Staphylococcus aureus enterotoxin B (SEB) was purchased from Dianotech.

[0208] FBS was purchased from Excell bio.

[0209] Mitomycin C (MMC) was purchased from Stressmarq.

[0210] The sequence of the isotype control, human anti-hen egg lysozyme IgG (anti-HEL antibody, or human IgG, abbreviated as hIgG) is derived from the variable region sequence of the Fab F10.6.6 sequence in the study reported by Acierno et al., entitled “Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies” (Acierno et al., J Mol Biol., 2007; 374(1):130-146).

[0211] Anlotinib used in the examples is hydrochloride salt of anlotinib under the brand name Fukewei® and generic name anlotinib hydrochloride, and was purchased from CTTQ Pharma.

Preparation Example 1: Sequence Design of Anti-PD-1 Antibody 14C12 and its Humanized Antibody 14C12H1L1(hG1WT)

[0212] The amino acid sequences and encoding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1(hG1WT) are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication No. CN106967172A (or No. CN106977602A), respectively.

[0213] (1) Heavy and Light Chain Variable Region Sequences of 14C12

TABLE-US-00003 Nucleotide sequence of the heavy chain variable region of 14C12: (354 bp) (SEQ ID NO: 1) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGT CACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGA CATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCT ACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAG GGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCA GATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAAC CGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGA CAGTCTCTGCC Amino acid sequence of the heavy chain variable region of 14C12: (118 aa) (SEQ ID NO: 2) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVA TISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCAN RYGEAWFAYWGQGTLVTVSA Nucleotide sequence encoding the light chain variable region of 14C12: (321 bp) (SEQ ID NO: 3) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCG AGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCT GTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTAC CGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTG GCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGA CATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTT GGAGCAGGCACAAAACTGGAACTGAAG Amino acid sequence of the light chain variable region of 14C12: (107 aa) (SEQ ID NO: 4) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIY RANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTF GAGTKLELK

[0214] (2) Heavy and Light Chain Variable Region and Heavy and Light Chain Sequences of Humanized Monoclonal Antibody 14C12H1L1(hG1WT)

TABLE-US-00004 Nucleotide sequence of the heavy chain variable region of 14C12H1L1(hG1WT): (354 bp) (SEQ ID NO: 5) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGT CACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGA CATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCT ACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGG GCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAAC CGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGA CAGTCTCTAGT Amino acid sequence of the heavy chain variable region of 14C12H1L1(hG1WT): (118 aa) (SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVA TISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCAN RYGEAWFAYWGQGTLVTVSS Nucleotide sequence encoding the light chain variable region of 14C12H1L1(hG1WT): (321 bp) (SEQ ID NO: 7) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCG ACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCT GAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTAC CGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAG GGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGA CATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTT GGCGCCGGGACAAAACTGGAGCTGAAG Amino acid sequence of the light chain variable region of 14C12H1L1(hG1WT): (107 aa) (SEQ ID NO: 8) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIY RANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTF GAGTKLELK Nucleotide sequence of the heavy chain of 14C12H1L1(hG1WT): (1344 bp) (SEQ ID NO: 9) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGT CACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGA CATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCT ACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGG GCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAAC CGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGA CAGTCTCTAGTGCCAGCACCAAAGGACCTAGCGTGTTTCCTCTCGCCCC CTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTG AAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTC TGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACT CTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACC CAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGG ACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCC CTGTCCTGCTCCCGAACTCCTCGGAGGCCCTAGCGTCTTCCTCTTTCCT CCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCT GTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTG GTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAG GAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAA GGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAA CCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCA CCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAG CGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTAC AAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACA GCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTC CTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGC CTGTCCCTGAGCCCCGGCAAA Amino acid sequence of the heavy chain of 14C12H1L1(hG1WT): (448 aa) (SEQ ID NO: 10) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVA TISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCAN RYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK Nucleotide sequence of the light chain of 14C12H1L1(hG1WT): (642 bp) (SEQ ID NO: 11) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCG ACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCT GAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTAC CGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAG GGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGA CATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTT GGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCG TCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAG CGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAG TGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGA CTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGAC TCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTG ACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCG AATGT Amino acid sequence of the light chain of 14C12H1L1(hG1WT): (214 aa) (SEQ ID NO: 12) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIY RANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTF GAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Preparation Example 2: Sequence Design of Humanized Antibody 14C12H1L1(hG4)

[0215] The heavy and light chain variable regions are identical to those of 14C12H1L1(hG1WT). Ig gamma-4 chain C region (ACCESSION: P01861.1) was used as the heavy chain constant region, and Ig kappa chain C region (ACCESSION: P01834) was used as the light chain constant region, thus giving the antibody 14C12H1L1(hG4). The sequence of 14C12H1L1(hG4) is as follows:

TABLE-US-00005 Nucleotide sequence of the heavy chain of  14C12H1L1(hG4): (1335 bp) (SEQ ID NO: 13) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGT CACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGA CATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCT ACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGG GCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAAC CGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGA CAGTCTCTAGTGCCAGCACCAAAGGGCCCTCGGTCTTCCCCCTGGCGCC CTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTC AAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACG AAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGC ACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCC AAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGA TGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCC GTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAA CCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTG TCTCTGGGTAAA Amino acid sequence of the heavy chain of 14C12H1L1(hG4): (445 aa) (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVA TISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCAN RYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK

[0216] The nucleotide sequence of the 14C12H1L1(hG4) light chain is identical to SEQ ID NO: 11.

[0217] The amino acid sequence of the 14C12H1L1(hG4) light chain is identical to SEQ ID NO: 12.

Preparation Example 3: Sequence Design of Humanized Antibody 14C12H1L1(hG1TM)

[0218] On the basis of 14C12H1L1(hG1WT) obtained in Preparation Example 1, a humanized mutant 14C12H1L1(hG1TM) was obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A), a leucine-to-alanine point mutation at position 235 (L235A), and a glycine-to-alanine point mutation at position 237 (G237A) in the hinge region of the heavy chain according to the EU numbering system.

TABLE-US-00006 Nucleotide sequence of the heavy chain of 14C12H1L1(hG1TM): (1344 bp) (SEQ ID NO: 15) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGT CACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGA CATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCT ACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGG GCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAAC CGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGA CAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCC CTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTG AAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTC TGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACT CTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACC CAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGG ACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCC CTGTCCTGCTCCCGAAGCTGCTGGAGCCCCTAGCGTCTTCCTCTTTCCT CCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCT GTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTG GTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAG GAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAA GGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAA CCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCA CCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAG CGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTAC AAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACA GCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTC CTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGC CTGTCCCTGAGCCCCGGCAAA Amino acid sequence of the heavy chain of 14C12H1L1(hG1TM): (448 aa) (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVA TISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCAN RYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0219] The nucleotide sequence of the 14C12H1L1(hG1TM) light chain is identical to SEQ ID NO: 11.

[0220] The amino acid sequence of the 14C12H1L1(hG1TM) light chain is identical to SEQ ID NO: 12.

Preparation Example 4: Sequence Design of Humanized Antibody 14C12H1L1(hG1DM)

[0221] On the basis of 14C12H1L1(hG1WT), a humanized mutant antibody 14C12H1L1(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 hinge region of the heavy chain.

TABLE-US-00007 Nucleotide sequence of the heavy chain of 14C12H1L1(hG1DM): (1344 bp) (SEQ ID NO: 17) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGT CACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGA CATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCT ACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGG GCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCA GATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAAC CGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGA CAGTCTCTAGTGCTAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCC CTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTG AAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTC TGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACT CTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACC CAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGG ACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCC CTGTCCTGCTCCCGAAGCTGCTGGAGGCCCTAGCGTCTTCCTCTTTCCT CCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCT GTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTG GTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAG GAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAA GGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAA CCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCA CCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAG CGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTAC AAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACA GCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTC CTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGC CTGTCCCTGAGCCCCGGCAAA Amino acid sequence of the heavy chain of 14C12H1L1(hG1DM): (448 aa) (SEQ ID NO: 18) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVA TISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCAN RYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0222] The nucleotide sequence of the 14C12H1L1(hG1DM) light chain is identical to SEQ ID NO: 11.

[0223] The amino acid sequence of the 14C12H1L1(hG1DM) light chain is identical to SEQ ID NO: 12.

Experimental Example 1: Affinity Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to Fc Receptor FcγRI

[0224] 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. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRI were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies. The method for determining the affinity constant of the antibodies to FcγRI by the Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. A 1 μg/mL FcγRI solution (from Sinobio) was added to the HIS1K sensor to immobilize the FcγRI on the sensor surface for 50 s. The association and dissociation constants of the antibodies to FcγRI were both determined in the buffer with the antibody concentrations being 3.12-50 nM (serial two-fold dilution). The sensor with immobilized antigen was equilibrated in the buffer for 60 s, and then the binding of the immobilized FcγRI on the sensor to the antibodies was determined for 120 s; the dissociation of FcγRI from the antibodies was determined in 120 s. The temperature was 30° C. and the frequency was 0.3 Hz. The data were fitted and analyzed with a 1:1 model to obtain the affinity constants to FcγRI for the antibodies.

[0225] The results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRI are shown in Table 1 and FIGS. 1-5.

TABLE-US-00008 TABLE 1 Kinetics for binding of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRI Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG4) 5.80E−09 6.37E+05 2.21E+04 3.69E−03 1.05E−04 0.45-0.54 14C12H1L1(hG1WT) 2.52E−09 6.94E+05 2.19E+04 1.75E−03 9.14E−05 0.50-0.55 14C12H1L1(hG1TM) N/A N/A N/A N/A N/A N/A 5C10H2L2-IgG1mt 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.

[0226] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) bound to FcγRI with affinity constants of 5.80E-09 M and 2.52E-09 M, respectively; 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt had no binding or an extremely weak binding signal to FcγRI, and thus the results were not analyzed and no corresponding data was obtained.

[0227] The results suggested that the binding activities of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRI are effectively eliminated as compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

Experimental Example 2: Affinity Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to Fc Receptor FcγRIIIa and Subtypes Thereof

[0228] (1) Affinity Constant Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIIa_V158

[0229] The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIIa_V158 were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0230] The method for determining the affinity constant of the antibodies and the control antibody 5C10H2L2-IgG1mt to FcγRIIIa_V158 by the Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. 5 μg/mL FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized FcγRIIIa_V158 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 temperature was 30° C. and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0231] The results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRIIIa_V158 are shown in Table 2 and FIGS. 6-10.

TABLE-US-00009 TABLE 2 Kinetics for binding of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRIIIa_V158 Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) 6.12E−07 1.35E+05 3.74E+04 8.24E−02 8.34E−03 0.33-0.41 14C12H1L1(hG4) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG1WT) 6.54E−08 2.61E+05 2.10E+04 1.71E−02 6.47E−04 0.80-1.17 14C12H1L1(hG1TM) N/A N/A N/A N/A N/A N/A 5C10H2L2-IgG1mt 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.

[0232] The results showed that both 14C12H1L1(hG1DM) and 14C12H1L1(hG1WT) bound to FcγRIIIa_V158 with affinity constants of 6.21E-07M and 6.54E-08M, respectively; 14C12H1L1(hG4), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt had no binding or an extremely weak binding signal to FcγRIIIa_V158, and thus the results were not analyzed.

[0233] The results suggested that the binding activities of 14C12H1L1(hG4), 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγR IIIa_V158 are effectively eliminated as compared to 14C12H1L1(hG1DM) and 14C12H1L1(hG1WT).

[0234] (2) Affinity Constant Assay of 14C12H1L1(hG1DM), 14C12H1L1(Hg4), 14C12H1L1(Hg1Wt) and 14C12H1L1(hG1TM) to FcγRIIIa_F158

[0235] The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM) and the control antibody to FcγRIIIa_F158 were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0236] The method for determining the affinity constant of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIIa_F158 by the Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. 5 μg/mL FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized FcγRIIIa_F158 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 temperature was 30° C. and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0237] The results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRIIIa_F158 are shown in Table 3 and FIGS. 11-15.

TABLE-US-00010 TABLE 3 Kinetics for binding of 14C12H1L1 antibodies and isotypes thereof to FcγRIIIa_F158 Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG4) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG1WT) 1.02E−07 2.52E+05 2.93E+04 2.56E−02 1.12E−03 0.34-0.57 14C12H1L1(hG1TM) N/A N/A N/A N/A N/A N/A 5C10H2L2-IgG1mt 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.

[0238] The results showed that 14C12H1L1(hG1WT) bound to FcγRIIIa_F158 with an affinity constant of 1.02E-07M; 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt had no binding or an extremely weak binding signal to FcγRIIIa_F158, and thus the results were not analyzed and no corresponding data was obtained.

[0239] The results suggested that the binding activities of 14C12H1L1(hG1DM), 14C12H1L1(hG4) and 14C12H1L1(hG1TM) to FcγRIIIa_F158 are effectively eliminated as compared to 14C12H1L1(hG1WT).

Experimental Example 3: Affinity Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to Fc Receptor FcγRIIa and Subtypes Thereof

[0240] (1) Affinity Constant Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIa_H131

[0241] The Fc receptor FcγRIIa_H131, also known as CD32a_H131, 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. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIa_H131 were measured in this experiment using a Fortebio Octet system to evaluate the binding capacity of the antibodies to Fc receptor.

[0242] The method for determining the affinity constant of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIa_H131 by the 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 0.02% Tween-20, 0.02% casein and 0.1% BSA in PBS, pH 7.4. 5 μg/mL FcγRIIa_H131 was immobilized on the NTA sensor at an immobilization height of about 1.0 nm. The sensor was equilibrated in a buffer of 0.02% Tween-20, 0.02% casein and 0.1% BSA in PBS, pH 7.4 for 300 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 antibody was dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine pH 1.7 and 10 nM nickel sulfate. 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.

[0243] The results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRIIa_H131 are shown in Table 4 and FIGS. 16-20.

TABLE-US-00011 TABLE 4 Kinetics for binding of 14C12H1L1 antibodies and isotypes thereof to FcγRIIa_H131 Sample ID K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG4) 5.07E−08 2.57E+05 2.37E+04 1.30E−02 6.47E−04 0.18-0.35 14C12H1L1(hG1WT) 5.74E−08 4.65E+05 5.99E+04 2.67E−02 1.24E−03 0.82-1.12 14C12H1L1(hG1TM) N/A N/A N/A N/A N/A N/A 5C10H2L2-IgG1mt 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.

[0244] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) bound to FcγR11a_H131 with affinity constants of 5.07E-08M and 5.74E-08M, respectively; 14C12H1L1(hG1DM), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt had no binding or an extremely weak binding signal to FcγR11a_H131, and thus the results were not analyzed and no corresponding data was obtained.

[0245] The results suggested that the binding activities of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγR11a_H131 are effectively eliminated as compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0246] (2) Affinity Constant Assay of 14C12H1L1(hG1DM), 14C12H1L1(Hg4), 14C12H1L1(Hg1Wt) and 14C12H1L1(hG1TM) to FcγRIIa_R131

[0247] The Fc receptor FcγRIIa_R131, also known as CD32a_R131, 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. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIa_R131 were measured in this experiment using a Fortebio Octet system to evaluate the binding capacity of the antibodies to Fc receptor.

[0248] The method for determining the affinity constant of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIa_R131 by the 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 0.02% Tween-20, 0.02% casein and 0.1% BSA in PBS, pH 7.4. 5 μg/mL FcγRIIa_R131 was immobilized on the NTA sensor at an immobilization height of about 1.0 nm. The sensor was equilibrated in a buffer of 0.02% Tween-20, 0.02% casein and 0.1% BSA in PBS, pH 7.4 for 300 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 antibody was dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine pH 1.7 and 10 nM nickel sulfate. 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.

[0249] The results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRIIa_R131 are shown in Table 5 and FIGS. 21-25.

TABLE-US-00012 TABLE 5 Kinetics for binding of 14C12H1L1 antibodies and isotypes thereof to FcγRIIa_R131 Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG4) 3.13E−08 3.50E+05 3.05E+04 1.10E−02 6.43E−04 0.21-0.46 14C12H1L1(hG1WT) 3.46E−08 6.60E+05 9.46E+04 2.28E−02 1.33E−03 0.39-0.83 14C12H1L1(hG1TM) 2.32E−07 4.04E+05 9.45E+04 9.38E−02 4.89E−03 0.19-0.35 5C10H2L2-IgG1mt 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.

[0250] The results showed that 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) bound to FcγRIIa_R131 with affinity constants of 3.13E-08M, 3.46E-08M and 2.32E-07M, respectively; 14C12H1L1(hG1DM) and 5C10H2L2-IgG1mt had no binding or an extremely weak binding signal to FcγRIIa_R131, and thus the results were not analyzed and no corresponding data was obtained.

[0251] The results suggest that among the antibodies with binding activities, 14C12H1L1(hG1TM) has the weakest binding capacity and the lowest binding activity to FcγRIIa_R131 as compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

Experimental Example 4: Affinity Constant Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIb

[0252] The Fc receptor FcγRIIb (also known as CD32b), can bind to the Fc fragment of IgG antibodies, down-regulate functions of immune cells, inhibit the activation and proliferation of immune cells and inhibit the secretion of cytokines. The affinity constants of the antibodies to FcγRIIb were measured in this experiment using a Fortebio Octet system to evaluate the binding capacity of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to Fc receptor.

[0253] The method for determining the affinity constant of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to FcγRIIb by the 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 0.02% Tween-20, 0.02% casein and 0.1% BSA in PBS, pH 7.4. 5 μg/mL hFcγRIIb-his was immobilized on the NTA sensor at an immobilization height of about 1.0 nm. The sensor was equilibrated in a buffer of 0.02% Tween-20, 0.02% casein and 0.1% BSA in PBS, pH 7.4 for 300 s of blocking, and the binding of the immobilized hFcγRIIb-his on the sensor to the antibodies at concentrations of 12.5-200 nM (serial two-fold dilution) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine pH 1.7 and 10 nM nickel sulfate. 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 results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγRIIb are shown in Table 6 and FIGS. 26-30.

TABLE-US-00013 TABLE 6 Kinetics for binding of 14C12H1L1 antibodies and isotypes thereof to FcγRIIb Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG4) 5.62E−08 2.88E+05 2.94E+04 1.62E−02 7.63E−04 0.22-0.33 14C12H1L1(hG1WT) 6.13E−08 3.18E+05 3.48E+04 1.95E−02 8.96E−04 0.16-0.37 14C12H1L1(hG1TM) N/A N/A N/A N/A N/A N/A 5C10H2L2-IgG1mt 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.

[0254] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) bound to FcγRIIb with affinity constants of 5.62E-08M and 6.13E-08M, respectively; 14C12H1L1(hG1DM), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt had no binding or an extremely weak binding signal to FcγRIIb, and thus the results were not analyzed and no corresponding data was obtained.

[0255] The results suggested that the binding activities of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRIIb are effectively eliminated as compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

Experimental Example 5: Affinity Assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to C1q

[0256] 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. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to C1q were measured in this experiment using a Fortebio Octet system to evaluate the CDC activity of the antibodies.

[0257] The method for determining the affinity constants of the antibodies to C1q by the Fortebio Octet system is briefly described as follows: the sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. 50 μg/mL antibody was immobilized on the FAB2G sensor at an immobilization height of about 2.0 nm. The sensor was equilibrated in a buffer for 60 s for blocking, and the binding of the immobilized antibody on the sensor to the antigen C1q at concentrations of 1.25-20 nM (serial two-fold dilution) was determined for 60 s. The antigen and antibody 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.

[0258] The results of affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) and the control 5C10H2L2-IgG1mt to C1q are shown in Table 7 and FIGS. 31-35.

TABLE-US-00014 TABLE 7 Kinetics for binding of 14C12H1L1 antibodies and isotypes thereof to C1q Antibody K.sub.D (M) kon (1/Ms) SE (kon) kdis (Vs) SE (kdis) Rmax (nm) 14C12H1L1(hG1DM) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG4) N/A N/A N/A N/A N/A N/A 14C12H1L1(hG1WT) 1.35E−09 4.83E+06 4.24E+05 6.54E−03 5.33E−04 0.32-0.53 14C12H1L1(hG1TM) N/A N/A N/A N/A N/A N/A 5C10H2L2-IgG1mt 4.43E−09 2.38E+06 4.21E+05 1.05E−02 1.10E−03 0.19-0.26 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.

[0259] The results showed that 14C12H1L1(hG1WT) bound to C1q with an affinity constant of 1.35E-09M; 14C12H1L1(hG1DM), 14C12H1L1(hG4) and 14C12H1L1(hG1TM) had no binding or an extremely weak binding signal to C1q, and thus the results were not analyzed and no corresponding data was obtained.

[0260] The results also showed that 5C10H2L2-IgG1mt bound to C1q with an affinity constant of 4.43E-09, indicating that it has binding activity to C1q and can cause CDC effects.

Experimental Example 6: Pharmacodynamic Activities of 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) in Co-Culture System of Peripheral Blood Mononuclear Cells and Raji-PDL1 Cells

[0261] In this experiment, the pharmacodynamic activity of anti-PD-1 antibodies 14C12H1L1 (hG1WT) and 14C12H1L1 (hG1TM), and the control anti-PD-L1 antibodies 5C10H2L2-IgG1mt and nivolumab in relieving immunosuppression mediated by PD-1/PD-L1 were detected in a co-culture system of peripheral blood mononuclear cells and Raji-PDL1 cells. In the mixed lymphocyte reaction, when isolated peripheral blood mononuclear cells (containing immune cells expressing immunocompetent PD-1) and the Raji-PDL1 cells expressing PD-L1 are co-cultured, the interaction of PD-1 and PD-L1 can mediate the function inhibition of the immune cells, showing reduced secretion of cytokines IFN-γ and IL-2. Anti-PD-1 or anti-PD-L1 antibodies can relieve such immunosuppression of immune cells, leading to increased cytokine secretion. Raji is a B cell line. As described above, B cells can be used as antigen presenting cells to mediate the immune response of immune cells to tumor cells. In the present invention, the Raji-PDL1 and PBMCs co-culture system was used to evaluate the pharmacological activity of the anti-PD-1 antibodies, and a Raji-PDL1, PBMCs and tumor cell co-culture system was used to evaluate the pharmacological activity of the anti-PD-1 antibodies in different tumors.

[0262] Peripheral blood mononuclear cells were isolated by the Ficoll-Paque Plus (GE Healthcare, Cat No.: 171440-02), and then stimulated with SEB (0.5 μg/mL) for two days. The stimulated mature peripheral blood mononuclear cells (1×10.sup.5 cells/well) and Raji-PDL1 cells (1×10.sup.5 cells/well) treated with MMC (Mito-mycin C with a treatment concentration of 2 μg/mL) for 1 h were added to a 96-well plate before 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), the control antibody nivolumab or the control anti-PD-L1 antibody 5C10H2L2-IgG1mt was added. The mixture was well mixed and incubated. After 3 days, the culture supernatant was collected and detected for IFN-γ secretion and IL-2 secretion by an ELISA kit (purchased from Dakewe Biotech Co., Ltd.).

[0263] The results of the IFN-γ secretion in the mixed lymphocyte reaction are shown in FIG. 36. The results showed that in the co-culture system of PBMCs and Raji-PDL1, 14C12H1L1(hG1TM) induced a significantly higher IFN-γ secretion than those induced by 14C12H1L1(hG1WT), nivolumab or 5C10H2L2-IgG1mt at the same dose level.

[0264] The results of the IL-2 secretion in mixed lymphocyte reaction are shown in FIG. 37. The results showed that in the co-culture system of PBMCs and Raji-PDL1, 14C12H1L1(hG1TM) induced a significantly higher IL-2 secretion than those induced by 14C12H1L1(hG1WT), nivolumab or 5C10H2L2-IgG1mt at the same dose level.

[0265] The results suggested that the pharmacodynamic activity of 14C12H1L1(hG1TM) in relieving the immunosuppression mediated by PD-1/PD-L1 is significantly superior to that of nivolumab, 14C12H1L1(hG1WT) or 5C10H2L2-IgG1mt.

Experimental Example 7: Antibody-Mediated Phagocytic Activities of Nivolumab, 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) on CHO-K1-PD1

[0266] To detect the antibody dependent cellular phagoxytosis (ADCP) activity, murine macrophages were used as effector cells and cell lines overexpressing PD1 were used as target cells. The femoral bone marrow of Blab/c 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% CO.sub.2 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 0.05% trypsin. Macrophages were collected, and centrifuged at 750×g for 5 min. The supernatant was discarded and the cells were suspended in DMEM complete medium (containing 10% FBS) and counted. The cell was adjusted to a proper density and filled into sterile EP tubes for further use. CHO-K1-PD1 cells (a CHO-K1 cell line overexpressing PD1) were centrifuged at 170×g for 5 min, washed once with PBS, resuspended and counted. The viability was determined. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM with PBS to resuspend the cells (staining density: 10 million cells/mL). A proper amount of the cells were incubated in a cell incubator for 20 min. 6 mL of DMEM complete medium was added to stop the staining. The cells were centrifuged at 170×g for 5 min, and the supernatant was discarded. 1 mL of DMEM complete medium was added. The cells were incubated in an incubator for 10 min, and adjusted to the experiment density. The cells were coded as CHO-K-PD1-CFSE.

[0267] The test antibodies were diluted in DMEM complete medium to 20, 2 and 0.2 μg/mL (the working concentrations were 10, 1 and 0.1 μg/mL). An anti-HEL IgG1 antibody and a medium were used as the isotype control group and a blank control group. According to the study design, the diluted antibodies and CHO-K1-PD1-CFSE cells were added into 1.5-mL EP tubes containing macrophages (the final volume was 100 μL and the effector-to-target ratio was 50,000:150,000). The mixture was well mixed for resuspension and incubated in an incubator at 37° C. for 2 h. 800 μL of PBS containing 1% bovine serum albumin (BSA) was added at room temperature to each tube. The mixture was centrifuged at 500×g for 5 min, and the supernatant was discarded. The cells were washed once with 800 μL of 1% PBSA. APC anti-mouse/human CD11b antibody (Biolegend, Cat. No.: 101212) was diluted 400-fold with PBSA and added to the corresponding samples at 100 μL/sample. The mixture was mixed well, incubated on ice for 40 min, and washed with 800 μL of 1% PBSA and centrifuged at 1200×g for 5 min twice, and the supernatant was discarded. 200 μL of 1% PBSA was added to each tube to resuspend the cells. The cells were transferred to loading tube and analyzed by BD FACS Calibur flow cytometer. 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-mediated ADCP activity was evaluated. The ADCP activity of each group, represented by P %, was calculated according to the following formulas:

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

[0268] The results are shown in FIG. 38.

[0269] The results showed that at the same concentration, the phagocytic rates of 14C12H1L1(hG1WT) and nivolumab were 3.94 and 4.26 times that of the isotype control anti-HEL antibody, respectively, indicating that 14C12H1L1 (hG1WT) and nivolumab have ADCP effects; and at the same concentration, the phagocytic rate of 14C12H1L1(hG1TM) was comparable to that of the isotype control antibody, indicating that 14C12H1L1(hG1TM) has no ADCP effect.

[0270] The results suggest that the amino acid mutation introduced by 14C12H1L1(hG1TM) can effectively eliminate the ADCP effect, and a surprising technical effect is obtained.

Experimental Example 8: Pharmacodynamic Evaluation of 14C12H1L1(hG1Tm)+Anlotinib Hydrochloride in Scid/Beige Immunodeficient Mouse Model Bearing Human Non-Small Cell Lung Cancer HCC827 Subcutaneous Xenograft Tumor

[0271] Female Scid/beige immunodeficient mice (purchased from Vital River) were divided into groups of 8. 0.2 μg/mL Staphylococcus aureus enterotoxin B (SEB) was added to 1 million/mL PBMC suspension. PBMC s were incubated for 3 days for activation to increase the expression of PD1 on PBMCs. Mice were grafted subcutaneously with a mixture of 800,000 SEB-activated PBMCs and 6,000,000 HCC827 human non-small cell lung cancer cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) on day 0, and divided into 2 groups, the isotype control antibody group (i.e., the anti-HEL antibody, prepared by Zhongshan Akeso Biopharma as described above) and the 14C12H1L1(hG1TM)+anlotinib hydrochloride group. The 14C12H1L1(hG1TM) was administered through the tail vein once weekly (the first dose was co-administered subcutaneously with the cells), and anlotinib was administered by oral gavage once daily for 30 days. The specific protocol is shown in Table 8. Tumors were measured continuously in the experiment, and the volume was calculated according to the formula: a (tumor length)×b(tumor width)×b(tumor width)/2.

TABLE-US-00015 TABLE 8 Experimental planning and grouping Group n Model Dosage information Isotype control 7 6,000,000 Anti-HEL antibody, 10 mg/kg, HCC827 cells administered via tail vein once weekly +800,000 for 9 doses (the first dose was co- PBMCs (SEB- administered subcutaneously with the activated for 3 cells) 14C12H1L1 8 days) 14C12H1L1(hG1TM), 10 mg/kg, (hG1TM) Subcutaneous administered via tail vein once weekly + anlotinib xenograft (d0) for 9 doses (the first dose was co- administered subcutaneously with the cells) Anlotinib hydrochloride, 3 mg/kg, administered by oral gavage daily for 30 days

[0272] The experimental results are shown in FIG. 39.

[0273] The results showed that 14C12H1L1(hG1TM)+anlotinib hydrochloride significantly inhibited the increase in tumor volume of human non-small cell lung cancer cells, indicating good tumor killing effects.

Experimental Example 9: Pharmacodynamic Evaluation of 14C12H1L1(hG1TM) in C57BL/6-hPD1/hPDL1/hCD73 Mouse Model Bearing Colon Cancer MC38-hPDL1/hCD73 Subcutaneous Graft Tumor

[0274] The mouse MC38 cell line is a mouse colorectal cancer cell line. It has been demonstrated that the MC38 cells line is a useful model for studying human MSI-H/dMMR tumors (Efremova M et al., Nat Commun., 2018; 9(1):32).

[0275] Female C57BL/6-hPD1/hPDL1/hCD73 mice (purchased from Nanjing GemPharmatech Co., Ltd.) were divided into groups of 8 and grafted subcutaneously on the right forelimb with colon cancer MC38-hPDL1/hCD73 cells (purchased from Nanjing GemPharmatech Co., Ltd.) (2×10.sup.6 cells/100 μL/mouse). The day of grafting was defined as D0. The dosing volume was adjusted according to the body weight: 10 μL/g mouse body weight (g). The anti-HEL antibody (the preparation and the source are the same as those of the Experimental Example 8) or 14C12H1L1(hG1TM) was administrated intraperitoneally twice weekly for 3 weeks, in a total of 6 doses. The specific protocol is shown in Table 9. Tumors were measured continuously in the experiment, and the volume was calculated as the following formula:

tumor volume (mm.sup.3)=(tumor length×(tumor width).sup.2)/2.

TABLE-US-00016 TABLE 9 Protocol and grouping Group N Dose (mg/kg) Frequency Cycle Isotype Control 8 10 BIW*3 6 doses in total 14C12H1L1 8 1 BIW*3 6 doses in (hG1TM) total

[0276] The experimental results are shown in FIG. 40.

[0277] The results showed that as compared to the isotype control antibody, the tumor growth was inhibited, indicating that 14C12H1L1(hG1TM) can significantly inhibit the proliferation of MC38 cells, and can effectively treat solid tumors of the MSI-H/dMMR phenotype, such as colon and/or rectal cancers.

Experimental Example 10: Effectively Enhanced Immune Response of Immune Cells to Human Gastric Cancer KATO III Cells by 14C12H1L1(hG1TM)

[0278] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instruction, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640+10% FBS complete medium, and KATO III cells (purchased from Chinese Academy of Sciences Shanghai Cell Bank) were cultured in DMEM+10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg/mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg/mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640+10% FBS complete medium and counted. Raji-PDL1 and PBMC cells were seeded on 96-well plates at 1×10.sup.5 cells/well. KATO III cells in logarithmic growth phase were collected and seeded on the 96-well plate at 5×10.sup.4 cells/well. The diluted antibody was added according to the study design. The mixture was mixed evenly and incubated in a 5% CO.sub.2 incubator at 37° C. for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to ELISA KIT instruction. The media in this experiment were all 10% FBS+RPMI 1640.

[0279] The experimental results are shown in FIG. 41.

[0280] The results showed that 14C12H1L1(hG1TM) co-cultured with human gastric cancer KATO III cells exhibited higher pharmacological activity as compared to 14C12H1L1(hG1WT) or nivolumab. 14C12H1L1(hG1TM) can stimulate PBMCs to secrete more IL-2 at the same concentration level, indicating potential for treating gastric cancer.

Experimental Example 11: Effectively Enhanced Immune Response of Immune Cells to Nasopharyngeal Cancer CNE-2Z Cells by 14C12H1L1(hG1TM)

[0281] Raji-PDL1, CNE-2Z cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) and PBMCs were thawed, wherein the PBMCs were stimulated with SEB (0.5 μg/mL) for two days after 2 hours of thawing. On the day of the experiment, Raji-PDL1 cells were treated with MMC (Mito-mycin C with a treatment concentration of 2 μg/mL and a cell treatment density of 200×10.sup.4 cells/mL) for 1 h. PBMCs were collected, and the treated Raji-PDL1 cells were washed twice with PBS. The PBMCs and the Raji-PDL1 cells were added to the cell plate at 10×10.sup.4 cells/well, and CNE-2Z cells were added at 3×10.sup.4 cells/well. The antibodies (with a final concentration of 300 nM and a final volume of 200 μL) were added according to the experimental design, and co-cultured with the cells for 3 days. The culture supernatant was collected and assayed for IL-2. The media in this experiment were all 10% FBS+RPMI 1640.

[0282] The results are shown in FIG. 42.

[0283] The results showed that 14C12H1L1(hG1TM) co-cultured with human nasopharyngeal cancer CNE-2Z cells exhibited higher pharmacological activity as compared to 14C12H1L1(hG1WT). 14C12H1L1(hG1TM) can stimulate PBMCs to secrete more IL-2 at the same concentration level, indicating potential for treating nasopharyngeal cancer.

Experimental Example 12: Effectively Enhanced Immune Response of Immune Cells to Mesothelioma NCI-112452 Cells by 14C12H1L1(hG1TM)

[0284] Raji-PDL1, NCI-112452 cells (purchased from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) and PBMCs were thawed, wherein the PBMCs were stimulated with SEB (0.5 μg/mL) for two days after 2 hours of thawing. On the day of the experiment, Raji-PDL1 cells were treated with MMC (Mito-mycin C with a treatment concentration of 2 μg/mL and a cell treatment density of 200×10.sup.4 cells/mL) for 1 h. PBMCs were collected, and the treated Raji-PDL1 cells were washed twice with PBS. The PBMCs and the Raji-PDL1 cells were added to the cell plate at 10×10.sup.4 cells/well, and NCI-112452 cells were added at 3×10.sup.4 cells/well. The antibodies (with a final concentration of 300 nM and a final volume of 200 μL) were added according to the experimental design, and co-cultured with the cells for 3 days. The culture supernatant was collected and assayed for IL-2. The media in this experiment were all 10% FBS+RPMI 1640.

[0285] The results are shown in FIG. 43.

[0286] The results showed that 14C12H1L1(hG1TM) co-cultured with human mesothelioma NCI-112452 cells exhibited higher pharmacological activity as compared to 14C12H1L1(hG1WT). 14C12H1L1(hG1TM) can stimulate PBMCs to secrete more IL-2 at the same concentration level, indicating potential for treating mesothelioma.

Experimental Example 12: Effectively Enhanced Immune Response of Immune Cells to Small Cell Lung Cancer NCI-11446 Cells by 14C12H1L1(hG1TM)

[0287] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instruction, and the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-11446 cells (purchased from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were cultured in RPMI 1640+10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg/mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg/mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640+10% FBS complete medium and counted. Raji-PDL1 and PBMC cells were seeded on 96-well plates at 1×10.sup.5 cells/well. NCI-11446 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 8×10.sup.4 cells/well. The diluted antibody was added according to the study design. The mixture was mixed evenly and incubated in a 5% CO.sub.2 incubator at 37° C. for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to ELISA KIT instruction. The media in this experiment were all 10% FBS+RPMI 1640.

[0288] The results are shown in FIG. 44.

[0289] The results showed that 14C12H1L1(hG1TM) co-cultured with human small cell lung cancer NCI-H446 cells exhibited equivalent or higher pharmacological activity as compared to 14C12H1L1(hG1WT) and nivolumab on the basis of effectively eliminated ADCC, CDC and ADCP activities. 14C12H1L1(hG1TM) can stimulate PBMCs to secrete equivalent or more IL-2 at the same concentration level, indicating potential for treating small cell lung cancer.

Experimental Example 13: Effectively Enhanced Immune Response of Immune Cells to Human Nasopharyngeal Cancer CNE-2Z Cells by 14C12H1L1(hG1TM) in Combination with Anlotinib Hydrochloride

[0290] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instruction, and the isolated PBMCs were counted and frozen. Raji-PDL1 and CNE-2Z cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in RPMI 1640+10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg/mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg/mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640+10% FBS complete medium and counted. Raji-PDL1 and PBMC cells were seeded on 96-well plates at 1×10.sup.5 cells/well. CNE-2Z cells in logarithmic growth phase were collected and seeded on the 96-well plate at 3×10.sup.4 cells/well. The diluted antibodies and anlotinib were added according to the study design. The mixture was mixed evenly and incubated in a 5% CO.sub.2 incubator at 37° C. for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to ELISA KIT instruction. The media in this experiment were all 10% FBS+RPMI 1640.

[0291] The results are shown in FIG. 45. As compared to the anti-HEL antibody and the anlotinib monotherapies, 14C12H1L1(hG1TM), 14C12H1L1(hG1WT) and nivolumab significantly enhanced the immune response of immune cells to human nasopharyngeal cancer CNE-2Z cells characterized by significantly increased IL-2 secretion level. 14C12H1L1(hG1TM) has superior pharmacological activity than those of 14C12H1L1(hG1WT) and nivolumab. Moreover, the pharmacological activity of 14C12H1L1(hG1TM) in combination with anlotinib in stimulating immune cell activation was superior to those of 14C12H1L1(hG1TM) monotherapy, 14C12H1L1(hG1WT) monotherapy, and nivolumab monotherapy and was also superior to those of 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib.

[0292] The above results indicated that 14C12H1L1(hG1TM) in combination with anlotinib has potential for treating human nasopharyngeal cancer.

Experimental Example 14: Significantly Enhanced Immune Response of Immune Cells to MSI-H/dMMR Tumor SW48 Cells by 14C12H1L1(hG1TM) in Combination with Anlotinib

[0293] SW48 is a human colorectal cancer cell line and is identified with MSI-H/dMMR phenotype (Branch P et al., (1995), Cancer Res, 55(11): 2304-2309). It was used for detecting the enhanced immune cell response to tumor of MSI-H/dMMR phenotype by 14C12H1L1(hG1TM).

[0294] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instruction, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640+10% FBS complete medium, and SW48 cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in DMEM+10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg/mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg/mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640+10% FBS complete medium and counted. Raji-PDL1 and PBMC cells were seeded on 96-well plates at 1×10.sup.5 cells/well. SW48 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 2×10.sup.5 cells/well. The diluted antibody and anlotinib was added according to the study design. The mixture was mixed evenly and incubated in a 5% CO.sub.2 incubator at 37° C. for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to ELISA KIT instruction. The media in this experiment were all 10% FBS+RPMI 1640.

[0295] The results are shown in FIG. 46.

[0296] The results showed that 14C12H1L1(hG1TM), 14C12H1L1(hG1WT) and nivolumab significantly enhanced the immune response of immune cells to human colorectal cancer cells SW48 cells of MSI-H/dMMR phenotype characterized by significantly increased secretion level of IL-2 as compared to anti-HEL antibody. 14C12H1L1(hG1TM) has superior pharmacological activity than that of 14C12H1L1(hG1WT).

[0297] Moreover, the pharmacological activity of 14C12H1L1(hG1TM) in combination with anlotinib in stimulating immune cell activation was superior to those of 14C12H1L1(hG1WT) monotherapy, 14C12H1L1(hG1TM) monotherapy, and nivolumab monotherapy and was also superior to those of 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib.

[0298] The above results showed that 14C12H1L1(hG1TM) in combination with anlotinib has potential for treating solid tumor of MSI-H/dMMR phenotype, particularly colon cancer and/or rectal cancer of MSI-H/dMMR phenotype.

Experimental Example 15: Significantly Enhanced Immune Response of Immune Cells to Human Colorectal Cancer SW837 Cells of MSI-H/dMMR Phenotype by 14C12H1L1(hG1TM)

[0299] SW837 is a human colorectal cancer cell line of non-MSI-H/dMMR (i.e., MSS) phenotype (Guo J et al., Cancer Res., 2011; 71(8):2978-2987), and was used for detecting the enhanced immune cell response to tumor of non-MSI-H/dMMR (i.e., MSS) phenotype by 14C12H1L1(hG1TM) in this example.

[0300] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instruction, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640+10% FBS complete medium, and SW837 cells (purchased from Shanghai Honsun Biological Technology Co., Ltd) were cultured in 10% FBS+Leibovitz's L-15 complete medium (purchased from Gibco). PBMCs were thawed and activated with 0.5 μg/mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg/mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640+10% FBS complete medium and counted. Raji-PDL1 and PBMC cells were seeded on 96-well plates at 1×10.sup.5 cells/well. SW837 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 5×10.sup.4 cells/well. The diluted antibody was added according to the study design. The mixture was mixed evenly and incubated in a 5% CO.sub.2 incubator at 37° C. for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to ELISA KIT instruction.

[0301] The results are shown in FIG. 47.

[0302] The results showed that 14C12H1L1(hG1TM), 14C12H1L1(hG1WT) and nivolumab significantly enhanced the immune response of immune cells to human colorectal cancer cells SW837 cells of non-MSI-H/dMMR phenotype. The pharmacological activity of 14C12H1L1(hG1TM) in the medium and high dose groups was superior to that of 14C12H1L1(hG1WT), characterized by significantly increased secretion level of IL-2. The above results showed that 14C12H1L1(hG1TM) had better or equivalent pharmacological activity relative to 14C12H1L1(hG1WT) and nivolumab on the basis of effectively eliminating ADCC, CDC or ADCP effects, indicating the potential for treating solid tumor of non-MSI-H/dMMR (i.e., MSS) phenotype, particularly colon cancer and/or rectal cancer of non-MSI-H/dMMR phenotype.

Experimental Example 16: Significantly Enhanced Immune Response of Immune Cells to Human Colorectal Cancer SW837 Cells of MSI-H/dMMR Phenotype by 14C12H1L1(hG1TM) in Combination with Anlotinib

[0303] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instruction, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640+10% FBS complete medium and SW837 cells were cultured in Leibovitz's L-15+10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg/mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg/mL MMC for 1 h. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were collected, washed twice with PBS, resuspended in RPMI 1640+10% FBS complete medium and counted. Raji-PDL1 and PBMC cells were seeded on 96-well plates at 1×10.sup.5 cells/well. SW837 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 5×10.sup.4 cells/well. The diluted antibody was added according to the study design. The mixture was mixed evenly and incubated in a 5% CO.sub.2 incubator at 37° C. for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to ELISA KIT instruction.

[0304] The results are shown in FIG. 48.

[0305] The results show that as compared to 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib, 14C12H1L1(hG1TM) in combination with anlotinib significantly enhanced the immune response of immune cells to human colorectal cancer SW837 cells of the non-MSI-H/dMMR phenotype characterized by significantly increased IL-2 secretion level, indicating a superior therapeutic effect on solid tumors of non-MSI-H/dMMR phenotype, particularly colon cancer and/or rectal cancer of non-MSI-H/dMMR phenotype.

[0306] 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.