ANTI-CTLA4-ANTI-PD-1 BISPECIFIC ANTIBODY AND USES THEREOF

Abstract

The present invention relates to the field of tumor treatment and molecular immunology, and particularly, to an anti-CTLA4/anti-PD-1 bispecific antibody and use thereof. Specifically, the anti-CTLA4/anti-PD-1 bifunctional antibody comprises a first protein functional region targeting PD-1 and a second protein functional region targeting CTLA4, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprised in the bispecific antibody 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. The bifunctional antibody of the present invention can well and specifically bind to CTLA4 and PD-1, specifically relieve immunosuppression of CTLA4 and PD-1 in an organism, and activate T lymphocytes, thus having good application prospect.

Claims

1. A bispecific antibody, comprising: a first protein functional region targeting PD-1, and a second protein functional region targeting CTLA4; 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 of amino acid sequences set forth in SEQ ID NOs: 27-29 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 30-32 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 33-35 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 36-38 respectively; or, for the immunoglobulin, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 33-35 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 36-38 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 27-29 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 30-32 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; L234A and G237A; 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 CTLA4; 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 of amino acid sequences set forth in SEQ ID NOs: 27-29 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 30-32 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 33-35 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 36-38 respectively; or, for the immunoglobulin, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 33-35 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 36-38 respectively; for the single chain antibody, the heavy chain variable region comprises HCDR1-HCDR3 of amino acid sequences set forth in SEQ ID NOs: 27-29 respectively, and the light chain variable region comprises LCDR1-LCDR3 of amino acid sequences set forth in SEQ ID NOs: 30-32 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; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

4. The bispecific antibody according to any of claims 1-3, 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 any of claims 1-4, wherein, the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 14 and SEQ ID NO: 18; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 16 and SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41 and SEQ ID NO: 43; the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 and SEQ ID NO: 44; or, the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41 and SEQ ID NO: 43; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 and SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO: 14 and SEQ ID NO: 18; the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO: 16 and SEQ ID NO: 20.

6. The bispecific antibody according to any of claims 1-5, wherein the bispecific antibody is selected from any one of the following (1)-(20): (1) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 4; (2) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 8; (3) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 12; (4) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 4; (5) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 8; (6) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 12; (7) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (8) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20; (9) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (10) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20; (11) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (12) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20; (13) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 42; (14) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44; (15) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 42; (16) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44; (17) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (18) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 16; (19) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 20; and, (20) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ HD NO: 20.

7. The bispecific antibody according to any of claims 1-6, wherein, the amino acid sequence of the heavy chain of the immunoglobulin is set forth in SEQ ID NO: 40, and the amino acid sequence of the light chain of the immunoglobulin is set forth in SEQ ID NO: 24.

8. The bispecific antibody according to any of claims 1-7, wherein the immunoglobulin or the antigen-binding fragment thereof 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 immunoglobulin or the antigen binding fragment thereof has no binding signal or a binding signal of less than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and/or FcγRIIb; preferably, the binding signal refers to a response measured by a Fortebio Octet system.

9. The bispecific antibody according to any of claims 1-8, wherein the immunoglobulin or the antigen-binding fragment thereof 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 immunoglobulin or the antigen binding fragment thereof 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. The bispecific antibody of any of claims 1-9, 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 of 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 any of claims 1-11, 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 any of claims 1-12, wherein the single chain antibody is linked to the 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 CTLA4; the number of the first protein functional region is 1, and the number of the second protein functional region is 2; wherein 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: 40, and the amino acid sequence of the light chain of the immunoglobulin is set forth in SEQ ID NO: 24; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44; 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: 25 and SEQ ID NO: 26; preferably, the amino acid sequences of the first linker fragment and second linker fragments are set forth in SEQ ID NO: 26.

15. An isolated nucleic acid molecule, encoding the bispecific antibody according to any of claims 1-14.

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 or the vector according to claim 16.

18. A conjugate comprising an antibody or antigen-binding fragment thereof, and a conjugated moiety, wherein the immunoglobulin is the bispecific antibody according to any of claims 1-14, 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 any of claims 1-14 or the conjugate according to claim 18; 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. Use of the bispecific antibody according to any of claims 1-14 or the conjugate according to claim 18 in preparing a kit for detecting the presence or level of PD-1 and/or CTLA4 in a sample.

21. A pharmaceutical composition, comprising the bispecific antibody according to any of claims 1-14 or the conjugate according to claim 18, wherein, optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

22. The pharmaceutical composition according to claim 21, 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).

23. The pharmaceutical composition according to claim 21 or 22, wherein the unit dose of the pharmaceutical composition is 100-1000 mg, 200-800 mg, 200-500 mg, 300-400 mg, 400-500 mg, or 450 mg, based on the mass of the bispecific antibody.

24. A combination product comprising a first product and a second product in separate packages, wherein, the first product comprises the bispecific antibody according to any of claims 1-14, the conjugate according to claim 18, or the pharmaceutical composition according to any of claims 21-23; the second product comprises 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); preferably, the first product and the second product further independently comprise one or more pharmaceutically acceptable excipients; preferably, the combination product further comprises a package insert.

25. The combination product according to claim 24, wherein the unit dose of the first product 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 bispecific antibody.

26. The combination product according to claim 24 or 25, wherein the unit dose of the second product is 0.1-100 mg, 0.5-50 mg, 1-20 mg, 2-15 mg, 4-12 mg, or 8-12 mg, based on the mass of the active ingredient.

27. Use of the bispecific antibody according to any of claims 1-14 or the conjugate according to claim 18 in preparing a medicament for treating and/or preventing a tumor or anemia, or in preparing a medicament for diagnosing a tumor or anemia; 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, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal 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. Use of the bispecific antibody according to any of claims 1-14 or the conjugate according to claim 18 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; and/or a medicament for blocking the binding of CTLA4 to B7, a medicament for down-regulating the activity or level of CTLA4, a medicament for relieving the immunosuppression of CTLA4 in an organism, or a medicament for elevating IL-2 expression in T lymphocytes.

29. The bispecific antibody according to any of claims 1-14, the conjugate according to claim 18, the pharmaceutical composition according to any of claims 21-23 or the combination product according to any of claims 24-26 for use in treating and/or preventing a tumor or anemia, or for use in diagnosing a tumor or anemia; 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, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal 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.

30. A method for preventing and/or treating a tumor, comprising: administering to a subject in need an effective amount of the bispecific antibody according to any of claims 1-14, the conjugate according to claim 18, the pharmaceutical composition according to any of claims 21-23 or the combination product according to any one of claims 24-26; 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, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal 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.

31. The method according to claim 30, wherein the administration is before or after a surgical treatment and/or before or after a radiotherapy.

32. The method according to claim 30 or 31, wherein the unit dose of the bispecific antibody is 0.1-100 mg per kg body weight, preferably 1-10 mg per kg body weight; alternatively, the unit dose of the bispecific 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.

33. The method according to any of claims 30-32, wherein the administration of the bispecific antibody is performed in cycles of 2 or 3 weeks, and preferably, the bispecific antibody is administered intravenously on the first day of each cycle; preferably, the bispecific antibody is administered once every two or three weeks.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0256] FIG. 1: Affinity constant assay of BiAb004(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.

[0257] FIG. 2: Affinity constant assay of BiAb004(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.

[0258] FIG. 3: Affinity constant assay of BiAb004(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.

[0259] FIG. 4: Affinity constant assay of BiAb004(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.

[0260] FIG. 5: Affinity constant assay of BiAb004(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.

[0261] FIG. 6: Affinity constant assay of BiAb004(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.

[0262] FIG. 7: Affinity constant assay of BiAb004(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.

[0263] FIG. 8: Affinity constant assay of BiAb004(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.

[0264] FIG. 9: Affinity constant assay of BiAb004(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.

[0265] FIG. 10: Affinity constant assay of BiAb004(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.

[0266] FIG. 11: Affinity constant assay of BiAb004(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.

[0267] FIG. 12: Affinity constant assay of BiAb004(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.

[0268] FIG. 13: Affinity constant assay of BiAb004(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.

[0269] FIG. 14: Affinity constant assay of BiAb004(hG1WT) 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.

[0270] FIG. 15: 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.

[0271] FIG. 16: ADCC activity assay of BiAb004(hG1WT) and BiAb004(hG1TM) on 293T-CTLA4-PD1 cells expressing CTLA-4 and PD-1 antigens.

[0272] FIG. 17: Inhibition of subcutaneous human lung cancer HCC827 cell tumor xenograft by BiAb004(hG1TM) in combination with lenvatinib.

[0273] FIG. 18: Inhibition of subcutaneous colon cancer MC38-hPDL1/hCD73 tumor graft by BiAb004(hG1TM) in C57BL/6-hPD1/hPDL1/hCD73 mice.

[0274] FIG. 19: Antibody-mediated phagocytic activity of BiAb004(hG1TM) on CHO-K1-PD1.

[0275] FIG. 20: Significantly enhanced immune response of immune cells to human gastric cancer KATO III cells by BiAb004(hG1TM).

[0276] FIG. 21: Significantly enhanced immune response of immune cells to human cervical cancer Hela cells by BiAb004(hG1TM).

[0277] FIG. 22: Significantly enhanced immune response of immune cells to human T cell lymphomas Jurkat cells by BiAb004(hG1TM).

[0278] FIG. 23: Significantly enhanced immune response of immune cells to human nasopharyngeal cancer CNE-2Z cells by BiAb004(hG1TM).

[0279] FIG. 24: Significantly enhanced immune response of immune cells to human breast cancer MDA-MB-231 cells by BiAb004(hG1TM).

[0280] FIG. 25: Significantly enhanced immune response of immune cells to human mesothelioma NCI-H2452 cells by BiAb004(hG1TM).

[0281] FIG. 26: Significantly enhanced immune response of immune cells to human non-small cell lung cancer (human lung adenocarcinoma) A549 cells by BiAb004(hG1TM) in combination with anlotinib.

[0282] FIG. 27: Significantly enhanced immune response of immune cells to human small cell lung cancer NCI-H446 cells by BiAb004(hG1TM) in combination with anlotinib.

[0283] FIG. 28: Significantly enhanced immune response of immune cells to human squamous cell lung cancer NCI-H226 cells by BiAb004(hG1TM) in combination with anlotinib.

[0284] FIG. 29: Significantly enhanced immune response of immune cells to human colorectal cancer SW48 cells of MSI-H/dMMR phenotype by BiAb004(hG1TM).

[0285] FIG. 30: Significantly enhanced immune response of immune cells to human colorectal cancer SW837 cells of non-MSI-H/dMMR phenotype by BiAb004(hG1TM).

DETAILED DESCRIPTION

[0286] 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 used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. In the cases where the techniques or conditions are not specified, the examples were carried out 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.

[0287] In the following examples of the present invention:

[0288] BALB/c mice were purchased from Guangdong Medical Laboratory Animal Center.

[0289] Human peripheral blood mononuclear cells were isolated and prepared in Akeso Biopharma, Inc., with informed consent of the donor.

[0290] Raji-PDL1 is a cell expressing human PD-L1 constructed by Akeso Biopharma on the basis of human B cells Raji via transfection.

[0291] Ficoll-Paque™ PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare.

[0292] Human IL-2 ELISA kit was purchased from Dakewe Biotech Co., Ltd.

[0293] RPMI 1640 medium, DMEM medium, Trypsin-EDTA (0.25%) phenol red and Blastidin were all purchased from Gibco.

[0294] Staphylococcus aureus enterotoxin B (SEB) was purchased from Dianotech.

[0295] FBS was purchased from Excel bio.

[0296] Mitomycin C (MMC) was purchased from Stressmarq.

[0297] 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).

[0298] 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.

[0299] Lenvatinib used in the examples is lenvatinib mesylate under the brand name Lenvima®, and was purchased from Eisai (China).

Preparation Example 1: Sequence Design of Anti-CTLA4 Antibodies

[0300] The amino acid sequences and encoding nucleotide sequences of the heavy and light chains of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to those of 4G10, 4G10H1L1 and 4G10H3L3 in Chinese Patent Publication No. CN106967172A, respectively.

[0301] (1) Heavy and Light Chain Variable Region Sequences of 4G10

TABLE-US-00007 Nucleotide sequence of the heavy chain variable region: (372 bp) (SEQ ID NO: 1) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTC AATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCA TGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTT ATTAATCCTTACAATAATATTACTAACTACAACCAGAAGTTCATGGGCAA GGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCC TCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGAC TATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAA AACGACACCCCCATCTGTCTAT Encoded amino acid sequence: (124 aa) (SEQ ID NO: 2) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGL INPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLD YRSYWGQGTLVTVSAAKTTPPSVY Nucleotide sequence of the light chain variable region: (378 bp) (SEQ ID NO: 3) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAAC AGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACT TTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATA GGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTC CCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGG ATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTC GGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATC AGTCACCCTGTTTCAAGGGCAATTCTGC Encoded amino acid sequence: (126 aa) (SEQ ID NO: 4) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLI GGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVF GGGTKLTVLGQPKSSPSVTLFQGQFC

[0302] (2) Heavy and Light Chain Variable Region Sequences of Humanized Monoclonal Antibody 4G10H1L1

TABLE-US-00008 Nucleotide sequence of the heavy chain variable region (4G10H1V): (345 bp) (SEQ ID NO: 5) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTC CATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCA TGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTG ATTAATCCTTACAACAACATCACCAACTACAACCAGAAGTTCATGGGAAA AGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGA GCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGAT TATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT Encoded amino acid sequence: (115 aa) (SEQ ID NO: 6) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGL INPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLD YRSYWGQGTLVTVSA Nucleotide sequence of the light chain variable region (4G10L1V): (327 bp) (SEQ ID NO: 7) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAAC TGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACT TCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATC GGAGGCACAAACAATCGGGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAG CCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGG ACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTT GGCGGGGGAACAAAGCTGACTGTGCTG Encoded amino acid sequence: (109 aa) (SEQ ID NO: 8) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLI GGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVF GGGTKLTVL

[0303] (3) Heavy and Light Chain Variable Region Sequences of Humanized Monoclonal Antibody 4G10H3L3

TABLE-US-00009 Nucleotide sequence of the heavy chain variable region (4G10H3V): (345 bp) (SEQ ID NO: 9) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTC AGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCA TGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTG ATTAACCCTTACAACAACATCACTAACTACGCACAGAAGTTCCAGGGGAG AGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGT CCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGAT TACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA Encoded amino acid sequence (4G10H3V): (115 aa) (SEQ ID NO: 10) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGL INPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLD YRSYWCQGTLVTVSA Nucleotide sequence of the light chain variable region (4G10L3V): (327 bp) (SEQ ID NO: 11) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGAC TGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACT TCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATC GGAGGCACCAACAACAAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAG CCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGG ACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTC GGCGGCGGCACCAAGCTGACTGTGCTG Encoded amino acid sequence (4G10L3V): (109 aa) (SEQ ID NO: 12) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLI GGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVF GGGTKLTVL

Preparation Example 2: Sequence Design of Anti-PD-1 Antibody 14C12 and Its Humanized Antibody 14C12H1L1

[0304] 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 are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication No. CN106967172A, respectively.

[0305] (1) Heavy and Light Chain Variable Region Sequences of 14C12

TABLE-US-00010 Nucleotide sequence of the heavy chain variable region: (354 bp) (SEQ ID NO: 13) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTC ACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACA TGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACT ATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAG ATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGT CTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTAC GGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTC TGCC Encoded amino acid sequence: (118 aa) (SEQ ID NO: 14) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAT ISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRY GEAWFAYWGQGTLVTVSA Nucleotide sequence of the light chain variable region: (321 bp) (SEQ ID NO: 15) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGA GCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGT CTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGG GCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAG TGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGG GCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCA GGCACAAAACTGGAACTGAAG Encoded amino acid sequence: (107 aa) (SEQ ID NO: 16) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYR ANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGA GTKLELK

[0306] (2) Heavy and Light Chain Variable Region and Heavy and Light Chain Sequences of Humanized Monoclonal Antibody 14C12H1L1

TABLE-US-00011 Nucleotide sequence of the heavy chain variable region: (354 bp) (SEQ ID NO: 17) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTC ACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACA TGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACT ATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCG GTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGA ACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTAC GGGGAAGCATGGTTTGCCTATTGGGGGCAGGCAACCCTGGTGACACTCTC TAGT Encoded amino acid sequence: (118 aa) (SEQ ID NO: 18) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVAT ISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRY GEAWFAYWGQGTLVTVSS Nucleotide sequence of the light chain variable region: (321 bp) (SEQ ID NO: 19) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGA CAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGA GCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGG GCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAG CGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGG CAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCC GGGACAAAACTGGAGCTGAAG Encoded amino add sequence: (107 aa) (SEQ ID NO: 20) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYR ANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGA GTKLELK DNA sequence of 14C12H1L1 heavy chain (14C12H1): (1344 bp) (SEQ ID NO: 21) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTC ACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACA TGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACT ATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCG GTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGA ACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTAC GGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTC TAGTGCCAGCACCAAAGGACCTAGCGTGTTTCCTCTCGCCCCCTCCTCCA AAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTAC TTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGG AGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGT CCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATC TGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGA GCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCG AACTCCTCGGAGGCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGAC ACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGT CAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCG AGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACC TACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGG CAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTG AGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTAT ACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGAC CTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGT CCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGAC TCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCAG GTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTCC ACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA Encoded amino acid sequence: (448 aa) (SEQ ID NO: 22) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVAT ISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRY GEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  DNA sequence of 14C12H1L1 light chain (14C12L1): (642 bp) (SEQ ID NO: 23) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGA CAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGA GCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGG GCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAG CGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGG CAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCC GGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCAT TTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCT GTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTC GATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGA CTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGG CTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGA CTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT Encoded amino acid sequence: (214 aa) (SEQ ID NO: 24) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYR ANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGA GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Preparation Example 3: Sequence Design of Bifunctional Antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M)

[0307] The structural patterns of the bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) are in the Morrison format (IgG-scFv), i.e., C termini of two heavy chains of one IgG antibody are separately linked to the scFv fragment of another antibody via linker fragments. The components for the heavy and light chain design are shown in Table A below.

TABLE-US-00012 TABLE A Sequence design of BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) immunoglobulin moiety Bifunctional Heavy Light Linker antibody chain chain fragment scfv part BiAh001(M) 14C12H1 14C12L1 Linker1 4G10H1V(M)-Linker2- 4G10L1V(M) BiAh002(M) 14C12H1 14C12L1 Linker2 4G10H1V(M)-Linker2- 4G10L1V(M) BiAh003(M) 14C12H1 14C12L1 Linker1 4G10H3V(M)-Linker2- 4G10L3V(M) BiAh004(M) 14C12H1 14C12L1 Linker2 4G10H3V(M)-Linker2- 4G10L3V(M)

[0308] In Table A above:

TABLE-US-00013 (SEQ ID NO: 25) the amino acid sequence of Linker 1 is (GGGGS).sub.3, and (SEQ ID NO: 26) the amino acid sequence of Linker 2 is (GGGGS).sub.4.

[0309] In Table A above, scFv fragments 4G10H1V(M), 4G10L1V(M), 4G10H3V(M) and 4G10L3V(M) of BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) antibodies comprised mutations in individual amino acids of framework regions on the basis of 4G10H1V, 4G10L1V, 4G10H3V and 4G10L3V, respectively, which effectively optimized the structure of the antibodies and improved their efficacy.

TABLE-US-00014 (1) 4G10H1V(M): (115 aa, mutation positions underlined in the amino acid sequence based on 4G10H1V) (SEQ ID NO: 41) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ LEWIGL INPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLD YRSYWGQGTLVTVSA (2) 4G10L1V(M): (110 aa, mutation positions underlined in the amino acid sequence based on 4G10L1V) (SEQ ID NO: 42) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLI GGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVF G GTKLTVL (3) 4G10H3V(M): (115 aa, mutation positions underlined in the amino acid sequence based on 4G10H3V) (SEQ ID NO: 43) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ LEWIGL INPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLD YRSYWGQGTLVTVSA (4) 4G10L3V(M): (110 aa, mutation positions underlined in the amino acid sequence based on 4G10L3V) (SEQ ID NO: 44) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLI GGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVF G GTKLTVL

[0310] For distinguishment from the mutated antibodies hereinafter, BiAb004(M) is also referred to as BiAb004(hG1WT) in the this example. BiAb004(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.

Preparation Example 4: Non-Variable Region Amino Acid Mutation Design Based on Humanized Bifunctional Antibody BiAb004

[0311] On the basis of BiAb004(hG1WT) obtained in Preparation Example 3, BiAb004(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 heavy chain.

TABLE-US-00015 DNA sequence of heavy chain of the immunoglobulin moiety in BiAb004(hG1TM): (1344 bp, mutation positions underlined) (SEQ ID NO: 39) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTC ACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACA TGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACT ATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCG GTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGA ACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTAC GGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTC TAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCA AAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTAC TTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGG AGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGT CCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATC TGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGA GCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCG AA GGA CCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGA CACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATG TCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTC GAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCAC CTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACG GCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATT GAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTA TACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGA CCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAG TCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGA CTCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCA GGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTC CACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA Amino acid sequence of heavy chain of the immunoglobulin moiety in BiAb004(hG1TM): (448 aa, mutation positions underlined) (SEQ ID NO: 40) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVAT ISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRY GEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE G APSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWVQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

[0312] BiAb004(hG1TM) and BiAb004(hG1WT) share the same DNA sequence of light chain and the same encoded amino acid sequence. The specific sequence is shown in Preparation Example 3.

Experimental Example 1: Affinity Assay of BiAb004(hG1WT) and BiAb004(hG1TM) to Receptor FcγRI

[0313] 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.

[0314] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRI were measured in this experiment using a Fortebio Octet system to evaluate the potential ADCC and ADCP activities of the antibodies.

[0315] 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 PBS, 0.02% Tween-20 and 0.1% BSA, 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.

[0316] The results of affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) to FcγRI are shown in Table 1 and FIGS. 1 and 2 below.

TABLE-US-00016 TABLE 1 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγR1 Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 N/A N/A N/A N/A N/A N/A (hG1TM) BiAb004 5.92E−09 3.06E+05 8.35E+03 1.81E−03 5.75E−05 0.53-0.62 (hG1WT)

[0317] The results showed that BiAb004(hG1WT) bound to FcγRI with an affinity constant of 5.92E-09 M; while for BiAb004(hG1TM), no corresponding data was obtained as no binding signal or an extremely weak signal to FcγRI was detected, Indicating no binding to FcγRI.

[0318] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRI is effectively eliminated as compared to BiAb004(hG1WT).

Experimental Example 2: Affinity Constant Assay of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_V158

[0319] The Fc receptor FcγRIIa_V158 (also known as CD16a_V158), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0320] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIa_V158 were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0321] The method for determining the affinity constant of the antibodies to FcγRIIa_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 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.

[0322] The results of affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) to FcγRIIa_V158 are shown in Table 2 and FIGS. 3 and 4 below.

TABLE-US-00017 TABLE 2 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_V158 Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 N/A N/A N/A N/A N/A N/A (hG1TM) BiAb004 1.77E−07 1.21E+05 1.64E+04 2.14E−02 3.71E−03 1.56-4.61 (hG1WT)

[0323] The results showed that BiAb004(hG1WT) bound to FcγRIIIa_V158 with an affinity constant of 1.77E-07 M; while for BiAb004(hG1TM), no corresponding data was obtained as no binding signal or an extremely weak signal to FcγRIIIa_V158 was detected, indicating no binding to FcγRIIIa_V158.

[0324] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIIa_V158 is effectively eliminated as compared to BiAb004(hG1WT).

Experimental Example 3: Affinity Constant Assay of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_F158

[0325] The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0326] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_F158 were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0327] The method for determining the affinity constant of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_F158 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_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.

[0328] The results of affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) to FcγRIIIa_F158 are shown in Table 3 and FIGS. 5 and 6 below.

TABLE-US-00018 TABLE 3 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_F158 Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 N/A N/A N/A N/A N/A N/A (hG1TM) BiAb004 2.21E−07 1.12E+05 1.39E+04 2.47E−02 3.43E−03 0.39-0.64 (hG1WT)

[0329] The results showed that BiAb004(hG1WT) bound to FcγRIIIa_F158 with an affinity constant of 2.21E-07 M; while for BiAb004(hG1TM), no corresponding data was obtained as no binding signal or an extremely weak signal to FcγRIIIa_F158 was detected, indicating no binding to FcγRIIIa_F158.

[0330] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIIa_F158 is effectively eliminated as compared to BiAb004(hG1WT).

Experimental Example 4: Affinity Constant Assay of BiAb004(hG1WT) an BiAb004(hG1TM) to FcγRIIa_H131

[0331] The Fc receptor FcγRIIa_H131 (also known as CD32a_H131), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0332] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIa_H131 were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0333] The method for determining the affinity constant of BiAb064(hG1WT) and BiAb004(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 dilation buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, 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 PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, pH 7.4 for 300 s of blocking, and the binding of the immobilized FcγRIIIa_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.

[0334] The results of affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) to FcγRIIa_H131 are shown in Table 4 and FIGS. 7 and 8 below.

TABLE-US-00019 TABLE 4 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIIa_H131 Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 N/A N/A N/A N/A N/A N/A (hG1TM) BiAb004 2.22E−08 3.83E+05 4.03E+04 8.49E−03 7.44E−04 0.96-1.63 (hG1WT)

[0335] The results showed that BiAb004(hG1WT) bound to FcγRIIa_H131 with an affinity constant of 2.22E-08 M; while for BiAb004(hG1TM), no corresponding data was obtained as no binding signal or an extremely weak signal to FcγRIIa_H131 was detected, indicating no binding to FcγRIIa_H131.

[0336] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIa_H131 is effectively eliminated as compared to BiAb004(hG1WT).

Experimental Example 5: Affinity Constant Assay of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIa_R131

[0337] The Fc receptor FcγRIIa_R131 (also known as CD32a_R131), can bind to the Fc fragment of IgG antibodies and mediate ADCC effects.

[0338] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIa_R131 were measured in this experiment using a Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0339] The method for determining the affinity constant of BiAb004(hG1WT) and BiAb004(hG1TM) 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 PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, 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 PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, 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.

[0340] The results of affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) to FcγRIIa_R131 are shown in Table 5 and FIGS. 9 and 10 below.

TABLE-US-00020 TABLE 5 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIa_R131 Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 4.20E−08 3.72E+05 4.19E+04 1.56E−02 8.99E−04 0.16-0.36 (hG1TM) BiAb004 1.43E−08 3.58E+05 3.13E+04 5.10E−03 5.87E−04 0.66-1.34 (hG1WT)

[0341] The results showed that both BiAb004(hG1WT) and BiAb004(hG1TM) bound to FcγRIIa_R131 with affinity constants of 1.43E-08 M and 4.20E-08 M, respectively.

[0342] The results suggest that both BiAb004(hG1WT) and BiAb004(hG1TM) have binding activity to FcγRIIa_R131. However, the results in FIGS. 9 and 10 showed that BiAb004(hG1WT) had a stronger binding signal and thus a higher affinity than BiAb004(hG1TM).

Experimental Example 6: Affinity Constant Assay of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIb

[0343] 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.

[0344] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIb were measured in this experiment using a Fortebio Octet system to evaluate binding capacity of BiAb004(hG1WT) and BiAb004(hG1TM) to the Fc receptor.

[0345] The method for determining the affinity constant of BiAb004(hG1WT) and BiAb004(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 PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, pH 7.4. 5 μg/mL FcγRIIb was immobilized on the NTA sensor at an immobilization height of about 1.0 nm. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein and 0.1% BSA, pH 7.4 for 300 s of blocking, and the binding of the immobilized FcγRIIb 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.

[0346] The results of affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) to FcγRIIb are shown in Table 6 and FIGS. 11 and 12 below.

TABLE-US-00021 TABLE 6 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) to FcγRIIb Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 N/A N/A N/A N/A N/A N/A (hG1TM) BiAb004 5.61E−08 2.07E+05 1.54E+04 1.16E−02 4.33E−04 0.68-0.83 (hG1WT)

[0347] The results showed that BiAb004(hG1WT) bound to FcγRIIb with an affinity constant of 5.61E-08 M; while for BiAb004(hG1TM), no corresponding data was obtained as no binding signal or an extremely weak signal to FcγRIIb was detected, indicating no binding to FcγRIIb.

[0348] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIb is effectively eliminated as compared to BiAb004(hG1WT).

Experimental Example 7: Affinity Assay of BiAb004(hG1WT) and BiAb004(hG1TM) to C1q

[0349] 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.

[0350] The affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) to C1q were measured in this experiment using a Fortebio Octet system to evaluate the CDC activity of the antibodies. In this experimental example, an anti-PDL1 antibody 5C10H2L2-IgG1mt was used as a control, of which the preparation is described in PCT Publication No. WO2017148424A.

[0351] 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 PBS, 0.02% Tween-20 and 0.1% BSA, pH 7.4. 50 μg/mL antibody was immobilized on the FAB2G sensor at an immobilization height of about 2.0 am. 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.

[0352] The results of affinity constant assay of BiAb004(hG1TM), BiAb004(hG1WT) and 5C10H2L2-IgG1mt to C1q are shown in Table 7 and FIGS. 13, 14 and 15 below.

TABLE-US-00022 TABLE 7 Kinetics for binding of BiAb004(hG1WT) and BiAb004(hG1TM) and 5C10H2L2-lgG1mt to C1q Sample ID K.sub.D (M) kon (1/Ms) S E (kon) kdis (1/s) S E (kdis) Rmax (nm) BiAb004 N/A N/A N/A N/A N/A N/A (hG1TM) BiAb004 2.53E-09 2.05E+06 2.10E+05 5.17E−03 5.81E−04 0.05-0.23 (hG1WT) 5C10H2L2- 4.43E−09 2.38E+06 4.21E+05 1.05E−02 1.10E−03 0.19-0.26 lgG1mt

[0353] The results showed that BiAb004(hG1WT) bound to C1q with an affinity constant of 2.53E-09 M; while for BiAb004(hG1TM), no corresponding data was obtained as no binding signal or an extremely weak signal to C1q was detected, indicating no binding to C1q.

[0354] The results suggest that the binding activity of BiAb004(hG1TM) to C1q is effectively eliminated as compared to BiAb004(hG1WT).

Experimental Example 8: ADCC Activity Assay of BiAb004(hG1WT) and BiAb004(hG1TM) on 293T-CTLA4-PD1 Cells Expressing CTLA4 and PD-1 Antigens

[0355] The ADCC effect refers to that effector immune cells with killing activity recognize Fe fragments of antibodies bound to antigens of target cells through Fe receptors (FcR) expressed on their surfaces, and directly kill the target cells. To test the ADCC effect of the anti-CTLA4/anti-PD-1 bifunctional antibodies BiAb004(hG1WT) and BiAb004(hG1TM), the inventors constructed a co-culture system of 293T-CTLA4-PD1 cells expressing PD-1 and CTLA4 antigens and primary PBMCs to test the ADCC activity of the antibodies.

[0356] The method for ADCC activity assay of BiAb004(hG1WT) and BiAb004(hG1TM) on 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens is as follows:

[0357] Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation instruction. The isolated PBMCs were resuspended in RPMI-1640 complete medium, stained with AO/PI, counted and frozen. One day before the experiment, PBMCs were thawed and counted, and the viability was determined. The cells were incubated overnight in a carbon dioxide incubator at 5% CO.sub.2 and 37° C. On the day of the experiment, 293T-CTLA4-PD1 cells and PBMCs were collected, centrifuged at 800 rpm or 1200 rpm for 5 min, resuspended in RPMI-1640 (containing 1% FBS; hereinafter referred to as the medium) and washed twice. The cells were counted and viability was determined. The cell density was adjusted to a proper range using the medium. 100 μL of 293T-CTLA4-PD1 cells was added to a 96-well plate at a density of 3.0E+04 cells/well according to the experimental design. 50 μL of antibody was added, and the cells were pre-incubated for 1 h at room temperature; after 1 h, 50 μL of PBMCs were added at 9.0E+05 cells/well and the mixture was mixed well, incubated at 37° C. for 4 h in a carbon dioxide incubator at 5% CO.sub.2. The cells were centrifuged at 250×g for 5 min. 100 μL of supernatant was 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. ADCC percentage 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%.

[0358] The ADCC activity of BiAb004(hG1WT) and BiAb004(hG1TM) on 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens was expressed as ADCC percentages, and the results are shown in FIG. 16.

[0359] The results showed that in the mixed culture system of PBMC and 293T-CTLA4-PD1, the ADCC percentage induced by BiAb004(hG1TM) was significantly lower than that induced by BiAb004(hG1WT) at the same level.

[0360] The results suggest that BiAb004(hG1TM) has no ADCC activity.

Experimental Example 9: Growth Inhibition of Colorectal Cancer MC3S Cell Graf in Mice by BiAb004(hG1TM) in Combination with Lenvatinib

[0361] 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).

[0362] MC-38 cells (purchased from Shanghai Ruilu Biotech Co., Ltd.) were collected, adjusted to a density of 5 million cells/mL, and subcutaneously grafted into PD-1 transgenic mice (purchased from Shanghai Model Organisms Center, Inc.) in a volume of 200 μL/mouse at the right flank. 12 mice were grafted, each bearing 1×10.sup.6 cells. When the tumor volume reached about 60-150 mm.sup.3, the mice were divided by tumor volume (with similar average volume of approximately 94 mm.sup.3) into 2 groups, model group and BiAb004(hG1TM)/lenvatinib treatment group, each containing 6 mice. The day of grouping was set as D0. 6 doses of antibody were intraperitoneally administered on D0, D4, D7, D10, D13 and D17, respectively; lenvatinib was administered daily by oral gavage for 20 days. Tumor volume was measured by vernier caliper. Tumor dimensions were measured twice weekly after grouping using vernier caliper, and tumor volume was calculated according to the formula TV=0.5×ab.sup.2, wherein a is the longest diameter of the tumor, b is the shortest diameter of the tumor, and TV is the volume of the tumor. The specific protocol is shown in Table 8.

TABLE-US-00023 TABLE 8 Study design Number of Model Group animals establishment Regimen Model group 6 MC-38 cells Anti-HEL antibody (i.e., isotype were grafted control antibody) at 1 mg/kg was subcutaneously injected intraperitoneally twice at a dose of 1 weekly in a total of 6 doses BiAb004(hG1TM) 6 million cells/200 BiAb004(hG1TM) at 1.33 mg/kg was Ienvatinib group μL/mouse injected intraperitoneally twice weekly in a total of 6 doses; Lenvatinib at 5 mg/kg was administered by oral gavage once daily in 20 doses

[0363] The sequence of the isotype control in this experiment, 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):130-46).

[0364] The experimental results are shown in FIG. 17.

[0365] The results showed that BiAb004(hG1TM) in combination with lenvatinib significantly inhibited the growth of MC38 tumors; BiAb004(hG1TM) in combination with lenvatinib demonstrated significantly efficacy against rectal and/or colon cancers of MSI-H/dMMR phenotype.

Experimental Example 10: Pharmacodynamic Evaluation of BiAb004(hG1TM) in C57BL/6-hPD1/hPDL1/hCD73 Mouse Colon Cancer MC38-hPDL1/hCD73 Subcutaneous Graft Tumor Model

[0366] 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 isotype control antibody (the preparation and the source are the same as those of the Experimental Example 9) or BiAb004(hG1TM) was administrated intraperitoneally twice weekly for 3 weeks, in a total of 6 doses. 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.

[0367] The results are shown in FIG. 18 and in Table 9 below.

TABLE-US-00024 TABLE 9 Protocol and grouping Dose Group (mg/kg) Route Frequency Cycle Isotype Control 10 Intraperitoneal Twice weekly, 6 doses in BiAb004 1 injection for a total of total (hG1TM) three weeks

[0368] The results showed that the tumor volume increase in the BiAb004(hG1TM) group was inhibited relative to the isotype control antibody group, indicating that BiAb004(hG1TM) significantly inhibits the proliferation of mouse MC38 cells, and can effectively treat colon cancer and/or rectal cancer.

Experimental Example 11: Antibody-Mediated Phagocytic Activity Study of BiAb004(hG1WT) and BiAb004(hG1TM) on CHO-K1-PD1

[0369] Zhang et al. (Zhang T et al, Cancer Immunol Immunother., 2018: 67(7):1079-1090.) and Dahan et al. (Dahan R et al., Cancer cell, 2015, 28(3):285-95.) reported that the binding of Fc fragments of antibodies targeting immune checkpoints such as PD-1 and CTLA-4 to Fc receptors negatively affects antibody-mediated anti-cancer activity, possibly due to Fc-dependent effector function-induced immune cell damage, where antibody-dependent cellular phagocytosis (ADCP) is an important mechanism leading to immune cell damage.

[0370] To test the ADCP activity of BiAb004(hG1TM), marine macrophages were used as effector cells and cell lines overexpressing the corresponding antigens were used as target cells. The ADCP effect mediated by the cells was tested. The femoral bone marrow of C57BL/6 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 macrophage colony stimulating factor (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 0.25% trypsin. Macrophages were collected, and centrifuged at 170×g for 5 min. The supernatant was discarded and the cells were suspended in DMEM complete medium and counted. The cell was adjusted to a proper density and filled into sterile EP tubes for further use.

[0371] CHO-K1-CTLA4-PD1 cells (a cell line based on CHO-K1 cells overexpressing both human CTLA4 and PD1 antigens, constructed by Akeso Biopharma, Inc.) at logarithmic phase were collected, centrifuged at 170×g for 5 min, washed with PBS, resuspended and counted, and 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). 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 for resuspension. The cells were incubated in an incubator for 10 min, and adjusted to the experiment density. The cells were coded as CHO-K1-PD1-CTLA4-CFSE. The test antibodies were diluted in DMEM complete medium. An isotype control antibody anti-HEL antibody and a medium were used as the isotype control group and a blank control group. According to the study design, the diluted antibody and CHO-K1-PD1-CTLA4-CFSE cells were added into 1.5 mL EP tubes containing macrophages (the final volume was 100 μL, the effector-to-target ratio was 50,000:150,000, the working concentrations of the antibody were 50, 5 and 0.5 nM). The mixture was resuspended, mixed evenly 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 1200×g for 5 min, and the supernatant was discarded. The cells were washed once with 800 μL of 1% PBSA. APC anti-mouse/human CDIIb antibody (Biolegend, Cat. No.: 101212) was diluted 400-fold with 1% PBSA and added to the corresponding samples at 100 μL/sample. The mixture was mixed well, incubated on ice for 30 min, washed once with 800 μL of 1% PBSA, and centrifuged at 1200×g for 5 min, 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 FACSCalibur 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.

[0372] The ADCP activity of each group, expressed as P %, was calculated as follows:

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

[0373] The results are shown in FIG. 19.

[0374] The results showed that in the antibody validation system for mediating phagocytic activity, the phagocytic activity of CHO-K1-PD1-CTLA4 cells by macrophages mediated by BiAb004(hG1TM) had no significant difference from that of the isotype control antibody anti-HEL antibody in comparison with BiAb004(hG1WT), indicating that BiAb004(hG1TM) had no ADCP activity.

[0375] The results suggest that the amino acid mutation introduced by BiAb004(hG1TM) can effectively eliminate the ADCP effect, and a surprising technical effect is obtained.

Experimental Example 12: Significantly Enhanced Immune Response of Immune Cells to Human Gastric Cancer KATO III Cells by BiAb004(hG1TM)

[0376] 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 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 mitomycin C (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. Ater 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.

[0377] The results are shown in FIG. 20.

[0378] The results showed that BiAb004(hG1TM) more significantly enhanced the immune response of immune cells to human gastric cancer cells KATO III characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT), and thus BiAb004(hG1TM) is prospective for treating gastric cancer.

Experimental Example 13: Significantly Enhanced Immune Response of Immune Cells to Human Cervical Cancer Hela Cells by BiAb004(hG1TM)

[0379] 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 Hela cells (purchased from Chinese Academy of Sciences Cell Bank) 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. Hela 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.

[0380] The results are shown in FIG. 21.

[0381] The results showed that BiAb004(hG1TM) more significantly enhanced the immune response of immune cells to human cervical cancer Hela cells characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT), and thus BiAb004(hG1TM) is prospective for treating cervical cancer.

Experimental Example 14: Significantly Enhanced Immune Response of Immune Cells to Human T Cell Lymphomas Jurkat Cells by BiAb004(hG1TM)

[0382] 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 human T-cell lymphoma Jurkat cells (purchased from Chinese Academy of Sciences Cell Bank) 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. Jurkat cells in logarithmic growth phase were collected and seeded on the 96-well plate at 1×10.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.

[0383] The results are shown in FIG. 22.

[0384] BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human T-cell lymphoma Jurkat cells characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT), while eliminating the ADCC and CDC (i.e., ADCP); the antibody has an equivalent or higher pharmacological activity at doses of 1.34 nM and 20 nM as compared to BiAb004(hG1WT) and is prospective for treating cervical cancer.

Experimental Example 15: Significantly Enhanced Immune Response of Immune Cells to Human Nasopharyngeal Cancer CNE-2Z Cells by BiAb004(hG1TM)

[0385] 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 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.

[0386] The results are shown in FIG. 23.

[0387] The results showed that BiAb004(hG1TM) equivalently and/or more significantly enhanced the immune response of immune cells to human nasopharyngeal cancer cells CNE-2Z characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT), and that BiAb004(hG1TM) especially had significant superiority at 20 nM.

[0388] The above results showed that BiAb004(hG1TM) had better or equivalent pharmacological activity relative to BiAb004(hG1WT) on the basis of effectively eliminating ADCC, CDC and ADCP effects, indicating the potential for treating human nasopharyngeal cancer.

Experimental Example 16: Significantly Enhanced Immune Response of Immune Cells to Human Breast Cancer MDA-MB-231 Cells by BiAb004(hG1TM)

[0389] 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 MDA-MB-231 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. MDA-MB-231 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 3×10.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+RPM 11640.

[0390] The results are shown in FIG. 24.

[0391] BiAb004(hG1TM) equivalently or more significantly enhanced the immune response of immune cells to human breast cancer MDA-MB-231 cells characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT).

[0392] The above results showed that BiAb004(hG1TM) had better or equivalent pharmacological activity relative to BiAb004(hG1WT) on the basis of effectively eliminating ADCC, CDC and ADCP effects, indicating the potential for treating human breast cancer.

Experimental Example 17: Significantly Enhanced Immune Response of Immune Cells to Human Mesothelioma NCI-112452 Cells by BiAb004hG1TM)

[0393] 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-H2452 cells (purchased from Chinese Academy of Sciences Shanghai Cell Bank) 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-H2452 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 3×10.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.

[0394] The results are shown in FIG. 25.

[0395] The results showed that BiAb004(hG1TM) more significantly enhanced the immune response of immune cells to human mesothelioma cells NCI-H2452 characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT), and thus BiAb004(hG1TM) is prospective for treating mesothelioma.

Experimental Example 18: Significantly Enhanced Immune Response of Immune Cells to Human Non-Small Cell Lune Cancer (Human Lune Adenocarcinoma) A549 Cells by BiAb004(hG1TM) in Combination with Anlotinib

[0396] 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 A549 cells 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. A549 cells (purchased from Chinese Academy of Sciences Cell Bank) 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.

[0397] As shown in FIG. 26, BiAb004(hG1TM) in combination with anlotinib hydrochloride more significantly enhanced the immune response of immune cells to human non-small cell lung cancer (human lung adeaocarcinoma) A549 cells characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1TM) monotherapy, BiAb004(hG1WT) monotherapy and BiAb004(hG1VT) in combination with anlotinib hydrochloride, and thus BiAb884(hG1TM) In combination with anlotinib hydrochloride is prospective for treating human non-small cell lung cancer or human lung adenocarcinoma.

Experimental Example 19: Significantly Enhanced Immune Response of Immune Cells to Human Small Cell Lone Cancer NCI-H446 Cells by BiAb004(hG1TM) in Combination with Anlotinib

[0398] 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-H446 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-H446 cells (purchased from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) in logarithmic growth phase were collected and seeded on the 96-well plate at 8×10.sup.4 cells/well. The diluted antibody 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.

[0399] The results are shown in FIG. 27.

[0400] BiAb004(hG1TM) in combination with anlotinib hydrochloride significantly enhanced the Immune response of immune cells to human small cell lung cancer NCI-H446 cells characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1MT) monotherapy. BiAb004(hG1WT) monotherapy and BiAb004(hG1WT) in combination with anlotinib hydrochloride, and thus BiAb004(hG1TM) in combination with anlotinib hydrochloride is prospective for treating human small cell lung cancer.

Experimental Example 20: Significantly Enhanced Immune Response of Immune Cells to Human Squamous Cell Lune Cancer NCI-H226 Cells by BiAb004(hG1TM) in Combination with Anlotinib

[0401] 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-H226 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-H226 cells in logarithmic growth phase were collected and seeded on the 96-well plate at 5×10.sup.4 cells/well. The diluted antibody 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.

[0402] The results are shown in FIG. 28.

[0403] BiAb004(hG1TM) monotherapy more effectively and significantly enhanced the immune response of immune cells to human squamous cell lung cancer NCI-H226 ceils characterized by significantly increased secretion level of IL-2 as compared to BiAb004(hG1WT) monotherapy.

[0404] BiAb004(hG1TM) in combination with anlotinib hydrochloride more significantly enhanced the immune response of immune cells to human squamous cell lung cancer NCI-H226 cells as compared to BiAb004(hG1WT) monotherapy and BiAb004(hG1TM) monotherapy, and demonstrated a comparable pharmacological activity with BiAb004(hG1WT) in combination with anlotinib.

[0405] The above results showed that BiAb004(hG1TM) had better or equivalent pharmacological activity relative to BiAb004(hG1WT) monotherapy or BiAb004(hG1WT) in combination with anlotinib on the basis of effectively eliminating ADCC, CDC and ADCP effects, Indicating a better efficacy for treating human squamous cell lung cancer.

Experimental Example 21: Significantly Enhanced Immune Response of Immune Cells to Human Colorectal Cancer SW48 Cells of MSI-H1/dMMR Phenotype by BiAb004(hG1TM)

[0406] 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 BiAb004(hG1TM).

[0407] In the experiment, 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 sg/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 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.

[0408] The results are shown in FIG. 29.

[0409] Both BiAb004(hG1WT) and BiAb004(hG1TM) 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.

[0410] The above results showed that BiAb004(hG1TM) had better or equivalent pharmacological activity relative to BiAb004(hG1WT) on the basis of effectively eliminating ADCC, CDC and ADCP effects, indicating the potential for treating solid tumor of MSI-H/dMMR phenotype, particularly colon cancer and/or rectal cancer of MSI-H/dMMR phenotype.

Experimental Example 22: Significantly Enhanced Immune Response of Immune Cells to Human Colorectal Cancer SW837 Cells of MSI-H/dMMR Phenotype by BiAb004(hG1TM)

[0411] 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 phenotype by BiAb004(hG1TM) in this example.

[0412] 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 (purchased from Gibco) 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% CO2 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.

[0413] The media in this experiment were all 10% FBS+RPMI 1640.

[0414] The results are shown in FIG. 30.

[0415] Both BiAb004(hG1WT) and BiAb004(hG1TM) significantly enhanced the immune response of immune cells to human colorectal cancer cells SW837 cells of non-MSI-H/dMMR phenotype as compared to anti-HEL antibody. The pharmacological activity of BiAb004(hG1TM) in the high dose group was superior to that of BiAb004(hG1WT), characterized by significantly increased secretion level of IL-2.

[0416] The above results showed that BiAb004(hG1TM) bad better or equivalent pharmacological activity relative to BiAb004(hG1WT) on the basis of effectively eliminating ADCC, CDC or ADCP effects, indicating the potential for treating solid tumor of non-MSI-H/dMMR phenotype, particularly colon cancer and/or rectal cancer of non-MSI-H/dMMR phenotype.

[0417] 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.