Anti-CTLA4 and anti-PD-1 bifunctional antibody, pharmaceutical composition thereof and use thereof

Abstract

An anti-CTLA4 (cytotoxic T lymphocyte associated antigen 4) and anti-PD-1 (programmed cell death 1) bifunctional antibody. a pharmaceutical composition thereof and use thereof. Particularly, the anti-CLTA4 and anti-PD-1 bifunctional antibody comprises a first protein functional domain that targets PD-1 and a second protein functional domain that targets CTLA-4. The bifunctional antibody can bind to CTLA-4 and PD-1 specifically, relieve immunosuppression of CTLA4 and PD-1 on an organism specifically, activate T lymphocytes, and thus has good application prospects.

Claims

1. A bispecific antibody, or an antigen-binding fragment thereof, comprising: (a) a first protein functional area that binds to PD-1 and comprises six CDRs with the amino acid sequences of SEQ ID NOs: 29-34, and (b) a second protein functional area that binds to CTLA-4 and comprises six CDRs with the amino acid sequences selected from the group consisting of: (i) SEQ ID NOs: 35-40, (ii) SEQ ID NOs: 35, 41 and 37-40, and (iii) SEQ ID NOs: 42-47.

2. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the first protein functional area and the second protein functional area are directly connected or connected by a connecting fragment.

3. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the first protein functional area and the second protein functional area are individually contained in immunoglobulins, half antibodies, Fab, F(ab′)2, or single-chain antibodies.

4. The bispecific antibody, or the antigen-binding fragment thereof, of claim 3, wherein the immunoglobulin is IgG, IgA, IgD, IgE, or IgM.

5. The bispecific antibody, or the antigen-binding fragment thereof, of claim 4, wherein the immunoglobulin is IgG.

6. The bispecific antibody, or the antigen-binding fragment thereof, of claim 5, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

7. The bispecific antibody, or the antigen-binding fragment thereof, of claim 3, wherein the single-chain antibody is attached at the c-terminus of the heavy chain of the immunoglobulin.

8. The bispecific antibody, or the antigen-binding fragment thereof, of claim 3, wherein the immunoglobulin, or the antigen-binding fragment thereof, comprises non-CDR regions from species other than mouse.

9. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, comprising one, two, or more first protein functional areas and one, two, or more second protein functional areas.

10. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein: the first protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 29-31, and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 32-34; and/or, the second protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 35-37, or SEQ ID NOs: 35, 41, and 37, or SEQ ID NOs: 42-44; and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 38-40 or SEQ ID NOs: 45-47.

11. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein: the first protein functional area comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 16 or 20, and a light chain variable region with the amino acid sequence of SEQ ID NO: 18 or 22; and the second protein functional area comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, or 25, and a light chain variable region with the amino acid sequence of SEQ ID NO: 4, 8, 12, or 27.

12. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the bispecific antibody, or the antigen-binding fragment thereof, binds to CTLA-4 protein and/or PD-1 protein with a binding affinity (K.sub.D) that is less than 10.sup.−5M.

13. A composition comprising one or more isolated nucleic acid molecules, wherein the one or more isolated nucleic acid molecules comprises one or more nucleotide sequences encoding the first protein functional area and the second protein functional area of the bispecific antibody of claim 1.

14. A vector comprising one or more isolated nucleic acid molecules, wherein the one or more isolated nucleic acid molecules comprises one or more nucleotide sequences encoding the first protein functional area and the second protein functional area of the bispecific antibody of claim 1.

15. A host cell line comprising one or more isolated nucleic acid molecules, wherein the one or more isolated nucleic acid molecules comprises one or more nucleotide sequences encoding the first protein functional area and the second protein functional area of the bispecific antibody of claim 1.

16. A method for preparing a bispecific antibody, or an antigen-binding fragment thereof, comprising culturing the host cell line of claim 15 under appropriate conditions, and recovering a bispecific antibody, or an antigen-binding fragment thereof, from the cell culture.

17. A conjugate, comprising the bispecific antibody, or the antigen-binding fragment thereof, of claim 1 and a conjugating partner as a detectable marker.

18. A reagent kit, comprising the bispecific antibody, or the antigen-binding fragment thereof, of claim 1.

19. A method to detect the existence or the levels of CTLA-4 and/or PD-1 in a sample, comprising contacting the sample with the bispecific antibody, or the antigen-binding fragment thereof, of claim 1.

20. A pharmaceutical composition comprising the bispecific antibody, or the antigen-binding fragment thereof, of claim 1 and a pharmaceutically acceptable carrier or excipient.

21. A method for treating or diagnosing tumors in a subject, wherein the method comprises administering to the subject an effective dose of the bispecific antibody, or the antigen-binding fragment thereof, of claim 1, or a conjugate thereof.

22. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein: the first protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 29-31 and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 32-34, and the second protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 35-37 and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 38-40.

23. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein: the first protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 29-31 and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 32-34, and the second protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 35, 41, and 37 and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 38-40.

24. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein: the first protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 29-31 and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 32-34, and the second protein functional area comprises a heavy chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 42-44 and a light chain variable region comprising CDRs with the amino acid sequences of SEQ ID NOs: 45-47.

25. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the first protein functional area comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 20 and a light chain variable region with the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 22.

26. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the second protein functional area comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 and a light chain variable region with the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 12.

27. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the second protein functional area comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 14 and a light chain variable region with the amino acid sequence of SEQ ID NO: 12.

28. The bispecific antibody, or the antigen-binding fragment thereof, of claim 1, wherein the second protein functional area comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO: 25 and a light chain variable region with the amino acid sequence of SEQ ID NO: 27.

29. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody or the antigen-binding fragment thereof of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 SDS-PAGE Results of Monoclonal Antibody 4G10. From left to right: 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 5 μL Marker; 1 μg BSA.

(2) FIG. 2 SDS-PAGE Results of Monoclonal Antibody 4G10H1L1. From left to right: 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 5 μL Marker.

(3) FIG. 3 SDS-PAGE Results of Monoclonal Antibody 4G10H3L3. From left to right: 1 μg antibody in reduced loading buffer; 5 μL Marker.

(4) FIG. 4 SDS-PAGE Results of Monoclonal Antibody 14C12H1L1. From left to right: 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 5 μL Marker; 1 μg BSA.

(5) FIG. 5 SDS-PAGE Results of Bispecific Antibody BiAb001. From left to right: 5 μL Marker; 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 1 μg BSA.

(6) FIG. 6 SDS-PAGE Results of Bispecific Antibody BiAb002. From left to right: 5 μL Marker; 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 1 μg BSA.

(7) FIG. 7 SDS-PAGE Results of Bispecific Antibody BiAb003. From left to right: 5 μL Marker; 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 1 μg BSA.

(8) FIG. 8 SDS-PAGE Results of Bispecific Antibody BiAb004. From left to right: 5 μL Marker; 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 1 μg BSA.

(9) FIG. 9 SDS-PAGE Results of Bispecific Antibody BiAb007. From left to right: 5 μL Marker; 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 1 μg BSA.

(10) FIG. 10 SDS-PAGE Results of Bispecific Antibody BiAb010. From left to right: 5 μL Marker; 1 μg antibody in non-reduced loading buffer; 1 μg antibody in reduced loading buffer; 1 μg BSA.

(11) FIG. 11. Binding kinetics of antibody 4G10

(12) FIG. 12. Binding kinetics of antibody 4G10 H1L1

(13) FIG. 13. Binding kinetics of antibody 4G10H3L3

(14) FIG. 14. Binding kinetics of antibody 4G10H4L3

(15) FIG. 15. Binding kinetics of antibody 14C12

(16) FIG. 16. Binding kinetics of antibody 14C12 H1L1

(17) FIG. 17. Binding kinetics of CTLA4 and antibody BiAb001

(18) FIG. 18. Binding kinetics of CTLA4 and antibody BiAb002

(19) FIG. 19. Binding kinetics of CTLA4 and antibody BiAb003

(20) FIG. 20. Binding kinetics of CTLA4 and antibody BiAb004

(21) FIG. 21. Binding kinetics of CTLA4 and antibody BiAb007

(22) FIG. 22. Binding kinetics of PD-1 and antibody BiAb001

(23) FIG. 23. Binding kinetics of PD-1 and antibody BiAb002

(24) FIG. 24. Binding kinetics of PD-1 and antibody BiAb003

(25) FIG. 25. Binding kinetics of PD-1 and antibody BiAb004

(26) FIG. 26. Binding kinetics of PD-1 and antibody BiAb007

(27) FIG. 27. Binding kinetics of PD-1 and antibody BiAb010

(28) FIG. 28. Indirect ELISA results of 4G10H1L1 and 4G10H3L3 binding to CTLA4.

(29) FIG. 29. Competition ELISA results of 4G10H1L1 and 4G10H3L3 binding to CTLA4 against B7.

(30) FIG. 30. Indirect ELISA results of 14C12 and 14C12H1L1 binding to PD-1.

(31) FIG. 31. Competition ELISA results of 14C12 and 14C12H1L1 binding to PD-1 against PDL1.

(32) FIG. 32. Indirect ELISA results of BiAb001, BiAb002, BiAb003, and BiAb004 binding to CTLA4.

(33) FIG. 33. Indirect ELISA results of BiAb001, BiAb002, BiAb003, and BiAb004 binding to PD-1.

(34) FIG. 34. Competition ELISA results of BiAb001, BiAb002, BiAb003, and BiAb004 binding to CTLA4 against B7.

(35) FIG. 35. Competition ELISA results of BiAb001, BiAb002, BiAb003, and BiAb004 binding to PD-1 against PDL1.

(36) FIG. 36. EC50 of 4G10H1L1 binding to CTLA4 on the Surface of 293T-CTLA4 Cells.

(37) FIG. 37. EC50 of 4G10H3L3 binding to CTLA4 on the Surface of 293T-CTLA4 Cells.

(38) FIG. 38. EC50 of 14C12H1L1 binding to PD-1 on the Surface of 293T-PD-1 Cells.

(39) FIG. 39. EC50 of BiAb001 binding to CTLA4 on the Surface of 293T-CTLA4 Cells.

(40) FIG. 40. EC50 of BiAb002 binding to CTLA4 on the Surface of 293T-CTLA4 Cells.

(41) FIG. 41. EC50 of BiAb003 binding to CTLA4 on the Surface of 293T-CTLA4 Cells.

(42) FIG. 42. EC50 of BiAb004 binding to CTLA4 on the Surface of 293T-CTLA4 Cells.

(43) FIG. 43. EC50 of BiAb001 binding to PD-1 on the Surface of 293T-PD-1 Cells.

(44) FIG. 44. EC50 of BiAb002 binding to PD-1 on the Surface of 293T-PD-1 Cells.

(45) FIG. 45. EC50 of BiAb003 binding to PD-1 on the Surface of 293T-PD-1 Cells.

(46) FIG. 46. EC50 of BiAb004 binding to PD-1 on the Surface of 293T-PD-1 Cells.

(47) FIG. 47. Binding activity of 4G10H3L3 to T Cell Surface Antigen CTLA4.

(48) FIG. 48. Binding activity of 14C12H1L1 to T Cell Surface Antigen PD-1.

(49) FIG. 49. Binding activity of BiAb003 and BiAb004 to T Cell Surface Antigens compared with those of 14C12H1L1 and 4G10H3L3.

(50) FIG. 50. Effects of 4G10H1L1 and 4G10H3L3 on IFN-γ Secretion of Mixed Lymphocytes.

(51) FIG. 51. Effect of 14C12H1L1 on IFN-γ Secretion of Mixed Lymphocytes.

(52) FIG. 52. Effects of BiAb001 and BiAb002 on IFN-γ Secretion of Mixed Lymphocytes compared with those of 14C12H1L1 and 4G10H1L1.

(53) FIG. 53. Effects of BiAb003 and BiAb004 on IFN-γ Secretion of Mixed Lymphocytes compared with effects of 14C12H1L1 and 4G10H3L3.

(54) FIG. 54. Effect of 4G10H3L3 on IL-2 Secretion of Mixed Lymphocytes.

(55) FIG. 55. Effect of 14C12H1L1 on IL-2 Secretion of Mixed Lymphocytes.

(56) FIG. 56. Effects of BiAb003 and BiAb004 on IL-2 Secretion of Mixed Lymphocytes compared with those of 14C12H1L1 and 4G10H3L3.

(57) FIG. 57. Effects of 4G10H1L1 and 4G10H3L3 on IL-2 Secretion induced by co-culturing of PBMC, MDA-MB-231 and Raji cells.

(58) FIG. 58. Effect of 14C12H1L1 on IL-2 Secretion induced by co-culturing of PBMC, MDA-MB-231 and Raji cells.

(59) FIG. 59. Effect of BiAb001, BiAb002, BiAb003, and BiAb004 on IL-2 Secretion induced by co-culturing of PBMC, MDA-MB-231 and Raji cells, compared with those of 4G10H1L1, 4G10H3L3, and 14C12H1L1.

(60) FIG. 60. Effect of BiAb004 on the Tumor Growth of MC38 Tumor Model in PD-1 HuGEMM Mice.

DESCRIPTION OF THE DEPOSITED BIOLOGICAL MATERIALS

(61) LT002 (CTLA4-4G10), a hybridoma cell line, was preserved in China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015. Deposit Accession NO.: C201587, Depository address: Wuhan university, Wuhan, China, zip code: 430072.

(62) LT003 (PD-1-14C12), a hybridoma cell line, was preserved in China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015. Deposit Accession NO.: C2015105, Depository address: Wuhan university, Wuhan, China, zip code: 430072.

DETAILED DESCRIPTION OF THE INVENTION

(63) The invention will now be described in detail. As will be appreciated by one skilled in the art, the following examples are only used for the description of the invention, and not to be deemed to limit the scope of the invention. The cases without the specific descriptions of techniques or conditions were carried out in accordance with the literature in the field (e.g., Guide to Molecular Cloning, written by J Sambrook, et al, translated by Peitang Huang, et al, third Edition, Science Press) or in accordance with the product instruction manual. The reagents or instruments with no specified manufacturer were all conventional products available commercially.

(64) In the embodiments of the present invention, the T cells used were from Akeso Biopharma, Inc., the BALB/C mice were purchased from the Guangdong Medical Laboratory Animal Center. The PD-1 HuGEMM mice used were from Nanjing Galaxy Biopharma Co., Ltd.; MC38 cells were from Shanghai Fudan IBS Cell Center.

Example 1: Preparation of Anti-CTLA4 Antibody 4G10

(65) 1. Establishment of Hybridoma Cell Line LT002

(66) Using CTLA4-mFc (a fusion protein of human CTLA4 protein (GenbankID: NP 005205.2) extracellular region and mouse IgG1Fc protein) as the antigen, the hybridoma cells were obtained by fusing the splenocytes of immunized BALB/C mice (purchased from Guangdong Medical Laboratory Animal Center) and mouse myeloma cells with currently established method (for example, Stewart, S. J., “Monoclonal Antibody Production”, in Basic Methods in antibody Production and Characterization, Eds. G. C. Howard and D. R. Bethell, Boca Raton: CRC Press, 2000).

(67) The CTLA4 protein was generated by digesting the fusion protein CTLA4-mFc with TEV protease, and further purified by purification column. Microplate was coated with CTLA4 as the antigen, and the above hybridoma cells were screened by indirect ELISA to select those secreting new antibodies specifically binding to CTLA4. The hybridoma cells screened via indirect ELISA were further screened by competitive ELISA against ligand B7-1 (CD80, NCBI Gene ID: 941) and B7-2 (CD86, NCBI Gene ID: 942) to select those secreting monoclonal antibodies that competitively bind to CTLA4, and then a stable hybridoma cell line was obtained by limited dilution method. This hybridoma cell line was named LT002 (CTLA4-4G10), and its secreted monoclonal antibody is named 4G10.

(68) LT002 (CTLA4-4G10), the hybridoma cell line, was preserved in China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015. Deposit Accession NO.: C201587, Depository address: Wuhan university, Wuhan, China, postcode: 430072.

(69) 2. Preparation of Anti-CTLA4 Antibody 4G10

(70) The LT002 cells in the present invention were cultured using IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium containing 1% streptomycin, cultured in cell incubator with 5% CO.sub.2, 37° C.), and then the cell culture supernatant was harvested and purified by high-speed centrifugation after 7 days culture, filtration through microporous membrane, and HiTrap protein A HP column to get the antibody 4G10. The purified 4G10 were identified on SDS-PAGE electrophoresis, and the result was shown in FIG. 1.

Example 2: Sequence Analysis of Anti-CTLA4 Antibody 4G10

(71) Sequence Analysis of Antibody 4G10

(72) mRNA was extracted from the hybridoma cell line LT002 prepared in Example 1 above according to the manual of the cell/bacterial total RNA extraction reagent kit (Tiangen, Product No DP430).

(73) cDNA was synthesized using Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR, and amplified by PCR.

(74) TA cloning was directly carried out on the PCR amplified product according to the instructions of pEASY-T1 Cloning Kit (Transgen CT101).

(75) The products of TA cloning were directly sequenced, and the sequencing results were as follows:

(76) Nucleic Acid Sequence of Heavy Chain Variable Region: (372 bp)

(77) TABLE-US-00012 (SEQ ID NO: 1) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTC AATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCA TGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTT ATTAATCCTTACAATAATATTACTAACTACAACCAGAAGTTCATGGGCAA GGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCC TCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGAC TATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAA AACGACACCCCCATCTGTCTAT

(78) Encoded Amino Acid Sequence: (124 aa)

(79) TABLE-US-00013 (SEQ ID NO: 2) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGL INPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLD YRSYWGQGTLVTVSAAKTTPPSVY

(80) Nucleic Acid Sequence of the Light Chain Variable Region: (378 bp)

(81) TABLE-US-00014 (SEQ ID NO: 3) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAAC AGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACT TTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATA GGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTC CCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGG ATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTC GGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATC AGTCACCCTGTTTCAAGGGCAATTCTGC

(82) Encoded Amino Acid Sequence: (126 aa)

(83) TABLE-US-00015 (SEQ ID NO: 4) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLI GGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVF GGGTKLTVLGQPKSSPSVTLFQGQFC

Example 3: Design and Preparation of Humanized Antibodies 4G10H1L1, 4G10H3L3 and 4G10H4L3 Against CTLA4

(84) 1. Design of Light and Heavy Chain Sequences of Anti-CTLA4 Humanized Antibodies 4G10H1L1, 4G10H3L3 and 4G10H4L3

(85) Based on the three-dimensional crystal structure of CTLA4 protein (Nat. Struct. Biol., (1997) 4 p. 527) and the amino acid sequence of antibody 4G10 obtained in the Example 2, antibody in silico modeling was performed and mutations of amino acids from mouse-like to human-like were engineered to obtain the amino acid sequences of variable regions of antibody 4G10H1L1, 4G10H3L3 and 4G10H4L3 (the constant region of heavy chain was Ig gamma-1 chain C region, ACCESSION: P01857 and the constant region of light chain was Ig kappa chain C region, ACCESSION: P01834)

(86) The designed sequences of variable regions are as follows

(87) (1) The Heavy Chain and Light Chain Sequences of the Humanized Monoclonal Antibody 4G10H1L1

(88) Nucleic Acid Sequence of the Heavy Chain Variable Region: (345 bp)

(89) TABLE-US-00016 (SEQ ID NO: 5) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTC CATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCA TGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTG ATTAATCCTTACAACAACATCACCAACTACAACCAGAAGTTCATGGGAAA AGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGA GCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGAT TATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT

(90) Encoded Amino Acid Sequence: (115 aa)

(91) TABLE-US-00017 (SEQ ID NO: 6) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGL INPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLD YRSYWGQGTLVTVSA

(92) Nucleic Acid Sequence of the Light Chain Variable Region: (327 bp)

(93) TABLE-US-00018 (SEQ ID NO: 7) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAAC TGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACT TCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATC GGAGGCACAAACAATCGGGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAG CCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGG ACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTT GGCGGGGGAACAAAGCTGACTGTGCTG

(94) Encoded Amino Acid Sequence: (109 aa)

(95) TABLE-US-00019 (SEQ ID NO: 8) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLI GGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVF GGGTKLTVL

(96) (2) The Heavy Chain and Light Chain Sequences of the Humanized Monoclonal Antibody 4G10H3L3

(97) Nucleic Acid Sequence of the Heavy Chain Variable Region: (345 bp)

(98) TABLE-US-00020 (SEQ ID NO: 9) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTC AGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCA TGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTG ATTAACCCTTACAACAACATCACTAACTACGCACAGAAGTTCCAGGGGAG AGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGT CCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGAT TACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA

(99) Encoded Amino Acid Sequence: (115 aa)

(100) TABLE-US-00021 (SEQ ID NO: 10) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGL INPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLD YRSYWGQGTLVTVSA

(101) Nucleic Acid Sequence of the Light Chain Variable Region: (327 bp)

(102) TABLE-US-00022 (SEQ ID NO: 11) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGAC TGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACT TCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATC GGAGGCACCAACAACAAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAG CCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGG ACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTC GGCGGCGGCACCAAGCTGACTGTGCTG

(103) Encoded Amino Acid Sequence: (109 aa)

(104) TABLE-US-00023 (SEQ ID NO: 12) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLI GGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVF GGGTKLTVL

(105) (3) The Heavy and Light Chain Sequences of the Humanized Monoclonal Antibody 4G10H4L3

(106) Nucleic Acid Sequence of the Heavy Chain Variable Region: (345 bp)

(107) TABLE-US-00024 (SEQ ID NO: 13) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTC AGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCA TGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTG ATTAACCCTTACAACGACATCACTAACTACGCACAGAAGTTCCAGGGGAG AGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGT CCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGAT TACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA

(108) Encoded Amino Acid Sequence: (115 aa)

(109) TABLE-US-00025 (SEQ ID NO: 14) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGL INPYNDITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLD YRSYWGQGTLVTVSA

(110) The nucleic acid and encoded amino acid sequences of the light chain variable region are the same as those of 4G10H3L3.

(111) 2. Preparation of Humanized Antibodies 4G10H1L1, 4G10H3L3 and 4G10H4L3

(112) The constant region of heavy chain was Ig gamma-1 chain C region, ACCESSION: P01857. The constant region of light chain was Ig kappa chain C region, ACCESSION: P01834.

(113) The heavy chain cDNAs and light chain cDNAs of 4G10H1L1, 4G10H3L3, 4G10H4L3 were separately cloned into pUC57simple vectors to obtain pUC57simple-4G10H1 and pUC57simple-4G10L1, pUC57simple-4G10H3 and pUC57simple-4G10L3, and pUC57simple-4G10H4 and pUC57simple-4G10L3, respectively. They were subcloned into pcDNA3.1 vectors. The recombinant plasmids were transfected into 293F cells and the culture medium was harvested and purified to obtain humanized antibodies 4G10H1L1, 4G10H3L3 and 4G10H4L3. The purified 4G10H1L1 and 4G10H3L3 were identified by SDS-PAGE electrophoresis, and the result was shown in FIG. 2 and FIG. 3 respectively.

Example 4: Preparation of Anti-PD-1 Antibody 14C12

(114) 1. Establishment of Hybridoma Cell Line LT003

(115) Using PD-1-mFc as the antigen, the hybridoma cells were obtained by fusing the splenocytes of immunized BALB/C mice (purchased from Guangdong Medical Laboratory Animal Center) and mice myeloma cells with currently established method (for example, Stewart, S. J., “Monoclonal Antibody Production”, in Basic Methods in antibody Production and Characterization, Eds. G. C. Howard and D. R. Bethell, Boca Raton: CRC Press, 2000).

(116) Microplate was coated with PD-1-mFc as the antigen, and indirect ELISA was used to screen those hybridoma cells secreting new antibodies specifically binding to PD-1.

(117) The hybridoma cells were further screened by competitive ELISA to select those secreting antibodies that competitively bind to PD-1 against ligand PDL1-hFc (PDL1 Genbank ID:NP_054862.1), and then a stable hybridoma cell line LT003(PD-1-14C12) was obtained by limited dilution method, and its secreted monoclonal antibody is named 14C12.

(118) LT003 (PD-1-14C12), a hybridoma cell line, was deposited in China Center for Type Culture Collection (CCTCC) on Jun. 16, 2015. Deposit Accession NO.: C2015105, depository address: Wuhan university, Wuhan, China, zip code: 430072.

(119) 2. Preparation of Anti-PD-1 Antibody 14C12

(120) The LT003 cells in the present invention were cultured using IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium containing 1% streptomycin, cultured in cell incubator with 5% CO.sub.2, 37° C. incubator), and after 7 days culture the cell culture supernatant was harvested and purified to get the antibody 14C12.

Example 5: Acquisition of the Sequence of Antibody 14C12

(121) Acquisition of the Sequence of Antibody 14C12

(122) mRNA was extracted from the hybridoma cell line LT003 prepared in Example 4 above according to the manual of the cell/bacterial total RNA extraction reagent kit (Tiangen, Product No. DP430).

(123) cDNA was synthesized using Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR, and amplified by PCR.

(124) TA cloning was directly carried out on the PCR amplified product according to the instructions of pEASY-T1 Cloning Kit (Transgen CT101).

(125) The products of TA cloning were directly sequenced, and the sequencing results were as follows:

(126) Nucleic Acid Sequence of Heavy Chain Variable Region: (354 bp)

(127) TABLE-US-00026 (SEQ ID NO: 15) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTC ACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACA TGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACT ATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAG ATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGT CTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTAC GGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTC TGCC

(128) Encoded Amino Acid Sequence: (118 aa)

(129) TABLE-US-00027 (SEQ ID NO: 16) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAT ISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRY GEAWFAYWGQGTLVTVSA

(130) Nucleic Acid Sequence of the Light Chain Variable Region: (318 bp)

(131) TABLE-US-00028 (SEQ ID NO: 17) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGA GCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGT CTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGG GCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAG TGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGG GCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCA GGCACAAAACTGGAACTG

(132) Encoded Amino Acid Sequence: (106 aa)

(133) TABLE-US-00029 (SEQ ID NO: 18) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYR ANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGA GTKLEL

Example 6: Design, Preparation and Assay of Humanized Antibody 14C12H1L1 Against PD-1

(134) 1. Design of the Light and Heavy Chain Sequences of the Humanized Antibody 14C12H1L1

(135) Based on the three-dimensional crystal structure of PD-1 protein (Shinohara T, et al., Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1995, 23 (3): 704-6) and the amino acids sequence of antibody 14C12 obtained in the Example 5, antibody in silico modeling was performed and mutations of amino acids from mouse-like to human-like were engineered to obtain the amino acid sequences of variable regions of antibody 14C12H1L1.

(136) The designed sequences of variable regions are as follows:

(137) Nucleic Acid Sequence of the Heavy Chain Variable Region: (354 bp)

(138) TABLE-US-00030 (SEQ ID NO: 19) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTC ACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACA TGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACT ATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCG GTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGA ACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTAC GGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTC TAGT

(139) Encoded Amino Acid Sequence: (118 aa)

(140) TABLE-US-00031 (SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVAT ISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRY GEAWFAYWGQGTLVTVSS

(141) Nucleic Acid Sequence of the Light Chain Variable Region: (321 bp)

(142) TABLE-US-00032 (SEQ ID NO: 21) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGA CAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGA GCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGG GCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAG CGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGG CAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCC GGGACAAAACTGGAGCTGAAG

(143) Encoded Amino Acid Sequence: (107 aa)

(144) TABLE-US-00033 (SEQ ID NO: 22) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYR ANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGA GTKLELK

(145) 2. Preparation and SDS-PAGE Electrophoresis of Humanized Antibody 14C12H1L1

(146) The constant region of heavy chain is Ig gamma-1 chain C region, ACCESSION: P01857; and the constant region of light chain is Ig kappa chain C region, ACCESSION: P01834.

(147) The heavy chain cDNA and light chain cDNA of 14C12H1L1 were separately cloned into pcDNA3.1 vector to obtain the recombinant expression plasmids. The recombinant plasmids were transfected into 293F cells. The 293F cell culture medium was purified and tested. As shown in FIG. 4, the reduced target protein appeared at approximately 24.5 kD and 49 kD, while the non-reduced target protein appeared at approximately 147 kD.

Example 7: Sequence Design, Expression and Assay of Heavy Chains and Light Chains of Bispecific Antibody BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010

(148) 1. Sequence Design

(149) Bispecific antibody BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 in the present invention all belong to Morrison design (IgG-scFv), in which each heavy chain of an IgG antibody are connected with a scFv fragment of another antibody. The configurations of the heavy chains and light chains are shown in Table 1 below.

(150) TABLE-US-00034 TABLE 1 The configurations of BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 heavy chains and light chains Heavy chains Bispecific Linker Light Antibody IgG fragment scFv chains BiAb001 14C12H1 Linker 1 4G10H1v-Linker 2- 14C12L1 4G10L1v BiAb002 14C12H1 Linker 2 4G10H1v-Linker 2- 14C12L1 4G10L1v BiAb003 14C12H1 Linker 1 4G10H3v-Linker 2- 14C12L1 4G10L3v BiAb004 14C12H1 Linker 2 4G10H3v-Linker 2- 14C12L1 4G10L3v BiAb007 14C12H1 Linker 2 4G10H4v-Linker 2- 14C12L1 4G10L3v BiAb010 14C12H1 Linker 2 8D2H14v-Linker 2- 14C12L1 8D2L2v

(151) In Table 1:

(152) (1) The antibody sequences marked with subscript “V” refer to the variable region of heavy chains or light chains. Those with no subscript “V” are full-length heavy chains or light chains with constant region. These variable regions or full-length sequences of amino acids and their coding nucleic acid sequences embody the corresponding sequences recorded in the examples above.

(153) (2) Linker 1 Amino Acid Sequence is (GGGGS)3 (SEQ ID NO: 23)

(154) Linker 2 amino acid sequence is (GGGGS)4 (SEQ ID NO: 24)

(155) (3) Amino Acid Sequence of the Heavy Chain Variable Region of 8D2H14L2 (8D2H14v):

(156) TABLE-US-00035 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSSRLSCAASGFTFSDNWMNWVRQAPGKGLEWLAQ IRNKPYNYETYYSASVKGRFTISRDDSKNSVYLQMNSLKTEDTGVYYCTA QFAYWGQGTLVTVSS

(157) Encoded Nucleic Acid Sequence of 8D2H14v:

(158) TABLE-US-00036 (SEQ ID NO: 26) GAGGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGCAGCCTGGAGGAAG CTCCCGGCTGTCATGTGCCGCTAGCGGCTTCACCTTTTCCGACAACTGGA TGAATTGGGTGCGACAGGCACCAGGCAAAGGACTGGAGTGGCTGGCTCAG ATCCGGAACAAGCCCTACAATTATGAAACATACTATAGCGCCTCCGTGAA AGGCCGGTTCACTATTAGTAGAGACGATTCTAAGAACAGCGTGTACCTGC AGATGAATAGCCTGAAGACAGAGGATACTGGCGTCTACTATTGCACAGCA CAGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTAGT

(159) (4) Amino Acid Sequence of the Light Chain Variable Region of 8D2H14L2 (8D2L2v):

(160) TABLE-US-00037 (SEQ ID NO: 27) DIQMTQSPSSLSASVGDRVTITCRTSENIYGGLNWYQRKPGKSPKLLIYG ATNLASGVSSRFSGSGSGTDYTLTISSLQPEDVATYYCQNVLRSPFTFGS GTKLEIK

(161) Encoded Nucleic Acid Sequence of 8D2L2v:

(162) TABLE-US-00038 (SEQ ID NO: 28) GACATCCAGATGACTCAGAGCCCCTCAAGCCTGTCTGCAAGTGTGGGCGA TAGGGTCACCATCACATGTCGCACCTCCGAAAACATCTACGGGGGACTGA ATTGGTATCAGCGCAAGCCCGGCAAATCCCCTAAGCTGCTGATCTACGGC GCTACCAACCTGGCATCTGGGGTGTCCTCTCGATTTTCAGGGAGCGGCAG CGGCACCGACTATACTCTGACCATTAGTTCACTGCAGCCTGAGGATGTGG CCACATACTATTGCCAGAATGTCCTGAGATCACCATTCACTTTTGGGAGC GGAACCAAACTGGAAATTAAG

(163) 2. Expression and Purification of Antibody BiAb001

(164) cDNAs of heavy chain and light chain of BiAb001 were separately cloned into pUC57simple vectors (provided by GenScript) to obtain plasmids pUC57simple-BiAb001H and pUC57simple-BiAb001L, respectively.

(165) pUC57simple-BiAb001H and pUC57simple-BiAb001L were individually digested with enzymes (HindIII&EcoRI), and genes of heavy chain and light chain recovered via electrophoresis were sub-cloned into pcDNA3.1 vector, respectively. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days culture, the culture supernatant was harvested by high-speed centrifugation and concentration, and purified by loading onto HiTrap protein A HP column and eluting with Elution Buffer in one step to obtain the antibody and stored in PBS.

(166) The purified antibody samples were added to reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer, respectively. After being boiled, the samples were examined on SDS-PAGE electrophoresis. The results of BiAb001 electrophoresis was shown in FIG. 5, in which the reduced protein sample appeared at 23.6 kD and 75.8 kD, and the non-reduced protein sample (individual antibody) appeared at 199 kD.

(167) 3. Expression and Purification of Antibody BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010

(168) Purified antibodies of BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 were obtained according to the aforementioned methods used for preparation of BiAb001.

(169) The purified antibodies samples were added into of reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer, respectively. After being boiled, the samples were examined on SDS-PAGE electrophoresis. The results of BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 electrophoresis were shown in FIGS. 6, 7, 8, 9 and 10, respectively, in which the reduced protein sample appeared at 23.6 kD and 75.8 kD, and the non-reduced protein sample (individual antibody) appeared at 199 kD.

Example 8: Determination of Antibody Binding Kinetics

(170) The binding kinetics of antigen and antibody were measured by Fortebio molecular interaction instrument.

(171) 1. Binding kinetics of antibody 4G10 and its humanized antibody 4G10H1L1, 4G10H3L3, and 4G10H4L3 to antigen CTLA4 were measured

(172) 1.1 CTLA4 antigen was obtained by digesting CTLA4-mFc with TEV protease and column purification

(173) 1.2 Antibody 4G10 was immobilized to AR2G Biosensors by amine coupling method, and then blocked with ethanolamine and equilibrating in PBST, and then bound to CTLA4. CTLA4 was double gradient diluted with PB ST to the concentrations of 268.1, 134.1, 67, 33.5, 16.8, 8.38, 4.19, and 0 nM. The dissociation was also in PBST. Humanized antibodies 4G10H1L1, 4G10H3L3 and 4G10H4L3 were measured with similar methods to that of 4G10, with antigen concentrations of 180, 90, 45, 22.5, 11.25, 5.625, 2.813 and 0 nM.

(174) 1.3 The binding kinetics of antibody 4G10 and its humanized antibodies 4G10H1L1, 4G10H3L3, and 4G10H4L3 to antigen CTLA4 are shown in Table 1 below, and in FIG. 11, FIG. 12, FIG. 13 and FIG. 14, respectively.

(175) 2. Binding kinetics of antibody 14C12 and its humanized antibody 14C12H1L1 to antigen PD-1

(176) 2.1 PD-1 antigen was obtained by digesting PD-1-mFc with TEV protease and column purification

(177) 2.2 The antigen PD-1 (antigen concentration of 1 μg/ml) was immobilized on the surface of SA sensor after being labeled with biotin, and after equilibrating in PBST it bind to antibodies 14C12 and 14C12H1L1, respectively. The antibodies were diluted with PBST from 200 nM down three fold each time, and the dissociation was also in PBST.

(178) 2.3 The binding kinetics of antibodies 14C12 and 14C12H1L1 to antigen are shown in Table 1 below and in FIGS. 15 and 16.

(179) 3. Binding kinetics of antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 to antigen CTLA4.

(180) 3.1 CTLA4 (antigen concentration of 1 μg/ml) was immobilized on the surface of SA sensor after being labeled with biotin, and after equilibrating in PBST, it binds to antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010, respectively. The antibodies were diluted with PBST from 200 nM down three fold each time. The dissociation was also in PBST.

(181) 3.2 Binding kinetics of antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 to antigen CTLA4 are shown in Table 1 and in FIGS. 17-21, respectively.

(182) 4. Binding kinetics of antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 to antigen to antigen PD-1

(183) 4.1 The antigen PD-1 (antigen concentration of 1 μg/ml) was immobilized on the surface of SA sensor after being labeled with biotin, and after equilibrating in PBST, it binds to antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010, respectively. The antibodies were diluted with PBST from 200 nM down three fold each time. The dissociation was also in PBST.

(184) 4.2 Binding kinetics of antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 to antigen PD-1 are shown in Table 2, and in FIG. 22-FIG. 27, respectively.

(185) TABLE-US-00039 TABLE 2 Kinetic parameters of antibody binding to antigen Antibody Antigen K.sub.D (M) Kon(1/Ms) Kon Error Kdis(1/s) Kdis Error 4G10 CTLA4 3.01E−10 3.78E+05 4.36E+03 1.14E−04 5.33E−06 4G10 H1L1 1 μg/ml 1.52E−09 1.86E+05 3.26E+03 2.82E−04 9.23E−06 4G10 H3L3 4.14E−09 2.09E+05 3.81E+03 8.64E−04 1.11E−05 4G10H4L3 9.67E−10 1.37E+05 2.22E+03 1.32E−04 8.69E−06 14C12 PD-1 1.81E−11 3.38E+05 8.23E+03 6.12E−06 1.04E−05 14C12H1L1 1 μg/ml 2.42E−11 3.17E+05 5.90E+03 7.66E−06 8.70E−06 BIAb001 CTLA4 1.67E−10 2.33E+05 4.45E+03 3.89E−05 8.75E−06 BIAb002 1 μg/ml 9.69E−11 2.37E+05 5.32E+03 2.30E−05 9.97E−06 BIAb003 3.95E−10 3.60E+05 7.10E+03 1.42E−04 9.99E−06 BIAb004 5.66E−10 2.20E+05 3.89E+03 1.24E−04 8.27E−06 BiAb007 2.72E−10 1.58E+06 5.17E+04 4.28E−04 1.12E−05 BiAb010 3.22E−10 1.08E+06 1.99E+04 3.47E−04 7.28E−06 BIAb001 PD-1 4.16E−11 2.97E+05 4.96E+03 1.24E−05 8.36E−06 BIAb002 1 μg/ml 3.33E−11 2.20E+05 5.93E+03 7.32E−06 1.15E−05 BIAb003 4.12E−11 2.64E+05 5.49E+03 1.09E−05 9.82E−06 BIAb004 4.82E−11 2.47E+05 5.45E+03 1.19E−05 9.61E−06 BiAb007 1.40E−11 4.52E+05 9.23E+03 6.30E−06 7.85E−06 BiAb010 2.97E−11 2.28E+05 4.40E+03 6.79E−06 8.70E−06 K.sub.D Is the affinity constant; K.sub.on is the association rate of antigen-antibody K.sub.dis is the dissociation rate of antigen-antibody; K.sub.D = K.sub.dis/K.sub.on.

(186) The results showed that:

(187) the antibody 4G10 and its humanized antibodies have good affinity to the antigen CTLA4. Both antibodies 14C12 and 14C12H1L1 have good affinity to antigen PD-1.

(188) Bispecific antibodies have good affinity to antigen CTLA4 and PD-1.

Example 9: The Binding Activity of Antibody to Antigen Measured by ELISA

(189) 1. The binding activity of humanized antibodies 4G10H1L1 and 4G10H3L3 to antigen CTLA4

(190) 1.1 The binding activity of humanized antibodies 4G10H1L1 and 4G10H3L3 to CTLA4 was determined by indirect ELISA.

(191) After incubated with antigen at 4° C. overnight, the microplate was blocked with 1% BSA at 37° C. for 2 h, and then the antibodies were added and incubated at 37° C. for 30 min, and then HRP-labeled secondary antibody (goat anti-human IgG (H+L)) (Jackson, 109-035-088) was added and incubated at 37° C. for 30 min. TMB (Neogen, 308177) was added to react for 5 mins. The absorbance was read at the wavelength of 450 nm in a microplate reader.

(192) The binding results were shown in FIG. 28. As shown in the figure, both 4G10H1L1 and 4G10H3L3 can bind to CTLA4 protein effectively with dose-dependency. The absorbance intensities at different doses were shown in Table 3. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of 4G10H1L1 and 4G10H3L3 were then determined to be 0.048 nM and 0.067 nM, respectively.

(193) TABLE-US-00040 TABLE 3 The binding activity of 4G10H1L1 and 4G10H3L3 to CTLA4 was measured by indirect ELISA Coating Antigen: CTLA4 0.5 μg/ml Serial dilution of antibody 4G10 H1L1 4G10 H3L3 6 μg/ml 2.926 2.946 2.809 2.764 1:5 2.784 2.732 2.729 2.739 1:25 2.729 2.688 2.668 2.617 1:125 2.490 2.469 2.367 2.309 1:625 1.736 1.709 1.498 1.357 1:3126 0.607 0.663 0.513 0.432 1:16525 0.198 0.225 0.175 0.149 1:78125 0.096 0.115 0.089 0.087 1:390625 0.075 0.087 0.075 0.072 1:1953125 0.071 0.090 0.066 0.077 1:9765625 0.066 0.087 0.078 0.089 0 0.073 0.079 0.079 0.068 Secondary antibody Goat anti-Human IgG, HRP (1:5000)

(194) 1.2. The binding activity of humanized antibodies 4G10H1L1 and 4G10H3L3 to CTLA4 by competition ELISA against B7

(195) Coating antigen to microplate with B7/1-hFc (B7/1 genbank ID: NP 005182.1) 4° C. overnight, and then after blocked with 1% BSA for 2 hours, mixtures of antibodies and CTLA4-mFc antibody were added (dilute concentrations are shown in table 4) and incubate for 30 min at 37° C.; and then secondary antibody labeled with enzyme was added and then incubated for 30 mins at 37° C. The absorption value of 450 nm was measured on the enzyme-labeled instrument (see table 4).

(196) The binding results of antibodies to CTLA4 competing against B7-1 were shown in FIG. 29. As shown in the figure, the antibodies 4G10H1L1 and 4G10H3L3 can compete against B7-1 and bind to CTLA4 protein effectively with dose-dependency. The absorbance at different doses were shown in Table 4. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of 4G10H1L1 and 4G10H3L3 binding with CTLA4 were then determined to be 1.297 nm and 1.229 nm, respectively.

(197) TABLE-US-00041 TABLE 4 The binding activity of humanized antibodies 4G10H1L1 and 4G10H3L3 to CTLA4 by competition ELISA against B7 Coating Antigen: B7/1-hFc 0.5 μg/ml Serial dilution of Antibody 4G10H1L1 4G10H3L3 receptor 3 μg/ml 0.132 0.121 0.146 0.185 CTLA4-mFc 1:3 0.120 0.170 0.159 0.182 0.3 μg/ml 1:9 0.260 0.343 0.382 0.340 1:27 0.399 0.593 0.570 0.507 1:81 0.565 0.614 0.642 0.642 1:243 0.628 0.753 0.784 0.773 1:729 0.573 0.760 0.768 0.702 1:2187 0.553 0.824 0.741 0.788 1:6561 0.661 0.844 0.824 0.679 1:19683 0.555 0.834 0.742 0.699 1:59049 0.552 0.725 0.773 0.770 0 0.610 0.665 0.822 0.717 Secondary antibody Goat anti-Mouse IgG, HRP (1:5000)

(198) 2. The binding activities of monoclonal antibody 14C12 and its humanized antibody 14C12H1L1 to antigen PD-1 measured by ELISA

(199) 2.1 The binding activity of monoclonal antibodies 14C12 and 14C12H1L1 to antigen PD-1 was determined by indirect ELISA as follows:

(200) After incubated with PD-1-mFc at 4° C. overnight, the microplate was blocked with 1% BSA at 37° C. for 2 h, and the antibodies were added, incubated at 37° C. for 30 min, and HRP-labeled secondary antibody (goat anti-human IgG (H+L)) (Jackson, 109-035-088) was added and incubated at 37° C. for 30 min. TMB (Neogen, 308177) was added to react for 5 mins. The absorbance was read at the wavelength of 450 nm in a microplate reader.

(201) The binding results of antibodies 14C12 and 14C12H1L1 to PD-1 were shown in FIG. 30. Evidently, both 14C12 and 14C12H1L1 can bind to PD-1 protein effectively with dose-dependency. The absorbance at different doses were shown in Table 5. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of 14C12 and 14C12H1L1 binding to PD-1 were then determined to be 0.175 nM and 0.043 nM, respectively.

(202) TABLE-US-00042 TABLE 5 The binding activities of antibodies 14C12 and 14C12H1L1 to PD-1, respectively Coating Antigen: PD-1-mFc (0.5 μg/mL) Antibody concentration (μg/mL) 14C12 14C12H1L1 1 2.463 2.439 2.643 2.557 0.3 2.572 2.380 2.734 2.586 0.1 2.118 2.126 2.633 2.535 0.03 1.607 1.438 2.384 2.335 0.01 0.930 0.809 1.892 1.839 0.003 0.407 0.346 1.115 1.011 0.001 0.167 0.150 0.503 0.455 0 0.062 0.047 0.068 0.064 Secondary antibody Goat anti-Mouse secondary antibody, HRP Conjugate

(203) 2.2. The binding activity of monoclonal antibody 14C12 produced by hybridoma and its humanized antibody 14C12H1L1 to antigen PD-1 by competition ELISA against PDL1 was measured as follows:

(204) After incubated with PD-1-hFc or PD-1-mFc at 4° C. overnight, the microplate was blocked with 1% BSA at 37° C. for 2 h; and then mixtures of individual antibody, 14C12 or 14C12H1L1, at different concentrations (see Table 6 for dilution gradient) and PDL1-hFc or PDL-1-mFc were added into the microplate to react for 10 mins; and then HRP-labeled secondary antibody was added and incubated at 37° C. for 30 min. The absorbance was read at the wavelength of 450 nm in a microplate reader (see Table 6).

(205) The binding results of antibodies to PD-1 competing against PDL1 were shown in FIG. 31. the antibody 14C12 and its humanized antibody 14C12H1L1 can compete against PDL1 to bind to PD-1 protein effectively with dose-dependency. The absorbance intensities at different doses were shown in Table 6. By using quantitative analyses of absorbance values, EC50 of 14C12 and 14C12H1L1 binding with PD-1 that were calculated via Curve Simulation were then determined to be 0.853 nM and 0.37 nM, respectively.

(206) TABLE-US-00043 TABLE 6 The binding activity of 14C12 and 14C12H1L1 to PD-1 by competition ELISA against PDL1 Coating antigen: Antibody PD-1-mFc 0.2 μg/mL concentration (μg/mL) 14C12 14C12H1L1 1.5 μg/ml 0.111 0.088 0.135 0.113 1:3 0.100 0.116 0.130 0.131 1:9 0.645 0.643 0.260 0.185 1:27 1.463 1.614 0.257 0.218 1:81 1.841 1.686 0.355 0.350 1:243 1.983 1.769 0.399 0.364 1:729 1.789 1.770 0.417 0.411 0 1.791 1.790 0.430 0.402 PDL1-hFc 2 μg/ml Secondary antibody Goat anti-Mouse secondary antibody HRP Conjugate

(207) 3. The binding activity of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to antigens measured by ELISA

(208) 3.1 The binding activity of antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to antigen CTLA-4 was determined by indirect ELISA (Refer to methods described in 1.1 of the present Example)

(209) The binding results of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to antigen CTLA4 were shown in FIG. 32. Evidently, antibodies BiAb001, BiAb002, BiAb003 and BiAb004 can bind to CTLA4 protein effectively with dose-dependency. The absorbance at different doses were shown in Table 7. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 binding to CTLA4 were then determined as shown in Table 7 below.

(210) TABLE-US-00044 TABLE 7 The binding activity of bispecific antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to antigen CTLA4 (Indirect ELISA) Serial dilution of Coating Antigen: CTLA4 0.5 μg/ml Antibody BiAb001 BiAb002 BiAb003 BiAb004 6 μg/ml 2.425 2.098 2.334 2.120 2.179 2.076 2.243 2.251 1:3 2.299 2.234 2.204 2.257 2.141 2.138 2.198 2.319 1:9 2.265 2.188 2.168 2.186 2.012 2.086 2.207 2.254 1:27 2.245 2.215 2.174 2.043 1.814 1.811 1.982 1.907 1:81 1.859 1.856 1.717 1.609 1.438 1.410 1.534 1.640 1:243 1.494 1.511 1.221 1.136 0.933 0.899 1.070 1.108 1:729 0.818 0.922 0.644 0.610 0.451 0.414 0.567 0.548 0 0.048 0.048 0.048 0.047 0.047 0.045 0.049 0.050 Secondary antibody: Goat anti-human IgG, HRP (1:5000) EC50 (nM) 0.105 0.12 0.189 0.154

(211) 3.2 The binding activity of antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to antigen PD-1 was determined by indirect ELISA. (Refer to methods described in 2.1 of the present Example)

(212) The binding results of antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to antigen PD-1 were shown in FIG. 33. Evidently, antibodies BiAb001, BiAb002, BiAb003, and BiAb004 can bind to PD-1 protein effectively with dose-dependency. The absorbance intensities at different doses were shown in Table 7. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 binding to PD-1 were then determined as shown in Table 8 below.

(213) TABLE-US-00045 TABLE 8 The binding activity of bispecific antibodies to antigen CTLA4 (Indirect ELISA) Serial dilution of Coating Antigen: PD-1-mFc 0.5 μg/ml Antibody BiAb001 BiAb002 BiAb003 BiAb004 6 μg/ml 2.400 2.360 2.370 2.314 2.332 2.290 2.347 2.343 1:3 2.450 2.426 2.290 2.388 2.271 2.326 2.410 2.458 1:9 2.402 2.457 2.372 2.346 2.279 2.351 2.390 2.505 1:27 2.409 2.467 2.332 2.348 2.350 2.243 2.414 2.396 1:81 2.375 2.254 2.084 1.990 1.996 1.928 2.197 2.175 1:243 1.871 1.725 1.627 1.544 1.414 1.419 1.573 1.560 1:729 1.067 1.047 0.954 0.814 0.746 0.719 0.920 0.865 0 0.085 0.067 0.065 0.068 0.055 0.055 0.056 0.058 Secondary antibody: Goat anti-human IgG, HRP (1:5000)

(214) 3.3 The binding activity of the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to CTLA4 respectively by competition ELISA against B7/1-hFc (Refer to methods described in 1.2 of the present Example).

(215) The binding results were shown in FIG. 34. As shown in the figure, the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 can effectively bind antigen CTLA4 and inhibit CTLA4 binding to B7/1 with dose-dependency. The absorbance intensities at different doses were shown in Table 9. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 were then determined as shown in Table 9 below.

(216) TABLE-US-00046 TABLE 9 The binding activity of antibodies to CTLA4 by competition ELISA against B7/1-hFc Serial dilution of Coating Antigen: B7/1-hFc 0.5 μg/ml Antibody BiAb001 BiAb002 BiAb003 BiAb004 3 μg/ml 0.076 0.072 0.078 0.095 0.074 0.080 0.095 0.076 1:3 0.081 0.076 0.079 0.079 0.095 0.086 0.097 0.100 1:9 0.748 0.706 1.040 1.031 1.029 1.049 0.907 0.973 1:27 1.153 1.129 1.076 1.152 1.125 1.361 1.010 1.056 1:81 1.121 1.241 1.153 1.315 1.241 1.198 1.121 1.206 1:243 1.261 1.236 1.047 1.266 1.333 1.335 1.231 1.235 1:729 1.063 1.077 1.085 1.337 1.210 1.323 1.157 1.287 0 1.0476 0.9808 0.9131 1.0762 1.067 1.074 1.032 0.966 Receptor: CTLA4-mFc 0.3 μg/ml Secondary antibody: Goat anti-Mouse IgG, HRP Conjugate (1:5000) EC50 (nM) 2.758 1.797 2.197 2.256

(217) 3.4 The binding activity of antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to antigen PD-1 by competition ELISA against PDL1 (Refer to methods described in 2.2 of the present Example)

(218) The binding results were shown in FIG. 35. Evidently, the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 can effectively bind antigen PD-1 and inhibit PD-1 binding to PDL1 with dose-dependency. The absorbance intensities at different doses were shown in Table 10. Through Curve Simulation using quantitative analyses of absorbance values, EC50 of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to CTLA4 were then determined as shown in Table 10 below.

(219) TABLE-US-00047 TABLE 10 The binding activity of antibodies to PD-1 by competition ELISA against PDL1. Antibody Coating antigen: PD-1-hFc 0.5 μg/ml concentration BiAb001 BiAb002 BiAb003 BiAb004 3 μg/ml 0.347 0.348 0.369 0.353 0.074 0.075 0.078 0.075 1:3 0.314 0.326 0.348 0.350 0.071 0.081 0.073 0.074 1:9 0.332 0.330 0.340 0.340 0.095 0.095 0.093 0.095 1:27 0.542 0.775 0.758 0.733 0.695 0.737 0.639 0.643 1:81 1.041 1.009 1.018 1.063 0.983 1.010 0.954 1.019 1:243 1.131 1.117 1.149 1.186 1.070 1.165 1.009 1.082 1:729 1.186 1.129 1.072 1.199 1.093 1.029 1.032 1.080 0 1.2345 1.1091 1.1243 1.1759 1.101 1.140 1.178 1.153 Receptor: PDL1-mFc 0.3 μg/ml Secondary antibody: Goat anti-human IgG, HRP (1:5000) EC.sub.50 (nM) 0.685 0.543 0.665 0.62

Example 10: The Binding Activity of Antibodies to Cell Surface Antigen by Flow Cytometry Method

(220) Host cells 293T expressing CTLA4 or PD-1 antigens were constructed respectively, and labeled with the humanized antibodies prepared in the present invention. The ability of the antibodies to bind specifically to corresponding cell surface antigens in its native conformation was analyzed and validated by flow cytometry.

(221) 1. Construction of 293T Host Cell Expressing CTLA4 or PD-1

(222) 293T cells were transfected with the CTLA4 containing plasmid pLenti6.3-CTLA4 or PD-1-containing plasmid pLenti6.3-PD-1 (vector pLenti6.3 was purchased from Invitrogen Corporation) and screened to obtain the stable pools of 293T-CTLA4 or 293T-PD-1 expressing CTLA4 or PD-1, respectively.

(223) 2. Antibody Binding to Cell Surface Antigens

(224) The host cells obtained above that express individual antigen were digested by using trypsin, and distributed into tubes each containing 2×10.sup.5 cells. Antibodies were diluted in gradient using PBSA buffer (1% BSA) and incubated with 293T cells that express corresponding antigens on ice for 2 h. 100 μL of FITC-labeled goat anti-human IgG (1:500) was added into each tube and incubated on ice for 1 h. After being washed with PBS for 3 times, cells were re-suspended in 300 μL of PBS, and fluorescence signals were measured on the flow cytometer using the FITC channel.

(225) 2.1 Binding Activity of Antibodies to Cell Surface Antigens

(226) The binding results of humanized antibodies 4G10H1L1 and 4G10H3L3 to 293T-CTLA4 cells were shown in FIG. 36 and FIG. 37. As shown in the figure, the antibodies 4G10H1L1 and 4G10H3L3 can effectively bind to target protein CTLA4 expressed on the surface of host cells 293T-CTLA4 with dose-dependency. The fluorescence intensities at different doses were shown in Table 11. Through Curve Simulation using quantitative analyses of absorbance values, EC.sub.50 of 4G10H1L1 and 4G10H3L3 binding to CTLA4 were determined to be 7.58 nM and 10.54 nM, respectively.

(227) TABLE-US-00048 TABLE 11 The fluorescence intensities of antibodies 4G10H1L1 and 4G10H3L3 binding to CTLA4 expressed on 293T-CTLA4 cell surface by Flow Cytometry 4G10H1L1 4G10H2L2 Antibody concentration (nM) fluorescence intensity 0.01 14.93 15.13 0.1 24.79 47.05 1 106.77 97.27 2.5 272.24 236.66 5 547.76 465.54 10 1080.91 788 20 1568.19 1296.95 50 1652.26 1539.24

(228) 2.2 The binding results of humanized antibody 14C12H1L1 to 293T-PD-1 cells were shown in FIG. 38. As shown in the figure, the antibody 14C12H1L1 can effectively bind to target protein PD-1 expressed on the surface of host cells 293T-PD-1 with dose-dependency. The fluorescence intensities at different doses were shown in Table 12. Through Curve Simulation using quantitative analyses of fluorescence intensity, EC.sub.50 of 14C12H1L1 binding to PD-1 was determined to be 1.89 nM.

(229) TABLE-US-00049 TABLE 12 The fluorescence intensity of antibody 14C12H1L1 binding to PD-1 expressed on 293T-PD-1 cell surface by Flow Cytometry. Antibody concentration (nM) 0.01 0.1 1 5 10 50 fluorescence 8.32 20.31 174.62 579.41 686.49 669.54 intensity

(230) 2.3 The binding results of the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to 293T-CTLA4 cells were shown in FIGS. 39-42. As shown in the figures, the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 can effectively bind to target protein CTLA4 expressed on the surface of host cell 293T-CTLA4 with dose-dependency. The fluorescence intensities at different doses were shown in Table 13. Through Curve Simulation using quantitative analyses of fluorescence intensities, EC.sub.50 of BiAb001, BiAb002, BiAb003, and BiAb004 were determined as shown in Table 13 below.

(231) TABLE-US-00050 TABLE 13 The fluorescence intensities and EC.sub.50 of the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 binding to CTLA4 expressed on 293T-CTLA4 cell surface by Flow Cytometry Antibody concentration BIAb001 BIAb002 BIAb003 BIAb004 (nM) MFI (fluorescence intensity) 0.0 — — 12.9 13.0 0.1 10.5 10.5 14.2 15.6 1.0 21.2 20.4 28.7 34.8 2.5 46.8 43.8 49.6 77.4 5.0 92.9 93.5 101.0 129.6 10.0 181.9 171.2 245.3 313.0 20.0 312.8 282.1 487.4 608.6 50.0 469.5 466.5 899.8 1260.8 100.0 423.0 435.3 937.5 1020.6 200.0 381.6 408.2 — — EC50 (nM) 11.9 13.7 19.9 17.8

(232) 2.4 The binding results of the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 to 293T-PD-1 cells are shown in FIGS. 43-46. Evidently, the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 can effectively bind to PD-1 expressed on the surface of host cell 293T-PD-1 with dose-dependency. The fluorescence intensities at different doses were shown in Table 14. Through Curve Simulation using quantitative analyses of absorbance values, EC.sub.50 of BiAb001, BiAb002, BiAb003, and BiAb004 were determined as shown in Table 14 below.

(233) TABLE-US-00051 TABLE 14 The fluorescence intensities of the antibodies BiAb001, BiAb002, BiAb003, and BiAb004 binding to PD-1 expressed on 293T-PD-1 cell surface by Flow Cytometry BIAb001 BIAb002 BIAb003 BIAb004 Antibody MFI (fluorescence Antibody MFI (fluorescence concentration/nM intensity) concentration/nM intensity) 0.01 10.18 11 0.01 8.56 8.89 0.08 13.92 14.09 0.1 14.57 13.46 0.75 36.44 36.11 1 70.76 50.8 1.88 74.27 70.97 2.5 143.24 100.83 3.75 148.28 142.37 5 332.5 204.83 7.5 223.29 204.22 10 632.57 378.31 15 337.03 302.05 20 1026.03 535.69 37.5 358.78 290.92 50 958.92 734.73 100 943.77 682.25 EC50 (nM) 5.69 4.61 7.18 10

(234) 3. The binding activity of antibodies to T cell surface antigens CTLA4 and PD-1 PBMC was isolated by Ficoll-Paque Plus (GE Healthcare LOT No.:171440-02), and further isolated to get CD4+ cells, and then cells were stimulated with PHA for three days and then cells were washed once with PBS and mixed with antibodies at different concentrations, and then incubated on ice for 1.5 h. The cells were then washed with PBS once after incubation, and the FITC-labeled anti-human IgG (Jackson immunoresearch lot. 102155). Then the cells were incubated on ice in the dark for 1 h, washed with PBS for once, and then the fluorescence signals were measured on the flow cytometer.

(235) The control anti-PD-1 antibody Nivolumab is commercially available, and its information could also be found in http://www.drugbank.ca/drugs/DB09035;

(236) The control anti-CTLA4 antibody Ipilimumab is commercially available, and its information can be found in http://www.drugbank.ca/drugs/DB06186.

(237) 3.1 The binding results of humanized antibody 4G10H3L3 to T cells were shown in FIG. 47. As shown in figure, the antibody 4G10H3L3 can effectively bind to the target protein CTLA4 expressed on the surface of T cells with dose-dependency.

(238) 3.2 The binding results of humanized antibody 14C12H1L1 to T cells were shown in FIG. 48. As shown in figure, the antibody 14C12H1L1 can effectively bind to the target PD-1 expressed on the surface of T cells with dose-dependency.

(239) 3.3 The binding activity to T cells of the antibodies BiAb003 and BiAb004 compared with that of 14C12H1L1 and 4G10H3L3 were shown in FIG. 49. As shown in figure, the antibodies BiAb003, BiAb004, 14C12H1L1, and 4G10H3L3 can effectively bind to the target protein PD-1 expressed on the surface of T cells with dose-dependency. Furthermore, the binding activity of the antibodies BiAb003, BiAb004, and 14C12H1L1 to T cells were stronger than those of the antibodies 4G10H3L3, Nivolumab, and Ipilimumab. The fluorescence intensity was shown in Table 15.

(240) TABLE-US-00052 TABLE 15 The fluorescence intensities of the antibodies 14C12H1L1, 4G10H3L3, BiAb003, and BiAb004 binding to T cells Antibody concentration/nM 100 10 1 Name of antibody MFI (fluorescence intensity) PBS 8.39 — — hIgG 10.15 — — Nivolumab 22.88 — — Ipilimumab 8.35 — — 14C12H1L1 48.94 29.93 19.97 4G10H3L3 14.11 9.78 8.62 BIAb003 49.09 23.67 14.65 BIAb004 47.54 22.85 14.66

Example 11: Mixed Lymphocyte Reaction: Secretion of Cytokine IFN-γ and IL-2

(241) PBMC was isolated by Ficoll-Paque Plus (GE Healthcare LOT No.: 171440-02), then mixed with IL-4 (Peprotech K2513, 1000 U/ml) and GM-CSF (Peprotech H1513, 1000 U/ml) to induce for 6 days, and then TNF-α (Peprotech G1513, 200 U/ml) was added to induce for 3 days to obtain DC cells.

(242) T cells were isolated from PBMC and mixed with the DC cells ousedbtained above in the ratio of 1:10 to culture together with each antibody (hIgG was control) in different ratios for 5-6 days. The secretions of IFN-γ or IL-2 were measured with ELISA reagent kits (both purchased from Dakewe), respectively.

(243) The secretions of IFN-γ after mixed culture of DC cells and T cells were shown in FIG. 50-FIG. 53. The secretions of IL-2 after mixed culture of DC cells and T cells were shown in FIGS. 54-56.

(244) As shown in figures, the antibodies 4G10H1L1, 4G10H3L3, and 14C12H1L1, as well as bispecific antibodies BiAb001, BiAb002, BiAb003, and BiAb004 all can effectively induce the secretion of IFN-γ and IL-2 in mixed lymphocytes. The IFN-γ secretion induced by 1 nM or 10 nM anti-PD-1 antibody 14C12H1L1 were comparable with that of 100 nM control antibody Nivolumab. The IFN-γ secretion induced by 100 nM anti-CTLA4 antibodies 4G10H1L1 and 4G10H3L3 were better than that of 100 nM control antibody Ipilimumab (FIG. 52).

Example 12: Induced IL-2 Secretion

(245) The isolated PBMCs (the same method as in Example) was stimulated with PHA (Shanghai Shenqi Biotech Co., Ltd, 50 μl/ml) for 3 days, and then PBMCs (from volunteer blood donors, 5×10.sup.4 cells/well) mixed with Raji cells (from ATCC, 5×10.sup.4 cells/well) and MDA-MB-231 cells (from ATCC, 1×10.sup.4 cells/well) in a 96-well plate. Antibodies (100 nM) were added and mixed and cultured together. After co-culture for 3 days, secretion of IL-2 was measured with ELISA reagent kit (purchased from Dakewe) according to the instructions.

(246) The IL-2 secretion after mixed cell culture was shown in FIG. 57, FIG. 58, and FIG. 59, respectively. As shown in the figures, the antibodies 4G10H1L1, 4G10H3L3, and 14C12H1L1, as well as bispecific antibodies BiAb001, BiAb002, BiAb003, and BiAb004 can effectively induce the secretion of IL-2 by PBMCs. The anti-PD-1 antibody 14C12H1L1 can induce a higher IL-2 secretion than the control antibody Nivolumab (FIG. 58), and bispecific antibodies BiAb001, BiAb002, BiAb003, and BiAb004 have the same effects on IL-2 secretion as 14C12H1L1+4G10H1L1 or 14C12H1L1+4G10H3L3 (FIG. 59).

Example 13: Impact of Antibody BiAb004 on the Tumor Growth of MC38 Tumor Model in PD-1 HuGEMM Mice

(247) MC38 tumor cells were inoculated subcutaneously on the right side of PD-1 HuGEMM mice (1×10.sup.6 cells/mouse, human PD-1 transgenic mice). When the mean tumor volume reached approximately 144 mm.sup.3, the mice were randomly divided into 4 experimental groups per tumor volume with 8 mice in each group. Antibodies were given through abdominal administration, the specific grouping and dosages were as follows:

(248) Isotype Control group (dose: 2.67 mg/kg),

(249) BiAb004 high-dose group (dose: 2.67 mg/kg),

(250) BiAb004 low-dose group (dose: 0.267 mg/kg),

(251) The above 3 groups were injected with antibodies twice weekly, 5 times in total. After injection, the tumor sizes were measured twice weekly.

(252) The results were presented in FIG. 60.

(253) Evidently:

(254) The tumor sizes in the BiAb004 high-dose, and BiAb004 low-dose groups were all significantly smaller than those in the Isotype control group statistically (P<0.001, P<0.05, respectively). BiAb004 low-dose groups showed a statistically significant antitumor effect on the MC38 tumor model in the PD-1 HuGEMNI mice.

(255) Although specific embodiments of the present invention have been described in detail, as will be appreciated by one skilled in the art, these details may incur various modifications and substitutions according to all the teachings we have disclosed. These changes are all covered by the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents.