CTLA4 ANTIBODY, PHARMACEUTICAL COMPOSITION AND USE THEREOF

Abstract

Provided is an isolated CTLA4 monoclonal antibody. The antibody can highly-specifically bind to CTLA4, and can effectively block the binding of CTLA4 to B7, reduce the activity or expression level of CTLA4, relieve the inhibition of CTLA4 on the immune function of an organism, activate T lymphocytes, and effectively treat tumors and immune system diseases. Also provided are a monoclonal antibody conjugate comprising the pharmaceutical composition and diagnostic composition of the antibody, and the use thereof in the preparation of a drug for preventing and/or treating and/or treating in combination tumors and immune system diseases.

Claims

1. An isolated anti-CTLA4 antibody or antigen-binding fragments thereof inhibiting the binding of CTLA4 to human B7, the anti-CTLA4 antibody or antigen-binding fragments thereof comprises heavy and light chain variable regions, wherein: the heavy chain variable region comprises a heavy chain CDR1 as set forth in SEQ ID NO: 13, a heavy chain CDR2 as set forth in SEQ ID NO: 14, and a heavy chain CDR3 as set forth in SEQ ID NO: 15; and/or the light chain variable region comprises a light chain CDR1 as set forth in SEQ ID NO: 16, a light chain CDR2 as set forth in SEQ ID NO: 17, and a light chain CDR3 as set forth in SEQ ID NO: 18; or the heavy chain variable region comprises a heavy chain CDR1 as set forth in SEQ ID NO: 19, a heavy chain CDR2 as set forth in SEQ ID NO: 20, and a heavy chain CDR3 as set forth in SEQ ID NO: 21; and/or the light chain variable region comprises a light chain CDR1 as set forth in SEQ ID NO: 22, a light chain CDR2 as set forth in SEQ ID NO: 23, and a light chain CDR3 as set forth in SEQ ID NO: 24; or the heavy chain variable region comprises a heavy chain CDR1 as set forth in SEQ ID NO: 25, a heavy chain CDR2 as set forth in SEQ ID NO: 26, and a heavy chain CDR3 as set forth in SEQ ID NO: 27; and/or the light chain variable region comprises a light chain CDR1 as set forth in SEQ ID NO: 28, a light chain CDR2 as set forth in SEQ ID NO: 29, and a light chain CDR3 as set forth in SEQ ID NO: 30.

2. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, the antibody is a full-length antibody.

3. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, the antigen-binding fragment is Fab or F(ab)′2 or scFv.

4. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, the antibody is a chimeric antibody or a humanized antibody.

5. The anti-CTLA4 antibody or antigen-binding fragments thereof of claims 1, wherein the heavy chain variable region comprises amino acid sequences having at least 75% identity to the sequences as set forth in SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

6. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, wherein the light chain variable region comprises amino acid sequences having at least 75% identity to the sequences as set forth in SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

7. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 6, wherein the heavy chain variable region comprises the sequence as set forth in SEQ ID NO: 7, and the light chain variable region comprises the sequence as set forth in SEQ ID NO: 8.

8. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 6, wherein the heavy chain variable region comprises the sequence as set forth in SEQ ID NO: 9, and the light chain variable region comprises the sequence as set forth in SEQ ID NO: 10.

9. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 6, wherein the heavy chain variable region comprises the sequence as set forth in SEQ ID NO: 11, and the light chain variable region comprises the sequence as set forth in SEQ ID NO: 12.

10. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, wherein the heavy chain variable region comprises amino acid sequences as set forth in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

11. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, wherein the light chain variable region comprises amino acid sequences as set forth in SEQ ID NO: 39 or SEQ ID NO: 40.

12. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, the anti-CTLA4 antibody or antigen-binding fragments thereof comprises a heavy chain constant region and/or a light chain constant region; wherein the heavy chain constant region of the antibody is a heavy chain constant region of a mouse antibody or a heavy chain constant region of a human antibody, and/or the light chain constant region of the antibody is a light chain constant region of a mouse antibody or a light chain constant region of a human antibody.

13. The anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, the anti-CTLA4 antibody or antigen-binding fragments thereof inhibits the binding of CTLA4 to human B7-1 and/or human B7-2.

14. An isolated anti-CTLA4 antibody or antigen-binding fragments thereof inhibiting the binding of CTLA4 to human B7, the anti-CTLA4 antibody or antigen-binding fragments thereof binds the same epitopes as the anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, or competes for binding human B7 with the anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1.

15. An isolated nucleic acid encoding the anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15

18. An immunoconjugate, comprising an antibody portion and a conjugation portion, wherein the antibody portion is the anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, and the conjugation portion is a therapeutic reagent or a diagnostic reagent.

19. The immunoconjugate of claim 18, wherein the conjugation portion is selected from one or more of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, and a biotin.

20. A pharmaceutical composition, comprising the anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1, and pharmaceutically acceptable carriers.

21. Use of the anti-CTLA4 antibody or antigen-binding fragments thereof of claim 1 in the manufacture of medicaments for specifically binding CTLA4.

22. The use of claim 21, wherein the medicaments specifically binding CTLA are used for blocking the binding of CTLA4 to B7, down-regulating the activity or level of CTLA4, relieving the immunosuppression of CTLA4 to an organism, activating T lymphocytes, or improving the expression of IL-2 in T lymphocytes.

23. The use of claim 21, wherein the medicaments specifically binding CTLA are used for prevention and/or treatment and/or adjuvant treatment of cancers and immune system diseases.

24. The use of claim 23, wherein the cancers are selected from breast cancer, lung cancer, colorectal cancer, stomach cancer, colon cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, glioma or melanoma.

25. A method of prevention and/or treatment and/or adjuvant treatment of cancers and immune system diseases, comprising administering an effective amount of pharmaceutical compositions of claim 20 to subjects in need thereof.

26. The use of claim 25, wherein the cancers are selected from breast cancer, lung cancer, colorectal cancer, stomach cancer, colon cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, glioma or melanoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1: The detection of binding of murine anti-CTLA4 antibodies to hCTLA4-Fc protein by ELISA.

[0057] FIG. 2: The detection of binding of murine anti-CTLA4 antibodies to 293T-hCTLA4 cells by FACS.

[0058] FIG. 3: The detection of murine anti-CTLA4 antibodies blocking the binding of CD80 or CD86 to CTLA4 by ELISA.

[0059] FIG. 4: The detection of binding of CTLA4 chimeric antibodies to the activated T cells by FACS.

[0060] FIG. 5: The detection of epitope competition between the CTLA4 chimeric antibodies and other antibodies by ELISA.

[0061] FIG. 6: The detection of CTLA4 chimeric antibodies dose-dependently enhancing IL-2 secretion in Jurkat cells by ELISA.

[0062] FIG. 7: The detection of CTLA4 chimeric antibodies dose-dependently enhancing IL-2 secretion in Jurkat-hCTLA4 cells by ELISA.

[0063] FIG. 8: The detection of CTLA4 chimeric antibodies enhancing IFN-γ secretion in MLR experiments by ELISA.

[0064] FIG. 9: Tumor growth inhibition in the human CTLA4 knock-in mice by CTLA4 chimeric antibodies.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The embodiments of the present invention will hereinafter be described in detail with reference to Examples. Those skilled in the art will understand that the Examples below are only intended to illustrate the invention, but not to limit the scope of the invention. Examples in which specific techniques or conditions are not indicated are carried out according to the techniques or conditions described in the literatures in the art (e.g., with reference to “Molecular Cloning: A Laboratory Manual”, Sambrook. J. et al. (authors), Huang Peitang et.al. (translators), the 3.sup.rd edition, Science Press) or according to the product specifications. Reagents or instruments in which the manufacturers are not indicated are conventional products that are commercially available.

Example 1: Animal Immunization and Screening of Mouse Anti-CTLA4 Antibodies

[0066] Age-appropriate Balb/c mice were selected and subjected immune injection. The hCTLA4-mFc fusion protein was used as an antigen and mixed with complete Freund's adjuvant (Sigma-Aldrich), and then injected into the immunized mice by subcutaneous injection to stimulate the cloning of corresponding B-lymphocytes. Then the immunized mice were boosted by the intraperitoneal injection of 100 μg of CTLA4-mFc emulsified with incomplete Freund's adjuvant (Sigma-Aldrich) at a ratio of 1:1 about every two to three weeks. Spleen lymphocytes taken from the mice by sterile operations were mixed with the prepared SP2/0 myeloma cells in a certain ratio (1×10.sup.8 of spleen cells and 2×10.sup.7 of myeloma cells) with the addition of polyethylene glycol (Sigma, P7181).

[0067] After fusion, the fused cells were added into 96-well plates with 0.1 mL of HAT medium per well and were cultured in the CO.sub.2 incubator at 37° C. On day 4, 0.1 mL of HT medium per well was added. On day 7, the culture medium was completely replaced with HT medium. The screening was carried out at days 8-12. The positive cells were sub-cloned by limiting dilution, and were expanded and tested by ELISA and FACS as described below.

[0068] The 2 μg/mL of hCTLA4-hFc was coated on the 96-well plates (Corning, catalog #9018) followed by overnight incubation at 4° C. The plates were washed with PBST (PBS containing 0.05% of Tween-20) three times, and were blocked with 5% skimmed milk powder (Oxoid, catalog #LP0031B). After washing the plates with PBST three times, the hybridoma supernatants were added and incubated for 2 hours at 37° C. The plates were washed and incubated for 1 hour at 37° C. with 1/5000 diluted HRP-labeled goat anti-mouse IgG second antibodies (BioLegend, catalog #405306). The plates were washed again, and were developed by adding TMB (Tiangen catalog #PA107-02).

[0069] The binding of antibodies to 293T-hCTLA4 cells was detected by FACS. 1×10.sup.5 of 293T-hCTLA4 cells per well were added into 96-well plates (Corning, catalog #3799) and were incubated with the hybridoma supernatants for 30 minutes at 4° C. After the cells were washed twice with buffer (PBS containing 1% BSA and 5 mM EDTA), the PE-labeled goat anti-mouse IgG secondary antibodies were added and incubated for 30 minutes at 4° C. The cells were washed with buffer and analyzed by Attune Nxt flow cytometer (Thermo Fisher).

[0070] The hybridoma supernatants were further screened by employing ligand blocking experiments, since CTLA4 antibodies can block the binding of CTLA4 to CD80. The 2 μg/mL goat anti-mouse IgG antibodies (Sigma, catalog #M4280) were coated on the 96-well plates (Corning, catalog #9018) followed by overnight incubation at 4° C. The plates were washed three times and blocked with 5% skimmed milk powder (Oxoid, catalog #LP0031B) for 2 hours at 37° C. The plates were washed three times and were incubated for 1 hour at room temperature with the pre-mixture of 20 μg/mL hCD80-Fc and 20 ng/mL biotinylated hCTLA4-His. The plates were washed and incubated with HRP-labeled streptavidin (BD, catalog #554066) for 1 hour at room temperature. The plates were washed and developed by adding TMB.

Example 2: Physicochemical and Binding Properties of Purified Hybridoma Supernatants Anti-CTLA4 Antibodies

[0071] Monoclonal cells of the hybridomas were obtained by three or more rounds of limiting dilution. Stable monoclonal cells of the hybridomas were carried out the serum-free domestication (Gibco, catalog #12045-076) for 8 to 9 days and were expanded to T-175 culture flask (NUNC, catalog #159910). On days 8-9, the supernatant was collected and filtered using a 0.22 μm filter membrane. The supernatant was passed through the Protein A column (GE Healthcare, catalog #17549112) to isolate and purify the antibodies, which were adsorbed on the column. The Protein A column was washed with 1 mM PBS, and the antibodies were eluted from the column using 50 mM PBS (pH3.0). The eluate was adjusted to neutrality with a 0.5 M of sodium hydroxide solution and filtered using a 0.22 μm filter membrane. The solution of antibodies was concentrated by Ultra-15 centrifugal concentrators (Millipore, catalog #ACS500024), and the concentration of the antibodies was detected using Nanodrop spectrophotometry (Thermo Scientific, NanoDrop 2000). The content of endotoxin in the solution of purified antibodies was detected using the Gel Clot TAL kit (Xiamen Bioendo Technology, catalog #010250) with a standard content of less than 1 EU/mL.

[0072] ELISA was used for detecting the binding of purified antibodies to CTLA4-hFc. The hCTLA4-hFc was coated on 96-well plates (Corning, catalog #9018) followed by incubation overnight at 4° C. The plates were washed with PBST (PBS containing 0.05% of Tween-20) three times, and were blocked with 5% skimmed milk powder (Oxoid, catalog #LP0031B) for 2 hours at 37° C. The plates were washed three times with PBST and were incubated for 1 hour at room temperature with antibodies to be detected. The plates were washed three times and incubated for 1 hour at room temperature with 1/5000 diluted HRP-labeled goat anti-mouse IgG secondary antibodies (Invitrogen, catalog #31432). The plates were washed with PBST and were developed by adding TMB. The results are shown in FIG. 1, and the median effective concentrations of binding are shown in Table I.

[0073] The purified antibodies were detected for the binding to 293T-hCTLA4 cells by employing FACS. 293T-hCTLA4 cells were resuspended to 4×10.sup.6 cells/mL with FACS buffer (PBS containing 1% BSA) and added to the 96-well round-bottom plates (Corning, catalog #3795) at 50 μL/well. The purified antibodies to be detected were added to wells at a certain concentration (50 μL/well) and incubated for 1 hour at 4° C. The cells were washed three times with buffer and were incubated for 0.5 hour at 4° C. with PE-labeled fluorescent secondary antibodies. The cells were washed three times with FACS buffer and re-suspended to 200 μL/well. The fluorescence signals were detected using Attune Nxt flow cytometer (Thermo Fisher). The results are shown in FIG. 2, and the median effective concentrations of binding are shown in Table I.

TABLE-US-00001 TABLE I binding force analysis of murine anti-CTLA4 monoclonal antibody ELISA EC.sub.50 FACS EC.sub.50 (nM) (nM) Antibody ID hCTLA4-hFc 293T-hCTLA4 9B8-H2-B6-G5 0.025 1.08 9E6-A7-E5-A11 0.023 2.28 4H9-G5-D5-B1 0.023 1.35 21H5-H9-D1-G10 0.026 5.52 26E3-H9-B9-C12 0.022 1.15 2E12-F11-E9-G9 0.028 4.11 3D3-G6-H5-C7 0.029 3.49 7D5-H2-C8-F9 0.047 13A2-H5-D6-A3 0.032 3.56 17D11-H9-B4-E9 0.059 18H11-H2-H2-H9 0.021 2.02 19C5-G11-A7-G5 0.036 22H12-H10-A1-B1 0.025 3.27 30F5-H4-G4-F9 2.34

Example 3: Blocking of Interactions Between CD80 or CD86 and CTLA4 by Murine CTLA4 Antibody

[0074] ELISA was used to detect whether the antibody blocked the binding between CD80 or CD86 and CTLA4. The hCTLA4-hFc protein was labeled with biotin using the Biotin Labeling Kit-NH.sub.2 (Dojindo, catalog#LK03). The purified antibodies were coated on 96-well plates (Corning, catalog #9018) followed by incubation overnight at 4° C., and were blocked with 5% skimmed milk powder (Oxoid, catalog #LP0031B) for 2 hours at 37° C. The plates were washed three times, and incubated for 1 hour at room temperature with the pre-mixture of biotinylated hCTLA4-hFc and hCD80-Fc or hCD86-Fc. The plates were washed with PBST and incubated with HRP-labeled streptavidin (BD, catalog #554066) for 1 hour at room temperature. The plates were washed and developed by adding TMB. The results are shown in FIG.3, and the results are summarized in Table II.

TABLE-US-00002 TABLE II ligand blocking experiments of murine anti-CTLA4 antibodies ELISA IC.sub.50 (ng/mL) Antibody ID CD80 CD86 9B8-H2-B6-G5 2.917 1079 9E6-A7-E5-A11 32.58 5824 4H9-G5-D5-B1 69.06 7244 21H5-H9-D1-G10 26.27 4089 26E3-H9-B9-C12 52.26 10058 2E12-F11-E9-G9 14.41 3D3-G6-H5-C7 23.62 2442 7D5-H2-C8-F9 272.5 13A2-H5-D6-A3 29.5 2091 17D11-H9-B4-E9 44.46 2286 18H11-H2-H2-H9 46.73 7293 19C5-G11-A7-G5 35.69 3145 22H12-H10-A1-B1 62.36 8395

Example 4: Species Cross Reactions of Murine CTLA4 Antibodies

[0075] Species cross reactions of the purified antibodies were detected using ELISA. The quantified purified antibodies were coated on 96-well plates (Corning, catalog #9018) followed by incubation overnight at 4° C., and were blocked with 5% skimmed milk powder (Oxoid, catalog #LP0031B) for 2 hours at 37° C. The biotin-labeled mouse CTLA4-His (SinoBiological, catalog #50503-M08H) or monkey CTLA4-Fc (SinoBiological, catalog #90213-C02H) was added to the plates and incubated for 1 hour at room temperature. The plates were washed three times with PBST and incubated with 1/1000 diluted HRP-labeled streptavidin (BD, catalog #554066) for 1 hour at room temperature. The plates were washed and developed by adding TMB. The results are shown in Table III.

TABLE-US-00003 TABLE III cross reactions of murine anti-CTLA4 antibodies ELISA (OD) Antibody ID mouse monkey 9B8-H2-B6-G5 0.06 0.37 9E6-A7-E5-A11 0.05 0.87 4H9-G5-D5-B1 0.05 2.79 21H5-H9-D1-G10 0.06 2.06 26E3-H9-B9-C12 0.06 1.83 2E12-F11-E9-G9 0.05 0.58 3D3-G6-H5-C7 0.06 2.42 13A2-H5-D6-A3 0.05 2.36 17D11-H9-B4-E9 0.05 0.62 18H11-H2-H2-H9 0.06 2.93 19C5-G11-A7-G5 0.06 0.64 22H12-H10-A1-B1 0.05 2.91 30F5-H4-G4-F9 0.05 2.64

Example 5: Cloning of cDNAs of Murine Antibodies and Construction of Chimeric Antibodies

[0076] Using degenerate primer PCR-based methods, the gene sequences of variable regions of the heavy and light chains of the hybridoma antibodies are obtained. Monoclonal cells of hybridoma were lysed using Trizol (Invitrogen, catalog #15596-018) to isolate total RNA, and reverse transcription was performed using RNA as a template with SuperScript III First-Strand Synthesis System (Invitrogen, catalog #18080-051) to obtain a cDNA bank. The resulting cDNA bank was used as a template, and PCR was performed using degenerate primers (Zhou H, et al., Nucleic Acids Research 22: 888-889 (1994), Chardes T et al., FEBS Letters 452: 386-394 (1999)). PCR products were detected by agarose gel electrophoresis. The size of the PCR amplification product of the heavy and light chain variable regions is expected to be 400 base pairs. The PCR products were cloned to the pClone007 vector (Tsingke, catalog #TSV-007BS) and transformed to Trans 5α, a competence of Escherichia coli, (Transgen, catalog# CD201-02). 3-6 monoclonal Escherichia colis were selected on agar plates for gene sequencing. In some cases, PCR products may also be directly used for gene sequencing. The full-length gene sequences of the variable regions of the heavy and light chains of antibodies 4H9-G5-D5-B1, 9B8-H2-B6-G5, and 19C5-G11-A7-G5 were finally obtained through the methods above. The gene and amino acid sequences of the complementarity determining regions and framework regions of the heavy and light chains of the antibodies were further obtained through analysis using NCBI Ig-BLAST (https://www.ncbi.nlm.nih.gov/projects/igblast/) (Kabat E.A., et al., 1991, Sequences of proteins of immunological interest, in: NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, Md.). The detailed information was disclosed in the sequencing listing of antibodies and Table IV.

[0077] The light and heavy chains of chimeric antibody were constructed by linking the variable regions of murine CTLA4 antibody to human IgG1 and kappa constant regions. The appropriate restriction sites were introduced into the variable regions of the heavy and light chains by PCR, and cloned into the expression vectors of corresponding chimeric antibodies, respectively. The plasmids of heavy and light chains of chimeric antibodies were introduced into 293F cells by lipofection and cultured for 6 days. The antibodies in the cell culture supernatant were purified with Protein A column (GE Healthcare, catalog #17549112). The Protein A column was washed with 1 mM PBS, and the antibodies were eluted from the column using 50 mM of PBS (pH3.0). The eluate was adjusted to neutrality with a 0.5 M of sodium hydroxide solution and filtered using a 0.22 μm filter membrane. The solution of antibodies was concentrated by Ultra-15 centrifugal concentrators (Millipore, catalog #ACS500024), and the concentration of the antibodies was detected using Nanodrop spectrophotometry. The content of endotoxin in the solution of purified antibodies was detected using the Gel Clot TAL kit (Xiamen Bioendo Technology, catalog #010250) with a standard content of less than 1 EU/mL. The binding activity of the chimeric antibodies produced by the transfected 293F cells was detected by ELISA and FACS (see Example 2 for specific steps). The results showed that chimeric antibodies bind to CTLA4 with comparable affinity to those of the murine antibodies.

TABLE-US-00004 TABLE IV Sequence ID number of anti-CTLA4 antibodies in this invention SEQ ID NO: Description 1 9B8-H2-B6-G5 heavy chain variable region (DNA) 2 9B8-H2-B6-G5 light chain variable region (DNA) 3 4H9-G5-D5-B1 heavy chain variable region (DNA) 4 4H9-G5-D5-B1 light chain variable region (DNA) 5 19C5-G11-A7-G5 heavy chain variable region (DNA) 6 19C5-G11-A7-G5 light chain variable region (DNA) 7 9B8-H2-B6-G5 heavy chain variable region (AA) 8 9B8-H2-B6-G5 light chain variable region (AA) 9 4H9-G5-D5-B1 heavy chain variable region (AA) 10 4H9-G5-D5-B1 light chain variable region (AA) 11 19C5-G11-A7-G5 heavy chain variable region (AA) 12 19C5-G11-A7-G5 light chain variable region (AA) 13 9B8-H2-B6-G5 heavy chain CDR1 (AA) 14 9B8-H2-B6-G5 heavy chain CDR2 (AA) 15 9B8-H2-B6-G5 heavy chain CDR3 (AA) 16 9B8-H2-B6-G5 light chain CDR1 (AA) 17 9B8-H2-B6-G5 light chain CDR2 (AA) 18 9B8-H2-B6-G5 light chain CDR3 (AA) 19 4H9-G5-D5-B1 heavy chain CDR1 (AA) 20 4H9-G5-D5-B1 heavy chain CDR2 (AA) 21 4H9-G5-D5-B1 heavy chain CDR3 (AA) 22 4H9-G5-D5-B1 light chain CDR1 (AA) 23 4H9-G5-D5-B1 light chain CDR2 (AA) 24 4H9-G5-D5-B1 light chain CDR3 (AA) 25 19C5-G11-A7-G5 heavy chain CDR1 (AA) 26 19C5-G11-A7-G5 heavy chain CDR2 (AA) 27 19C5-G11-A7-G5 heavy chain CDR3 (AA) 28 19C5-G11-A7-G5 light chain CDR1 (AA) 29 19C5-G11-A7-G5 light chain CDR2 (AA) 30 19C5-G11-A7-G5 light chain CDR3 (AA) 31 9B8-VH4 heavy chain variable region (DNA) 32 9B8-VH5 heavy chain variable region (DNA) 33 9B8-VH6 heavy chain variable region (DNA) 34 9B8-VL4 light chain variable region (DNA) 35 9B8-VL5 light chain variable region (DNA) 36 9B8-VH4 heavy chain variable region (AA) 37 9B8-VH5 heavy chain variable region (AA) 38 9B8-VH6 heavy chain variable region (AA) 39 9B8-VL4 light chain variable region (AA) 40 9B8-VL5 light chain variable region (AA) 41 9B8-VH4 full length heavy chain (AA) 42 9B8-VH5 full length heavy chain (AA) 43 9B8-VH6 full length heavy chain (AA) 44 9B8-VL4 full length light chain (AA) 45 9B8-VL5 full length light chain (AA)

Example 6: Binding of Human-mouse CTLA4 Chimeric Antibodies to Activated Human T Cells

[0078] Binding of the CTLA4 chimeric antibodies to activated human T cells was detected by FACS. Human peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood using density gradient centrifugation (Ficoll-Paque Premium, GE Healthcare, catalog #17-5442-02). T cells were further purified from PBMC using the Pan T Isolation Kit (Miltenyi, catalog #130-096-535). T cells were resuspended in the medium (RPMI-1640 medium (Gibco, catalog #C22400500BT), 10% inactivated FBS (Gibco, catalog #10099-141) and penicillin/penicillin diabody (Gibco, catalog #15140-122)), and were activated using Dynabeads® Human T-Activator CD3/CD28 (Gibco, catalog #11132D) according to instructions. After 3 days, the activated T cells were incubated with 10 μg/mL of chimeric antibodies in buffer (PBS containing 1% BSA) for 1 hour at 4° C. The cells were washed three times and incubated with the PE-labeled mouse anti-human IgG Fc secondary antibodies (BioLegend, catalog #409304) for 1 hour at 4° C. The T cells were washed three times with buffer and resuspended in 200 μL/well buffer prior to detecting the fluorescence signal on Attune Nxt flow cytometer (Thermo Fisher). The results are shown in FIG. 4.

Example 7: Kinetic Detections of Human-mouse CTLA4 Chimeric Antibodies

[0079] The kinetic constants (k.sub.assoc and k.sub.dissoc) of binding of the antibody and the antigen were measured using the biomolecular interaction system Octet-96 (Pall Life Sciences, S-000959), and the equilibrium binding constant K.sub.D was further calculated. The CTLA4-mFc antigen proteins were coupled to the surfaces of an AMC sensors (Pall Life Sciences, PN18-5099), and antibodies of different concentrations were added to measure the binding and dissociation between the CTLA4 chimeric antibodies and the CTLA4-mFc proteins on the sensor surfaces. Specifically, AMC sensors were pre-wet in the buffer (PBS containing 0.02% Tween-20 and 0.1% BSA) for 10 minutes and then equilibrated in CTLA4-mFc sample buffer for 5 minutes to such that the CTLA4-mFc proteins were coupled to the surfaces of sensors. The CTLA4-mFc-coupled AMC sensors were first equilibrated in the buffer for 2 minutes and then incubated for 5 minutes in buffer containing different concentrations of antibody (3-200 nM) to measure the binding of antibodies to the CTLA4-mFc protein. Finally, the antigen and antibody-bound sensors were repositioned in the sample buffer and waited for 10 minutes to measure the dissociation of antibodies from the CTLA4-mFc proteins. The measured data were globally fitted using a 1:1 model to obtain kinetic constants of binding and dissociation. On this basis, an equilibrium binding constant K.sub.D was further obtained. Experimental results are presented in Table V.

TABLE-US-00005 TABLE V Kinetic Analysis (ForteBio) k.sub.assoc (1/s) k.sub.dissoc (1/Ms) K.sub.D (M) 4H9-G5-D5-B1 1.10E+06 <1.0E−07 <1.0E−12 9B8-H2-B6-G5 4.78E+05 4.14E−05 8.68E−11

Example 8: Epitope Analysis of Human-mouse CTLA4 Chimeric Antibodies

[0080] ELISA was used to detect the epitope competition between the CTLA4 chimeric antibodies and reference antibodies. The 2 μg/mL of Ipilimumab was coated on the 96-well plates (Corning, catalog #9018) followed by incubation overnight at 4° C., blocked with 5% skimmed milk powder (Oxoid, catalog #LP0031B) for 2 hours at 37° C., and washed three times with PBST. The biotin-labeled CTLA4-hFc were pre-incubated with gradient-diluted chimeric antibodies for 30 minutes at room temperature, added to the 96-well plates and incubated for 1 hour at room temperature. The plates were washed three times with PBST and incubated with 1/1000 diluted HRP-labeled streptavidin (BD, catalog #554066) for 1 hour at room temperature. The plates were washed and developed by adding TMB. The results are shown in FIG. 5. The binding of Ipilimumab to CTLA4-hFc can be completely blocked by chimeric antibody 4H9-G5-D5-B1. In contrast, the binding of Ipilimumab to CTLA4-hFc cannot be completely blocked by chimeric antibody 9B8-H2-B6-G5, indicating that these two antibodies have different binding epitopes to antigen CTLA4-hFc.

Example 9: Enhancement of Immune Function of Jurkat Cells by Human-mouse CTLA4 Chimeric Antibodies

[0081] Jurkat cells were used to detect the cellular biological activity of human-mouse CTLA4 chimeric antibodies. One experiment used Raji cells as the antigen presenting cells to stimulate Jurkat cells, which could be blocked by adding exogenous CTLA4-hFc protein. The addition of CTLA4 antibody in the systems above could neutralize the exogenous CTLA4-hFc protein and recover the function of Jurakt cells to activate Jurkat cells. Specifically, Raji cells were treated with 25 μg/mL mitomycin C (MedChem Express, catalog #HY-13316) for 1 hour at 37° C., and were washed 4 times with PBS. Jurkat cells and treated Raji cells were resuspended in medium (RPMI-1640 with 10% inactivated FBS) and added to 96-well round-bottom plates (Corning, catalog #3799) with an equal amount of anti-CD3 antibody (BioLengend, catalog #317326), CTLA4-hFc fusion protein, as well as test antibodies at varying concentrations. The supernatants were collected after 2 days of culture of cells, and the content of IL-2 in supernatants was quantified using ELISA (antibodies were purchased from BD). The results showed that CTLA4 antibodies could enhance the secretion of IL-2 and showed a dose-dependent effect as shown in FIG. 6.

[0082] In another experiment, a Jurkat-hCTLA4 cell line was used to evaluate the in vitro biological activity of anti-CTLA4 antibodies. The hCTLA4 plasmid was introduced into Jurkat cells by electroporation (Lonza), and then cloned and screened with hygromycin to obtain a Jurkat-hCTLA4 stable cell line. Jurkat-hCTLA4 cells and mitomycin C-treated Raji cells were added into 96-well round-bottom plates with CTLA4 antibodies at varying concentrations. The supernatants were collected after 2 days of culture of cells, and IL-2 was quantitatively detected using ELISA as mentioned above. The results showed that CTLA4 antibodies could enhance the secretion of IL-2 and showed a dose-dependent effect as shown in FIG. 7.

[0083] Example 10: Allogeneic Mixed Lymphocyte Reaction (MLR) of Human-mouse CTLA4 Chimeric Antibodies

[0084] Mixed lymphocyte reaction was used to detect the in vitro biological activity of CTLA4 antibodies. Peripheral blood monocytes (PBMCs) were isolated from human peripheral blood using density gradient centrifugation (Ficoll-Paque Premium, GE Healthcare, catalog #17-5442-02). Monocytes were further purified from PBMCs using CD14 Cell Isolation Kit (Miltenyi, catalog #130-050-201). Cytokines 1000 U/mL GM-CSF (Prospec, catalog #CYT-221) and 1000 U/mL IL-4 (Prospec, catalog #CYT211) were added to the medium to induce monocyte differentiation into dendritic cells. Cytokines were supplemented every 2-3 days, and immature dendritic cells were harvested after 5-6 days for subsequent experiments.

[0085] Human CD3.sup.+T cells were isolated from PBMCs using Pan T Cell Isolation Kit (Miltenyi, catalog #130-096-535). The CD3.sup.+T cells, immature dendritic cells and CTLA4 antibodies of various concentrations were added to 96-well round-bottom plates (Corning, catalog #3799). After 5-6 days of cell culture, the supernatants were harvested, and the content of cytokines was quantitatively detected using ELISA. The results were shown in FIG. 8 that human-mouse CTLA4 chimeric antibodies could enhance the secretion of IFN-γ in MLR.

Example 11: Tumor Growth Inhibition of Human-mouse CTLA4 Chimeric Antibodies

[0086] The tumor animal model in the present invention was a special tumor animal model, which was constructed by inoculating MC38 murine colon cancer cells (NCI) in CTLA4 humanized transgenic mice (developed by Nanjing Galaxy Biopharm Co, Ltd) to test the druggability and the in vivo efficacy of CTLA4 antibodies in mice.

[0087] CTLA4 humanized transgenic mice (7-11 weeks old) were injected subcutaneously in the flank with 3×10.sup.5 MC38 tumor cells in 0.1 mL volume. After 6 days, when the tumor volume reached 70 mm.sup.3, the CTLA4 antibody, reference antibody and control PBS were injected biweekly at a dose of 10 mg/kg (dosing volume 10 mL/kg) for a total of 6 doses. The volume of subcutaneous tumor was measured biweekly using vernier calipers during the course of the experiment. Animals were followed until day 34 after injection of tumor cells. As shown in FIG. 9, after the 6 doses, the tumor growth is significantly inhibited by the antibody 9B8-H2-B6-G5 to be detected, and some tumor is nearly disappeared after the treatment of drug.

Example 12: Humanization of Human-mouse Chimeric CTLA4 Antibodies

[0088] Murine antibody 9B8-H2-B6-G5 was humanized by CDR Grafting techniques. According to the literature reports, framework regions of subtype III of heavy chain and subtype I of kappa light chains in human antibodies have been widely used in antibody humanization (Carter P et al., PNAS 89: 4285-4289 (1992); Presta LG et al., Cancer Research 57: 4593-4599 (1997)). The CDR regions of murine antibody 9B8-H2-B6-G5 were grafted onto the framework regions of human antibodies above, and the amino acid residues in the framework regions were mutated to enhance the affinity of the antibodies. The affinity of the resultant humanized antibody for CTLA4-mFc was detected using Octet system and the AMC sensor so as to screen the best one. DNA and amino acid sequences of the heavy chains and the light chains (9B8-VH4, 9B8-VH5, 9B8-VH6, 9B8-VL4, 9B8-VL5) of the humanized antibody are shown in table IV.

[0089] Although the present invention has been described with reference to specific embodiments, those skilled in the art will recognize that, according to all the disclosed teachings, various changes and replacements may be made thereto within the scope of the present invention as set forth in the appended claims and any equivalent thereof.