Anti-CTLA4 monoclonal antibody or its antigen binding fragments, pharmaceutical compositions and uses

1. A monoclonal antibody or antigen binding fragment thereof that binds to human CTLA4, comprising a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable region comprises:

an HCDR1 comprising the amino acid sequence of SEQ ID NO: 27,

an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and

an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and

(b) the light chain variable region comprises:

an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30,

an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and

an LCDR3 comprising the amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

2. The monoclonal antibody or antigen binding fragment thereof of claim 1 , wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of:

(a) the heavy chain variable region comprising the HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 29 and the light chain variable region comprising the LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 32;

(b) the heavy chain variable region comprising the HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprising the LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, the LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 33; and

(c) the heavy chain variable region comprising the HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, the HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and the HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprising the LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, the LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and the LCDR3 comprising the amino acid sequence of SEQ ID NO: 34.

3. The monoclonal antibody or antigen binding fragment thereof of claim 1 , wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 10, and any of SEQ ID NO: 14, SEQ ID NO: 6, and SEQ ID NO: 10 wherein the methionine at amino acid position 18 of SEQ ID NO: 14, SEQ ID NO: 6, and SEQ ID NO: 10 is substituted with an amino acid selected from the group consisting of leucine, valine, isoleucine and alanine; and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 8, and SEQ ID NO: 12.

4. The monoclonal antibody or antigen binding fragment thereof of claim 3 , wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of:

(a) the heavy chain variable region of SEQ ID NO: 6 and the light chain variable region of SEQ ID NO: 8;

(b) the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region of SEQ ID NO: 12;

(c) the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 16;

(d) the heavy chain variable region of SEQ ID NO: 18 and the light chain variable region of SEQ ID NO: 20;

(e) the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 22;

(f) the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO 24;

(g) the heavy chain variable region of SEQ ID NO: 6 wherein the methionine at amino acid position 18 is substituted with leucine, valine, isoleucine, or alanine, and the light chain variable region of SEQ ID NO: 8;

(h) the heavy chain variable region of SEQ ID NO: 10 wherein the methionine at amino acid position 18 is substituted with leucine, valine, isoleucine, or alanine and the light chain variable region of SEQ ID NO: 12;

(i) the heavy chain variable region of SEQ ID NO: 14 wherein the methionine at amino acid position 18 is substituted with leucine, valine, isoleucine, or alanine, and the light chain variable region of SEQ ID NO: 16;

(j) the heavy chain variable region of SEQ ID NO: 14 wherein the methionine at amino acid position 18 is substituted with leucine, valine, isoleucine, or alanine, and the light chain variable region of SEQ ID NO: 22; and

(k) the heavy chain variable region of SEQ ID NO: 14 wherein the methionine at amino acid position 18 is substituted with leucine, valine, isoleucine, or alanine, and the light chain variable region of SEQ ID NO: 24.

5. The monoclonal antibody or antigen binding fragment thereof of claim 4 , wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of:

(a) the heavy chain variable region of SEQ ID NO: 6 wherein the methionine at amino acid position 18 is substituted with leucine, and the light chain variable region of SEQ ID NO: 8;

(b) the heavy chain variable region of SEQ ID NO: 10 wherein the methionine at amino acid position 18 is substituted with leucine, and the light chain variable region of SEQ ID NO: 12;

(c) the heavy chain variable region of SEQ ID NO: 14 wherein the methionine at amino acid position 18 is substituted with leucine, and the light chain variable region of SEQ ID NO: 16;

(d) the heavy chain variable region of SEQ ID NO: 14 wherein the methionine at amino acid position 18 is substituted with leucine, and the light chain variable region of SEQ ID NO: 22; and

(e) the heavy chain variable region of SEQ ID NO: 14 wherein the methionine at amino acid position 18 is substituted with leucine, and the light chain variable region of SEQ ID NO: 24.

6. A monoclonal antibody that binds to human CTLA4, comprising a heavy chain variable region of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 16, wherein the methionine at amino acid position 18 of SEQ ID NO: 14 is substituted with leucine.

7. The monoclonal antibody or antigen binding fragment thereof according to claim 1 , wherein the monoclonal antibody or antigen binding fragment thereof is humanized.

8. The monoclonal antibody or antigen binding fragment thereof according to claim 1 , wherein the monoclonal antibody or antigen binding fragment thereof binds to human CTLA4 with a K D less than about 10 −5 M, as determined by surface plasmon resonance.

9. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain variable region of a monoclonal antibody or antigen binding fragment thereof, wherein

(a) the nucleotide sequence encodes a heavy chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 18;

(b) the nucleotide sequence encodes a heavy chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14 wherein the methionine at amino acid position 18 of each of SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14 is substituted with leucine, valine, isoleucine, or alanine; or

(c) the nucleotide sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17.

10. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a light chain variable region of a monoclonal antibody or antigen binding fragment thereof, wherein

(a) the nucleotide sequence encodes a light chain variable region comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; or

(b) the nucleotide sequence is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23.

11. A method of preparing the monoclonal antibody or antigen binding fragment thereof of claim 1 , comprising:

culturing a host cell under suitable conditions, wherein the host cell comprises:

(i) a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof, wherein

(a) the nucleotide sequence encodes the heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof as set forth by the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 18; or

(b) the nucleotide sequence encodes the heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof as set forth by the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14, wherein the methionine at amino acid position 18 of each of SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14 is substituted with leucine, valine, isoleucine, or alanine; or

(c) the nucleotide sequence is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO 17; and

(ii) a nucleotide sequence encoding the light chain variable region of the monoclonal antibody or antigen binding fragment thereof, wherein

(a) the nucleotide sequence encodes the light chain variable region of the monoclonal antibody or antigen binding fragment thereof as set forth by the amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22and SEQ ID NO 24; or

(b) the nucleotide sequence is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23; and

recovering the monoclonal antibody or antigen binding fragment thereof from the cell culture.

12. A pharmaceutical composition comprising a monoclonal antibody or antigen binding fragment thereof and at least one of a pharmaceutically acceptable carrier and an excipient, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CTLA4 and comprises a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable region comprises:

an HCDR1 comprising the amino acid sequence of SEQ ID NO: 27,

an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and

an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and

(b) the light chain variable region comprises:

an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30,

an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and

an LCDR3 comprising the amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

13. A method of treating a tumor in a human subject, comprising administering to the subject an effective amount of a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CTLA4 and comprises a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable region comprises:

an HCDR1 comprising the amino acid sequence of SEQ ID NO: 27,

an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and

an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and

(b) the light chain variable region comprises:

an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30,

an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and

an LCDR3 comprising the amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

14. A monoclonal antibody comprising:

(a) a heavy chain variable region comprising:

the HCDR comprising the amino acid sequence of SEQ ID NO: 27,

the HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and

the HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and

(b) a light chain variable region comprising:

the LCDR1 comprising the amino acid sequence of SEQ ID NO: 30,

the LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and

the LCDR3 comprising the amino acid sequence of SEQ ID NO: 32.

15. The monoclonal antibody according to claim 6 , wherein the monoclonal antibody binds to human CTLA4 with a K D less than about 10 −5 M, as determined by surface plasmon resonance.

16. The pharmaceutical composition of claim 12 , wherein the monoclonal antibody binds to human CTLA4 and comprises the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 16, wherein the methionine at amino acid position 18 of SEQ ID NO: 14 is substituted with leucine.

17. The method of claim 13 , wherein the monoclonal antibody binds to human CTLA4 and comprises the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 16, wherein the methionine at amino acid position 18 of SEQ ID NO: 14 is substituted with leucine.

18. The method of claim 13 , wherein the tumor is melanoma, a kidney tumor, a renal tumor, a prostate cancer, a bladder cancer, a colorectal cancer, a cancer of the gastrointestinal tract, a liver cancer, or a hepatic cancer.

19. The monoclonal antibody of claim 6 , wherein the monoclonal antibody has an IgG isotype selected from the group consisting of IgG1, IgG2, IgG3, or IgG4.

20. The monoclonal antibody of claim 19 , wherein the monoclonal antibody has an IgG isotype of IgG1.