ANTI-TIM-3 ANTIBODIES AND METHODS OF USE THEREOF

• US 20170368168A1
• Filed: 05/26/2017
• Published: 12/28/2017
• Est. Priority Date: 05/27/2016
• Status: Active Grant

• Current Assignee: Agenus Inc.
• Original Assignee: Agenus Inc.
• Inventors: van Dijk, Marc, Breous-Nystrom, Ekaterina Vladimirovna, Wilson, Nicholas Stuart, Waight, Jeremy Dale, Underwood, Dennis John
• Granted Patent: US 10,639,368 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/71 Decreased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin

Abstract

The instant disclosure provides antibodies that specifically bind to TIM-3 (e.g., human TIM-3) and antagonize TIM-3 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

Claims

1. An isolated antibody that specifically binds to human TIM-3, the antibody comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein:

(a) CDRH1 comprises the amino acid sequence of X₁X₂X₃X₄X₅S (SEQ ID NO;

     48), wherein

X₁ is R, S, A, G, K, M, or T,

X₂ is Q, S, A, G, R, or T,

X₃ is N, Y, G, or Q,

X₄ is A or Q, and

X₅ is W, M, A, S, or T;

(b) CDRH2 comprises the amino acid sequence of WVSAISGSGGSTY (SEQ ID NO;

     2);

(c) CDRH3 comprises the amino acid sequence of AKGGDYGGNYFD (SEQ ID NO;

     3);

(d) CDRL1 comprises the amino acid sequence of X₁ASQSVX₂SSYLA (SEQ ID NO;

     52), wherein

X₁ is R or G, and

X₂ is absent or S;

(e) CDRL2 comprises the amino acid sequence of X₁ASX₂RAT (SEQ ID NO;

     53), wherein

X₁ is D or G, and

X₂ is N, S, or T; and

(f) CDRL3 comprises the amino acid sequence of QQYGSSPX₁T (SEQ ID NO;

     54), wherein X₁ is L or I.

4. The isolated antibody of claim 1, wherein:

(a) CDRH1 comprises the amino acid sequence of X₁X₂NAWS (SEQ ID NO;

     49), wherein;

X₁ is R or A; and

X₂ is Q or R;

(b) CDRH1 comprises the amino acid sequence of X₁X₂GQX₃S (SEQ ID NO;

     50), wherein;

X₁ is K, M, or G;

X₂ is A or S; and

X₃ is S or T;

(c) CDRH1 comprises the amino acid sequence of X₁X₂QQAS (SEQ ID NO;

     51), wherein;

X₁ is S, R, T, or G; and

X₂ is A, S, T, or G;

(d) CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

1 and 4-12;

(e) CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

13-16;

(f) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

17-21; and

/or

(g) CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

22 and 23.

11. The isolated antibody of claim 1, wherein CDRH1, CDRH2 and CDRH3 comprise the CDRH1, CDRH2 and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs:

1, 2, and 3;

4, 2, and 3;

5, 2, and 3;

6, 2, and 3;

7, 2, and 3;

8, 2, and 3;

9, 2, and 3;

10, 2, and 3;

11, 2, and 3;

or 12, 2, and 3, and/or wherein CDRL1, CDRL2 and CDRL3 comprise the CDRL1, CDRL2 and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs;

13, 17, and 22;

14, 17, and 22;

15, 18, and 22;

14, 19, and 22;

14, 20, and 22;

14, 21, and 22;

16, 20, and 22;

or 14, 17, and 23.

13. The isolated antibody of claim 1, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs:

1, 2, 3, 14, 21, and 22;

4, 2, 3, 14, 21, and 22;

5, 2, 3, 14, 21, and 22;

6, 2, 3, 14, 21, and 22;

7, 2, 3, 14, 21, and 22;

8, 2, 3, 14, 21, and 22;

9, 2, 3, 14, 21, and 22;

10, 2, 3, 14, 21, and 22;

11, 2, 3, 14, 21, and 22;

12, 2, 3, 14, 21, and 22;

or 1, 2, 3, 15, 18, and 22, respectively.

18. The isolated antibody of claim 1, wherein the antibody is internalized upon binding to cells expressing human TIM-3.

20. The isolated antibody of claim 18, wherein a lower percentage of the cells expressing human TIM-3 survive in the presence of the antibody than in the presence of pab1944w (IgG₁ N297A) or Hum11 (IgG₄ S228P) in an assay comprising the following steps:

(a) plating the cells expressing human TIM-3 at 2×

10⁴ cells per well in a tissue culture plate;

(b) adding 1111 ng/ml of α

HFc-NC-DM1 and 1111 ng/ml of the antibody or pab1944w (IgG₁ N297A) or Hum11 (IgG₄ S228P) at a final volume of 100 μ

l/well;

(c) incubating at 37°

C. and 5% CO₂ for 72 hours;

(d) measuring survival of the cells expressing human TIM-3; and

(e) calculating percentage of cell survival relative to untreated cells expressing human TIM-3 optionally wherein the percentage of cell survival in the presence of the antibody is at least 50% lower than the percentage of cell survival in the presence of pab1944w (IgG1 N297A) or Hum11 (IgG4 S228P),

and optionally wherein the cells expressing human TIM-3 are Kasumi-3 cells or Jurkat cells engineered to express human TIM-3.

26. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

55, and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO;

56.

27. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:

24-35, and/or wherein the antibody comprises a light chain variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs;

36-47.

40. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:

58, 61, or 65, and/or a light chain comprising the amino acid sequence of SEQ ID NO;

69.

46. The isolated antibody of claim 1, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs:

24 and 36;

24 and 38;

26 and 42;

24 and 42;

24 and 46;

24 and 43;

26 and 43;

26 and 46;

26 and 41;

24 and 41;

25 and 39;

24 and 47;

25 and 40;

26 and 47;

25 and 37;

25 and 45;

25 and 44;

25 and 46;

25 and 42;

25 and 41;

25 and 43;

25 and 47;

27 and 46;

28 and 46;

29 and 46;

30 and 46;

31 and 46;

32 and 46;

33 and 46;

34 and 46;

or 35 and 46.

50. The isolated antibody of claim 1, the antibody comprising a heavy chain and a light chain comprising the amino acid sequences of SEQ ID NOs:

58 and 69, 61 and 69, or 65 and 69.

53. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region having an amino acid sequence derived from a human IGHV3-23 germline sequence, and/or a light chain variable region having an amino acid sequence derived from a human germline sequence selected from the group consisting of IGKV1-27, IGKV3-11, IGKV3-20, and IGKV3D-20.

56. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain constant region selected from the group consisting of human IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂, and optionally wherein the heavy chain constant region is:

(a) a variant of a wild type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to a human Fc gamma receptor with lower affinity than the wild type human IgG heavy chain constant region binds to the human Fc gamma receptor, optionally wherein the human Fc gamma receptor is selected from the group consisting of Fcγ

RI, Fcγ

RII, and Fcγ

RIII;

(b) IgG₁ comprising a N297A mutation, numbered according to the EU numbering system, optionally comprising the amino acid sequence of SEQ ID NO;

72;

(c) IgG₁ comprising a N297Q mutation, numbered according to the EU numbering system;

(d) non-fucosylated IgG₁; and

/or

(e) IgG₄ comprising a S228P mutation, numbered according to the EU numbering system, optionally comprising the amino acid sequence of SEQ ID NO;

74.

65. The isolated antibody of claim 1, wherein the antibody comprises a light chain constant region selected from the group consisting of human IgGκ

and IgGλ

, and optionally wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO;

76.

68. An isolated antibody that cross-competes for binding to human TIM-3 with the antibody of claim 1.

69. An isolated antibody that binds to the same epitope of human TIM-3 as the antibody of claim 1.

84. The isolated antibody of claim 1, wherein the antibody:

(a) is a human antibody;

(b) is antagonistic to human TIM-3;

(c) deactivates, reduces, or inhibits an activity of human TIM-3;

(d) inhibits binding of human TIM-3 to phosphatidylserine;

(e) induces IFNγ

production by peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA); and

/or

(f) induces IFNγ

or TNFα

production by tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies.

91. The isolated antibody of claim 1 conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label.

92. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier or excipient.

93. An isolated polynucleotide encoding a heavy and/or light chain of the antibody of claim 1.

94. A vector comprising the polynucleotide of claim 93.

95. A recombinant host cell comprising the polynucleotide of claim 93 or the vector of claim 94.

96. A method of producing an antibody that binds to human TIM-3, the method comprising culturing the host cell of claim 95 so that the polynucleotide is expressed and the antibody is produced.

97. A method of increasing T cell activation in response to an antigen in a subject, the method comprising administering to the subject an effective amount of the antibody of claim 1.

98. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody of claim 1.

112. A method of treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the isolated antibody of claim 1.

2. An isolated antibody that specifically binds to human TIM-3, the antibody comprising a heavy chain variable region having complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region having complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein the antibody is internalized upon binding to cells expressing human TIM-3, and wherein CDRH3 comprises the amino acid sequence of AKGGDYGGNYFD (SEQ ID NO:

3).

3. (canceled)

5-10. -10. (canceled)

12. (canceled)

14-17. -17. (canceled)

19. (canceled)

21-25. -25. (canceled)

28-35. -35. (canceled)

36. An isolated antibody that specifically binds to human TIM-3, the antibody comprising (a) a heavy chain variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:

24-35; and

/or (b) a light chain variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs;

36-47.

37-39. -39. (canceled)

41-45. -45. (canceled)

47-49. -49. (canceled)

51-52. -52. (canceled)

54. (canceled)

55. An isolated antibody that specifically binds to human TIM-3, the antibody comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-23 germline sequence, and a light chain variable region having an amino acid sequence derived from a human germline sequence selected from the group consisting of IGKV1-27, IGKV3-11, IGKV3-20, and IGKV3D-20.

57-64. -64. (canceled)

66-67. -67. (canceled)

70. An isolated antibody that specifically binds to human TIM-3, wherein:

(a) the antibody specifically binds to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO;

101 with a lower affinity than to a wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO;

79;

(b) the antibody does not specifically bind to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO;

101;

(c) the antibody binds to residue 40 of SEQ ID NO;

79;

(d) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

93;

(e) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

94;

(f) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

95;

(g) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

96;

(h) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

97;

(i) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

98;

(j) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

99; and

/or

(k) the antibody binds to an epitope located within a region of human TIM-3 consisting of the amino acid sequence of SEQ ID NO;

100.

71-83. -83. (canceled)

85-90. -90. (canceled)

99-111. -111. (canceled)

113. (canceled)