EA041979B1 - ANTIBODIES AGAINST PD-1 AND METHODS FOR THEIR APPLICATION - Google Patents

Description

This application claims the benefit of U.S. Provisional Applications No.

62/212851, filed September 1, 2015; 62/216043, filed September 9, 2015; and 62/257195, filed November 18, 2015, each of which is incorporated herein by reference in its entirety.

1. Technical field

The present disclosure relates to antibodies that specifically bind to human PD-1 and methods of using the same.

2. State of the art

Programmed cell death protein 1 (PD-1) is an inhibitory member of the CD28 receptor family. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. (1996), Int. Immunol., 8: 765-72; Okazaki et al. (2002), Curr. Opin. Immunol. , 14: 391779-82; Bennett et al. (2003), J. Immunol., 170: 711-8). PD-1 is a 55 kDa type 1 transmembrane protein that is part of the Ig superfamily and contains a membrane proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal tyrosine tyrosine switching motif (ITSM) (Thomas M. L. (1995), J. Exp. Med., 181: 1953-6; Vivier E. and Daeron M. (1997), Immunol. Today, 18: 286-91). Two ligands for PD-1 PD-L1 and PD-L2 have been identified that have been shown to downregulate T cell activation after binding to PD-1 (Freeman et al. (2000), J. Exp. Med., 192: 1027 -34 Latchman et al (2001) Nat Immunol 2: 261-8 Carter et al (2002) Eur J Immunol 32: 634-43). PD-L1 is frequently found in many human cancers (Dong et al. (2002) Nat. Med. 8: 787-9). The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and cancer cell avoidance of immune surveillance (Dong et al. (2003), J. Mol. Med., 81: 281-7 Blank et al (2005) Cancer Immunol Immunother 54: 307-314 Konishi et al (2004) Clin Cancer Res 10: 5094-100). This immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is also blocked (Iwai et al. (2002), Proc. Nat'1. Acad. Sci USA 99: 12293-7 Brown et al (2003) J. Immunol. 170: 1257-66).

PD-1 is an inhibitory molecule of immune cells. Animals deficient in PD-1 develop various autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999), Immunity, 11: 141-51; Nishimura et al. (2001), Science, 291 : 319-22). In addition, PD-1 has also been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft versus walking disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003), J. Exp. Med. , 198: 71-78; Prokunina and Alarcon-Riquelme (2004), Hum. Mol. Genet., 13: R143; Nielsen et al. (2004), Lupus, 13: 510). In the mouse tumor B cell line, ITSM PD-1 has been shown to be essential for blockade of BCR-mediated Ca ²⁺ flux and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001), PNAS, 98: 13866-71).

Given the role of human PD-1 in modulating immune responses, therapeutics designed to antagonize PD-1 signaling are considered highly promising for the treatment of diseases that involve PD-1 mediated immunosuppression.

3. The essence of the invention

The present disclosure relates to antibodies that specifically bind to human PD-1 and antagonize PD-1 function, such as PD-1 mediated immunosuppression. The present disclosure also relates to pharmaceutical compositions comprising such antibodies, nucleic acids encoding such antibodies, expression vectors and host cells for producing such antibodies, and methods of treating a patient using such antibodies. The antibodies disclosed herein are particularly useful for enhancing T cell activation in response to an antigen (e.g., tumor antigen or infectious disease antigen) and/or attenuating Treg-mediated immunosuppression and, therefore, for treating or preventing an infectious disease in a subject.

Accordingly, in one aspect, the present disclosure relates to an antibody, or an isolated antibody, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2, and CDRH3, a light chain variable region comprising the complementarity determining regions of CDRL1, CDRL2, and CDRL3, wherein: (a) CDRH1 includes the amino acid sequence SYGMH (SEQ ID NO: 1); (b) CDRH2 includes the amino acid sequence VIWX1DGSNX2YYADSVX3G (SEQ ID NO: 32), where X1 is Y or F; X ₂ is K or E and X ₃ is K or M; (c) CDRH3 includes the amino acid sequence NX1DX2 (SEQ ID NO: 33), where X1 is G or V and X ₂ is H or Y; (d) CDRL1 includes the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4); (e) CDRL2 includes the amino acid sequence GASTRAT (SEQ ID NO: 5) and (f) CDRL3 includes the amino acid sequence QQYNNWPRT (SEQ ID NO: 6).

In some embodiments, CDRH2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 34-46.

In some embodiments, the CDRH3 includes an amino acid sequence selected from

- 1 041979 group comprising SEQ ID NO: 3, 7 and 37.

In some embodiments, CDRH1, CDRH2, and CDRH3 include the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, shown in SEQ ID NOs: 1, 2, and 3; 1,2 and 7; 1, 2 and 37;,

1, 34 and 7; 1, 35 and 7 or 1, 36 and 7.

In some embodiments, the antibody comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise amino acids the sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise amino acids the sequences shown in SEQ ID NOs: 1, 2, 7, 4, 5 and 6, respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49.

In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15 , 17 and 26-31. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17 and 2 6-31. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26. in embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody generally comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, in embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24. In In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 53. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody comprises a heavy chain comprising the amino acid the sequence of SEQ ID NO: 56. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 58.

In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence derived from a human IGHV3-33 germline sequence (eg, IGHV3-33*01, eg, having the amino acid sequence of SEQ ID NO: 50).

In some embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the variable region the light chain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence derived from a human IGKV3-15 germline sequence (e.g., IGKV3-15*01, e.g., having the amino acid sequence of SEQ ID

- 2 041979

NO: 51).

In some embodiments, the antibody comprises 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: 15 and 16; 17 and 16; 26 and 16; 27 and 16; 28 and 16; 29 and 16; 30 and 16 or 31 and 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody, or an isolated antibody, comprising: (a) a heavy chain variable region having an amino acid sequence derived from a human IGHV3-33 germline sequence (e.g., IGHV3-33*01, for example, having the amino acid sequence of SEQ ID NO: 50), and (b) a light chain variable region having an amino acid sequence derived from a human IGKV3-15 germline sequence (eg, IGKV3-15*01, eg, having the amino acid sequence of SEQ ID NO: 51).

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or an isolated antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17 and 26-31.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:21, and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

- 3 041979

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:52 and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:53 and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:54 and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:55, and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the present disclosure relates to an antibody or isolated antibody comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:56, and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region, including complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region, including complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein: (a) CDRH1 includes the amino acid sequence SYGMH (SEQ ID NO: 1); (b) CDRH2 includes the amino acid sequence VIWX ₁ DGSNX ₂ YYADSVX ₃ G (SEQ ID NO: 32), where X ₁ is Y or F; X ₂ is K or E; and X ₃ is K or M; (c) CDRH3 includes the amino acid sequence NX ₁ DX ₂ (SEQ ID NO: 33), where X ₁ is G or V; and X ₂ is H or Y; (d) CDRL1 includes the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4); (e) CDRL2 includes the amino acid sequence GASTRAT (SEQ ID NO: 5) and (f) CDRL3 includes the amino acid sequence QQYNNWPRT (SEQ ID NO: 6).

In some embodiments, CDRH2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 34-36.

In some embodiments, CDRH3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, and 37.

In some embodiments, CDRH1, CDRH2, and CDRH3 include the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, as shown in SEQ ID NOs: 1, 2, and 3; 1, 2 and 7; 1, 2 and 37; 1, 34 and 7; 1, 35 and 7 or 1, 36 and 7.

In some embodiments, the antibody comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise amino acids the sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise amino acids the sequences shown in SEQ ID NOs: 1, 2, 7, 4, 5 and 6, respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49.

In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15 , 17 and 26-31. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17, and 26-31. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26. in some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the heavy chain variable region heavy chain region includes

- 4 041979 amino acid sequence of SEQ ID NO: 30. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy a chain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, in embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 54. In In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 58.

In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence derived from a human IGHV3-33 germline sequence (eg, IGHV3-33*01, eg, having the amino acid sequence of SEQ ID NO: 50).

In some embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the variable region the light chain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence derived from a human IGKV3-15 germline sequence (eg, IGKV3-15*01, eg, having the amino acid sequence of SEQ ID NO: 51).

In some embodiments, the antibody comprises 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: 15 and 16; 17 and 16; 26 and 16; 27 and 16; 28 and 16; 29 and 16; 30 and 16 or 31 and 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

- 5 041979

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and (b) a light chain variable region comprising amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain variable region having an amino acid sequence derived from the human IGHV3-33 germline sequence (e.g., IGHV3-33* 01, for example, having the amino acid sequence of SEQ ID NO: 50), and (b) a light chain variable region having an amino acid sequence derived from the human germline sequence of IGKV3-15 (for example, IGKV3-15*01, for example, having the amino acid sequence of SEQ ID NO: 51).

In another aspect, the present disclosure provides an antibody or isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 15, 17, and 26-31.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 52 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 53, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 54, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In another aspect, the present disclosure relates to an antibody or isolated antibody that specifically binds to human PD-1, comprising: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 56, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 19.

- 6 041979

In another aspect, the present disclosure provides an antibody or isolated antibody that cross-competes for binding to human PD-1 with any antibody disclosed herein. In another aspect, the present disclosure relates to an antibody or isolated antibody that binds, eg, specifically binds, to an epitope of human PD-1. In some embodiments, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 107-122 of SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope consisting of residues 107-122 of SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 5-22 of SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope consisting of residues 5 -22 SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 6-15 of SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope, consisting of residues 6-15 of SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 130-138 of SEQ ID NO: 74. In some embodiments, In some embodiments, the antibody binds to a human PD-1 epitope consisting of residues 130-138 of SEQ ID NO: 74. In some embodiments, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 10 6-113 of SEQ ID NO: : 74. In some embodiments, the antibody binds to a human PD-1 epitope consisting of residues 106-113 of SEQ ID NO: 74.

In another aspect, the present disclosure relates to an antibody for which, after binding the antibody to a human PD-1 protein followed by the addition of deuterium, the exchange of hydrogen in the human PD-1 protein for deuterium in a region comprising residues 107-122 of SEQ ID NO: 74, significantly reduced relative to the exchange of hydrogen in the human PD-1 protein for deuterium in the same region in the absence of antibody as determined by hydrogen/deuterium exchange. In another aspect, the present disclosure relates to an antibody for which, after binding the antibody to a human PD-1 protein followed by the addition of deuterium, the exchange of hydrogen in the human PD-1 protein for deuterium in a region comprising residues 5-22 of SEQ ID NO: 74, significantly reduced relative to the exchange of hydrogen in the human PD-1 protein for deuterium in the same region in the absence of antibody as determined by hydrogen/deuterium exchange.

In another aspect, the present disclosure relates to an antibody or isolated antibody that binds, for example, specifically binds to the same human PD-1 epitope as any antibody of the present invention. In a preferred embodiment, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 107122 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human PD-1 epitope consisting of residues 107-122 of SEQ ID NO: : 74. In another preferred embodiment, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 5-22 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human PD-1 epitope consisting of residues 5-22 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 6-15 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human PD epitope -1, consisting of residues 6-15 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to an epitope of human PD-1, comprising, with consisting essentially of or consisting of residues 130-138 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human PD-1 epitope consisting of residues 106-138 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human epitope PD-1 comprising, consisting essentially of, or consisting of residues 106-113 of SEQ ID NO: 74. In another preferred embodiment, the antibody binds to a human PD-1 epitope consisting of residues 106-113 of SEQ ID NO: 74.

In another preferred embodiment, the antibody binds to a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 6-15 of SEQ ID NO: 74 and/or a human PD-1 epitope comprising, consisting essentially of, or consisting of residues 130 -138 SEQ ID NO: 74, and/or comprising, consisting essentially of, or consisting of residues 106-113 of SEQ ID NO: 74. Epitope binding is preferably determined by a Pepscan assay, particularly as described in the examples. For example, binding to more than one of the above human PD-1 epitope sequences may occur in the case of discontinuous epitopes.

In another aspect, the present disclosure relates to an antibody or an isolated antibody that binds, e.g., specifically binds to the same human PD-1 epitope as any antibody of the present invention, for which, after binding of the antibody to a human PD-1 protein followed by by adding deuterium, the exchange of hydrogen in the human PD-1 protein for deuterium in the region including residues 107-122 of SEQ ID NO: 74 is significantly reduced relative to the exchange of hydrogen in the human PD-1 protein for deuterium in the same region in the absence of antibody as determined by hydrogen/deuterium exchange. In another aspect, the present disclosure relates to an antibody or

- 7 041979 an isolated antibody that binds, for example, specifically binds to the same epitope of human PD-1 as any antibody of the present invention, for which, after binding of the antibody to the human PD-1 protein, followed by the addition of deuterium, the exchange of hydrogen in human PD-1 protein for deuterium in the region comprising residues 5-22 of SEQ ID NO: 74 is significantly reduced relative to the exchange of hydrogen in human PD-1 protein for deuterium in the same region in the absence of antibody as determined by hydrogen/deuterium exchange. In a more preferred embodiment, upon binding of the antibody to the human PD-1 protein followed by the addition of deuterium, the exchange of hydrogen in the human PD-1 protein for deuterium in the region comprising residues 107-122 of SEQ ID NO: 74 is significantly reduced relative to the exchange of hydrogen in the human protein PD-1 for deuterium at the same site in the absence of antibody as determined by hydrogen/deuterium exchange, and after binding of the antibody to human PD-1 protein followed by addition of deuterium, exchange of hydrogen in human PD-1 protein for deuterium at the site including residues 5-22 of SEQ ID NO: 74 is significantly reduced relative to the exchange of hydrogen in the human PD-1 protein for deuterium in the same region in the absence of antibody as determined by hydrogen/deuterium exchange. For example, binding to more than one of the above human PD-1 epitope sequences may occur in the case of discontinuous epitopes.

In another aspect, the present disclosure relates to an antibody or isolated antibody that binds, e.g., specifically binds to the same human PD-1 epitope as any antibody of the present invention, the epitope being determined by hydrogen-deuterium exchange (HDX), in particular , as described in the examples, or Pepscan analysis, in particular, as described in the examples, preferably by hydrogen-deuterium exchange.

The following embodiments apply to all of the above aspects.

In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG ₂ , IgG3, IgG ₄ , IgA ₁ and IgA ₂ . In some embodiments, the antibody comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody comprises a human IgG1 heavy chain constant region that lacks a glycan group at position N297 according to the EU numbering system. In some embodiments, the antibody comprises a human IgG1 heavy chain constant region comprising the N297A mutation according to the EU numbering system. In some embodiments, the antibody comprises a human IgG1 heavy chain constant region comprising the N297Q mutation according to the EU numbering system. In some embodiments, the antibody comprises a human IgG1 heavy chain constant region comprising the D265A mutation according to the EU numbering system. In some embodiments, the antibody comprises a human IgG ₄ heavy chain constant region comprising the S228P mutation according to the EU numbering system.

In some embodiments, the antibody comprises a human IgG heavy chain constant region that is a wild-type variant human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to the human Fc receptor with less affinity than the human IgG heavy chain constant region binds. wild type with human Fc receptor. In some embodiments, the human Fc receptor is FcyR. In some embodiments, FcyR is FcyRIIB. In some embodiments, FcyR is expressed on a cell selected from the group consisting of dendritic cells, monocytes, macrophages, neutrophils, granulocytes, B cells, and natural killer cells. In some embodiments, the variant human IgG heavy chain constant region is a human IgG1 heavy chain constant region variant, a human IgG ₂ heavy chain constant region variant, or a human IgG ₄ heavy chain constant region variant. In some embodiments, the antibody comprises a light chain constant region selected from the group consisting of human IgGK IqGλ.

In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is antagonistic to human PD-1. In some embodiments, the antibody deactivates, reduces or inhibits the activity of human PD-1. In some embodiments, the antibody inhibits the binding of human PD-1 to human PD-L1 or to human PD-L2. In some embodiments, the antibody increases the production of IL-2 by peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin A (SEA). In some embodiments, the antibody increases the production of IFNy by a co-culture of human T cells and allogeneic dendritic cells. In some embodiments, the antibody increases the proliferation of anti-CD3 antibody-stimulated CD4+ or CD8+ T cells co-cultured with ascitic fluid from ovarian cancer. In some embodiments, the antibody enhances NFAT signaling in PD-1-expressing NFAT-luciferase reporter cells co-cultured with PDL1-expressing target cells.

In some embodiments, an antibody disclosed herein is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label.

- 8 041979

In another aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody disclosed in the present description, and a pharmaceutically acceptable carrier or excipient.

In another aspect, the present disclosure relates to a polynucleotide or an isolated polynucleotide encoding the heavy and/or light chain of an antibody disclosed herein. In another aspect, the present disclosure relates to a vector comprising a polynucleotide. In yet another aspect, the present disclosure relates to a recombinant host cell comprising a polynucleotide or vector. In another aspect, the present disclosure provides a method for producing an antibody that binds to human PD-1, the method comprising culturing a host cell such that a polynucleotide is expressed and an antibody is produced. In a preferred embodiment, the method is an in vitro method.

In one embodiment, the present invention relates to an antibody of the invention or a pharmaceutical composition of the invention or a polynucleotide of the invention or a vector of the invention or a recombinant host cell of the invention for use as a drug.

In one embodiment, the present invention relates to an antibody of the invention or a pharmaceutical composition of the invention or a polynucleotide of the invention or a vector of the invention or a recombinant host cell of the invention for use as a diagnostic tool.

In one embodiment, the present invention relates to the use of an antibody of the present invention for the preparation of pharmaceutical compositions or medicaments for immunotherapy. Preferably, the immunotherapy is for increasing T cell activity, optionally for treating cancer or treating or preventing an infectious disease.

In another aspect, the present disclosure provides a method for increasing T cell activation in response to an antigen in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein.

In another aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein. In some embodiments, the cancer is selected from the group consisting of melanoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), breast cancer (e.g., Herceptin-resistant breast cancer). and trastuzumab-DM1 (T-DM1) resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancers, vaginal cancers, vulvar cancers, cancers of the penis, cancers of the anus, cancers of the rectum, cancers of the oropharynx, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular carcinoma, endometrial cancer, pancreatic cancer, lymphoma, and leukemia (eg, geriatric leukemia, acute myeloid leukemia (AML) ) and AML in the elderly). In some embodiments, the antibody or pharmaceutical composition is administered subcutaneously or intravenously. In some embodiments, the antibody or pharmaceutical composition is administered intratumorally. In some embodiments, the antibody or pharmaceutical composition is delivered to a tumor-draining lymph node. In some embodiments, the antibody or pharmaceutical composition is administered intra-arterially.

In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for use in a method of increasing T cell activation in response to an antigen.

In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for use in a method of increasing T cell activation in response to an antigen in a subject.

In one aspect, the present invention relates to the use of an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for the preparation of a pharmaceutical composition for increasing T cell activation in response to an antigen in a subject.

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or a pharmaceutical composition of the present invention for preparing a pharmaceutical composition for use in a method for increasing T cell activation in response to an antigen in a subject, comprising administering to the subject an effective amount an antibody, a polynucleotide, a vector, a recombinant host cell, and/or a pharmaceutical composition of the invention.

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for use in a method of treating cancer, preferably the cancer being selected from the group consisting of melanoma, head and neck cancer (e.g., squamous cell head and neck cancer), lung cancer (eg, non-small cell lung cancer and small cell lung cancer), breast cancer (eg, Herceptin-resistant breast cancer and trastuzumab-DM1 (T-DM1) resistant breast cancer ), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancers, vaginal cancers, vulvar cancers, penis cancers, anus cancers, rectal cancers, cancers oropharynx, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular carcinoma, e endometrial cancer, pancreatic cancer, lymphoma, and leukemia (eg, leukemia in the elderly, acute myeloid leukemia (AML) and AML in the elderly).

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject, preferably the cancer being selected from the group consisting of melanoma, head and neck cancer (e.g. , head and neck squamous cell carcinoma), lung cancer (eg, non-small cell lung cancer and small cell lung cancer), breast cancer (eg, Herceptin-resistant breast cancer and trastuzumab-DM1 (T-DM1) resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancers, vaginal cancers, vulvar cancers, penis cancers, anus cancers, rectal cancers, oropharyngeal cancers, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular carcinoma, e endometrial cancer, pancreatic cancer, lymphoma, and leukemia (eg, leukemia in the elderly, acute myeloid leukemia (AML) and AML in the elderly).

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or a pharmaceutical composition of the present invention for use in a method of treating cancer in a subject, comprising administering to the subject an effective amount of the antibody, polynucleotide, vector, recombinant host cell, and / or a pharmaceutical composition according to the invention, and preferably the cancer is selected from the group including melanoma, head and neck cancer (for example, squamous cell head and neck cancer), lung cancer (for example, non-small cell lung cancer and small cell lung cancer), breast cancer (for example , Herceptin-resistant breast cancer and trastuzumab-DM1 (T-DM1) resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancers, cancers vaginal cancer, vulvar cancer, penis cancer, cancerous anus, rectal cancers, oropharyngeal cancers, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular cancer, endometrial cancer, pancreatic cancer, lymphoma, and leukemia (eg, geriatric leukemia, acute myeloid leukemia (AML) and AML in the elderly).

In a preferred embodiment of an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition for use in the present invention, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition, preferably the antibody or pharmaceutical composition, is administered subcutaneously or intravenously. In another preferred embodiment of the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition for use according to the present invention, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition, preferably the antibody or pharmaceutical composition is administered intratumorally or intraarterially. .

In some embodiments, the above methods also include administering an additional therapeutic agent to the subject. Therefore, in one preferred embodiment of the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition for use in the method of the present invention, the method also includes administering an additional therapeutic agent to the subject.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent for use as a medicine.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent for use in a method of treating cancer.

In one aspect, the present invention relates to a pharmaceutical composition, kit or kit of parts, comprising (a) an antibody, a polynucleotide, a vector, a recombinant host cell and/or a pharmaceutical composition of the present invention and (b) an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a chemotherapeutic or checkpoint targeting agent. In some embodiments, the checkpoint targeting agent is selected from the group consisting of an anti-PD-1 antagonist antibody, an anti-PD-L1 antagonist antibody, an anti-PD-L2 antagonist antibody, an anti-CTLA-4 antagonist antibody, an anti-PD-L2 antagonist antibody, -TIM-3 antibody, anti-LAG-3 antagonist antibody, anti-CEACAM1 antagonist antibody, antagonists

- 10 041979 an anti-TIGIT antibody, an aHTu-CD137 antagonist antibody, an anti-ICOS antagonist antibody, an anti-GITR antagonist antibody, and an anti-OX40 antagonist antibody. In some embodiments, the additional therapeutic agent is an indolamine 2,3-dioxygenase (IDO) inhibitor. In some embodiments, the inhibitor is selected from the group consisting of epacadostat, F001287, indoxymod, and NLG919. In some embodiments, the additional therapeutic agent is a vaccine. In some embodiments, the vaccine comprises a heat shock protein-peptide complex (HSPPC) comprising a heat shock protein in combination with an antigenic peptide. In some embodiments, the heat shock protein is hsc70 and is complexed with a tumor-associated antigenic peptide. In some embodiments, the heat shock protein is gp9b protein and is complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a subject-derived tumor. In some embodiments, the additional therapeutic agent includes TCR. In some embodiments, the additional therapeutic agent is a soluble TCR. In some embodiments, the additional therapeutic agent is a TCR expressing cell. In some embodiments, the additional therapeutic agent is a cell expressing a chimeric antigen receptor. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to the peptide-MHC complex. In some embodiments, the additional therapeutic agent is an adjuvant. In one aspect, the present invention relates to (a) an antibody, a polynucleotide, a vector, a recombinant host cell and/or a pharmaceutical composition of the present invention, as well as their use in the manufacture of medicines, and (b) a vaccine for use as a medicine, in in particular for use in a method of treating cancer, wherein preferably the vaccine comprises a heat shock protein-peptide complex (HSPPC) comprising a heat shock protein in complex with an antigenic peptide. In one aspect, the present invention relates to a pharmaceutical composition, kit or kit of parts, comprising (a) an antibody, a polynucleotide, a vector, a recombinant host cell and/or a pharmaceutical composition of the present invention, and (b) a vaccine, and preferably the vaccine comprises a thermal protein complex. shock with a peptide (HSPPC), which includes a heat shock protein in combination with an antigenic peptide.

4. Brief description of the drawings

Fig. 1A, 1B, 1C and 1D are graphs showing the binding of anti-PD-1 antibodies to activated primary human or cynomolgus monkey T cells as measured by flow cytometry. Calculate the average fluorescence intensity (MFI) and build a plot of dependence on the range of antibody concentrations. In FIG. 1A measures the binding of AGEN2046w, AGEN2047w and isotype control human IgG1 to human CD4+ T cells stimulated with staphylococcal enterotoxin A (SEA). AGEN2034w and isotype control human IgG ₄ were tested against SEA stimulated human CD4+ T cells (FIGS. IB and 1D) and cynomolgus monkey CD4+ T cells stimulated with staphylococcal enterotoxin B (SEB) (FIG. 1C).

Fig. 2 is a graph showing the binding of AGEN2034w to human PD-1Fc, human ROBO2-Fc, human B7-H7-Fc, or SIRPy-His. The interaction is measured by suspension array technology and a graph of mean fluorescence intensity (MFI) versus antibody concentration is plotted.

Fig. 3A, 3B, 3C, 3D, 3E and 3F are graphs showing the percent binding of recombinant PD-L1-Fc and/or PD-12-Fc to PD-1 bound beads in the presence of anti-PD-1 dose titration. antibodies. In FIG. 3A, 3B, 3C, and 3D, the anti-PD-1 antibodies tested are AGEN2033w, AGEN2034w, AGEN2046w, and AGEN2047w, respectively. In FIG. 3E and 3F show similar results for AGEN2034w and an isotype control antibody.

Fig. 4A, 4B, 4C, 4D, 4E and 4F are graphs showing the functional activity of anti-PD-1 antibodies on cultures of primary human PBMCs after SEA stimulation. Fig. 4A is a graph showing IL-2 production induced by anti-PD-1 antibodies AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w and isotype control IgG1. Average values (error values) of secreted IL-2 are shown. Fig. 4B is a graph showing IL-2 production in the presence of a dose titration of AGEN2034w compared to an isotype control. The error bars represent the standard deviation. AGEN2034w or an isotype control antibody is tested in the presence or absence of ipilimumab anti-CTLA-4 antibody (Figure 4C), pab2197 or pab2196 anti-TIGIT antibody (Figure 4D), pab2225 anti-CD137 antibody (Figure 4E), or anti- OX40 antibody pab1928 (Fig. 4F).

Fig. 5A, 5B and 5C are the results of studies testing the effect of Fc-gamma receptor (FcyR) linkage on the antagonistic activity of anti-PD-1 antibodies against primary human PBMCs after SEA stimulation. Fig. 5A is a graph showing IL-2 production stimulated by a reference anti-PD-1 antibody or isotype control in the presence or absence of an anti-CD16 antibody. Fig. 5B is the result of a similar study to test

- 11 041979 ke secretion of IL-2 induced by AGEN2034w or an isotype control in the presence of an isotype control, an anti-CD16 antibody, an anti-CD32 antibody, or an anti-CD64 antibody. Fig. 5C is a graph showing IL-2 production induced by AGEN2047w with wild-type human IgG1 Fc region, three respective Fc mutants (N297A, S267E/L328F and S239D/A330L/I332E) and their respective isotype controls. Average values (error values) of secreted IL-2 are shown.

Fig. 6 is a graph showing IFNy production by a co-culture of human T cells and allogeneic dendritic cells in the absence of any antibody or in the presence of an isotype control antibody or anti-PD-1 antibody AGEN2034w. The mean values (error values) of IFNy are shown.

Fig. 7A, 7B and 7C are the results of assays measuring the proliferation of anti-CD3 stimulated T cells after co-culture with ascitic fluid in ovarian cancer in the presence of AGEN2034w or an isotype control antibody. Fig. 7A and 7B are representative histograms showing CFSE fluorescence from CD4+ and CD8+ T cells, respectively, in the presence of AGEN2034w or an isotype control antibody at 10 μg/ml. Fig. 7C is a graph showing the results of such a study. In FIG. 7C CD4+ T cell proliferation as measured by CFSE dilution is normalized to CD4+ T cell proliferation in the absence of ascitic fluid in ovarian cancer and plotted against antibody concentrations.

Fig. 8 is a graph showing the response in the Jurkat-NFATluciferase reporter assay elicited by the anti-PD-1 antibody AGEN2034w or the IgG ₄ isotype control antibody. The response measured by luciferase expression is normalized to the response elicited in the presence of the isotype control antibody at the lowest concentration tested (folded induction) and plotted against antibody concentrations.

Fig. 9A, 9B, 9C, 9D, 9E and 9F are graphs showing the percent binding of recombinant PD-L1-Fc and PD-12-Fc to PD-1 coupled beads in the presence of dose titration of AGEN2001w anti-PD-1 antibodies. (Fig. 9A), AGEN2002W (Fig. 9B), EP11_p11_B03 (Fig. 9C), EP11_p11_B05 (Fig. 9D), EP11_p11_C02 (Fig. 9E), or EP11_p11_C03 (Fig. 9F).

Fig. 10 is a graph showing the functional activity of anti-PD-1 antibody AGEN2002w or isotype control IgG1 on cultures of primary human PBMCs after SEA stimulation, indicated by IL-2 production. Average values (error values) of secreted IL-2 are shown.

5. Detailed description

The present disclosure relates to antibodies that specifically bind to human PD-1 and antagonize PD-1 function, eg, PD-1 mediated immunosuppression. The invention also relates to pharmaceutical compositions comprising such antibodies, nucleic acids encoding such antibodies, expression vectors and host cells for producing such antibodies, and methods of treating a subject using such antibodies. The antibodies disclosed herein are particularly useful for enhancing T cell activation in response to an antigen (eg, a tumor antigen or infectious disease antigen) and, therefore, for treating cancer in a subject or treating or preventing an infectious disease in a subject. All examples of isolated antibodies described in the present description, are further considered as antibodies that can be isolated, but not without necessity. All examples of isolated polynucleotides described herein are further considered as polynucleotides that can be isolated, but not unnecessarily. All examples of antibodies described in the present description, are further considered as antibodies that can be isolated, but not without necessity. All examples of polynucleotides described herein are further considered as polynucleotides that can be isolated, but not unnecessarily.

5.1. Definitions

As used herein, the terms roughly and approximately, when used to modify a numerical value or range, indicate that deviations of up to 5%10% above and up to 5%-10% below the value or range remain within the intended value of the named value or range. .

As used herein, the term PD-1 refers to programmed cell death protein 1. The nucleotide and amino acid sequences of PD-1 are well known in the art. An example of the amino acid sequence of human PD-1 is provided in the GenBank GI deposit: 167857792.

As used herein, the terms antibody and antibodies include full length antibodies, full length antibody antigen-binding fragments, and molecules comprising antibody CDR, VH, or VL regions. Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific

- 12 041979 human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies, including molecules with two heavy chains and two light chains, antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer , light-chain-heavy-chain antibody pair, intrabodies, antibody heteroconjugates, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFV), camelized antibodies, affibodies, Fab fragments, F(ab)'2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above antibodies. In some embodiments, the antibodies described herein refer to populations of polyclonal antibodies. Antibodies can be any type (eg IgG, IgE, IgM, IgD, IgA or IgY), any class (eg IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 or IgA2) or any subclass (eg IgG _2a or IgG _2b ) immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies or a class (eg, human IgG1 or IgG ₄ ) or subclass. In a specific embodiment, the antibody is a humanized monoclonal antibody. In a specific embodiment, the antibody is a human monoclonal antibody. In some embodiments, the antibody described herein is an IgG1 or IgG2 antibody.

As used herein, the terms VH region and VL region refer to the individual heavy and light chain variable regions of an antibody, respectively, including FR (framework regions) 1, 2, 3 and 4 and CDR (complementarity determining regions) 1, 2 and 3 (see Kabat et al., (1991), Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), incorporated herein by reference in its entirety).

As used herein, the term CDR or complementarity determining region refers to the inconsistent antigen-binding sites found in the variable region of both heavy chain and light chain polypeptides. Such defined sites are described in Kabat et al., J. Biol. Chem., 252, 6609-6616 (1977), and Kabat et al., Sequences of protein of immunological interest (1991); Chothia et al., J. Mol. Biol., 196: 901-917 (1987), and MacCallum et al., J. Mol. Biol., 262: 732-745 (1996), incorporated herein by reference in its entirety, where definitions include overlapping or subsets of amino acid residues when compared to one another. Preferably, the term CDR refers to CDR as defined by Kabat based on sequence comparisons. CDRH1, CDRH2 and CDRH3 are heavy chain CDRs and CDRL1, CDRL2 and CDRL3 are light chain CDRs.

As used herein, the term EU numbering system refers to the EU numbering solution for antibody constant regions described in Edelman G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and Kabat et al., in Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, incorporated herein by reference in their entirety.

As used herein, the term backbone amino acid residues (FR) refers to amino acids in the backbone region of an immunoglobulin chain. The term framework region or FR region as used herein includes amino acid residues that are part of the variable region but not part of the CDR (eg, using the Kabat definition of CDR).

As used herein, the terms variable region and variable domain are used interchangeably and are common in the art. Variable region typically refers to a portion of an antibody, typically a portion of a light or heavy chain, typically about 110-125 amino-terminal amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which vary considerably in sequence among antibodies and are used in binding and the specificity of a particular antibody for its particular antigen. Variability in the sequence is concentrated in such regions, called complementarity determining regions (CDRs), while the more conserved regions in the variable domain are called framework regions (FRs). In some embodiments, the variable region is a human variable region. In some embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (eg, non-human primate) variable region. In some embodiments, the variable region includes a rodent or mouse CDR and a primate (FR) framework (eg, a non-human primate).

The terms VL and VL domain are used interchangeably in relation to the variable region of the light chain of an antibody.

The terms VH and VH domain are used interchangeably with respect to the variable region of an antibody heavy chain.

As used herein, the terms constant region and constant domain are used interchangeably and are common in the art. A constant region is a portion of an antibody, such as the carboxy-terminal portion of the light and/or heavy chain, that is not involved

- 13 041979 directly into the binding of the antibody to the antigen, but which may exhibit various effector functions, such as interaction with the Fc receptor (for example, with the Fc-gamma receptor). The constant region of an immunoglobulin molecule generally has a more conservative amino acid sequence relative to the variable domain of an immunoglobulin.

As used herein, the term heavy chain, when used in relation to an antibody, can refer to any specific type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain that gives rise to the IgA, IgD, IgE, IgG and IgM antibody classes, respectively, including IgG subclasses, eg IgG1, IgG ₂ , IgG ₃ and IgG ₄ .

As used herein, the term light chain, when used in relation to an antibody, can refer to any specific type, such as kappa (k) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain. Binding affinity generally refers to the strength of the sum of all non-covalent interactions between a particular binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, binding affinity refers to true binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, the dissociation equilibrium constant (KD), and the association equilibrium constant (K _A ). KD is calculated from the ratio koff/kon while K _A is calculated from the ratio k _on /ko _ff . ko _n refers to the association rate constant of eg an antibody to an antigen, and k _off refers to the dissociation rate constant of eg an antibody to an antigen. Determine ko _n and ko _ff can be methods known to the person skilled in the art, such as BIAcore® or KinExA.

As used herein, the term specifically binds to refers to the ability of an antibody to bind to an antigen with a dissociation constant (K _d ) of at least about ^1x10'6M , ^1x10'7M , 1x10'8M, ^1x10'9M , ^1x10'10M , 1x10 ^-11 M, 1x10'12 M or less, and/or bind to an antigen with an affinity of at least two times greater than its affinity for a non-specific antigen. In one embodiment, a molecule that specifically binds to an antigen can bind to other peptides or polypeptides, typically with lower affinity when determined, for example, by immunoassays, BIAcore®, KinExA 3000 (Sapidyne Instruments, Boise, ID) or other assays. known in the art. In one embodiment, a molecule that specifically binds to an antigen binds to the antigen with an association constant (K _A ) that is at least log 2, log 2.5, log 3, log 4 or more times greater than K _A when the molecule binds nonspecifically to another antigen. In one embodiment, a molecule that specifically binds to an antigen binds to an antigen with a K _{d of} 1x10' ⁶ M or less, 1x10' ⁷ M or less, 1x10' ⁸ M or less, 1x10' ⁹ M or less, 1x10' ¹⁰ M, or less, 1x10 ^-11 M or less or 1x10' ¹² M or less.

In another specific embodiment, molecules that specifically bind to an antigen do not cross-react with other proteins under similar binding conditions. In another specific embodiment, molecules that specifically bind to PD-1 do not cross-react with other non-PDn proteins. In a specific embodiment, the present invention provides an antibody that binds to PD-1 (eg, human PD-1) with greater affinity than to another unrelated antigen. In some embodiments, the present invention relates to an antibody that binds to PD-1 (e.g., human PD-1) with an affinity of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more higher than with another unrelated antigen when measured, for example, by radioimmunoassay, surface plasmon resonance or kinetic exclusion analysis. In a specific embodiment, the degree of binding of an anti-PD-1 antibody described herein to an unrelated non-PDn protein is less than 10%, 15%, or 20% of the binding of the antibody to the PD-1 protein as measured, for example, by radioimmunoassay.

As used herein, an epitope is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, consecutive amino acids of a polypeptide (linear or sequential epitope), or an epitope may, for example, be derived together from two or more non-consecutive regions of a polypeptide or polypeptides (conformational, non-linear or non-sequential epitope). In some embodiments, the epitope to which the antibody binds can be determined using, for example, NMR spectroscopy, X-ray crystallography studies, ELISA assays, hydrogen/deuterium exchange combined with mass spectrometry (for example, liquid chromatography with electron sputter mass spectrometry ), scanning analysis with template-based oligopeptides (e.g., limiting peptides using CLIPS (Chemical Linkage of Peptides onto Scaffolds) to map discontinuous or conformational epitopes), and/or mapping by mutagenesis (e.g., mapping by site-directed mutagenesis) . In the case of X-ray crystallography, crystallization can be carried out using any method known in the art (for example, Giege R. et al. (1994), Acta Crystallogr D Biol Crystallogr, 50 (Pt 4): 339-350; McPherson A. (1990) , Eur. J. Biochem., 189: 1-23; Chayen NE (1997), Structure 5: 1269-1274; McPherson A. (1976), J. Biol. Chem., 251: 6300-6303, all papers in full incorporated herein by reference). Antibody:antigen crystals can be examined using well-known X-ray techniques and can be refined using a computer program such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Meth. Enzymol. (1985) , volumes 114 & 115, eds Wyckoff HW et al.; U.S. 2004/0014194), and BUSTER (Bricogne G. (1993), Acta Crystallogr. D. Biol. Crystallogr., 49(Pt 1): 37-60; Bricogne G. (1997), Meth. Enzymol., 276A: 361-423, ed Carter CW Roversi P. et al. (2000), Acta Crystallogr. D. Biol. Crystallogr., 56(Pt 10): 1316- 1323, all works are incorporated herein by reference in their entirety). Mutagenesis mapping study can be performed using any method known to the person skilled in the art. See, for example, Champe M. et al. (1995), J. Biol. Chem., 270: 1388-1394; and Cunningham BC & Wells JA (1989), Science, 244: 1081-1085, both of which are hereby incorporated by reference in their entirety, for descriptions of mutagenesis methods, including alanine scanning methods for mutagenesis. CLIPS (Chemical Linkage of Peptides onto Scaffolds) is a technology for presenting one or more peptides in a structurally restricted configuration, with the behavior of functional mimetics of complex protein domains. See, for example, US Patent Publication Nos. US 2008/0139407 Al and US 2007/099240 A1, and US Patent No. 7,972,993, incorporated herein by reference in their entirety. In a specific embodiment, an antibody epitope is determined using alanine scan mutagenesis assays. In a specific embodiment, an antibody epitope is determined using hydrogen/deuterium exchange in combination with mass spectrometry. In a specific embodiment, the antibody epitope is determined using CLIPS Epitope Mapping Technology from Pepscan Therapeutics.

As used herein, the terms T cell receptor and TCR are used interchangeably and refer to full-length αβ or γδ TCRs, antigen-binding fragments of full-length TCRs, and molecules comprising CDRs or TCR variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of full-length TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing TCR variable regions connected by a flexible linker, TCR chains connected by an engineered disulfide bond, monospecific TCRs, multispecific TCRs (including bispecific TCRs), TCR hybrids, human TCRs, humanized TCRs, recombinant TCRs, and synthetic TCRs. The term encompasses wild-type TCRs and genetically engineered TCRs (eg, chimeric TCRs comprising a chimeric TCR chain that includes a first portion from a first species TCR and a second portion from a second species TCR).

As used herein, the terms major histocompatibility complex and MHC are used interchangeably and refer to an MHC class I molecule and/or an MHC class II molecule.

As used herein, the term peptide-MHC complex refers to an MHC molecule (MHC class I or MHC class II) with a peptide tethered to a recognizable MHC peptide-binding site.

As used herein, the terms treat, curative, and treatment refer to the therapeutic or preventive measures described herein. Methods of treatment use the administration of an antibody to a subject with or suspected of having a disease or disorder to prevent, cure, delay, lessen the severity or lessen one or more symptoms of the disease or disorder or a recurrent disease or disorder, or to prolong the duration of the existence of the subject beyond the expected in the absence of such treatment.

As used herein, the term effective amount in the context of administering therapy to a subject refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

As used herein, the term subject includes any non-human human or animal. In a preferred embodiment, the subject is a human or non-human mammal, preferably a human.

Determination of percent identity between two sequences (eg, amino acid sequences or nucleotide sequences) can be performed using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is Karlin S. & Altschul SF (1990), PNAS, 87: 2264-2268, modified in Karlin S. & Altschul SF (1993), PNAS, 90: 5873- 5877, included in its entirety in the present description as a reference. Such an algorithm is included in the NBLAST and XBLAST programs, Altschul SF et al., (1990), J. Mol. Biol., 215: 403, included in full in

- 15 041979 this description as a reference. Nucleotide searches in BLAST can be performed with the NBLAST nucleotide program parameter set, eg, score=100, wordlength=12, and obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST search for proteins can be performed with a set of XBLAST program parameters, eg score 50, word length=3, and obtain amino acid sequences homologous to the protein molecules described herein. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul SF et al. (1997), Nuc. Acids Res., 25: 3389-3402, incorporated herein by reference in its entirety. On the other hand, PSI BLAST can be used to perform repeated searches that detect weak relationships between molecules (ibid.). When using BLAST, gapped BLAST and PSI Blast programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) can be used (see e.g. National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih .gov). Another specific non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS, 4:11-17, the work is incorporated herein by reference in its entirety. Such an algorithm is included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment package. When the ALIGN program is used to compare amino acid sequences, a weighted PAM120 residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Percent identity between two sequences can be determined using methods similar to those described above, with or without gaps. When calculating percent identity, only exact matches are typically counted.

5.2. Anti-PD-l antibodies

In one aspect, the present disclosure relates to antibodies that specifically bind to human PD-1 and antagonize PD-1 function. Amino acid sequences of examples of antibodies are given in the present description in tables 1-6.

Table 1 Variable region sequences, CDRs, and FRs of examples of anti-PD-1 antibodies

SEQ ID NO: Description Amino acid sequence 1 AGEN2033w, Kabat CDRH1 SYGMH 2 AGEN2033W, Kabat CDRH2 VIWYDGSNKYYADSVKG 3 AGEN2033W, Kabat CDRH3 NVDY 4 AGEN2033W, Kabat CDRL1 RASQSVSSNLA 5 AGEN2033W, Kabat CDRL2 GAST RAT 6 AGEN2033W, Kabat CDRL3 QQYNNWPRT 7 AGEN2034W, Kabat CDRH3 NGDH 8 AGEN2033w IMGT CDRH1 GFTFSSYG 9 AGEN2033W IMGT CDRH2 IWYDGSNK 10 AGEN2033w IMGT CDRH3 ASNVDY eleven AGEN2033w IMGT CDRL1 QSVSSN 12 AGEN2033w IMGT CDRL2 GAS 13 AGEN2033w IMGT CDRL3 QQYNNWPRT 14 AGEN2034w IMGT CDRH3 ASNGDH 15 AGEN2033w, AGEN2046w VH QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNVDYWGQ GTLVTVSS

- 16 041979

16 AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w VL EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAW YQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVE IK 17 AGEN2034w, AGEN2047w VH QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSS 18 AGEN2033w, heavy chain IgG4 S228P QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNVDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 52 AGEN2033w, heavy chain IgG4 S228P (no C-terminal lysine) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNVDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG 19 AGEN2033W, AGEN2034W, AGEN2046W, AGEN2047w light chain EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAW YQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACENSFTHQGL

- 17 041979

20 AGEN2034w, heavy chain IgG4 S228P QVQLVE S GGGWQPGRS LRL S CAAS G FT FS S YGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 53 AGEN2034w, heavy chain IgG4 S228P (no C-terminal lysine) QVQLVE S GGGWQPGRS LRL S CAAS G FT FS S YGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG 21 AGEN2046W IgGl heavy chain QVQLVE S GGGWQPGRS LRL S CAAS G FT FS S YGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNVDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 54 AGEN2046w IgGl heavy chain (no C-terminal lysine) QVQLVE S GGGWQPGRS LRL S CAAS G FT FS S YGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNVDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

- 18 041979

22 AGEN2047w IgGl heavy chain QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 55 AGEN2047w IgGl heavy chain (no C-terminal lysine) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 23 AGEN2047W, heavy chain IgGl N297A QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 56 AGEN2047w IgGl heavy chain N297A (no C-terminal lysine) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

- 19 041979

24 AGEN2047w IgGl heavy chain S267E/L328F QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVEHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 57 AGEN2047w, heavy chain IgGl S267E/L328F (no C-terminal lysine) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVEHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 25 AGEN2047W IgGl heavy chain S239D/A330L/I332E QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 58 AGEN2047w IgGl heavy chain S239D/A330L/I332E (no C-terminal lysine) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

- 20 041979

26 AGEN2001W VH (BADD426-2614) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCATNGDYWGQ GTLVTVSS 27 AGEN2002w VH (BADD426-2615) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDYWGQ GTLVTVSS 28 EPll_pll_B03 (BADD438-2743) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNEYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSS 29 EPll_pll_B05 (BADD438-2745) QVQLVESGGGMVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWFDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSS thirty EPll_pll_C02 (BADD438-2746) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGH GTLVTVSS 31 EPll_pll_C03 (BADD438-2747) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVMGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQ GTLVTVSS 32 CDRH2 consensus VIWXiDGSNX ₂ YYADSVX ₃ G Xi is Y or F; X ₂ is K or E; and Χ ₃ is K or M 33 CDRH3 Consensus NXiDX ₂ XI is G or V; and X2 is H or Y 34 CDRH2 VIWYDGSNEYYADSVKG 35 CDRH2 VIWFDGSNKKYYADSVKG 36 CDRH2 VIWYDGSNKYYADSVMG 37 CDRH3 NGDY 38 VH-ERI QVQLVE S GGGWQPGRS LRL S CAAS G FT FS 39 VH-ERI QVQLVESGGGMVQPGRSLRLSCAASGFTFS 40 VH FR2 WVRQAPGKGLEWVA 41 VH FR3 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS 42 VH FR3 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT 43 VH FR4 WGQGTLVTVSS 44 VH FR4 WGHGTLVTVSS 45 VL-FR1 EIVMTQSPATLSVSPGERATLSC 46 VL FR2 WYQQKPGQAPRLLIY 47 VL FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 48 VL FR4 FGQGTKVEIK

- 21 041979

49 VH consensus sequence QVQLVESGGGXiVQPGRSLRLSCAASGFTFSSYGM hwvrqapgkglewvaviwx ₂ dgsnx ₃ yyadsvx ₄ gr FTIsrdnskntlylqmnslraedtavyycax ₅ nx ₆ dx ₇ wgx ₈ gtlvtvss Xi is V or M, X ₂ is Y or F, X ₃ is K or M, X 4 is K or M, X ₄ is K or M, X ₄ is , X ₆ is G or V, X ₇ is H or Y, and X ₈ is Q or H 50 IGHV3-33*01, germline sequence QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAR 51 IGKV3-15*01, germline sequence EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAW YQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQQYNNWP 59 Allotype Glm3 human IgGl (without C- terminal lysine) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP Е VT С VWDVS НЕ D Р Е VK FNW YVD GVE VHNAKT KPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG 60 Allotype Glm3 human IgGl ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP Е VT С VWDVS НЕ D Р Е VK FNW YVD GVE VHNAKT KPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 61 IgGl N297A (without C-terminal lysine) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP Е VT С VWDVS НЕ D Р Е VK FNW YVD GVE VHNAKT KPR EEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG

- 22 041979

62 IgGl N297A ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 63 IgG4 S228P (without C-terminal lysine) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLG 64 IgG4 S228P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 65 Constant human kappa light chain region IGKC*01, Kt3 allotype RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLS S PVTKS FNRG EU

Table 2. Heavy chain CDR sequences of examples of anti-PD-l antibodies ¹

Antibody CDRH1 (SEQ ID NO:) CDRH2 (SEQ ID NO:) CDRH3 (SEQ ID NO:) AGEN2033W SYGMH (1) VIWYDGSNKKYYADSVKG (2) NVDY (3) AGEN2034W SYGMH (1) VIWYDGSNKKYYADSVKG (2) NGDH (7) AGEN2001W SYGMH (1) VIWYDGSNKKYYADSVKG (2) NDDY (37) AGEN2002W SYGMH (1) VIWYDGSNKKYYADSVKG (2) NDDY (37) EP11_r11_B0 3 SYGMH (1) VIWYDGSNEYYADSVKG (34) NGDH (7) EP11_r11_B0 5 SYGMH (1) VIWFDGSNKKYYADSVKG (35) NGDH (7) EP11_p11_C0 2 SYGMH (1) VIWYDGSNKKYYADSVKG (2) NGDH (7) EP11_r11_C0 3 SYGMH (1) VIWYDGSNKYYADSVMG (36) NGDH (7)

The 1CDRs VH in Table 2 are determined by Kabat.

Table 3. Light chain CDR sequences of examples of anti-PD-1 antibodies ²

Antibody CDRL1 (SEQ ID NO:) CDRL2 (SEQ ID NO:) CDRL3 (SEQ ID NO:) AGEN2033w RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) AGEN2034W RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) AGEN2001W RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) AGEN2002w RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) EPll_pll_B03 RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) EPll_pll_B05 RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) EPll_pll_C02 RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6) EPll_pll_C03 RASQSVSSNLA (4) GASTRAT (5) QQYNNWPRT(6)

^{The 2} VL CDRs in Table 3 were determined by Kabat.

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Table 4. Framework (FR) sequences of VH examples of anti-PD-l antibodies ³

Antibody VH FR1 (SEQ ID NO:) VH FR2 (SEQ ID NO:) VH FR3 (SEQ ID NO:) VH FR4 (SEQ ID NO:) AGEN2033 W QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (4 0) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGQGTLVTV SS (43) AGEN2034 W QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (40) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGQGTLVTV SS (43) AGEN2001 W QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (4 0) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAT (4 2) WGQGTLVTV SS (43) AGEN2002 W QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (40) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGQGTLVTV SS (43) EP11_r11 _B03 QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (40) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGQGTLVTV SS (43) EP11_r11 _B05 QVQLVESGGGMVQPGRS LRLSCAASGFTFS (39) WVRQAPGKGL EWVA (4 0) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGQGTLVTV SS (43) EP11_r11 _C02 QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (4 0) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGHGTLVTV SS (44) EP11_r11 _C03 QVQLVESGGGWQPGRS LRLSCAASGFTFS (38) WVRQAPGKGL EWVA (40) RFTISRDNSKNTLYLQMNS LRAEDTAVYYCAS (41) WGQGTLVTV SS (43)

³ The VH framing sections described in Table 4 are defined based on the boundaries of the Kabat numbering system for CDRs. In other words, the VH CDRs are defined by Kabat and the framework regions are the amino acid sequences surrounding the CDRs in the variable region in the format FR1, CDRH1, FR2, CDRH2, FR3, CDRH3 and FR4.

Table 5 Framework (FR) sequences of VL examples of anti-PD-1 antibodies ⁴

Antibody VL FR1 (SEQ ID NO:) VL FR2 (SEQ ID NO:) VL FR3 (SEQ ID NO:) VL FR4 (SEQ ID NO:) AGEN2033w EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48) AGEN2034 w EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48) AGEN2001 w EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48) AGEN2002 w EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48) EPll_pll _B03 EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48) EPll_pll _B05 EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC FGQGTKVEIK (48) (47) EPll_pll _C02 EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48) EPll_pll _C03 EIVMTQSPATLSVSP GERATLSC (45) WYQQKPGQAPR LLIY (46) GIPARFSGSGSGTEFTL TISSLQSEDFAVYYC (47) FGQGTKVEIK (48)

⁴ The VL framing sections described in Table 5 are defined based on the boundaries of the Kabat numbering system for CDRs. In other words, the VL CDRs are defined by Kabat and the framework regions represent

- 24 041979 are the amino acid sequences surrounding the CDR in the variable region in FR1 format,

CDRL1, FR2, CDRL2, FR3, CDRL3 and FR4.

Table 6. VH and VL sequences of examples of anti-PD-1 antibodies

Antibody Variable heavy chain region SEQ ID NO: light chain variable region SEQID NO: AGEN2033W BADD438-2742 15 3738 16 AGEN2034W BADD438-2744 17 3738 16 AGEN2001W BADD426-2614 26 3738 16 AGEN2002W BADD426-2615 27 3738 16 EP11_r11_B03 BADD438-2743 28 3738 16 EP11_r11_B05 BADD438-2745 29 3738 16 EP11_r11_C02 BADD438-2746 thirty 3738 16 EP11_r11_C03 BADD438-2747 31 3738 16

Table 7 Example PD-1 sequences

SEQID NO: Description Amino acid sequence 74 An example of a mature PD-1 sequence PGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFR VTQLPNGRDFHMSWRARRNDSGTYLCGAISLAPKAQI KESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLWG WGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLK EDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEY ATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSW PL 75 Epitope PD-1 (residue 107122) SLAPKAQIKESLRAEL 76 Epitope PD-1 (residues 5-22) LDSPDRPWNPPTFSPALL 77 Epitope PD-1 (residues 6-15) DSPDRPWNPP 78 Epitope PD-1 (residue 130138) EVPTAHPSP 79 Epitope PD-1 (106IZ residues) ISLAPKAQ

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, and (a) CDRH1 includes the amino acid sequence SYGMH (SEQ ID NO: 1); and/or (b) CDRH2 comprises the amino acid sequence VIWX1DGSNX ₂ YYADSVX ₃ G (SEQ ID NO: 32) wherein

X ₁ is Y or F;

X ₂ is K or E and

X ₃ is K or M and/or (c) CDRH3 includes the amino acid sequence NX1X2 (SEQ ID NO: 33) wherein

X ₁ is G or V and

X ₂ is H or Y and/or (d) CDRL1 includes the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4); and/or (e) CDRL2 includes the amino acid sequence GASTRAT (SEQ ID NO: 5); and/or (f) CDRL3 includes the amino acid sequence QQYNNWPRT (SEQ ID NO: 6).

In some embodiments, the antibody includes one, two, or all three of the VH CDRs described above. In some embodiments, the antibody comprises a CDRH1 of one of the antibodies in Table. 2. In some embodiments, the antibody includes the CDRH2 of one of the antibodies from table. 2. In some embodiments, the antibody includes the CDRH3 of one of the antibodies from table. 2. In some embodiments, the antibody includes one, two, or all three VH CDRs of one of the antibodies in Table. 2 (eg VH CDRs from one row in Table 2, eg all VH CDRs from AGEN2033w or AGEN2034w). In some embodiments, the antibody comprises the VH frameworks described herein. In some embodiments, the antibody includes the framework regions of the VH antibodies presented in table. 4 (for example, one, two, three or four frame sections from one row in table. 4).

In some embodiments, the antibody includes one, two, or all three VL CDRs described above. In some embodiments, the antibody includes CDRL1 of one of the antibodies from table. 3. In some embodiments, the antibody includes CDRL2 of one of the antibodies from table. 3. In some embodiments, the antibody comprises CDRL3 of one of the antibodies in Table. 3. In some embodiments, the antibody includes one, two, or all three VL CDRs of one of the antibodies in Table. 3 (eg VL CDRs from one row in Table 3, eg all VL CDRs from AGEN2033w or AGEN2034w). In some embodiments, the antibody comprises the VL frameworks described herein. In some embodiments, the antibody includes framework regions (FR) VL antibodies presented in table. 5 (for example, one, two, three or four frame sections from one row in table. 5).

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region, including the complementarity determining regions of CDRH1, CDRH2, and CDRH3, and a light chain variable region, including the complementarity determining regions of CDRL1, CDRL2, and CDRL3 , and:

(a) CDRH1 includes the amino acid sequence SYGMH (SEQ ID NO: 1);

(b) CDRH2 includes the amino acid sequence VIWX1DGSNX ₂ YYADSVX ₃ G (SEQ ID NO: 32), in which

X1 is Y or F;

X ₂ is K or E and

X ₃ is K or M;

(c) CDRH3 includes the amino acid sequence NX1DX2 (SEQ ID NO: 33) in which

X1 is G or V and

X ₂ is H or Y;

(d) CDRL1 includes the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4);

(e) CDRL2 includes the amino acid sequence GASTRAT (SEQ ID NO: 5) and (f) CDRL3 includes the amino acid sequence QQYNNWPRT (SEQ ID NO: 6).

In some embodiments, CDRH2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 34-36. In some embodiments, CDRH3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, and 37.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region including the complementarity determining regions CDRH1, CDRH2 and CDRH3, where CDRH1, CDRH2 and CDRH3 include the amino acid sequences CDRH1, CDRH2 and CDRH3, respectively presented in SEQ ID NO: 1, 2 and 3; 1, 2 and 7 and 1, 2 and 37; 1, 34 and 7; 1, 35 and 7 or 1, 36 and 7.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region, including the complementarity determining regions of CDRH1, CDRH2, and CDRH3, and a light chain variable region, including the complementarity determining regions of CDRL1, CDRL2, and CDRL3 , where CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 include the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 respectively shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6; 1, 2, 7, 4, 5 and 6; 1, 2, 37, 4, 5 and 6; 1, 34, 7, 4, 5 and 6; 1, 35, 7, 4, 5 and 6 or 1, 36, 7, 4, 5 and 6, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, including:

(a) CDRH1 comprising the amino acid sequence SYGMH (SEQ ID NO: 1) and/or (b) CDRH2 comprising the amino acid sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 2); and/or (c) a CDRH3 comprising the amino acid sequence of NVDY (SEQ ID NO: 3) or NGDH (SEQ ID NO: 7); and/or (d) CDRL1 comprising the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4); and/or (e) CDRL2 comprising the amino acid sequence GASTRAT (SEQ ID NO: 5); and/or (f) a CDRL3 comprising the amino acid sequence QQYNNWPRT (SEQ ID NO: 6).

In some embodiments, the antibody includes one, two, or all three VH CDRs shown in SEQ ID NOs: 1, 2, and 3. In some embodiments, the antibody includes one, two, or all three VH CDRs shown in SEQ ID NOs: 1, 2, and 7. In some embodiments, the antibody comprises one, two, or all three of the VL CDRs shown in SEQ ID NOS: 4, 5, and 6.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region including the complementarity determining regions CDRH1, CDRH2 and CDRH3, where CDRH1, CDRH2 and CDRH3 include the amino acid sequences CDRH1, CDRH2 and CDRH3, presented in SEQ ID NO: 1, 2 and 3, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region, including complementarity determining regions CDRH1, CDRH2 and CDRH3, where CDRH1,

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CDRH2 and CDRH3 include the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs: 1, 2 and 7, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a light chain variable region including the complementarity determining regions of CDRL1, CDRL2 and CDRL3, where CDRL1, CDRL2 and CDRL3 include the amino acid sequences of CDRL1, CDRL2 and CDRL3, presented in SEQ ID NO: 4, 5 and 6, respectively.

In some embodiments, the antibody includes a heavy chain variable region, including the CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region, including the CDRL1, CDRL2, and CDRL3 regions, where the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively.

In some embodiments, the antibody includes a heavy chain variable region, including the CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region, including the CDRL1, CDRL2, and CDRL3 regions, where the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 7, 4, 5 and 6, respectively.

In some embodiments, the CDR of an antibody can be determined according to the Chothia numbering scheme referring to the location of the immunoglobulin structural loops (see, for example, Chothia C. & Lesk AM, (1987), J. Mol. Biol., 196: 901-917; Al- Lazikani B. et al (1997), J. Mol. Biol., 273: 927-948 Chothia C. et al. (1992), J. Mol. Biol., 227: 799-817, Tramontano A. et al (1990), J. Mol. Biol., 215(1): 175-82, and US Pat. Typically, when using the Kabat numbering system, a CDRH1 Hotia loop is present at heavy chain amino acids 26-32, 33, or 34, a CDRH2 Hotia loop is present at heavy chain amino acids 52-56, and a CDRH3 Hotia loop is present at heavy chain amino acids 95-102, while while the Hotia CDRL1 loop is present at light chain amino acids 24-34, the Hotia CDRL2 loop is present at light chain amino acids 50-56, and the Hotia CDRL3 loop is present at light chain amino acids 89-97. The end of the loop of Hotia CDRH1, when numbered using the Kabat numbering system, varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places inserts in H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising the VL CDR of the Hotia VL of the antibody disclosed herein in Table 6 (eg, AGEN2033w or AGEN2034w). In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, the antibody comprising the Hotia VH CDR of the antibody disclosed herein in Table B (eg, AGEN2033w or AGEN2034w). In some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises the Hotia VH CDR and the Hotia VL CDR of the antibody disclosed herein in Table 6 (e.g., AGEN2033w or AGEN2034w). In some embodiments, antibodies that specifically bind to human PD-1 include one or more CDRs wherein the Hotia and Kabat CDRs share the same amino acid sequence. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and includes combinations of the Hotia CDR and the Kabat CDR.

In some embodiments, the CDR of an antibody can be determined according to the IMGT numbering system described in Lefranc M-P, (1999), The Immunologist., 7: 132-136, and in Lefranc M-P et al. (1999), Nucleic Acids Res., 27: 209-212, incorporated herein by reference in their entirety. According to the IMGT numbering system, CDRH1 is at positions 26-35, CDRH2 is at positions 51-57, CDRH3 is at positions 93-102, CDRL1 is at positions 27-32, CDRL2 is at positions 50-52, and CDRL3 is at positions 89-97. In a separate embodiment, the present disclosure relates to antibodies that specifically bind to human PD-1 and include the CDR of the antibody disclosed in the present description in table. 6 (eg AGEN2033w or AGEN2034w) defined by the IMGT numbering system, eg as described in Lefranc M-P (1999), op. above, and in Lefranc M-P et al. (1999) op. higher.

In some embodiments, the CDR of an antibody can be determined according to MacCallum RM et al. (1996), J. Mol. Biol., 262: 732-745, incorporated herein by reference in its entirety. See also, for example, Martin A. Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), incorporated herein by reference in its entirety. In a separate embodiment, the present disclosure relates to antibodies that specifically bind to human PD-1 and include the CDR of the antibody disclosed in the present description in table. 6 (eg AGEN2033w or AGEN2034w) determined by the method of MacCallum RM et al. (1996), op. higher.

In some embodiments, the CDR of an antibody can be determined according to the AbM numbering system, which refers to the AbM hypervariable regions, which is a compromise between

- 27 041979

CDR by Kabat and structural loops by Hotia, and used in the Oxford molecular modeling program AbM (Oxford Molecular Group, Inc.), incorporated herein by reference in its entirety. In a separate embodiment, the present disclosure relates to antibodies that specifically bind to human PD-1 and include the CDR of the antibody disclosed in the present description in table. 6 (for example, AGEN2033w or AGEN2034w) defined by the AbM numbering scheme.

Accordingly, in some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions shown in SEQ ID NO: 15 and the amino acid sequences the CDRL1, CDRL2, and CDRL3 regions shown in SEQ ID NO: 16, with each CDR defined by Kabat definition, Hotia definition, combination of Kabat definition and Hotia definition, IMGT numbering system, AbM definition, or contact CDR definition.

Accordingly, in some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions shown in SEQ ID NO: 17 and the amino acid sequences of the regions CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NO: 16, each CDR defined by Kabat definition, Hotia definition, combination of Kabat definition and Hotia definition, IMGT numbering system, AbM definition, or CDR contact definition.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NOs: 8, 9, 14, 11, 12, and 13, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, the antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 49.

In some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence derived from the human IGHV3-33 germline sequence. One or more regions selected from framework 1, framework 2, framework 3, CDRH1 and CDRH2 (eg two, three, four or five such regions) can be derived from the human IGHV3-33 germline sequence. In one embodiment, framework 1, framework 2, framework 3, CDRH1 and CDRH2 are all derived from the human IGHV3-33 germline sequence. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100 % (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 15, 17, and 2631. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17, and 26-31. In some embodiments, the antibody includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the antibody includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the antibody includes a variable heavy chain a heavy chain region having the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the antibody includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the antibody includes a heavy chain variable region having the amino acid the sequence shown in SEQ ID NO: 28. In some embodiments, the antibody includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the antibody includes a heavy chain variable region having ami the acid sequence set forth in SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the antibody comprises a heavy chain having the amino acid sequence set forth in in SEQ ID NO: 18. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 23. in S EQ ID NO: 24. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 53. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 54. SEQ ID NO: 55. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, the antibody includes a heavy chain having amino the acid sequence shown in SEQ ID NO: 58.

In some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1, comprising a light chain variable region having an amino acid sequence derived from the human IGKV3-15 germline sequence. One or more regions selected from framework 1, framework 2, framework 3, CDRL1 and CDRL2 (eg two, three, four or five such regions) can be derived from the human IGKV3-15 germline sequence. In one embodiment, framework 1, framework 2, framework 3, CDRL1 and CDRL2 are all derived from the human IGKV3-15 germline sequence. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a light chain variable region having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100 % (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence shown in SEQ ID NO:16. In some embodiments, the antibody includes a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody includes a light chain having the amino acid sequence shown in SEQ ID NO: 19.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100 % (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17, and a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) is identical to the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17 and 26-31, and a light chain variable region having containing the amino acid sequence shown in SEQ ID NO: 16.

In some embodiments, the antibody comprises the heavy chain variable region and light chain variable region amino acid sequences shown in SEQ ID NOs: 15 and 16; 17 and 16; 26 and 16; 27 and 16; 28 and 16; 29 and 16; 30 and 16 or 31 and 16, respectively. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the antibody comprises a heavy chain variable region, having the amino acid sequence shown in SEQ ID NO:17; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2 to 6 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: :28, and a light chain variable region,

- 29 041979 having the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 30 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 19 In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain a chain containing an amino acid sequence SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 52, and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55 and a light chain comprising the amino acid the sequence of SEQ ID NO: 19. In some embodiments the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a light chain comprising the amino acid the sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a light chain comprising the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 17 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 17 and 16, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 26 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 26 and 16

- 30 041979 respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NO:

and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 27 and 16, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOS: 28 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 28 and 16, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOS: 29 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 29 and 16, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOS: 30 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 30 and 16, respectively.

In some embodiments, the present disclosure relates to an isolated antibody that cross-competes for binding to human PD-1 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 31 and 16, respectively.

In some embodiments, the present disclosure provides an isolated antibody that binds to the same human PD-1 epitope as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 31 and 16, respectively.

Any Ig constant region can be used in the antibodies disclosed herein. In some embodiments, the Ig region is a human heavy chain constant region of IgG1, IgG ₂ , IgG ₃ , IgG ₄ , IgAi and IgA ₂ . In some embodiments, the Ig region is human IgG1. In some embodiments, the Ig region is human IgG ₄ . In some embodiments, the Ig (eg, IgG1) lacks the N-linked glycan group that is normally present at position N297 (according to the EU numbering system) in mature wild-type IgG1 antibodies in vivo. The absence of the N297 glycan leads to a significant loss of effector function. Elimination of the N297 glycan can be achieved using any methods known in the art. For example, in some embodiments, elimination of the N297 glycan is achieved by mutating the N297 residue to remove the glycosylation site. Accordingly, in some embodiments, the antibodies disclosed herein comprise a heavy chain constant region (eg, a human IgG1 heavy chain constant region) comprising the N297A mutation according to the EU numbering system. In some embodiments, the antibodies disclosed herein comprise a heavy chain constant region (eg, human IgG1 heavy chain constant region) comprising the N297Q mutation according to the EU numbering system. In some embodiments, the antibodies disclosed herein comprise a heavy chain constant region (eg, a human IgG1 heavy chain constant region) comprising the D265A mutation according to the EU numbering system. In some embodiments, the antibodies disclosed herein comprise a heavy chain constant region (eg, human IgG1 heavy chain constant region) comprising the S228P mutation according to the EU numbering system.

In some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NOs: 59, 60, 61, 62, 63, or 64 In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds with human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region, including th amino acid sequence of SEQ ID NO: 61. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the present disclosure refers to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region th chain, including the amino acid sequence of SEQ ID NO: 65.

In some embodiments, the IgG regions of the antibodies described herein have increased affinity for CD32B (also known as FcyRIIB or FCGR2B), for example, compared to an antibody with a wild-type Fc region, for example, IgG1 Fc. In some embodiments, the antibodies described herein have a selectively increased affinity for CD32B (FcyyRIIB) over both CD32A (FcyRIIA) and CD16 (FcyRIIIA). Sequence changes that result in increased affinity for CD32B are known in the art, see, for example, Mimoto et al., Protein Engineering,. Design & Selection, 10: 589-598 (2013); Chu et al., Molecular Immunology, 45: 3926-3933 (2008), and Strohl, Current Opinion in Biology 20: 685-691 (2009), all of which are hereby incorporated by reference in their entirety. In some embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1 constant region, or a fragment thereof, comprising a mutation selected from the group consisting of G236D, P238D, S239D, S267E, L328F, and L328E, and combinations thereof, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, eg, an IgG1 constant region, or a fragment thereof, comprising substitutions S267E and L328F, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region, or a fragment thereof, comprising P238D and L328E substitutions, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region, or a fragment thereof, comprising a P238D substitution and substitutions selected from the group consisting of E233D, G237D, H268D, P271G, A330R, and combinations thereof, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1 constant region, or a fragment thereof, comprising substitutions P238D, E233D, G237D, H268D, P271G, and A330R, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region, or a fragment thereof, comprising G236D and S267E, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region, or a fragment thereof, comprising S239D and S267E, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region, or a fragment thereof, including V262E, S267E, and L328F, numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region, or a fragment thereof, including V264E, S267E, and L328F, numbered according to the EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., CH2 domain (human IgG1 residues 231-340) and/or CH3 domain (human IgG1 residues 341-447) IgG1) and/or hinge region, numbered according to the EU numbering system), to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular toxicity.

In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc hinge region (CH1 domain) such that the number of cysteine residues in the hinge region changes (e.g., increases or decreases), as described, for example, in US Pat. No. 5,677,425, incorporated herein by reference in its entirety. Number of cysteine residues

- 32 041979 in the hinge region can be changed in order, for example, to facilitate the assembly of light and heavy chains or to change (for example, increase or decrease) the stability of the antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g., amino acid substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn-binding fragment (preferably an Fc hinge domain or an Fc hinge fragment) to change (e.g., reduce or increase) in vivo half-life of the antibody. See, for example, International Publication Nos. WO 02/060919, WO 98/23289 and WO 97/34631 and US Patent Nos. the subject of examples of mutations that will alter (eg, decrease or increase) the in vivo half-life of an antibody. In some embodiments, one, two, or more amino acid mutations (e.g., amino acid substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn-binding fragment (preferably the Fc hinge domain or Fc hinge fragment) to reduce the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., amino acid substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn-binding fragment (preferably the Fc hinge domain or Fc hinge fragment) to increase the in vivo half-life of the antibody. . In a specific embodiment, antibodies may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (human IgG1 residues 231-340) and/or the third (CH3) domain (human IgG1 residues 341447), numbered according to numbering. EU systems. In a specific embodiment, the constant region of an IgG1 antibody described herein comprises a change from methionine (M) to tyrosine (Y) at position 252, a change from serine (S) to threonine (T) at position 254, and a change from threonine (T) to glutamic acid. (E) at 256, when numbered according to the EU numbering system. See US Pat. No. 7,658,921, incorporated herein by reference in its entirety. This type of IgG mutant, called the YTE mutant, has been shown to display a four-fold increased half-life compared to wild-type versions of the same antibody (see Dall'Acqua WF et al. (2006), J. Biol. Chem. , 281: 23514-24, incorporated herein by reference in its entirety). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions for amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., CH2 domain (human IgG1 residues 231-340) and/or CH3 domain (human IgG1 residues 341-447) IgG1) and/or hinge region, numbered according to the EU numbering system), to increase or decrease the affinity of the antibody for the Fc receptor (eg, activated Fc receptor) on the surface of the effector cell. Mutations in the Fc region of an antibody that reduce or increase the affinity of the antibody for the Fc receptor, and methods for introducing such mutations into the Fc receptor or fragment thereof, are known to those skilled in the art. Examples of antibody Fc receptor mutations that can be made to change the affinity of an antibody for the Fc receptor are described in, for example, Smith P. et al. (2012), PNAS, 109: 6181-6186; US Pat. No. 6,737,056 and International Publication Nos. WO 02/060919, WO 98/23289 and WO 97/34631, all of which are incorporated herein by reference in their entirety.

In another embodiment, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the function(s) of the antibody. For example, one, two or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has an altered affinity for the effector ligand, but retains the antigen-binding capacity of the original antibody. The effector ligand for which the affinity has changed may be, for example, the Fc receptor or the C1 component of complement. This approach is described in detail in US Pat. Nos. 5,624,821 and 5,648,260, both of which are incorporated herein by reference in their entirety. In some embodiments, deletion or inactivation (through point mutation or other means) of the constant domain can impair circulating antibody binding to the Fc receptor, thereby enhancing tumor localization. See for example US Pat. In some embodiments, one, two, or more amino acid substitutions can be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region that may impair Fc receptor binding (see, e.g., Shields RL et al (2001) J Biol Chem 276: 6591-604, incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations can be made in the constant region of an antibody described herein: N297A substitution; N297Q replacement; replacement of L235A and L237A; replacement of L234A and L235A; replacement for E233P; replacement L234V; replacement L235A; deletion C236; replacement R238A; replacement D265A; A327Q replacement or P329A replacement, numbered according to numbering

- 33 041979 of the EU system. In some embodiments, the constant region of an antibody described herein may carry a mutation selected from the group consisting of D265A, P329A, and a combination thereof, numbered according to the EU numbering system.

In a specific embodiment, an antibody described herein comprises an IgG1 constant domain with the amino acid substitution N297Q or N297A, numbered according to the EU numbering system. In one embodiment, an antibody described herein comprises an IgG1 constant domain with a mutation selected from the group consisting of D265A, P329A, and a combination thereof, when numbered according to the EU numbering system. In another embodiment, an antibody described herein comprises an IgG1 constant domain with a mutation selected from the group consisting of L234A, L235A, and a combination thereof, when numbered according to the EU numbering system. In some embodiments, amino acid residues in the constant region of an antibody described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain, when numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO 14/108483, which is incorporated herein by reference in its entirety. In a specific embodiment, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively, when numbered according to the EU numbering system.

In some embodiments, one or more amino acids selected from amino acid residues 329, 331, and 433 in the constant region of an antibody described herein, when numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has an altered Clq binding and/ or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more detail in US patent No. 6194551 (Idusogie et al.), which is fully incorporated into the present description by reference. In some embodiments, one or more amino acid residues at amino acid positions 231-238 in the N-terminal portion of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement, when numbered according to the EU numbering system. This approach is further described in International Publication No. WO 94/29351, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region of an antibody described herein is modified to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or increase the affinity of the antibody for the Fcγ receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) into following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292 , 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337 , 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 numbered according to the EU numbering system. This approach is further described in International Publication No. WO 00/42072, which is incorporated herein by reference in its entirety.

In some embodiments, an antibody described herein comprises an IgG ₄ antibody constant region and the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, has been replaced with a proline. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 64.

In some embodiments, any of the constant region mutations or modifications described herein can be made to one or two constant regions of an antibody described herein having two heavy chain constant regions.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and functions as an antagonist.

In some embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and reduces PD-1 activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, when assessed by the methods described in this description, and/or known to one of skill in the art regarding PD-1 activity without any antibody or with an irrelevant antibody (eg, an antibody that non-specifically binds to human PD-1). In some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1 and reduces PD-1 activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, as assessed by the methods described herein and/or known to one of skill in the art, for PD-1 activity without any antibody or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1). Non-limiting examples of PD-1 activity may include PD-1 signaling, binding of PD-1 to a PD-1 ligand (e.g., PD-11 or PD-12), inhibition of cytokine production (e.g., IL-2 or IFNy), and inhibition of proliferation. T cells. In some embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1 and inactivates, reduces, or inhibits PD-1 activity. In specific embodiments, the activity of PD-1 is evaluated as described in the examples, see below.

In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and reduces the binding of PD-1 to its ligand (e.g., PD-11 or PD-12) by at least about 5%, 10%, 15 %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, as assessed by methods described herein (see examples below) or known to one of skill in the art, for binding of PD-1 to its ligand (e.g., PD-11 or PD-12) without any either antibodies or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1). In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and reduces the binding of PD-1 to its ligand (e.g., PD-11 or PD-12) by at least about 1.2-fold, 1, 3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold as measured by the methods described herein (see examples below), or known to one of skill in the art regarding the binding of PD-1 to its ligand (e.g., PD-11 or PD-12) without any antibody, or to an irrelevant antibody (e.g., an antibody that does not specifically bind to human PD-1) .

In specific embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1 and increases cytokine (e.g., IL-2 or IL-Ny) production by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% , as assessed by methods described herein (see examples below) or known to one of skill in the art, for cytokine production without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to human PD-1) . In specific embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1 and increases cytokine (e.g., IL-2 or IFNy) production by at least about 1.2-fold, 1.3-fold, 1.4-fold , 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times , 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, when assessed by the methods described in the present description (see examples below) or known to a person skilled in the art, regarding the production of cytokines without any antibody or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1).

In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1, and either alone or in combination with an anti-CTLA-4 antibody (e.g., ipilimumab or tremelimumab), an anti-TIGIT antibody, an anti-CD137 antibody ( e.g., urelumab or utomilumab) or an anti-OX40 antibody (e.g., pogalizumab or tavolixizumab) increases IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to staphylococcal enterotoxin A (SEA) stimulation by at least about 1, 2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times , 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, when assessed by the methods described in this description (see . examples below), or known to a person skilled in the art, regarding the production of IL-2 without any antibody or with an irrelevant antibody m (for example, an antibody that does not specifically bind to human PD-1).

In some embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA) in the presence of an antibody described herein that specifically binds to human PD-1, either alone or in combination with an anti-CTLA-4 antibody (eg, ipilimumab or tremelimumab), anti-TIGIT antibody, anti-CD137 antibody (eg, urelumab or utomilumab), or anti-OX40 antibody (eg, pogalizumab or tavolixizumab) have approximately 1.2-fold increased IL-2 production , 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold relative to PBMC stimulated with SEA alone without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to human PD-1), when assessed by the methods described in this opis sled (see examples below), or known to a person skilled in the art.

In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and enhances co-culture IFNy production.

- 35 041979 human T cells and allogeneic dendritic cells at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 fold, 90 fold, or 100 fold, as assessed by methods described herein (see examples below), or known to one skilled in the art, for IFNy production from a co-culture of human T cells and allogeneic dendritic cells without any antibody, or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1).

In some embodiments, a co-culture of human T cells and allogeneic dendritic cells in the presence of an antibody described herein that specifically binds to human PD-1 has IFNy production increased by at least about 1.2-fold, 1.3-fold , 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold relative to co-culture of human T cells and allogeneic dendritic cells without any antibody or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1), as assessed by the methods described herein (see examples below), or known to those skilled in the art.

In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and increases T cell proliferation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as measured by the methods described herein (see examples below) or known to one of skill in the art regarding T cell proliferation without any antibody or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1). In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and increases T cell proliferation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, as assessed by the methods described in the present description (see examples below), or known to a person skilled in the art, regarding the proliferation of T cells without any antibody, or with an irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1).

In specific embodiments, the present disclosure relates to an isolated antibody that specifically binds to human PD-1 and increases the proliferation of anti-CD3 stimulated CD4+ or CD8+ T cells co-cultured with ascitic fluid in ovarian cancer by at least about 1.2-fold , 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, when assessed by the methods described in this description (see examples below), or known to one of skill in the art regarding the proliferation of anti-CD3 antibody-stimulated CD4+ or CD8+ T cells co-cultured with ovarian cancer ascitic fluid without any antibody or with an irrelevant antibody (e.g., an antibody that does not bind specifically to human PD-1).

In specific embodiments, the present disclosure provides an isolated antibody that specifically binds to human PD-1 and enhances NFAT signaling in PD-1 luciferase NFAT-expressing reporter cells co-cultured with PD-1 expressing target cells in at least about 1, 2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times , 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, when assessed by the methods described in this description (see examples below) or known to one skilled in the art regarding NFAT signaling in PD-1-expressing NFAT-luciferase reporter cells co-cultured with PD-1 expressing target cells without any antibody or with irrelevant antibody (eg, an antibody that does not specifically bind to human PD-1).

5.3. Pharmaceutical compositions

The present invention relates to compositions comprising an anti-PD-1 antibody described herein, having the desired degree of purity, in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers at the concentrations used are non-toxic to recipients and include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechin, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight (less than about 10 residues

- 36 041979 cov) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg protein-Zn complexes); and/or non-ionic surfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG).

In a specific embodiment, the pharmaceutical compositions comprise an anti-PD-1 antibody as described herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical compositions comprise an effective amount of an antibody described herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, only the antibody is the active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein can be used in the inhibition of PD-1 activity and the treatment of a condition such as cancer or an infectious disease. In a preferred embodiment, the present invention relates to a pharmaceutical composition of the present invention, comprising an anti-PD-1 antibody of the present invention, for use as a medicine. In another preferred embodiment, the present invention relates to a pharmaceutical composition of the present invention for use in a method of treating cancer or an infectious disease. In another preferred embodiment, the present invention relates to the use of an anti-PD-1 antibody of the present invention for the preparation of a pharmaceutical composition for the treatment of cancer or an infectious disease.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous media, non-aqueous media, antimicrobials, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering or chelating agents, and other pharmaceutically acceptable agents. Examples of aqueous vehicles include sodium chloride injection, Ringer's, isotonic dextrose solutions, sterile water for injection, dextrose solution, and lactated Ringer's solution for injection. Non-aqueous parenteral media include fixed vegetable oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents may be added to parenteral preparations packaged in multi-dose containers at bacteriostatic or fungistatic concentrations, which include phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl and propyl esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspension and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifiers include Polysorbate 80 (Tween™ 80). The metal ion sequestering or chelating agent includes EDTA.

Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible media, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

The pharmaceutical composition can be formulated for any route of administration to a subject. Specific routes of administration include intranasal, oral, pulmonary, transdermal, intradermal and parenteral. Parenteral administration, as described by subcutaneous, intramuscular or intravenous injection, is also intended in the present description. Formulations for injection may be prepared in the usual forms as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or emulsions. Formulations for injection, rasters and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, pharmaceutical compositions for administration may also contain small amounts of non-toxic excipients such as wetting agents or emulsifiers, pH buffering agents, stabilizers, solubility enhancers and other agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate. and cyclodextrins.

Preparations for parenteral administration of antibodies include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized products ready to be combined with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products, ready for connection with the medium immediately before use, and sterile emulsions. Solutions may be either aqueous or non-aqueous.

- 37 041979

When administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS) and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Topical mixtures containing the antibody are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion, or the like, and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, washes, sprays, suppositories, bandages, dermal patches or any other preparation suitable for topical administration.

The anti-PD-I antibody described herein can be prepared as an aerosol for topical use, for example, by inhalation (see, for example, US Pat. treatment of inflammatory diseases, in particular asthma, and which are incorporated herein by reference in their entirety). Such compositions for administration to the respiratory tract may be in the form of an aerosol or nebulizer solution, or as a microfine powder for insufflation alone or in combination with an inert carrier such as lactose. In such a case, the particles of the composition will in one embodiment have a diameter of less than 50 microns, in one embodiment less than 10 microns.

The anti-PD-I antibody described herein can be prepared for topical or topical application, for example, for topical application to the skin and mucous membranes, for example, the eyes, in the form of gels, creams and lotions, and for application to the eye or for intracisternal or intraspinal application. Topical administration is considered for transdermal delivery and also for administration to the eyes or mucous membranes or for inhalation therapies. It is also possible to administer nasal solutions of the antibody alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer an antibody. For example, such patches are disclosed in US Pat. Nos. 6,267,983; 6,261,595; 6,256,533; 6,167,301;

In some embodiments, a pharmaceutical composition comprising an antibody described herein is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and made into solids or gels. The lyophilized powder is prepared by dissolving the antibody described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain an excipient that improves stability, or another pharmacological component of the powder or reconstituted solution obtained from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other similar buffer known to those skilled in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, yields the desired composition. In one embodiment, the resulting solution will be dispensed into lyophilization vials. Each vial will contain one dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions such as from about 4° C. to room temperature. Reconstitution of such a lyophilized powder with water for injection provides a composition for parenteral use. For reconstitution, the lyophilized powder is added to sterile water or other suitable vehicle. The exact amount depends on the selected compound. This number can be determined empirically.

The anti-PD-I antibodies described herein and other compositions described herein can also be formulated to target a specific tissue, receptor, or other area of the body of the subject being treated. Many such targeting techniques are well known to those skilled in the art. All such targeting methods are discussed herein for use in the compositions of the present invention. Как примеры методов направленного воздействия, не являющиеся ограничивающими, см., например, патенты США №№ 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975, 6004534, 5985307, 5972366, 5900252, 5840674 , 5,759,542 and 5,709,874, which are all incorporated herein by reference in their entirety. In a specific embodiment, the antibody described herein targets a tumor.

Compositions used for in vivo administration may be sterile. This is easily accomplished by filtration through, for example, sterile filtration membranes.

5.4. Methods of application and application

In another aspect, the present disclosure relates to a method of treating a subject using

- 38 041979 anti-PD-l antibodies disclosed in the present description. Any disease or disorder that would benefit from inhibition of PD-1 function can be treated using the anti-PD-1 antibodies disclosed herein. The anti-PD-I antibodies disclosed herein are particularly useful for inhibiting the tolerance of the immune system to tumors, and accordingly can be used as an immunotherapy for a subject with cancer. For example, in some embodiments, the present disclosure relates to a method of enhancing T-cell activation in a subject in response to an antigen, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or pharmaceutical composition disclosed herein. In some embodiments, the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or pharmaceutical composition disclosed herein. Cancers that can be treated with the anti-PD-1 antibodies or pharmaceutical composition disclosed herein include, without limitation, melanoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer). and small cell lung cancer), breast cancer (eg, Herceptin-resistant breast cancer and trastuzumab-DM1 (T-DM1) resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancers, vaginal cancers, vulvar cancers, penile cancers, anus cancers, rectal cancers, oropharyngeal cancers, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular cancer, endometrial cancer , pancreatic cancer, lymphoma, and leukemia (eg, leukemia in the elderly, acute myeloid leukemia (AML) and AML in the elderly). Therefore, the present invention relates in one embodiment to an antibody and/or pharmaceutical composition of the present invention for use as a medicine. In a preferred embodiment, the present invention relates to the use of an antibody and/or a pharmaceutical composition of the present invention for the manufacture of a medicament for use in a method of treating cancer in a subject and/or for use in inhibiting immune system tolerance to tumors and/or for use in immunotherapy for a subject having cancer and/or for use in a method of enhancing T cell activation in a subject in response to an antigen. In a preferred embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), breast cancer (e.g., Herceptin-resistant breast cancer). and trastuzumab-DM1 (T-DM1) resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancers, vaginal cancers, vulvar cancers, cancers of the penis, cancers of the anus, cancers of the rectum, cancers of the oropharynx, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular carcinoma, endometrial cancer, pancreatic cancer, lymphoma, and leukemia (eg, geriatric leukemia, acute myeloid leukemia (AML) and AML in the elderly).

Other cancers that can be treated with anti-PD-1 antibodies or pharmaceutical compositions disclosed herein include, without limitation, melanoma (e.g., metastatic malignant melanoma and skin malignant melanoma or intraocular malignant melanoma), kidney cancer (e.g., clear cell cancer), prostate cancer (eg, hormone-refractory adenocarcinoma of the prostate), breast cancer, colon cancer, lung cancer (eg, non-small cell lung cancer), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, p penis, chronic or acute leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, childhood solid tumors, lymphocytic leukemia, bladder cancer, kidney or ureter cancer, renal pelvis carcinoma, central nervous system neoplasm (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmental cancers including asbestos-induced diseases, esophageal cancer , liver cancer, refractory or recurrent malignancies, metastatic cancers, cancers in which PD-11 is expressed, and combinations of these cancers.

In some embodiments, the present disclosure relates to a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an anti-PD-1 antibody or pharmaceutical composition disclosed herein. One embodiment relates to methods for preventing and/or treating an infection (e.g., a viral

- 39 041979 infection, bacterial infection, fungal infection, protozoal infection or parasitic infection). The infection prevented or treated according to the methods may be caused by the infectious agent identified herein. In a specific embodiment, the anti-PD-1 antibody described herein, or composition thereof, are the only active agents administered to a subject. In some embodiments, an anti-PD-1 antibody described herein, or a composition thereof, is used to treat infectious diseases in combination with anti-infective corrective agents (eg, antiviral, antibacterial, antifungal, or anthelmintic). Therefore, in a preferred embodiment, the present invention relates to an antibody, the use of such an antibody for the preparation of a pharmaceutical composition and/or pharmaceutical compositions of the present invention for use in a method for the prevention and/or treatment of an infectious disease, more preferably the antibody or pharmaceutical composition being the only active agents administered to the subject. , or the antibody or pharmaceutical composition is used in combination with anti-infective corrective agents.

Infectious diseases that can be treated and/or prevented with anti-PD-1 antibodies or pharmaceutical compositions disclosed herein are caused by infectious agents including, but not limited to, bacteria, parasites, fungi, protozoa, and viruses. In a specific embodiment, the infectious disease being treated and/or prevented by the anti-PD-1 antibodies or pharmaceutical compositions disclosed herein is caused by a virus. Viral diseases or viral infections that can be prevented and/or treated according to the methods described herein include, but are not limited to, diseases caused by hepatitis A, hepatitis B, hepatitis C, influenza (e.g., influenza A or influenza B), chickenpox, adenovirus, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory sensitial virus, papillomavirus, papovavirus, cytomegalovirus , Echinovirus, arbovirus, hantavirus, Coxsackievirus, mumps, measles, rubella measles, polio, variola, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and agents of viral diseases such as viral meningitis, encephalitis, dengue or smallpox.

Preventable and/or treatable bacterial infections include Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa infections. Bacterial diseases caused by bacteria (eg, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridians, and Pseudomonas aeruginosa) that can be prevented and/or treated according to the methods described herein include, but are not limited to , mycobacterial rickettsiosis, mycoplasmosis, neisseriasis, pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus, Streptococcus, Staphylococcus, mycobacteria, whooping cough, cholera, plague, diphtheria, chlamydia , S. aureus disease, and Legionnaires' disease.

Protozoal diseases or protozoal infections caused by protozoa that can be prevented and/or treated by the methods described herein include, but are not limited to, leishmaniasis, coccidiosis, trypanosomiasis, schistosomiasis, or malaria. Parasitic diseases or parasitic infections that can be prevented and/or treated according to the methods described herein include, but are not limited to, chlamydia and rickettsiosis.

Fungal diseases or fungal infections that can be prevented and/or treated by the methods described herein include, but are not limited to, diseases caused by Candida infections, zygomycosis, Candida mastitis, progressive disseminated trichosporonosis with latent trichosporonemia, disseminated candidiasis, paracoccidioidomycosis of the lungs, pulmonary aspergillosis, Pneumocystis carinii pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis, tibial dyschondroplasia, vaginitis, Xandoropharyngeal glaucoma linked chronic granulomatous disease, athlete's foot, skin candidiasis, mycotic placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic keratitis, Cryptococcus neoformans infection, fungal peritonitis , Curvularia geniculata infection, staphylococcal endophthalmin, sporotrichosis and dermatophytosis.

In some embodiments, such methods also include administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic, radiotherapeutic, or checkpoint targeting agent. In some embodiments, the checkpoint targeting agent is selected from the group consisting of an anti-CTLA-4 antagonist antibody, an anti-PD-11 antagonist antibody, an anti-PD-2 antagonist antibody, an anti-PD-1 antagonist antibody, an anti-PD-1 antagonist

40 041979 anti-T1M-3 antagonist antibody, aHTu-LAG-3 antagonist antibody, antiCEACAM-1 antagonist antibody, anti-TIGIT antagonist antibody, anti-CD137 agonist antibody, anti-ICOS agonist antibody, anti-GITR agonist antibody and agonist anti-OX40 antibody. In some embodiments, an anti-PD-l antibody disclosed herein is administered to a subject in combination with an anti-CTLA-4 antagonist antibody and an anti-ICOS agonist antibody. In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with an anti-CTLA-4 antagonist antibody. In some embodiments, the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject an anti-PD-I antibody or pharmaceutical composition disclosed herein in combination with an anti-CTLA-4 antagonist antibody or pharmaceutical composition thereof, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), e.g., first-line NSCLC), melanoma (e.g., first-line melanoma), and head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN), for example, SCCHN of the first line).

In a preferred embodiment, the present invention relates to an antibody, the use of such an antibody for the preparation of pharmaceutical compositions, and/or a pharmaceutical composition of the present invention for use in the method of the present invention, the method also comprising administering to the subject an additional therapeutic agent. In another preferred embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use as a medicine. In another preferred embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent for use in a method of treating cancer. In another embodiment, the present invention relates to a pharmaceutical composition, kit or kit of parts, comprising (a) an antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent. In one more preferred embodiment, the additional therapeutic agent is a chemotherapeutic, radiotherapeutic, or checkpoint targeting agent.

In some embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, developed by Bristol-Myers Squibb. In some embodiments, the anti-CTLA-4 antibody is tremelimumab, developed by Pfizer and Medimmune. In some embodiments, the anti-CTLA-4 antibody is a CTLA-4 targeting Probody developed by CytomX and Bristol-Myers Squibb.

Examples of anti-CTLA-4 antibodies that can be used in the treatments disclosed herein are disclosed, without limitation, in the following patents and patent applications, which are all incorporated herein in their entirety: US Pat. No. 6,984,720; US patent No. 7411057; US patent No. 7034121; US patent No. 8697845; US patent No. 8518404; US Patent Application Publication No. US 2009/0123477 A1; U.S. Patent Application Publication No. US 2014/0105914 A1; U.S. Patent Application Publication No. US 2013/0267688 A1; U.S. Patent Application Publication No. US 2016/0145355 A1; PCT Publication No. WO 2014/207064 A1 and PCT Publication No. WO 2016/015675 A1.

In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with a compound that targets immunomodulatory enzyme(s), such as IDO (indolamine-(2,3)-dioxygenase) and /or TDO (tryptophan-2,3-dioxygenase). Therefore, in another more preferred embodiment, the additional therapeutic agent is a compound that targets an immunomodulatory enzyme(s), even more preferably an indolamine-(2,3)-dioxygenase (IDO) inhibitor. In some embodiments, such a compound is selected from the group consisting of epacadostat (Incyte Corp; see e.g. WO 2010/005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences/Bristol-Myers Squibb), indoxymod (NewLink Genetics) and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indoxymod. In another embodiment, the compound is NLG919.

In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with a vaccine. The vaccine may be, for example, a peptide vaccine, a DNA vaccine or an RNA vaccine. In some embodiments, the vaccine is a heat shock protein-based antitumor vaccine or a heat shock protein-based pathogen vaccine. In a specific embodiment, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with a heat shock protein based antitumor vaccine. Heat shock proteins (HSPs) are a family of highly conserved proteins found ubiquitously in all species. Their expression can be strongly induced to higher levels by heat shock or other forms of stress, including exposure to toxins, oxidative stress, or glucose deprivation. Five families are classified by molecular weight: HSP-110, -90, -70, -60 and -28. HSP

- 41 041979 deliver immunogenic peptides by way of cross-presentation in antigen-presenting cells (APC), such as macrophages and dendritic cells (DC), leading to T-cell activation. HSPs function as chaperone carriers of tumor-associated antigenic peptides that form complexes capable of inducing tumor-specific immunity. After being released from dying tumor cells, the HSP-antigen complexes are processed into peptides that bind class I and class II MHC molecules, leading to the activation of antitumor CD8+ and CD4+ T cells. The immunity elicited by HSP complexes derived from tumor preparations is specifically directed against the unique repertoire of antigenic peptides expressed in cancer in each subject. Therefore, in another preferred embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention and (b) a vaccine for use as a drug, in particular for use in a method of treating cancer. In another preferred embodiment, the present invention relates to a pharmaceutical composition, kit or kit of parts, comprising (a) an antibody and/or pharmaceutical composition of the present invention and (b) a vaccine. In another preferred embodiment, the vaccine is a heat shock protein based antitumor vaccine or a heat shock protein based pathogen vaccine, preferably a heat shock protein based antitumor vaccine.

The heat shock protein complex with peptides (HSPPC) is a protein-peptide complex consisting of a heat shock protein in a non-covalent complex with antigenic peptides. HSPPC detects both innate and acquired immune responses. In a specific embodiment, the antigenic(e) peptide(s) display(s) antigenicity for the cancer being treated. HSPPCs are efficiently taken up by APCs via membrane receptors (mainly CD91) or by binding to toll-like receptors. Internalization of HSPPC leads to functional maturation of APC with production of chemokines and cytokines leading to activation of natural killer (NK) cells, monocytes and Th-1 and Th-2 mediated immune responses. In some embodiments, the HSPPCs used in the methods disclosed herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 stress protein families in combination with antigenic peptides. In some embodiments, HSPPCs include hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or combinations of two or more of them.

In a specific embodiment, the heat shock protein complex with peptides (HSPPC) comprises recombinant heat shock proteins (eg hsp70 or hsc70) or their peptide-binding domains in complex with recombinant antigenic peptides. Recombinant heat shock proteins can be produced by recombinant DNA technology, for example using the hsc70 sequence as described in Dworniczak and Mirault, Nucleic Acids Res., 15: 5181-5197 (1987), and GenBank accession number P11142 and/or Y00371, all are incorporated herein by reference in their entirety. In some embodiments, the Hsp70 sequences are as described in Hunt and Morimoto, Proc. Natl. Acad. sci. U.S.A., 82 (19), 6455-6459 (1985) and GenBank accession number P0DMV8 and/or M11717, all of which are hereby incorporated by reference in their entirety. Antigenic peptides can also be obtained by recombinant DNA methods known in the art.

In some embodiments, the antigenic peptides include a modified amino acid. In some embodiments, the modified amino acid includes a post-translational modification. In some embodiments, the modified amino acid includes mimicking a post-translational modification. In some embodiments, the modified amino acid is Tyr, Ser, Thr, Arg, Lys, or His that is phosphorylated at the side chain hydroxyl or amine. In some embodiments, the modified amino acid is a Tyr, Ser, Thr, Arg, Lys, or His amino acid mimetic that is phosphorylated at the side chain hydroxyl or amine.

In a specific embodiment, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with a heat shock protein-peptide complex (HSPPC), e.g., heat shock protein-peptide complex 96 (HSPPC-96), for the treatment of cancer. HSPPC-96 includes a 96 kDa heat shock protein (Hsp), gp96, in complex with antigenic peptides. HSPPC-96 is an anti-cancer immunotherapy derived from a subject's tumor and contains an antigenic fingerprint of the cancer. In some embodiments, such a fingerprint contains unique antigens that are present only in specific cancer cells of a particular subject, and it is assumed that the injection of the vaccine stimulates the subject's immune system to recognize and attack any cells with a specific cancerous fingerprint. Therefore, in another preferred embodiment, the present invention provides an antibody and/or a pharmaceutical composition of the present invention in combination with a heat shock protein-peptide complex (HSPPC) for use as a drug and/or for use in a method of treating cancer.

In some embodiments, HSPPC, for example, HSPPC-96, is obtained from the tumor tissue of the subject. In a specific embodiment, the HSPPC (eg, HSPPC-96) is derived from a tumor of the type of cancer being treated or a metastasis thereof. In another specific embodiment, the HSPPC (eg, HSPPC-96) is autologous to the subject being treated. In some embodiments, the tumor tissue is non-necrotic tumor tissue. In some embodiments, at least 1 g (for example, at least

- 42 041979 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 g) of non-necrotic tumor tissue is used to prepare the vaccine regimen. In some embodiments, after surgical resection, the non-necrotic tumor tissue is frozen prior to use in preparing the vaccine. In some embodiments, HSPPCs, such as HSPPC-96, are isolated from tumor tissue by purification techniques, filtered, and prepared for injectable vaccine. In some embodiments, the subject is administered 6-12 doses of HSPPC, such as HSPCC-96. In such embodiments, doses of HSPPC, eg HSPPC-96, may be administered weekly for the first 4 doses and then biweekly for an additional 2-8 doses.

Other examples of HSPPCs that can be used according to the methods described herein are disclosed in the following patents and patent applications: US Pat. description as links.

In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with an adjuvant. Various adjuvants may be used depending on the context of treatment. Non-limiting examples of suitable adjuvants include, but are not limited to, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Montanide ISA (Incomplete Adjuvant from Seppic), Ribi Adjuvant System (RAS), Titer Max, Muramyl Peptides, Syntex Adjuvant (SAF) ), alum (aluminum hydroxide and/or aluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvants, nitrocellulose absorbed antigen, encapsulated or trapped antigen, 3-de-O-acylated monophosphoryl lipid A (3D-MPL), immunostimulatory oligonucleotides, toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, immunostimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX and others. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(C). Combinations of the above adjuvants can also be used. See, for example, US Pat. In one embodiment, the adjuvant used in the present invention is QS-21 STIMULON.

In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with an additional therapeutic agent comprising a TCR. In some embodiments, the additional therapeutic agent is soluble TCR. In some embodiments, the additional therapeutic agent is a TCR expressing cell. Therefore, in yet another preferred embodiment, the present invention relates to an antibody and/or pharmaceutical composition of the present invention in combination with an additional therapeutic agent comprising a TCR for use as a drug and/or for use in a method of treating cancer.

In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with a cell expressing a chimeric antigen receptor (CAR). In some embodiments, the cell is a T cell.

In some embodiments, an anti-PD-1 antibody disclosed herein is administered to a subject in combination with a TCR mimic antibody. In some embodiments, the TCR mimic antibody is an antibody that specifically binds to a peptide-MHC complex. For examples of TCR-mimicking antibodies, but without limitation, see, for example, US Patent No. 9,074,000 and US Patent Application Publication Nos. US 2009/0304679 A1 and US 2014/0134191 A1, which are all incorporated herein by reference in their entirety.

Anti-PD-I antibody and additional therapeutic agent (eg, chemotherapeutic, radiotherapeutic, checkpoint targeting agent, IDO inhibitor, vaccine, soluble TCR, TCR expressing cell, chimeric antigen receptor expressing cell, and/or mimicking TCR antibody) can be administered separately, sequentially or simultaneously as separate dosage forms. In one embodiment, the anti-PD-1 antibody is administered parenterally and the IDO inhibitor is administered orally.

The antibody or pharmaceutical composition described herein can be delivered to a subject in a variety of ways. These include, without limitation, parenteral, intranasal, intrathecal, oral, intradermal, conjunctival, intraarterial, and subcutaneous routes. Pulmonary administration may also be used, for example using an inhaler or nebulizer, and a formulation with an aerosolizing agent for spray application. In some embodiments, the antibody or pharmaceutical composition described herein is delivered subcutaneously or intravenously. In some embodiments, the antibody or pharmaceutical composition described herein is delivered intra-arterially. In some embodiments, the antibody or pharmaceutical composition described herein is delivered intratumorally. In some embodiments, the antibody or pharmaceutical composition described herein is delivered to a lymph node draining a tumor.

- 43 041979

The amount of antibody or composition that will be effective in treating and/or preventing the condition will depend on the nature of the disease and can be determined by conventional clinical methods.

The exact dose used in the composition will also depend on the route of administration and the severity of the infection or disease caused by it, and should be determined according to the opinion of the attending physician and the characteristics of each subject. For example, effective dosages may also vary depending on the routes of administration, the target site, the physiological state of the patient (including age, body weight and health status), whether the patient is a human or animal, other drugs administered, or whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Therapeutic dosages are optimally titrated to optimize safety and efficacy.

The anti-PD-I antibody described herein can also be used to analyze PD-1 protein levels in a biological sample using classical immunohistological methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or Western blotting. Suitable labels for antibody assays are known in the art and include enzyme labels such as glucose oxidase, radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Ts); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibody described herein. Alternatively, a second antibody that recognizes the anti-PD-1 antibody described herein can be labeled and used in combination with an anti-PD-1 antibody to detect PD-1 protein levels. Therefore, in one embodiment, the present invention relates to the use of an antibody of the present invention for in vitro detection of human PD-1 protein in a biological sample. In another embodiment, the present invention relates to the use of an anti-PD-1 antibody of the present invention for the analysis and/or detection of human PD-1 protein levels in an in vitro biological assay, preferably the anti-PD-1 antibody is conjugated to a radionuclide or a detectable label and / or contains the label described in the present description, and / or using an immunohistological method.

Analysis of the level of PD-1 protein expression is intended to include a qualitative or quantitative measurement or assessment of the level of PD-1 protein in the first biological sample, either directly (for example, by determining or assessing the absolute level of the protein) or relatively (for example, by comparing with the level of protein associated with the disease, in the second biological sample). The level of expression of the PD-1 polypeptide in the first biological sample can be measured or estimated and compared with a standard level of PD-1 protein, and the standard level is taken from a second biological sample obtained from an individual without a disorder, or determined by averaging the level in a population of individuals, without disorder. As will be appreciated in the art, since the standard level of PD-1 polypeptide is known, it can be reused as a standard for comparison. Therefore, in another embodiment, the present invention relates to an in vitro method for the analysis and/or detection of PD-1 protein levels, in particular human PD-1 protein levels, in a biological sample, comprising qualitatively or quantitatively measuring or assessing the level of PD-1 protein, in particular, human PD-1 protein, in a biological sample by immunohistological method.

As used herein, the term biological sample refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing PD-1. Methods for obtaining biopsies of tissues and body fluids from animals (eg, humans) are well known in the art. Biological samples include peripheral blood mononuclear cells.

The anti-PD-I antibody described herein can be used for predictive, diagnostic, monitoring and screening applications, including in vitro and in vivo applications well known and standard to those skilled in the art and based on the present description. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro judgment and assessment of immune system status and/or immune response can be used for prediction, diagnosis, and monitoring to evaluate patient samples, including patients known to have or suspected to have immune dysfunction. system, or in relation to an expected or desired immune system response, response to an antigen, or response to a vaccine. Judgment and assessment of immune system status and/or immune response is also applicable in determining a patient's eligibility for a clinical drug trial or for administration of a particular chemotherapeutic agent, radiotherapeutic agent, or antibody, including combinations thereof, against another agent or antibody. This type of prognostic and diagnostic monitoring and evaluation is already in practice using antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), where the assay is also used to evaluate patients for therapy.

- 44 041979 antibodies using Herceptin®. In vivo applications include targeted cell therapy and modulation of the immune system and radio imaging of immune responses. Therefore, in one embodiment, the present invention relates to an anti-PD-1 antibody, the use of such an antibody for the preparation of a pharmaceutical composition, and/or pharmaceutical compositions of the present invention for use as a diagnostic agent. In a preferred embodiment, the present invention relates to an anti-PD-1 antibody, the use of such an antibody for the preparation of a pharmaceutical composition and/or pharmaceutical compositions of the present invention for use in a method for predicting, diagnosing and/or monitoring a subject having or suspected of having an immune system dysfunction, and/or in relation to an expected or desired immune system response, response to an antigen, or response to a vaccine. In another embodiment, the present invention relates to the use of an anti-PD-1 antibody of the invention for predicting, diagnosing and/or monitoring a subject having or suspected of having an immune system dysfunction and/or in relation to an expected or desired immune system response, response to an antigen, or response to the vaccine, by analyzing and/or detecting in vitro levels of human PD1 protein in a biological sample of the subject.

In one embodiment, an anti-PD-1 antibody can be used in immunohistochemistry of biopsy specimens.

Preferably the method is an in vitro method. In another embodiment, an anti-PD-1 antibody can be used to detect levels of PD-1, or levels of cells that contain PD-1 on the surface of their membranes, and such levels can then be associated with symptoms of certain diseases. The anti-PD-1 antibodies described herein may contain a detectable or functional label and/or may be conjugated to a radionuclide or a detectable label. When fluorescent labels are used, currently available microscopy and fluorescent activated cell sorter (FACS) analysis, or a combination of both techniques known in the art, can be used to identify and quantify specific binding participants. The anti-PD-I antibodies described herein may contain or may be conjugated with a fluorescent label. Examples of fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein, and Texas Red, Alexa Fluor, Cy and DyLight dyes. Anti-PD-I antibodies may contain or may be conjugated with a radioactive label or radionuclide, such as the isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ^{57 Co, 58 Co} , ⁵⁹ Fe, ⁶⁷ Cu , ⁹⁰ Y, ⁹⁹ Tc, ¹ⁿ In, ¹¹⁷ Lu, ¹²¹ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac, and ¹⁸⁶ Re. When radioactive labels are used, currently available scoring procedures known in the art can be used to identify and quantify the specific binding of an anti-PD-1 antibody to PD-1 (eg, human PD-1). In the case where the label is an enzyme, detection can be performed by any currently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric methods known in the art. This can be achieved by contacting the sample or control sample with an anti-PD-1 antibody under conditions that allow the formation of a complex between the antibody and PD-1. Any complexes formed between the antibody and PD-1 are detected in the sample and in the control and compared. In light of the specific binding of antibodies for PD-1 described herein, antibodies can be used specifically to detect cell surface expression of PD-1. The antibodies described herein can also be used to purify PD-1 by immunoaffinity purification. Also included herein is an assay system that can be obtained in the form of an assay kit, a kit, or a set of parts for quantitative analysis of the presence limit, for example, for PD-1 or PD-1/PD-1 ligand complexes. The system, assay kit, kit, or kit may include a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents.

5.5. Polynucleotides, vectors and methods for producing anti-PD-1 antibodies

In another aspect, the present invention provides polynucleotides comprising a nucleotide sequence encoding an antibody as described herein, or a fragment thereof (e.g., a light chain variable region and/or a heavy chain variable region) that specifically binds to a PD-1 antigen (e.g. , human PD-1), and vectors, eg, vectors comprising such polynucleotides for recombinant expression in host cells (eg, E. coli and mammalian cells). The present invention relates to polynucleotides comprising nucleotide sequences encoding the heavy and/or light chain of any of the antibodies of the present invention, as well as vectors comprising such nucleotide sequences, for example, expression vectors, for their efficient expression in host cells, for example, cells mammals.

As used herein, an isolated polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in a natural source (eg, mouse or human) of nucleic acid molecules. In addition, an isolated nucleic acid molecule, such as a DNA molecule, can

- 45 041979 be essentially free from other cellular material or culture medium when obtained by recombinant methods, or essentially free from chemical precursors or other chemical compounds when synthesized chemically. For example, the term substantially free includes preparations of a polynucleotide or nucleic acid molecule having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular, less than about 10%) other material, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemical compounds. In a specific embodiment, the nucleic acid molecule(s) encoding(s) the antibody described in the present description is(are) isolated(s) or purified(s).

In certain aspects, the present invention relates to polynucleotides comprising nucleotide sequences encoding antibodies that specifically bind to a PD-1 polypeptide (e.g., human PD-1) and include the amino acid sequence described herein, as well as antibodies that compete with such antibodies for binding to a PD-1 polypeptide (eg, depending on the dose), or which bind to the same epitope as such antibodies.

In some aspects, the present invention provides polynucleotides comprising a nucleotide sequence encoding a light chain or heavy chain of an antibody described herein. The polynucleotides may include nucleotide sequences encoding a light chain comprising the FR and CDR VL of the antibodies described herein (see, for example, Tables 3 and 5) or nucleotide sequences encoding a heavy chain comprising the FR and CDR VH of the antibodies described in the present description (see, for example, tables 2 and 4).

The present invention also relates to anti-PD-1 antibodies that are optimized, for example, by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA fragility elements. Methods for obtaining optimized nucleic acids encoding an anti-PD-1 antibody or fragment (e.g., light chain, heavy chain, VH domain, VL domain) for recombinant expression by incorporating codon changes and/or elimination of inhibitory sites in mRNA can be carried out by adapting the methods described in, for example, US Pat.

For example, potential splice sites and fragility elements (eg, enriched in A/T or A/U elements) in RNA can be mutated without changing the amino acids encoded by the nucleotide sequences to enhance the stability of the RNA for recombinant expression. The changes exploit the degeneration of the genetic code, for example by using an alternative codon for an identical amino acid. In some embodiments, it may be desirable to change one or more codons to encode a conservative mutation, for example, a similar amino acid with similar chemical structure and properties and/or function as the original amino acid. Such methods can increase the expression of an anti-PD-1 antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold , 70 times, 80 times, 90 times, or 100 times or more relative to the expression of an anti-PD-1 antibody encoded by polynucleotides that are not optimized.

In some embodiments, an optimized nucleotide sequence encoding an anti-PD-I antibody described herein, or a fragment thereof (e.g., VL domain and/or VH domain), can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized nucleotide sequence encoding an anti-PD-1 antibody as described herein, or a fragment thereof (eg, VL domain and/or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an anti-PD-1 antibody described herein, or a fragment thereof, hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-PD-1 antibody described herein, or its fragment. In a specific embodiment, an optimized nucleotide sequence encoding an anti-PD-1 antibody described herein hybridizes under high, intermediate, or lower stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-PD-1 antibody described herein. description, or a fragment of it. Information regarding hybridization conditions is available, see, for example, US Patent Application Publication No. US 2005/0048549 (eg, paragraphs 72-73), incorporated herein by reference in its entirety.

Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. Nucleotide sequences encoding the antibodies described in the present description, for example, the antibodies described in table. 1-6, and modified versions of such antibodies can be determined using methods known in the art, i.e., codons of nucleotides known to encode certain amino acids are assembled such that a nucleic acid is obtained that encodes an antibody. A polynucleotide encoding an antibody

- 46 041979 can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G.

et al. (1994), BioTechniques, 17: 242-6, herein incorporated by reference in its entirety) when, briefly, it involves the synthesis of overlapping oligonucleotides containing portions of an antibody coding sequence, annealing and ligation of such oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR. .

On the other hand, a polynucleotide encoding an antibody described herein can be obtained from a nucleic acid from a suitable source (eg, hybridoma) using methods well known in the art (eg, PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers capable of hybridizing at the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification techniques can be used to generate nucleic acids comprising a sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification techniques can be used to generate nucleic acids comprising a sequence encoding a light chain variable region and/or an antibody heavy chain variable region. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example to produce chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the immunoglobulin-encoding nucleic acid can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or a nucleic acid preferably poly A+ RNA isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express the antibody described herein) by PCR amplification using synthetic primers capable of hybridization at 3'- and 5'- ends of the sequence, or by cloning using a sequence-specific oligonucleotide probe to identify, for example, a cDNA clone from a cDNA library that encodes an antibody. The resulting PCR amplified nucleic acids can then be cloned into replicable cloning vectors using any method well known in the art.

DNA encoding an anti-PD-1 antibody described herein can be easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are able to bind specifically to genes encoding the heavy and light chains of anti-PD-1 antibodies) . Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors which are then transfected into host cells such as E. coli cells, COS monkey cells, Chinese Hamster Ovary (CHO) cells (e.g., CHO cells from CHO GS System™ (Lonza)) or myeloma cells that do not otherwise produce immunoglobulin protein, and to synthesize anti-PD-1 antibodies in recombinant host cells.

In order to obtain whole antibodies, PCR primers comprising VL or VH nucleotide sequences, a restriction site, and flanking sequences to protect the restriction site can be used to amplify VL or VH sequences in scFv clones. Using cloning techniques known to those skilled in the art, PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, such as the human kappa or lambda constant regions. In some embodiments, vectors for expressing VH or VL domains include an EF-la promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the required constant regions. Heavy chain conversion vectors and light chain conversion vectors are then transfected into cell lines using methods known to those skilled in the art to produce stable or transient cell lines that express full-length antibodies, eg, IgG.

The DNA can also be modified, for example, by replacing the coding sequence for the human heavy and light chain constant domains with mouse sequences, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

The invention also relates to polynucleotides that hybridize under very strong, moderate or milder hybridization conditions with polynucleotides that encode an antibody described herein. In specific embodiments, the polynucleotides described herein are hybridized under very strong, moderate, or milder hybridization conditions with polynucleotides encoding the VH domain and/or VL domain disclosed herein.

Hybridization conditions are described in the art and known to the person skilled in the art. For example, stringent hybridization may involve hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes at 0.2xSSC/0.1% SDS at about 50-65°C; hybridization under highly stringent conditions may include hybridization to the filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC/0.2% SDS at about 68°C. Hybridizations under other stringent conditions are known to those skilled in the art and are described, see, for example, Ausubel FM et al., eds. (1989), Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3, incorporated herein by reference in their entirety.

Certain aspects of the present invention relate to cells (e.g., host cells) expressing (e.g., recombinantly) the antibodies described herein that specifically bind to PD-1 (e.g., human PD-1), and related polynucleotides and expression vectors. . The present invention provides vectors (eg, expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-PD-1 antibodies or fragments for recombinant expression in host cells, preferably mammalian cells. The present invention relates to host cells, including such vectors for recombinant expression of anti-PD-1 antibodies described in the present description (for example, human or humanized antibodies). A separate aspect relates to methods for producing an antibody described in the present description, including the expression of such an antibody in a host cell.

Recombinant expression of an antibody described herein (e.g., full length antibody, heavy and/or light chain antibody, or single chain antibody described herein) that specifically binds to PD-1 (e.g., human PD-1) involves constructing a vector containing a polynucleotide that encodes an antibody. Once a polynucleotide encoding an antibody molecule, an antibody heavy and/or light chain, or a fragment thereof (e.g., heavy and/or light chain variable regions) described herein has been obtained, a vector for producing an antibody molecule can be obtained using recombinant DNA technology with using methods known in the art. Thus, the present disclosure describes methods for producing a protein by expressing a polynucleotide containing an antibody or an antibody fragment (eg, light chain or heavy chain) encoding a nucleotide sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing an antibody or antibody fragment (eg, light chain or heavy chain), coding sequences, and appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA methods, in vivo synthesis and genetic recombination methods. The invention also provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule as described herein, an antibody heavy or light chain, an antibody heavy or light chain variable region or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding a constant region of an antibody molecule (see, for example, International Publications Nos. WO 86/05807 and WO 89/01036; and US Pat. No. 5,122,464, which are incorporated herein by reference in their entirety. ), and the variable regions of the antibody can be cloned into such a vector to express the full heavy chain, the full light chain, or both the full heavy and the full light chain.

The expression vector can be transferred into a cell (eg, a host cell) by conventional methods, and then the resulting cells can be cultured by conventional methods to obtain an antibody described herein, or a fragment thereof. Thus, the present invention relates to host cells containing a polynucleotide encoding an antibody described herein, or fragments thereof, or a heavy or light chain or fragments thereof, or a single chain antibody described herein, operably linked to a promoter, for expression of such sequences in the host cell. In some embodiments, for the expression of double chain antibodies, vectors encoding both the heavy and light chains can be separately co-expressed in a host cell to express the entire immunoglobulin molecule, as detailed below. In some embodiments, the host cell contains a vector comprising a polynucleotide encoding both the heavy and light chain of the antibody described herein, or a fragment thereof. In specific embodiments, the host cell comprises two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of the antibody described herein, or a fragment thereof, and the second vector comprising a polynucleotide encoding the light chain or light chain variable region. an antibody described herein, or a fragment thereof. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody described herein, or a fragment thereof, and the second host cell comprises a second vector comprising

- 48 041979 polynucleotide encoding the light chain or variable region of the light chain of the antibody described in the present description. In specific embodiments, the heavy chain/heavy chain variable region expressed by the first cell associates with the light chain/light chain variable region of the second cell to form the anti-PD-1 antibody described herein. Some embodiments relate to a population of host cells, including such a first host cell and such a second host cell.

A separate embodiment relates to a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain/variable region of the light chain of an anti-PD-1 antibody described herein, and a second vector comprising a polynucleotide encoding the heavy chain/variable region of the anti-PD-1 heavy chain. -PD-1 antibodies described in the present description.

A number of host-expression vector systems can be used to express the antibody molecules described herein (see, for example, US Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression vector systems are media with which coding sequences of interest can be generated and then purified, but are also cells that can, when transformed or transfected with the appropriate nucleotide sequences, express in situ the antibody molecules described herein. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody-coding sequences; yeast (eg Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing antibody-coding sequences; plant cell systems (e.g. green algae such as (Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g. cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing antibody-coding sequences or mammalian cell systems (e.g., COS cells (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH 3T3, HEK- 293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10) containing recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example , adenoviral late promoter; vaccinia virus 7.5K promoter.) In a particular embodiment, the cells for expressing the antibodies described herein are CHO, for example, CHO cells from the CHO GS System™ (Lonza). In a particular embodiment, the cells for expressing the antibodies described herein are human cells, eg human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a specific embodiment, bacterial cells, such as Escherichia coli cells, or eukaryotic cells (eg, mammalian cells) are used to express the recombinant antibody molecule, especially for expression of the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, when conjugated to a vector such as the major early gene promoter intermediate from human cytomegalovirus, are an efficient expression system for antibodies (Foecking MK & Hofstetter H. (1986), Gene 45: 101-5 and Cockett MI et al (1990), Biotechnology, 8(7): 662-7, both of which are hereby incorporated by reference in their entirety). In some embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of nucleotide sequences encoding antibodies described herein that specifically bind PD-1 (eg, human PD-1) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, the number of expression vectors can be advantageously chosen depending on the intended use for the antibody molecule that is being expressed. For example, when a large amount of such an antibody is to be prepared in order to prepare a pharmaceutical composition with antibody molecules, vectors that drive the expression of high levels of fusion protein products that are easy to purify may be desirable. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruether U. & Mueller-Hill B. (1983), EMBO J., 2: 17911794), into which an antibody coding sequence can be ligated separately into a framed vector. with a lac Z coding region such that a fusion protein is produced; pIN vectors (Inouye S & Inouye M. (1985), Nuc. Acids Res., 13: 3101-3109; Van Heeke G. & Schuster SM (1989), J. Biol. Chem., 24: 5503-5509) and etc., and all of these works are fully incorporated in the present description as a reference. For example, pGEX vectors can also be used to express foreign polypeptides as glutathione-5-transferase (GST) fusion proteins. Typically, such fusion proteins are soluble and can be readily purified from lysed cells by absorption and binding to glutathione-agarose matrix beads followed by elution in the presence of free glutathione.

The pGEX vectors are designed to include cleavage sites for thrombin or factor Xa proteases so that the cloned target gene product can be released from the GST particle.

In the insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector for the expression of foreign genes. The virus replicates in the cells of Spodoptera frugiperda. The antibody coding sequence can be cloned separately into non-essential regions (eg the poledrin gene) of the virus and placed under the control of an AcNPV promoter (eg the polyhedrin promoter).

A number of virus-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as the expression vector, the antibody coding sequence of interest can be ligated to an adenoviral transcription/translation control complex, eg a late promoter, and a tripartite leader sequence. Such a chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (eg, see Logan J. & Shenk T. (1984), PNAS, 81 (12): 3655-9, incorporated herein by reference in its entirety). Efficient translation of inserted antibody coding sequences may also require specific initiation signals. Such signals include the start codon ATG and neighboring sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the complete insert. Such exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by the inclusion of appropriate transcriptional enhancer elements, transcription terminators, etc. (See, for example, Bitter G. et al. (1987), Methods Enzymol., 153: 516-544, incorporated herein by reference in its entirety).

In addition, a host cell strain can be selected that modulates the expression of the inserted sequences, or modifies and processes a gene product of a certain desired type. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Various host cells and specific mechanisms of post-translational processing and modification of proteins and gene products have been characterized. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of foreign expressed proteins. For this purpose, eukaryotic host cells that possess the cellular mechanics for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 cells (a mouse myeloma cell line that is not endogenously produces any immunoglobulin chains), CRL7O3O, COS (eg COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 , BMT10 and HsS78Bst. In some embodiments, the anti-PD-1 antibodies described herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein are fucose-reduced or fucose-free. Such antibodies can be obtained using methods known to those skilled in the art. For example, antibodies can be expressed in cells deficient or lacking the ability to fucosylate. In a specific example, cell lines that knock out both α-1,6-fucosyltransferase alleles can be used to generate fucose-reduced antibodies. The Potelligent® system (Lonza) is an example of such a system that can be used to generate fucose-reduced antibodies.

It is possible to obtain cells that stably express recombinant proteins in high yield for a long time. For example, cell lines can be engineered that stably express the anti-PD-1 antibody described herein. In specific embodiments, the cell of the present invention stably expresses a light chain/light chain variable region and a heavy chain/heavy chain variable region that are associated to form an antibody described herein.

In some aspects, host cells can be transformed not with expression vectors that contain viral origins of replication, but with DNA regulated by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenellation sites, etc.) and selectable marker. After the introduction of foreign DNA/polynucleotide engineered cells can be left to grow for 1-2 days in enriched media and then transferred to selective media. The selectable marker in the recombinant plasmid confers selection resistance and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and propagated into cell lines. Such a method can be advantageously used to engineer cell lines that express the anti-PD-1 antibody described herein, or a fragment thereof. Such engineered cell lines can be used especially in the screening and evaluation of compositions that directly or indirectly interact with the antibody molecule.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase genes (Wigler M. et al. (1977), Cell, 11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W. (1962 ), PNAS, 48(12): 2026-2034) and adenine phosphorylribosyltransferase (Lowy I. et al. (1980), Cell, 22(3): 817-23) in tk, hgprt or aprt cells, respectively, all of these works are fully incorporated in the present description as a reference. Antimetabolite resistance can also be used as a selection basis for the following genes: dhfr, which confers resistance to methotrexate (Wigler M. et al. (1980), PNAS, 77(6): 3567-70; O'Hare K. et al. (1981), PNAS, 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P. (1981), PNAS, 78(4): 2072-6); neo which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991), Biotherapy, 3: 87-95; Tolstoshev P. (1993), Ann. Rev. Pharmacol. Toxicol., 32: 573-596; Mulligan RC (1993), Science, 260: 926-932; and Morgan RA & Anderson WF (1993), Ann. Rev. Biochem., 62: 191-217; Nabel GJ & Feigner PL (1993), Trends Biotechnol., 11(5): 211-5); and hygro, which confers hygromycin resistance (Santerre RF et al. (1984), Gene, 30(1-3): 147-56), all of which are incorporated herein by reference in their entirety. One can traditionally apply methods commonly known in recombinant DNA technology to select the desired recombinant clone, and such methods are described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M., Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F. et al. (1981), J. Mol. Biol., 150: 1-14, incorporated herein by reference in their entirety.

Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York , 1987), incorporated herein by reference in its entirety). When a marker in a vector system expressing an antibody can be amplified, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also be increased (Crouse GF et al. (1983), Mol. Cell. Biol., 3: 257-66, the article is incorporated herein by reference in its entirety).

The host cell can be co-transfected with two or more expression vectors described herein, wherein the first vector encodes a heavy chain derived from a peptide and the second vector encodes a light chain derived from a peptide. The two vectors may contain identical selectable markers that allow equal expression of the heavy and light chain polypeptides. Host cells can be co-transfected with varying amounts of two or more expression vectors. For example, host cells can be co-transfected with any ratio of the first expression vector to the second expression vector of the following: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1: 8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

Alternatively, a single vector can be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such situations, the light chain must be placed before the heavy chain in order to avoid an excess of toxic free heavy chain (Proudfoot NJ (1986), Nature, 322: 562-565; and Kohler G. (1980), PNAS, 77: 2197-2199 , both works are incorporated herein by reference in their entirety). Coding sequences for heavy and light chains may include cDNA or genomic DNA. The expression vector may be monocistronic or multicistronic. A multicistronic nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10, or 10-20 gene/nucleotide sequences. For example, a bicistronic nucleic acid construct may include, in the following order, a promoter, a first gene (eg, the heavy chain of an antibody described herein), and a second gene (eg, a light chain of an antibody described herein). In such an expression vector, transcription of both genes can be driven by a promoter, while translation of mRNA from the first gene can be driven by a cap dependent scanning mechanism, and translation of mRNA from the second gene can be driven by a cap dependent mechanism, such as by IRES.

Once an antibody molecule described herein has been obtained by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, e.g. column chromatography), centrifugation, differential dissolution, or any other standard protein purification method. Additionally, antibodies described herein can be fused to heterologous polypeptide sequences described herein, or otherwise known in the art, to facilitate purification.

In specific embodiments, the antibody described herein is isolated or purified. Typically, an isolated antibody is an antibody that is substantially free of other antibodies with different antigenic specificity than the isolated antibody. For example, in a particular embodiment, an antibody preparation described herein is substantially free of cellular material and/or chemical precursors. The term essentially free of cellular material includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it was isolated, or from which it was produced recombinantly. Thus, an antibody that is substantially free of cellular material includes antibody preparations having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (dry basis) mass) of a heterologous protein (also referred to herein as a contaminating protein) and/or variants of an antibody, eg, various post-translationally modified forms of an antibody, or other versions of an antibody (eg, antibody fragments). When an antibody is produced recombinantly, it is also typically substantially free of the culture medium, i.e., the culture medium is less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% by volume. protein drug. When an antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that were involved in protein synthesis. Accordingly, such antibody preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to PD-1 (eg, human PD-1) can be made by any method known in the art for antibody synthesis, eg, chemical synthesis or recombinant expression methods. The methods described herein use, unless otherwise indicated, conventional techniques in molecular biology, methodology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related arts. . Such methods are described, for example, in the references cited in the present description, and fully explained in the literature. See, for example, Maniatis T. et al. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J. et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J. et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 annual news); Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991); Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999; Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all incorporated herein by reference in their entirety.

In a specific embodiment, an antibody as described herein (eg, a recombinant antibody) is produced, expressed, generated, or purified by any means that includes the creation, for example, through synthesis, of DNA sequences by genetic engineering. In some embodiments, such an antibody includes sequences (eg, DNA sequences or amino acid sequences) that do not naturally exist in the animal or mammalian (eg, human) germline antibody repertoire in vivo.

One aspect of the present invention relates to a method for producing an antibody that specifically binds to PD-1 (eg, human PD-1), including culturing the cell or host cell described in the present description. An aspect of the present invention relates to a method for producing an antibody that specifically binds to PD-1 (e.g., human PD-1) comprising expression (e.g., recombinant expression) of the antibody using a cell or host cell described herein (e.g., cells or host cells comprising polynucleotides encoding an antibody described herein). In a particular embodiment, the cell is an isolated cell. In a separate embodiment, the method further comprises the step of purifying an antibody derived from a host cell or cell.

Methods for making polyclonal antibodies are known in the art (see, for example, chapter 11 in Short Protocols in Molecular Biology (2002), 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, incorporated herein in full. description as reference).

Monoclonal antibodies can be obtained using a wide variety of techniques known in the art, including the use of hybridomas, recombinants, and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be prepared using hybridoma techniques, including those known in the art and described, for example, in Harlow E & Lane D., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in Monoclonal Antibodies and T-Cell Hybridomas, 563, 681 (Elsevier, N.Y., 1981), incorporated herein by reference in its entirety. The term monoclonal antibody as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein, or a fragment thereof, such as the light chain and/or heavy chain of such an antibody.

In a specific embodiment, a monoclonal antibody as used herein is an antibody produced by a single cell (e.g., a hybridoma or a recombinant antibody-producing host cell), wherein the antibody specifically binds to PD-1 (e.g., human PD-1) as determined by ELISA or other antigen binding or competitive binding assay known in the art or described in the examples herein. In certain embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent antibody or a polyvalent (eg, bivalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). The monoclonal antibodies described herein can, for example, be prepared by the hybridoma method described in Kohler G. & Milstein C. (1975), Nature, 256:495, incorporated herein by reference in its entirety, or can, for example, be isolated from phage libraries using the methods described herein, for example. Other methods of obtaining cloned cell lines and monoclonal antibodies expressed through them are well known in the art (see Chapter 11 in Short Protocols in Molecular Biology (2002), 5th Ed., Ausubel FM et al., op. supra).

Methods for obtaining and screening specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, a mouse or other appropriate host animal such as a sheep, goat, rabbit, hamster, or monkey is immunized to detect lymphocytes that produce or are capable of producing antibodies that will specifically bind to a protein (e.g., PD-1 ( eg human PD-1)) used for immunization. On the other hand, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), incorporated herein in its entirety). description as reference). In addition, the RIMMS (multiple boost site) method can be used to immunize an animal (Kilpatrick KE et al. (1997) Hybridoma 16: 381-9, incorporated herein by reference in its entirety).

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., PD-1 (e.g., human PD-1)), and once the immune response is detected eg, antibodies specific for the antigen are detected in mouse serum, mouse spleens are harvested, and splenocytes are isolated. The splenocytes are then fused by well-known methods with any suitable myeloma cells, for example, cells from the SP20 cell line available from the American Type Culture Collection (ATCC) (Mannassas, VA) to form hybridomas. Hybridomas are selected and cloned by the limiting dilution method. In some embodiments, lymph nodes from immunized mice are harvested and fused with NS0 myeloma cells.

The resulting hybridoma cells are then seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

In specific embodiments, myeloma cells are used that fuse efficiently, maintain a sustained high level of antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Such cell lines include murine myeloma cells such as the NS0 cell line, or cells derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP- 2 or X63-Ag8.653, available from the American Type Culture Collection, MD, USA. Human myeloma cell lines and mouse-human heteromyeloma cells have also been described for the production of human monoclonal antibodies (Kozbor D. (1984), J. Immunol., 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) are incorporated herein by reference in their entirety).

The culture medium in which the hybridoma cells are grown is evaluated for the production of monoclonal antibodies directed against PD-1 (eg, human PD-1). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

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Once hybridoma cells have been identified that produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, op. supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. In addition, hybridoma cells can be grown in vivo as ascitic tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

The antibodies described herein include antibody fragments that recognize a specific PD-1 (eg, human PD-1) and can be generated by any method known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be prepared by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of an antibody molecule linked by disulfide bonds at a hinge region.

In addition, the antibodies described herein can also be obtained using various phage display methods known in the art. In phage display methods, the functional domains of an antibody are displayed on the surface of phage particles that contain the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human and mouse cDNA libraries of diseased tissues). The DNA encoding the VH and VL domains are recombined with the scFv PCR linker and cloned into a phagemid vector. The vector is electroporated into E. coli and E. coli is infected with helper phage. The phages used in such methods are typically filamentous phages, including fd and M13, and the VH and VL domains are typically fused recombinantly to the phage gene III or gene VIII. A phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified by the antigen, for example, using a labeled antigen or an antigen bound to or captured on a solid surface or bead. Examples of phage display methods that can be used to generate the antibodies described herein include those disclosed in Brinkman U. et al. (1995), J. Immunol. Methods, 182: 41-50; Ames RS et al. (1995), J. Immunol. Methods, 184: 177-186; Kettleborough CA et al. (1994), Euro. J. Immunol., 24: 952-958; Persic L. et al. (1997), Gene, 187: 9-18; Burton D.R. & Barbas C.F. (1994), Advan. Immunol., 57: 191-280; PCT Application No. PCT/GB91/001134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844 and US Patent Nos. 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5969108, which are all incorporated herein by reference in their entirety.

As described in the above references, once the phage has been selected, the antibody-coding regions can be isolated from the phage and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, such as those described below. Methods for recombinant production of antibody fragments such as Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art such as those described in PCT Publication No. WO 92/22324; in Mullinax RL et al. (1992), BioTechniques, 12(6): 864-9; Sawai H. et al. (1995), Am. J. Reprod. Immunol., 34: 26-34; and in Better M. et al. (1988), Science, 240: 1041-1043, which are all incorporated herein by reference in their entirety.

In some embodiments, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site to amplify VH or VL sequences from a template, such as scFv clones, can be used to generate whole antibodies. Using cloning techniques known to those skilled in the art, PCR-amplified VH domains can be cloned into vectors expressing the VH constant region, and PCR-amplified VL domains can be cloned into vectors expressing the VL constant region, such as the human kappa or lambda constant regions. . The VH and VL domains can also be cloned into a single vector expressing the required constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, eg, IgG, using methods known to those skilled in the art.

- 54 041979

A chimeric antibody is a molecule in which the different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise a murine or rat antibody variable region fused to a human antibody constant region. Methods for obtaining chimeric antibodies are known in the art. See, for example, Morrison SL (1985), Science, 229: 1202-7; Oi VT & Morrison SL (1986), BioTechniques, 4: 214-221; Gillies SD et al. (1989), J. Immunol. Methods, 125: 191-202 and US Pat.

The humanized antibody is capable of binding to a given antigen and includes a framework region having essentially the amino acid sequence of a human immunoglobulin and CDRs having essentially the amino acid sequence of a non-human immunoglobulin (eg, mouse immunoglobulin). In certain embodiments, the humanized antibody also includes at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. The antibody may also include CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG ₂ , IgG ₃ and IgG ₄ . Humanized antibodies can be obtained using a number of methods known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239400; International Publication No. WO 91/09967; and US Pat. Nos. or surface alteration (EP 592106 and EP 519596; Padlan EA (1991), Mol. Immunol., 28(4/5): 489-498; Studnicka GM et al. (1994), Prot. Engineering, 7 (6): 805-814 and Roguska MA et al (1994) PNAS 91: 969-973), strand permutation (US Pat. No. 5,565,332), and methods described in, for example, US Pat. No. 5766886, international publication No. WO 93/17105; Tan P. et al. (2002), J. Immunol., 169: 1119-25; Caldas C. et al. (2000), Protein Eng., 13(5): 353-60; Morea V. et al. (2000), Methods, 20(3): 267-79; Baca M. et al. (1997), J. Biol. Chem., 272(16): 10678-84; Roguska M.A. et al. (1996), Protein Eng., 9(10): 895 904; Couto JR et al. (1995), Cancer Res., 55 (23 Supp): 5973s-5977s; Couto JR et al. (1995), Cancer Res., 55(8): 1717-22; Sandhu JS (1994), Gene 150(2): 409-10, and Pedersen JT et al. (1994), J. Mol. Biol., 235(3): 959-73, which are all incorporated herein by reference in their entirety. See also US Patent Application Publication No. US 2005/0042664 Al (Feb. 24, 2005), which is hereby incorporated by reference in its entirety.

Methods for preparing multispecific (e.g., bispecific) antibodies are described, see, for example, US Pat.

Single domain antibodies, for example, antibodies lacking light chains, can be prepared by methods well known in the art. See Riechmann L. & Muyldermans S. (1999). J. Immunol., 231: 25-38; Nuttall SD et al. (2000), Curr. Pharm. Biotechnol., 1(3): 253-263; Muyldermans S. (2001), J. Biotechnol., 74(4): 277302; US patent No. 6005079 and international publication No. WO 94/04678, WO 94/25591 and WO 01/44301, which are all incorporated into the present description by reference.

In addition, antibodies that specifically bind to the PD-1 antigen can in turn be used to generate anti-idiotypic antibodies that mimic the antigen using techniques well known to those skilled in the art. See, for example, Greenspan NS & Bona CA (1989), FASEB J., 7(5): 437-444; and Nissinoff A. (1991), J. Immunol., 147(8): 24292438, which are all incorporated herein by reference in their entirety.

In certain embodiments, an antibody described herein that binds to the same PD-1 epitope (eg, human PD-1) as an anti-PD-1 antibody described herein is a human antibody. In certain embodiments, an antibody described herein that competitively blocks (eg, dose dependent) any of the antibodies described herein from binding to PD-1 (eg, human PD-1) is a human antibody. Human antibodies can be obtained using any method known in the art. For example, transgenic mice that are unable to express functional endogenous immunoglobulins but that can express human immunoglobulin genes can be used. In particular, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or by homologous recombination. On the other hand, in mouse embryonic stem cells, in addition to the human heavy and light chain genes, a human variable region, a constant region, and an additional region can be introduced. Mouse immunoglobulin heavy and light chain genes can be rendered non-functional alone or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are grown and injected into blastocytes to give chimeric mice. The chimeric mice are then propagated to produce homozygous offspring that express antibodies. Transgenic mice are routinely immunized with a selected antigen, eg all or part of the antigen (eg PD-1). Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional

- 55 041979 hybridoma technology. Human immunoglobulin transgenes harbored by transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutation. Thus, using such a method, it is possible to obtain therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of such technology for the production of human antibodies, see Lonberg N. & Huszar D. (1995), Int. Rev. Immunol., 13: 65-93, incorporated herein by reference in its entirety. For a detailed disclosure of such technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735 and US Patent Nos. 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of producing human antibodies include Xenomouse™ (Abgenix, Inc.; US Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Mederex, Inc./Gen Pharm; US Pat. Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin) and KM Mouse™ (Medarex/Kirin), where all works are incorporated herein by reference in their entirety.

Human antibodies that specifically bind to PD-1 (eg, human PD-1) can be generated by a number of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also US Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98/4 6645, WO 98/504 33, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, all of which are incorporated herein in their entirety in as links.

In some embodiments, human antibodies can be generated using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and such mouse-human hybridomas can be screened to identify hybridomas that secrete human monoclonal antibodies. antibodies that specifically bind to the target antigen (eg PD-1 (eg human PD1)). Such methods are known and described in the art, see, for example, Shinmoto H. et al. (2004), Cytotechnology, 46: 19-23; Naganawa Y. et al. (2005), Human Antibodies, 14:27-31, which are incorporated herein by reference in their entirety.

5.6. Sets

The invention also relates to kits comprising one or more antibodies described in the present description, or their pharmaceutical compositions or conjugates. A specific embodiment relates to a pharmaceutical package or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more of the antibodies described herein. In some embodiments, the kits contain a pharmaceutical composition described in the present description, and any prophylactic or therapeutic agent, such as described in the present description. In some embodiments, the kits may contain a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or an antibody that stimulates the TCR complex, such as an anti-CD3 antibody and an anti-CD28 antibody. Optionally, associated with such container(s) may be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, indicating the agency's approval of the manufacture, use, or sale for human administration.

The invention also relates to kits that can be used in the methods described above. In one embodiment, the kit includes an antibody described herein, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits described herein contain essentially isolated PD-1 antigen (eg, human PD-1) as a control. In another specific embodiment, the kits described herein contain a control antibody that does not interact with the PD-1 antigen. In another specific embodiment, the kits described herein contain one or more elements for detecting the binding of an antibody to a PD-1 antigen (for example, the antibody can be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody may be conjugated to a detectable substrate). In specific embodiments, the kit described herein may include a PD-1 antigen obtained recombinantly or chemically synthesized. The PD-1 antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detection means in the above kit includes a solid support to which the PD-1 antigen is attached. Such a kit may also include an unattached labeled anti-human or anti-mouse/rat antibody. In such an embodiment, binding of the antibody to the PD-1 antigen can be detected by binding to said reporter labeled antibody. In one embodiment, the present invention relates to the use of the kit of the present invention for the in vitro assay and/or detection of a PD-1 antigen (eg, human PD-1) in a biological sample.

6. Examples

The examples in this section (ie, section 6) are offered to illustrate, but not to limit the invention.

6.1. Example 1 Characterization of Anti-PD-1 Antibodies

This example describes the characterization of antibodies that specifically bind to human PD-1, in particular the antibodies designated AGEN2033w, AGEN2034w, AGEN204 6w, and AGEN2047w. AGEN2033w and AGEN204 6w share the same heavy chain variable region amino acid sequence (SEQ ID NO: 15) and the same light chain variable region amino acid sequence (SEQ ID NO: 16). AGEN2034w and AGEN2047w share the same heavy chain variable region amino acid sequence (SEQ ID NO: 17) and the same light chain variable region amino acid sequence (SEQ ID NO: 16). AGEN2033w and AGEN2034w are human IgG ₄ antibodies containing the S228P mutation (i.e., a serine to proline change at position 228 relative to the wild-type IgG ₄ constant region) according to the EU numbering system, while AGEN2046w and AGEN2047w are human IgG1 antibodies . In addition, three Fc AGEN2047w mutants are also characterized: the N297A mutant, the S267E/L328F double mutant, and the S239D/A330L/I332E triple mutant, numbered according to the EU numbering system.

6.1.1. Antibodies binding to PD-1 expressed by activated T cells

Anti-PD-I antibodies AGEN2046w, AGEN2047w and AGEN2034w are tested for binding to activated peripheral blood mononuclear cells (PBMCs) by flow cytometry. Cryopreserved human PBMCs prepared from crude buffy coats (Research Blood Components, cat. no. 002) or cryopreserved cynomolgus monkey PBMCs (Worldwide Primates Inc., on order) are plated at 105 cells/well in RPMI1640 supplemented with Normocin™ (InvivoGen, cat. no. ant-nr-1) and 10% heat-inactivated FBS (Gibco, cat. no. 16140063) in 96-well NUNCLON delta surface plates (NUNC™) Cells cultured in the presence of 100 ng/ml staphylococcal enterotoxin A (SEA; for human PBMC) (Toxin Technologies, cat. no. at101red) or staphylococcal enterotoxin B (SEB; for cynomolgus monkey PBMC) (Toxin Technology, cat. no. bt202red) for 5 days at 37°C, 5% CO ₂ and 97% humidity Cells are then washed once with sample buffer (PBS + 2% FBS + 0.09% sodium azide) and incubated in the dark on ice with 100 µl of serially diluted antibodies or isotype controls (10, 1, 0, 1, 0.01, 0.001 and 0.0001 µg/mL AGEN2046w, AGEN2047w or isotype control human IgG1 (LifeTein LLC, cat. No. LT12031); or 25, 5, 1, 0.2, 0.04, 0.008, 0.0016, 0.00032, and 0.000064 μg/mL of AGEN2034w or isotype control human IgG ₄ (LifeTein LLC, cat. no. LT12034)). After 45 min, cells are washed twice with sample buffer and then incubated with LIVE/DEAD® Fixable Near-IR dead cell dye (Life Technologies, cat. N L10119), CD4-BV421 (Biolegend, cat. 1 317434) and goat F( ab') with ₂ anti-human IgG+A+M, R-PE (Life Technologies cat. 1 AHH707) for 30 min. The cells are washed twice with sample buffer and then resuspended in sample buffer and analyzed with a Fortessa FACS cytometer (Becton Dickinson). CD4+ T cells are captured and the mean fluorescence intensity (MFI) is recorded.

Anti-PD-I antibodies AGEN2046w and AGEN2047w bind to activated human CD4+ T cells (Figure 1A). AGEN2034w binds to activated human and cynomolgus monkey CD4+ T cells (Figures 1B and 1C).

Binding of AGEN2034w to activated primary human T cells is measured again in a similar assay. Briefly, human PBMCs were cultured in the presence of 100 ng/ml SEA peptide for 5 days and then stained serially with (50, 10, 2, 0.4, 0.080, 0.016, 0.0032, 0.00064, 0.000128, 0 .0000256, 0.00000512 and 0.000001024 µg/ml) AGEN2034w or isotype control human IgG ₄ . Cells are analyzed using a FACS Fortessa cytometer (Becton Dickinson). CD4+ T cells are captured and the mean fluorescence intensity (MFI) is recorded.

As seen in FIG. 1D, AGEN2034w binds to activated primary human CD4+ T cells.

6.1.2. Anti-PD-1 antibody selectivity assay

The selectivity of AGEN2034w for PD-1 was assessed against homologous proteins using suspension array technology.

Based on the homology of their amino acid sequences with PD-1, Ig superfamily proteins ring homologue 2 (ROBO2), B7 homologue 7 (B7-H7) and signal regulatory protein gamma (SIRPyyy) are selected to evaluate AGEN0234w binding using suspension array technology. ROBO2, B7-H7, and SIRPy are identified as PD-1 homologues by protein alignment using the Basic Local Alignment (BLAST; NCBI) finder. Sequence homology of human PD-1 with its

- 57 041979 homologues as follows: human PD-1 vs. (vs) human ROBO2: 27.9%; human

PD-1 vs human SIRPy (SIRPG_HUMAN): 24.8%; and human PD-1 vs human B7-H7:

22.6%.

Recombinant proteins ROBO2-Fc human chimera (R&D Systems, cat. no. 3147-RB-050), B7-H7-FC human chimera (R&D Systems, cat. no. 8084-B7-050), SIRPy-His human chimera (Sino Biologicals , cat. no. 11828-H08H) and human PD-1-Fc chimera (R&D Systems, cat. no. 1086-PD) are combined with Luminex® microspheres (Luminex Corp., cat. no. LC10005-01, LC10022-01, LC10046-01, LC10048-01 and LC10059-01) using N-hydroxysuccinimide (NHS) ester chemistry and incubated with dose titration (7.5, 2.5, 0.833, 0.277, 0.0926, 0.0309, 0 .0103, 0.0034, 0.0011, 0.0004, 0.0001 and 0.00004 µg/mL) AGEN2 034w. An anti-human IgG antibody labeled with phycoerythrin (PE) is then added to detect AGEN2 034w. Binding is assessed using the Luminex® 200 detection system.

The AGEN2034w antibody shows specific binding to human PD-1 and, at the concentrations tested, does not show significant binding to ROBO2, B7-H7, or SIRPy (FIG. 2).

6.1.3. Ligand-blocking activity determined using suspension array technology

In order to determine whether anti-PD-1 antibodies block the binding of PD-11 and PD12 ligands, the suspension array assay described above is performed. 1200 Luminex® beads in 5 µl of assay buffer (Luminex Corp, cat. no. 48 LC10014-48) are added to each well in half area of 96-well plates (Corning, Inc., cat. no. 3884). The beads are coupled to the PD-1 PD-1-Fc antigen chimera (R&D Systems cat. no. 1086-PD) via amino coupling with COOH on the surface of the beads. The coupling reaction is performed using 50 μg/ml of PD-1 antigen and 1 x 10 ⁷ Luminex beads per ml. Use standard NHS ester chemistry to form carbodiimide bonds between the antigen's primary amino groups and carboxyl groups on the surface of the beads (Luminex Xmap cookbook, chapter 3).

The coupling of antigen to proteins is a simple two-step procedure for the formation of carbodiimide bonds, during which the carboxyl groups of the microspheres are first activated with an EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) reagent in the presence of sulfo-NHS (Nhydroxysulfosuccinimide) to form an intermediate complex sulfo-NHS-ether. The reactive intermediate is then reduced by reacting with the primary amine of the target molecule (antibody, protein, or peptide) to form a covalent amide bond. Coupling beads are incubated with various concentrations of anti-PD-1 antibodies in triplicate (final concentrations 7.5 to 0.01 μg/ml per well) for 1 hour at 20° C. and 650 rpm. The antibodies tested are AGEN2033w, AGEN2034w, AGEN2046w and AGEN2047w, an IgG1 isotype control and an IgG4 isotype control. Then, 30 µl of R-PE-labeled PD-L1-Fc (R&D systems, cat. no. 156-B7) or PD-L2-Fc (R&D systems, cat. no. 1224-PL) at a concentration of 1 nM is added to each well, obtaining total volume per well 60 μl (1200 beads per well and a final concentration of 0.5 nM labeled PD-11 or PD-12). Ligand labeling is performed using RPE labeling kits (AbDSerotec, LYNX Rapid RPE Antibody Conjugation Kit, Cat. 1 LNK023RPE) according to the manufacturer's protocol. The plates are analyzed using the Luminex® 200 system (Millipore). Count 100 beads per well in a 50 µl sample. The ligand-blocking potential is calculated using the MFI values of the non-competing signal (100% binding) of the control ligand only. The detected PE signal indicates the binding of the ligand to the antigen.

All tested anti-PD-1 antibodies inhibit the binding of PD-11 and PD-12 to PD-1 (Fig. 3A3D).

The measurement of the ligand-blocking activity of AGEN2034w is repeated in a similar assay. Briefly, a recombinant PD-1-Fc chimera (R&D Systems, cat. no. 1086-PD) is coupled with Luminex® microspheres (Luminex Corp, cat. no. LC10048-01) using N-hydroxysuccinimide (NHS) ester chemistry. PD-1 attached beads are incubated with dose titration (4.0x10'5'7.5 µg/mL) of AGEN2034w or isotype control antibody followed by incubation of fluorescently labeled PD-L1-Fc (R&D Systems cat. no. 156-B7) or PD-12-Fc (R&D Systems, cat. no. 1224-PL). The binding of PD-11 or PD-12 to PD-1 attached beads is then assessed using the Luminex® 200 detection system and the mean fluorescence intensity (MFI) is recorded.

The AGEN2034w antibody effectively blocks the coupling of PD-1 to its PD-11 (FIG. 3E) and PD-12 (FIG. 3F) ligands.

6.1.4. Effect of anti-PD-1 antibodies on human PBMCs after stimulation with staphylococcal enterotoxin A (SEA)

The functional activity of anti-PD-1 antibodies on primary human T cells was assessed using SEA stimulation. Cryopreserved human PBMCs prepared from crude buffy coats (Research Blood Components, cat. no. 002) are plated at 105 cells/well in RPMI1640 supplemented with normocin™ (InvivoGen, cat. no. ant-nr-1) and 10% heat-inactivated FBS (Gibco, cat. no. 16140063) in 96-well NUNCLON delta plates

- 58 041979 (NUNC™). Cells are cultured in the presence of a fixed concentration (10 μg/ml) or dose amounts of antibodies (50, 10, 2, 0.4, 0.08, 0.016, and 0.0032 μg/ml) and a fixed amount of SEA (100 ng/ml, Toxin Technology, Cat No. at101red) for 5 days at 37°C, 5% CO ₂ and 97% humidity. The antibodies tested are AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w and an IgG1 isotype control. The supernatant is collected and stored at -80°C until analysis. IL-2 titers are measured by electrochemiluminescence (MSD).

As seen in FIG. 4A and 4B, anti-PD-1 antibodies AGEN2033w, AGEN2034w, AGEN2046w, and AGEN2047w increase IL-2 production of PBMC relative to isotype control in the presence of SEA stimulation.

Next, the antagonistic activity of AGEN2034w, either alone or in combination with antu-CTLA-4, anti-TIGIT, anti-CD137, or anti-OX40 antibodies, was tested in the primary PBMC assay described above. Briefly, cryopreserved human PBMCs prepared from crude buffy coats (Research Blood Components, cat. no. 002) were cultured with SEA superantigen (Toxin Technology, cat. no. at101red) (100 ng/ml in Figs. 4C, 4d and 4F; 200 ng/ml in Fig. 4E) and AGEN2034w (10 μg/ml in Figs. 4C and 4E; 5 μg/ml in Fig. 4D; dose interval 12, 6, 3, 0.3, 0.03, 0.003, 0 .0003 and 0.0001 µg/mL in Fig. 4F) or an isotype control antibody in the presence of 5 µg/mL anti-CTLA-4 antibody ipilimumab (Myoderm) (Fig. 4C), 10 µg/mL anti-TIGIT antibody pab2197, or pab2196 (Figure D 4), 5 μg/ml anti-CD137 antibody pab2225 (Figure 4E) or dose range (12, 6, 3, 0.3, 0.03, 0.003, 0.0003, and 0.0001 μg /ml) anti-OX40 antibody pab1928 for 5 days. Supernatants are harvested and IL-2 titers are measured using AlphaLISA (Perkin Elmer, cat. no. AL221C). The anti-TIGIT antibodies pab2197 and pab2196 are derived from the variable region sequences of antibodies 10A7 and 1F4, respectively, as described in US Patent Application Publication No. US 2013/0251720 (incorporated herein by reference in its entirety). the anti-CD137 antibody pab2225 is derived from the sequences of the variable region of the 20H4 antibody described in US Pat. No. 8,137,667 (incorporated herein by reference in its entirety). The anti-OX40 antibody pab1928 is derived from the sequences of the variable region of the Hu106-122 antibody described in US Patent Application Publication No. US 2013/0280275 (incorporated herein by reference in its entirety). The sequence of anti-TIGIT, anti-CD137 and anti-OX40 antibodies are given in table. 8.

- 59 041979

Table 8. Anti-TIGIT, anti-CD137, and anti-OX40 antibody sequences

SEQ ID NO: Description Amino acid sequence 66 pab2197, heavy chain EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGK GLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDL KSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 67 pab2197 light chain DIVMTQSPSSLAVSPGEKVMTMTCKSSQSLYYSGVKENLLAWYQ QKPGQSPKLLIYYASIRFTGVPDRFTGSGTDYTLTITSVQA 68 pab2196 heavy chain EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGK NLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSL TSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG

- 60 041979

69 pab2196 light chain DWLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLH KPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPE 70 pab2225 heavy chain QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEK GLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVT AADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG 71 pab2225 light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLGESFSKADYEKHKVYACEVTHQС 72 pahl928 heavy chain QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQ GLKWMGWINTETGEPTYADDFKGRFVFSLDTSVSTAYLQISSL KAEDTAVYYCANPYYDYVSYYAMDYWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 73 pahl928 light chain DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKA PKLLIYSASYLYTGVPSRFSGSGSGTDFTFTISSLQPEDIATY YCQQHYSTPRTFGQGTKLEIKRSVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTKTLTLSKADYEKHKSSVY

Anti-PD-l antibody AGEN2034w, either alone or in combination with aHTu-CTLA-4 antibody ipilimumab (Fig. 4C), anti-TIGIT antibody pab2197 or pab2196 (Fig. 4D), anti-CD137 antibody pab2225 (Fig. 4E) or the anti-OX40 antibody pab1928 (FIG. 4F), enhances IL-2 production in human PBMCs in the presence of the SEA superantigen.

6.1.5. Effect of Fc Receptor Binding on Antagonistic Activity of Anti-PD-1 Antibodies

In this example, the effect of FcyR binding on the antagonistic activity of anti-PD-1 antibodies is tested.

First, the antagonistic activity of a reference αanti-PD-1 antibody is tested in the presence or absence of FcyR blockers. Cryopreserved human PBMCs prepared from crude buffy coats (Research Blood Components, cat. no. 002) were plated at 10 ⁵ cells/well in RPMI1640 medium supplemented with normocin™ (InvivoGen, cat. no. ant-nr-1) and 10% inactivated by heating FBS (Gibco cat. no. 16140063) into 96-well NUNCLON delta (NUNC™) surface plates. Cells are cultured with 100 ng/mL of SEA peptide (Toxin Technology, Cat # at101red) and 10 μg/mL of anti-PD-1 reference antibody or isotype control in the presence or absence of a fixed concentration of an Fc receptor blocking cocktail containing anti-CD16 antibody (1 µg/ml, R&D systems, cat. no. AF1330) and irrelevant IgG1 (25 µg/ml, LifeTein, cat. no. LT12031), for 5 days at 37°C, 5% CO ₂ and 97% humidity. Supernatants are collected and stored at -80°C until ana

- 61 041979 Lisa. IL-2 titers are measured by electrochemiluminescence (MSD).

Blockade of FcyR using an anti-CD16 antibody enhances the ability of the reference anti-PD-1 antibody to induce IL-2 secretion in this primary human PBMC assay (FIG. 5A).

A similar study was performed to test the effect of FcyR blockade on the antagonistic activity of AGEN2034w. Briefly, human PBMCs are cultured with 100 ng/ml SEA peptide (Toxin Technology, cat. no. at101red), 10 μg/ml AGEN2034w or an isotype control antibody (HEL IgG1, LifeTein, cat. no. LT12031), and 20 μg/ml anti- CD16 antibody (Biolegend, cat. no. 302013), anti-CD32 antibody (eBioscience, cat. no. 16-0329-81) that binds to both CD32A and CD32B, anti-CD64 antibody (Biolegend, cat. no. 305016 ) or isotype control (Biolegend cat. no. 400543) for 5 days at 37°C, 5% CO ₂ and 97% humidity. Supernatants are collected and stored at -80°C until analysis. IL-2 titers are measured by electrochemiluminescence (MSD) .

According to the result in FIG. 5A, FcyR blockade using an anti-CD16 antibody, antu-CD32 antibody, or an anti-CD64 antibody increases AGEN2034w-induced IL-2 secretion in this primary human PBMC assay (FIG. 5B).

Next, three IgG1 AGEN2047w Fc mutants (N297A mutant, S267E/L328F double mutant, and S239D/A330L/I332E triple mutant, numbered according to the EU numbering system) were prepared and compared against AGEN2047w, which includes a wild-type IgG1 constant region, in a human PBMC assay with SEA described above. Briefly, cryopreserved human PBMCs are incubated with 100 ng/mL SEA and 10 μg/mL anti-PD-1 antibodies AGEN2047w, AGEN2047w-N297A, AGEN2047wS267E/L328F, AGEN2047w-S239D/A330L/I332E, or isotype control antibodies with corresponding wild-type Fc regions. or mutants.

As seen in FIG. 5C, the N297A mutant with reduced FcyR binding further enhances IL-2 secretion. In contrast, the double mutant S267E/L328F and the triple mutant S239D/A330L/I332E, which both have enhanced FcyR binding, induce less IL-2 production than wild-type AGEN2047w.

6.1.6. Effect of Anti-PD-1 Antibody on the Response in Mixed Lymphocyte Culture

Next, the anti-PD-1 antibody AGEN2034w was tested in a mixed lymphocyte culture reaction. Dendritic cells derived from isolated CD14+ cells (Stemcell Technologies, cat. no. 18058) obtained from cryopreserved HLA-A2+ human PBMCs and differentiated first in the presence of 500 U/ml IL-4 (Peprotech, cat. no. 200-04-20UG) and 1000 U/ml GM-CSF (Peprotech, cat. no. 30003-20UG) for 24 h and then in the presence of 1000 U/ml TNFa (Peprotech, cat. no. 300-01A-50UG), 10 ng/ml IL- 1e (Peprotech, cat. no. 200-01B-10UG), 10 ng/ml IL-6 (Peprotech, cat. no. 200-06-20UG) and 1 μM PGE2 (Sigma, cat. no. PO4O9-5MG) for another 24 h Selected 50,000 T cells purified from allogeneic HLA-A2 donor human PBMC by MACS column purification (Miltenyi Biotec, cat. no. µg/mL human IgG ₄ isotype control antibody (Biolegend, cat. no. 403402) or 10 µg/ml1 AGEN2034w in RPMI (Corning, cat. no. 10-040-CM) containing 5% human AB serum (Corning, cat. no. 3 5-060-CI) and penicillin/streptomycin (Gibco, Cat. No. 15140-122). Cultures are incubated for 5 days at 37°C and 5% CO2. Supernatants are assessed for steady-state IFNy concentrations using AlphaLISA (Perkin Elmer, cat. no. AL217C).

As seen in FIG. 6, AGEN2034w induces IFNy production in a co-culture of purified human T cells and in vitro derived allogeneic dendritic cells.

6.1.7. Effect of anti-PD-1 antibody in the ascitic fluid suppression assay

In this example, the anti-PD-1 antibody AGEN2034w is tested for its ability to attenuate the suppression of T cell proliferation caused by ascitic fluid in ovarian cancer. Briefly, primary human PBMCs are labeled with CFSE (Biolegend, cat. no. 423801) and then stimulated with 1 μg/ml anti-CD3 antibody (eBioscience, cat. no. 16-0037-85) and 50% v/v cancer ascitic fluid. ovaries in the presence of increasing concentrations (0.00000102-50 μg/ml) of AGEN2034w or an isotype control IgG ₄ antibody (Biolegend cat. no. 317434) for 4-5 days. Cells are stained with immuno-anti-human CD4 antibody (Biolegend, cat. no. 317434) or anti-human CD8 antibody (Biolegend, cat. no. 344710) and LIVE-DEAD viability dye (Life Technologies, cat. no. L10119). Proliferation of CD4+ or CD8+ T cells, as illustrated by CFSE dilution, was measured by flow cytometry using a BD Fortessa (Becton Dickinson).

As seen in FIG. 7A-7C, co-cultivation with ascitic fluid in ovarian cancer reduces the proliferation of anti-CD3 antibody stimulated T cells, and this decrease can be partially attenuated by the anti-PD-1 antibody AGEN2034w.

6.1.8. Action of anti-PD-1 antibody in NFAT-luciferase reporter analysis of Yurkat cells

In addition, a reporter assay is used to probe the antagonistic activity of AGEN2034w. Specifically, in such a reporter assay, co-culture of PD-11 expressing target cells and PD-1 expressing reporter cells inhibits the expression of the NFAT-luciferase reporter gene in the reporter cells. Blockade of the PD-1/PD-11 interaction with anti-PD-62 041979 antibody can attenuate the inhibition signal, leading to luciferase expression.

Briefly, PD-11+ CHOK1 target cells (Promega, cat. no. CS187108) are co-cultured with GloResponse™ NFAT-luc2/PD-1 Jurkat reporter cells (Promega, cat. no. CS187102) in the presence of increasing concentrations (0-50 μg /ml) AGEN2034w or isotype control antibody in RPMI-1640 medium (Corning, cat. no. 21-040-CV) supplemented with 2% heat-inactivated FBS (Gemini, cat. no. 100-106). After 6 hours of incubation, the effectiveness of AGEN2 034w in attenuating reporter gene suppression induced by PD-11 binding to PD-1 is determined by detecting luciferase using the Bio-Glo™ Luciferase Assay System (Promega, cat. 1 G7941).

As seen in FIG. 8, the anti-PD-1 antibody AGEN2034w enhances TCR signaling in a dose dependent manner in such a Jurkat-NEAT-luciferase reporter assay.

6.2. Example 2 Characterization of Additional Anti-PD-1 Antibodies

In this example, the following six additional anti-PD-1 antibodies are characterized: AGEN2001w, AGEN2002w, EP11_pl1_B03, EP11_pl1_B05, EP11_pI_C02, and EP11_pI_C03. The variable heavy chain and variable light chain sequences of these antibodies are disclosed in Table 1. 6.

6.2.1. Analysis of anti-PD-1 antibodies for ligand binding and blockade

The affinity of the six anti-PD-1 antibodies described above was analyzed by surface plasmon resonance. All six antibodies bind to recombinant human PD-1 (data not shown).

Ligand blocking activity of six anti-PD-1 antibodies is tested using suspension array technology in an assay similar to that described in section 6.1.3. The pooled beads are incubated with various concentrations of anti-PD-1 antibodies in duplicate (final concentrations from 7.5 to 0.001 μg/ml per well) for 1 hour at 20° C. and 650 rpm. R-PE labeled PD-L1-Fc (R&D Systems cat. no. 156-B7) or PD-12-Fc (R&D Systems cat. no. 1224-PL) is then added. The anti-PD-1 antibodies tested are AGEN2001w, AGEN2002w, EP11_pl1_B03, EP11_p1_B05, EP11_p11_C02 and EP11_p11_C03. As seen in FIG. 9A-9F, all anti-PD-1 antibodies tested block PD-1 binding to PD-11 and PD-12 in a dose dependent manner.

6.2.3. Effect of anti-PD-1 antibodies on human PBMCs after staphylococcal enterotoxin A (SEF) stimulation

The functional activity of the anti-PD-1 antibody AGEN2002w on human PBMCs is tested in an assay similar to that described in section 6.1.4. Briefly, cryopreserved human PBMCs prepared from crude buffy coats (Research Blood Components, cat. no. 002) are cultured in the presence of 10 μg/ml of AGEN2002w anti-PD-1 antibody or an isotype control antibody (HEL IgG1, LifeTein, cat. no. LT12031 ) and 100 ng/ml of SEA peptide (Toxin Technologies, cat. no. at101red) for 4 days at 37°C, 5% CO ₂ and 97% humidity. Supernatants are collected and stored at 80°C until analysis. IL-2 titers are measured by electrochemiluminescence (MSD).

As seen in FIG. 10, the anti-PD-1 antibody AGEN2002w increases IL-2 production in primary human PBMCs in the presence of SEA stimulation.

6.3. Example 3 Anti-PD-1 Antibody Epitope Mapping

The anti-PD-1 epitope of the AGEN2034w antibody is characterized using hydrogen-deuterium exchange (HDX) mass spectrometry and Pepscan analysis.

6.3.1. Anti-PD-1 Fab epitope mapping using hydrogen-deuterium exchange (HDX) mass spectrometry

The interaction of the AGEN2034w Fab fragment (AGEN2034w-Fab) with the extracellular domain of human PD-1 is examined by hydrogen-deuterium exchange (HDX) mass spectrometry.

Recombinant His-tagged human PD-1 was obtained from Sino Biological Inc (Cat# 10377H08H). When deglycosylated PD-1 is used, it is prepared from 300 μg of recombinant His-tagged human PD-1 protein by incubation with 6 μl of PNGase F at 37° C. for 4 hours. An anti-PD-1 antibody Fab fragment is prepared from AGEN2034w by protease treatment.

For pepsin/protease XVIII digestion, 4.0 µg of native or deglycosylated human PD-1 in 125 µl of control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) is denatured by adding 135 µl of 4 M guanidine hydrochloride, 0. 85 M TCEP buffer (final pH 2.5) and incubate the mixture for 3 min at 11°C. The mixture is then digested on a pepsin/protease XVIII column using a column packed with pepsin/protease XVIII in situ, and the resulting peptides are analyzed using an UPLC-MS system consisting of a Waters Acquity UPLC combined with a quadrupole orbital trap mass spectrometer Q Exactive™ Hybrid (Thermo). The peptides were separated on a 50 mm x 1 mm column, C8, with a 19 min gradient of 2-27% solvent B (0.2% formic acid in acetonitrile). The identification of peptides is carried out by searching for MS/MS data against the sequence of human PD-1 using Mascot. The mass tolerance for the precursor and product ions is 10 ppm and 0.05 Da, respectively.

Incubate 10 µl of human PD-1 (4.0 µg) or 10 µl of a mixture of human PD-1 and Fab (4.0 µg, 4.0 µg) with 125 µl of deuterium oxide labeling buffer (50 mM sodium phosphate, 100 mM chloride

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Claims (27)

sodium at pD 7.4) for 0, 60, 600 and 3600 s at 11°C. The hydrogen/deuterium exchange was quenched by adding 135 μl of a solution of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). The quenched samples are then subjected to pepsin/protease XVIII column digestion and LCMS analysis as described above. Mass spectra are recorded only in the MS mode. Raw MS data is processed using HDX WorkBench to analyze H/D exchange MS data (J. Am. Soc. Mass Spectrom., 2012, 23(9), 1512-1521, incorporated herein by reference in its entirety). Deuterium levels are calculated using the median difference in weight between the deuterated peptide and its native form (to). Sequence coverage of 85.4% is achieved for deglycosylated human PD-1 without His tag. Most PD-1 peptides display identical or similar levels of deuterium in the presence and absence of anti-human PD-1 Fab. However, some peptide segments have been found to have significantly reduced levels of deuterium incorporation after Fab binding. All residues in this paragraph are numbered according to SEQ ID NO: 74. Deglycosylated human PD-1 shows a strong reduction in deuterium uptake upon binding to anti-human PD-1 Fab at residues 107-122 (SLAPKAQIKESLRAEL) (SEQ ID NO: 75) . In addition, a decrease in deuterium uptake after binding to an anti-human PD-1 Fab can be observed at residues 5-22 (LDSPDRPWNPPTFSPALL) (SEQ ID NO: 76). 6.3.2. Anti-PD-1 antibody epitope mapping using Pepscan assay The binding of anti-PD-1 antibody AGEN2034w is measured against fragments of the synthesized PD-1 peptide obtained as a peptide matrix associated with the chip. The analysis is performed at Pepscan Presto BV, Lelystad, The Netherlands. Briefly, a peptide library is synthesized to reconstruct human PD-1 epitopes. The amino-functionalized polypropylene carrier is prepared by grafting a proprietary hydrophilic polymer preparation followed by reaction with tert-butoxycarbonylhexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with Nhydroxybenzotriazole (HOBt), and then Boc cleavage using trifluoroacetic acid (TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on an amino-functionalized solid support with custom-modified JANUS positioning fluid (Perkin Elmer). The synthesis of structural mimetics is carried out using Pepscan's patented technology of chemically linked peptides on scaffolds (CLIPS). CLIPS technology allows peptides to be structured into single loops, double loops, triple loops, leaf folds, helical folds, and combinations thereof. The binding of the antibody to each of the synthesized peptides is tested in a PEPSCAN-based ELISA. The peptide matrices are incubated with the primary antibody solution overnight at 4°C. After washing, the peptide arrays are incubated with goat anti-human HRP conjugate (Southern Biotech, cat. no. 2010-05) for 1 hour at 25°C. After washing, peroxidase substrate 2,2'-azinodia-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml 3% H ₂ O ₂ are added. After 1 hour, color development is noted and quantified using a charge-coupled device (CCD)-camera and image processing system. The Pepscan study shows that the anti-PD-1 antibody AGEN2034w recognizes stretches of human PD-1, including residues 6-15 (DSPDRPWNPP) (SEQ ID NO: 77), residues 130-138 (EVPTAHPSP) (SEQ ID NO: 78) and residues 106-113 (ISLAPKAQ) (SEQ ID NO: 79), numbered according to SEQ ID NO: 74. The invention is not limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention other than those described will become apparent to those skilled in the art from the above description and accompanying figures. Such modifications are intended to be within the scope of the appended claims. All references (e.g., publications or patents or patent applications) cited in this specification are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or application for patent) has been specifically and separately incorporated by reference in its entirety for all purposes. Other embodiments are within the scope of the following claims. CLAIM 1. An isolated antibody that specifically binds to human PD-1, containing a heavy chain variable region containing complementarity-determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity-determining regions CDRL1, CDRL2 and CDRL3, where: (a) CDRH1 contains the amino acid sequence SYGMH (SEQ ID NO: 1); (b) CDRH2 contains the amino acid sequence VIWX1DGSNX2YYADSVX3G (SEQ ID NO: 32), where X1 is Y or F; X2 is K or E and - 64 041979 X ₃ is K or M; (c) CDRH3 contains the amino acid sequence NX1X2 (SEQ ID NO: 33), where X1 is G or V and X2 is H or Y; (d) CDRL1 contains the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4); (e) CDRL2 contains the amino acid sequence GASTRAT (SEQ ID NO: 5) and (f) CDRL3 contains the amino acid sequence QQYNNWPRT (SEQ ID NO: 6). 2. An antibody conjugate containing an isolated antibody that specifically binds to human PD1, containing a heavy chain variable region containing complementarity-determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity-determining regions CDRL1, CDRL2 and CDRL3, where: (a) CDRH1 contains the amino acid sequence SYGMH (SEQ ID NO: 1); (b) CDRH2 contains the amino acid sequence VIWX1DGSNX2YYADSVX3G (SEQ ID NO: 32), where X1 is Y or F; X ₂ is K or E and X ₃ is K or M; (c) CDRH3 includes the amino acid sequence NX1DX2 (SEQ ID NO: 33), where X ₁ is G or V and X ₂ is H or Y; (d) CDRL1 contains the amino acid sequence RASQSVSSNLA (SEQ ID NO: 4); (e) CDRL2 contains the amino acid sequence GASTRAT (SEQ ID NO: 5) and (f) CDRL3 contains the amino acid sequence QQYNNWPRT (SEQ ID NO: 6) wherein the isolated antibody is conjugated to a cytotoxic agent, cytostatic agent, or toxin. 3. An isolated antibody according to claim 1 or an antibody conjugate according to claim 2, where: (a) CDRH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 34-36; (b) CDRH3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, and 37 and/or (c) CDRH1, CDRH2, and CDRH3 contain the amino acid sequences CDRH1, CDRH2, and CDRH3 of SEQ ID NOs: 1, 2 and 3; 1, 2 and 7; 1, 2 and 37; 1, 34 and 7; 1, 35 and 7 or 1, 36 and 7 respectively. 4. An isolated antibody according to claim 1 or an antibody conjugate according to claim 2, where: (a) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, or (b) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 contain the amino acid sequence SEQ ID NO: 1, 2, 7, 4, 5 and 6, respectively. 5. An isolated antibody or antibody conjugate according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49. 6. The isolated antibody or antibody conjugate of any one of claims 1 to 4, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of from SEQ ID NOs: 15, 17 and 26-31. 7. An isolated antibody or antibody conjugate according to any one of claims 1 to 6, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 52, 53, 54, 55, or 56. 8. An isolated antibody or antibody conjugate according to any one of claims 1 to 6, wherein the antibody comprises a heavy chain variable region with an amino acid sequence derived from a human IGHV3-33 germline sequence and/or a light chain variable region with an amino acid sequence derived from a human germline. human IGKV3-15 sequences. 9. An isolated antibody or antibody conjugate according to any one of claims 1 to 8, wherein the antibody contains a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. 10. An isolated antibody or antibody conjugate according to any one of claims 1-9, wherein the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 19. 11. The selected antibody according to claim 1 or the antibody conjugate according to claim 2, where the variable region of the heavy chain and the variable region of the light chain, respectively, contain the amino acid sequence of SEQ ID NO: 15 and 16; 17 and 16; 26 and 16; 27 and 16; 28 and 16; 29 and 16; 30 and 16 or 31 and 16. 12. An isolated antibody that specifically binds to human PD-1, comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, comprise the amino acid sequences of SEQ ID NOs: 15 and 16, 17 and 16, 52 and 19 , 53 and 19, 54 and 19, 55 and 19 or 56 and 19. 13. The isolated antibody of claim 11, wherein the antibody contains: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID - 65 041979 NO: 17, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. 14. An isolated antibody according to claim 12, wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 53 and the light chain contains the amino acid sequence of SEQ ID NO: 19. 15. The isolated antibody of claim 14, wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 20. 16. An isolated antibody or antibody conjugate according to any one of claims 1-15, where: (a) the antibody binds to an epitope consisting of residues 107-122 of SEQ ID NO: 74; (b) the antibody binds to an epitope consisting of residues 5-22 of SEQ ID NO: 74; (c) the antibody binds to an epitope consisting of residues 6-15 of SEQ ID NO: 74; (d) the antibody binds to an epitope consisting of residues 130-138 of SEQ ID NO: 74; or (e) the antibody binds to an epitope consisting of residues 106-113 of SEQ ID NO: 74. 17. An isolated antibody or antibody conjugate according to any one of claims 1-6, 11, or 13, wherein the antibody contains a heavy chain constant region selected from the group consisting of IgG1, IgG ₂ , IgG ₃ , IgG4, IgA1 and IgA2 human and/ or a light chain constant region selected from the group consisting of human kappa light chain and human lambda light chain. 18. An isolated antibody or antibody conjugate according to any one of claims 1-6, 11 or 13, where: (a) the antibody contains a human IgG1 heavy chain constant region that lacks a glycan group at position N297 according to the EU numbering system; (b) the antibody contains a human IgG1 heavy chain constant region containing the N297A mutation according to the EU numbering system; (c) the antibody contains a human IgG1 heavy chain constant region containing the N297Q mutation according to the EU numbering system; (d) the antibody contains a human IgG1 heavy chain constant region containing the D265A mutation according to the EU numbering system; and/or (e) the antibody comprises a human IgG4 heavy chain constant region containing the S228P mutation according to the EU numbering system. 19. The isolated antibody or antibody conjugate of any one of claims 1-6, 11, or 13, wherein the antibody comprises a human IgG heavy chain constant region that is a wild-type human IgG heavy chain constant region variant, wherein the human IgG heavy chain constant region variant : (a) binds to the human Fc receptor with less affinity than the corresponding wild-type human IgG heavy chain constant region; or (b) is a heavy chain constant region of a human IgG1 variant, a human IgG ₂ variant, or a human IgG ₄ variant. 20. The isolated antibody or antibody conjugate of claim 19, wherein the human Fc receptor is FcyR. 21. The isolated antibody or antibody conjugate of claim 19, wherein the human Fc receptor is FcyRIIB. 22. An isolated antibody or antibody conjugate according to any one of claims 1-19, where: (a) the antibody is a human antibody; (b) the antibody exhibits antagonism against human PD-1; (c) the antibody deactivates, reduces or inhibits the activity of human PD-1; (d) the antibody inhibits the binding of human PD-1 to human PD-L1 or human PD-L2; (e) increases the production of IL-2 by peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin A (SEA); (f) the antibody increases the production of IFNy by a co-culture of human T cells and allogeneic dendritic cells; (g) the antibody enhances the proliferation of anti-CD-3 antibody-stimulated CD4+ or CD8+ T cells co-cultured with ovarian cancer ascites; and/or (h) the antibody enhances luciferase NFAT signaling by reporter cells co-cultured with PD-L1 expressing target cells. 23. A pharmaceutical composition comprising an antibody or antibody conjugate according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier or excipient. 24. An isolated polynucleotide encoding the heavy chain of an antibody according to any one of claims 1 or 3-22. 25. An isolated polynucleotide encoding an antibody light chain according to any one of claims 1 or 3-22. 26. An isolated polynucleotide encoding the heavy chain and light chain of an antibody according to any one of claims 1 or 3-22. 27. An isolated polynucleotide encoding the heavy chain variable region of an antibody according to any one of claims 1 or 3-22. -