NZ714537B2 - Antibodies directed against programmed death-1 (pd-1) - Google Patents

Abstract

The invention relates to an isolated immunoglobulin heavy chain polypeptide and an isolated immunoglobulin light chain polypeptide that bind to a programmed death-1 (PD-1) protein. The invention provides a PD-1-binding agent that comprises the immunoglobulin heavy chain CDRs sequences of GFTFSSYDMS, TISGGGSYTY, PYYAMDY, and the immunoglobulin light chain CDRs sequences of KASQDVGTAVA, WASTLHT, QHYSSYPWT. The invention also provides related vectors, compositions, and methods of using the PD-1-binding agent to treat a cancer or an infectious disease. , TISGGGSYTY, PYYAMDY, and the immunoglobulin light chain CDRs sequences of KASQDVGTAVA, WASTLHT, QHYSSYPWT. The invention also provides related vectors, compositions, and methods of using the PD-1-binding agent to treat a cancer or an infectious disease.

Description

ANTIBODIES DIRECTED AGAINST PROGRAMMED DEATH—l (PD-l) INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its ty herein is a computer-readable nucleotide/amino acid sequence listing submitted rently herewith and identified as follows: One 45,084 Byte ASCII (Text) file named "716746_ST25.TXT," created on May 1, 2014.

OUND OF THE INVENTION Programmed Death 1 (PD-l) (also known as Programmed Cell Death 1) is a type I transmembrane n of 268 amino acids originally fied by subtractive hybridization of a mouse T cell line undergoing apoptosis a et al., Embo J., I I : 3887—95 (1992)). PD— 1 is a member of the CD28/CTLA-4 family of T-cell regulators, and is expressed on activated T-cells, B-cells, and myeloid lineage cells (Greenwald et al., Annu. Rev. Immunol., 23: 515- 548 (2005); and Sharpe et al., Nat. Immanol., 8: 5 ).

Two ligands for PD-l have been identified, PD ligand 1 (PD-Ll) and PD ligand 2 (PD-L2), both of which belong to the B7 protein superfamily (Greenwald et al., supra). PD- L1 is expressed in a variety of cell types, including cells of the lung, heart, thymus, spleen, and kidney (see, e.g., Freeman et al., J. Exp. Med, 192(7): 1027-1034 (2000); and Yamazaki et al., J. Immunol, [69(10): 5538-5545 (2002)). PD-Ll expression is upregulated on macrophages and dendritic cells (DCs) in response to lipopolysaccharide (LPS) and GM-CSF treatment, and on T-cells and B-cells upon signaling via T-cell and B—cell receptors. PD—Ll also is expressed in a variety of murine tumor cell lines (see, e.g., Iwai et al., Proc. Natl.

Acad. Sci. USA, 99(19): 12293—12297 (2002); and Blank et al., Cancer Res., 64(3): 1140- 1145 (2004)). In contrast, PD—L2 exhibits a more restricted expression n and is sed primarily by antigen presenting cells (e.g., dendritic cells and macrophages), and some tumor cell lines (see, e.g., Latchman et al., Nat. Immunol, 2(3): 261—238 (2001)). High PD-Ll expression in tumors, whether on the tumor cell, stroma, or other cells within the tumor microenvironment, correlates with poor al prognosis, presumably by inhibiting or T cells and upregulating regulatory T cells (Treg) in the tumor.

PD-l negatively regulates T-cell activation, and this inhibitory function is linked to an immunoreceptor tyrosine—based switch motif (ITSM) in the cytoplasmic domain (see, e.g., Greenwald et al., supra; and Parry et al., Mol. Cell. Biol, 25: 9543-9553 (2005)). PD-1 deficiency can lead to munity. For example, C57BL/6 PD-l knockout mice have been shown to develop a lupus—like me (see, e.g., Nishimura et al., ty, 11: 141-1151 (1999)). In humans, a single nucleotide polymorphism in the PD-l gene is ated with higher incidences of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and progression of multiple sis (see, e. g., Nielsen et al., Tissue Antigens, 62(6): 7 ; Bertsias et al., Arthritis Rheum, 60(1): 207-218 (2009); Ni et al., Hum. Genet, : 223—232 (2007); Tahoori et al., Clin. Exp. tol., 29(5): 763-767 (2011); and Kroner et al., Ann. Neurol, 58(1): 50-57 (2005)). Abnormal PD-l expression also has been implicated in T—cell dysfunctions in several pathologies, such as tumor immune evasion and chronic viral infections (see, e.g., Barber et al., Nature, 439: 682-687 (2006); and Sharpe et al., supra).

Recent studies demonstrate that T—cell suppression induced by PD-l also plays a role in the suppression of anti-tumor immunity. For example, PD-Ll is expressed on a variety of human and mouse tumors, and binding of PD-l to PD-Ll on tumors results in T— cell suppression and tumor immune evasion and protection (Dong et al., Nat. Med., 8: 793- 800 (2002)). Expression of PD-Ll by tumor cells has been directly associated with their resistance to lysis by anti-tumor T-cells in vitro (Dong et al., supra; and Blank et al., Cancer Res, 64: 1140-1145 (2004)). PD—l knockout mice are resistant to tumor challenge (Iwai et al., Int. Immunol, 1 7: 133-144 (2005)), and T-cells from PD-l ut mice are highly effective in tumor rejection when adoptively transferred to tumor-bearing mice (Blank et al., supra). Blocking PD-l inhibitory signals using a monoclonal antibody can potentiate host anti-tumor immunity in mice (Iwai et al., supra; and Hirano et al., Cancer Res, 65: 1089- 1096 (2005)), and high levels of PD-Ll expression in tumors are associated with poor prognosis for many human cancer types (Hamanishi et al., Proc. Natl. Acad. Sci. USA, 104: 3360-335 (2007), Brown et al., J. Immunol, 170: 1257-1266 (2003); and Flies et al., Yale Journal ofBiology and Medicine, 84(4): 409-421 (2011)).

In view of the ing, strategies for inhibiting PD—1 activity to treat various types of cancer and for immunopotentiation (e.g., to treat infectious diseases) have been ped (see, e.g., Ascierto et al., Clin. Cancer. Res, 19(5): 1009-1020 (2013)). In this t, monoclonal antibodies targeting PD-l have been developed for the ent of cancer (see, e.g., Weber, Semin. 0nc0l., 37(5): 430-4309 (2010); and Tang et al., Current Oncology Reports, 15(2): 98—104 (2013)). For example, nivolumab (also known as BMS- 936558) produced complete or partial responses in non-small-cell lung cancer, ma, and renal-cell cancer in a Phase I clinical trial (see, e.g., Topalian, New England J. Med., 366: 2443-2454 (2012)), and is currently in Phase III clinical trials. 5 is a zed monoclonal antibody directed t PD-l that has shown evidence of antitumor activity in Phase I clinical trials (see, e.g., Patnaik et al., 2012 American Society of Clinical Oncology (ASCO) Annual Meeting, Abstract # 2512). In addition, recent evidence suggests that therapies which target PD-l may enhance immune responses against pathogens, such as HIV (see, e.g., Porichis et al., Carr. HIV/AIDS Rep, 9(1): 81-90 (2012)). Despite these advances, however, the y of these potential therapies in humans may be limited.

Therefore, there is a need for a PD—l—binding agent (e.g., an antibody) that binds PD-l with high affinity and effectively neutralizes PD-I activity. The ion provides such PDbinding agents.

BRIEF SUMMARY OF THE INVENTION The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, wherein optionally (a) e 9 of SEQ ID NO: 1 is replaced with a different amino acid residue, (b) one or more of es 7, 8, and 9 of SEQ ID NO: 2 is replaced with a different amino acid residue, (0) one or more of residues 1, 2, and 5 of SEQ ID NO: 3 is replaced with a different amino acid residue, or (d) any combination of (a)- (c).

The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, wherein optionally (a) residue 9 of SEQ ID NO: 12 is replaced with a different amino acid residue, (b) residue 8 and/or e 9 of SEQ ID NO: 13 is replaced with a different amino acid residue, (c) e 5 of SEQ ID NO: 14 is replaced with a different amino acid residue, or (d) any combination of (a)-(c).

The ion provides an isolated immunoglobulin heavy chain polypeptide which comprises a mentarity determining region 1 (CDR) amino acid ce of SEQ ID NO: 19, a CDR2 amino acid sequence of SEQ ID NO: 20, and a CDR3 amino acid sequence of SEQ ID NO: 21.

The invention also provides an isolated immunoglobulin heavy chain ptide which comprises an amino acid ce that is at least 90% identical to any one of SEQ ID NOs: 4-11, SEQ ID NOs: 15-18, and SEQ ID NOs: 22-25.

The invention es an ed immunoglobulin light chain polypeptide which comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 26 and a CDR2 amino acid sequence of SEQ ID NO: 27.

The invention provides an ed immunoglobulin light chain polypeptide which ses a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31, wherein optionally residue 12 of SEQ ID NO: 30 is replaced with a different amino acid residue.

The invention provides an isolated immunoglobulin light chain polypeptide which comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37, n optionally (a) residue 5 of SEQ ID NO: 36 is replaced with a different amino acid residue, and/or (b) residue 4 of SEQ ID NO: 37 is replaced with a different amino acid e.

The ion provides an isolated immunoglobulin light chain polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

In addition, the invention provides isolated or purified nucleic acid sequences encoding the foregoing immunoglobulin polypeptides, vectors sing such nucleic acid sequences, isolated PD-I-binding agents sing the foregoing immunoglobulin polypeptides, nucleic acid sequences encoding such PD-I-binding agents, vectors comprising such nucleic acid sequences, isolated cells comprising such vectors, compositions comprising such PD-I-binding agents or such vectors with a pharmaceutically acceptable carrier, and s of treating cancer or infectious diseases in mammals by administering effective amounts of such compositions to mammals.

BRIEF DESCRIPTION THE DRAWINGS Figure 1 is a diagram which tically depicts different PD-I antigen constructs utilized to generate anti-PD-I monoclonal antibodies as described in Example 1.

The constructs are numbered as follows: 1. Full-length human PD-1, expressed on the surface on CHO cells 2. Human PD-1 extracellular domain with C-terminal tags G/S-avi-(His)6 3. Human PD-1 extracellular domain - Mouse lgG2a Fc 4. Human PD-L1 ellular domain – mouse IgG1 Fc . Human PD-L2 extracellular domain - mouse IgG1 Fc 6. Full-length cynomolgus monkey PD-1 expressed on the surface of CHO cells Figure 2 is a graph which illustrates experimental results demonstrating increased activity of an anti-TIM-3 antagonist dy in a human CD4+ T-cell MLR assay in the ce of low levels of anti—PD—l antibody APE2058.

Figure 3 is a graph which illustrates experimental results demonstrating sed activity of an anti-LAG-3 antagonist antibody in a human CD4+ T—cell MLR assay in the presence of low levels of anti-PD-l APE2058.

DETAILED DESCRIPTION OF THE INVENTION The invention es an isolated immunoglobulin heavy chain polypeptide and/or an isolated immunoglobulin light chain polypeptide, or a fragment (e.g., antigen- binding fragment) thereof. The term "immunoglobulin" or ody," as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and Viruses. The polypeptide is "isolated" in that it is removed from its natural environment. In a preferred embodiment, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the variable region" of an antibody, which is responsible for antigen binding (discussed further . A whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain ptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal le (VH) region and three C-terminal constant (CH1, CH2, and CH3) s, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda (9»), based upon the amino acid sequences of their constant domains. In a typical immunoglobulin, each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds. The light chain variable region is aligned with the variable region ofthe heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

The variable s of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL s have the same general structure, with each region comprising four framework (FW or FR) regions. The term "framework region," as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located n the hypervariable or complementary determining regions (CDRs). There are four framework regions in each variable , which are ated FRl, FR2, FR3, and FR4. The ork regions form the B sheets that provide the structural framework of the variable region (see, e. g., C.A. Janeway et al. (eds.), Immunobz’ology, 5th Ed., Garland Publishing, New York, NY (2001)).

The framework regions are ted by three complementarity determining regions (CDRs). As discussed above, the three CDRs, known as CDRl, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.

The CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions. While the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions. The constant regions also exhibit various effector ons, such as participation in antibody-dependent complement-mediated lysis or dy-dependent cellular toxicity Via interactions with effector molecules and cells.

The isolated immunoglobulin heavy chain polypeptide and the isolated immunoglobulin light chain ptide of the invention desirably bind to PD-l. As discussed above, programmed death 1 (PD-1) (also known as programmed cell death 1) is a 268 amino acid type I transmembrane protein (Ishida et al., supra). PD-l is a member ofthe CD28/CTLA-4 family of T-cell regulators and is expressed on activated T-cells, B-cells, and myeloid lineage cells (Greenwald et al., supra; and Sharpe et al., supra). PD—l es an extracellular IgV domain followed by short extracellular stalk, a transmembrane region and an ellular tail. The intracellular tail contains two orylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which play a role in the ability of PD-l to negatively te T-cell receptor signaling (see, e.g., Ishida et al., supra; and Blank et al., supra). The inventive isolated immunoglobulin heavy chain polypeptide and the inventive ed immunoglobulin light chain polypeptide can form an agent that binds to PD-l and another antigen, resulting in a "dual reactive" binding agent (e.g., a dual reactive antibody). For example, the agent can bind to PD—l and to another negative regulator of the immune system such as, for example, lymphocyte-activation gene 3 (LAG-3) and/or T-cell immunoglobulin domain and mucin domain 3 n (TIM—3).

Antibodies which bind to PD-l, and components thereof, are known in the art (see, e.g., US. Patent 8,168,757; Topalian et al., supra; and k et al., supra). Anti-PD-l antibodies also are commercially ble from sources such as, for example, Abcam (Cambridge, MA).

An amino acid "replacement" or "substitution" refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or e within a polypeptide sequence.

Amino acids are broadly grouped as tic" or "aliphatic." An aromatic amino acid includes an ic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as "aliphatic." Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), ine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).

Aliphatic amino acids may be sub-divided into four sub-groups. The "large aliphatic non-polar sub-group" ts of valine, leucine, and isoleucine. The atic slightly-polar sub-group" consists of methionine, serine, threonine, and cysteine. The "aliphatic polar/charged sub-group" consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine. The "small-residue sub-group" consists of glycine and alanine. The group of charged/polar amino acids may be sub-divided into three sub-groups: the "positively-charged sub-group" consisting of lysine and ne, the "negatively-charged sub-group" consisting of glutamic acid and aspartic acid, and the "polar sub-group" consisting of asparagine and ine.

Aromatic amino acids may be sub-divided into two sub-groups: the "nitrogen ring sub-group" consisting of histidine and tryptophan and the l sub-group" consisting of phenylalanine and tyrosine.

The amino acid replacement or substitution can be conservative, semi— conservative, or non—conservative. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to e the normalized frequencies of amino acid changes between corresponding ns of gous organisms (Schulz and Schirmer, Principles of Protein ure, Springer—Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).

Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups bed above, for example, lysine for arginine and vice versa such that a positive charge may be ined, glutamic acid for ic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained.

"Semi—conservative mutations" include amino acid tutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. "Non-conservative mutations" involve amino acid substitutions n different groups, for example, lysine for phan, or phenylalanine for serine, etc.

The invention provides an immunoglobulin heavy chain ptide that comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3. In one embodiment of the invention, the isolated immunoglobulin heavy chain ptide comprises, consists of, or consists essentially of a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, wherein optionally (a) residue 9 of SEQ ID NO: 1 is ed with a different amino acid residue, (b) one or more of residues 7, 8, and 9 of SEQ ID NO: 2 is replaced with a different amino acid residue, (c) one or more of residues 1, 2, and 5 of SEQ ID NO: 3 is replaced with a different amino acid residue, or (d) any combination of (a)-(c). When the inventive immunoglobulin heavy chain polypeptide consists essentially of a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid ce of SEQ ID NO: 3 and optional amino acid replacements, additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation). When the ive immunoglobulin heavy chain polypeptide consists of a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3 and optional amino acid ements, the polypeptide does not comprise any additional ents (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).

In one embodiment ofthe invention, the isolated immunoglobulin polypeptide comprises a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, except that (a) residue 9 of SEQ ID NO: 1 is replaced with a different amino acid residue, (b) one or more of residues 7, 8, and 9 of SEQ ID NO: 2 is replaced with a different amino acid residue, (c) one or more ofresidues 1, 2, and 5 of SEQ ID NO: 3 is replaced with a different amino acid residue, or (d) any combination of (a)-(c). For example, the isolated immunoglobulin heavy chain polypeptide can se a CDRI amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, except that residue 9 of SEQ ID NO: I is ed with a different amino acid residue and one or more of residues 7, 8, and 9 of SEQ ID NO: 2 is replaced with a different amino acid residue. atively, the isolated immunoglobulin heavy chain polypeptide can comprise a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, except that residue 9 of SEQ ID NO: 1 is replaced with a different amino acid residue, one or more of residues 7, 8, and 9 of SEQ ID NO: 2 is replaced with a different amino acid residue, and one or more of residues 1, 2, and 5 of SEQ ID NO: 3 is replaced with a different amino acid residue. In another embodiment, the isolated immunoglobulin heavy chain polypeptide can comprise a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, except that one or more of residues 1, 2, and 5 of SEQ ID NO: 3 is replaced with a different amino acid e. Each of residue 9 of SEQ ID NO: 1, residues 7, 8, and 9 of SEQ ID NO: 2, and residues 1, 2, and 5 of SEQ ID NO: 3 can be replaced with any le amino acid residue that can be the same or different in each on. For example, the amino acid residue of a first position can be replaced with a first different amino acid e, and the amino acid residue of a second position can be replaced with a second different amino acid e, wherein the first and second different amino acid residues are the same or different.

In one embodiment, the isolated immunoglobulin heavy chain polypeptide comprises a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, except that residue 9 of SEQ ID NO: 1 is replaced with a methionine (M) e. In another embodiment, the isolated immunoglobulin heavy chain polypeptide comprises a CDR1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, except that (a) residue 7 of SEQ ID NO: 2 is replaced with an asparagine (N) residue, (b) residue 8 of SEQ ID NO: 2 is replaced with a serine (S) residue, (c)residue 9 of SEQ ID NO: 2 is replaced with a threonine (T) residue, or (d) any combination of (a)-(c). In another embodiment, the isolated globulin heavy chain polypeptide comprises a CDRl amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid ce of SEQ ID NO: 3, except that (a) residue 1 of SEQ ID NO: 3 is replaced with a glutamic acid (E) residue, (b) e 2 of SEQ ID NO: 3 is replaced with a tyrosine (Y) residue, (0) residue 5 of SEQ ID NO: 3 is replaced with a serine (S) e, or (d) any combination of (a)-(c).

Exemplary immunoglobulin heavy chain polypeptides as described above can se any one of the following amino acid sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

The invention provides an isolated immunoglobulin heavy chain polypeptide ses, consists essentially of, or consists of a complementarity ining region 1 (CDR) amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, wherein optionally (a) residue 9 of SEQ ID NO: 12 is replaced with a different amino acid residue, (b) residue 8 and/or residue 9 of SEQ ID NO: 13 is replaced with a different amino acid residue, (c) residue 5 of SEQ ID NO: 14 is replaced with a different amino acid residue, or (d) any ation of (a)-(c). When the inventive immunoglobulin heavy chain polypeptide consists essentially of a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14 and optional amino acid replacements, additional components can be included in the polypeptide that do not materially affect the ptide (e. g., protein moieties such as biotin that facilitate purification or isolation). When the inventive globulin heavy chain polypeptide consists of a CDR] amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14 and optional amino acid replacements, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).

In one embodiment, the isolated immunoglobulin heavy chain polypeptide can se a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that (a) residue 9 of SEQ ID NO: 12 is replaced with a different amino acid residue, (b) residue 8 and/or residue 9 of SEQ ID NO: 13 is replaced with a different amino acid residue, (c) residue 5 of SEQ ID NO: 14 is ed with a ent amino acid residue, or (d) any combination of (a)-(c). For e, the isolated immunoglobulin heavy chain polypeptide can comprise a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that residue 9 of SEQ ID NO: 12 is replaced with a different amino acid residue, residue 8 of SEQ ID NO: 13, and residue 9 of SEQ ID NO: 13 is replaced with a different amino acid residue. Alternatively, the isolated immunoglobulin heavy chain polypeptide can comprise a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that e 9 of SEQ ID NO: 12 is replaced with a different amino acid residue and residue 5 of SEQ ID NO: 14 is replaced with a ent amino acid residue. In another embodiment, the isolated immunoglobulin heavy chain polypeptide can comprise a CDRI amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that residue 9 of SEQ ID NO: 12 is replaced with a different amino acid e, residue 8 of SEQ ID NO: 13 is replaced with a ent amino acid residue, residue 9 of SEQ ID NO: 13 is replaced with a different amino acid residue, and residue 5 of SEQ ID NO: 14 is replaced with a different amino acid residue. Each of residue 9 of SEQ ID NO: 12, es 8 and 9 of SEQ ID NO: 13, and residue 5 of SEQ ID NO: 14 can be replaced with any suitable amino acid residue that can be the same or different in each position. For example, the amino acid residue of a first position can be replaced with a first different amino acid residue, and the amino acid residue of a second position can be replaced with a second different amino acid residue, wherein the first and second different amino acid es are the same or different. In one embodiment, the isolated immunoglobulin heavy chain polypeptide comprises a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that e 9 of SEQ ID NO: 12 is replaced with a leucine (L) residue. In another embodiment, the isolated immunoglobulin heavy chain polypeptide comprises a CDRl amino acid ce of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that (a) residue 8 of SEQ ID NO: 13 is replaced with a tyrosine (Y) residue, and/or (b) residue 9 of SEQ ID NO: 13 is replaced with an alanine (A) e. In another embodiment, the isolated immunoglobulin heavy chain polypeptide comprises a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14, except that residue 5 of SEQ ID NO: 14 is replaced with a threonine (T) residue.

Exemplary immunoglobulin heavy chain polypeptides as described above can comprise any one of the following amino acid sequences: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

The invention provides an ed immunoglobulin heavy chain polypeptide comprises, ts essentially of, or ts of a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 19, a CDR2 amino acid sequence of SEQ ID NO: 20, and a CDR3 amino acid ce of SEQ ID NO: 21. When the inventive immunoglobulin heavy chain polypeptide consists essentially of a CDRl amino acid sequence of SEQ ID NO: 19, a CDR2 amino acid sequence of SEQ ID NO: 20, and a CDR3 amino acid sequence of SEQ ID NO: 21, additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation). When the inventive immunoglobulin heavy chain polypeptide consists of a CDRI amino acid sequence of SEQ ID NO: 19, a CDR2 amino acid sequence of SEQ ID NO: 20, and a CDR3 amino acid sequence of SEQ ID NO: 21, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide). Exemplary immunoglobulin heavy chain ptides as described above can comprise any one of the ing amino acid sequences: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

In addition, one or more amino acids can be inserted into the aforementioned globulin heavy chain polypeptides. Any number of any suitable amino acids can be ed into the amino acid sequence of the immunoglobulin heavy chain polypeptide. In this respect, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e. g., 18 or less, 15 or less, or 12 or less amino acids), can be inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide. ably, 1—10 amino acids (e.g., l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are inserted into the amino acid sequence of the globulin heavy chain polypeptide. In this respect, the amino acid(s) can be ed into any one of the aforementioned immunoglobulin heavy chain polypeptides in any suitable location. Preferably, the amino acid(s) are inserted into a CDR (e.g., CDRl or CDR3) ofthe immunoglobulin heavy chain ptide.

, CDR2, The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs: 4-11, SEQ ID NOs: 15-18, and SEQ ID NOs: 22—25. Nucleic acid or amino acid sequence "identity," as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). A number of atical algorithms for obtaining the optimal alignment and calculating ty between two or more sequences are known and incorporated into a number of available re programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, , and later versions thereof) and FASTA ms (e.g., FASTA3X, FASTM, and SSEARCH) (for ce alignment and sequence similarity es). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol, 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, [06(10): 775 (2009), Durbin et al., eds., ical Sequence Analysis: Probalistic Models ofProteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res, 25(17): 3389-3402 (1997), and Gusfield, thms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK ).

The invention provides an immunoglobulin light chain polypeptide that comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 26 and a CDR2 amino acid sequence of SEQ ID NO: 27. In one embodiment of the invention, the isolated immunoglobulin light chain polypeptide comprises, consists essentially of, or consists of a CDRl amino acid sequence of SEQ ID NO: 26 and a CDR2 amino acid sequence of SEQ ID NO: 27. When the inventive immunoglobulin light chain polypeptide consists essentially of a CDRl amino acid sequence of SEQ ID NO: 26 and a CDR2 amino acid sequence of SEQ ID NO: 27, additional ents can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that tate purification or isolation). When the inventive immunoglobulin light chain polypeptide consists of a CDRl amino acid sequence of SEQ ID NO: 26 and a CDR2 amino acid sequence of SEQ ID NO: 27, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin light chain polypeptide). Exemplary immunoglobulin light chain polypeptides as described above can comprise SEQ ID NO: 28 or SEQ ID NO: 29.

The invention provides an ed immunoglobulin light chain polypeptide comprises a complementarity ining region 1 (CDR) amino acid ce of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31. In one ment of the invention, the isolated immunoglobulin light chain polypeptide comprises, consists of, or ts essentially of a CDRl amino acid sequence of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31, wherein optionally residue 12 of SEQ ID NO: 30 is replaced with a different amino acid residue. When the inventive immunoglobulin light chain polypeptide ts essentially of a CDRl amino acid sequence of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 3land optional amino acid replacements, additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).

When the ive immunoglobulin light chain polypeptide consists of a CDRl amino acid sequence of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31 and optional amino acid replacements, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin light chain polypeptide).

In this respect, for example, the isolated immunoglobulin light chain polypeptide can comprise a CDRl amino acid sequence of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31, except that residue 12 of SEQ ID NO: 30 is replaced with a different amino acid residue. Residue 12 of SEQ ID NO: 30 can be replaced with any suitable amino acid residue. In one ment, the isolated immunoglobulin light chain polypeptide can comprise a CDRl amino acid ce of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31, except that residue 12 of SEQ ID NO: 30 is replaced with a threonine (T) e. Exemplary immunoglobulin light chain polypeptides as described above can comprise any one of the following amino acid sequences: SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.

The invention provides an isolated immunoglobulin light chain polypeptide comprises a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37. In one embodiment, the immunoglobulin light chain polypeptide comprises, consists essentially of, or ts of a CDRl amino acid ce of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37, wherein optionally (a) residue 5 of SEQ ID NO: 36 is replaced with a different amino acid residue, and/or (b) residue 4 of SEQ ID NO: 37 is replaced with a different amino acid residue. When the inventive immunoglobulin light chain ptide consists essentially of a CDRI amino acid sequence of SEQ ID NO: 35, a CDR2 amino acid ce of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37 and optional amino acid replacements, additional components can be included in the polypeptide that do not materially affect the ptide (e.g., protein moieties such as biotin that facilitate purification or isolation). When the inventive immunoglobulin light chain polypeptide ts of a CDRl amino acid sequence of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37 and optional amino acid replacements, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin light chain ptide). In this respect, for example, the isolated immunoglobulin light chain polypeptide can comprise a CDRI amino acid sequence of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid ce of SEQ ID NO: 37.

Alternatively, the isolated immunoglobulin light chain polypeptide can comprise a CDRl amino acid ce of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37, except that (a) residue 5 of SEQ ID NO: 36 is replaced with a different amino acid residue, and/or (b) residue 4 of SEQ ID NO: 37 is replaced with a different amino acid residue. Each of residue 5 of SEQ ID NO: 36 and residue 4 of SEQ ID NO: 37 can be replaced with any suitable amino acid residue that can be the same or different in each position. For example, the amino acid residue of a first position can be ed with a first different amino acid residue, and the amino acid residue of a second position can be replaced with a second ent amino acid residue, wherein the first and second different amino acid residues are the same or different.

In one embodiment, the isolated immunoglobulin light chain polypeptide comprises a CDRI amino acid sequence of SEQ ID NO: 35, a CDR2 amino acid sequence of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37, except that (a) residue of SEQ ID NO: 36 is ed with a leucine (L) residue, and/or (b) residue 4 of SEQ ID NO: 37 is replaced with an asparagine (N) residue. Exemplary immunoglobulin light chain polypeptides as described above can comprise any one of the following amino acid ces: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

In on, one or more amino acids can be inserted into the aforementioned globulin light chain polypeptides. Any number of any suitable amino acids can be ed into the amino acid sequence of the immunoglobulin light chain polypeptide. In this respect, at least one amino acid (e. g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e. g., 18 or less, 15 or less, or 12 or less amino acids), can be inserted into the amino acid sequence of the immunoglobulin light chain polypeptide.

Preferably, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are inserted in to the amino acid sequence of the immunoglobulin light chain polypeptide. In this respect, the amino acid(s) can be inserted into any one of the aforementioned immunoglobulin light chain polypeptides in any suitable location. ably, the amino acid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of the immunoglobulin light chain polypeptide.

The invention provides an isolated immunoglobulin light chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. Nucleic acid or amino acid sequence "identity," as described herein, can be determined using the methods described herein.

The invention provides an isolated mmed death 1 (PD-l)—binding agent comprising, ting essentially of, or consisting of the inventive isolated amino acid sequences described herein. By "programmed death 1 (PD-1)-binding agent" is meant a molecule, preferably a proteinaceous molecule, that binds specifically to the programmed death 1 protein . Preferably, the PD-l-binding agent is an antibody or a fragment (e.g., immunogenic fragment) thereof. The isolated PD-l-binding agent of the invention comprises, consists essentially of, or consists of the inventive isolated immunoglobulin heavy chain polypeptide and/or the inventive isolated immunoglobulin light chain polypeptide. In one embodiment, the isolated PD—l—binding agent comprises, consists essentially of, or consists of the inventive immunoglobulin heavy chain polypeptide or the ive immunoglobulin light chain polypeptide. In another embodiment, the isolated PD-l-binding agent comprises, consists essentially of, or consists of the inventive immunoglobulin heavy chain polypeptide and the inventive immunoglobulin light chain polypeptide.

The invention is not limited to an isolated PD-l-binding agent that comprises, ts ially of, or ts of an globulin heavy chain polypeptide and/or light chain polypeptide having replacements, insertions, and/or deletions of the specific amino acid residues disclosed . , any amino acid residue of the inventive immunoglobulin heavy chain polypeptide and/or the inventive immunoglobulin light chain ptide can be replaced, in any combination, with a different amino acid residue, or can be deleted or inserted, so long as the biological activity of the PD-l-binding agent is enhanced or ed as a result of the amino acid replacements, insertions, and/or deletions. The gical activity" of an PD-l-binding agent refers to, for example, binding affinity for PD-l or a particular PD-l epitope, neutralization or inhibition of PD-l protein binding to its s PD-Ll and PD-Ll neutralization or inhibition of PD-l protein activity in vivo (e.g., IC50), cokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of the PD-l protein, or with other proteins or tissues). Other biological properties or characteristics of an antigen—binding agent recognized in the art include, for example, avidity, ivity, solubility, folding, immunotoxicity, expression, and formulation. The aforementioned properties or characteristics can be observed, measured, and/or assessed using standard techniques including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORETM), or KINEXATM, in vitro or in viva neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and/or secretion assays, and signal transduction and immunohistochemistry assays.

The terms "inhibit" or "neutralize," as used herein with t to the activity of a PD-l-binding agent, refer to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of, for example, the biological activity of a PD-l protein, or a e or condition associated with an PD-l protein. The isolated PD—l—binding agent of the invention preferably inhibits or neutralizes the activity of a PD-l protein by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or a range defined by any two of the foregoing values.

The isolated PD-l-binding agent of the invention can be a whole antibody, as described herein, or an dy nt. The terms ent of an antibody,77 LLantibody fragment," and "functional fragment of an antibody" are used interchangeably herein to mean one or more fragments of an antibody that retain the y to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech, 23(9): 1126-1129 (2005)). The ed PD-l binding agent can contain any PD-l-binding antibody fragment. The antibody nt desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or ns thereof), or ations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent nt consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild ng conditions, (v) a disulfide-stabilized Fv fragment (dst), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.

In embodiments where the isolated PD—l—binding agent comprises a fragment of the immunoglobulin heavy chain or light chain polypeptide, the fragment can be of any size so long as the fragment binds to, and preferably ts the activity of, a PD-l protein. In this respect, a fragment of the immunoglobulin heavy chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, ll, 12, 13, 14, 15,16,17,18,or a range defined by any two of the foregoing values) amino acids. Similarly, a fragment of the immunoglobulin light chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 1,12,13,14,l5,16,l7,18, or arange defined by any two ofthe foregoing values) amino acids.

When the PD-l-binding agent is an antibody or antibody fragment, the antibody or antibody fragment desirably comprises a heavy chain constant region (Fe) of any suitable class. Preferably, the antibody or antibody fragment comprises a heavy chain constant region that is based upon wild-type IgGl or IgG4 dies, or variants thereof.

, IgG2, The PD—l—binding agent also can be a single chain antibody fragment. Examples of single chain antibody fragments include, but are not limited to, (i) a single chain FV (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879—5883 (1988); and Osbourn et al., Nat. hnol, 16: 778 (1998)) and (ii) a diabody, which is a dimer of polypeptide chains, wherein each ptide chain comprises a VH connected to a VL by a e linker that is too short to allow pairing between the VB and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH —VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, e.g., US. Patent Application Publication 2009/0093024 A1.

The isolated PD—l—binding agent also can be an intrabody or fragment thereof An intrabody is an antibody which is expressed and which functions intracellularly. Intrabodies lly lack disulfide bonds and are capable of modulating the sion or activity of target genes through their specific binding activity. Intrabodies include single domain fragments such as isolated VH and VL domains and scFvs. An intrabody can include sub- cellular trafficking signals attached to the N or C terminus of the intrabody to allow expression at high concentrations in the sub-cellular compartments where a target protein is located. Upon interaction with a target gene, an intrabody tes target protein function and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein ation and sequestering the target protein in a non-physiological sub-cellular tment. Other mechanisms of ody-mediated gene inactivation can depend on the epitope to which the intrabody is ed, such as binding to the catalytic site on a target protein or to es that are involved in protein—protein, protein—DNA, or protein-RNA interactions.

The isolated PD-l-binding agent also can be an antibody conjugate. In this respect, the isolated PD-l-binding agent can be a ate of (1) an antibody, an alternative scaffold, or fragments thereof, and (2) a n or non—protein moiety comprising the PD—l— binding agent. For e, the PD-l-binding agent can be all or part of an antibody conjugated to a peptide, a fluorescent molecule, or a chemotherapeutic agent.

The isolated PD-l-binding agent can be, or can be obtained from, a human antibody, a non-human antibody, or a chimeric antibody. By "chimeric" is meant an antibody or fragment thereof comprising both human and non-human regions. Preferably, the isolated PD-l-binding agent is a humanized antibody. A "humanized" antibody is a monoclonal antibody sing a human antibody scaffold and at least one CDR ed or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non— human antibody. In a preferred embodiment of the invention, CDRH3 of the ive PD binding agent is obtained or derived from a mouse monoclonal antibody, while the remaining variable s and nt region of the inventive PDbinding agent are obtained or d from a human monoclonal antibody.

A human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody inantly) and in vivo sources (e.g., rodents). Methods for ting antibodies are known in the art and are described in, for example, Kohler and Milstein, Eur. J. Immunol, 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immanobiology, 5th Ed., Garland Publishing, New York, NY (2001)). In certain embodiments, a human antibody or a chimeric dy can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples oftransgenic mice wherein endogenous antibody genes are effectively ed with human antibody genes include, but are not limited to, the Medarex HUMAB—MOUSETM, the Kirin TC MOUSETM, and the Kyowa Kirin KM- MOUSETM (see, e.g., Lonberg, Nat. Biotechnol, 23(9): 5 , and Lonberg, Handb. Exp. Pharmacol, 181: 69-97 (2008)). A humanized dy can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey ), including, e. g., grafting of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. m., 144(1): 115-120 (2008)). In one embodiment, a humanized antibody can be produced using the methods described in, e.g., US. Patent Application Publication 287485 A1.

In a preferred embodiment, the PD—1-binding agent binds an epitope of a PD-l protein which blocks the binding of PD-1 to PD-L1. The invention also provides an isolated or purified epitope of a PD-1 protein which blocks the binding of PD-1 to PD-L1 in an indirect or allosteric manner.

The invention also provides one or more isolated or purified nucleic acid sequences that encode the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and the inventive PD—l—binding agent.

The term "nucleic acid sequence" is intended to encompass a polymer ofDNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms "nucleic acid" and "polynucleotide" as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include - and single-stranded DNA, and double- and single- stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides. Nucleic acids are lly linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., orothioates, boranophosphates, and the like). Nucleic acid sequences ng the inventive immunoglobulin heavy chain polypeptides include, for example, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55. Nucleic acid sequences ng the inventive immunoglobulin light chain ptides e, for example, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64.

The invention further provides a vector sing one or more nucleic acid sequences encoding the ive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and/or the inventive PD-l—binding agent. The vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage. Suitable vectors and methods of vector preparation are well known in the art (see, e.g., ok et al., Molecular Cloning, a tory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY. (2001), and l et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY. (1994)).

In addition to the nucleic acid sequence encoding the inventive immunoglobulin heavy polypeptide, the inventive immunoglobulin light chain polypeptide, and/or the inventive PD-l-binding agent, the vector preferably comprises expression control sequences, such as promoters, enhancers, enylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the sion of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

A large number of promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources of promoters include for example, Virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily ble, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other cial or dual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3’ or 5 ’ direction). Non-limiting examples oters include, for example, the T7 bacterial sion , pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include, for example, the Tet system (US. Patents 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci, 93: 3346-3351 (1996)), the T-REXTM system (Invitrogen, Carlsbad, CA), TCHTM system agene, San Diego, CA), and the Cre-ERT tamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); US. Patent 715; and Kramer & Fussenegger, Methods Mol. Biol, 308: 123-144 (2005)).

The term "enhancer" as used herein, refers to a DNA ce that increases ription of, for example, a nucleic acid sequence to which it is operably linked.

Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the g of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer ces. Enhancers can be located upstream, within, or downstream of coding sequences.

The vector also can comprise a "selectable marker gene." The term "selectable marker gene," as used herein, refers to a nucleic acid ce that allow cells expressing the nucleic acid sequence to be specifically selected for or against, in the presence of a corresponding selective agent. Suitable selectable marker genes are known in the art and described in, e.g., International Patent Application Publications and WO 1994/028143; Wigler eta1.,Pr0c. Natl. Acad. Sci. USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci.

USA, 78: 2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol, 150: 1-14 ; Santerre eta1., Gene, 30: 147-156 (1984); Kent et al., Science, 237: 3 (1987); Wigler eta1., Cell, 11: 223-232 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026-2034 (1962); Lowy eta1., Cell, 22: 817-823 (1980); and US. Patents 5,122,464 and 5,770,359.

In some embodiments, the vector is an "episomal expression vector" or "episome," which is able to replicate in a host cell, and persists as an extrachromosomal segment ofDNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11 : 1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNAl) and the Epstein Barr Virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu l and oriP.

Other suitable vectors include integrating expression vectors, which may randomly integrate into the host cell’s DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell’s chromosome.

Such integrating expression vectors may e the endogenous expression control ces of the host cell’s chromosomes to effect sion of the desired protein. Examples of vectors that ate in a site specific manner e, for example, components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., MS/FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, CA). Examples ofvectors that randomly integrate into host cell somes include, for example, pcDNA3.1 (when introduced in the absence of gen) from Invitrogen bad, CA), UCOE from ore (Billerica, MA), and pCI or pFNlOA (ACT) M from Promega (Madison, WI).

Viral vectors also can be used. entative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentiviral—based pLPl from Invitrogen (Carlsbad, CA), and the retroviral vectors pFB—ERV plus pCFB-EGSH from Stratagene (La Jolla, CA).

Nucleic acid sequences encoding the inventive amino acid sequences can be ed to a cell on the same vector (i.e., in cis). A unidirectional promoter can be used to control expression of each nucleic acid sequence. In another embodiment, a ation of bidirectional and unidirectional promoters can be used to control expression of multiple nucleic acid sequences. Nucleic acid ces encoding the inventive amino acid sequences alternatively can be provided to the population of cells on separate s (i.e., in trans).

Each of the nucleic acid sequences in each of the separate vectors can comprise the same or different expression control ces. The separate vectors can be provided to cells simultaneously or sequentially.

The (s) sing the nucleic ) encoding the inventive amino acid sequences can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby, including any suitable prokaryotic or otic cell. As such, the invention provides an isolated cell comprising the inventive . Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently.

Examples of suitable prokaryotic cells include, but are not d to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and a. Particularly usefial prokaryotic cells include the various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 33694), DHSa, DH10, MC1061 (ATCC No. 53338), and CC102).

Preferably, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and e, for example, yeast cells, insect cells, and mammalian cells. es of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.

Suitable insect cells are described in, for example, Kitts et al., Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol, 4: 564-572 ; and Lucklow et al., J.

Virol, 67: 4566-4579 (1993). Preferred insect cells include Sf—9 and H15 (Invitrogen, Carlsbad, CA).

Preferably, ian cells are utilized in the invention. A number of suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, as, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL6l), CHO DHFR— cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human nic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92).

Other suitable mammalian cell lines are the monkey COS-l (ATCC No. CRL1650) and COS- 7 cell lines (ATCC No. CRL1651), as well as the CV—l cell line (ATCC No. CCL70). r exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of y tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines e, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK r cell lines, all of which are available from the ATCC. Methods for selecting suitable mammalian host cells and methods for transformation, culture, cation, screening, and purification of cells are known in the art.

Most ably, the ian cell is a human cell. For example, the mammalian cell can be a human lymphoid or lymphoid derived cell line, such as a cell line of pre-B lymphocyte origin. es ofhuman lymphoid cells lines include, without limitation, RAMOS (CRL-1596), Daudi (CCL-213), EB—3 (CCL—85), DT40 (CRL-21 l 1), 18- 81 (Jack et al., Proc. Natl. Acad. Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86), and derivatives thereof.

A nucleic acid sequence encoding the inventive amino acid sequence may be introduced into a cell by "transfection,,3 "transformation," or "transduction." "Transfection," "transformation," or "transduction," as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE—dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol, 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells, after growth of ious particles in suitable packaging cells, many ofwhich are commercially available.

The invention provides a composition comprising an effective amount of the inventive imrnunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive PD-l-binding agent, the ive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive c acid sequence. Preferably, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) ition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen- g agent, or . Any suitable carrier can be used within the context of the invention, and such rs are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The ition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable e carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and ce ofPharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

The invention further provides a method of treating any disease or disorder in which the improper sion (e.g., overexpression) or increased activity of a PD-l protein causes or contributes to the pathological effects of the disease, or a se in PD-l protein levels or activity has a therapeutic t in mammals, preferably humans. The invention also provides a method of treating a cancer or an infectious disease in a mammal. The method comprises administering the aforementioned composition to a mammal having a cancer or an infectious disease, whereupon the cancer or infectious disease is treated in the mammal. As discussed herein, PD-l is abnormally expressed in a variety of cancers (see, e.g., Brown et al., J. Immunol, 170: 1257-1266 (2003); and Flies et. al., Yale Journal of Biology and Medicine, 84: 409-421 ), and PD-Ll expression in some renal cell carcinoma patients correlates with tumor aggressiveness. The inventive method can be used to treat any type of cancer known in the art, such as, for example, melanoma, renal cell carcinoma, lung cancer, r , breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, ry gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma (see, e.g., Bhatia et al., Carr. Oncol. Rep. 488-497 (2011)). The inventive method can be used to treat any , 13(6): type of infectious disease (i.e., a disease or disorder caused by a bacterium, a virus, a fungus, or a parasite). Examples of infectious diseases that can be treated by the inventive method include, but are not limited to, diseases caused by a human immunodeficiency virus (HIV), a respiratory syncytial virus (RSV), an influenza virus, a dengue virus, a hepatitis B virus (HBV, or a hepatitis C virus . Administration of a composition comprising the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive PD-l-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence induces an immune response against a cancer or infectious disease in a mammal.

An "immune response" can entail, for e, antibody tion and/or the tion of immune or cells (e.g., s).

As used herein, the terms "treatmen ," "treating," and the like refer to obtaining a desired pharmacologic and/or physiologic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a e and/or adverse symptom attributable to the disease. To this end, the inventive method ses administering a "therapeutically effective amount" of the PD-l-binding agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the PD-l-binding agent to elicit a desired response in the individual. For example, a therapeutically effective amount of a PD-l-binding agent of the invention is an amount which decreases PD—l protein bioactivity in a human and/or enhances the immune response t a cancer or infectious Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or lly prevents a disease or symptom thereof. In this respect, the inventive method comprises administering a "prophylactically effective amount" of the PD-l-binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a d prophylactic result (e.g., tion of disease onset).

A typical dose can be, for example, in the range of l pg/kg to 20 mg/kg of animal or human body weight; however, doses below or above this exemplary range are within the scope of the invention. The daily parenteral dose can be about 1 ug/kg to about 20 mg/kg of total body weight (e.g., about 0.001 ug /kg, about 0.1 ug /kg about 1 ug /kg, about ug /kg, about 10 ug/kg, about 100 ug /kg, about 500 ug/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two of the foregoing values), preferably from about 0.1 ug/kg to about 10 mg/kg of total body weight (e. g., about 0.5 ug/kg, about 1 ug/kg, about 50 ug/kg, about 150 ug/kg, about 300 ug/kg, about 750 ug/kg, about 1.5 mg/kg, about mg/kg, or a range defined by any two of the foregoing values), more preferably from about 1 ug/kg to 5 mg/kg of total body weight (e. g., about 3 ug/kg, about 15 ug/kg, about 75 ug/kg, about 300 ug/kg, about 900 ug/kg, about 2 mg/kg, about 4 mg/kg, or a range defined by any two ofthe foregoing values), and even more preferably from about 0.5 to 15 mg/kg body weight per day (e.g., about 1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 11 mg/kg, about 13 mg/kg, or a range defined by any two of the foregoing values). Therapeutic or prophylactic efficacy can be red by ic ment of treated patients. For repeated administrations over several days or longer, ing on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infiasion administration of the composition.

The composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive PDbinding agent, the inventive nucleic acid sequence ng any of the foregoing, or the ive vector comprising the inventive nucleic acid sequence can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, ermal, intramuscular, asal, buccal, sublingual, or suppository administration. The ition preferably is suitable for parenteral administration. The term teral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

Once administered to a mammal (e.g., a human), the biological activity of the inventive PD-l-binding agent can be measured by any suitable method known in the art. For e, the biological activity can be assessed by determining the stability of a particular PD-l-binding agent. In one embodiment of the invention, the PD—1-binding agent (e.g., an antibody) has an in viva half life between about 30 minutes and 45 days (e.g., about 30 s, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values). In another embodiment, the inding agent has an in viva half life between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values). In another embodiment, the PDbinding agent has an in viva half life between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).

The ical activity of a particular PD-l-binding agent also can be assessed by determining its binding y to a PD—1 protein or an epitope thereof. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (KD). Affinity of a binding agent to a ligand, such as affinity of an dy for an epitope, can be, for example, from about 1 picomolar (pM) to about 100 micromolar (uM) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), from about 1 nM to about 1 micromolar (nM), or from about 1 uM to about 100 uM). In one embodiment, the PD-l-binding agent can bind to an PD-l protein with a KD less than or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two ofthe foregoing values). In another embodiment, the PD-l-binding agent can bind to PD-l with a KD less than or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values). Immunoglobulin affinity for an antigen or e of interest can be measured using any art—recognized assay. Such s include, for e, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), solution phase competition (KINEXATM), antigen panning, and/or ELISA (see, e.g., Janeway et al. (eds), Immunobz'ology, 5th ed., Garland Publishing, New York, NY, 2001).

The PD—l—binding agent of the invention may be administered alone or in combination with other drugs (e.g., as an adjuvant). For e, the inding agent can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein. In this respect, the PD-l-binding agent can be used in combination with at least one other anticancer agent including, for example, any chemotherapeutic agent known in the art, ionization radiation, small molecule anticancer agents, cancer vaccines, biological therapies (e. g., other monoclonal dies, cancer—killing Viruses, gene therapy, and adoptive T-cell transfer), and/or y. When the inventive method treats an infectious disease, the PD-l-binding agent can be administered in combination with at least one anti- bacterial agent or at least one anti-viral agent. In this respect, the acterial agent can be any suitable antibiotic known in the art. The anti-viral agent can be any vaccine of any suitable type that specifically targets a particular virus (e.g., ttenuated vaccines, t vaccines, recombinant vector es, and small molecule anti-viral therapies (e. g., viral replication inhibitors and nucleoside analogs).

In another ment, the inventive PD-l binding agent can be stered in combination with other agents that inhibit immune checkpoint pathways. For example, the inventive PD-1 binding agent can be administered in ation with agents that inhibit or antagonize the CTLA-4, TIM-3 or LAG-3 pathways. Combination treatments that simultaneously target two or more of these immune checkpoint ys have demonstrated improved and potentially synergistic antitumor activity (see, e.g., Sakuishi et al., J. Exp.

Med., 207: 2187—2194 (2010); Ngiow et al., Cancer Res., 71: 3540-3551 (2011); and Woo et al., Cancer Res., 72: 917-927 ). In one embodiment, the inventive PD-l binding agent is administered in ation with an antibody that binds to TIM—3 and/or an antibody that binds to LAG-3. In this respect, the inventive method of treating a cancer or an infectious disease in a mammal can further comprise administering to the mammal a composition comprising (i) an antibody that binds to a TIM-3 protein and (ii) a pharmaceutically acceptable carrier or a composition comprising (i) an antibody that binds to a LAG-3 protein and (ii) a pharmaceutically acceptable carrier.

In addition to therapeutic uses, the PD-l-binding agent described herein can be used in diagnostic or ch applications. In this respect, the PD-l-binding agent can be used in a method to diagnose a cancer or infectious disease. In a similar manner, the PD-l- binding agent can be used in an assay to monitor PD—l protein levels in a subject being tested for a disease or disorder that is associated with abnormal PD-l expression. ch applications include, for example, methods that utilize the PD-l-binding agent and a label to detect a PD-l protein in a sample, e. g., in a human body fluid or in a cell or tissue extract.

The inding agent can be used with or without modification, such as covalent or non- covalent labeling with a detectable moiety. For example, the detectable moiety can be a radioisotope (e.g., 3H, 14C, 32P, 358, or 125I), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, alactosidase, or horseradish peroxidase), or prosthetic groups. Any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be employed in the context of the invention (see, e.g., Hunter et al., Nature, 194: 495-496 (1962); David et al., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Meth, 40: 219-230 (1981); and Nygren, J. hem. and Cytochem., 30: 407-412 ).

PD-1 protein levels can be measured using the inventive PD-l-binding agent by any suitable method known in the art. Such methods include, for example, radioimmunoassay (RIA), and FACS. Normal or standard expression values of PD-l protein can be established using any suitable technique, e.g., by combining a sample comprising, or suspected of comprising, a PD-l polypeptide with a PD-l-specific antibody under conditions suitable to form an antigen-antibody complex. The antibody is directly or indirectly labeled with a detectable substance to tate detection of the bound or unbound antibody. le detectable substances include various enzymes, prosthetic groups, fluorescent als, luminescent materials, and radioactive materials (see, e.g., Zola, Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc. (1987)). The amount of PD-l polypeptide expressed in a sample is then compared with a standard value.

The PDbinding agent can be provided in a kit, i.e., a ed combination of reagents in predetermined amounts with instructions for performing a diagnostic assay. If the PD-l-binding agent is labeled with an enzyme, the kit desirably includes ates and ors required by the enzyme (e.g., a substrate precursor which provides a detectable chromophore or fluorophore). In addition, other additives may be included in the kit, such as izers, buffers (e.g., a blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents can be varied to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. The reagents may be provided as dry s (typically lyophilized), including excipients which on dissolution will provide a reagent on having the appropriate concentration.

The ing es further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1 This example demonstrates a method of generating monoclonal antibodies ed against human PD-l.

Several forms of genes encoding human PD-l and its ligands PD-Ll and PD-L2 were generated as antigens for use in mouse immunization, hybridoma ing, and affinity maturation of CDR-grafted antibodies, and are schematically depicted in Figure 1. Full- length human and cynomolgus monkey PD-l genes were expressed with their native leader sequence and no added tags using a ubiquitous chromatin opening element (UCOE) single expression vector with ycin selection pore, Billerica, MA). CHO—K1 cells were stably transfected with Lipofectamine LTX (Life Technologies, Carlsbad, CA) ing to the manufacturer’s instructions. Following selection with hygromycin, cells expressing PD—l on the cell surface were identified by flow cytometry using a PE-conjugated mouse antibody to human PD-l (BD Bioscience, Franklin Lakes, NJ) and subcloned. Subclones were then selected for high-level and uniform PD—l expression.

Nucleic acid sequences encoding soluble monomeric forms of the extracellular domain (ECD) of human and lgus monkey PD-l were constructed with His tags appended to the C-terminus of the ECD or as soluble dimeric fusion proteins with mouse IgG2a Fc as indicated in Figure 1. Nucleic acid sequences encoding soluble c forms of the ECDs ofhuman PD-Ll and PD-L2 were constructed as fiasion proteins with mouse IgGl Fc as indicated in Figure 1. Soluble proteins were expressed transiently in HEK 293 cells or in stable CHO cell lines using rd techniques. His-tagged proteins were purified from cell culture supernatant via Ni-affinity column chromatography followed by size exclusion chromatography. IgG-Fc fusion ns were purified using protein A/G affinity chromatography. d proteins were analyzed by SDS-PAGE and size-exclusion chromatography to ensure homogeneity. Additionally, identity and size were ed by mass spectrometry.

For FACS sorting experiments, purified proteins were labeled with biotin using an NHS ester crosslinker (Thermo-Fisher Scientific, Inc., Waltham, MA) or the fluorescent dye DyLight 650 (Thermo-Fisher Scientific, Inc., Waltham, MA) using rd techniques.

Mice were immunized with either CHO cells expressing full—length PD-l on the cell surface or the PD-l ECD His protein. Specifically, female BALB/c mice (7 weeks old) were purchased from Harlan tories, Inc. (Indianapolis, IN) and divided into two groups. After six days of acclimatization, one group of animals was immunized with four weekly doses of purified human PD—l s at 50 ug/mouse, as a 1:1 emulsion with TITERMAX GOLDTM (Sigma h, St. Louis, MO). Immunization was carried out subcutaneously around the armpits and inguinal regions. The second group of animals was injected with four weekly doses of CHO-K1 cells stably expressing full length human PD-l (5 X 106 cells/mouse) subcutaneously around the inguinal regions. After ten days, animals were bled for measurement of the serum titer to PD-l and one animal from each group was d with soluble human PD-l after a 3-week rest. After three days, spleens, axillary/brachial lymph nodes, and inguinal lymph nodes were collected from each animal.

Single cell suspensions of cells from all tissues collected from both animals were pooled and used for generation of hybridomas by cell fusion using standard techniques. Two different myeloma cell lines were used for fusion, F0 (as described in de St. Groth and Scheidegger, J.

Immunol. Methods, 35: 1—21 (1980)) and P3X63Ag8.653 (as bed in Kearney et al., J.

Immunol., 123: 1548-1550 (1979)).

Hybridoma supernatants from ten 96-well plates were screened for binding to a CHO-Kl cell clone stably transfected with a nucleic acid sequence encoding full length human PD-l and compared to binding to untransfected CHO-K1 cells. Specifically, hybridoma supernatants were diluted 1:1 with PBS/2% FBS and ted with an equal volume of PD-l CHO-K1 cells (2.5x105 cells in PBS, 2% FBS) for 30 minutes at 4 °C. Cells were centrifuged, washed once with PBS/1% FBS, and incubated with AFC-conjugated goat anti-mouse IgG (H+L) (Southern Biotechnology, Birmingham, AL) for 30 minutes at 4 CC.

Cells were washed twice in PBS/2% FBS, ended in PBS, 2% FBS, 1% rrnaldehyde, and cence analyzed on a BD FACSARRAYTM lyzer (BD Biosciences, in Lakes, NJ). Mouse IgG levels were quantified by ELISA.

Based on strong g to PD-l CHO cells, 46 parental wells were expanded, and the supernatants were tested for the ability to block binding of DyL650-labeled PD—Ll- mIgGl Fc fusion protein to PD-l CHO cells. Specifically, purified mouse monoclonal antibodies were incubated in a dose response with the E030 concentration of PD-Ll-DyL650 (10 nM), and tion was quantified by flow cytometry. Cells from wells showing the best PD-Ll blocking activity and highest levels of mouse IgG were subcloned for r analysis, including purification and heavy and light chain (VH and VL) sequencing. Eleven of the strongest blockers of PD-l/PD—Ll interaction were selected for subcloning. Following re- confirmation of PD-l binding and PD—Ll blocking, selected subclones were scaled up, and supernatant was submitted for antibody purification. Purified antibodies were verified for binding to both human and cynomolgus monkey PD—l and for PD-Ll blocking activity. KD values were determined by surface plasmon resonance on a ETM T200 instrument (GE Healthcare, Waukesha, WI), and kinetic constants were determined using the E TM T200 evaluation software (GE Healthcare, Waukesha, WI). In this respect, antibodies TM CMS chip to which GE anti-mouse IgG were captured on a BIACORE was coupled. PD- l-His monomer was flowed over the captured dy using two— or three-fold serial ons beginning with 500 nM at the highest concentration. The resulting sensorgrams were fit globally using a 1:1 g model to calculate on- and off-rates and the uent affinities (KD).

The results of this example demonstrate a method of producing monoclonal antibodies that bind to human and cynomolgus monkey PD-l and block PD-l ligand binding.

EXAMPLE 2 This example describes the design and generation of CDR-grafted and chimeric anti—PD-l monoclonal dies.

Subclones of the hybridomas which ed PD-l-binding antibodies with PD- Ll blocking activity as described in Example 1 were isotyped, subjected to RT-PCR for cloning the antibody heavy chain variable region (VH) and light chain variable region (VL), and sequenced. Specifically, RNA was isolated from cell pellets of hybridoma clones (5 x 105 pellet) using the RNEASYTM kit (Qiagen, Venlo, Netherlands), and cDNA was prepared using oligo-dT-primed SUPERSCRIPTTM III First-Strand Synthesis System (Life Technologies, Carlsbad, CA). PCR amplification of the VL utilized a pool of 9 or 11 degenerate mouse VL forward primers (see Kontermann and Dubel, eds., Antibody Engineering, Springer-Verlag, Berlin (2001)) and a mouse K constant region reverse primer.

PCR amplification of the VH utilized a pool of 12 degenerate mouse VH forward primers (Kontermann and Dubel, supra) and a mouse yl or y2a constant region reverse primer (based on isotyping of d antibody from each clone) with the protocol recommended in the SUPERSCRIPTTM III Stand Synthesis System (Life Technologies, Carlsbad, CA). PCR products were purified and cloned into .3-TOPO (Life Technologies, ad, CA).

Individual colonies from each cell pellet (24 heavy chains and 48 light chains) were selected and sequenced using standard Sanger sequencing methodology (Genewiz, Inc., South Plainfield, NJ). Variable region sequences were examined and aligned with the closest human heavy chain or light chain V-region germline sequence. Three antibodies were selected for CDR—grafting: (1) 9A2, comprising a VH of SEQ ID NO: 4 and a VL of SEQ ID NO: 28, (2) 10B11, comprising a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 32, and (3) 6E9, comprising a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 38.

CDR-grafted antibody sequences were designed by grafting CDR residues from each of the above-described mouse antibodies into the closest human germline gue.

CDR-grafted antibody variable regions were synthesized and expressed with human IgGl/K constant regions for analysis. In addition, mouse:human chimeric antibodies were constructed using the variable regions of the above—described mouse antibodies linked to human IgGl/K constant regions. Chimeric and CDR-grafted antibodies were characterized for binding to human and cynomolgus monkey PD-l antigens and for activity in the PD- l blocking assay as described above.

The functional antagonist activity of chimeric and CDR-grafted dies also was tested in a human CD4+ T-cell mixed lymphocyte reaction (MLR) assay in which activation of CD4+ s in the presence of anti-PD-l antibodies is assessed by ing IL-2 secretion. Because PD—l is a negative regulator of T-cell function, antagonism of PD—1 was expected to result in increased T-cell activation as measured by increased IL—2 production. The 9A2, 10B11, and 6E9 CDR-grafted antibodies demonstrated nistic activity and were ed for affinity maturation.

The results of this example demonstrate a method of generating chimeric and CDR-grafted monoclonal antibodies that specifically bind to and t PD-l.

EXAMPLE 3 This example demonstrates affinity maturation of monoclonal antibodies directed t PD- 1.

CDR—grafted antibodies derived from the original murine monoclonal dies, (9A2, 10B11, and 6E9) were subjected to affinity maturation via in vitro somatic utation. Each antibody was displayed on the surface of HEK 293018 cells using the SHM-XEL deciduous system (see Bowers et al., Proc. Natl. Acad. Sci. USA, 108: 20455- 20460 (201 1); and US. Patent Application Publication No. 2013/0035472). After establishment of stable episomal lines, a vector for expression of activation-induced cytosine deaminase (AID) was transfected into the cells to te somatic hypermutation as bed in Bowers et al., supra. After le rounds of FACS sorting under conditions of increasing antigen binding stringency, a number of mutations in the variable region of each antibody were identified and recombined to produce mature humanized antibodies with improved properties.

A panel of six affinity—matured humanized heavy and light chain variable region sequences were paired (denoted APE1922, APE1923, APE1924, APE1950, APE1963 and APE2058) and selected for terization, and are set forth in Table l. The PD-l binding properties of each of these antibody sequences were d using surface plasmon resonance (SPR) and solution-based affinity analysis. Antibodies were expressed from HEK 293 cells as human IgG] antibodies and compared to the reference antibody, a human IgG1 version of BMS-936558, ated BMS.

SPR analyses were carried out using a BIACORETM T200 ment, and c constants were determined using the BIACORETM T200 evaluation software. Experimental parameters were chosen to ensure that saturation would be reached at the highest antigen trations and that Rmax values would be kept under 30 RU. GE anti-Human IgG (Fc- specific, approximately 7,000 RU) was immobilized on a BIACORETM CMS chip using EDC-activated amine coupling chemistry. Antibodies (0.5 ug/mL, 60 second capture time) were then captured using this surface. Next, monomeric soluble human PDl—Avi-His was flowed over captured dy (300 second association, 300 second dissociation) using a three-fold serial on series from 500 nM to 2 nM. Captured dy and antigen were removed between each cycle using 3 M MgClz (60 second t time) in order to ensure a fresh g e for each concentration of antigen. The resulting sensorgrams were fit globally using a 1:1 binding model in order to calculate on- and off-rates (ka and kd, respectively), as well as affinities (KD).

Solution-based affinity analyses were carried out using a KINEXATM 3000 assay (Sapidyne Instruments, Boise, Idaho), and results were analyzed using KINE)Q%TM Pro Software 3.2.6. Experimental parameters were selected to reach a maximum signal with antibody alone between 0.8 and 1.2 V, while limiting nonspecific g signal with buffer alone to less than 10% of the maximum signal. Azlactone beads (50 mg) were coated with antigen by diluting in a solution of PD-l-Avi-His (50 ug/mL in 1 mL) in 50 mM N32C03.

The solution was rotated at room temperature for 2 hours, and beads were pelleted in a picofuge and washed twice with blocking solution (10 mg/mL BSA, 1 M Tris-HCl, pH 8.0).

Beads were resuspended in blocking solution (1 mL), rotated at room temperature for 1 hour, and diluted in 25 s PBS/0.02% NaNg. For affinity measurement, the secondary antibody was ALEXFLUORTM 647 dye-anti-human IgG (500 ng/mL). Sample antibody concentrations were held constant (50 pM or 75 pM), while antigen PD l—Avi—His was titrated using a three-fold dilutions series from 1 uM to 17 pM. All samples were diluted in PBS, 0.2% NaNg, 1 mg/mL BSA and allowed to equilibrate at room ature for 30 hours.

Additionally, samples containing only antibody and only buffer were tested in order to determine maximum signal and nonspecific binding signal, respectively. The results of the affinity es are set forth in Table 1. All of the ed antibodies exhibited higher affinities for PD-l than the BMS reference antibody, with the highest affinity antibody being Table l vH SEQ vL SEQ BIACORETM BIACORETM BIACORETM KINEXATM Antibody ID NO: ID NO: ka(Ms)'l kd(s'l) KD(nM) KD(nM) 88x10 2.1x10‘ APE1922 __1.3x105 1.8x10‘3 _— APE1923 1.9x10 1.7x10' _— APE1924 __———— APE1950 __———_ APE1963 __———_ APE2058 To assess g of the antibodies to cell surface PD-l, binding to CHO cells expressing either human or cnyomolgus monkey PD-l was determined by flow cytometry analysis as described above. In addition, blocking of the PD—l/PD—Ll interaction was assessed using DyL650 labeled PD-Ll (mouse IgG] Fc fusion protein) and PD-l-expressing CHO cells as described above. High binding affinities for cell-surface PD-l were observed for all tested affinity-matured antibody sequences, with reactivity to cynomolgus monkey PD-l within a factor of 3-4 fold of human. Blocking of the PD-l/PD-Ll ction was also efficient with all of the tested affinity-matured antibody sequences, with IC50 values in the low nM range. These results were consistent with g affinities assayed both by the BIACORETM and KINEXATM systems as well as cell surface ECso values.

Thermal stability of the selected antibodies was assessed using a fluor assay as described in McConnell et al., n Eng. Des. Sel., 26: 151 . This assay assesses stability through the ability of a hydrophobic fluorescent dye to bind to hydrophobic patches on the protein surface which are exposed as the protein s. The temperature at which 50% of the protein unfolds is determined (Tm) to measure thermal stability. This assay trated that all of the tested affinity-matured antibody sequences had high thermal ity, and all were more stable than the reference antibody. APE2058 was the most stable antibody, exhibiting a Tm more than 10 °C greater than the Tm of the IgGl version of BMS-936558.

De-risking of potential issues related to in viva pharmokinetics of the tested antibodies was undertaken through (a) assessment of non-specific binding to target negative cells (see, e.g., Hotzel eta1., mAbs, 4: 753-760 (2012)) and (b) measurement of differential neonatal Fc receptor (FcRn) dissociation properties (see, e.g., Wang et al., Drug Metab.

Disp, 39: 1469-1477 (2011)). To assess non—specific binding, antibodies were tested for binding to HEK 293f cells using a flow cytometry-based assay. The tested antibodies were ed to two FDA-approved antibodies, infliximab and denosumab. The results indicated that ecific binding was low for all of the antibodies. To assess FcRn g and dissociation, both human FcRn and cynomolgus FcRn were tested in a BIACORETM- based assay. Antibodies were bound to FcRn at pH 6.0, and after pH adjustment to 7.4, residual bound antibody was ined. The results of this assay are shown in Table 2.

Table 2 Antibod % Residual Bindin_ at H 7.4 APE1922 APE1923 APE1924 APE1950 APE1963"— The results of this example demonstrate a method of generating the ive immunoglobulin heavy and light chain polypeptides, which exhibit thermostability and high affinity for PD-l.

EXAMPLE 4 This example demonstrates the activity of the inventive immunoglobulin heavy and light chain polypeptides in vitro.

Functional antagonist activity of the VH and VL sequences described in Example 3 was tested in a human CD4+ T-cell MLR assay as bed above. For determination of functional potency, the EC50 for each antibody was determined in five separate experiments using different human donors. The results are shown in Table 3 and demonstrate potent activity for each of the selected antibodies, which was indistinguishable from the ty of the reference antibody.

Table 3 EC50 Values (ug/mL) BMS APE2058 APE1922 APE1923 APE1924 APE1950 APE1963 Reference 0.01 0.01 0.01 0.01 0.01 0.01 Each line represents an independent experiment using different human donors for the responder CD4+ T cells.

Shaded line with one responder produced higher IL-2 levels in the presence of the affinity-matured mAbs than in the other ments, artificially raising the ECSO values.

The results of this example demonstrate that the inventive imrnunoglobulin heavy and light chain ptides can nize PD-l signaling, resulting in increased T-cell activation.

EXAMPLE 5 This example trates that a combination of the inventive PD-l binding agent and either an anti-LAG-3 antibody or an anti-TIM-3 antibody enhances T-cell activation in vitro.

To establish ters for combination studies, the anti—PD-l dy APE2058 was titrated in a dose response in the human CD4+ T-cell MLR assay described above.

Antagonism of PD-l ing resulted in increased T-cell activation and a corresponding 4- to 5-fold increase in the production of IL-2.

Based on the results from titrating the APE2058 antibody in multiple MLR assays, an EC50 value of 20 ng/mL and a concentration 10-fold lower that represents an approximate EC10 value (2 ng/mL) were selected for combination studies with antagonist antibodies to the TIM-3 or LAG-3 checkpoint molecules.

A fully human anti-TIM—3 antibody was characterized in a CD4+ T cell in vitro assay as having antagonist activity as measured by increased IL-2 production in the presence of low levels of D3 and anti-CD28 antibodies. The anti-TIM-3 antibody demonstrated activity in the MLR assay with an EC50 value of approximately 0.3 ug/mL, as shown in Figure 2 and Table 4, which is approximately d less ty than the anti—PD—l 8 antibody alone (EC50 approximately 0.02 ug/mL). In ation with 0.02 ug/mL ofAPE2058, the anti-TIM-3 antagonist antibody stimulated increased amounts of IL- 2 production as compared to APE2058 or anti-TIM—3 alone, resulting in a lO-fold decrease in the EC50 values, as shown in Figure 2 and Table 4. These results demonstrate that enhanced T-cell activation occurs with combination inhibition of the PD—l and TIM-3 checkpoint pathways.

A fully human antagonist anti-LAG-3 antibody (described in US. Patent Application Publication 201 1/0150892) has demonstrated potent activity in blocking g ofrecombinant soluble LAG-3 to MHC Class 11 positive cells. This antibody, designated herein as APE03109, was evaluated for functional activity in the human CD4+ T-cell MLR assay. APE03109 demonstrated activity in the MLR with an EC50 value of approximately 0.05 , as shown in Figure 3 and Table 4, which was similar to the activity of the anti- PD-l dy alone. In combination with 0.02 ug/mL ofthe anti-PD-l APE2058 antibody, the APE03109 antibody stimulated increased amounts of IL-2 production over 8 or APE03109 alone, resulting in a 5-fold se in the EC50 values.

A time course of IL-2 production with the anti-LAG-3 APE03109 dy alone and the combination of APE2058 with APE03109 also was characterized in a human CD4+ MLR assay. A similar decrease in EC50 value for the combination of 0.02 ug/mL APE2058 and APE03109 was observed after 72 hours of culture, as shown in Figure 3. After 96 hours of culture the differences in EC50 values were not as nced; however, the levels of IL-2 produced in the cultures treated with 0.02 ug/mL of the anti-PD-l APE2058 antibody and the anti-LAG-3 APE03109 antibody almost doubled as compared to cultures treated with APE03109 alone (2,200 pg/mL versus 1,200 pg/mL). Consistent with the time course of LAG-3 expression, no increased IL-2 production from adding APE03109 to APE2058 was observed after 24 hours, although 8 alone produced a dose responsive increase in IL- 2 production at this time. In separate MLR ments it was also demonstrated that the combination ofAPE2058 and APE03109 enhanced the levels of production ofthe T-cell cytokine IFN-y by over 50% after 48 hours.

To demonstrate that the ed effects of the anti-TIM—3 antibody or the anti— LAG-3 antibody in the CD4+ T-cell MLR were due to target specificity, an irrelevant human IgG1 antibody, APE0422, was tested in combination with 0.02 ug/mL anti—PD—l antibody APE2058. At the highest concentration tested (30 ug/mL), the APE0422 antibody exhibited no effect on IL—2 production over anti-PD-l alone.

Table 4 MLR Assay EC50 MLR Assay EC50 MLR Assay EC50 Fold Antibody Sin_1e aent with 2 n_/mL anti—PD—l with 20 n/mL anti-PD-l Imorovement Anti-LAG-3 53 ng/mL 44 ng/mL 11 ng/mL The results of this example demonstrate that the inventive PD-l-binding agent combined with antagonistic antibodies directed against TIM-3 or LAG-3 enhances CD4+ T- cell activation in vitro.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the t of describing the invention (especially in the context of the following ) are to be ued to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" ed by a list of one or more items (for e, "at least one ofA and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more ofthe listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not d to,") unless otherwise noted. Recitation ofranges of values herein are merely intended to serve as a shorthand method of ing individually to each te value falling within the range, unless ise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary ge (e.g., "such as") ed herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No ge in the ication should be ued as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, ing the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. er, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly dicted by context.

In a first aspect, the invention relates to a Programmed Death 1 (PD-1) binding agent comprising: an immunoglobulin light chain variable (VL) region sing a CDR 1 comprising the amino acid sequence of SEQ ID NO: 35; a CDR 2 comprising the amino acid sequence of SEQ ID NO: 36 except that residue 5 of SEQ ID NO: 36 is replaced with a leucine (L) residue; and a CDR 3 comprising the amino acid ce of SEQ ID NO: 37; and an immunoglobulin heavy chain variable (VH) region comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR 2 comprising the amino acid sequence of SEQ ID NO: 20; and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 21.

In a second aspect, the invention relates to a nucleic acid encoding the heavy chain immunoglobulin polypeptide or light chain immunoglobulin polypeptide of the first aspect.

In a third aspect, the invention relates to a nucleic acid encoding the heavy chain immunoglobulin polypeptide and light chain immunoglobulin polypeptide of the first aspect.

In a fourth aspect, the invention relates to a nucleic acid encoding the PD-1 binding agent of the first aspect.

In a fifth aspect, the invention relates to a vector comprising the c acid of any one of aspects 2-4.

In a sixth , the invention relates to an in vitro host cell comprising the vector of the fifth aspect.

In a seventh , the invention relates to a pharmaceutical composition comprising the PD-1 binding agent of the first aspect, and a pharmaceutically able carrier or diluent.

In an eighth aspect, the invention relates to a method of producing the PD-1 binding agent of the first aspect, the method comprising expressing a nucleic acid encoding the PD-1 binding agent in a cell in vitro.

In a ninth aspect, the invention relates to use of the PD-1 binding agent of the first aspect in the manufacture of a medicament for treating a cancer in a human.

In a tenth aspect, the invention relates to use of the PD-1 binding agent of the first aspect in the manufacture of a medicament for treating an infectious disease in a human.

In an eleventh aspect, the invention relates to use of the PD-1 binding agent of the first aspect in the manufacture of a medicament for enhancing an immune response or increasing the activity of an immune cell in a human.

In a twelfth aspect, the invention s to use of the PD-1 binding agent of the first aspect in the cture of a medicament for enhancing an immune response or increasing the activity of an immune cell in combination with an IM-3 binding agent or an anti-LAG-3 binding agent in a human.

Claims (28)

CLAIMS :

1. A Programmed Death 1 (PD-1) binding agent comprising: an immunoglobulin light chain variable (VL) region comprising a CDR 1 comprising the amino acid sequence of SEQ ID NO: 35; a CDR 2 comprising the amino acid sequence of SEQ ID NO: 36 except that residue 5 of SEQ ID NO: 36 is replaced with a leucine (L) residue; and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 37; and an immunoglobulin heavy chain variable (VH) region comprising a complementarity ining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR 2 comprising the amino acid sequence of SEQ ID NO: 20; and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 21.

2. The PD-1 binding agent of claim 1, wherein the VL region comprises an amino acid ce that is at least 90% identical to SEQ ID NO:40; and the VH region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:23.

3. The PD-1 binding agent of either claim 1 or 2, wherein the VL region comprises SEQ ID NO: 40, and the VH region ses SEQ ID NO: 23.

4. The PD-1 binding agent of any one of claims 1-3, n the PD-1 binding agent is an antibody or an n-binding antibody fragment.

5. The PD-1 binding agent of any one of claims 1-4, wherein the PD-1 binding agent comprises an IgG1, IgG2, or IgG4 heavy chain constant region (Fc).

6. A nucleic acid encoding the heavy chain immunoglobulin ptide and light chain immunoglobulin polypeptide of any one of claims 1-5.

7. A nucleic acid encoding the PD-1 binding agent of any one of claims 1-5.

8. The PD-1 binding agent of claim 1, wherein the PD-1 binding agent is an antibody comprising a heavy chain immunoglobulin polypeptide comprising SEQ ID NO: 23, a light chain immunoglobulin polypeptide comprising SEQ ID NO: 40, and an lgG4 heavy chain constant region (Fc).

9. A vector sing the nucleic acid of any one of claims 6-7.

10. An in vitro host cell comprising the vector of claim 9.

11. The in vitro host cell of claim 10, wherein the host cell is a mammalian host cell.

12. A pharmaceutical composition comprising the PD-1 binding agent of any one of claims 1-5 or 8, and a pharmaceutically acceptable carrier or diluent.

13. The pharmaceutical composition of claim 12, n the pharmaceutical composition is formulated for parenteral administration.

14. A method of producing the PD-1 binding agent of any one of claims 1-5 or 8, the method comprising expressing a nucleic acid ng the PD-1 binding agent in a cell in vitro.

15. Use of the PD-1 binding agent of any one of claims 1-5 or claim 8, in the manufacture of a medicament for enhancing an immune response or increasing the ty of an immune cell in a human.

16. The use of claim 15, wherein the immune response or activity is a cellmediated immune response.

17. The use of claim 15, wherein the immune cell is a T cell.

18. The use of any one of claims 15-17, wherein the immune response or activity is a T cell response.

19. The use of any one of claims 15-18, wherein the medicament is for treating a cancer in a human.

20. The use of any one of claims 15-18, wherein the medicament is for ng an infectious disease in a human.

21. The use of any one of claims 15-18, wherein the human has an infectious disease.

22. The use of any one of claims 15-18, wherein the human has cancer.

23. The use of either claim 19 or claim 22, n the cancer is characterized by expression of PD-1 or PD-L1.

24. The use of claim 23, wherein the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast , cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, and Merkel cell carcinoma.

25. The use of any one of claims 15-24, wherein the medicament is for enhancing an immune response or increasing the activity of an immune cell in combination with an anti-TIM-3 binding agent or an anti-LAG-3 binding agent in a human.

26. The use ing to claim 25, wherein the TIM-3 binding agent is an antibody, an dy conjugate, or an antigen-binding fragment thereof.

27. The use according to claim 25, wherein the anti-LAG-3 binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof.

28. The use of any one of claims 15-27 wherein the PD-1 g agent binds to PD-1 with a KD between about 1 picomolar (pM) and about 100 micromolar (μM). AnaptysBio, Inc. By the Attorneys for the ant SPRUSON & FERGUSON Per: