NZ753073B2 - 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 aforementioned immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide. The invention also provides related vectors, compositions, and methods of using the PD-1-binding agent to treat a cancer or an infectious disease. nd immunoglobulin light chain polypeptide. 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 I (PD-I) INCORPORATION-BY-REFERENCE OFMATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety herein is a computer-readable nucleotide/ amino acid sequence listing submitted concurrently herewith and identified as follows: One 45,084 Byte ASCII (Text) fi le named "716746_ST25.TXT," created on May 1, 2014. The present application is a onal of New Zealand Patent Application No.714537, which is incorporated herein by reference OUND OF THE INVENTION Programmed Death 1 (PD-I) (also known as Programmed Cell Death 1) is a type I transmembrane protein of268 amino acids originally identified by subtractive hybridization ofa mouse T cell line undergoing apoptosis (Ishida et al., Embo J., 11: 3887-95 (1992)). PDI is a member ofthe CD28/CTLA-4 fa mily ofT-cell regulators, and is expressed on activated T-cells, B-cells, and d e cells wald et al., Annu. Rev. l., 23: 515-548 (2005); and Sharpe et al., Nat. Immunol., 8: 239-245 (2007)).

Two ligands for PD-I have been identified, PD ligand 1 (PD-LI) and PD ligand 2 (PD-L2), both ofwhich belong to the B7 protein superfamily (Greenwald et al., supra). PDLI 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., 169(10): 5538-5545 (2002)). PD-LI 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-LI 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): 145 (2004)). In st, PD-L2 ts a more restricted expression pattern and is expressed primarily by antigen presenting cells (e.g., dendritic cells and macrophages), and some tumor cell lines (see, e.g., Latchman et al., Nat. l., 2(3): 261-238 (2001)). High PD-LI expression in tumors, whether on the tumor cell, stroma, or other cells within the tumor microenvironment, correlates with poor clinical prognosis, presumably by inhibiting effector T cells and upregulating regulatory T cells (Treg) in the tumor.

PD-I 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 autoimmunity. For example, C57BL/6 PD-l knockout mice have been shown to develop a lupus—like me (see, e.g., Nishimura et al., Immunity, 11: 141-1151 (1999)). In humans, a single nucleotide polymorphism in the PD-l gene is associated with higher incidences of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and progression of multiple sclerosis (see, e. g., Nielsen et al., Tissue Antigens, 62(6): 492-497 (2003); Bertsias et al., Arthritis Rheum, 60(1): 207-218 (2009); Ni et al., Hum. Genet, 121(2): 223—232 (2007); Tahoori et al., Clin. Exp. Rheumatol., 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 n and c viral infections (see, e.g., Barber et al., Nature, 439: 682-687 (2006); and Sharpe et al., supra).

Recent studies demonstrate that T—cell ssion induced by PD-l also plays a role in the suppression of anti-tumor ty. 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 ). 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. l, 1 7: 133-144 (2005)), and T-cells from PD-l knockout mice are highly effective in tumor rejection when adoptively transferred to tumor-bearing mice (Blank et al., . 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 ne, 84(4): 409-421 (2011)).

In view of the foregoing, gies for inhibiting PD—1 ty to treat various types of cancer and for immunopotentiation (e.g., to treat infectious diseases) have been developed (see, e.g., Ascierto et al., Clin. Cancer. Res, 19(5): 1009-1020 ). In this respect, 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 l responses in non-small-cell lung cancer, melanoma, 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. MK-3575 is a humanized monoclonal dy directed against PD-l that has shown evidence of antitumor activity in Phase I al trials (see, e.g., Patnaik et al., 2012 American Society of Clinical Oncology (ASCO) Annual g, Abstract # 2512). In on, recent evidence suggests that therapies which target PD-l may enhance immune ses 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 efficacy of these potential therapies in humans may be d.

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

BRIEF SUMMARY OF THE INVENTION The ion 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) 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, (0) one or more of es 1, 2, and 5 of SEQ ID NO: 3 is ed 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 residue 9 of SEQ ID NO: 13 is replaced with a different amino acid e, (c) residue 5 of SEQ ID NO: 14 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: 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 polypeptide which comprises an amino acid sequence 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 provides an isolated globulin 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 isolated globulin light chain polypeptide which comprises a complementarity ining 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 ce of SEQ ID NO: 36, and a CDR3 amino acid sequence of SEQ ID NO: 37, wherein ally (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.

The invention provides an ed 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 on, the invention provides isolated or purified nucleic acid sequences ng the ing immunoglobulin ptides, vectors comprising such nucleic acid sequences, isolated PD-I-binding agents comprising the foregoing immunoglobulin polypeptides, nucleic acid sequences encoding such PD-I-binding agents, vectors comprising such nucleic acid ces, isolated cells comprising such vectors, compositions comprising such PD-I-binding agents or such vectors with a pharmaceutically acceptable carrier, and methods of ng cancer or infectious es in mammals by administering effective amounts of such compositions to mammals.

BRIEF DESCRIPTION THE DRAWINGS Figure 1 is a diagram which schematically 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 extracellular domain – mouse IgG1 Fc . Human PD-L2 extracellular domain - mouse IgG1 Fc 6. Full-length cynomolgus monkey PD-1 sed on the surface of CHO cells Figure 2 is a graph which illustrates experimental results demonstrating increased activity of an anti-TIM-3 antagonist antibody in a human CD4+ T-cell MLR assay in the presence of low levels of anti—PD—l antibody 8.

Figure 3 is a graph which illustrates experimental results demonstrating increased ty 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 ION The invention provides an isolated immunoglobulin heavy chain polypeptide and/or an isolated immunoglobulin light chain polypeptide, or a nt (e.g., antigen- binding fragment) f. The term “immunoglobulin” or “antibody,” as used herein, refers to a n 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 “hypervariable ” of an antibody, which is responsible for antigen binding (discussed further below). A whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three inal constant (CH1, CH2, and CH3) regions, 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 hide bonds. The light chain variable region is d with the variable region ofthe heavy chain, and the light chain nt region is aligned with the first nt region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

The le regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions 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 between the hypervariable or complementary determining regions (CDRs). There are four framework regions in each variable domain, which are designated FRl, FR2, FR3, and FR4. The framework s form the B sheets that provide the structural framework of the variable region (see, e. g., C.A. Janeway et al. (eds.), bz’ology, 5th Ed., Garland Publishing, New York, NY (2001)).

The framework regions are connected by three complementarity ining regions (CDRs). As sed above, the three CDRs, known as CDRl, CDR2, and CDR3, form the variable region” of an antibody, which is responsible for antigen g.

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 g of the dy to an antigen, the constant regions can influence the orientation of the variable regions. The constant regions also exhibit various effector fianctions, 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 polypeptide 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 TLA-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 intracellular tail. The intracellular tail contains two phosphorylation 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 regulate T-cell receptor signaling (see, e.g., Ishida et al., supra; and Blank et al., supra). The inventive ed immunoglobulin heavy chain polypeptide and the inventive isolated immunoglobulin light chain polypeptide can form an agent that binds to PD-l and r 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 tor of the immune system such as, for example, lymphocyte-activation gene 3 (LAG-3) and/or T-cell immunoglobulin domain and mucin domain 3 protein (TIM—3). dies 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 Patnaik et al., supra). Anti-PD-l antibodies also are commercially available from sources such as, for example, Abcam (Cambridge, MA).

An amino acid “replacement” or “substitution” refers to the ement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide ce.

Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. es 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.” es 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), nine (M or Met), serine (S or Ser), threonine (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” consists of valine, leucine, and isoleucine. The “aliphatic slightly-polar sub-group” consists of methionine, serine, threonine, and cysteine. The “aliphatic polar/charged sub-group” consists of ic acid, aspartic acid, asparagine, glutamine, , 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 arginine, 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 “phenyl sub-group” consisting of phenylalanine and tyrosine.

The amino acid replacement or substitution can be conservative, semi— conservative, or non—conservative. The phrase rvative 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 ties n dual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, 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 ure (Schulz and Schirmer, supra).

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

“Semi—conservative ons” include amino acid substitutions 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 ent sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

The invention es an immunoglobulin heavy chain ptide that comprises a mentarity 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 polypeptide 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 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 of residues 1, 2, and 5 of SEQ ID NO: 3 is replaced with a different amino acid e, or (d) any combination of (a)-(c). When the inventive immunoglobulin heavy chain polypeptide consists essentially of a CDRl amino acid ce of SEQ ID NO: 1, a CDR2 amino acid ce of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3 and al 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 inventive 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 replacements, the polypeptide does not comprise any additional components (i.e., components that are not nous to the inventive immunoglobulin heavy chain ptide).

In one embodiment ofthe invention, the isolated immunoglobulin polypeptide ses 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 e, (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 replaced 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.

Alternatively, 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 es 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 se a CDRl amino acid ce of SEQ ID NO: 1, a CDR2 amino acid ce 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 residue. Each of residue 9 of SEQ ID NO: 1, es 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 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 residues are the same or different.

In one ment, 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) residue. 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 ed 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 ation of (a)-(c). In another 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 (a) residue 1 of SEQ ID NO: 3 is replaced with a glutamic acid (E) residue, (b) residue 2 of SEQ ID NO: 3 is ed with a ne (Y) residue, (0) residue 5 of SEQ ID NO: 3 is replaced with a serine (S) residue, or (d) any combination of (a)-(c).

Exemplary immunoglobulin heavy chain polypeptides as described above can comprise 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 ion provides an isolated immunoglobulin heavy chain polypeptide comprises, consists essentially of, or consists of a mentarity 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 residue 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 ). 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, onal 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 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 se any onal 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 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 (a) residue 9 of SEQ ID NO: 12 is replaced with a ent amino acid residue, (b) residue 8 and/or e 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 ent amino acid residue, or (d) any combination of (a)-(c). For example, 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 ed 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 e. Alternatively, the isolated immunoglobulin heavy chain polypeptide can comprise a CDRl amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid ce 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 and residue 5 of SEQ ID NO: 14 is replaced with a different 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 residue, residue 8 of SEQ ID NO: 13 is replaced with a different 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, residues 8 and 9 of SEQ ID NO: 13, and residue 5 of SEQ ID NO: 14 can be ed 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 ent 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 residues are the same or different. In one ment, the isolated immunoglobulin heavy chain ptide 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 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 8 of SEQ ID NO: 13 is replaced with a tyrosine (Y) residue, and/or (b) residue 9 of SEQ ID NO: 13 is ed with an alanine (A) residue. In r 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 se 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 isolated globulin heavy chain polypeptide comprises, consists essentially of, or consists 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., ents that are not endogenous to the inventive globulin heavy chain polypeptide). Exemplary immunoglobulin heavy chain polypeptides as bed above can comprise any one of the following 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 immunoglobulin heavy chain polypeptides. Any number of any suitable amino acids can be inserted into the amino acid sequence of the globulin 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.

Preferably, 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 t, the amino acid(s) can be inserted 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 polypeptide.

, 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 es 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 ). A number of mathematical algorithms for ing the optimal alignment and calculating ty between two or more ces are known and incorporated into a number of available software ms. Examples of such programs e CLUSTAL-W, ee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3X, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol, 215(3): 403-410 (1990), t et al., Proc. Natl. Acad. Sci. USA, [06(10): 3770—3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models ofProteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 , Altschul et al., Nucleic Acids Res, 25(17): 3389-3402 (1997), and Gusfield, Algorithms on s, Trees and ces, Cambridge University Press, Cambridge UK (1997)).

The invention provides an immunoglobulin light chain ptide 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 facilitate purification or isolation). When the inventive globulin 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 isolated immunoglobulin light chain polypeptide comprises 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. In one embodiment of the invention, the isolated immunoglobulin light chain polypeptide ses, consists of, or consists essentially of a CDRl amino acid sequence of SEQ ID NO: 30 and a CDR2 amino acid sequence of SEQ ID NO: 31, n optionally residue 12 of SEQ ID NO: 30 is replaced with a ent amino acid residue. When the inventive immunoglobulin light chain polypeptide consists 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 es such as biotin that facilitate purification or ion).

When the inventive globulin 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 se 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 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 threonine (T) residue. 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 ed 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 ses, consists essentially of, or consists 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, 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 polypeptide consists essentially of 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 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 inventive immunoglobulin light chain ptide consists 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 al 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 t, 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 sequence of SEQ ID NO: 37.

Alternatively, the ed globulin light chain polypeptide can comprise 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, 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 e 5 of SEQ ID NO: 36 and residue 4 of SEQ ID NO: 37 can be replaced with any suitable amino acid e 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 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 residues are the same or different.

In one ment, the isolated globulin 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 replaced with a leucine (L) residue, and/or (b) residue 4 of SEQ ID NO: 37 is replaced with an asparagine (N) residue. ary immunoglobulin light chain polypeptides as bed above can comprise any one of the following amino acid sequences: 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 immunoglobulin light chain polypeptides. Any number of any suitable amino acids can be inserted 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. Preferably, the amino ) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of the immunoglobulin light chain ptide.

The ion 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 bed herein.

The invention provides an isolated programmed death 1 (PD-l)—binding agent comprising, consisting essentially of, or consisting of the inventive isolated amino acid sequences described . 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 (PD-1). 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 ive immunoglobulin heavy chain polypeptide or the inventive 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, consists essentially of, or consists of an immunoglobulin heavy chain polypeptide and/or light chain polypeptide having replacements, insertions, and/or deletions of the specific amino acid residues disclosed herein. Indeed, any amino acid residue of the inventive globulin heavy chain polypeptide and/or the inventive immunoglobulin light chain polypeptide can be replaced, in any ation, with a different amino acid residue, or can be deleted or inserted, so long as the ical activity of the inding agent is enhanced or improved as a result of the amino acid replacements, insertions, and/or deletions. The “biological activity” of an PD-l-binding agent refers to, for example, g affinity for PD-l or a particular PD-l epitope, neutralization or tion of PD-l protein binding to its ligands PD-Ll and PD-Ll neutralization or inhibition of PD-l protein activity in vivo (e.g., IC50), pharmacokinetics, 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, selectivity, solubility, g, 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, e plasmon resonance analysis (BIACORETM), or KINEXATM, in vitro or in viva neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and/or ion , and signal uction and immunohistochemistry assays.

The terms “inhibit” or “neutralize,” as used herein with respect to the activity of a inding 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 ty of a PD-l protein, or a disease or condition associated with an PD-l protein. The isolated inding agent of the ion 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 bed herein, or an antibody fragment. The terms “fragment of an antibody,77 LLantibody fragment,” and “functional fragment of an antibody” are used interchangeably herein to mean one or more nts of an antibody that retain the ability to cally bind to an antigen (see, generally, Holliger et al., Nat. Biotech, 23(9): 1126-1129 (2005)). The isolated PD-l binding agent can contain any inding antibody fragment. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or ations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 s, (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 dy, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv nt (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 inhibits the activity of, a PD-l protein. In this respect, a nt of the globulin 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 ing values) amino acids. Similarly, a fragment of the immunoglobulin light chain ptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9,10,11,12,13,14,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 dy 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 antibodies, 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 s 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. Biotechnol, 16: 778 (1998)) and (ii) a diabody, which is a dimer of polypeptide chains, n each ptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VB and VL on the same polypeptide chain, thereby g the pairing between the complementary s 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 ed 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 typically lack de bonds and are capable of modulating the expression 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 ody can include sub- cellular trafficking s ed 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 d. Upon interaction with a target gene, an intrabody modulates target protein function and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein degradation and sequestering the target protein in a non-physiological sub-cellular compartment. Other mechanisms of intrabody-mediated gene inactivation can depend on the epitope to which the intrabody is directed, such as binding to the catalytic site on a target protein or to epitopes that are involved in n—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 conjugate of (1) an antibody, an alternative scaffold, or fragments thereof, and (2) a protein or non—protein moiety comprising the PD—l— binding agent. For example, the PD-l-binding agent can be all or part of an antibody ated 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 man dy, or a chimeric antibody. By “chimeric” is meant an antibody or fragment thereof comprising both human and man regions. Preferably, the isolated PD-l-binding agent is a zed antibody. A “humanized” antibody is a monoclonal antibody comprising a human antibody ld and at least one CDR obtained 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 inventive PD binding agent is ed or derived from a mouse onal antibody, while the remaining variable regions and constant region of the inventive PDbinding agent are ed or derived from a human monoclonal antibody.

A human antibody, a non-human antibody, a chimeric dy, 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 dy recombinantly) 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 , Antibodies: A Laboratory , CSH Press (1988); and Janeway et al. (eds.), biology, 5th Ed., Garland Publishing, New York, NY (2001)). In certain embodiments, a human antibody or a chimeric antibody 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 replaced with human antibody genes e, but are not limited to, the Medarex MOUSETM, the Kirin TC MOUSETM, and the Kyowa Kirin KM- MOUSETM (see, e.g., Lonberg, Nat. Biotechnol, 23(9): 1117-25 , 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 (2009)), 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. Biochem., 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 2011/0287485 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 ive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and the inventive inding 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 double- 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 cleotides such as, though not limited to, ated and/or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like). Nucleic acid sequences encoding the inventive immunoglobulin heavy chain ptides 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 ces encoding the inventive immunoglobulin light chain polypeptides include, 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 comprising one or more nucleic acid sequences encoding the inventive immunoglobulin heavy chain polypeptide, the ive immunoglobulin light chain polypeptide, and/or the ive inding 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 ation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring , NY. (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing ates and John Wiley & Sons, New York, NY. (1994)).

In addition to the nucleic acid sequence encoding the inventive globulin 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, polyadenylation signals, ription terminators, internal ribosome entry sites , and the like, that provide for the expression of the coding sequence in a host cell. Exemplary sion control sequences are known in the art and described in, for example, l, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

A large number of ers, including constitutive, inducible, and repressible promoters, from a variety of different s are well known in the art. entative s of promoters include for example, Virus, mammal, insect, plant, yeast, and bacteria, and suitable ers from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources. ers can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate ription in either a 3’ or 5 ’ direction). Non-limiting examples ofpromoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include, for example, the Tet system (US. s 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), LACSWITCHTM system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen inducible inase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); US. Patent 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol, 308: 123-144 (2005)).

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

Enhancers can be d many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of ers 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 sequences. Enhancers can be located upstream, within, or downstream of coding sequences.

The vector also can comprise a “selectable marker gene.” The term table marker gene,” as used herein, refers to a nucleic acid sequence 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., ational Patent ation 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); an & Berg, Proc. Natl. Acad. Sci.

USA, 78: 2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol, 150: 1-14 (1981); Santerre eta1., Gene, 30: 147-156 (1984); Kent et al., Science, 237: 901-903 (1987); Wigler eta1., Cell, 11: 223-232 ; 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 mal expression vector” or “episome,” which is able to replicate in a host cell, and persists as an hromosomal segment ofDNA within the host cell in the presence of appropriate selective re (see, e.g., Conese et al., Gene Therapy, 11 : 1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, al plasmids that utilize Epstein Barr Nuclear n 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 ofEBNAl 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 utilize the endogenous expression control sequences of the host cell’s chromosomes to effect sion of the desired protein. Examples of s that integrate in a site specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., pcDNATMS/FRT), or the cre-lox , such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, CA). Examples ofvectors that randomly ate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen (Carlsbad, CA), UCOE from Millipore rica, MA), and pCI or pFNlOA (ACT) FLEX]TM from Promega (Madison, WI).

Viral vectors also can be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system ble from l, 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 provided 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 combination of bidirectional and ectional promoters can be used to control expression of multiple nucleic acid sequences. Nucleic acid sequences encoding the inventive amino acid sequences alternatively can be provided to the population of cells on separate vectors (i.e., in trans).

Each of the nucleic acid sequences in each of the separate vectors can se the same or different expression control sequences. The separate vectors can be provided to cells aneously or tially.

The vector(s) sing the nucleic acid(s) encoding the ive amino acid sequences can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby, including any suitable prokaryotic or eukaryotic cell. As such, the invention es an isolated cell comprising the inventive vector. 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 limited to, cells from the genera Bacillus (such as us 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 .

Preferably, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and ian cells. Examples of suitable yeast cells e 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 (1993); and Lucklow et al., J.

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

Preferably, mammalian 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, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, e 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).

Further exemplary ian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell s derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the ATCC. Methods for selecting suitable mammalian host cells and methods for transformation, culture, amplification, ing, and purification of cells are known in the art.

Most preferably, the mammalian 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. Examples ofhuman id cells lines include, t limitation, RAMOS (CRL-1596), Daudi (CCL-213), EB—3 (CCL—85), DT40 1 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 formation,” 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 y, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE—dextran; oporation; cationic liposome-mediated transfection; tungsten particle-facilitated article bombardment (Johnston, Nature, 346: 776-777 ); 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 infectious 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 ive globulin 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. Preferably, the composition is a ceutically able (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the ive amino acid sequences, antigen- binding agent, or vector. Any suitable carrier can be used within the context of the invention, and such carriers 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 composition optionally can be e. 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 Practice ofPharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

The invention further es 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 decrease in PD-l protein levels or activity has a therapeutic benefit in mammals, preferably humans. The invention also es 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: 266 ; and Flies et. al., Yale Journal of y and Medicine, 84: 409-421 (2011)), and PD-Ll expression in some renal cell carcinoma ts correlates with tumor siveness. 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, bladder cancer, breast cancer, cervical cancer, colon , gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach , salivary 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 ious 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 (HCV)). Administration of a composition comprising the inventive globulin 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 se” can entail, for example, antibody production and/or the activation of immune effector cells (e.g., T-cells).

As used herein, the terms “treatmen ,” “treating,” and the like refer to obtaining a desired pharmacologic and/or logic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease. To this end, the ive method comprises administering a “therapeutically effective amount” of the inding agent. A peutically 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 inding agent of the invention is an amount which decreases PD—l n bioactivity in a human and/or enhances the immune response against a cancer or infectious disease.

Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a e or symptom thereof. In this respect, the inventive method comprises administering a “prophylactically effective amount” of the PD-l-binding agent. A “prophylactically effective ” refers to an amount effective, at dosages and for s of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease .

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 0.00001 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 ably 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 monitored by periodic assessment of treated ts. 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 ive immunoglobulin light chain polypeptide, the inventive PDbinding agent, the inventive nucleic acid sequence ng any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository stration. The composition preferably is suitable for eral administration. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is stered to a mammal using peripheral systemic ry by intravenous, intraperitoneal, or subcutaneous injection.

Once stered 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 example, the biological ty 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 minutes, 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 PD-l-binding 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 ing values).

The biological activity of a particular PD-l-binding agent also can be assessed by ining its binding affinity 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 y of an antibody 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 ing ). Immunoglobulin affinity for an n or epitope of interest can be measured using any art—recognized assay. Such methods include, for e, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface n 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 PD-l-binding agent can be administered in combination with other agents for the treatment or prevention of the es 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 antibodies, cancer—killing Viruses, gene therapy, and adoptive T-cell er), and/or surgery. When the inventive method treats an infectious disease, the PD-l-binding agent can be administered in ation with at least one anti- bacterial agent or at least one iral agent. In this t, the anti—bacterial 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., live-attenuated vaccines, subunit vaccines, recombinant vector vaccines, and small molecule anti-viral therapies (e. g., viral replication inhibitors and nucleoside analogs).

In another embodiment, the inventive PD-l g 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 combination with agents that inhibit or antagonize the , TIM-3 or LAG-3 pathways. Combination treatments that simultaneously target two or more of these immune checkpoint pathways have demonstrated improved and potentially istic 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 (2012)). In one embodiment, the inventive PD-l binding agent is administered in combination with an dy 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 r or a composition comprising (i) an antibody that binds to a LAG-3 protein and (ii) a pharmaceutically able carrier.

In addition to therapeutic uses, the PD-l-binding agent described herein can be used in diagnostic or research 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 t being tested for a disease or er that is associated with abnormal PD-l expression. Research 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 cent or uminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, beta-galactosidase, 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. l. Meth, 40: 219-230 (1981); and , J. Histochem. and Cytochem., 30: 407-412 (1982)).

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 facilitate detection of the bound or unbound dy. le detectable substances include s enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, e.g., Zola, Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc. (1987)). The amount of PD-l polypeptide sed in a sample is then compared with a standard value.

The PDbinding agent can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a diagnostic assay. If the PD-l-binding agent is d with an enzyme, the kit bly es substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides a able chromophore or fluorophore). In addition, other additives may be included in the kit, such as stabilizers, 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 powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

The following es further rate 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 dies directed against human PD-l.

Several forms of genes encoding human PD-l and its ligands PD-Ll and PD-L2 were ted as antigens for use in mouse immunization, hybridoma screening, and affinity maturation of CDR-grafted dies, 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 tous chromatin opening element (UCOE) single expression vector with ycin selection (Millipore, Billerica, MA). CHO—K1 cells were stably transfected with Lipofectamine LTX (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Following selection with hygromycin, cells expressing PD—l on the cell e were identified by flow cytometry using a jugated 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 cynomolgus monkey PD-l were constructed with His tags appended to the C-terminus of the ECD or as e dimeric fusion proteins with mouse IgG2a Fc as indicated in Figure 1. Nucleic acid sequences encoding soluble dimeric 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 standard techniques. His-tagged proteins were purified from cell culture supernatant via Ni-affinity column chromatography followed by size exclusion chromatography. IgG-Fc fusion proteins were purified using protein A/G y chromatography. Purified proteins were analyzed by SDS-PAGE and size-exclusion chromatography to ensure homogeneity. Additionally, ty and size were confirmed by mass spectrometry.

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

Mice were immunized with either CHO cells expressing ength 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 Laboratories, 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 ECD—His 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 al 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 boosted 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 described in Kearney et al., J.

Immunol., 123: 550 (1979)).

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

Cells were washed twice in PBS/2% FBS, resuspended in PBS, 2% FBS, 1% paraforrnaldehyde, and fluorescence ed on a BD FACSARRAYTM Bioanalyzer (BD Biosciences, Franklin Lakes, NJ). Mouse IgG levels were quantified by ELISA.

Based on strong binding to PD-l CHO cells, 46 parental wells were expanded, and the atants were tested for the ability to block binding of DyL650-labeled PD—Ll- mIgGl Fc fusion n 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 inhibition was quantified by flow cytometry. Cells from wells showing the best PD-Ll ng activity and highest levels of mouse IgG were subcloned for further 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 ation. 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 BIACORETM T200 instrument (GE Healthcare, Waukesha, WI), and kinetic constants were determined using the BIACORE TM T200 evaluation software (GE Healthcare, Waukesha, WI). In this respect, antibodies TM CMS chip to which GE ouse IgG were captured on a BIACORE was coupled. PD- l-His monomer was flowed over the captured antibody using two— or three-fold serial dilutions 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 subsequent ies (KD).

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

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

Subclones of the hybridomas which produced PD-l-binding antibodies with PD- Ll ng activity as described in Example 1 were isotyped, subjected to RT-PCR for g 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 cells/pellet) using the RNEASYTM kit (Qiagen, Venlo, Netherlands), and cDNA was prepared using oligo-dT-primed SUPERSCRIPTTM III First-Strand Synthesis System (Life Technologies, ad, CA). PCR cation 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 rate mouse VH forward primers (Kontermann and Dubel, supra) and a mouse yl or y2a nt region e primer (based on isotyping of purified antibody from each clone) with the protocol recommended in the SUPERSCRIPTTM III First-Stand Synthesis System (Life Technologies, Carlsbad, CA). PCR products were purified and cloned into pcDNA3.3-TOPO (Life Technologies, Carlsbad, CA).

Individual es 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 ng CDR residues from each of the above-described mouse antibodies into the closest human germline gue. afted antibody variable regions were synthesized and expressed with human IgGl/K constant regions for analysis. In on, mouse:human chimeric antibodies were constructed using the variable regions of the above—described mouse antibodies linked to human IgGl/K constant regions. ic and CDR-grafted antibodies were characterized for binding to human and cynomolgus monkey PD-l antigens and for activity in the PD- l/PD-Ll blocking assay as described above.

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

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

EXAMPLE 3 This example demonstrates affinity maturation of monoclonal antibodies ed against PD- 1. afted antibodies derived from the original murine monoclonal antibodies, (9A2, 10B11, and 6E9) were ted to affinity maturation via in vitro somatic hypermutation. 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 ishment of stable episomal lines, a vector for expression of activation-induced cytosine deaminase (AID) was transfected into the cells to initiate somatic hypermutation as described in Bowers et al., supra. After multiple 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 ed properties.

A panel of six y—matured humanized heavy and light chain variable region sequences were paired (denoted APE1922, APE1923, APE1924, APE1950, APE1963 and 8) and selected for characterization, and are set forth in Table l. The PD-l binding properties of each of these antibody sequences were assayed using surface plasmon resonance (SPR) and solution-based affinity is. Antibodies were expressed from HEK 293 cells as human IgG] antibodies and compared to the reference dy, a human IgG1 version of BMS-936558, designated BMS.

SPR analyses were d out using a BIACORETM T200 instrument, and kinetic constants were determined using the ETM T200 evaluation software. Experimental parameters were chosen to ensure that saturation would be d at the highest antigen concentrations and that Rmax values would be kept under 30 RU. GE uman IgG (Fc- c, 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 antibody (300 second association, 300 second dissociation) using a three-fold serial dilution series from 500 nM to 2 nM. Captured antibody and antigen were removed between each cycle using 3 M MgClz (60 second contact time) in order to ensure a fresh binding e for each concentration of antigen. The resulting sensorgrams were fit globally using a 1:1 binding model in order to ate 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. mental 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 .

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 volumes PBS/0.02% NaNg. For affinity measurement, the secondary antibody was ALEXFLUORTM 647 dye-anti-human IgG (500 ng/mL). Sample antibody trations 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 , tively. The results of the affinity analyses are set forth in Table 1. All of the selected antibodies exhibited higher affinities for PD-l than the BMS reference dy, with the t affinity antibody being APE2058.

Table l vH SEQ vL SEQ BIACORETM ETM BIACORETM KINEXATM Antibody ID NO: ID NO: '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 binding 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 interaction 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 binding affinities assayed both by the ETM and KINEXATM systems as well as cell surface ECso values.

Thermal stability of the ed antibodies was assessed using a Thermofluor assay as described in McConnell et al., Protein Eng. Des. Sel., 26: 151 (2013). This assay assesses stability through the ability of a hydrophobic fluorescent dye to bind to hobic patches on the protein surface which are exposed as the n unfolds. The ature at which 50% of the protein unfolds is determined (Tm) to measure thermal stability. This assay demonstrated that all of the tested affinity-matured antibody sequences had high thermal stability, and all were more stable than the reference antibody. 8 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 dies 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 proved antibodies, infliximab and denosumab. The results indicated that non-specific binding was low for all of the antibodies. To assess FcRn binding 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 determined. The results of this assay are shown in Table 2.

Table 2 Antibod % Residual Bindin_ at H 7.4 APE1922 APE1923 APE1924 APE1950 APE1963“— APE2058 The results of this e demonstrate a method of generating the inventive 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 globulin heavy and light chain ptides in vitro.

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

Table 3 EC50 Values ) 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 ment 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 experiments, artificially raising the ECSO values.

The results of this example demonstrate that the inventive imrnunoglobulin heavy and light chain polypeptides can antagonize PD-l ing, resulting in sed T-cell activation.

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

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

Antagonism of PD-l signaling resulted in increased T-cell activation and a corresponding 4- to 5-fold se 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 ation 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 tion 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 15-fold less activity than the anti—PD—l APE2058 antibody alone (EC50 approximately 0.02 . In combination 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, ing 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 ibed in US. Patent Application Publication 201 1/0150892) has demonstrated potent activity in blocking binding ofrecombinant e LAG-3 to MHC Class 11 positive cells. This antibody, designated herein as APE03109, was ted for functional ty in the human CD4+ T-cell MLR assay. APE03109 demonstrated activity in the MLR with an EC50 value of approximately 0.05 ug/rnL, as shown in Figure 3 and Table 4, which was similar to the activity of the anti- PD-l antibody 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 APE2058 or APE03109 alone, ing in a 5-fold decrease in the EC50 values.

A time course of IL-2 production with the anti-LAG-3 APE03109 antibody 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 pronounced; r, the levels of IL-2 produced in the cultures treated with 0.02 ug/mL of the anti-PD-l APE2058 antibody and the AG-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 sion, no sed IL-2 production from adding APE03109 to APE2058 was observed after 24 hours, although APE2058 alone produced a dose responsive increase in IL- 2 tion at this time. In separate MLR experiments 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 combined 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 ement 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 ations, 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 .

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be ued to cover both the singular and the plural, unless ise indicated herein or clearly contradicted by context. The use of the term “at least one” followed 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,” g,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation es of values herein are merely intended to serve as a shorthand method of referring individually to each te value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually d 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 language (e.g., "such as") provided , is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the ce of the invention.

Preferred embodiments of this invention are described , including the best mode known to the inventors for carrying out the invention. Variations of those preferred ments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled ns 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 es all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or ise clearly dicted by t.

In a first aspect, the ion relates to a Programmed Death 1 (PD-1) binding agent comprising: a heavy chain immunoglobulin variable region (VH) polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:6-10; and a light chain immunoglobulin variable region (VL) polypeptide comprising the amino acid sequence of SEQ ID NO:29.

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

In a third aspect, the invention relates to a nucleic acid encoding the VL polypeptide of the PD-1 binding agent of one the first aspect.

In a fourth aspect, the invention relates to a nucleic acid ng the VH polypeptide and the VL polypeptide of the PD-1 binding agent of the first aspect.

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

In a sixth aspect, the ion relates to a host cell expressing the nucleic acid of any one of aspects 2-4.

In a seventh aspect, the invention relates to a method of producing the PD-1 binding agent of the first aspect, the method comprising expressing a nucleotide sequence encoding the immunoglobulin heavy and light chains of the PD-1 binding agent in a cell in vitro.

In an eighth aspect, the invention s to a pharmaceutical composition comprising an effective amount of the PD-1 binding agent of the first aspect and a pharmaceutically acceptable carrier or diluent.

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

In a tenth aspect, the invention s 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 subject.

In an eleventh aspect, the invention relates to a method of treating a cancer associated with overexpression of PD-1 protein, comprising administering the PD-1 binding agent of the first aspect or the pharmaceutical composition the eighth aspect to a non-human subject in need thereof.

In a twelfth aspect, the ion relates to a method for enhancing an immune response or increasing the activity of an immune cell in a non-human subject comprising administering the PD-1 binding agent of the first aspect or the pharmaceutical composition of the eighth aspect to the man subject.

Claims (26)

CLAIMS :

1. A mmed Death 1 (PD-1) binding agent comprising: a heavy chain immunoglobulin variable region (VH) polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:6-10; and a light chain immunoglobulin variable region (VL) polypeptide sing the amino acid sequence of SEQ ID NO:29.

2. The PD-1 binding agent of claim 1, wherein the PD-1 binding agent is an antibody or antigen-binding dy fragment.

3. The PD-1 binding agent of claim 1 or 2, wherein the PD-1 binding agent comprises an IgG1, IgG2, or IgG4 heavy chain constant region (Fc).

4. The PD-1 binding agent of any one of claims 1-3, wherein the PD-1 binding agent comprises a VH polypeptide comprising the amino acid sequence of SEQ ID NO:6, and a VL ptide comprising the amino acid sequence of SEQ ID NO:29.

5. The PD-1 g agent of any one of claims 1-3, wherein the PD-1 binding agent comprises a VH polypeptide comprising the amino acid sequence of SEQ ID NO:7, and a VL polypeptide comprising the amino acid sequence of SEQ ID NO:29.

6. The PD-1 binding agent of any one of claims 1-3, wherein the PD-1 g agent comprises a VH polypeptide comprising the amino acid sequence of SEQ ID NO:8, and a VL polypeptide comprising the amino acid sequence of SEQ ID NO:29.

7. The PD-1 binding agent of any one of claims 1-3, wherein the PD-1 binding agent comprises a VH polypeptide comprising the amino acid sequence of SEQ ID NO:9, and a VL polypeptide comprising the amino acid sequence of SEQ ID NO:29.

8. The PD-1 binding agent of any one of claims 1-3, wherein the PD-1 binding agent ses a VH polypeptide comprising the amino acid sequence of SEQ ID NO:10, and a VL polypeptide comprising the amino acid sequence of SEQ ID NO:29.

9. A nucleic acid encoding the VH polypeptide and the VL polypeptide of the PD-1 binding agent of any one of claims 1-3.

10. An expression vector comprising the nucleic acid of claim 9.

11. An ex vivo host cell sing the nucleic acid of claim 9.

12. A method of producing the PD-1 binding agent of any one of claims 1-8, the method comprising expressing a nucleotide sequence ng the immunoglobulin heavy and light chains of the PD-1 binding agent in a cell in vitro.

13. A pharmaceutical composition comprising an effective amount of the PD-1 binding agent of any one of claims 1-8 and a pharmaceutically acceptable carrier or diluent.

14. Use of the PD-1 g agent of any one of claims 1-8 in the manufacture of a ment for treating cancer in a subject.

15. The use according to claim 14 n the medicament is formulated for use in ation with a TIM-3 binding agent or a LAG-3 binding agent.

16. The use according to claim 14 or claim 15, wherein the cancer is associated with overexpression of PD-1 protein.

17. The use according to claim 14, wherein the medicament enhances an immune response or increases the activity of an immune cell in a subject.

18. The use according to claim 17 wherein the medicament is formulated for use in combination with a TIM-3 binding agent or a LAG-3 binding agent.

19. A method for treating a cancer associated with overexpression of PD-1 protein comprising administering the PD-1 binding agent of any one of claims 1-8 or the pharmaceutical composition of claim 13 to a non-human subject in need thereof.

20. The method of claim 19, wherein the cancer is selected from the group consisting of: ma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, 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.

21. The method of claim 19 or claim 20, wherein the PD-1 binding agent or the pharmaceutical ition enhanced an immune response or increases the ty of an immune cell in the t.

22. The method of claim 21, wherein the subject has an infectious disease.

23. The method of claim 22, n the infectious disease is caused by a virus or a bacterium.

24. The method of claim 23, wherein the virus is human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), influenza virus, dengue virus, or tis B virus (HBV).

25. The method of any one of claims 19-24, further comprising administering to the subject an antibacterial agent and/or an anti-viral agent.

26. The method of any one of claims 19-25, further comprising administering to the subject a TIM-3 binding agent and/or a LAG-3 binding agent.