BR112017010762B1 - ANTI-PD-1 ANTAGONIST ANTIBODY, PHARMACEUTICAL COMPOSITION, KIT, ITS USES AND METHOD OF PRODUCING AN ANTI-PD-1 ANTAGONIST ANTIBODY - Google Patents

Abstract

ANTI-PD-1 ANTAGONIST ANTIBODY, ISOLATED CELL LINE, ISOLATED NUCLEIC ACID, RECOMBINANT EXPRESSION VECTOR, HOST CELL, HYBRIDOMA, PHARMACEUTICAL COMPOSITION, KIT, ITS USES AND METHOD OF PRODUCING AN ANTI-PD-1 ANTAGONIST ANTIBODY. The present invention relates to antagonist antibodies that bind to programmed cell death protein 1 (PD-1) and methods of using the same. Anti-PD-1 antibodies can be used therapeutically alone or in combination with other therapeutics to treat cancer and other diseases.

Description

FIELD

[001] The present invention relates to antibodies, for example, full-length antibodies that bind to PD-1. The invention further relates to compositions comprising antibodies to PD-1, and methods of using anti-PD-1 antibodies as a medicament. Certain embodiments refer to methods of using anti-PD-1 antibodies for the treatment, prevention and/or diagnosis of various diseases, including hyperproliferative disease such as cancer.

BACKGROUND

[002] PD-1 is a 50 to 55 kDa type I transmembrane receptor that was originally identified in a T cell line that undergoes activation-induced apoptosis. PD-1 is expressed on T cells, B cells and macrophages. The ligands for PD-1 are the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).

[003] PD-1 is a member of the immunoglobulin (Ig) superfamily that contains a single Ig type V domain in its extracellular region. The cytoplasmic domain of PD-1 contains two tyrosines, with the most membrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM (immunoreceptor tyrosine-based inhibitory motif). The presence of an ITIM on PD-1 indicates that this molecule functions to attenuate antigen receptor signaling through the recruitment of cytoplasmic phosphatases. Human and murine PD-1 proteins share approximately 60% amino acid identity with conservation of four potential N-glycosylation sites, and residues defining the Ig V domain. The ITIM in the cytoplasmic region and the ITIM-like motif involving the carboxy-terminal tyrosine are also conserved between human and murine orthologs.

[004] Cancer immunotherapy has traditionally involved complicated methods using individualized and time-consuming cells and preparations. Recently, monoclonal antibody-based cancer immunotherapy based on disruption of suppressive signals that are delivered to the adaptive immune system has shown promise in the clinic within the off-the-shelf systemic immunotherapy setting. However, there is a continuing need in the art for safer and more effective cancer treatments.

SUMMARY

[005] Antibodies that selectively interact with PD-1 are provided. Certain anti-PD-1 antibodies have been shown to be effective in vivo to prevent and/or treat cancer. Advantageously, the anti-PD-1 antibodies provided herein bind to human, cynomolgus monkey and mouse PD-1. Also advantageously, the anti-PD-1 antibodies provided here are effective in vivo for stimulating T cell proliferation.

[006] Isolated antagonist antibodies that specifically bind to PD-1 and prevent or reduce the biological effect of PD-1 are provided here. In some embodiments, the antagonist antibody may be, for example, a human, humanized or chimeric antibody.

[007] The invention disclosed here is directed to antibodies that bind to PD-1.

[008] In one aspect, the invention provides an isolated antagonist antibody that specifically binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR1), CDR2 of VH and VH CDR3 of VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6; is a light chain variable region (VL) comprising a VL CDR1, VL CDR2 and VL CDR3 that has an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO:7; SEQ ID NO: 8; and SEQ ID NO: 9.

[009] In some embodiments, the VH region comprises the amino acid sequence shown in SEQ ID NO: 3, 4, 5 or 6, or a variant with one or more conservative amino acid substitutions at residues that are not within a CDR and /or the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 8 or 9, or a variant thereof with one or more amino acid substitutions in amino acids that are within a CDR. In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO: 39 and/or a heavy chain comprising the sequence shown in SEQ ID NO: 29 or 38. In some embodiments, the antibody comprises a VH region produced by the expression vector with ATCC Accession No. PTA-121183. In some embodiments, the antibody comprises a VL region produced by the expression vector with ATCC Accession No. PTA-121182.

[0010] In another aspect, the invention provides an isolated antibody that specifically binds to PD-1, wherein the antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15, a CDR2 of VH comprising the amino acid sequence of SEQ ID NO: 16, 17, 24, 25, 27, 28, 35 or 36, a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, 23, 2, or 37 , a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, 22, 30 or 32, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, 20 or 33 and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, 21, 31 or 34.

[0011] In some embodiments, the antibody may be a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody.

[0012] In some embodiments, the antibody comprises a constant region. In some embodiments, the antibody is of the subclass of human IgG1, IgG2, IgG2Δa, IgG3, IgG4, IgG4Δb, IgG4Δc, IgG4 S228P, IgG4Δb S228P, and IgG4Δc S228P. In some embodiments, the antibody is of the IgG4 isotype and comprises a stabilized hinge, e.g., S228P.

[0013] In another aspect, the invention provides an isolated antibody that specifically binds to PD-1 and competes with and/or binds to the same epitope of PD-1 as the antibodies as described herein.

[0014] In some embodiments, an anti-PD-1 antibody provided herein promotes IFN and/or TNF secretion from T cells.

[0015] In some embodiments, an anti-PD-1 antibody provided here promotes T cell proliferation.

[0016] In some embodiments, an anti-PD-1 antibody provided herein inhibits tumor growth.

[0017] In some embodiments, an anti-PD-1 antibody provided herein binds to human PD-1 and mouse PD-1.

[0018] In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a PD-1 antibody as described herein and a pharmaceutically acceptable carrier.

[0019] In another aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a PD-1 antibody as described herein. In another aspect, the invention provides a vector comprising the polynucleotide.

[0020] In another aspect, the invention provides an isolated host cell that recombinantly produces a PD-1 antibody as described herein.

[0021] In another aspect, the invention provides a method of producing an anti-PD-1 antagonist antibody, the method comprising: cultivating a cell line that recombinantly produces the antibody as described herein under conditions in which the antibody is produced; and recover the antibody.

[0022] In another aspect, the invention provides a method of producing an anti-PD-1 antagonist antibody, the method comprising: cultivating a cell line comprising nucleic acid encoding an antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 29 or 38 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39 under conditions in which the antibody is produced; and recover the antibody.

[0023] In some embodiments, the heavy and light chains are encoded in separate vectors. In other embodiments, the heavy and light chains are encoded in the same vector.

[0024] In another aspect, the invention provides a method for treating a condition in an individual comprising administering to the individual in need an effective amount of the pharmaceutical composition as described herein. In some embodiments, the condition is cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer and melanoma. In some embodiments, the subject is a previously treated adult patient with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian carcinoma, sarcoma, or relapsed or refractory classical Hodgkin Lymphoma (cHL). In some embodiments, the cancer may be a platinum-refractory and/or platinum-resistant cancer, such as, for example, platinum-refractory and/or platinum-resistant ovarian cancer, platinum-refractory and/or platinum-resistant breast cancer, or breast cancer. refractory and/or platinum-resistant lung. In some embodiments, an anti-PD-1 antibody is administered at a dosage of about 0.5 mg/kg, about 1.0 mg/kg, about 3.0 mg/kg, or about 10 mg/kg. . In some embodiments, the anti-PD-1 antibody is administered once every 7, 14, 21 or 28 days. In some embodiments, the anti-PD-1 antibody is administered intravenously or subcutaneously.

[0025] In another aspect, the invention provides a method of inhibiting tumor growth or progression in an individual who has a tumor, comprising administering to the individual an effective amount of the pharmaceutical composition as described herein.

[0026] In another aspect, the invention provides a method of inhibiting or preventing metastasis of cancer cells in an individual, comprising administering to the individual in need an effective amount of the pharmaceutical composition as described herein.

[0027] In another aspect, the invention provides a method of inducing tumor regression in an individual who has a PD-1 expressing tumor, comprising administering to the individual an effective amount of the pharmaceutical composition as described herein.

[0028] In some embodiments, the antibody herein can be administered parenterally to an individual. In some embodiments, the individual is a human.

[0029] In some embodiments, the method may further comprise administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is, for example, crizotinib, palbociclib, an anti-CTLA4 antibody, an anti-4-1BB antibody, or a second PD-1 antibody.

[0030] Also provided is the use of any of the anti-PD-1 antagonist antibodies provided herein in the manufacture of a medicament for the treatment of cancer or for inhibiting tumor growth or progression in an individual in need thereof. In some embodiments, the anti-PD-1 antagonist antibody reduces weight gain in the individual.

[0031] Also provided here are anti-PD-1 antagonist antibodies for use in treating cancer or for inhibiting tumor growth or progression in an individual in need. In some embodiments, the cancer is, for example without limitation, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, cancer thyroid, lung cancer (including, for example, non-small cell lung carcinoma), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer and melanoma.

[0032] In another aspect, the present description provides a method for increasing the immunogenicity or therapeutic effect of a vaccine for the treatment of cancer in a mammal, particularly a human being, which method comprises administering to the mammal receiving the vaccine an amount effective anti-PD-1 antagonist antibody provided by the present disclosure.

[0033] In another aspect, the present description provides a method for treating cancer in a mammal, particularly a human being, which method comprises administering to the mammal (1) an effective amount of a vaccine capable of inducing an immune response against cells of the cancer and (2) an effective amount of an anti-PD-1 antagonist antibody provided by the present disclosure.

BRIEF DESCRIPTION OF FIGURES/DRAWINGS

[0034] Figure 1A depicts a graph that summarizes the body weight of mice treated with anti-PD-1 antagonist antibody.

[0035] Figure 1B depicts a graph that summarizes the body weight of mice treated with anti-PD-1 antagonist antibody.

[0036] Figure 1C depicts a graph that summarizes the body weight of mice treated with anti-PD-1 antagonist antibody.

[0037] Figure 1D depicts a graph that summarizes the body weight of mice treated with anti-PD-1 antagonist antibody.

[0038] Figure 1E depicts a graph that summarizes the body weight of mice treated with anti-PD-1 antagonist antibody.

[0039] Figure 2A depicts a graph summarizing EC50 for anti-PD-1 antibody that binds to primary activated human T cells.

[0040] Figure 2B depicts a graph summarizing EC50 for anti-PD-1 antibody that binds to primary activated cynomolgus T cells.

[0041] Figure 3 depicts a bar graph that summarizes the proliferation of cultured activated T cells treated as follows (a) in the antibody; (b) isotype control; (c) EH12.1; (d) C1; (e) C2; (f) C3; (g) mAb1; (h) mAbX; (i) mAb4; (j) mAb5; (k) mAb6; (l) mAb7; (m) mAb9; (n) mAb10; (o) mAb11; (p) mAb14; (q) mAb15; (r) mAb16.

[0042] Figure 4 depicts a bar graph that summarizes the proliferation of cultivated activated CD8 T cells treated as follows (a) in the antibody; (b) isotype control; (c) EH12.1; (d) C1; (e) C2; (f) C3; (g) mAb1; (h) mAbX; (i) mAb4; (j) mAb5; (k) mAb6; (l) mAb7; (m) mAb9; (n) mAb10; (o) mAb11; (p) mAb14; (q) mAb15; (r) mAb16.

DETAILED DESCRIPTION

[0043] Here antibodies that specifically bind to PD-1 are revealed. Methods of manufacturing anti-PD-1 antibodies, compositions comprising such antibodies, and methods of using such antibodies as a medicament are provided. Anti-PD-1 antibodies can be used to inhibit tumor progression, and can be used in the prevention and/or treatment of cancer and/or other diseases.

GENERAL TECHNIQUES

[0044] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

DEFINITIONS

[0045] The following terms, unless otherwise indicated, should be understood to have the following meanings: the term "isolated molecule", as referring to a molecule, is, for example, a polypeptide, a polynucleotide or an antibody that , by virtue of its origin or source of derivation (1) is not associated with naturally associated components accompanying it in its natural state, (2) is substantially free of other molecules of the same source, e.g., species, cells from from which it is expressed, library, etc., (3) is expressed by a cell of a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system other than the system from which it naturally originates, will be "isolated" from its naturally associated components. A molecule can also be processed substantially free of naturally associated components by isolation using purification techniques known in the art. The purity or homogeneity of the molecule can be assessed in several ways well known in the art. For example, the purity of a polypeptide sample can be assessed using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be provided through the use of HPLC or other means well known in the art for purification.

[0046] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise indicated, any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising a portion of antigen binding, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen-binding moieties include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single-chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer binding of specific antigen to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA or IgM (or subclass thereof), and the antibody need not be any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0047] A "variable region" of an antibody refers to the variable region of the light chain of the antibody or the variable region of the heavy chain of the antibody, whether alone or in combination. As known in the art, the variable regions of the light and heavy chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions and contribute to the formation of the antigen binding site. of antibodies. If variants of an individual's variable region are desired, particularly with substitutions at amino acid residues outside of a CDR region (i.e., in the framework region), the appropriate amino acid substitution, preferably conservative amino acid substitution, can be identified. by comparing the subject's variable region to the variable regions of other antibodies that contain CDR1 and CDR2 sequence in the same canonical class as the subject's variable region (Chothia and Lesk, J Mol Biol 196(4): 901-917, 1987) .

[0048] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. In certain embodiments, which may be performed by any variety of techniques known to those skilled in the art, such as x-ray crystallography. In certain embodiments, various analytical methods may be employed to identify or approximate the CDR regions. In certain embodiments, various analysis methods can be employed to identify or approximate CDR regions. Examples of these methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the conformational definition.

[0049] The Kabat definition is a standard for numbering residues in an antibody and is typically used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. Chothia's definition is similar to Kabat's definition, but Chothia's definition takes into account positions of certain structural mesh regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated set of computer programs produced by the Oxford Molecular Group that model the antibody structure. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of a primary sequence antibody using a combination of knowledge base and ab initio methods, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The definition of contact is based on an analysis of the available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to here as the "conformational definition" of CDRs, the positions of the CDRs can be identified as the residues that make antigen-binding enthalpic contributions. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still other definitions of CDR limits may not strictly follow one of the above approaches, but will nevertheless overlap with at least a portion of Kabat's CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that residues Particulars or groups of residues do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used here can utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.

[0050] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, whether alone or in combination.

[0051] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in the Patent. US No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. As used herein, "humanized" antibody refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab ')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues of a CDR of the recipient are replaced by residues of a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the specificity, affinity and desired capacity. The humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequence, but are included to further refine and optimize the performance of the antibody.

[0052] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for manufacturing human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0053] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the region sequences variable are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0054] The term "epitope" refers to that portion of a molecule capable of being recognized or bound by an antibody in one or more of the antibody's antigen-binding regions. Epitopes often consist of a surface cluster of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, the epitope may be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" comprises noncontiguous polypeptides (or amino acids) within the antigenic protein to which an epitope-specific antibody binds. The term "antigenic epitope" as used herein is defined as a portion of an antigen to which an antibody can specifically bind as determined by any method well known in the art, for example, by conventional immunoassays. Once the desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, for example, using the techniques described in the present patent application. Alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information about desirable epitopes. Using this information, it is then possible to competitively screen antibodies for binding to the same epitope. One approach to achieving this is to conduct competition and cross-competition studies to find antibodies that compete or cross-compete with each other for binding to PD-1, e.g., the antibodies compete for antigen binding.

[0055] As used herein, the term "PD-1" refers to any form of PD-1 and variants thereof that retain at least part of the activity of PD-1. Unless otherwise indicated, such as specific reference to human PD-1, PD-1 includes all mammalian species of natural PD-1 sequence, e.g., human, canine, feline, equine and bovine. An exemplary human PD-1 is found as Uniprot Accession Number Q15116 (SEQ ID NO: 1).

[0056] The term "agonist" refers to a substance that promotes (that is, induces, causes, enhances or increases) the biological activity or effect of another molecule. The term agonist encompasses substances that bind the receptor, such as an antibody, and substances that promote receptor function without binding to them (for example, by activating an associated protein).

[0057] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes or reduces a biological activity or effect of another molecule, such as a receptor.

[0058] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of that target. In some embodiments, the term may indicate a target-preventing antibody, e.g., PD-1, to which it is bound to perform a biological function.

[0059] As used herein, an "anti-PD-1 antagonist antibody" refers to an antibody that is capable of inhibiting the biological activity of PD-1 and/or PD-mediated downstream event(s). -1. Anti-PD-1 antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) the biological activity of PD-1, including PD-1-mediated downstream events such as PD-L1 binding. and downstream signaling, PD-L2 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10 and inhibition of antitumor immune responses. For the purposes of the present invention, it will be explicitly understood that the term "anti-PD-1 antagonist antibody" (indistinctly referred to as "PD-1 antagonist antibody", "anti-PD-1 antagonist antibody" or "PD-1 antagonist antibody" 1") encompasses all previously identified terms, titles, and functional states and characteristics by which PD-1 itself, a biological activity of PD-1, or the consequences of the biological activity, are substantially nullified, reduced, or neutralized to any significant degree . In some embodiments, an anti-PD-1 antagonist antibody binds to PD-1 and upregulates an antitumor immune response. Examples of anti-PD-1 antagonist antibodies are provided herein.

[0060] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably here to refer to amino acid chains of any length. The chain may be linear or branched, it may comprise modified amino acids and/or it may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides that contain one or more analogues of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It is understood that polypeptides can occur as single chains or associated chains.

[0061] As known in the art, "polynucleotide" or "nucleic acid", as used interchangeably herein, refers to chains of nucleotides of any length, and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogues, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and analogues thereof. If present, the modification to the nucleotide structure can be imparted before or after chain assembly. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "capsules", replacement of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as, for example, those with uncharged linkages (e.g. methyl phosphonates, phosphotriesters , phosphoamidates, carbamates, etc.) and with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing dangling moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Furthermore, any of the hydroxyl groups commonly present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups or activated to prepare additional bonds for additional nucleotides, or can be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido -ribose, carboxylic sugar analogues, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acrylic analogues and abasic nucleoside analogues such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linkage groups. Such alternative linkage groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate "), P(O)R, P(O)OR', CO or CH2 ("formacetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing a ether bond (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide need to be identical. The previous description applies to all polynucleotides referred to here, including RNA and DNA.

[0062] As used herein, an antibody "interacts with" PD-1 when the equilibrium dissociation constant is equal to or less than 20 nM, preferably less than about 6 nM, more preferably less than about 1 nM, more preferably even less than about 0.2 nM, as measured by the methods disclosed herein in Example 7.

[0063] An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more quickly, longer lastingly and/or with greater affinity with a particular cell or substance than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" a target if it binds with greater affinity, avidity, more readily, and/or longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a PD-1 epitope is an antibody that binds that epitope with greater affinity, avidity, more readily, and/or longer duration than it binds to other PD-1 epitopes. or non-PD-1 epitopes. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferred binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0064] As used herein, "substantially pure" refers to material that is at least 50% pure (i.e., free from contaminants), more preferably, at least 90% pure, more preferably, at least 95% pure, even more preferably, at least 98% pure, and most preferably, at least 99% pure.

[0065] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide insertions. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in complement of genomic DNA) to the original progenitor cell due to natural, accidental or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

[0066] As known in the art, the term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus thereof. The numbering of residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. As is known in the art, an Fc region can be present in dimeric or monomeric form.

[0067] As used in the art, the "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors from the FCYRI, FCYRII and FCYRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FCYRII receptors include FCYRIIA (an "activating receptor") and FCYRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[0068] The term "compete", as used herein with respect to an antibody, means that a first antibody, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody. , or an antigen-binding portion thereof, such that the binding result of the first antibody with its cognate epitope is detectably reduced in the presence of the second antibody compared to binding of the first antibody in the absence of the second antibody. The alternative, where binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, may, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater or lesser extent, the antibodies are said to be "cross-competing" with each other for binding to their respective ) epitope(s). Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), one skilled in the art would appreciate, based on the teachings provided herein, that such competing antibodies and/or cross competitors are encompassed and may be useful for the methods disclosed herein.

[0069] A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor), etc. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays known in the art for evaluating such antibody effector functions.

[0070] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification still retaining at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably, from about from one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably have at least about 80% sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and more preferably at least about 90% sequence identity with that, more preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[0071] As used herein, "treatment" is an approach to achieving beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of a tumor, remission of cancer, decrease symptoms resulting from cancer, increase the quality of life of those suffering from cancer, decrease the dose of other medications required to treat cancer, delay the progression of cancer, cure cancer and/or prolong the survival of cancer patients.

[0072] "Improvement" means a decrease or improvement in one or more symptoms as compared to not administering an anti-PD-1 antagonist antibody. "Improvement" also includes shortening or reducing the duration of a symptom.

[0073] As used herein, an "effective dosage" or "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. In more specific aspects, an effective amount prevents, alleviates or improves the symptoms of the disease and/or prolongs the survival of the individual being treated. For prophylactic use, the beneficial or desired results include eliminating or reducing the risk, decreasing the severity or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes that present during development. of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms of a disease such as, for example, cancer including, for example, without limitation, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer , esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer and melanoma, decreasing the dose of other medications required to treat the disease, increasing the effect of other medication and/or delaying the progression of cancer in patients. An effective dosage may be administered in one or more administrations. For purposes of this invention, an effective dosage of the drug, compound or pharmaceutical composition is an amount sufficient to carry out prophylactic or therapeutic treatment directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be achieved. be or is achieved.

[0074] A "person" or an "individual" is a mammal, more preferably a human being. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sporting animals, pets, primates, horses, dogs, cats, mice, and rats.

[0075] As used herein, "vector" means a construct, which is capable of preferentially delivering, expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA expression vectors or RNA encapsulated in liposomes and certain eukaryotic cells, such as producer cells.

[0076] As used herein, "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. An expression control sequence may be a promoter, such as a constitutive or inducible promoter or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0077] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and be non-reactive with the individual's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal saline (0.9%). Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0078] The term "kon", as used herein, refers to the rate constant for association of an antibody to an antigen. Specifically, rate constants (kon and koff) and equilibrium dissociation constants are measured using full-length antibodies and/or Fab (i.e., univalent) and PD-1 antibody fragments.

[0079] The term "koff", as used herein, refers to the rate constant for dissociation of an antibody from the antibody/antigen complex.

[0080] The term "KD", as used herein, refers to the equilibrium dissociation constant of an antibody-antigen interaction.

[0081] Reference to "about" a value or parameter herein includes (and describes) modalities that are directed to that value or parameter per se. For example, description that refers to "about X" includes description of "X." Numeric ranges are inclusive of the numbers that define the range.

[0082] The term "immune effector cell enhancer" or "IEC enhancer" refers to a substance capable of increasing or improving the number, quality or function of one or more types of immune effector cells in a mammal. Examples of immune effector cells include cytolytic CD8 T cells, CD4 T cells, NK cells, and B cells.

[0083] The term "immune modulator" refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing or stimulating) the immune response (as defined here) or the work of any component of the innate immune system , humoral, or cellular of a mammalian host. Thus, the term "immune modulator" encompasses "immune effector cell enhancer" as defined herein and "immune suppressor cell inhibitor" as defined herein, as well as substances that affect other components of a mammal's immune system.

[0084] The term "immune response" refers to any detectable response to a particular substance (such as an antigen or immunogen) by the immune system of a host mammal, such as innate immune responses (e.g., activation of a signaling cascade Toll receptor receptor), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells and non-specific immune cells), and humoral immune responses (e.g., B cell-mediated responses, such as generation and secretion of antibodies into plasma, lymph and/or tissue fluids).

[0085] The term "immunogenic" refers to the ability of a substance to cause, extract, stimulate or induce an immune response or improve, enhance, increase or prolong a pre-existing immune response, against a particular antigen, whether alone or when connected to a vehicle, in the presence or absence of an adjuvant.

[0086] The term "immune suppressor cell inhibitor" or "ISC inhibitor" refers to a substance capable of reducing or suppressing the number or fraction of immune suppressor cells in a mammal. Examples of immune suppressor cells include regulatory T cells ("T regs"), myeloid-derived suppressor cells, and tumor-associated macrophages.

[0087] The term "intradermal administration" or "intradermally administered", in the context of administering a substance to a mammal including a human, refers to the distribution of the substance in the dermis layer of the mammal's skin. A mammal's skin is made up of an epidermis layer, a dermis layer, and a subcutaneous layer. The epidermis is the outermost layer of the skin. The dermis, which is the middle layer of the skin, contains nerve endings, sweat glands and oil (sebaceous) glands, hair follicles and blood vessels. The subcutaneous layer is made of fat and connective tissue that houses the largest blood vessels and nerves. In contrast to intradermal administration, "subcutaneous administration" refers to the administration of a substance into the subcutaneous layer and "topical administration" refers to the administration of a substance to the surface of the skin.

[0088] The term "neoplastic disorder" refers to a condition in which cells proliferate at an abnormally high and uncontrolled rate, the rate exceeding and not coordinated with that of the surrounding normal tissues. It usually results in a solid lesion or lump known as a "tumor." This term encompasses benign and malignant neoplastic disorders. The term "malignant neoplastic disorder", which is used interchangeably with the term "cancer" in the present description, refers to a neoplastic disorder characterized by the ability of tumor cells to spread to other locations in the body (known as "metastasis"). . The term "benign neoplastic disorder" refers to a neoplastic disorder in which tumor cells lose the ability to metastasize.

[0089] The term "preventing" or "preventing" refers to (a) preventing a disorder from occurring or (b) delaying the onset of a disorder or onset of symptoms of a disorder.

[0090] The term "tumor-associated antigen" or "TAA" refers to an antigen that is specifically expressed by tumor cells or expressed at a higher frequency or density on tumor cells than by non-tumor cells of the same type of tissue. Tumor-associated antigens may be antigens not normally expressed by the host; they may be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they may be identical to normally expressed molecules but expressed at abnormally high levels; or they may be expressed in a context or medium that is abnormal. Tumor-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these and other biological molecules.

[0091] The term "vaccine" refers to an immunogenic composition for administration to a mammal to induce an immune response against a particular antigen in the mammal. A vaccine typically contains an agent (known as an "antigen" or "immunogen") that resembles, or is derived from, the target of the immune response, such as a disease-causing microorganism or tumor cells. A vaccine intended for the treatment of a tumor, such as cancer, typically contains an antigen that is derived from a TAA found in the target tumor and is capable of inducing immunogenicity against the TAA in the target tumor.

[0092] The term "vaccine-based immunotherapy regimen" refers to a therapeutic regimen in which a vaccine is administered in combination with one or more immune modulators. The vaccine and immune modulators can be administered together in a single formulation or administered separately.

[0093] It is understood that whenever embodiments are described herein with the language "comprising", otherwise analogous embodiments described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[0094] Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group missing one or more of the group members. The present invention also provides for the explicit exclusion of one or more of any of the members of the group in the claimed invention.

[0095] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. In the event of a conflict, this patent application, including definitions, will control. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer. or group of integers. Unless otherwise required by the context, singular terms shall include pluralities and plural terms include the singular. Any example(s) that follow the term "for example" or "for example" are not intended to be exhaustive or limiting.

[0096] Exemplary methods and materials are described here, although similar or equivalent methods and materials to those described here may be used in the practice or testing of the present invention. The materials, methods and examples are illustrative only and are not intended to be limiting.

ANTI-PD-1 ANTAGONIST ANTIBODIES

[0097] Herein anti-PD-1 antagonist antibodies are provided that block, suppress or reduce (including significantly reduce) the biological activity of PD-1, including downstream events mediated by PD-1. An anti-PD-1 antagonist antibody must exhibit any one or more of the following characteristics: (a) bind to PD-1 and block downstream signaling events; (b) block PD-L1 from binding to PD-1; (c) upregulate a T cell-mediated immune response; (d) stimulate IFNY secretion; (e) stimulate the secretion of TNF; (f) increase T cell proliferation; and (g) reduce inhibitory signal transduction through PD-1.

[0098] For purposes of this invention, the antibody preferentially reacts with PD-1 in a manner that inhibits the signaling function of PD-1. In some embodiments, the anti-PD-1 antagonist antibody specifically binds to primate PD-1.

[0099] Antibodies useful in the present invention may encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain antibodies (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., an antibody domain), humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises a ScFv recognition site. antigen of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. Antibodies may be of murine, rat, human or any other origin (including chimeric or humanized antibodies). In some embodiments, the anti-PD-1 antagonist antibody is a monoclonal antibody. In some embodiments, the antibody is a human or humanized antibody.

[00100] Anti-PD-1 antagonist antibodies can be made by any method known in the art. General techniques for producing human and mouse antibodies are known in the art and/or are described herein.

[00101] Anti-PD-1 antagonist antibodies can be identified or characterized using methods known in the art, whereby the reduction, improvement or neutralization of PD-1 biological activity is detected and/or measured. In some embodiments, an anti-PD-1 antagonist antibody is identified by incubating a candidate agent with PD-1 and monitoring the binding and/or reduction or neutralization of PD-1 biological activity. The binding assay can be performed with, for example, purified PD-1 polypeptide(s) or with cells that naturally express (e.g., various strains), or transfected to express, PD-1 polypeptide(s). In one embodiment, the binding assay is a competitive binding assay, where the ability of a candidate antibody to compete with a known anti-PD-1 antagonist antibody for PD-1 binding is evaluated. The assay can be performed in several formats, including the ELISA format. In some embodiments, an anti-PD-1 antagonist antibody is identified by incubating a candidate antibody with PD-1 and monitoring ligation.

[00102] After initial identification, the activity of a candidate anti-PD-1 antagonist antibody can be further confirmed and refined by bioassays, known for testing targeted biological activities. In some embodiments, an in vitro cell assay is used to further characterize a candidate anti-PD-1 antagonist antibody. For example, a candidate antibody is incubated with primary human T cells and PD-L1 is added, and IFNY secretion is monitored. Alternatively, bioassays can be used to screen candidates directly.

[00103] The anti-PD-1 antagonist antibodies of the invention exhibit one or more of the following characteristics: (a) bind to PD-1 and block downstream signaling events; (b) block PD-L1 which binds to PD-1; (c) upregulate a T cell-mediated immune response; (d) stimulate the secretion of IFNY; (e) stimulate the secretion of TNF; (f) increase T cell proliferation; (g) reduce inhibitory signal transduction through PD-1; and (h) blocking PD-L2 from binding to PD-1. Preferably, anti-PD-1 antibodies have two or more of these characteristics. More preferably, the antibodies have three or more of the characteristics. More preferably, the antibodies have four or more of the characteristics. More preferably, the antibodies have five or more of the characteristics. More preferably, the antibodies have six or more of the characteristics. More preferably, the antibodies have seven or more of the characteristics. More preferably, the antibodies have all eight characteristics.

[00104] Anti-PD-1 antagonist antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope to which it binds or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays. as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In a further example, epitope mapping can be used to determine the sequence in which an anti-PD-1 antagonist antibody binds. Epitope mapping is commercially available from several sources, e.g. Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope can be a linear epitope, that is, contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that cannot necessarily be contained in a single stretch. Peptides of variable lengths (e.g., at least 4 to 6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an anti-PD-1 antagonist antibody. In another example, the epitope to which the anti-PD-1 antagonist antibody binds can be determined in a systematic screen by using overlapping peptides derived from the PD-1 sequence and determining binding by the anti-PD-1 antagonist antibody. According to gene fragment expression assays, the open reading frame encoding PD-1 is fragmented, either randomly or by specific genetic constructs, and the reactivity of the expressed fragments of PD-1 with the antibody to be tested is determined. Gene fragments can, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of reactive amino acids. Antibody binding to radioactively labeled PD-1 fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in single binding assays. In a further example, mutagenesis of an antigen, domain swap experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient and/or necessary for epitope binding. For example, alanine scanning mutagenesis experiments can be performed using a PD-1 mutant in which several residues of the PD-1 polypeptide have been replaced with alanine. By evaluating antibody binding to mutant PD-1, the importance of particular PD-1 residues for antibody binding can be assessed.

[00105] Yet another method that can be used to characterize an anti-PD-1 antagonist antibody is to use competition assays with other antibodies known to bind to the same antigen, i.e., multiple fragments of PD-1, to determine whether The anti-PD-1 antagonist antibody binds to the same epitope as other antibodies. Competition assays are well known to those skilled in the art, including in an ELISA format.

[00106] The binding affinity (KD) of an anti-PD-1 antagonist antibody for PD-1 can be from about 0.001 to about 200 nM. In some embodiments, the binding affinity is any of about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM , about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM or about 1 pM. In some embodiments, the binding affinity is less than any of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM or about 2 pM.

[00107] Accordingly, the invention provides any of the following or compositions (including pharmaceutical compositions) comprising an antibody having a partial light chain sequence and a partial heavy chain sequence as found in Table 1, or variants thereof. In Table 1, the underlined sequences are CDR sequences. In Table 1, KD indicates affinity for human PD-1 as measured using surface plasmon resonance at 25°C unless otherwise noted. Table 1: Variable Region Sequences of anti-PD-1 antagonist antibodies

[00108] The invention also provides CDR portions of antibodies to PD-1. Determination of CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of the Kabat and Chothia CDR (also referred to as "combined CDRs" or "extended CDRs"). In another approach, referred to here as the "conformational definition" of CDRs, the positions of the CDRs can be identified as the residues that make enthalpic contributions to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. In general, "conformational CDRs" include the residue positions in the Kabat CDRs and Vernier zones that are constrained in order to maintain the proper loop structure for the antibody to bind a specific antigen. Determination of conformational CDRs is well within the skill of the art. In some embodiments, the CDRs are the Kabat CDRs. In other embodiments, the CDRs are the Chothia CDRs. In other embodiments, the CDRs are extended, AbM, conformational or contact CDRs. In other words, in embodiments with more than one CDR, the CDRs can be any of the Kabat, Chothia, extended, AbM, conformational, contact CDRs or combinations thereof.

[00109] In some embodiments, the antibody comprises three CDRs from any of the heavy chain variable regions shown in Table 1. In some embodiments, the antibody comprises three CDRs from any of the light chain variable regions shown in Table 1. In In some embodiments, the antibody comprises three CDRs of any of the heavy chain variable regions shown in Table 1, and three CDRs of any of the light chain variable regions shown in Table 1.

[00110] Table 2 provides examples of CDR sequence of anti-PD-1 antagonist antibodies provided here. Table 2. Anti-PD-1 antagonist antibodies (mAbs) and their antigen-binding CDR sequences according to Kabat (underlined) and Chothia (bold)

[00111] In some embodiments, the antibody comprises three light chain CDRs and three heavy chain CDRs from Table 2.

[00112] An alignment of light chain CDRs of anti-PD-1 antibodies is provided in Table 3. Variable residues are shown in bold. The consensus light chain CDR sequences are provided in the last row of Table 3. Table 3. Alignment of anti-PD-1 light chain CDRs

[00113] An alignment of anti-PD-1 antibody heavy chain CDRs is provided in Table 4. Variable residues are shown in bold. The consensus heavy chain CDR sequences are provided in the last row of Table 4. Table 4. Alignment of anti-PD-1 heavy chain CDRs

[00114] In some embodiments, the antibody comprises three light chain CDRs from Table 3 and three heavy chain CDRs from Table 4.

[00115] In some embodiments, the antibody comprises the full-length heavy chain, with or without the C-terminal lysine and/or the full-length light chain of the anti-PD-1 antagonist antibody mAb7 or mAb15. The amino acid sequence of full-length mAb7 heavy chain (SEQ ID NO: 29) is shown below: VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 29) The amino acid sequence of mAb7 full-length heavy chain without the C-terminal lysine (SEQ ID NO: 38) is shown below: VHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:38)

[00116] The mAb7 full-length light chain amino acid sequence (SEQ ID NO: 39) is shown below: DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKP GQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY CQNDYFYPHTFGGGTKVEIKRGTVAAPSVFIFPPSDEQLKSGTASV V CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39)

[00117] The invention also provides methods for generating, selecting and manufacturing anti-PD-1 antagonist antibodies. The antibodies of this invention can be made by procedures known in the art. In some embodiments, the antibodies can be made recombinantly and expressed using any method known in the art.

[00118] In some embodiments, antibodies can be prepared and selected by phage display technology. See, for example, US Patent No. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, phage display technology (McCafferty et al., Nature 348:552-553, 1990) can be used to produce human antibodies and antibody fragments in vitro from gene repertoires of variable domain (V) of immunoglobulin from non-immunized donors. According to this technique, antibody V domain genes are cloned in frame, either into a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection for the gene encoding the antibody that exhibits those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571, 1993. Various sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628, 1991, isolated on a diverse array of antioxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of human donor V genes can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Mark et al., J. Mol. Biol. 222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993. In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity and B cells displaying high affinity immunoglobulin are preferentially replicated and differentiated during subsequent antigenic challenge. This natural process can be imitated by employing a technique known as "chain dragging." (Marks et al., Bio/Technol. 10:779-783, 1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing light and heavy chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from donors. not immunized. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for manufacturing very broad phage antibody repertoires (also known as "the master libraries of them all") has been described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene drag can also be used to derive human antibodies from rodent antibodies, where the human antibody has similar affinities and specificities to the rodent starting antibody. According to this method, which is also referred to as "epitope imprinting", the light or heavy chain V domain gene of rodent antibodies obtained by the phage display technique is replaced with a repertoire of human V domain genes, creating rodent-human chimeras. Selection on antigen results in the isolation of human variable regions capable of restoring a functional antigen-binding site, i.e., the epitope governs (records) mate choice. When the process is repeated in order to replace the remaining rodent V domain, a human antibody is obtained (see PCT Publication No. WO 93/06213). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides completely human antibodies, which have no structure or CDR residues of rodent origin.

[00119] In some embodiments, antibodies can be made using hybridoma technology. It is contemplated that any individual mammal including humans or antibody producing cells thereof may be manipulated to serve as the basis for producing mammalian hydridoma cell lines including humans. The route and schedule of immunization of the host animal are generally in accordance with established and conventional techniques for antibody stimulation and production, as further described herein. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly and/or intradermally with an amount of immunogen, including as described herein.

[00120] Hybridomas can be prepared from immortalized lymphocytes and myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., 1975, Nature 256:495-497 or as modified by Buck , D. W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines, including, but not limited to, X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif. ., USA, can be used in hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using fusogen such as polyethylene glycol or by electrical means well known to those skilled in the art. After fusion, cells are separated from the fusion medium and cultured in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized stem cells. Any of the media described here, supplemented with or without serum, can be used to cultivate hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells can be used to produce the PD-1 monoclonal antibodies of the subject matter of the invention. Hybridomas or other immortalized B cells are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogenic activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[00121] Hybridomas that can be used as a source of antibodies encompass all derivatives, progenitor cells of parental hybridomas that produce PD-1-specific monoclonal antibodies, or a portion thereof.

[00122] Hybridomas that produce such antibodies can be cultured in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration, if desired. Unwanted activity, if present, can be removed, for example, by running the preparation on adsorbents made from the immunogen attached to a solid phase and eluting or releasing the desired antibodies from the immunogen. Immunization of a host animal with a PD-1 polypeptide, or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R1N=C =NR, where R and R1 are different alkyl groups, can produce a population of antibodies (e.g., monoclonal antibodies).

[00123] If desired, the anti-PD-1 antagonist antibody (monoclonal or polyclonal) of interest can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in the vector in a host cell and the host cell can then be expanded and frozen for future use. The production of recombinant monoclonal antibodies in cell culture can be accomplished by cloning the B cell antibody genes by means known in the art. See, for example, Tiller et al., 2008, J. Immunol. Methods 329, 112; US Patent No. 7,314,622.

[00124] In some embodiments, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or improve the affinity, or other characteristics of the antibody. Antibodies can also be customized for use, for example, in dogs, cats, primates, horses and cattle.

[00125] In some embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are engineered to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used to generate humanized or human antibodies. Examples of such technology are Xenomouse™ from Abgenix, Inc. (Fremont, CA) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ).

[00126] Antibodies can be made recombinantly by first isolating the antibodies and antibody producing cells from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in the host cells (e.g. CHO cells). Another method that can be employed is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Methods for expressing the antibodies recombinantly in plants or milk have been disclosed. See, for example, Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods 231:147, 1999. Methods for manufacturing antibody derivatives, e.g., domain, single chain, etc. are known in the art.

[00127] Immunoassay and flow cytometry sorting techniques such as fluorescence-activated cell sorting (FACS) can also be employed to isolate antibodies that are specific to PD-1.

[00128] The DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (for example, by the use of oligonucleotide probes that are capable of specifically binding to the genes encoding the light and heavy chains of monoclonal antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors (such as expression vectors disclosed in PCT Publication No. WO 87/04462), which are then transfected into host cells such as E. coli cells, simian COS cells, , Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to achieve synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87/04462. DNA can also be modified, for example, by substituting the coding sequence for human light and heavy chain constant domains in place of the homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, "chimeric" or "hybrid" antibodies are prepared having the binding specificity of a PD-1 monoclonal antibody herein.

[00129] Antibody fragments can be produced by proteolytic or other degradation of the antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above or by chemical synthesis. Antibody polypeptides, specifically shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an antibody could be produced by an automatic polypeptide synthesizer employing the solid phase method. See also US Patent No. 5,807,715; 4,816,567; and 6,331,415.

[00130] In some embodiments, a polynucleotide comprises a sequence encoding the heavy chain and/or light chain variable regions of antibody mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, mAb14, mAb15 or mAb16. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[00131] The invention includes affinity-matured modalities. For example, affinity matured antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91 :3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; 154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889896;

[00132] The following methods can be used to adjust the affinity of an antibody and to characterize a CDR. One way to characterize a CDR of an antibody and/or alter (such as improve) the binding affinity of a polypeptide, such as an antibody, is called "library scanning mutagenesis." Generally, library scanning mutagenesis works as follows. One or more amino acid positions in the CDR are replaced with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids using methods recognized in the art. This generates small clone libraries (in some embodiments, one for each amino acid position that is analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at each position). Generally, the library also includes a clone comprising the native (unsubstituted) amino acid. A small number of clones, for example, about 20 to 80 clones (depending on library complexity) from each library, are selected for binding affinity to the target polypeptide (or other binding agent), and candidates with increased binding, to same, diminished or none are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using, for example, Biacore™ surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or more, Kinexa® Biosensor, scintillation proximity assays, ELISA, immunoassay ORIGEN®, fluorescence quenching, fluorescence transfer and/or yeast display. Binding affinity can also be selected using a suitable bioassay. Biacore™ is particularly useful when the starting antibody already binds with a relatively high affinity, for example, a KD of about 10 nM or less.

[00133] In some embodiments, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 natural amino acids using art-recognized mutagenesis methods (some of which are described here). This generates small clone libraries (in some embodiments, one for each amino acid position that is analyzed), each with a complexity of 20 members (if all 20 amino acids are substituted at each position).

[00134] In some embodiments, the library to be selected comprises substitutions at two or more positions, which may be in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions at two or more positions in a CDR. The library may comprise substitutions at two or more positions in two or more CDRs. The library may comprise substitution at 3, 4, 5, or more positions, said positions found in two, three, four, five or six CDRs. The substitution can be prepared using low-redundancy codons. See, for example, Table 2 of Balint et al., 1993, Gene 137(1):109-18.

[00135] The CDR can be the heavy chain variable region (VH) CDR3 and/or light chain variable region (VL) CDR3. The CDR may be one or more of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3. The CDR can be a Kabat CDR, a Chothia CDR, an extended CDR, an AbM CDR, a contact CDR, or a conformational CDR.

[00136] Candidates with improved binding can be sequenced, thereby identifying a CDR substitution mutant that results in improved affinity (also called an "improved" substitution). Candidates that bind can also be sequenced, thereby identifying a CDR substitution that retains binding.

[00137] Multiple rounds of screening can be conducted. For example, candidates (each comprising an amino acid substitution at one or more positions of one or more CDRs) with enhanced binding are also useful for designing a secondary library containing at least the original and substituted amino acid at each enhanced CDR position. (i.e., amino acid position in the CDR at which a substitution mutant showed improved binding). Preparation, and scanning and selection, of this library is discussed further below.

[00138] Library scanning mutagenesis also provides a means for characterizing a CDR, in that the frequency of clones with enhanced binding, the same binding, decreased binding or no binding also provides information related to the importance of each position of amino acid for the stability of the antibody-antigen complex. For example, if a CDR position retains binding when changed to all 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a CDR position retains binding in only a small percentage of substitutions, that position is identified as a position that is important for the CDR to function. Thus, library scanning mutagenesis methods generate information relating to positions in the CDRs that can be changed for many different amino acids (including all 20 amino acids), and positions in the CDRs that cannot be changed or that can only be changed in a few amino acids.

[00139] Candidates with improved affinity may be combined into a second library, which includes the improved amino acid, the original amino acid at that position, and may further include additional substitutions at that position, depending on the complexity of the library that is desired or permitted using the method. desired scan or selection. Furthermore, if desired, the adjacent amino acid position can be randomized to at least two or more amino acids. Randomization of adjacent amino acids may allow additional conformational flexibility in the mutant CDR, which may in turn allow or facilitate the introduction of a large number of improved mutations. The library may also comprise substitution at positions that did not show improved affinity in the first round of scanning.

[00140] The second library is screened or selected for library members with improved and/or altered binding affinity using any method known in the art, including scanning using Kinexa™ biosensor analysis, and selection using any method known in the art for selection, including phage display, yeast display and ribosome display.

[00141] To express the anti-PD-1 antibodies of the present invention, DNA fragments encoding VH and VL regions can first be obtained using any of the methods described above. Various modifications, for example mutations, deletions and/or additions, can also be introduced into DNA sequences using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods, such as PCR-mediated mutagenesis, in which the mutated nucleotides are incorporated into the PCR primers so that the PCR product contains the desired mutations, or site-directed mutagenesis.

[00142] The invention encompasses modifications to the variable regions shown in Table 1 and the CDRs shown in Tables 2, 3 or 4. For example, the invention includes antibodies that comprise functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants that have improved or reduced activity and/or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for PD-1. Modification of polypeptides is routine practice in the art and does not need to be described in detail here. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more amino acid deletions or additions which do not significantly alter the functional activity, or which mature (improve) the affinity of the polypeptide for its ligand, or use of chemical analogues.

[00143] Amino acid sequence insertions include terminal amino and/or carboxyl fusions varying in length of one residue for polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertion variants of the antibody molecule include the fusion to the N or C terminus of the antibody of an enzyme or a polypeptide that increases the half-life of the antibody in the blood circulation.

[00144] Substitutive variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include hypervariable regions, but structure changes are also contemplated. Conservative substitutions are referred to in Table 5 under the heading "conservative substitutions". If such substitutions result in a change in biological activity, then more substantial changes, termed "exemplary substitutions" in Table 5, or as further described below with reference to amino acid classes, can be introduced and the products screened. Table 5: Amino Acid Substitutions

[00145] Substantial modifications to the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of substitution, for example, as a β-sheet or helical conformation , (b) the charge or hydrophobicity of the molecule at the target site, or (c) the majority of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile; (2) Uncharged polar: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His.

[00146] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[00147] One type of substitution, for example, that can be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there may be a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody. In some embodiments, cysteine is canonical. Any cysteine residue not involved in maintaining the proper conformation of the antibody can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine linkage(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[00148] Antibodies can also be modified, for example, in the variable domains of the light and/or heavy chains, for example, to change a binding property of the antibody. Changes in the variable region can alter binding affinity and/or specificity. In some embodiments, no more than five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR domain. For example, a mutation can be made in one or more of the CDR regions to increase or decrease the KD of the antibody to PD-1, to increase or decrease koff, or to change the binding specificity of the antibody. Techniques in site-directed mutagenesis are well known in the art. See, for example, Sambrook et al. and Ausubel et al., supra.

[00149] A modification or mutation can also be made in a framework region or constant region to increase the half-life of an anti-PD-1 antibody. See, for example, PCT Publication No. WO 00/09560. A mutation in a framework region or constant region can also be made to alter the immunogenicity of the antibody, to provide a site for covalent or noncovalent binding to another molecule, or to alter such properties as complement fixation, FcR binding, and cytotoxicity. cell-mediated depending on the antibody. In some embodiments, no more than one to five conservative amino acid substitutions are made within the framework region or constant region. In other embodiments, no more than one to three conservative amino acid substitutions are made within the framework region or constant region. According to the invention, a single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain or the constant region.

[00150] Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:41754180) and the intramolecular interaction between portions of the glycoprotein , which can affect the conformation and three-dimensional surface appearance of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides can also serve to target a given glycoprotein to certain molecules based on specific recognition structures. Antibody glycosylation has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, antibodies produced by CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisealing GlcNAc, has been reported to have enhanced ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

[00151] Antibody glycosylation is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid except proline, are the recognition sequences for attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[00152] The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more tripeptide sequences described above (for N-linked glycosylation sites). The change can also be made by adding, or substituting, one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).

[00153] The glycosylation pattern of antibodies can also be altered without altering the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics is rarely the native cell, variations in the glycosylation pattern of antibodies can be expected (see, e.g. Hse et al., 1997 , J. Biol. 272:9062-9070).

[00154] In addition to the choice of host cells, factors that affect glycosylation during recombinant antibody production include growth mode, medium formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in the production of oligosaccharides (US Patent Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. Furthermore, the recombinant host cell can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

[00155] Other modification methods include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. The modifications can be used, for example, to attach immunoassay labels. Modified polypeptides are made using procedures established in the art and can be screened using standard assays known in the art, some of which are described below and in the Examples.

[00156] In some embodiments, the Fc may be human IgG2 or human IgG4. In some embodiments, the antibody comprises an IgG4 constant region comprising the following mutations (Armour et al., 2003, Molecular Immunology 40 585-593): E233F234L235 to P233V234A235 (IgG4Δc), wherein the numbering is with reference to the IgG4 type wild. In yet another embodiment, the Fc is human IgG4 E233F234L235 to P233V234A235 with G236 deletion (IgG4Δb). In some embodiments, the Fc is any human IgG4 Fc (IgG4, IgG4Δb, or IgG4Δc) containing the S228 to P228 hinge-stabilizing mutation (Aalberse et al., 2002, Immunology 105, 919). In other embodiments, the Fc may be human IgG2 containing the A330P331 to S330S331 (IgG2Δa) mutation, in which the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 1999, 29:2613-2624.

[00157] In some embodiments, the antibody comprises a modified constant region that has increased or decreased binding affinity for a human Fc gamma receptor, is immunologically inert or partially inert, for example, does not trigger complement-mediated lysis, does not stimulate mediated cytotoxicity by antibody-dependent cell (ADCC), or does not activate microglia; or reduced activities (compared to the unmodified antibody) in any one or more of the following: triggering complement-mediated lysis, stimulating ADCC, or activating microglia. Different modifications of the constant region can be used to achieve optimal level and/or combination of effector functions. See, for example, Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:5976, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publication No. WO99/058572.

[00158] In some embodiments, a constant region of the antibody can be modified to avoid interaction with the Fc gamma receptor and the complementary and immune systems. Techniques for preparing such antibodies are described in WO 99/58572. For example, the constant region can be designed to better resemble human constant regions to avoid immune response if the antibody is used in human clinical trials and treatments. See, for example, US Patent Nos. 5,997,867 and 5,866,692.

[00159] In still other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating the oligosaccharide linker residue and/or flanking residues that are part of of the N-glycosylation recognition sequence in the constant region. For example, the N297 N-glycosylation site can be mutated to, for example, A, Q, K, or H. See, Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically (such as by removing carbohydrate by enzyme PNGase), or by expression in a glycosylation-deficient host cell.

[00160] Other antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572. Such antibodies comprise, in addition to a binding domain directed to the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of a constant region of a human immunoglobulin heavy chain. These antibodies are capable of binding to the target molecule without triggering significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding to FcRn and/or FcYRIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly suitable for use in chronic antibody therapy to avoid inflammatory reactions and other adverse reactions to conventional antibody therapy.

[00161] In some embodiments, the antibody comprises a modified constant region that has increased binding affinity for FcRn and/or an increased serum half-life as compared to the unmodified antibody.

[00162] In a process known as "germination", certain amino acids in the VH and VL sequences can be mutated to match those found naturally in the VH and VL sequence. In particular, the amino acid sequence of the framework regions in the VH and VL sequences can be mutated to match the germline sequences to reduce the risk of immunogenicity when the antibody is administered. The germline DNA sequences for human VH and VL genes are known in the art (see, for example, the human germline sequence database "Vbase"; see, also Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; TomLinson et al., 1992, J. Mol. Biol. Eur. J. Immunol. 24:827-836).

[00163] Another type of amino acid substitution that can be made is to remove potential proteolytic sites on the antibody. Such sites may occur in a CDR or framework region of a variable domain or in the constant region of an antibody. Replacement of cysteine residues and removal of proteolytic sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by altering one or both residues. In another example, C-terminal lysis alters one or both residues. In another example, the C-terminal lysine of the heavy chain of an anti-PD-1 antibody of the invention can be cleaved. In various embodiments of the invention, the light and heavy chains of anti-PD-1 antibodies may optionally include a signal sequence.

[00164] Once the DNA fragments encoding the VH and VL segments of the present invention are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, for Fab fragment genes, or for a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[00165] Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably it is an IgG1 or IgG2 constant region. The IgG constant region sequence may be any of several alleles or allotypes known to occur among different individuals, such as Gm(1), Gm(2), Gm(3) and Gm(17). These allotypes represent naturally occurring amino acid substitutions in the constant regions of IgG1. For a Fab fragment heavy chain gene, the DNA encoding VH can be operatively linked to another DNA molecule encoding only the CH1 heavy chain constant region. The CH1 heavy chain constant region can be derived from any of the heavy chain genes.

[00166] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) through operative ligation of the DNA encoding VL to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region can be any of several alleles known to occur among different individuals, such as Inv(1), Inv(2), and Inv(3). The lambda constant region can be derived from any of the three lambda genes.

[00167] To create an Scfv gene, DNA fragments encoding Vh and Vl are operably linked to another fragment encoding a flexible linker so that the Vh and Vl sequences can be expressed as a contiguous single-chain protein, with The Vl and Vh Regions Joined by the Flexible Linker (See, for example, Bird Et Al., 1988, Science 242:423-426; Huston Et Al., 1988, Proc. Natl. Acad. Sci. Usa 85:5879-5883 ;Mccafferty et al., 1990, Nature 348:552-554. An example of a binding peptide is (Ggggs)3 (Seq Id No: 19), which spans approximately 3.5 Nm between the carboxy terminus of a variable region. and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). To Solid Supports. The Single Chain Antibody Can Be Monovalent, If Only A Single Vh And Vl Are Used, Bivalent, If Two Vh And Vl Are Used, Or Polyvalent, If More Than Two Vh And Vl Are Used. Bispecific or polyvalent antibodies can be generated, which specifically bind to pd-1 and another molecule. Single-chain variants can be produced either recombinantly or synthetically. For synthetic production of scfv, an automatic synthesizer can be used. For recombinant production of Scfv, a suitable plasmid containing polynucleotide encoding Scfv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. Coli. The polynucleotides that encode the scfv of interest can be made by routine manipulations such as polynucleotide ligation. The resulting Scfv can be isolated using standard protein purification techniques known in the art.

[00168] Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with complementary domains. of another chain and creating two antigen binding sites (see, e.g., Holliger, P., et al., 1993, Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et al., 1994, Structure 2:11211123).

[00169] Heteroconjugate antibodies, which comprise two covalently joined antibodies, are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (PCT Publications No. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies can be made using any convenient cross-linking methods. Suitable crosslinking agents and techniques are well known in the art, and are described in US Patent No. 4,676,980.

[00170] Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[00171] The invention also encompasses fusion proteins that comprise one or more fragments or regions of the antibodies disclosed herein. In some embodiments, a fusion antibody may be made that comprises all or a portion of an anti-PD-1 antibody of the invention linked to another polypeptide. In another embodiment, only the variable domains of the anti-PD-1 antibody are linked to the polypeptide. In another embodiment, the VH domain of an anti-PD-1 antibody is linked to a first polypeptide, while the VL domain of an anti-PD-1 antibody is linked to a second polypeptide that associates with the first polypeptide in a manner so that the VH and VL domains can interact with each other to form an antigen binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker so that the VH and VL domains can interact with each other. The VH ligand VL antibody is then linked to the polypeptide of interest. Additionally, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to each other. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody.

[00172] In some embodiments, there is provided a fusion polypeptide comprising at least 10 contiguous amino acids from the light chain variable region shown in SEQ ID NO: 2, 7, 8 or 9 and/or at least 10 amino acids from the light chain variable region. heavy chain shown in SEQ ID NO: 3, 4, 5 or 6. In other embodiments, there is provided a fusion polypeptide comprising at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous light chain variable region amino acids and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the heavy chain variable region. In another embodiment, the fusion polypeptide comprises a light chain variable region and/or a heavy chain variable region as shown in any of sequence pairs selected from SEQ ID NOs: 2 and 3, 7 and 3, 8 and 3, 8, 9 and 3, 2 and 4, 7 and 4, 8 and 4, 9 and 4, 2 and 5, 7 and 5, 8 and 5, 9 and 5, 2 and 6, 7 and 6, 8 and 6, and 9 and 6. In another embodiment, the fusion polypeptide comprises one or more CDR(s). In still other embodiments, the fusion polypeptide comprises VH CDR3 and/or VL CDR3. For the purposes of this invention, a fusion protein contains one or more antibodies and another amino acid sequence to which is not attached to the native molecule, for example, a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, a "tag", such as a FLAG tag or a 6His tag. Tags are well known in the art.

[00173] A fusion polypeptide can be created by methods known in the art, for example, synthetically or recombinantly. Typically, the fusion proteins of this invention are made by preparing and expressing a polynucleotide encoding them using recombinant methods described herein, although they can also be prepared by other means known in the art, including, for example, chemical synthesis.

[00174] In other embodiments, other modified antibodies can be prepared using anti-PD-1 antibodies that encode nucleic acid molecules. For example, "Kappa bodies" (Ill et al., 1997, Protein Eng. 10:949-57), "Minibodies" (Martin et al., 1994, EMBO J. 13:5303-9), "Diabodies" ( Holliger et al., supra), or "Janusins" (Traunecker et al., 1991, EMBO J. 10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer (Suppl.) 7:51-52 ) can be prepared using standard molecular biology techniques following the techniques in the specification.

[00175] For example, bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens, can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, for example, Suresh et al., 1986, Methods in Enzymology 121:210). For example, bispecific antibodies or antigen-binding fragments can be produced by hybridoma fusion or ligation of Fab' fragments. See, for example, Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553. Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two immunoglobulin light chain/heavy chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, 1983, Nature 305, 537-539). Furthermore, bispecific antibodies can be formed as "diabodies" or "Janusins". In some embodiments, the bispecific antibody binds to two different epitopes of PD-1. In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions of an anti-PD-1 antibody provided herein.

[00176] According to one approach for making bispecific antibodies, antibody variable domains with desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant region sequence. The fusion preferably is with an immunoglobulin heavy chain constant region, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1), containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding immunoglobulin heavy chain fusions and, if desired, immunoglobulin light chain, are inserted into separate expression vectors and are cotransfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of three polypeptide chains used in the construction provide optimal yields. Therefore, it is possible to insert the coding sequences for two or all three polypeptide chains into an expression vector when expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are not of particular significance.

[00177] In one approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin light/heavy chain pair (providing a second binding specificity) on the other arm. . This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in PCT Publication No. WO 94/04690.

[00178] This invention also provides compositions comprising antibodies conjugated (e.g., linked) to an agent that facilitates coupling to a solid support (such as biotin or avidin). For simplicity, reference will generally be made to antibodies with the understanding that these methods apply to any of the PD-1 binding and/or antagonist modalities described herein. Conjugation generally refers to the linking of these components as described here. Bonding (which is generally fixing these components in close association at least for administration) can be achieved in any number of ways. For example, a direct reaction between an agent and an antibody is possible when each has a substituent capable of reacting with each other. For example, a neophilic group, such as an amino or sulfhydryl group, may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g. , a halide) on the other.

[00179] Antibodies can be linked to many different vehicles. Vehicles can be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the vehicle may be soluble or insoluble for the purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to verify this using routine experimentation. In some embodiments, the vehicle comprises a portion that targets the lung, heart, or heart valve.

[00180] An antibody or polypeptide of this invention can be linked to a labeling agent such as a fluorescent molecule, a radioactive molecule or any other labels known in the art. Labels are known in the art, which generally provide (either directly or indirectly) a signal.

POLYNUCLEOTIDES, VECTORS AND HOST CELLS

[00181] The invention also provides polynucleotides that encode any of the antibodies, including antibody fragments and modified antibodies described herein, such as, for example, antibodies that have impaired effector function. In another aspect, the invention provides a method of manufacturing any of the polynucleotides described herein. Polynucleotides can be made and expressed by procedures known in the art. Accordingly, the invention provides polynucleotides or compositions, including pharmaceutical compositions, comprising polynucleotides, which encode any of the following: the antibodies mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13 , mAb14, mAb15, mAb16 and mAb17 or any fragment or part thereof having the ability to antagonize PD-1.

[00182] Polynucleotides complementary to any of these sequences are also encompassed by the present invention. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA molecules (genomic, cDNA or synthetic) or RNA. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

[00183] Polynucleotides may comprise a native sequence (that is, an endogenous sequence that encodes an antibody or a fragment thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. The variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity. for a polynucleotide sequence encoding a native antibody or a fragment thereof.

[00184] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over the comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein refers to a segment of at least about 20 contiguous positions, usually from 30 to about 75, or from 40 to about 50, at which a sequence can be compared to a reference sequence from the same number of contiguous positions after the two sequences are ideally aligned.

[00185] Optimal alignment of sequences for comparison can be conducted using the MegAlign® program in the Lasergene® bioinformatics suite software (DNASTAR®, Inc., Madison, WI), using standardized parameters. This program incorporates several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evolution. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Academic. Sci USA 80:726-730.

[00186] Preferably, the "percent sequence identity" is determined by comparing two ideally aligned sequences over a window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to reference sequences (which do not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residue occur in both sequences to produce the number of matching positions, then dividing the number of matching positions by the total number of positions in the reference sequence (i.e., window size) and multiplying the results by 100 to produce the percentage sequence identity.

[00187] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[00188] Suitable "moderately stringent conditions" include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C to 65°C, 5X SSC, overnight; followed by two washes at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[00189] As used herein, "highly stringent conditions" or "high stringency conditions" are those that: (1) employ low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0 M sodium citrate .0015 M/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone /50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate , 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. One skilled in the art will recognize how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[00190] It will be appreciated by those skilled in the art that as a result of the degeneration of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, the alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are changed as a result of one or more mutations, such as deletions, additions and/or nucleotide substitutions. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

[00191] The polynucleotides of this invention can be obtained using chemical synthesis, recombinant methods or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail here. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.

[00192] To prepare polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector, in turn, can be introduced into a suitable host cell for replication and amplification, with further discussed here. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by the introduction of an exogenous polynucleotide by direct absorption, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[00193] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in US Patents Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[00194] RNA can be obtained by using isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, as set forth in Sambrook et al., 1989, supra, for example.

[00195] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary depending on the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in the selection of clones containing the vector. Suitable examples include bacterial plasmids and viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors. such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene and Invitrogen.

[00196] Expression vectors are further provided. Expression vectors are generally polynucleotide constructs that contain a polynucleotide according to the invention. It is implied that an expression vector needs to be replicable in host cells, either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids and expression vector(s) disclosed in PCT Publication No. WO 87/04462. The components of the vector may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; appropriate transcription control elements (such as promoters, enhancers and terminators). For expression (i.e., translation), one or more translational control elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[00197] Vectors containing the polynucleotides of interest can be introduced into the host cell by any number of appropriate means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g. where the vector is an infectious agent such as vaccinia virus). The choice of introduction vectors or polynucleotides will often depend on the characteristics of the host cell.

[00198] The invention also provides host cells that comprise any of the polynucleotides described here. Any host cells capable of overexpressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cells express the cDNAs at a level of about 5 times greater, more preferably 10 times greater, even more preferably 20 times greater than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells. . Scanning host cells for specific binding to PD-1 or a domain of PD-1 is performed by an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.

[00199] An expression vector can be used to direct the expression of an anti-PD-1 antagonist antibody. One skilled in the art is familiar with administering expression vectors to obtain expression of an exogenous protein in vivo. See, for example, US Patent No. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration and topical administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle or pericardium.

[00200] Targeted release of therapeutic compositions containing an expression vector, or subgenomic polynucleotides can also be used. Receptor-mediated DNA release techniques are described in, for example, Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J. A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Academic. Sci. USA, 1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges are from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be constitutive or regulated.

[00201] Viral-based vectors for releasing a desired polynucleotide and expressing it in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, for example, PCT Publications No. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; US Patent Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; of Sindbis virus, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR - 1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, for example, PCT Publication No. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can also be employed.

[00202] Non-viral delivery vehicles and methods may also be employed, including, but not limited to, polycationic condensed DNA bound or unbound to killed adenovirus alone (see, for example, Curiel, Hum. Gene Ther., 1992, 3:147); DNA bound to the ligand (see, for example, Wu, J. Biol. Chem., 1989, 264:16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publications No. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and neutralization of nucleic loading or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publications No. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol., 1994, 14:2411, and in Woffendin, Proc. Natl. Academic. Sci., 1994, 91:1581.

COMPOSITIONS

[00203] The invention also provides pharmaceutical compositions comprising an effective amount of an anti-PD-1 antibody described herein. Examples of such compositions, as well as formulating them, are also described here. In some embodiments, the composition comprises one or more PD-1 antibodies. In other embodiments, the anti-PD-1 antibody recognizes PD-1. In other embodiments, the anti-PD-1 antibody is a human antibody. In other embodiments, the anti-PD-1 antibody is a humanized antibody. In some embodiments, the anti-PD-1 antibody comprises a constant region that is capable of triggering a desired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the anti-PD-1 antibody comprises a constant region that does not trigger an unwanted or undesirable immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the anti-PD-1 antibody comprises one or more antibody CDR(s) (such as one, two, three, four, five, or, in some embodiments, all six CDRs).

[00204] It is understood that the compositions may comprise more than one anti-PD-1 antibody (for example, a mixture of PD-1 antibodies that recognize different epitopes of PD-1). Other exemplary compositions comprise more than one anti-PD-1 antibody that recognizes the same epitope(s), or different species of anti-PD-1 antibodies that bind to different PD-1 epitopes. In some embodiments, the compositions comprise a mixture of anti-PD-1 antibodies that recognize different variants of PD-1.

[00205] The composition used in the present invention may further comprise pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients in dosages and concentrations, and may comprise buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenolic, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[00206] The anti-PD-1 antibody and compositions thereof can also be used together with, or administered separately, simultaneously or sequentially, other agents that serve to increase and/or complement the effectiveness of the agents.

[00207] The invention also provides compositions, including pharmaceutical compositions, comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the antibody as described herein. In another embodiment, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein.

METHODS FOR PREVENTION OR TREATMENT OF PD-1-MEDIATED CONDITIONS

[00208] The antibodies and antibody conjugates of the present invention are useful in various applications including, among others, therapeutic treatment methods and diagnostic treatment methods.

[00209] In one aspect, the invention provides a method for treating cancer. In some embodiments, the method of treating a cancer in an individual comprises administering to the individual in need thereof an effective amount of a composition (e.g., pharmaceutical composition) comprising any of the PD-1 antibodies as described herein. As used herein, cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, gastric cancer, glioblastoma, glioma, brain tumor, head and neck cancer, kidney cancer , lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, liver cancer, uterine cancer, bone cancer, leukemia, lymphoma, sarcoma, blood cancer, thyroid cancer, thymic cancer, eye cancer and cancer of skin. In some embodiments, there is provided a method of inhibiting tumor growth or progression in an individual, comprising administering to the individual in need thereof an effective amount of a composition comprising PD-1 antibodies or PD-1 antibody conjugates as described. here. In some embodiments, the tumor is a PD-L1 expressing tumor. In other embodiments, the tumor does not express PD-L1. In other embodiments, there is provided a method of inhibiting metastasis of cancer cells in an individual, comprising administering to the individual in need thereof an effective amount of a composition comprising any of the PD-1 antibodies as described herein. In other embodiments, there is provided a method of inducing regression of a tumor in an individual, comprising administering to the individual in need thereof an effective amount of a composition comprising any of the PD-1 antibodies as described herein.

[00210] In another aspect, a method of detecting, diagnosing and/or monitoring a cancer is provided. For example, PD-1 antibodies as described herein can be labeled with a detectable moiety such as an imaging agent and an enzyme substrate label. Antibodies as described herein can also be used for in vivo diagnostic assays, such as in vivo imaging (e.g., PET or SPECT), or a staining reagent.

[00211] In some embodiments, the methods described here further comprise a step of treating an individual with an additional form of therapy. In some embodiments, the form of additional therapy is additional anticancer therapy including, but not limited to, additional chemotherapy, radiation, surgery, hormonal therapy and/or immunotherapy.

[00212] With respect to all methods described here, reference to anti-PD-1 antagonist antibodies also includes compositions comprising one or more additional agents. Such compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art. The present invention can be used alone or in combination with other treatment methods.

[00213] The anti-PD-1 antagonist antibody can be administered to an individual via any suitable route. It should be apparent to one skilled in the art that the examples described here are not intended to be limiting, but to be illustrative of available techniques. Accordingly, in some embodiments, the anti-PD-1 antagonist antibody is administered to a subject in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneal, intracerebrospinal, transdermal, subcutaneous, intra-articular, sublingual, intrasynovial, through insufflation, intrathecal, oral, inhalation or topical. Administration can be systemic, for example, intravenous or localized administration. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the anti-PD-1 antagonist antibody can be in aerosol form using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[00214] In some embodiments, an anti-PD-1 antagonist antibody is administered via targeted or site-specific local delivery techniques. Examples of targeted or site-specific local delivery techniques include various implantable anti-PD-1 antagonist antibody depot sources or local delivery catheters, such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific vehicles, direct injection or direct application. See, for example, PCT Publication No. WO 00/53211 and U.S. Patent No. 5,981,568.

[00215] Various formulations of an anti-PD-1 antagonist antibody can be used for administration. In some embodiments, the anti-PD-1 antagonist antibody can be administered neat. In some embodiments, the anti-PD-1 antagonist antibody is a pharmaceutically acceptable excipient that can be in various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, an excipient may give shape or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers and skin penetration enhancers. Excipients as well as formulations for parenteral and non-parenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000.

[00216] In some embodiments, these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents can be combined with pharmaceutically acceptable carriers such as saline solution, Ringer's solution, dextrose solution and the like. The particular dosage regimen, i.e., dose, time and repetition, will depend on the particular individual and the individual's medical history.

[00217] An anti-PD-1 antagonist antibody can be administered using any suitable method, including by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Anti-PD-1 antibodies can also be administered topically or via inhalation, as described here. Generally, for administration of anti-PD-1 antibodies, an initial candidate dosage may be about 2 mg/kg. For purposes of the present invention, a typical daily dosage ranges from about 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg to 30 mg/kg to 100 mg/kg or more, depending of the factors mentioned above. For example, dosages of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg can be used. For repeated administrations over several days or more, depending on the condition, treatment is continued until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved, for example, to reduce symptoms associated with cancer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage regimen (including the anti-PD-1 antagonist antibody used) may vary over time.

[00218] For the purpose of the present invention, the appropriate dosage of an anti-PD-1 antagonist antibody will depend on the anti-PD-1 antagonist antibody (or compositions thereof) employed, the type and severity of the symptoms to be treated, if the agent is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the agent, the patient's clearance rate for the administered agent, and the discretion of the treating physician. Typically, the doctor will administer an anti-PD-1 antagonist antibody until a dosage is reached that achieves the desired result. Dose and/or frequency may vary during treatment. Empirical considerations, such as half-life, will generally contribute to dosage determination. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or improvement and/or delay of symptoms. Alternatively, prolonged continuous release formulations of anti-PD-1 antagonist antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[00219] In one embodiment, dosages for an antagonist antibody can be determined empirically in individuals who have received one or more administrations of an antagonist antibody. Subjects receive incremental dosages of an anti-PD-1 antagonist antibody. To assess effectiveness, a disease indicator can be followed.

[00220] The administration of an anti-PD-1 antagonist antibody, according to the method in the present invention can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to qualified professionals. Administration of an anti-PD-1 antagonist antibody may be essentially continuous over a pre-selected period of time or may be in a series of spaced doses.

[00221] In some embodiments, more than one anti-PD-1 antagonist antibody may be present. At least one, at least two, at least three, at least four, at least five different, or more antagonist antibodies may be present. Generally, those anti-PD-1 antagonist antibodies may have complementary activities that do not adversely affect each other. An anti-PD-1 antagonist antibody may also be used in conjunction with other antibodies and/or other therapies. An anti-PD-1 antagonist antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

[00222] In some embodiments, the anti-PD-1 antagonist antibody can be administered in combination with the administration of one or more additional therapeutic agents. These include, but are not limited to, administration of a chemotherapeutic agent, a vaccine, a CAR T cell-based therapy, radiotherapy, a cytokine therapy, a vaccine, an anti-PD-1 bispecific antibody, an inhibitor of other immunosuppressive pathways, an inhibitor of angiogenesis, a T cell activator, an inhibitor of a metabolic pathway, an inhibitor of mTOR, an inhibitor of an adenosine pathway, a tyrosine kinase inhibitor including, but not limited to, inite, ALK inhibitors and sunitinib, a BRAF inhibitor, an epigenetic modifier, an inhibitor or depleter of Treg cells and/or myeloid-derived suppressor cells, a JAK inhibitor, a STAT inhibitor, a cyclin-dependent kinase inhibitor, a biotherapeutic agent ( including, but not limited to, antibodies to VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, and ICOS), an agent immunogenic (e.g., attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells pulsed with antigen or tumor-derived nucleic acids, immune stimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immune stimulatory cytokines such as, among others, GM-CSF). Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CBI-TMI); eleutherobine; pancratistatin; a sarcodictiin; spongistatin; nitrogen mustards such as chlorambucil, chlornafazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediine antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin phiI1, see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemycin, including dynemycin A; bisphosphonates, such as clodronate; a esperamycin; as well as neocarzinostatin chromophore and chromoprotein-related enediine antibiotic chromophores), aclacinomysins, actinomycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin , 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorrubicin, idarubicin, marcelomycin, mitomycins such as mitomycin mycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubecidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; an epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pyrarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2'- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesina; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrona; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are antihormonal agents that act to regulate or inhibit hormone action on tumors such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene , ceoxifene, LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00223] In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with one or more other therapeutic agents that target an immune checkpoint modulator, such as, for example without limitation, an agent that targets PD-1. 1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC (PD-L2), B7-H5, B7-H6, B7-H8, B7-H2, B7-1 , B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD -H1), OX40, OX40L, GITR, GITRL, CD70, CD27, 4-1BB, 4-BBL, DR3, TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150), SLAMF2 (CD48 ), SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84), SLAMF6 (NTB-A), SLAMCF7 (CS1), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28, CEACAM1(CD66a ), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73 adenosine pathway (A2AR) , BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, VEGF pathway, CSF-1, or an innate immune response modulator. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with, for example, an anti-PD-L1 antagonist antibody such as, for example, BMS-936559 (MDX-1105) and MPDL3280A; an anti-PD-1 antagonist antibody such as, for example, nivolumab, pembrolizumab and pidilizumab; an anti-CTLA-4 antagonist antibody such as, for example, ipilimumab; an anti-LAG-3 antagonist antibody such as BMS-986016 and IMP701; an anti-TIM-3 antagonist antibody; an anti-B7-H3 antagonist antibody such as, for example, MGA271; an anti-VISTA antagonist antibody; an anti-TIGIT antagonist antibody; an anti-CD28 antagonist antibody; an anti-CD80 antibody; an anti-CD86 antibody; an anti-B7-H4 antagonist antibody; an anti-ICOS agonist antibody; an anti-CD28 agonist antibody; an innate immune response modulator (e.g., TLRs, KIR, NKG2A), and an IDO inhibitor. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with a 4-1BB (CD137) agonist such as, for example, PF-05082566 or BMS-663513. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with an OX40 agonist such as, for example, an anti-OX-40 agonist antibody. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with a GITR agonist such as, for example, an anti-GITR agonist antibody such as, for example, without limitation, TRX518. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with an IDO inhibitor. In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with a cytokine therapy such as, for example without limitation, IL-15, CSF-1, MCSF-1, etc.

[00224] In some embodiments, an anti-PD-1 antagonist antibody is used in conjunction with one or more other therapeutic antibodies, such as, for example without limitation, an antibody that targets CD19, CD22, CD40, CD52 or CCR4.

[00225] In some embodiments, anti-PD-1 antibody therapy may precede or follow the other treatment agent for intervals ranging from minutes to weeks. In embodiments where the other agents and/or proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time has not expired between each release such that the agent and composition of the present invention would still be able to exert an effect. advantageously combined in the individual. In such cases, it is contemplated that one can administer both modalities within about 12 to 24 hours of each. In some situations, it may be desirable to extend the time period for significant administration, however, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) pass between the respective administrations.

[00226] In some embodiments, an anti-PD-1 antagonist antibody composition comprises a second agent selected from crizotinib, palbociclib, gemcitabine, cyclophosphamide, fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib, sunitinib malate, tofacitinib, bevacizumab, rituximab and Trastuzumab.

[00227] In some embodiments, an anti-PD-1 antibody composition is combined with a treatment regimen further comprising a traditional therapy selected from the group consisting of: surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal, angiogenesis inhibition and palliative care.

USE OF PD-1 ANTIBODIES IN CANCER VACCINE-BASED IMMUNOTHERAPY REGIMES

[00228] In some particular embodiments, the present description provides a method for improving the immunogenicity or therapeutic effect of a vaccine for the treatment of cancer in a mammal, particularly a human being, which method comprises administering to the mammal receiving the vaccine a effective amount of anti-PD-1 antagonist antibody provided by the present disclosure. In some other particular embodiments, the present description provides a method for treating a cancer in a mammal, particularly a human being, which method comprises administering to the mammal (1) an effective amount of a vaccine capable of eliciting an immune response against cells of the cancer and (2) an effective amount of an anti-PD-1 antagonist antibody provided by the present disclosure. The method of treating a neoplastic disorder in a mammal and the method of improving the immunogenicity or therapeutic effect of a vaccine for treating a neoplastic disorder in a mammal described hereinabove are collectively referred to as "vaccine-based immunotherapy regimens for cancer” (or “VBIR for cancer”).

[00229] In VBIR for cancer, the vaccine may be in any form or formulations, such as (i) cell-based vaccines, (ii) subunit vaccines, (iii) protein-based vaccines, (iv) peptide-based or (v) nucleic acid-based vaccines (such as DNA-based vaccines, RNA-based vaccines, plasmid-based vaccines or viral vector-based vaccines).

[00230] The VBIR for cancer provided by the present description may be applicable for any type of cancer. Examples of specific cancers include: small cell lung cancer, non-small cell lung cancer, glioma, gastric cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, cancer prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, bladder cancer, breast cancer and head and neck cancer.

[00231] Vaccines intended to treat cancers typically contain an antigen (in the form of a peptide, protein, cell component, whole cell, or a nucleic acid molecule encoding a peptide-based antigen) that is capable of eliciting a immune response against a particular TAA expressed in or by target tumor cells. Many TAAs are known in the art. Examples of known TAAs include: PSA, PSCA, and PSMA for prostate cancer; CEA, MUC-1, Ep-CAM, 5T4, hCG-b, K-ras, and TERT for colorectal cancer; CEA, Muc-1, p53, mesothelin, Survivin and NY-ESO-1 for ovarian cancer; Muc-1, 5T4, WT-1, TERT, CEA, EGF-R and MAGE-A3 for non-small cell lung cancer; 5T4 for renal cell carcinoma; and Muc-1, mesothelin, K-Ras, Annexin A2, TERT, and CEA for pancreatic cancer. In some particular embodiments, the vaccine used in the cancer VBIR provided by the present disclosure is selected from the group consisting of: (1) a vaccine capable of eliciting an immune response against a TAA selected from PSA, PSCA, PSMA, CEA, MUC-1 , TERT, mesothelin, EGF-R or MAGE-A3; (2) a vaccine containing a peptide antigen derived from a TAA selected from PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R or MAGE-A3; and (3) a vaccine containing a nucleic acid molecule encoding a peptide antigen, wherein the peptide antigen is derived from a TAA selected from PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF- R or MAGE-A3.

[00232] In still other particular embodiments, the vaccine contains a nucleic acid molecule that encodes one or more immunogenic polypeptides derived from PSA, one or more immunogenic polypeptides derived from PSCA, or one or more immunogenic polypeptides derived from PSMA.

[00233] In a specific embodiment, the nucleic acid molecule is selected from the group consisting of: (1) a nucleic acid molecule encoding an immunogenic polypeptide derived from human PSMA of SEQ ID NO:42; (2) a nucleic acid molecule encoding an immunogenic polypeptide comprising amino acids 15-750 of SEQ ID NO:42; (3) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 43, or a degenerate variant thereof; (4) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 44, or a degenerate variant thereof; (5) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 45, or a degenerate variant thereof; (6) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46, or a degenerate variant thereof; (7) a nucleic acid molecule encoding an immunogenic polypeptide derived from human PSA of SEQ ID NO:47; (8) a nucleic acid molecule encoding an immunogenic polypeptide comprising amino acids 25-261 of SEQ ID NO:47; (9) a nucleic acid molecule encoding an immunogenic polypeptide derived from the human PSCA of SEQ ID NO:48; (10) a nucleic acid molecule encoding (i) an immunogenic polypeptide derived from the human PSMA of SEQ ID NO:42, (ii) an immunogenic polypeptide derived from the human PSA of SEQ ID NO:47, and (iii) a polypeptide human PSCA-derived immunogen of SEQ ID NO:48; and (11) a nucleic acid molecule encoding (i) an immunogenic polypeptide comprising amino acids 15-750 of SEQ ID NO:42, (ii) an immunogenic polypeptide comprising amino acids 25-261 of SEQ ID NO:47, and (iii ) an immunogenic polypeptide of SEQ ID NO:48.

[00234] Nucleic acid molecules encoding one or more immunogenic polypeptides derived from prostate-associated antigens may be in the form of plasmids or vectors. An example of such a plasmid is the nucleic acid construct of SEQ ID NO:46 (also referred to as Plasmid 458). The nucleotide sequence of a vector expressing an immunogenic polypeptide derived from human PSMA is set forth in SEQ ID NO:44 (also referred to as AdC68W vector). The nucleotide sequence of a vector expressing a human PSMA-derived immunogenic polypeptide, a human PSA-derived immunogenic polypeptide, and a human PSCA-derived immunogenic polypeptide is set forth in SEQ ID NO:45 and a human PSMA vector (AdC68W- vector 734). Various immunogenic polypeptides derived from human PSMA, PSA, and PSCA, nucleic acid constructs (including plasmids and vectors) encoding such immunogenic polypeptides, and methods for preparing the immunogenic polypeptides and nucleic acid constructs, including Plasmid 458, and vector AdC68W and AdC68W -734, are disclosed in International Patent Application Publications WO2013/164754 and WO 2015/063647, each of which is incorporated herein by reference in its entirety.

[00235] In one aspect, the invention provides an isolated antagonist antibody that specifically binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR1), CDR2 of VH and VH CDR3 of VH having a group selected from the amino acid sequence, the group consisting of SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6; and a light chain (VL) variable region comprising a VL CDR1, VL CDR2 and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO:7; SEQ ID NO: 8; and SEQ ID NO: 9.

[00236] Any anti-PD-1 antagonist antibodies disclosed in the present description can be used in VBIR for cancer. In some embodiments, the anti-PD-1 antagonist antibody comprises a VH region and/or a VL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 3, 4, 5, or 6, or a variant having one or more conservative amino acid substitutions at residues not within a CDR, and wherein the VL region comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 8, or 9, or a variant thereof with one or more amino acid substitutions in amino acids that are not within a CDR. In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO: 39 and/or a heavy chain comprising the sequence shown in SEQ ID NO: 29 or 38. In some particular embodiments, the antibody comprises a VH region produced by the expression vector with ATCC Accession No. PTA-121183. In some embodiments, the antibody comprises a VL region produced by the expression vector with ATCC Accession No. PTA-121182.

[00237] The VBIR for cancer provided by the present description may further comprise one or more other immune modulators (in addition to the PD-1 antagonist antibody provided by the present description). The other immune modulators may be an immune effector cell enhancer ("IEC enhancer") or an immune suppressor cell inhibitor ("ISC inhibitor"). Additional IEC enhancer or additional ISC inhibitor can be used alone in combination with VBIR for cancer. Additional IEC enhancer and additional ISC inhibitor can also be used together in combination with VBIRs for cancer.

[00238] Examples of classes of ISC inhibitors include protein kinase inhibitors, cyclooxygenase-2 (COX-2) inhibitors, phosphodiesterase type 5 (PDE5) inhibitors, and DNA crosslinkers. Examples of COX-2 inhibitors include celecoxib and rofecoxib. Examples of PDE5 inhibitors include avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, and zaprinast. An example of DNA cross-linkers is cyclophosphamide. The term "protein kinase inhibitors" refers to any substance that acts as a selective or non-selective inhibitor of a protein kinase. Examples of specific protein kinase inhibitors suitable for use in VBIR for cancer include Lapatinib, AZD 2171, ET18OCH 3, Indirubin-3'-oxime, NSC-154020, PD 169316, Quercetin, Roscovitine, Triciribine, ZD 1839, 5-Iodotubercidin, Adafostine, Aloisin, Alsterpaulone, Aminogenistein, API-2, Apigenin, Arctigenin, ARRY-334543, Axitinib (AG-013736), AY-22989, AZD 2171, Bisindolylmaleimide IX, CCl-779, Chelerythrine, DMPQ, DRB, Edelfosine, ENMD -981693, Erbstatin analogue, Erlotinib, Fasudil, Gefitinib (ZD1839), H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA- 1100, Hydroxifasudil, Kenpaullone, KN- 62, KY12420, LFM-A13, Luteolin, LY294002, LY-294002, Malotoxin, ML-9, MLN608, NSC- 226080, NSC-231634, NSC-664704, NSC-680410, NU6102, Olomoucine, Oxindol I, PD 153 035, PD 98059, Chloridzine, Piceatanol, Picropodophylline PKI, PP1, PP2, PTK787/ZK222584, PTK787/ZK- 222584, Purvalanol A, Rapamune, Rapamycin, Ro 31-8220, Rotlerin, SB202190, SB203580, Sirolimus, SL327, 00125, Staurosporine, STI-571, SU1498, SU4312, SU5416, SU5416 (Semaxanib), SU6656, SU6668, syk inhibitor, TBB, TCN, Tyrphostin AG 1024, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957, U0126, W-7, Wortmannin , Y-27632, Zactima (ZD6474), ZM 252868. gefitinib (Iressa.RTM.), sunitinib malate (SUTENT; SU11248), erlotinib (TARCEVA; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), sematinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec.RTM .; STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (ZACTIMA; ZD6474), and nilotinib.

[00239] In some particular embodiments, the tyrosine kinase inhibitor is sunitinib malate, Sorafenib tosilaro or Axitinib. Sunitinib malate, which is marketed by Pfizer Inc. under the trade name SUTENT, is chemically described as butanedioic acid, hydroxy-, (2S)-, compound with N-[2-(diethylamino)ethyl]-5-[(Z )-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide (1:1). The compound, its synthesis, and particular polymorphs are described in US Patent No. 6,573,293. Sunitinib malate is approved in the United States for the treatment of gastrointestinal stromal tumor, advanced renal cell carcinoma, and progressive, well-differentiated pancreatic neuroendocrine tumors in patients with unresectable locally advanced or metastatic disease. The recommended dose for gastrointestinal stromal tumor (GIST) and advanced renal cell carcinoma (RCC) for humans is 50 mg taken orally once daily on a schedule of 4 weeks on followed by 2 weeks off (Schedule 4 /two). The recommended dose of sunitinib malate for pancreatic neuroendocrine tumors is 37.5 mg taken orally once daily. In VBIR for cancer, sunitinib malate can be administered orally in a single dose or multiple doses. Typically, sunitinib malate is released for two, three, fourth, or more consecutive weekly doses followed by an "off" period of about 1 or 2 weeks or longer where no sunitinib malate is released. In one embodiment, doses are released for about 4 weeks, with 2 weeks off. The effective amount of sunitinib malate administered orally to a human is typically below 40 mg per person per day, such as 37.5, 31.25, 25, 18.75, 12.5, or 6.25 mg per person. per day. In some embodiments, sunitinib malate is administered orally in the range of 1 to 25 mg per person per day. In some other embodiments, sunitinib malate is administered orally in the range of 6.5, 12.5, or 18.75 mg per person per dose. Other dosage regimens and variations are predictable, and will be determined through medical advice.

[00240] Sorafenib tosylate, which is sold under the trade name NEXAVAR, has the chemical name 4-(4-{3-[4-Chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N-methylpyridine-2 -carboxamide. It is approved in the United States for the treatment of primary kidney cancer (advanced renal cell carcinoma) and primary advanced liver cancer (hepatocellular carcinoma). The recommended daily dose is 400 mg taken orally twice a day. In the VBIR for cancer provided by the present disclosure, the effective amount of sorafenib tosylate administered orally is typically below 400 mg per person per day. In some embodiments, the effective amount of sorafenib tosylate administered orally is in the range of 10 to 300 mg per person per day. In some other embodiments, the effective amount of sorafenib tosylate administered orally is between 10 to 200 mg per person per day, such as 10, 20, 60, 80, 100, 120, 140, 160, 180, or 200 mg per person per day. day.

[00241] Axitinib, which is sold under the trade name INLYTA, has the chemical name (N-Methyl-2-[3-((E)-2-pyridin-2-yl-vinyl)-1H- indazol-6- ilsulfanyl]-benzamide. It is approved for the treatment of advanced renal cell carcinoma after failure of previous systemic therapy. The starting dose is 5 mg orally twice daily. and tolerability. In the VBIR for cancer provided by the present disclosure, the effective amount of axitinib administered orally is typically below 5 mg twice daily. In some other embodiments, the effective amount of axitinib administered orally is between 1 and 5 mg twice daily. per day. In some other embodiments, the effective amount of axitinib administered orally is between 1, 2, 3, 4, or 5 mg twice daily.

[00242] Examples of IEC enhancers that can be used in the VBIR for cancer provided by the present disclosure include TNFR agonists, CTLA-4 antagonists, TLR agonists, other PD-1 antagonists (such as BMS-936558 and anti- - PD-1 CT-011), programmed cell death protein 1 (PD-L1) ligand 1 (PD-L1) antagonists (such as BMS-936559), lymphocyte activation gene 3 (LAG3) antagonists, and molecule-containing antagonists mucin domain and T cell immunoglobulin 3 (TIM-3). Examples of TNFR agonists include OX40, 4-1BB (such as BMS-663513), GITR (such as TRX518), and CD40 agonists. Examples of specific CD40 agonists are described in detail below.

[00243] In certain other embodiments, the additional immune modulator is an anti-CD40 agonist antibody. The antibody may be a human, humanized or partially human chimeric anti-CD40 antibody. Examples of specific anti-CD40 monoclonal antibodies include monoclonal antibody G28-5, mAb89, EA-5 or S2C6, and CP870893. In a particular embodiment, the anti-CD40 agonist antibody is CP870893 or dacetuzumab (SGN-40). 870893 or dacetuzumab (SGN-40).

[00244] CP-870,893 is a fully human agonistic CD40 monoclonal antibody that has been clinically investigated as an antitumor therapy. The structure and preparation of CP870,893 are disclosed in WO2003040170, in which the CP870,893 antibody is identified as antibody "21.4.1". The heavy chain and light chain amino acid sequences of CP-870,893 are set forth in SEQ ID NO: 46 and SEQ ID NO: 48, respectively, as well as in Table 7 in WO2003040170. In clinical trials, CP870,893 was administered by intravenous infusion at doses generally in the ranges of 0.05 and 0.25 mg/kg per infusion. In the VBIR for cancer provided by the present disclosure, CP-870,893 can be administered intradermally, subcutaneously or topically. The effective amount of CP870893 to be administered in the regimen is generally below 0.2 mg/kg, typically in the range of 0.01 mg to 0.15 mg/kg, or 0.05 to 0.1 mg/kg.

[00245] Dacetuzumab (also known as SGN-40 or huS2C6; CAS number 88-486-59-9) is another anti-CD40 agonist antibody that has been investigated in clinical trials for indolent lymphomas, diffuse large B-cell lymphomas, and multiple myeloma . In the VBIR for cancer provided by the present description, dacetuzumab can be administered intradermally, subcutaneously or topically. The effective amount of dacetuzumab to be administered is generally below 16 mg/kg, typically in the range of 0.2 mg to 14 mg/kg or 0.5 to 8 mg/kg, or 1 to 5 mg/kg.

[00246] In still other embodiments, the additional immune modulator is an anti-CTLA-4 antagonist. Examples of suitable anti-CTLA-4 antagonist include anti-CTLA-4 antibodies (such as anti-human CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, anti-CTLA-4 antibodies, 4, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, anti-CTLA-4 domain antibodies), and CTLA-4 inhibitors that agonize the co-stimulatory pathway. In some embodiments, the CTLA-4 inhibitor is Ipilimumab or Tremelimumab.

[00247] Ipilimumab (marketed as YERVOY; also known as MEX-010, MDX-101, or by its CAS Registry number 477202-00-9) is disclosed as antibody 10DI in PCT Publication No. WO 01/14424, incorporated herein by reference in its entirety and for all purposes. Examples of pharmaceutical composition comprising Ipilimumab are provided in PCT Publication No. WO 2007/67959. Ipilimumab is approved in the United States for the treatment of unresectable or metastatic melanoma. In the methods provided by the present invention, Ipilimumab can be administered intradermally or subcutaneously. The effective amount of locally administered ipilimumab is typically in the range of 5 to 200 mg/dose per person. In some embodiments, the effective amount of Ipilimumab is in the range of 10 to 150 mg/dose per person per dose. In some particular embodiments, the effective amount of Ipilimumab is about 10, 25, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

[00248] Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. Tremelimumab is disclosed as antibody 11.2.1 in US Patent No. 6,682,736, incorporated herein by reference in its entirety and for all purposes. In the VBIR for cancer provided by the present invention, Tremelimumab can be administered intravenously, intradermally or subcutaneously. The effective amount of Tremelimumab administered intradermally or subcutaneously is typically in the range of 5 to 200 mg/dose per person. In some embodiments, the effective amount of Tremelimumab is in the range of 10 to 150 mg/dose per person per dose. In some particular embodiments, the effective amount of Tremelimumab is about 10, 25, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

[00249] In still other modalities, the additional immune modulator is a Toll-Like Receptor (TLR) agonist. The term "toll-like receptor agonist" or "TLR agonist" refers to a compound that acts as an agonist of a toll-like receptor (TLR). These include agonists of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and TLR11 or a combination thereof.

[00250] TLR agonists useful in the method of the present invention include both small organic molecules and large biological molecules. Examples of small molecule TLR agonists include 4-amino-alpha, alpha,2-trimethyl-1H-imidazo[4,5-c]qumolin-1-ethanol, N-(2-{2-[4-amino- 2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy-}ethyl)-N-methylmorpholine-4-carboxamide, 1-(2-amino-2-methylpropyl)- 2-(ethoxymethyl-)-1H-imidazo[4,5-c]quinolin-4-amine, N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1 -yl)butyl]methanesulfonamide, N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide and imiquimod. Some TLR agonists particularly useful in the methods or regimen provided by the present disclosure are discussed in the review article: Folkert Steinhagen, et al.: TLR-based immune adjuvants. Vaccine 29 (2011): 3341-3355. In some embodiments, the TLR agonists are TLR9 agonists, particularly CpG oligonucleotides (or CpG.ODN). A CpG oligonucleotide is a short nucleic acid molecule containing a cytosine followed by a guanine linked by a phosphate bond in which the pyrimidine ring of the cytosine is unmethylated. Examples of particular CpG oligonucleotides useful in the methods provided by the present disclosure include: 5' TCGTCGTTTTGTCGTTTTGTCGTT3' (CpG 7909; SEQ ID NO:49); 5' TCGTCGTTTTTCGGTGCTTTT3' (CpG 24555; SEQ ID NO:50); and 5' TCGTCGTTTTTCGGTCGTTTT3' (CpG 10103; SEQ ID NO:51).

[00251] CpG7909, a synthetic single-chain 24mer, has been extensively investigated for the treatment of cancer as a monotherapy and in combination with chemotherapeutic agents, as well as an adjuvant for vaccines against cancer and infectious diseases. In the methods provided by the present disclosure, CpG7909 can be administered by injection into the muscle or any other suitable methods. For use with a nucleic acid-based vaccine, such as a DNA vaccine, a CpG can be co-formulated with the vaccine in a single formulation and administered by intramuscular injection coupled with electroporation. The effective amount of CpG7909 for intramuscular, intradermal, or subcutaneous administration is typically in the range of 10 μg/dose to 10 mg/dose. In some embodiments, the effective amount of CpG7909 is in the range of 0.05 mg to 14 mg/dose. In some particular embodiments, the effective amount of CpG7909 is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1 mg/dose. Other CpG oligonucleotides, including CpG 24555 and CpG 10103, can be administered at similar manner and dose levels.

[00252] In VBIR for cancer, the anti-PD-1 antagonist, the vaccine and additional immune modulators can be administered either simultaneously or sequentially. In some embodiments, a vaccine is administered sequentially with respect to the anti-PD-1 antagonist antibody, but simultaneously (e.g., in a mixture) with respect to one or more additional immune modulators. In cases where a nucleic acid vaccine is administered in combination with a CpG, the vaccine and CpG may be contained in a single formulation and administered together by any suitable method. In some embodiments, the nucleic acid and CpG vaccine in a coformulation (mixture) are administered by intramuscular injection in combination with electroporation.

FORMULATIONS

[00253] Therapeutic anti-PD-1 antagonist antibody formulations used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and may comprise buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

[00254] Liposomes containing the anti-PD-1 antagonist antibody are prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Academic. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and US Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in US Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes of the desired diameter.

[00255] Active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacile polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in drug delivery systems colloidal (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[00256] Extended-release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of molded articles, for example, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl -L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-lactic acid copolymers such as LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyrate acetate and poly acid -D-(-)-3-hydroxybutyric acid.

[00257] Formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic anti-PD-1 antagonist antibody compositions are generally placed in a container having a sterile access port, for example, an intravenous saline or vial having a stopper pierceable by a hypodermic injection needle.

[00258] The compositions according to the present invention may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or inflation.

[00259] To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, for example, conventional tablet ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums and other pharmaceutical diluents, for example water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically non-toxic salt thereof. When referring to these pre-formulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may comprise an internal dosage and external dosage component, the latter being in the form of an envelope over the former. The two components may be separated by an enteric layer that serves to resist disintegration in the stomach and allows the internal component to pass intact into the duodenum or be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including various polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[00260] Suitable surface active agents include, in particular, non-ionic agents, such as polyoxyethylene sorbitans (e.g., TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g., SpanTM 20, 40, 60, 80 or 85). Compositions with a surface active agent will conveniently comprise between 0.05 and 5% surface active agent and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example, mannitol or other pharmaceutically acceptable carriers, if necessary.

[00261] Suitable emulsions can be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient may be dissolved in a premixed emulsion composition or, alternatively, may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed by mixing with a phospholipid (for example, egg phospholipids, soy phospholipids and soy lecithin) and water. It will be appreciated that other ingredients may be added, for example, glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example between 5 and 20%. The fat emulsion may comprise fat droplets between 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and has a pH in the range of 5.5 to 8.0.

[00262] Emulsion compositions can be those prepared by mixing an anti-PD-1 antagonist antibody with IntralipidTM or components thereof (soybean oil, egg phospholipids, glycerol and water).

[00263] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered via the oral or nasal respiratory route for local or systemic effect. Compositions in sterile pharmaceutically acceptable solvents can be nebulized by the use of gases. Nebulized solutions can be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, positive pressure breathing machine, or intermittent. Solution, suspension or powder compositions can be administered, preferably orally or nasally, from devices that release the formulation in an appropriate manner.

KITS

[00264] The invention also provides kits comprising any or all of the antibodies described herein. Kits of the invention include one or more containers comprising an anti-PD-1 antagonist antibody described herein and instructions for use in accordance with any of the methods of the invention described herein. Generally, these instructions comprise a description of administration of the anti-PD-1 antagonist antibody for the therapeutic treatments described above. In some embodiments, kits are provided for producing a single dose administration unit. In certain embodiments, the kit may contain both a first container having a dry protein and a second container having an aqueous formulation. In certain embodiments, kits containing single- or multiple-chamber prefilled syringes (e.g., liquid syringes and lyosyringes) are included.

[00265] In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. Instructions regarding the use of an anti-PD-1 antibody generally include information such as dosage, dosing schedule, and route of administration for the intended treatment. Recipients may be unit doses, bulk packages (e.g., multidose packages) or subunit doses. Instructions provided in invention kits are typically instructions written on a label or leaflet (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions executed on a magnetic or optical storage disk) also are acceptable.

[00266] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The packages are also contemplated for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer), or an infusion device such as a minipump. A kit may have a sterile access port (for example, the container may be a bag of intravenous solution or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-PD-1 antibody. The container may further comprise a second pharmaceutically active agent.

[00267] Kits may optionally provide additional components such as buffers and interactive information. Typically, the kit comprises a container and a label or leaflet(s) on or associated with the container.

BIOLOGICAL DEPOSIT

[00268] Materials representative of the present invention were deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on April 29, 2014. The msb7-LC vector having ATCC Accession n ° PTA-121182 is a polynucleotide encoding the mAb7 light chain variable region, and mab7-HC vector having ATCC Accession No. PTA-121183 is a polynucleotide encoding the mAb7 heavy chain variable region. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This guarantees the maintenance of a viable deposit culture for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Pfizer, Inc. and ATCC, which ensures permanent and unrestricted availability of the deposit's crop progeny to the public upon issuance of the pertinent US patent or open to the public of any U.S. or foreign patent application, whichever comes first, and ensures availability of progeny for one determined by the Commissioner of United States Patents and Trademarks to be entitled thereto in accordance with 35 U.S.C. § 122 and the rules of the Commissioner pursuant thereto (including 37 C.F.R. § 1.14 with particular reference to 886 OG 638).

[00269] The assignee of this application has agreed that if a crop of materials in the deposit should die or be lost or destroyed when grown under suitable conditions, the materials will be promptly replaced on notice with others of the same. The availability of the deposited material should not be construed as a license to practice the invention in contravention of the rights granted under the authority of any government under its patent laws.

[00270] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

EXAMPLES Example 1: Effect of anti-PD-1 antibody on IFN-Y and TNF secretion

[00271] This example illustrates the effect of the anti-PD-1 antibody on the secretion of IFN-I and TNF in a mixed lymphocyte reaction (MLR) assay.

[00272] Primary human T cell isolated from whole blood (Stanford University Blood Bank) was activated by allogeneic dendritic cells (DC) expressing high levels of PD-L1 and PD-L2, which were previously differentiated using IL- 4 and GM-CSF from CD14+ myeloid cells. In this study, the following antibodies were used: isotype control (stabilized IgG4 kappa joint), anti-PD-1 antagonist antibody C1, anti-PD-1 antagonist antibody C2, anti-PD-1 antagonist antibody C3, EH12. 1 (BD Biosciences mouse anti-human PD-1 antibody, IgG1 mouse isotype Kappa), mAb7-G4, mAb15-G4, mAb- AAA, mAb15-AAA (G4 = stabilized IgG4 joint; AAA = mutant IgG1 which does not bind to FcyR). Antibodies were tested at the following concentrations: 0, 0.1, 1 or 10 Qg/mL).

[00273] For the MLR assay, cultures were incubated with test or control antibody in 96-well plates in triplicates at ratios of 1:10 DC: T cells and incubated in a humidified incubator at 37°C with 5% CO2 . Supernatants were harvested on day 5 and cytokines were measured using Cytometric Bead Array (CBA) using Human Soluble Protein Flex Set System kit (BD Biosciences, cat no. 558265) according to the manufacturer's protocol with the following human analytes: IFNY (BD Biosciences, cat no. 558269), TNF (BD Biosciences, cat no. 558273). Briefly, 96-well filter plates (Millipore, cat no. MSBVN1250) were washed with Wash Buffer (proprietary formula from BD Biosciences) and aspirated by vacuum collector. The standards provided by the kit and samples were diluted in Assay Diluent (proprietary formula from BD Biosciences) and added to the Capture Bead plates (capture beads are beads coated with antibodies to a specific soluble protein coated with a distinct fluorescence). Plates were mixed for 5 minutes at 500 rpm using a plate shaker, and incubated for 1 hour at room temperature. Detection Reagent (phycoerythrin (PE)-conjugated antibodies, provided by k) was added to the plates and the plates were mixed for 5 minutes. The plates were incubated for 2 hours at room temperature. Then, they were washed with Buffer for 5 minutes, and the samples were acquired on the BD Fortessa platforms. Data was analyzed using the FCAP v3 Matrix (BD). The MLR assay results are shown in Tables 6A and B below. Table 6A shows IFNγ levels (in pg/mL), and Table 6B shows TNF levels (in pg/mL). Data are presented as a mean ± SEM of biological triplicates. Samples are representative of an MLR experiment. Table 6A: IFNy secretion Table 6B: TNF Secretion

[00274] Treatment of activated T cells with anti-PD-1 antagonist antibodies resulted in increased levels of IFNγ compared to isotype control (Table 6A). For example, treatment with 0.1 μg/mL of mAb15-G4 and mAb7-G4 resulted in an IFNγ level of 4728.295 + 2.035 pg/mL and 8567.203 + 2085.826 pg/mL, respectively. Treatment with 1 μg/mL of mAb15-G4 and mAb7-G4 resulted in an IFNγ level of 8893.595 + 365.125 pg/mL and 11876.86 + 1259.788 pg/mL, respectively. Treatment with 10 μg/mL of mAb15-G4 and mAb7-G4 resulted in an IFNγ level of 7790.95 + 1700.012 pg/mL and 11794.82 + 1827.243 pg/mL, respectively. In contrast, treatment with 0.1, 1, or 10 qg/mL isotype control resulted in IFNγ levels of 3760 + 367.4262 pg/mL, 3693.972 + 1033.879 pg/mL, and 3525.655 + 744.676 pg/mL, respectively.

[00275] Treatment of activated T cells with anti-PD-1 antagonist antibodies resulted in increased levels of TNF compared to isotype control (Table 6B). For example, treatment with 0.1 qg/mL of mAb15-G4 or mAb7-G4 resulted in a TNF level of 743.1167 + 56.75547 pg/mL and 730.47 + 33.35488 pg/mL, respectively. . Treatment with 1 qg/mL of mAb15-G4 and mAb7-G4 resulted in a TNF level of 686.32 + 45.63348 pg/mL and 793.05 + 21.19019 pg/mL, respectively. Treatment with 10 □g/mL of mAb15-G4 or mAb7-G4 resulted in a TNF level of 798.853 + 9.14366 pg/mL and 930.623 + 16.0494 pg/mL, respectively. In contrast, treatment with 0.1, 1, or 10 qg/mL of isotype control resulted in TNF levels of 407.4133 + 49.58195 pg/mL, 486.7167 + 4.4241 pg/mL, and 501 .17 + 5.033334 pg/mL, respectively.

[00276] These results demonstrate that the anti-PD-1 antibodies mAb7 and mAb15 stimulate secretion of IFNγ and TNF from T cells at least as well as or better than the anti-PD-1 antibodies C1, C2 and C3.

[00277] A second MLR study was conducted to test the effect of lower antibody concentrations on T cell activation. Primary human T cell isolated from whole blood was activated as described above. The following antibodies were tested in the second study: mAb7 (G4), mAb15 (G4), C1 (G4), EH12.1, and G4 isotype control. Antibodies were tested at the following concentrations: 0.0001, 0.001, 0.01, 0.1, 1 and 10 μg/mL. The MLR assay was conducted as described above. The results are summarized in Tables 7A and 7B below. Table 7A: IFNy secretion Table 7B: TNF Secretion

[00278] Treatment of activated T cells with anti-PD-1 antagonist antibodies resulted in increased levels of IFNγ compared to isotype control (Table 7A). In cultures without antibodies, the level of IFNγ was 901,453 + 216,472 pg/mL. In cultures given 0.0001, 0.001, 0.01, 0.1, 1, or 10 μg/mL antibody isotype control, IFNγ levels were 558.9629 + 489.4828 pg/mL, 753.3767 + 291 .6092 pg/mL, 1074.37 + 324.2031 pg/mL, 1667.96 + 144.7286 pg/mL, 1867.96 + 282.7461 pg/mL, 2501.293 + 220.1829 pg/mL, respectively. In contrast, in cultures treated with 0.0001, 0.001, 0.01, 0.1, 1 or 10 dg/mL of mAb7 (G4), IFNγ levels were 2082.07 + 720.9931 pg/mL, 3062 .09 + 370.2791 pg/mL, 5067.823 + 111.4903 pg/mL, 7082.667 + 1336.082 pg/mL, 11928.81 + 1457.723 pg/mL, 11862.13 + 800.586 pg/mL mL, respectively. In cultures treated with 0.0001, 0.001, 0.01, 0.1, 1 or 10 Qg/mL of mAb15 (G4), IFNγ levels were 1678.27 + 233.82 pg/mL, 1410.758 + 439.9474 pg/mL, 4734.49 + 322.2087 pg/mL, 5416 + 1054.075 pg/mL, 9140.337 + 1320.499 pg/mL, and 10992.13 + 1008.533 pg/mL, respectively.

[00279] Treatment of activated T cells with anti-PD-1 antagonist antibodies resulted in increased levels of TNF compared to isotype control (Table 7B). In cultures without antibodies, the TNF level was 365.523 + 84.6607 pg/mL. In cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10 Dg/mL of isotype control antibody, TNF levels were 452.7133 + 62.85 pg/mL, 282.9287 + 56.77266 pg/mL, 310.9144 + 21.7811 pg/mL, 358.948 + 81.09122 pg/mL, 338.1107 + 46.88385 pg/mL and 331.008 + 31.35559 pg/mL, respectively. In contrast, in cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10 μg/mL of mAb7 (G4), TNF levels were 227.6 + 50.63436 pg/mL, 394 .4233 + 30.47005, 452.65 + 30.64335 pg/mL, 1089.377 + 174.7824 pg/mL, 1583.52 + 267.2131 pg/mL, and 1419.88 + 108.711 pg/mL, respectively. In cultures given 0.0001, 0.001, 0.01, 0.1, 1, or 10 dg/mL of mAb15 (G4), TNF levels were 494.7967 + 48.1810 pg/mL, 489.2333 + 30 .63302 pg/mL, 593.34 + 65.87622 pg/mL, 811.16 + 89.50238 pg/mL, 1143.54 + 136.3954 pg/mL, and 1109.063 + 57.70232 pg/mL , respectively.

[00280] These results demonstrate that the anti-PD-1 antibodies mAb7 and mAb15 block PD-1 signaling and promote secretion of IFNI and TNF from primary human T cells.

Example 2: Effect of anti-PD-1 antibodies on T cell proliferation

[00281] This example illustrates the effect of anti-PD-1 antibodies on T cell proliferation.

[00282] In this study, T cell proliferation was measured in an MLR assay in which T cells were cultured in the presence of anti-PD-1 antagonist antibodies or isotype control.

[00283] For MLR, primary human T cell isolates from whole blood (obtained from the Stanford University blood bank) were activated by allogeneic dendritic cells (DC) expressing high levels of PD-L1 and PD-L2, which were previously differentiated using IL-4 and GM-CSF from CD14+ myeloid cells. Two experiments were conducted.

[00284] In the first experiment, mAb7 (stabilized IgG4 kappa joint), mAb15 (stabilized IgG4 kappa joint), C1, EH12.1 and isotype control were compared. In the second experiment, clones mAb7, mAb15, C2, EH12.1 and isotype control were compared. In both experiments, antibodies were added at the following concentrations: 0, 0.0001; 0.001; 0.01; 0.1; 1 and 10 dg/mL.

[00285] For both experiments, cultures were incubated with antibody in 96-well plates in triplicates at 1:10 ratios of DC: T cells and incubated in the humidified incubator at 37°C with 5% CO2. On day 5, cultures were pulsed for 18 h with 1 μCi per well of [3H]-thymidine before harvesting. Plates are then harvested onto DNA-specific filter papers (Perkin Elmer) using Harvester96 (Tomtec Life Sciences). The radiolabeled filters were covered with beta scintillation liquid (Perkin Elmer) and read on Microbeta® counter plates (Perkin Elmer). Thymidine incorporation was analyzed as counts per minute (CPM). Results are shown as means of triplicates ± SEM. Table 8A Table 8B

[00286] Treatment of activated T cells with anti-PD-1 antagonist antibodies at a concentration of 0.01 Qg/mL or more resulted in significantly increased T cell proliferation compared to the isotype control (Tables 8A and 8B).

[00287] For example, treatment with 0.01, 0.1, 1 or 10 Qg/mL of mAb7 resulted in thymidine incorporation rates of 365625.3 + 30171.07 CPM, 380054.3 + 9774.328 CPM , 392256.7 + 15341.19 CPM, and 372889.7 + 14826.49 CPM, respectively (Table 8B). Treatment with 0.01, 0.1, 1, or 10 Qg/mL of mAb15-G4 and mAb7 resulted in thymidine incorporation rates of 317377 + 31915.29 CPM, 360226.3 + 1802.69 CPM, 421229 + 27865 .13 CPM, and 323441.3 + 64476.55 CPM, respectively (Table 8B). In contrast, treatment with 0.01, 0.1, 1, or 10 Qg/mL isotype control resulted in thymidine incorporation rates of 278072.3 + 32671.62 CPM, 268939.7 + 12332.06 CPM, 241164 + 13776.81 CPM, and 231897.7 + 25865.95 CPM, respectively (Table 8B).

[00288] These results demonstrate that the anti-PD-1 antibodies mAb7 and mAb15 block PD-1 signaling and promote the proliferation of primary human T cells.

Example 3: Effect of anti-PD-1 antibodies in a mouse model of GvHD

[00289] This example illustrates the effect of anti-PD-1 antibodies on T cell proliferation and body weight loss in a mouse model of graft versus host disease (GvHD).

[00290] NOD-scid-IL-2 receptor gamma chain (NSG) null mice were used in this study to test the effects of anti-PD-1 antagonist antibodies on T cell proliferation in vivo. Because NSG mice lose T cells and B cells and impair NK cells, high human cell engraftment is quickly achieved. When human PBMCs are engrafted into these mice, human T cell proliferation occurs and induces GvHD. GvHD involves the host myeloid compartment as well as human cells. Massive proliferation of human lymphocytes can be seen in the blood at early stages followed by high infiltration of these cells within the organs of the mouse such as liver, spleen, kidney, intestine etc. resulting in loss of body weight of the mice as well as injuries in the skin, hunchback and death. The severity of the models depends on the donor PBMCs and may differ between donors.

[00291] In this study, the following anti-PD-1 antagonist antibodies were used: mAb7 (stabilized human IgG4 joint or AAA), mAb15 (stabilized human IgG4 joint), C1, C2 and C3. For the negative control, a stabilized human IgG4 articulation antibody was used. Primary human PBMCs were isolated from whole blood (Stanford University Blood Bank) using the Ficoll gradient. 107 of human PBMCs were injected into NSG mice (8-week-old females, Jackson Laboratories). On day 0, mice were randomized based on body weight and PBMCs were injected intravenously. For experiments 1 to 4, on day 2 and day 8, antibodies were administered intraperitoneally at 10 mg/kg. For experiment 5, on day 2 and day 8, antibodies were administered intraperitoneally at 1 mg/kg or 10 mg/kg. Table 9 summarizes the antibodies used in each experiment. Table 9: Antibodies Used in Experiments 1 to 5

[00292] Body weight was measured periodically. The results are summarized in Figures 1A-1E. Mice were bled periodically to assess T cell proliferation. Anti-PD-1 antibody treatment accelerated disease cure as measured by the rate of body weight loss. Compared to control mice, mice treated with anti-PD-1 antagonist antibody had faster body weight loss (Figures 1A-1E).

[00293] Human T cell proliferation was measured by flow cytometry using CD45 as a marker (clone HI30; BD Biosciences). Flow cytometry results are summarized below in Table 10. T cell proliferation was greater in mice treated with anti-PD-1 antagonist antibody than in mice treated with isotype control. Higher percentage CD45 indicates high level of proliferation in CD45 cells and therefore more severe GvHD.

[00294] In Experiment 1, the percentage of CD45 positive blood cells was 63.86% in control mice (Table 10). In contrast, the percentage of CD45-positive blood cells in mice treated with anti-PD-1 antibody mAb7, mAb15, C1, or mAb7-AA was 80.34%, 77.62%, 77.26%, and 76.9%. %, respectively (Table 10). Table 10: T cell proliferation as measured by the presence of CD45, treated animals

[00295] In summary, mice treated with anti-PD-1 antibody had faster body weight loss and increased T cell proliferation compared to mice treated with isotype control. These results demonstrate that anti-PD-1 antibody treatment stimulates the proliferation of human T cells in vivo.

Example 4: Binding of anti-PD-1 antibodies

[00296] This example illustrates anti-PD-1 antibody binding to activated human T cells and cynomolgus monkey (cyno) T cells.

[00297] Primary human T cells were isolated from PBMCs (Stanford University blood bank) using a human PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-096-353). Cyno PBMCs were purchased from (BioreclamationIVT), and PAN T cells were isolated using the nonhuman primate PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-091-993). Human T cells were activated for 3 days with DYNABEADS™ Human CD3/CD28 T Activator for Cell Expansion and Activation (Life Technologies ; 11131D). Ratio of beads to cell used was 1:1 bead:T cell, respectively. Cyno T cells were activated for 3 days using T Cell Activation/Expansion Kit, non-human primate according to the manufacturer's protocol (Miltenyi Biotec; 130092-919). The bead to cell ratio used was 1:1 bead:T cell; respectively. After 3 days, the cultures were harvested, the spheres were separated from the activated T cell using magnetic force. Cells were washed and incubated with FACS buffer (including 2% FBS) and Human Fc Receptor binding inhibitor (Affymetrix eBioscience cat. no. 16-9161-73). For cyno cells, Fc Blocking reagent (BD Biosciences cat. no. 564765) was used. Cells were incubated for 10 minutes at room temperature and then stained with live dead color to exclude dead cells (LIVE/DEAD® Fixable Blue Dead Cell Staining Kit, for UV excitation; catalog no. A10346) for an additional 5 minutes . Anti-PD-1 antibodies were added (concentrations of anti-PD-1 clones were incubated in the cell at 1:3 serial dilution ratios starting from 10 Qg/mL to 0 Qg/mL) 1x106 cells were used in each reaction in total 100 Dl and the cells were incubated on ice for 30 minutes. The cells were then washed with FACS buffer to remove access of primary antibodies and incubated with secondary anti-human IgG (AffiniPure F(ab')2 (H+L) Donkey Fragment Anti-Human IgG conjugated to Allophycocyanin (APC); cat . no. 709136-149). Cells were stained for 30 minutes on ice. Cells were washed and kept on ice until reading using BD LSRFortessa Cell Analyzer, (BD Biosciences, cat. no. 647465). Data were analyzed using FlowJo™ software. The results are summarized in Figures 2A and 2B.

[00298] Figure 2A shows measured EC50 for anti-PD-1 antibody that binds to human activated cells, and Figure 2B shows measured EC50 for anti-PD-1 antibody that binds to activated cyno cells. Anti-PD-1 antibodies mAb7 and C1 bind to activated T cells with similar EC50 (Figures 2A and 2C).

Example 5: Inhibition of PD-L1 binding by anti-PD-1 antibody

[00299] This example illustrates the inhibition of PD-1 ligand binding (PD-L1) by the anti-PD-1 antibody.

[00300] Primary human T cells were isolated from PBMCs (Stanford University blood bank) using PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130-096-353). Cynomolgus Monkey PBMCs were purchased from (BioreclamationIVT), and PAN T cells were isolated using non-human primate PAN T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec; 130091-993). Human T cells were activated for 3 days with DYNABEADS™ Human CD3/CD28 T Activator (for cell expansion and activation, Life Technologies; 11131D). The ratio of beads to cell used was 1:1; respectively. Cyno T cells were activated for 3 days using T Cell Activation/Expansion Kit, non-human primate according to the manufacturer's protocol (Miltenyi Biotec; 130-092-919). The ratio of beads to cells used was 1:1; respectively. After 3 days, the cultures were harvested, the spheres were separated from the activated T cell using magnetic force. Cells were washed and incubated with FACS buffer (including 2% FBS) and Human Fc Receptor binding inhibitor (Affymetrix eBioscience; cat. no. 16-9161-73). For cyno cells, Fc blocking reagent (BD Biosciences; cat. no. 564765) was used. Cells were incubated for 10 minutes at room temperature and then stained with live dead color to exclude dead cells (LIVE/DEAD® Fixable Blue Dead Cell Staining Kit, for UV excitation; cat. no. A10346) for another 5 minutes. minutes. Human Recombinant PD-L1 Fc (R&D Systems, cat. no. 156-B7) or buffer alone was incubated with the cells at 10 ng/mL. Each ligand was incubated separately and incubated on ice for 30 minutes. Cells were washed and incubated with anti-PD-1 antibodies (concentrations of anti-PD-1 clones were incubated in the cell at 1:3 serial dilution ratios starting at 1 μg/mL to 0 μg/mL). 1x106 cells were used in each reaction totaling 100 μl and cells were incubated on ice for 30 minutes. Cells were then washed with FACS buffer to remove accession of primary antibodies and incubated with Allophycocyanin (APC)-conjugated anti-human kappa (Life Technologies; cat. no. MH10515). Cells were stained for 30 minutes on ice, then washed and kept on ice until reading using BD LSRFortessa Cell Analyzer, (BD Biosciences, cat. no. 647465). Data were analyzed using FlowJo™ software and Mean Fluorescence Intensity (MFI) and geometric means (Geo.M) of APC staining in live cells were calculated in FlowJo™ software. After geometric mean calculation, IC50 were calculated using GraphPD Prism software. The results are summarized in Tables 11 and 12 below. Table 11: Anti-PD-1 Blockade of PD-L1 Binding to PD-1 on Human T Cells Table 12: Anti-PD-1 Blockade of PD-L1 Binding to PD-1 on Cyno T Cells

[00301] These results demonstrate that the anti-PD-1 antibodies mAb7 and C1 inhibit the binding of PD-L1 to human and cyno T cells with similar IC50.

Example 6: Effect of anti-PD-1 antibody on T cell proliferation

[00302] This example illustrates the effect of anti-PD-1 antibody on T cell proliferation.

[00303] CD4 and CD8 (AllCells, LLC) were activated for 2 days with DYNABEADS™ CD3/CD28 Human T Activator (or cell expansion and activation (Life Technology; 11131D). The ratio of beads to cell used was 1: 1; respectively, to induce PD-1. On day 2, the cultures were harvested and the spheres were separated from the activated T cell using magnetic force. were incubated with different anti-PD-1 clones at 1 μg/mL in 96-well plates, in triplicates, in ratios of 1:10 DC:T cells and incubated in a humidified incubator at 37°C with 5% CO2. Day 3, cultures were pulsed for 18h with 1 μCi per well of [3H]-thymidine before harvesting. Plates were then harvested onto DNA-specific filter papers (PerkinElmer) using Harvester96 (Tomtec Life Sciences). Radiolabeled samples were covered with beta scintillation liquid (Perkin Elmer) and read on Microbeta® counterplates (Perkin Elmer). Thymidine incorporation was analyzed as counts per minute (CPM). Results are shown as mean of triplicates ± SEM in Figures 3 and 4.

Example 7: Determination of Kinetics and affinity of human, cynomolgus monkey and mouse PD-1 interacting with humanized anti-PD-1 antibodies

[00304] This example illustrates the binding of anti-PD-1 antibodies to human, kine or mouse PD-1.

[00305] All interaction analysis was performed on label-free biosensors at 25°C, unless otherwise stated. Surface plasmon resonance biosensors (ProteOn-XPR™ from BioRad™, and Biacore 2000™ and Biacore T200™ from GE Life Sciences) were used to study human and cynomolgus monkey PD-1 and a biolayer interferometry biosensor (Octet - Red384, Fortebio/Pall Life Sciences) was used to study mouse PD-1. ProteOn experiments were performed in PBS pH 7.4 + 0.01% running buffer Tween-20 (PBST). Biacore experiments were performed in 10 mM Hepes pH 7.4, 150 mM NaCl, 0.05% Tween-20 (HBST+) and Octet experiments were performed in HBST+ with 1 g/l BSA. ProteOn data was processed in ProteOn Manager software, Biacore data was processed in Biaevaluation, and Octet data was simply zero-aligned in the control software. The SPR data were double referenced (Myszka, 1999, J Mol Recognit12(5):279-284) and globally fitted to a simple Langmuir model to determine the equilibrium dissociation constant, KD, from the ratio of the kinetic rate constants (KD = kd/ka).

Calibration-free concentration analysis (CFCA)

[00306] The active concentration of human PD-1 monomer (hPD-1) (Sino Biologicals, cat no. 10377-H08H) for use as an analyte in kinetic experiments with immobilized IgGs was determined empirically using a CFCA test on a Biacore T200™ equipped with a CM5 sensor chip. To prepare the surfaces for these experiments, a high capacity (approximately 12,000 RU) of mAb15 hIgG4 (or in some experiments, competing antibody C2-hIgG1) was coupled to the amine in flow cell 2, leaving a control flow cell 1. (only "activated and blocked", without any IgG) to provide a reference surface. hPD-1 samples were injected at nominal concentrations of 0.1, 1, and 10 μg/mL for 36 s at both low (5 μL/min) and high (100 μL/min) flow rates. Surfaces were regenerated with a cocktail of 2:1 v/v Pierce IgG Elution Buffer (pH 2.8):4 M NaCl. Data was analyzed in the CFCA tool in T200 software to derive an activity value evident for the hPD-1 analyte, which was used to correct for its "nominal" protein concentration as determined by absorbance at 280 nm with extinction coefficient appropriate, at an "active" protein concentration. Some batches were observed to be 32% active, while others were 100% active.

[00307] Kinetic analysis of human PD-1 (hPD-1) binding to amine-coupled mAb7, mAb15, C1, C2, C3 and C4, mAbs

[00308] A ProteOn-XPR36 equipped with GLC sensor chips (BioRad™, Hercules, CA), was used to determine the kinetics and affinity of hPD-1 monomer binding to a panel of anti-hPD-1 mAbs coupled to the amine (mAb7, mAb15, C1, C2, C3 and C4) in standard PBST buffer. The surfaces for these experiments were prepared in three steps; (1) ligand channels were minimally activated for two minutes using a freshly prepared mixture of the activation reagents in 0.8 mM EDC and 0.2 mM sulfo-NHS final in water, (2) IgGs were coupled for three min at 15 μg/mL in 10 mM sodium acetate pH 4.5 and (3) excess reactive esters were blocked for three min with 1 M ethanolamine HCl, pH 8.5. Final coupled IgG levels ranged from 400 RU to 1157 RU. The hPD-1 monomer was injected in a one-step kinetic fashion (Bravman et al., 2006, Anal Biochem 358(2):281-288) along the "analyte" channels as a three-fold dilution series. with maximum "active" concentrations of 30, 44, or 36 nM, depending on the experiment. Association and dissociation times were 3 min and 20 min, respectively, and all analytes were injected in binding cycles in duplicates. Surfaces were regenerated with a cocktail of 2:1 v/v Pierce IgG elution buffer (pH 2.8):4 M NaCl. TABLE 13: Kinetic analysis of hPD-1 monomer binding to amine-coupled IgGs

[00309] The interactions of the hPD-1 monomer with mAb7 and mAb15 showed no visible decay in the binding response within the allowed dissociation phase, so an upper limit was placed on their kd and KD values, in accordance with the "rule of 5%" (Katsamba et al, 2008, Anal Biochem 352(2):208-221), was applied to place an upper limit on its kd and KD values. N = 2 refers to two independent experiments on different chips. Cross-reaction of mAb7 and mAb15 to cynomolgus monkey PD-1

[00310] The binding kinetics of the recombinant purified Fab fragments (mAb7 and mAb15) to both hPD-1-hFc1 (R&D Systems cat no. 1086PD) and cinoPD-1-hFc1 (prepared on site) were determined using a Biacore 2000™ equipped with a CM4 sensor chip and HBST+ current buffer. An anti-hFc polyclonal antibody was amine-coupled to the chip and used to capture approximately 90 RU of hPD-1-hFc1 and 125 RU of cinoPD-1-hFc1 in flow cells 2 and 3, leaving a control flow cell 1 (unprotected anti-hFc capture surface) to provide a reference channel. Recombinant purified Fabs were injected for two min as analyte at 0, 10, and 100 nM into freshly captured PD-1-hFc1 fusion proteins, allowing a dissociation time of 15 min. Capture surfaces were regenerated using 75 mM phosphoric acid and mAb7 Fab samples were injected into duplicate binding cycles. All of the Fab/PD-1 complexes were very stable, such that none of the interactions showed any visible decay in their binding responses within the allowed dissociation time, thus the "5% rule" (Katsamba et al, 2008) was applied to place an upper limit on its kd and KD values. TABLE 14: Determination of the affinity of mAb7 and mAb15 Fabs for the hPD-1-hFc1 and cinoPD-1-hFc1 fusion proteins

[00311] Temperature dependence of hPD-1 binding AI to mAb15, mAb7 and C3

[00312] A Biacore T200™ equipped with a CM4 sensor chip was used to determine the kinetics and affinities of hPD-1 monomer binding to a panel of hIgG4 molecules (mAb15, mAb7 and C3) that were captured at low levels using amine-coupled anti-hFc polyclonal antibody. hIgG4 mAbs were captured at 10 μg/mL on individual flow cells, leaving a control flow cell 1 to serve as a reference surface (unprotected capture surface). hPD-1 was injected at active concentrations of 0, 10, and 100 nM for three minutes, allowing a dissociation phase of 18 min. Trapping surfaces were regenerated with 75 mM phosphoric acid after each binding cycle. Table 15: Kinetic analysis of the binding of hPD-1 monomer as analyte to hIgG4 molecules captured with anti-hFc

Cross-reaction of mAb7 with mouse PD-1

[00313] An Octet-Red384 equipped with streptavidin sensor tips was used to determine whether mouse PD-1 binds to mAb7. An avidity-prone assay format was selected to increase the detection sensitivity of the assay. The sensors were coated with biotinylated polyclonal anti-human coat and used to capture a panel of hIgG4 anti-hPD-1 mAbs (mAb7, C1, C2, and C3) at 10 μg/mL; each mAb was captured on eight sensors. As a positive control, eight streptavidin sensors were coated with biotinylated J43 (eBioSciences), an anti-mouse PD-1 antibody. Each mAb-coated sensor was exposed to the following analytes: buffer, 1 μM mouse PD-1-hFc with binding sites, 1 μM hPD-1-hFc with binding sites (positive control), or 1 μM hGHR -hFc with binding sites (negative control). All recombinant Fc fusion proteins were from R&D Systems. Each analyte/mAb interaction was therefore tested on the sensors in duplicate.

[00314] Anti-PD-1 antibodies C1, C2 and C3 did not bind mouse-PD-1-hFc1 (data not shown). Weak mouse-PD-1-hFc1-linked anti-PD-1 antibody mAb7 (data not shown). All anti-PD-1 antibodies tested bound to hPD-1-hFc. These results demonstrate that mAb7 is weakly cross-reactive with mouse PD-1, whereas C1, C2, and C3 are not cross-reactive with mouse PD-1.

Example 8: Cancer treatment with anti-PD-1 antibodies

[00315] This is a prophetic example that illustrates use of the anti-PD-1 antibodies of the present invention to treat cancer.

[00316] Patients with histologically confirmed, previously untreated, measurable metastatic colorectal cancer are selected for treatment with an anti-PD-1 antibody. Patients are assigned to one of two treatment groups: chemotherapy plus placebo or chemotherapy plus mAb7. A dynamic randomization algorithm is used to achieve balance overall and within each of the following categories: study center, baseline ECOG performance status (0 vs. > 1), primary disease site (colon vs. rectum), and number of metastatic sites (1 vs. >1). Chemotherapy treatment is given weekly for the first 6 weeks of each 8-week cycle. Chemotherapy is continued until study completion (96 weeks) or disease progression. 5 mg/kg of Ab7 or placebo is administered every 2 weeks. Patients in the mAb7 arm who have a confirmed complete response or unacceptable toxicity experienced as a result of chemotherapy treatment are allowed to discontinue chemotherapy and continue receiving mAb7 alone, as first-line treatment. Only patients who are randomized to the mAb7 group can receive mAb7 as a component of second-line treatment. After completing the study, patients are followed for any subsequent treatment and survival every 4 months until death, loss to follow-up, after completing the study, or study termination.

[00317] Patients will undergo a tumor status assessment at baseline and at the conclusion of each 8-week cycle using radiographic techniques, typically spiral CT scanning. Tumor response, or progression, will be determined by both the investigator and an independent radiology center (IRF) using the Response Evaluation Criteria in Solid Tumors. Therasse et al. (2000). The IRF assessment will be performed without knowledge of treatment assignment or investigator assessment. Additionally, patients will complete the Functional Assessment of Cancer Therapy—Colorectal (FACT-C), Version 4, a validated instrument to assess quality of life (QOL) in patients with colorectal cancer, at baseline and before each cycle. treatment until disease progression. Ward et al. (1999) Qual. Life Res. 8: 181-195.

[00318] Safety is assessed from adverse event reports, laboratory test results, and vital sign measurements. Adverse events and abnormal laboratory results are evaluated according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC), Version 2. Prespecified safety measures include four adverse events of special interest (hypertension, proteinuria, thrombosis, and hemorrhage ).

[00319] The primary outcome measure is the duration of overall survival. Secondary outcome measures include: progression-free survival, objective response rate (complete and partial), duration of response, and changes in QOL FACT-C score. Duration of survival is defined as the time from randomization to death. For patients alive at the time of analysis, duration of survival will be censored at the date of last contact. Progression-free survival is defined as the time from randomization to the first of disease progression or death on study, defined as death from any cause within 30 days of the last dose of study drug or chemotherapy. For patients alive without disease progression at the time of analysis, progression-free survival at their last tumor assessment, or day 1 (the first day of study treatment) will be censored if no post-baseline assessment was performed. In objective response analysis, patients without tumor assessments are categorized as non-responders. Disease progression and response analyzes are based on IRF assessments. Changes in quality of life are analyzed as the time to deterioration in QOL (TDQ), defined as the period from randomization to the first of a >3-point decrease from baseline on the FACT-C subscale score specific to colon cancer (CCS), disease progression, or death in the study. TDQ will also be determined for TOI-C (sum of CCS, physical and functional well-being) and total FACT-C for changes from baseline.

Example 9: Cancer treatment with anti-PD-1 antibodies

[00320] This example illustrates use of the anti-PD-1 antibodies of the present invention to treat cancer.

[00321] The study, in this example, is a Phase 1, open-label, multicenter, multiple-dose, dose-escalation, safety, PK, and PD study of anti-PD-1 monoclonal antibody mAb7 administered intravenously to adult patients previously treated with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian carcinoma, sarcoma, or relapsed or refractory Classical Hodgkin Lymphoma (cHL). The study protocol is summarized below in Table 16. Table 16

[00322] Inclusion Criteria: - Histological or cytological diagnosis of locally advanced or metastatic melanoma, SCCHN, ovarian cancer, sarcoma, or relapsed or refractory cHL: - patient must have received at least 1 and no more than 5 prior lines of therapy for recurrent or metastatic disease, including both standards of care and investigational therapies. - At least one measurable lesion as defined by RECIST version 1.1, or (for cHL) at least 1 fluorodeoxyglucose positron emission tomography (FDG PET), avid measurable lesion (Deauville 4/5) >1.5 cm as defined per Response Criteria for Malignant Lymphoma, which has not been previously irradiated. - For Part 1B expansion and all Part 2 cohorts: patient has consented to undergo pre-treatment and on-treatment biopsy. - Appropriate exclusion criteria of kidney function, liver, bone marrow - Active brain or leptomeningeal metastasis. - Ocular melanoma - Active, known or suspected autoimmune disease. Patients with vitiligo, type I diabetes mellitus, residual hypothyroidism due to autoimmune condition only requiring hormone replacement, psoriasis not requiring systemic treatment, or conditions not expected to occur periodically in the absence of an external trigger are permitted to enroll. Diagnosis of previous immunodeficiency or immunosuppressive therapy requiring organ transplantation. - For Part 2: prior treatment with a PD 1 or PD L1 antibody. - History of grade >3 immune-mediated AE (including AST/ALT elevations that were considered drug-related and cytokine release syndrome) that was considered related to prior immunomodulatory therapy (e.g., immune checkpoint inhibitors, co-stimulators etc.) and required immunosuppressive therapy.

[00323] The number of patients with ORR (Objective Response Rate) will be measured at baseline and every six weeks until disease progression or unacceptable toxicity up to 24 months.

Example 9: Antagonistic activity of anti-PD-1 antibodies in human and cynomolgus monkey primary T cells

[00324] This example illustrates the activity of anti-PD-1 antibodies in human and cynomolgus monkey primary T cells.

[00325] In this study, the antagonistic activities of anti-PD-1 monoclonal antibody mAb7 were examined in vitro using a mixed lymphocyte reaction (MLR). Primary T cells were isolated from human and cynomolgus monkey peripheral blood mononuclear cells (PBMCs). After mAb7 exposure, cell proliferation and cytokine secretion were assessed in vitro under different activation conditions using an MLR for human and cynomolgus monkey and the cytokine release assay using cynomolgus monkey blood activated with the Staphylococcal superantigen enterotoxin B ( SEB).

Methods Human T Lymphocytes

[00326] Human buffy coat was purchased from Stanford Blood Center (Stanford, CA), diluted with phosphate buffered saline (PBS) and layered on Ficoll for isolation of PBMCs. hu-PBMCs were washed 4 times with PBS, and T lymphocytes were isolated using a Pan T cell isolation kit with negative selection as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA).

Cynomolgus Monkey T Lymphocytes

[00327] Fresh cynomolgus monkey PBMCs were purchased from Bioreclamation IVT (New York, NY) and washed twice with PBS. T lymphocytes were isolated using a nonhuman primate-specific Pan T cell isolation kit with negative selection as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA).

Generation of Human Dendritic Cells Expressing High Levels of PD-L1

[00328] Human buffy coat was purchased from Stanford Blood Center (Stanford, CA), diluted with PBS and layered on Ficoll for isolation of hu-PBMCs. hu-PBMCs were washed 4 times with PBS and monocyte differentiation cluster 14 (CD14+) was isolated using a positive selection human-specific CD14 cell isolation kit as described in the manufacturer's protocol (Miltenyi Biotec, San Diego , CA). Cells were then seeded at 5 x 105 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum (FBS) for 7 days. Cultures were supplemented with recombinant human (rh-)IL-4 (1000 U/mL) (R&D Systems, Minneapolis, MN) and rh-granulocyte-macrophage colony-stimulating factor (GM-CSF) (rh-GMCSF). (500 U/mL) (R&D Systems, Minneapolis, MN) on Days 0, 2, and 5. Immature DCs were harvested, washed, and counted on Day 7. One sample from each preparation was tested for PD-L1 expression using anti- r-phycoerythrin (RPE)-labeled hu-PD-L1 (eBioscience/Affymatrix, San Diego, CA) via flow cytometry using an LSRFortessa™ analyzer (BD Biosciences, San Jose, CA).

Generation of Cynomolgus Monkey Dendritic Cells Expressing High Levels of PD-L1

[00329] Fresh cynomolgus monkey PBMCs were purchased from Bioreclamation IVT (New York, NY) and washed twice with PBS. CD14 + monocytes were isolated using a positive selection nonhuman primate-specific CD14 cell isolation kit as described in the manufacturer's protocol (Miltenyi Biotech, San Diego, CA). Cells were then seeded at 5 x 105 cells/mL in RPMI complete 1640 media supplemented with 10% FBS for 7 days. Cultures were supplemented with rhIL-4 (1000 U/mL) (R&D Systems, Minneapolis) and rh-GMCSF (500 U/mL) (R&D Systems, Minneapolis, MN) on Days 0, 2, and 5. Immature DCs were collected, washed, and counted on Day 7. One sample from each preparation was tested for PD-L1 expression using RPE-labeled anti-hu-PD-L1 (eBioscience/Affymatrix, San Diego, CA) via flow cytometry using a LSRFortessa™ analyzer (BD Biosciences, San Jose, CA).

Generation of Clones of JeKo-1-Luc-Green Fluorescent Protein Cells Expressing High Levels of Human PD-L1

[00330] The JeKo-1 cell line (a Mantle Cell Lymphoma) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The JeKo-1-luc-2A-GFP cell line was produced by Pfizer (South San Francisco, CA) by a transduction process that used lentiviral particles which individually express firefly luciferase (luc2A) and green fluorescent protein (GFP). through a bicistronic system with a blasticidin tracer (AMSBIO, LVP323, 1 x 10E7 particles per 200 μL) according to the manufacturer's protocol. JeKo-1 cells were pelleted and diluted to 1 x 106 cells/mL in RPMI with 20% FBS medium. Lentiviral particles were added to the diluted cells at a ratio of 50 μL of virus per 0.5 mL of cells. To generate a JeKo-1 cell line expressing hu-PD-L1, hu-PD-L1 cDNA was synthesized as usual and cloned into generic expression vector (pcDNA3.1) through Life Technology (San Diego, CA) . A JeKo-1-Luc-GFP cell line stably expressing hu-PDL-1 was produced from Pfizer (South San Francisco, CA) via electroporation using the Amaxa® Nucleofector system (Lonza, Walkersville, MD) and Kit V used from according to the manufacturer's protocol (Lonza, Walkersville, MD). Cells were then cultured in the presence of 250 μg/mL hygromycin for 2 weeks and then selected by cell sorting using a BD FACSAria™ II cell sorter (BD Biosciences, San Jose, CA). Cell sorting was conducted with the anti-hu-PD-L1 antibody clone MIH-1 (Affymetrix/eBioscience, San Diego, CA) directly labeled with allophycocyanin (APC) tag. Positive clones were expanded and tested for high PD-L1 expression using flow cytometry (LSRFortessa™ analyzer, BD Biosciences, San Jose, CA). Clones that were generated from the single sorted cells and that contained high levels of PD-L1 expression were selected.

Antibody Generation

[00331] mAb7 was generated in CHO cells (Pharmaceutical Sciences, Pfizer Inc, Saint Louis, MO) using Good Laboratory Practices (GLP) material (Lot No. STL0005717) and supplied in 20 mM histidine, 85 mg/mL sucrose , 0.2 mg/mL polysorbate-80, 0.05 mg/mL disodium EDTA, buffer pH 5.5. Endotoxin was measured as <0.01 EU/mg.

[00332] The control antibody used in all in vitro assays was an anti-bovine herpes virus cloned into an IgG4-HG structure (the same structure as MAB7). Control antibody was generated at Pfizer (South San Francisco, CA), Lot No. 4945, with <0.3 EU/mg of endotoxin.

[00333] Two anti-hu-PD-1 antibodies were generated from sequences published in previous patents and expressed in the IgG4-HG structure that resembles that used for MAB7. Antibodies were generated at Pfizer (South San Francisco, CA) and were designated as positive control 1 and positive control 2 (Lot No. 5053 and 4255, respectively). Measured endotoxin was <0.13 EU/mg and <0.056 EU/mg, respectively.

Human Assay Using Dendritic Cells Expressing High Levels of PD-L1

[00334] The protocol was adapted from Kruisbeek et al, 2004, with some modifications. Differentiated primary hu-DCs were harvested on Day 7 and checked via flow cytometry for high levels of PD-L1 expression and co-stimulatory signals required for T cell activation; markers included CD80 and CD86 (antibodies purchased from BD Bioscience, San Jose, CA). Cells were counted and irradiated at 3000 radiation units (rads) using an RS2000 x-ray machine (Radsource, Brentwood, TN) to prevent DCs from secreting cytokines but functioning only as the Ag presentation scaffold for T cells. Thus, the assay result was only induced by T cells. On Day 7, freshly isolated hu-T cells were harvested from allogeneic donors. T cells were plated with irradiated DCs at a ratio of 10:1 (optimal assay conditions were determined as 2 x 105 T cells incubated with 2 x 104 DCs in 200 μL cultures) in the presence of different concentrations of mAb7, negative and positive control antibodies or media alone (to assess baseline reaction). All conditions were placed in a tissue culture-treated 96-well round-bottom plate (Fisher Scientific Pittsburg, PA). Cells were cultured using serum-free X-vivo15 media (Lonza, Walkersville, MD) to prevent human serum variability between experiments. Cultures were incubated at 37°C with 5% CO2 for 5 days. On Day 5, supernatants were harvested and cytokine concentrations were measured using cytometric bead array (CBA) (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Data were acquired using flow cytometry (LSRFortessa™ analyzer, BD Biosceinces, San Jose, CA) and data analysis was performed using BD FCAP Array Software Version 3.0 (BD Biosceinces, San Jose, CA). Proliferation was measured in parallel cultures by adding 1 μCi of thymidine titrated with 3H methyl (Perkin Elmer, Waltham, MA) to each well and further incubated for 16 to 18 hours. Cultures were then harvested onto deoxyribonucleic acid (DNA) incorporation filters (Perkin Elmer, Waltham, MA), and titrated thymidine incorporation provided an index of cell proliferation measured as counts per minutes (cpm) using the MicroBeta2 machine. (Perkin Elmer, Waltham, MA).

Human Assay Using JeKo1-PD-L1 Expression Cell Line

[00335] This assay was performed using a 5:1 ratio of T cells to JeKo-1-PD-L1 cell line and was used as this ratio provided a more optimal approach to capturing cytokine secretion on Day 5. Therefore, each well was incubated with 2 x 105 T cells with 4 x 104 JeKo-1-PD-L1. On Day 5, supernatants were harvested and cytokine concentrations were measured using CBA (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. This cell line expresses a very modest amount of co-stimulatory molecules (CD80 and CD86) and thus enhanced proliferation is very moderate following antibody treatment. In contrast, cytokine secretion, including IL-2, is robust, thus allowing measurement of cytokine secretion following mAb7 treatment and non-proliferation of T cells.

Cynomolgus Monkey Assay Using Dendritic Cells Expressing High Levels of PD-L1

[00336] Differentiated DCs were harvested on Day 7 and checked via flow cytometry (using LSRFortessa™ analyzer, BD Biosceinces, San Jose, CA) for high PD-L1 expression and co-stimulatory signals required for T cell activation ; Markers included CD80 and CD86 (antibodies from BD Bioscience, San Jose, CA). Cells were counted and irradiated at 3000 rads using the RS2000 X-ray machine (Radsource, Brentwood, TN). Freshly isolated cynomolgus monkey T cells were harvested from allogeneic donors. T cells were plated with irradiated DCs using a 10:1 ratio of T cells to DCs (optimal assay when 2 x 105 T cells were incubated with 2 x 104 DCs in 200 μL of culture) in the presence of different concentrations of mAb7, negative and positive control antibodies, or media alone to assess the baseline reaction. All conditions were placed in a tissue culture-treated 96-well round-bottom plate (Fisher Scientific, Pittsburg, PA). Cells were cultured using serum-free X-vivo15 media (Lonza, Walkersville, MD) to prevent serum variability between experiments. Cultures were incubated at 37°C with 5% CO2 for 5 days. On day 5, supernatants were harvested and cytokine concentrations were measured using CBA (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Data were acquired using flow cytometry (LSRFortessa™ analyzer, BD Biosceinces San Jose, CA), and data analysis was performed using BD FCAP Array Software Version 3.0 (BD Biosciences, San Jose, CA). At the same time and in similar cultures, 1 μCi of thymidine titrated with 3H methyl (Perkin Elmer, Waltham, MA) was added to each well; cells were further cultured for 16 to 18 hours to measure proliferation. Cultures were harvested under Glass Printed filtermate A DNA incorporation filters (Perkin Elmer, Waltham, MA) and titrated thymidine incorporation, providing an index of cell proliferation, was measured as cpm using the MicroBeta2 machine (Perkin Elmer, Waltham, MA ).

Cytokine Release from Cynomolgus Monkey Blood Induced by Superantigen (Staphylococcal enterotoxin B) Stimulation

[00337] Whole blood from a cynomolgus monkey was collected; 225 μL of blood was split into tissue culture-treated 96-well plates (Fisher Scientific, Pittsburg, PA). Samples were incubated in duplicate at 37°C between 5% CO2 in the presence of mAb7 or an isotype-equivalent negative control antibody at concentrations ranging from 0.1 to 100 μg/mL. One hour after antibody addition, samples were stimulated with 0.1 μg/mL SEB (Toxic Technologies, Sarasota, FL) and cultures were incubated for 3 days. On day 3, plasma was collected, pooled, and frozen at −80°C. Concentrations of IFN-Y, IL-2, and TNF-α in thawed serum samples were measured in duplicate according to the manufacturer's protocol using MSD immunoassay plates (Meso Scale Diagnostics, Rockville, MD) and an MSD reader. (Model 1200) with MSD Discovery Workbench software (Version 4.0.12). Means of duplicates were reported.

Results Antagonistic Activity of mAb7 in Primary T Cells Humanities

[00338] When primary human T cells were activated in MLR with allogeneic hu-DCs expressing PD L1, mAb7 increased T cell proliferation (measured through titrated thymidine incorporation) and T cell activation (measured through secretion of pro-inflammatory cytokine) in a dose-dependent manner. Treatment of T cells with mAb7 (10 μg/mL) resulted in an increase in T cell proliferation of up to 2.5-fold under treatment with a negative control antibody (10 μg/mL). The levels of IFN-Y and TNF-α were increased by up to 8- and 5-fold, respectively, when compared to the negative control antibody. Increase in IFN-Y was greater than increase in TNF-α. IL-2 expression was not detected in these cultures. When primary human T cells were activated in MLR, using the JeKo 1 PD-L1 tumor cell line as the allogeneic antigen presenting cells, mAb7 induced a dose-dependent increase in IFN-Y secretion (up to 2.5-fold ), TNF-α (up to 2-fold) and IL-2 (up to 5-fold) when compared to the negative control antibody. The effect of mAb7, in this assay, resembled the data obtained with both positive control antibodies. Enhanced T cell proliferation was not observed under these conditions. Cell proliferation was minimal with a weak signal provided by CD80 and CD86 compared to MLR mediated by primary DCs. IL-10, IL-4, IL-17A, and IL-6 were minimal to undetected in all assays described above.

Antagonistic Activity of mAb7 in Cynomolgus Monkey Primary T Cells

[00339] The binding affinity of mAb7, when in solution, for hu-PD-1 and cynomolgus monkey PD-1 was very similar in the kinetic exclusion assay (KinExA) (KD = 23 and 28 pM for PD-1 human and cynomolgus monkey, respectively). The EC50 of mAb7 in cells expressing human PD-1 and cynomolgus monkey PD-1 was also similar. In a functional MLR assay that used T cells and DCs isolated from different cynomolgus monkeys, mAb7 induced T cell proliferation and activation in a dose-dependent manner (measured through titrated thymidine incorporation). This effect was also observed with positive control (antibody 1 and antibody 2) but not with the negative control antibody. mAb7 also enhanced cytokine secretion (i.e., IFN-Y and TNF-α, up to 5-fold and 3-fold compared to negative control antibody treatment. IL-2 expression was not detected in these cultures. In one assay of different cytokine release using cynomolgus monkey whole blood stimulated with 0.1 μg/mL of SEB superantigen for 3 days, mAb7 (0.1-100 μg/mL) induced greater secretion of IFN-Y, IL-2 and TNF-α when compared to the negative control antibody.

[00340] MLR studies were used to create an in vitro scenario resembling the tumor microenvironment where T cells are activated and expressing high levels of PD-1 in the presence of allogeneic DCs or tumor cells expressing PD-L1. The PD-1/PD-L1 interaction further inhibits T cell proliferation and cytokine release. The addition of anti-PD-1 antibodies to these MLRs restored T cell activation and increased proliferation and cytokine secretion, especially IFN-Y, due to blockade of the PD-1/PD-L1 axis.

[00341] mAb7 accelerated the proliferation and secretion of IFN-Y, TNF-α and IL-2 by human T cells when cultured with allogeneic cells expressing high levels of PD-L1 (DCs or PD-L1 cell line from JeKo 1). In contrast, the negative control antibody, which proved similar to medium alone, did not enhance these effects. Similarly, mAb7 enhanced T cell proliferation, IFN-Y and TNF-α secretion in the cynomolgus monkey MLR system, as well as enhanced cytokine release from cynomolgus monkey whole blood using the SEB superantigen.

[00342] These results demonstrate that anti-PD-1 antibody mAb7 enhanced T cell proliferation and pro-inflammatory cytokine secretion including interferon-gamma (IFN-Y), tumor necrosis factor alpha (TNF-α) and interleukin (IL)-2. These activities were observed in both human and cynomolgus monkey primary T cells.

Example 10: Effect of anti-PD-1 antibodies on graft-versus-host disease

[00343] This example illustrates the effect of anti-PD-1 antibodies on T cell activation and expansion in vivo using a xeno-aGvHD model in NSG mice transferred with hu-PBMCs.

[00344] In this study, the effect of anti-PD-1 antibody mAb7 on T cell activation and expansion in vivo was studied in a xeno-model of acute graft vs host disease (aGvHD) in non-obese diabetic mice ( NOD), with severe combined immunodeficiency (SCID), with interleukin 2 receptor gamma null (IL2rynull) (NSG) using human peripheral blood mononuclear cells (PBMCs).

[00345] NSG immunocompromised female mice (5 per group) 8 to 10 weeks old (formal name, NOD, Cg-Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were purchased from Jackson Laboratory (Bar Harbor, ME). All animals were housed in a pathogen-free facility at Pfizer (South San Francisco, CA) in accordance with the Institutional Animal Care and Use Committee (IACUC).

[00346] Human buffy coat was purchased from the Stanford Blood Center (Standord, CA), diluted with phosphate buffered saline (PBS) and layered on Ficoll for isolation of PBMCs. PBMCs were washed 2 times with PBS. Red blood cells (RBCs) were lysed using ammonium-chloride-potassium (ACK) lysis buffer as indicated in the manufacturer's protocol (Life Technology; San Diego, CA). After RBC lysis, cells were washed once more and diluted in PBS at 5 x 10 cells per mL.

[00347] For the induction of xeno-aGvHD, NSG mice were intravenously injected via the tail vein with hu-PBMCs. Each mouse received 1 x 107 cells in 200 μL of PBS. In all experiments, mice were weighed 3 times per week and monitored for the appearance of xeno-aGvHD-like symptoms including weight loss, hunched posture, goose bumps, reduced mobility, and in some cases, diarrhea. Mice were euthanized after losing 20% of body weight or losing 1 g/day over 2 days; this time point was recorded as the survival time. mAb7, negative control antibody and positive control antibodies were generated as described above in Example 9.

[00348] NSG mice were treated with negative control antibodies, positive control antibodies or mAb7, in a dose range between 0.1-10 milligrams per kilogram (mg/kg). Antibodies were administered to mice using the appropriate vehicle on Day 2 and Day 8 after hu-PBMC transfer. The antibody administration route was intraperitoneal (ip).

[00349] Peripheral blood, spleens and livers were collected from mice transplanted with hu-PBMCs. Single cell suspensions from each organ were prepared as follows: peripheral blood was collected and RBCs were lysed using ACK-lysis buffer and washed with PBS. Spleens and livers were mechanically homogenized using the back of a syringe plunger to macerate cells through a 70 μM filter and washed once with PBS. RBCs were lysed and cells were washed 2 more times and macerated again through the 70 μM filter. At this stage, the spleen cells were ready. To isolate liver leukocytes, single cell suspensions were layered using a 30% to 80% Percoll gradient (Percoll® Plus, GE Healthcare Bio-Sciences, Pittsburgh, PA) and subjected to high-speed centrifugation. Leukocytes were harvested from the middle layer and washed twice with PBS. All single cell suspensions were counted and 1 x 106 cells from each sample were used for fluorescence-activated cell sorting (FACS). For human cytokine secretion assays, mouse CD45+ cells were depleted using a mouse-specific CD45 cell isolation kit through positive selection, as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA), to ensure that cytokine secretion was from human immune cells alone. Cells were counted and 1 x 106 cells from each sample were used in the assay.

[00350] Peripheral blood single cell suspensions were obtained from the spleens and livers of xeno-aGvHD mice. A total of 1 x 106 cells per sample were incubated for 30 minutes at 4°C under light protection with a mixture of appropriate fluorescently labeled monoclonal antibodies in FACS buffer, including 1 x PBS containing 2% fetal bovine serum (FBS). , then cells were washed with FACS buffer. For intracellular staining of Kiel protein 67 (Ki67, to measure cell proliferation), cells were fixed after surface staining using Intracellular Fixation & Permeabilization Buffer Set (Affymetrix/eBioscience, San Diego, CA) according to the protocol from the manufacturer. Intracellular staining was performed for 30 minutes at 4°C in darkness. Upon completion of staining, samples were washed and subjected to multicolor flow cytometry using the BD LSR Fortessa™ cell analyzer (BD Biosciences, San Jose, CA). Data analyzes were performed using FlowJo software (FLOWJO LLC, Ashland, OR). The antibodies used in different combinations for the recognition of cell surface molecules were: Pacific Blue (PB) or Amcyan anti-hu-CD45, CD45-Brilliant Violet anti-mouse (BV)-711, anti-hu-CD3-PerCPCy5. 5, anti-hu-CD8 phycoerythrin-Cy7 (PE-Cy7) or BV-786, anti-hu-CD4-fluorescein isothiocyanate (FITC) or BV-650, anti-hu-PD-1 (clone EH12.1) BV - 786 or PE, PE or FITC anti-hu-PD-1 (clone MIH-4), anti-hu-Ki67 allophycocyanin (APC) (all antibodies from BD Biosciences, San Jose, CA), anti-hu -PD-L1 (PE-Affymatrix-eBioscience, San Diego, CA) and blue fluorescent reactive dye for Live/Dead staining (Life Technology, Main Island, NY).

[00351] Single cell suspensions of human CD45-enriched population from spleens and livers were obtained from xeno-aGvHD mice (after depletion of mouse CD45). A total of 1 x 10 cells per sample were incubated in tissue culture-treated 96-well round-bottom plates (Fisher Scientific; Pittsburg, PA) in 200 μL of X-vivo 15 medium. stimulated or stimulated with phorbol myristate acetate (PMA) 10 ng/mL and ionomycin (iono) 125 ng/mL, as the low stimulation condition, or PMA 50 ng/mL and ionomycin 1 μg/mL, as the low stimulation condition. high stimulation (both obtained from Sigma-Aldrich, Saint Louis, MO). Cultures were incubated for 8 hours at 37°C in 5% CO2 to ensure maximal cytokine secretion. Supernatants were harvested and human cytokine concentrations were measured using human cytokine-specific cytometric bead array (CBA) (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Data were acquired using flow cytometry (LSRFortessa™ analyzer, BD Biosciences, San Jose, CA) and data analysis was performed using FCAP Array™ Software Version 3.0 (BD Biosciences, San Jose, CA). HU-IFN-Y, hu-TNF-α, and hu-IL-2 bead array kits were purchased from BD Biosciences (San Jose, CA), and were confirmed to not cross-react with mouse cytokines.

[00352] All analyzes involved a comparison of means using the independent sample t-test or two-way ANOVA using Graphpad Prism (Graphpad Software, San Diego, CA). P values <0.05 were considered statistically significant. Engraftment data are depicted in the figures as mean concentrations including the standard error of the mean (sem).

[00353] Treatment of immunocompromised NSG mice with anti-PD-1 antibody mAb7 accelerated body weight loss (Table 17) and induced other signs of disease that are commonly seen in this model, such as hunched posture, raised skin, mobility decreased and, in some cases, diarrhea. In this model, body weight loss is expected to accelerate between Day 20 and Day 30 post-transfer (see, for example, Schroeder and DiPersio, 2011). Because treatment with mAb7 and positive controls 1 and 2 accelerated xeno-aGvHD symptoms, mice had to be euthanized at the earliest time points and a survival curve could be obtained; therefore, body weight loss was the primary outcome measurement. In the first experiment, mice were treated with 0.1, 1, and 10 mg/kg mAb7 or negative control antibody at 10 mg/kg. In Table 17, body weight loss was calculated by normalizing body weight differences between groups treated on Day 23 relative to Day 0. Values indicate an average of 5 mice per group ± sem. Table 17

[00354] Mice were treated with the indicated antibodies on Day 2 and Day 8 post-hu-PBMC transfer. While body weight loss was evident in the control group on Day 23, body weight loss and disease progression were detected in individual mice from all groups treated with anti-PD-1 antibody mAb7 starting on Day 10-11 post transfer of hu-PBMCs. In both groups treated with 1 mg/kg and 10 mg/kg, body weight loss reached significance between Days 14 to 23, when compared to the group treated with negative control (p <0.0001). On day 23, a 15% weight loss was detected in the 1 mg/kg and 10 mg/kg treated groups and a 7.9% weight loss was detected in the 0.1 mg/kg treated group (Table 17).

[00355] FACS analyzes of peripheral blood, liver and spleen demonstrated an increase in the percentage of hu-CD45 positive cells and hu-CD3 positive T cells in the mAb7 treated group versus the control group. Representative data from spleens also showed an increase in hu-CD8 positive T cell count and hu-CD4 positive T cell count (but fewer) in mAb7 versus the negative control antibody treatment group. A similar increase was also observed in T-hu cell counts in the liver and blood. To assess whether the increase in T cells was due to PD-1 blockade through mAb7 binding to human T cells, T cells were stained with a commercial antibody to PD-1 (clone EH12.1) that competes with binding. of mAb7. mAb7-treated lymphocytes from all organs (analyzed by FACS) showed no binding to EH12.1. To confirm that T cells still treated with mAb7 express PD-1, T cells were stained with a different anti-hu-PD-1 clone (clone MIH4) that partially competes with mAb7 to bind to T cells. T cells treated with mAb7 partially bind MIH4; thereby, indicating that PD-1 was expressed on treated T cells and that blockade of PD-1 by mAb7 was evident (data not shown, maintained in internal Pfizer records). No detectable changes were observed for hu-PD-L1 expression in the spleen, in T cells (hu-CD3 positive) or in non-hu T cells (hu-CD3 negative). Ki67, a marker for lymphocyte proliferation, was elevated in hu-CD3 positive cells from the PF-0681591 treated group versus the negative control treated group in the spleen median panel). Similar results were seen in blood and liver.

[00356] To examine the effects of mAb7 on cytokine release during xeno-aGvHD, hu-CD45 positive cells (from livers and spleens) were further isolated from mouse CD45 positive cells. These lymphocytes were then treated with a mixture of PMA and ionomycin (in 2 concentrations: low versus high) or left untreated for 8 hours at 37°C. Cytokine secretion was measured in supernatants by CBA (Table 18). No detectable cytokines were obtained without stimulation (Table 18). Following mAb7 treatment, hu-IFN-Y, hu-IL-2, and hu-TNF-α were elevated in human lymphocytes isolated from both spleen and liver compartments of mice compared to control groups. Under weak ex vivo stimulation conditions, mAb7-treated T cells increased hu-IFN-Y secretion to levels that were significantly higher than those of T cells isolated from the negative control group (p < 0.05). Under strong ex vivo stimulation conditions, all cytokines were induced in all groups but still increased in mAb7-treated T cells compared to T cells isolated from negative controls. Table 18 shows data collected from 5 different mice in each group. In Table 18, *p < 0.05, **p < 0.01 in unpaired t-test comparing mAb7 versus the negative control group; aGvHD = acute graft versus host disease; Hu = Human; IFN-Y = Interferon gamma; IL-2 = Interleukin-2; Ino = Ionomycin; N = Number; ns = Not significant; P = P value; PMA = Phorbol myristate acetate; TNFα = tumor necrosis factor alpha; Xeno = Xenogenic. Table 18

[00357] These results demonstrate that treatment of xeno-aGvHD with anti-PD-1 antibody mAb7 accelerated the development of xeno-aGvHD in NSG mice, as measured by loss of body weight, T cell proliferation, and increased secretion of cytokines including interferon-gamma (IFN-Y) and interleukin-2 (IL-2).

Example 11: Characterization of anti-PD-1 antibodies

[00358] This example illustrates the binding affinity, specificity and binding activity of anti-PD-1 antibody mAb7 on cells expressing the PD-1 surface receptor.

[00359] mAb7, negative control antibody and positive control antibodies were generated, as described above in Example 9. Anti-hu-PD-1 antibody clone EH12.1, primary labeled with phycoerythrin (PE) or Brilliant Violet (BV )-786 and isotype control antibodies labeled with the same dyes were purchased from BD Biosciences (San Jose, CA). PE-labeled antimouse PD-1 clone J43 and antihamster IgG isotype control antibody was purchased from Affymetrix/eBiosciense (San Diego, CA). Biotinylated hu-PD-L1 and biotinylated hu-PD-L2 (CD273) were obtained from ACRO Biosystems (Newark, DE). Cynomolgus monkey PD-L1 and PD-L2 were purchased from Creative BioMart® Recombinant Proteins (Shirley, NY), and both were locally labeled with Alexa Fluor® 647 dye using an Alexa Fluor® 647 protein labeling kit (LifeTechnologies, San Diego, CA), as instructed by the manufacturer's protocol. Cynomolgus monkey PD-L1 and PD-L2 were tested to bind to cynomolgus monkey PD-1 expressing cell line. Rat and mouse PD-L1-Fc markers (human IgG1 Fc region to C terminus) were purchased from Creative BioMart® Recombinant Proteins (Shirley, NY). Detection of biotinylated PD-L1 and PD-L2 was achieved using streptavidin PE or allophycocyanin (APC) (Affymetrix/eBiosciense, San Diego, CA). Detection of rat and mouse PD-L1 was achieved using affiniPure donkey-anti-human F(ab')2 IgG specific for APC-labeled Fc Fragment gamma (FCY) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA). .

Vectors for Transient Transfection

[00360] Hu-PD-1 expression plasmid (in pCMV6-Entry vector) was purchased from OriGene Technologies, Inc (Rockville, MD), Catalog No. RC210364/Accession No. NM_005018.

[00361] Mouse PD-1 expression plasmid (in pCMV6-Entry vector) was purchased from OriGene Technologies, Inc (Rockville, MD), Catalog No. MR227347/Accession No. NM_008798. Cynomolgus monkey PD-1 was codon optimized and synthesized by Life Technology (San Diego, CA), Lot No. 1482149/Accession No. EF443145. It was cloned into a cytomegalovirus (CMV)-based expression plasmid owned by Pfizer (San Francisco, CA), in backbone with a cap mouse secretory signal sequence that added a C-terminal FLAG tag to the C terminus.

[00362] Rat PD-1 deoxyribonucleic acid (DNA) was synthesized as usual according to the sequence of Accession No. NM_001106927 and cloned into the BamHI-NotI sites of the expression vector pEF1V5-His from LifeTechnologies (San Diego, CA), Lot No. 1598305.

PD-1 Expression Vectors for Stable Transfection

[00363] Hu-PD-1 DNA was synthesized as usual according to the sequence of Accession No. NM_005018 and cloned into the BamHI-NotI sites of the InVitroGen expression vector, pEF1V5-His B; Lot No. 513478.

[00364] Cynomolgus monkey PD-1 DNA was synthesized as usual according to Accession No. EF443145 and cloned into the BamHI-NotI sites of the InVitroGen expression vector, pEF1V5-His B, Batch No. 1482149.

[00365] Mouse PD-1 DNA was synthesized as usual according to Accession No. NM_008798 and cloned into the BamHI-NotI sites of the InVitroGen Expression Vector, pEF1V5-his B, Lot No. 1513476.

[00366] All vectors were synthesized and cloned into the InVitroGen expression vector, pEF1V5-His B, through Life Technology (San Diego, CA). All vectors contain the complete PD-1 sequence including extracellular domain, membrane domain and cytosolic domain. All vectors encoded a V5-6His epitope tag at the C terminus of PD-1. The neomycin resistance gene was included in each vector for selection using G418 sulfate antibiotics.

Transient Transfection using HEK-293T Cell Line

[00367] HEK-293T cells were purchased from the American Type Culture Collection (ATCC®, Manassas, VA), and the cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Corning CellGro, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) and 1 x Penicillin/Streptomycin (Pen/Strep) and grown in 6% carbon dioxide (CO2) at 37°C. One day before transfection, cells were trypsinized and plated at 4 x 106 cells per T75 flask (Fisher Scientific, Pittsburg, PA). On the day of transfection, pre-warmed, antibiotic-free growth media were replaced for the old media and cells were further incubated for 2 hours at 37°C in 6% CO2. Expression vector or empty vector (10 μg) was added to 1.5 mL of OptiMEM medium (Life Technology, San Diego, CA). Lipofectamine 2000 reagent (20 μL) (Life Technology, San Diego, CA) was then added to the other 1.5 mL of OptiMEM. The OptiMEM plasmid and Lipofectamine OptiMEM tubes were then mixed and incubated at room temperature for 25 minutes. The OptiMEM mixture was then added dropwise to the appropriate culture flask, and the flask was left overnight at 37°C in 6% CO2. The next day, the media was removed from the flask and replaced with complete growth media. Forty-eight (48) hours after transfection, cells were harvested using StemPro-Accutase (Life Technology, San Diego, CA), thus allowing cells to be gently removed from the culture surface without affecting PD-1 surface expression. . The cells were then subjected to antibody binding and fluorescence-activated cell sorting (FACS) analyses.

Stable Transfection Using Jurkat Cell Line

[00368] The Jurkat cell line, (clone E6-1-TIB-152™, ATCC®, Manassas, VA), was used to stably express human and cynomolgus monkey PD-1. Cell lines were generated using electroporation via the Necluotransfector system from Amaxa (Lonza, Walkersville, MD). Transfections were performed at Pfizer (San Francisco, CA) using a V Kit as instructed by the manufacturer's protocol (Lonza, Walkersville, MD). Cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 growth media supplemented with 10% FBS and 1x L-glutamine (LifeTechnologies, San Diego, CA) at 37°C in 5% CO2 at a density between 0.3 to 1.0 x 106 cells/mL. For each of the vectors, 2 μg/mL was used to transfect 2 x 106 cells. After transfection, cells were grown in the presence of G418 Sulfate (600 μg/mL) for 2 weeks to select and maintain stably transfected cells. To prevent long-term contamination, 1 x Pen/Strep was added to the media. Single-cell clones were selected by culturing the cells at a 1:200 dilution (1 cell in 200 μL of complete media supplemented with G418 sulfate antibiotics). To select clones expressing high hu or cynomolgus monkey PD-1, PE-labeled anti-hu-PD-1 clone, EH12.1, was used in flow cytometry assays. Samples were collected using BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA). Data analyzes and mean fluorescence intensity (MFI) were calculated using Flowjo software (FLOWJO LLC, Ashland, OR). Cell lines with high MFI were chosen for assay development.

[00369] HEK-293T cells transfected with PD-1 hu, cynomolgus monkey, mouse or rat vectors, or empty vector were harvested 48 hours post transfection. Prior to binding with mAb7, cells from each specific transfection were probed with commercial antibodies or ligands to ensure that the appropriate PD-1 receptor was highly expressed on the cell surface. For human and cynomolgus monkey, commercial cross-reactive anti-PD-1 antibody (clone EH12.1) was used. For mouse, a commercial anti-mouse-PD-1 antibody (clone J43) was used. For rat, a rat-PD-L1 ligand was used (since there was no commercial cross-reactive rat anti-PD-1 antibody available). After appropriate expression of PD-1 was confirmed, cells were counted and incubated 2 x 105 cells with hu-Fc receptor binding inhibitor functional type mix (FC-Y receptor blockade at 1 μg/1 x 106 cells, Affymetrix/eBiosciense, San Diego, CA) for 20 minutes and blue fluorescent reactive dye, used for Live/Dead staining, was added to the mixture (Life Technology, San Diego, CA). Serial dilutions of mAb7 or negative control antibody (1-0.00001 μg/mL) were added to the cell mixtures and then allowed to incubate on ice for 1 hour. The cells were then washed and Fcy-specific APC-labeled Donkey-anti-human-affiniPure F(ab')2 Fragment IgG (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) was added to the mixture, and the cells were then incubated on ice for another 30 minutes. Data were acquired on the BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA) and analyzed using Flowjo software (FLOWJO LLC, Ashland, OR).

[00370] Stable Jurkat cell clones that were transfected with PD-1 from hu, cynomolgus monkey or mouse were generated. PD-1 cell clones selected for these assays from each species expressed similar amounts of PD-1 receptors (~400,000 receptors/cell; receptors were previously quantified during generation of these cell lines). After appropriate PD-1 expression was confirmed, cells were counted and incubated 2 x 105 cells with a Functional-type hu-Fc Receptor Binding inhibitor (Fc-γ receptor blockade at 1 μg/1 x 106 cells, Affymetrix/eBiosciense, San Diego, CA) for 20 minutes and blue fluorescent reactive dye, used for Live/Dead staining, was added to the mixture (Life Technology, San Diego, CA). Serial dilutions (1:3) of mAb7, positive control 1, positive control 2, or negative control antibodies (using an IgG4 construct) were added to the cell mixture and then allowed to incubate on ice for 1 hour. The cells were then washed and APC-labeled APC-specific Donkey-anti-human-affiniPure F(ab')2 Fragment IgG (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) was added to the mixture and the cells were then allowed to incubate on ice for another 30 minutes. Data were acquired on the BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA). Analyzes were completed using Flowjo software (FLOWJO LLC, Ashland, OR) and geometric means were calculated. EC50 values were calculated using Graph Pad Prism (Log agonistic versus response [binding]).

[00371] Activated T cells expressing high levels of PD-1 receptors were generated. Appropriate PD-1 expression was confirmed for T cells, obtained from each species, using commercial reagents (anti-hu-PD-1 clones EH12.1 for human and cynomolgus monkey, anti-mouse-PD-1 J43 clones for mouse -PD-1, and rat-PD-L1 to rat-PD-1). Cells were counted and incubated at 1 x 106 cells with Functional Type hu-Fc Receptor Binding Inhibitor (FC-Y receptor blockade at 1 μg/1 x 106 cells, Affymetrix/eBiosciense, San Diego, CA) for 20 minutes and blue fluorescent reactive dye, used for Live/Dead dyeing, was added to the mixture (Life Technology, San Diego, CA). Serial dilutions of mAb7 (1:3 dilution) were added to the cell mixture and then allowed to incubate on ice for 1 hour. The cells were then washed before adding APC-labeled Donkey-anti-human-affiniPure F(ab')2 Fragment IgG, Fcy-specific secondary antibody (1:100 dilutions) (Jackson ImmunoResearch Laboratories Inc, West Grove , PA), and the cells were allowed to incubate on ice for 30 minutes. Data were acquired on the BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA). Data analyzes were performed using Flowjo software (FLOWJO LLC, Ashland, OR) and geometric means were calculated. EC50 values were calculated using Graph Pad Prism (Log agonistic versus response [binding]).

[00372] Stable Jurkat cell clones expressing high levels of hu-PD-1 were chosen for the assay. Twenty (20) μg/mL of biotinylated hu-PD-L1 or biotinylated hu-PD-L2 saturated all PD-1 receptors in this cell line (after binding titration assays). Therefore, this concentration was chosen as the optimal concentration in our studies. Jurkat cells expressing Hu-PD-1 (2 x 105) were incubated with Functional Type hu-Fc Receptor Binding Inhibitor (Fc—Y receptor blockade at 1 μg/1 x 106 cells, Affymetrix/eBiosciense, San Diego, CA) for 20 minutes, and blue fluorescent reactive dye, used for Live/Dead dyeing, was added to the mixture as well (Life Technology, San Diego, CA). Biotinylated hu-PD-L1 or biotinylated hu-PD-L2 were added to cells at 20 μg/mL, immediately followed by addition of serial dilutions of mAb7, positive control 1, positive control 2, or negative control antibodies (all backbone of IgG4 and in serial dilutions 1:3). Cells were allowed to incubate on ice for 1 hour. The cells were then washed and PE Streptavidin (1:100 dilution) (Affymetrix/eBiosciense, San Diego, CA) was added and the cells allowed to incubate on ice for 30 minutes. Data were then acquired on the BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA) and analyzed using Flowjo software (FLOWJO LLC, Ashland, OR) and geometric means were calculated. IC50 values were calculated using Graph Pad Prism (Log inhibitory versus response [binding]).

[00373] Jurkat cells were stably transfected with high levels of hu or cynomolgus monkey PD-1 receptors. Cells expressing human and cynomolgus monkey PD-1 receptors (~400,000 receptors/cell) were chosen for this assay. Binding of cynomolgus monkey PD-L1 and PD-L2 was tested using these cells and the results showed that 20 μg/mL of cynomolgus monkey PD-L1 or PD-L2 was sufficient to saturate all PD-1 receptors with based on the geometric mean tested for different concentrations (50 ng/mL-50 μg/mL) of these ligands when bound to this cell line. Cynomolgus monkey PD-1 expressing Jurkat cells (2 x 105) was incubated with Functional Type hu-Fc Receptor Binding Inhibitor (Fc—Y Receptor Blockade in Cells 1 μg/1 x 106, Affymetrix/eBiosciense, San Diego, CA) for 20 minutes and blue fluorescent reactive dye, used for Live/Dead Dyeing, was added to the mixture (Life Technology, San Diego, CA). Alexa-Fluor® cynomolgus monkey 647 PD-L1 or Alexa-Fluor® cynomolgus monkey 647 PD-L2 were immediately added to the cells at 20 μg/mL followed by different concentrations of mAb7, positive control 1, positive control 2 or negative control antibodies (using the IgG4 backbone, in 1:3 serial dilution). The cells were then allowed to incubate on ice for 1 hour. After washing, cells were acquired on the BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA). Data analyzes were performed using Flowjo software (FLOWJO LLC, Ashland, OR), and geometric means were calculated. IC50 values were calculated with Graph Pad Prism (Log inhibitory versus response [binding]).

[00374] Human buffy coat, purchased from Stanford Blood Center (Stanford, CA), was diluted with PBS and layered on Ficoll for isolation of PBMCs. PBMCs were washed 4 times with PBS. RBCs are lysed using ACK lysis buffer as indicated in the manufacturer's protocol (Life Technology, San Diego, CA). After RBC lysis, cells were washed once more and diluted in PBS at 5 x 10 cells per mL. Half of the PBMCs were counted and iced in cold media (90% FBS with 10% dimethyl sulfoxide [DMSO]), and the remaining cells were subjected to T cell purification.

[00375] From the remaining PBMCs, T lymphocytes were isolated using a human-specific Pan t-cell isolation kit with negative selection as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA).

[00376] For the ADCC assay, freshly isolated T cells (target cells) were counted, and 1 x 106 T cells per mL were cultured in serum-free 15 X-vivos medium (Lonza, Walkersville, MD) and stimulated using beads coated with anti-CD3 and anti-CD28 (Dynabeads® Human T-Activator CD3/CD28 for T-Cell Expansion and Activation (Life Technologies, San Diego, CA) and 100 U/mL of recombinant (rh)-IL-2 -human (R&D Systems, Minneapolis, MN). Cultures were incubated at 37°C in 5% CO2 for 72 hours. When T cell activation reached the 48 hour period, PBMCs (effector cells derived from the same donor) were thawed, counted, and then stimulated with rh-IL-2 (50 U/mL at 5 x 106 cells/mL culture) for a total of 18 to 24 hours in complete RPMI-1640 media with 10% FBS. On the day of the assay, both cell types were counted and tested by flow cytometry to ensure appropriate activation. For T cells, high PD-1 expression was examined and for PBMCs the expression level of CD16, CD32 receptors. and CD64 (Fc-gamma receptor- [FCYR] III [a and b], FcYRIIa, FCYRI, respectively) that mediate ADCC was examined. Cells were plated at a 5:1 ratio of effector to target (as this was a great ratio) in round-bottom 96-well tissue culture treated plates (Fisher Scientific, Pittsburg, PA) following the addition of the antibodies: mAb7 (IgG4 and IgG1 structures), and positive control antibodies. All antibodies were tested at concentrations of 0.01 to 100 μg/mL (in 1:10 serial dilutions). Assay controls were added, including target cells alone to assess maximal lysis, effector cells alone, and effector to target at a ratio of 5:1 without addition of antibody. Assay plates were incubated at 37°C in 5% CO2 for 4 hours. Data were analyzed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega US, Madison, WI) as instructed by the manufacturer's protocol. The assay quantitatively measured lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. The percentage (%) of cytotoxicity was measured as = 100 x (Experimental LDH Release [OD490]/Maximum LDH Release [OD490]). Maximum LDH release was calculated when target cells alone (T cells) were chemically lysed to achieve maximum killing.

[00377] The ability of mAb7 to bind to human PD-1, cynomolgus monkey, mouse and rat was evaluated using flow cytometry (FACS) cell-based binding assays that included transiently transfected HEK-293T cell line , primary activated T cells and Jurkat cells stably transfected with hu-PD-1 or cynomolgus monkey and mouse PD-1. mAb7 bound to hu-PD-1 and cynomolgus monkey PD-1 with high affinity and showed similar EC50 values for the 2 species (the amino acid sequence for these isoforms is the most similar of the species analyzed). The data are summarized in Tables 19, 20 and 21. Binding of mouse PD-1 was only achieved at a high concentration of mAb7, which is biologically irrelevant and could be due to the affinity maturation process. No binding of mAb7 was detected to rat PD-1 when using transfected cells or activated primary T cells (Table 19). Table 19: Binding of mAB7 on FACS-Inactivated and Activated Primary T Cells Table 20: Binding of mAb7 to Activated Primary T Cells Obtained Expressing PD-1 from Individual Human and Cynomolgus Monkey (EC50)

[00378] In Table 20, each number represents the EC50 values for mAb7 binding to a different donor. The bottom row represents the mean ± sem. sem = standard error of the mean. Table 21: Binding of mAb7 to Jurkat Cell Lines Stably Transfected with Human PD-1 or Cynomolgus Monkey PD-1 (EC50)

[00379] In Table 21, each number represents the EC50 values for mAb7 binding in a single experiment. *indicates the mean ± sem. mAb (IgG4) = monoclonal antibody with IgG4 structure; PD- 1= Programmed Death-1; sem = standard error of the mean.

[00380] Binding of mAb7 to both Hu and cynomolgus monkey PD-1 was examined using different cell-based assay systems. In all systems, binding was observed to be with high affinity and specificity in both species. Using transiently transfected HEK-293T cell lines that expressed hu or cynomolgus monkey PD-1, mAb7 showed similar binding patterns as indicated by MFI. Minimal to no binding was observed in the parental cell line transfected with empty vector (vehicle).

[00381] EC50 values of mAb7 binding to activated primary hu and cynomolgus monkey T cells were determined at 72 hours post-activation, when PD-1 expression on the cell surface and cell viability were optimal (Table 19) . EC50 values were calculated for 2 different donors of each species (Table 20). In both hu and cynomolgus monkey activated T cells, low EC50 values were obtained and EC50 values were observed to be close between the 2 species, 51.04 ± 5.07 pM and 85.34 ± 11.73 pM, respectively (mean ± standard error of the mean [sem]). No binding was observed with the negative control antibody above baseline values.

[00382] The Jurkat T cell line that minimally expresses hu-PD-1 receptors was used to generate stably transfected hu-PD-1, cynomolgus monkey PD-1, and mouse PD-1 cell lines. Cell lines were subcloned and clones expressing high levels of hu-PD-1 and cynomolgus monkey PD-1 receptors were selected (~400,000 PD-1 receptors per cell was the highest expression obtained). In this system, mAb7 showed high affinity for hu-PD-1 and cynomolgus monkey PD-1 receptors. EC50 values for both species were similar to data from primary activated T cells (Table 21); EC50 = 64.725 ± 1.89 for hu-PD-1 and 222.55 ± 41.3 for cynomolgus monkey PD-1. EC50 values for cells expressing cynomolgus monkey PD-1 were more variable than human EC50 values between the 2 experimental runs. Data for the two species were similar to data obtained with the positive control antibodies used in any given replicate (Table 21). The negative control antibody did not exhibit binding above baseline values in any experimental repeat. The ability of mAb7 to block PD-L1 and PD-L2 ligands from interacting with the PD-1 receptor was examined in a Jurkat cell line transfected with PD-1 (expressing hu-PD-1 or PD-1 from cynomolgus monkey). In this assay, cells were incubated with the labeled ligands at saturating concentrations after incubation with unlabeled mAb7 at different concentrations. As shown in Table 22, mAb7 inhibited the binding of PD-L1 and PD-L2 to the PD-1 receptor in a dose-dependent manner. Positive control antibodies to PD-1 showed comparable inhibition. The IC50 of mAb7 was comparable between hu-PD-1 and cynomolgus monkey PD-1 (880.15 ± 289.85 and 1058 ± 355.4 for hu-PD-L1 and hu-PD-L2, respectively; and 942 .9 ± 110.1 and 839 ± 89.5 for cynomolgus monkey PD-L1 and cynomolgus monkey PD-L2, respectively). A summary of IC50 values is provided in Table 22. In Table 22, each number represents the IC50 values for mAb7 in a single experiment; *indicates the mean ± sem; Φ indicates the number repeated in parentheses; mAb IgG4 = monoclonal antibody with IgG4 structure; PD-1= Programmed Death-1; PD-L1 = Programmed Death Ligand 1; PD-L2 = Programmed Death Ligand 2; sem = standard error of the mean. Table 22: Inhibitory concentrations (IC50; pM) for blocking human PD-1 or cynomolgus monkey PD-1 binding to PD-L1 and PD-L2 using stably transfected Jurkat cell line system.

[00383] mAb7 showed weak binding with complementary component 1, subcomponent q (C1q) and CD64. C1q and CD64 (for IgG4 structure) are considered potential surrogates for complement-dependent cytotoxicity (CDC) and ADCC, respectively. To further investigate the lack of the ability of mAb7 to induce T cell death in vitro, an ADCC assay was performed with activated T cells (target cells) expressing high levels of PD-1 receptors and PBMCs (effector cells) that expressed high levels of Fcy receptors. Cells were selected from 2 healthy donors. mAb7 (in its original IgG4 structure) showed minimal ADCC activity in both donors. The lack of ADCC activity was in accordance with the activity exhibited by the positive control anti PD-1 antibody on the IgG4 structure and both were similar to the negative control IgG4 antibody. When anti-PD-1 (mAb7 antibody or positive control anti-PD-1) were evaluated in the structure of human IgG1, which is known to induce stronger ADCC, anti-PD-1 induced up to 4-fold higher ADCC than when the antibody is in the IgG4 structure. Maximum LDH release was calculated when target cells alone (T cells) were chemically lysed to obtain maximal killing (killing was estimated as 100% lysis for each donor according to assay calculation). T cell lysis using the IgG1 construct corresponds to the level of PD-1 on activated T cells (PD1 expression from donor q as well as lysis is higher than that from donor 2), confirming the accuracy of the assay.

[00384] These results demonstrate that the anti-PD-1 antibody mAb7 binds with high affinity to cynomolgus monkey and human PD-1 receptors expressed on cells with EC50 values ranging from 46 to 270 pM, depending on the test system. . mAb7 did not bind to cells expressing mouse PD-1 or rat PD-1 at physiological concentrations. mAb7 blocked PD-L1 and PD-L2 ligands from interacting with cell surface PD-1 receptors; IC50 values ranged from 500 to 1000 pM indicating its high antagonistic function in blocking PD-1 function induced through ligand binding. mAb7, in its IgG4 structure, elicited minimal to no antibody-induced cytotoxicity that is consistent with the properties of the IgG4 antibody.

Example 12: Characterization of anti-PD-1 antibody mAb7 binding to PD-1, FcRn, FCYRS, and C1Q using label-free biosensors and ELISA.

[00385] This example illustrates the in vitro binding affinities of mAb7 toward purified recombinant PD-1 from various species relevant to toxicology studies using SPR biosensors. The ability of the Fc region of mAb7 to bind to FCGRs and FcRn was also tested by SPR to confirm that it exhibited properties consistent with those of an isotype-matching control. By ELISA, mAb7 and an isotype-matched control were tested for binding to human C1q. The ability of mAb7 to block the binding interaction of PD-1 with its ligands, PD-L1 and PD-L2, was also tested by label-free biosensors to support its mechanism of action.

[00386] All kinetic and affinity experiments were conducted at 25°C in phosphate-buffered saline (PBS) + 0.01% Tween 20 running buffer, unless otherwise stated. Kinetic studies were performed on a ProteOn XPR36 SPR biosensor equipped with NLC (neutralvidin-coated) chips (BioRad, Hercules, CA). ProteOn was also used to determine the active concentrations of hu-PD-1 and cynomolgus monkey PD-1 analytes by titration against mAb7 as the reference standard. The concentrations of protein analytes used here refer to “active” or “nominal” values. The active concentration of human Fc gamma receptor (hu-FCGR) I, human neonatal Fc receptor (hu-FcRn) (Lot No R3091), and cynomolgus monkey neonatal Fc receptor (cynomolgus monkey-FcRn) (Lot No JCR) was determined using calibration-free concentration analysis (CFCA) experiments on a Biacore T200 equipped with CM5 sensor chips (GE Life Sciences, Marlborough, MA). All other analytes were used at their nominal concentrations as determined by light absorbance at A280 nm with an appropriate extinction coefficient. Solution affinities were determined at room temperature (approximately 23°C) using a KinExA 3000 or 3200 instrument equipped with an autosampler (Sapidyne). Secondary detection antibodies were labeled with DyLight 650 (Pierce Biotechnology, Grand Island, NY.) according to the manufacturer's instructions. Immunoglobulin G (IgG) biotinylations were performed at an equimolar ligand:IgG ratio using EZ-Link™ Sulfo-NHS-LC-LC-Biotin (Pierce Biotechnology Grand Island, NY) according to the manufacturer's instructions. Unless otherwise stated, immobilized IgGs were regenerated with a "Pierce/salt" cocktail comprising a 2:1 v/v mixture of Pierce IgG elution buffer (pH 2.8)/4 M sodium chloride (NaCl ).

[00387] Binding interactions of hu-PD-1 and cynomolgus monkey PD-1 (both His-tagged monomers) toward mAb7 were determined in solution using the KinExa method in a PBS + 0.01 running buffer % Tween 20 and a sample buffer of PBS + 0.01% Tween 20 + 1 g/l bovine serum albumin (BSA). Two different assay formats were employed. In the first assay format, mAb7 was titrated to a constant concentration of hu-PD-1 (nominal 200, 400, or 4000 pM) and samples were allowed to reach equilibrium. Free hu-PD-1 was captured on polymethylmethacrylate (PMMA) beads that were coated (by adsorption) with anti-hu-PD-1 mAb7 monoclonal antibody (prepared in-house under non-Good Laboratory Practice (GLP) conditions [Batch No R5432]). Bead-captured hu-PD-1 was then detected with 0.5 μg/mL of Dylight-labeled anti-His mAb (R&D Systems, Minneapolis, MN). In the second assay format, hu-PD-1 or cynomolgus monkey PD-1 were titrated to a constant concentration of mAb7 (20, 50, 100, or 500 pM) and these mixtures were allowed to equilibrate. Free mAb7 was captured on PMMA beads that were adsorbed with a blocking anti-idiotypic mouse mAb 1699.1H6 that specifically binds free mAb7 but not PD-1-saturated mAb7. Bead-captured mAb7 was then detected with Dylight (H+L)-labeled goat anti-hu-IgG (Jackson ImmunoResearch Inc, West Grove, PA). All titrations were prepared as a series of 12-member 2-fold dilutions by varying the concentration at the main nominal binding site to fall within the range of 1 nM to 10 nM, depending on the experiment. Samples were allowed to equilibrate for up to 48 hours and injection volumes were adjusted per experiment to give a total signal that fell within the range of 0.7 V to 1.9 V. All samples were injected in duplicate cycles. Data from up to 4 independent experiments per interaction were analyzed globally using the N curve tool in the analysis software and fitted to a simple bimolecular model where the concentration of mAb7 was used as the reference concentration. The overall analysis reports the best fit values (and 95% confidence interval) for the KD and concentration of PD-1 at the apparent active binding site.

[00388] The binding affinities of hu-PD-1 and cynomolgus monkey PD-1 toward mAb7 in solution were indistinguishable from each other when studied using a KinExA assay; apparent equilibrium dissociation constant (KD) values at 23°C were determined to be 17 pM or 23 pM for hu-PD-1 (when studied in opposite assay orientations) and 28 pM for cynomolgus monkey PD-1 ; these three values were statistically indistinguishable from each other. These values were recaptured by surface plasmon resonance (SPR) biosensor measurements, which yielded KD values of 42 pM for hu-PD-1 and 69 pM for cynomolgus monkey PD-1 at 25°C, and 109 pM for hu-PD-1 and 115 pM for cynomolgus monkey PD-1 at 37°C. mAb7 bound mouse PD-1 with a KD value of 0.9 μM and no binding was detected toward rat PD-1 when tested at 0.5 μM with SPR kinetic analysis. Label-free biosensors were used to demonstrate that mAb7 blocks hu-PD-1 from binding to its natural ligands, human programmed death ligand 1 (hu-PD-L1) and human programmed death ligand 2 (hu-PD- L2). SPR was further used to confirm that mAb7 bound a range of crystallizable fragment gamma (Fc) receptors (FCGR) and the neonatal Fc receptor (FcRn) from both human and cynomolgus monkey species with the same kinetics and specificity as a control. isotype matching. By ELISA, mAb7 showed negligible binding to complementary component 1, subcomponent q (C1q), consistent with the behavior of an isotype-matching control. In general, mAb7 binds both hu-PD-1 and cynomolgus monkey PD-1 with high binding affinity and specificity, but has low affinity toward mouse PD-1 and negligible binding toward rat PD-1.

[00389] Table 23 summarizes the kinetic and affinity data obtained for the analysis of the interaction of mAb7 with PD-1 from various species. mAb7 bound hu-PD-1 and cynomolgus monkey PD-1 with statistically indistinguishable apparent affinities when these interactions were measured in solution at 23°C using the KinExa method; the apparent KD values were 17 pM and 23 pM for hu-PD-1 (when studied in two opposing assay orientations) and 28 pM for cynomolgus monkey PD-1. KinExA values were corroborated by kinetic measurements performed by SPR, which provided apparent KD values at 25°C of 42 ± 11 pM (n = 10) for hu-PD-1 and 69 ± 24 pM (n = 10) for cynomolgus monkey PD-1. SPR measurements at 37°C showed 2-fold weaker affinities than those at 25°C; KD values at 37°C were determined to be 109 ± 8 pM (n = 15) for hu-PD-1 and 115 ± 15 pM (n = 8) for cynomolgus monkey PD-1. mAb7 bound mouse PD-1 with an apparent KD of 0.9 ± 0.1 μM (n = 5) when measured at 25°C by SPR, corresponding to an affinity 20,000 times weaker than that for hu-PD- 1. No binding was detected for rat PD-1 when tested at 0.5 μM at 25°C by SPR, although rat PD-1 proteins were confirmed active through their clear binding to positive controls, mouse PD-L1. and mouse PD-L2. The KD values for the KinExA measurements represent the best fit and 95% confidence interval from a global analysis of N independent experiments, although those for the SPR measurements represent the mean ± standard deviation of n independent experiments on a sensor chip. ProteOn NLC. In Table 23, h or hu = Human; ka = association rate constant; kd = dissociation rate constant; KD = equilibrium dissociation constant; KinExA = kinetic exclusion assay; mAb = monoclonal antibody; Ms = molar per second; n = number; ND = Not determined; PD-1 = programmed death-1; s = second; SPR = surface plasmon resonance; Temp = temperature; *the interaction of mAb7 with hu-PD-1 was studied in two opposing assay orientations as described in Bee et al, 2012. Table 23: Summary of kinetic and affinity constants obtained for the interactions of PF 06801591 with human PD-1 , cynomolgus monkey PD-1 and mouse PD-1

[00390] Table 24 shows that the kinetics and specificity of binding for mAb7 over a range of Fc receptors, including both human and cynomolgus monkey FCGRs and FcRn recapitulated those values expected for an isotype-matched, human, isotype control. IgG4. For example, mAb7 bound hu-FCGRI with an apparent KD value (mean ± standard deviation) at 25°C of 146 ± 42 pM (n = 14) compared with 177 ± 71 pM (n = 4) for the isotype control . All interactions were characterized by weak affinities (KD values ~1 μM or greater). Kinetic analysis was performed on a ProteOn NLC chip at pH 7.4 for FCGR and pH 5.9 for FcRn. In Table 24, CD = differentiation group; cy = cynomolgus monkey; FCGR = Fc gamma receptor; FcRn = neonatal Fc receptor; hu = human; ka = association rate constant; kd = dissociation rate constant; KD = equilibrium dissociation constant; Ms = molar per second; n = number; s = second. Table 24

[00391] In addition, mAb7 poorly bound C1q by ELISA, consistent with the behavior of the isotype-matching control, hu-IgG4 (data not shown). Its binding was even lower than the low binding of human immunoglobulin G2 (hu-IgG2). In contrast, the positive controls, hu-IgG1 and human immunoglobulin G3 (hu-IgG3), provided high binding signals. Using both SPR and Octet biosensors and different assay formats (both premix and classic sandwich assay formats), it was demonstrated that mAb7 blocked the binding of hu-PD-1 to its natural ligands, hu-PD-L1 and hu -PD-L2.

[00392] These Data Demonstrate That Mab7 Binds With High Affinity And High Specificity Towards Hu-pd-1. When Measured In Solution At 23°C Using The Kinexa Method, Mab7 Binded Hu-pd-1 And Cynomolgus Monkey Pd-1 With Statistically Indistinguishable Apparent Kd Values Of 17 Pm And 23 Pm For Hu-pd-1 (When Studied In Alternative Assay Guidelines) And 28 Pm For Cynomolgus Monkey Pd-1. Mab7 Showed 20,000 Times Weaker Affinity With Mouse Pd-1 (Apparent Kd Of 0.9 µm At 25°c Per Spr) And No Detectable Binding To Mouse Pd-1 When Tested At 0.5 µm. Mab7 Is A Linked Hu-igg4 Isotype Matching Control With Similar Kinetics And Specificity When Tested Against A Panel Of Human And Cynomolgus Monkey Fc Receptors When Measured At 25°C Per Spr. Mab7 bound Hu-c1q with negligible signal when tested by ELISA, consistent with the Hu-igg4 isotype controls, and its binding was even lower than that of Hu-igg2. Mab7 has been shown to block Hu-pd-1 from binding to its natural ligands, Hu-pd-l1 and Hu-pd-l2.

Example 13: Combination treatment with anti-PD-1 antibody and anti-OX40 antibody

[00393] This example illustrates the effects of treatment with anti-PD-1 antibody in combination with anti-OX40 antibody in a mouse model for colon cancer.

[00394] For this study, female C57B6/J mice (6-8 weeks old) were inoculated subcutaneously with the MC-38 murine colon tumor (a grade III adenocarcinoma) at 5x105 cells/mouse. On day 10, mice were randomized and treatment began at 10 mg/kg mouse anti-PD-1 and 1 mg/kg anti-mouse-OX-40. Treatments were administered on days 10, 12, and 14 for anti-OX40 antibody (1 mg/kg); and on days 10, 12, 14, 17, 23, and 25 for anti-PD-1 antibody (10 mg/kg). Tumor volume was measured twice a week. The study was terminated on day 34. Statistical analyzes were performed using two-way ANOVA to compare the means between each two groups at a single point in time. The results are summarized in Table 25. In Table 25, values indicate tumor volume at the indicated daily mean of 8 mice per group ± (mean standard error) sem; N = number per group., mm2= cubic millimeter. Isotype control included the appropriate isotype of each antibody at 1 mg/kg for anti-OX40 and 10 mg/kg for anti-PD-1. Table 25. Tumor volume in mice carrying MC38 after treatment with anti-PD-1 antibody and anti-OX40 antibody

[00395] Starting on Day 24, all groups treated with anti-PD-1 antibody and/or anti-OX-40 antibody showed significantly lower tumor growth when compared to the control group (Table 25). On Day 24, the combination treated group (anti-PD-1 antibody plus anti-OX40 antibody) showed significance of p<0.0001 when compared to the isotype control group, while each single antibody treatment showed significance of p< 0.01 when compared to the control group. On Day 34, all treated groups showed significance of p<0.0001 when compared to the control group. On Day 34, the group treated with the combination showed differences when compared to the group treated with anti-PD-1 antibody alone or group treated with anti-OX-40 antibody alone. These results demonstrate that treatment with both anti-PD-1 antibody and anti-OX40 antibody delayed tumor growth when compared to treatment with the antibody alone.

Example 14: Effect of vaccine on PD-1 expression on activated T cells

[00396] This example illustrates the effect of a DNA-based vaccine expressing a human prostate-specific membrane antigen (PSMA) on PD-1 expression on CD3+CD4 (Table 26) and CD3+CD8 (Table 26) T cells. Table 27) in the presence of anti-CTLA4 monoclonal antibody tremelimumab (a checkpoint inhibitor).

[00397] In vivo Study Procedures. Three groups of Chinese cynomolgus monkeys (n=8/group) were used in the study. Animals in groups 1 and 2 received intramuscular injection with a vaccine containing an AdC68 adenovirus vector (SEQ ID NO:44) encoding amino acids 15-750 of human PSMA (at a total dose of 2e11 VP) while animals in group 3 did not receive any vaccination. The complete sequence of the AdC68 adenovirus vector is provided in SEQ ID NO:44. In addition, immediately after vaccination, animals in group 2 were treated with 50 mg of anti-CTLA4 monoclonal antibody tremelimumab by subcutaneous injections. Whole blood samples from each animal before (Pre) and after vaccination were collected on Days 9, 15, and 29 and analyzed for the frequency of PD-1 expressing CD3+CD4 and CD3+CD8 T cells by flow cytometry.

[00398] CD3+ CD4 and CD8 T cell phenotype. Leukocyte populations were phenotyped by multicolor flow cytometry analysis modality on freshly collected whole blood. Briefly, whole blood was stained for 20 min at room temperature with an antibody cocktail containing CD3-V500 (clone SP34-2), CD4-BV605 (clone L200), CD8-APC.H7 (clone SK1), PD1- AF488 (clone EH12.2H7, Biolegend), PDL1-BV421 (clone MIH1), Lag3- PE.Cy7 (clone 11E3, Novus), CD69-PE.Texas Red (clone TP1.553, Beckman Coulter), TIM3-APC ( clone 344823, R&D Systems), CD20-PE.Cy5.5 (clone 2H7, eBioscience), CD14-AF700 (clone M5E2), CD16-PE.Cy5 (clone 3G8), CD56-PerCP.Cy5.5 (clone B159) , and CD11c-PE (clone SHCL3). All antibodies were purchased from BD unless otherwise noted. Samples were treated with RBC lysis buffer (BD), washed, mixed with 50 mL of liquid counting beads (BD), and acquired in LSR Fortessa (BD). Data were analyzed using FlowJo (Tree Star Inc, USA). The results of PD-1 expression on CD3+CD4 T cells are shown in Table 26 and the results of PD-1 expression on CD3+CD8 T cells are shown in Table 27. PD-1 induction was observed 9 days after vaccination and resolved to levels seen in unexposed animals (Group 3) by day 29 after vaccination. These results indicate that the vaccine vector provided with anti-CTLA4 antibody can provoke the expression of PD-1 on CD3+ CD4 and CD8 T cells in non-human primates. Table 26. Kinetics of PD-1 expression on CD3+CD4 T Cells in cynomolgus monkeys Table 27. Kinetics of PD-1 expression on CD3+CD8 T Cells in cynomolgus monkeys

Example 15. Effect of anti-PD-1 antibody on antigen-specific T cell response induced by a vaccine expressing human PSMA

[00399] This example illustrates the effect of anti-PD1 antibodies on CD4 IFNY and CD8 T cell responses induced by a vaccine containing an AdC68 adenovirus vector expressing human PSMA in cynomolgus monkeys.

[00400] Intracellular Cytokine Staining (ICS) Assay. Briefly, PBMCs from individual animals were coincubated with peptide pools from a 15-mer peptide library overlapping by 11 amino acids spanning the entire sequence of the respective antigen, each peptide at 2 μg/mL. Plates were incubated ~16 hours at 37°C, 5% CO2. The cells were then stained to detect intracellular expression of IFNγ from CD8+ and fixed T cells. Cells were acquired on a flow cytometer. Response frequency was normalized to the number of CD8+ IFNγ T cells per million CD8+ T cells, with responses in dimethyl sulfoxide control wells, which contained no peptide, subtracted. The antigen-specific responses in the tables represent the sum of the responses for the corresponding antigen-specific peptide pools.

[00401] In vivo Study Procedures. In brief, five groups of Chinese cynomolgus monkeys (n=8/group) were intramuscularly injected with a vaccine containing an adenovirus AdC68 (SEQ ID NO:44) encoding amino acids 15-750 of human PSMA (hPSMA) at a total dose of 2e11 VP . Vaccination in Groups 2, 4 and 5 was immediately followed by subcutaneous injections of 50 mg of anti-CTLA4 monoclonal antibody tremelimumab and/or anti-PD-1 mAb7 monoclonal antibody delivered subcutaneously (groups 3 and 4) or intravenously (group 5). at 10 mg/kg. PBMCs were isolated from each animal and subjected to an ICCS assay to measure human PSMA-specific IFN CD4 (Day 9 or Day 29) or CD8 T cell responses (Day 29). The amino acid sequence of full-length human PSMA is provided in SEQ ID NO:42. The sequence of the AdC68 adenovirus vector expressing amino acids 15-750 of human PSMA is provided in SEQ ID NO:44.

[00402] Results. The results are presented in Tables 29, 30, and 31. The data demonstrated that both anti-CTLA4 and anti-PD-1 individually can increase the frequency of vaccine-induced CD4 and CD8 IFNY T cell responses. Table 28. Peptide sequence information for peptide pools used in the ELISpot and ICCS assays to induce IFNγ antigen-specific T cell responses. Table 29. Vaccine-Induced CD8 IFNγ T Cell Responses on Day 9 Table 30. Vaccine-induced CD4 IFNY T cell responses at Day 9 Table 31. Vaccine-induced CD4 IFNγ T cell responses at Day 29

Example 16. Effect of Anti-PD-1 Antibodies on IFNY T Cell Responses Induced by a Vaccine Coexpressing a Human Prostate Specific Membrane Antigen (PSMA), Prostate Stem Cell Antigen (PSCA), and Prostate Specific Antigen (PSA)

[00403] In vivo Study Procedures. In brief, three groups of Chinese cynomolgus monkeys (n=4/group) were used in the study. Animals in Group 1 received intramuscular injection with vehicle. Animals in Group 2 were administered the anti-PD-1 monoclonal antibody mAb7 subcutaneously at 20 mg/kg. Animals in Group 3 were treated with a vaccine containing an AdC68 adenovirus vector coexpressing three human prostate-associated antigens (PSMA, PSCA, and PSA) (AdC68W-734 Vector), at a total dose of 6e11 VP, followed immediately by subcutaneous injections. of 150 mg of anti-CTLA4 antibody tremelimumab and anti-PD-1 mAb7 monoclonal antibody at 20 mg/kg. Twenty-eight days after injections, animals were boosted with vehicle (group 1), anti-PD-1 monoclonal antibody mAb7 subcutaneously at 20 mg/kg (group 2), or a DNA plasmid (Plasmid 458) expressing three antigens. of human prostate cancer (PSMA, PSCA and PSA) distributed by electroporation immediately followed by subcutaneous injections of 150 mg of anti-CTLA4 and anti-PD-1 monoclonal antibodies at 20 mg/kg (group 3). Forty-three days after the first main injections, PBMCs were isolated from each animal and subjected to ELISPOT assays to measure PSMA, PSCA, and PSA-specific IFNY T cell responses.

[00404] The complete sequence of the AdC68W-734 Vector used in the study is provided in SEQ ID NO:45 of the present description and in SEQ ID NO:63 of International Patent Application Publication WO2015/063647. The construction of the AdC68W-734 Vector is also described in WO2015/063647, the description of which is incorporated herein by reference. The AdC68W-734 vector and plasmid 458 each comprise (1) a nucleotide sequence encoding amino acids 15-750 of human PSMA having the sequence of SEQ ID NO:42, (2) a nucleotide sequence encoding amino acids 25-261 of human PSA having the sequence of SEQ ID NO:47, and (3) a nucleotide sequence encoding the full-length human PSCA of SEQ ID NO:48.

[00405] IFNY ELISPOT Assay. Briefly, peripheral blood mononuclear cells (PBMCs) from individual animals were coincubated in duplicate with peptide pools from a 15-mer peptide library overlapping by 11 amino acids spanning the entire sequence of the respective antigen, each peptide at 2 μg/mL. The plates were incubated for ~16 hours at 37°C, 5% CO2, then washed and developed, as per the manufacturer's instructions. The number of IFNγ spot forming cells (SFC) was counted with a CTL reader. The duplicates were averaged and the response from the negative control wells, which contained no peptide, subtracted. SFC counts were then normalized to describe the response by PBMCs 1e6. The antigen-specific responses in the tables represent the sum of the responses for the corresponding antigen-specific peptide pools.

[00406] Results. The ELISpot results, which are presented in Table 33, demonstrate that animals that received the subcutaneous anti-PD-1 antibody and anti-CTLA4 antibody vaccine exhibited a consistent increase in the IFNγ T cell response to at least one antigen from prostate cancer, while there were no IFNγ T cell responses to these antigens in the vehicle group (Group 1) or in the group administering anti-PD-1 antibody alone (Group 2). Table 32. Peptide sequence information for peptide pools used in the ELISpot and ICCS assays to induce antigen-specific IFNY T cell responses. Table 33. Effect of anti-PD-1 antibody on vaccine-induced antigen-specific IFNγ ELISpot T cell responses in cynomolgus monkeys.

[00407] Although the disclosed teachings have been described with reference to various applications, methods, kits and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings here and the invention claimed below. The foregoing examples are provided to further illustrate the teachings revealed and are not intended to limit the scope of the teachings presented here. Although the present teachings have described and described these exemplary embodiments, one skilled in the art will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of current teachings.

[00408] All references cited herein, including patents, patent applications, works, manuals and the like, and references cited therein, to the extent they are not already there, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differ from or contradict this patent application, including but not limited to, defined terms, use of the term, described techniques or the like, this patent application takes precedence.

[00409] The previous description and examples detail certain specific embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in the text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof. same.

Claims (15)

1. Isolated antagonist antibody that specifically binds to PD-1, characterized by the fact that it comprises: a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14, or 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 24, 25, 27, 28, 35, or 36, a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, 23, 26, or 37 , a light chain variable region (VL) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, 22, 30, or 32, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, 20, or 33 and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, 21, 31, or 34. 2. Isolated antagonist antibody according to claim 1, characterized by the fact that it comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 3, 4, 5, or 6. 3. Isolated antagonist antibody according to claim 2, characterized in that it comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 2, 7, 8, or 9. 4. Isolated antagonist antibody that specifically binds to PD-1, characterized by the fact that it comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 4 and a VL comprising the amino acid sequence shown in SEQ ID NO: 8. 5. Isolated antagonist antibody according to any one of claims 1 to 4, characterized in that the antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 17, a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: : 20 and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 21. 6. Isolated antagonist antibody according to any one of claims 1 to 5, characterized in that it comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 29 or 38 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39. 7. Isolated antagonist antibody according to any one of claims 1 to 5, characterized in that it comprises a constant region, optionally in which the antibody has an isotype that is selected from the group consisting of IgG2, IgG2Δa, IgG4, IgG4Δb, IgG4Δc, IgG4 S228P, IgG4Δb S228P and IgG4Δc S228P. 8. Isolated antagonist antibody according to claim 1, characterized by the fact that each CDR of the antibody is defined according to the Kabat definition, the Chothia definition or the combination of the Kabat definition and the Chothia definition of CDR. 9. Isolated antibody according to any one of claims 1 to 8, characterized by the fact that the antibody promotes the secretion of IFNY and/or TNF from T cells, promotes the proliferation of T cells; and/or inhibits tumor growth. 10. Isolated antibody according to any one of claims 1 to 9, characterized in that the antibody binds human PD-1 and mouse PD-1, optionally wherein the antibody binds human PD-1 with an affinity of about 0.73 nM at 25°C as measured by surface plasmon resonance. 11. Method of producing an anti-PD-1 antagonist antibody, characterized in that it comprises: culturing a cell line that recombinantly produces the antibody, as defined in any one of claims 1 to 10, under conditions in which the antibody is produced; and antibody recovery. 12. Pharmaceutical composition, characterized by the fact that it comprises the antibody, as defined in any one of claims 1 to 10, and a pharmaceutically acceptable carrier. 13. Kit for the treatment of cancer, characterized by the fact that it comprises the pharmaceutical composition, as defined in claim 12. 14. Use of the anti-PD-1 antibody, as defined in any one of claims 1 to 10, or of the pharmaceutical composition, as defined in claim 12, characterized by the fact that it is for the preparation of a medicine for the treatment of cancer , so that one or more symptoms associated with cancer are improved in the individual. 15. Use according to claim 14, characterized in that the cancer is selected from the group consisting of gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, head and neck cancer squamous cell cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer and melanoma.