Anti-PSGL-1 compositions and methods for modulating myeloid cell inflammatory phenotypes and uses thereof

Abstract

To provide anti-PSGL-1 compositions and methods for modulating myeloid cell inflammatory phenotypes and uses thereof.SOLUTION: The present invention is based, in part, on the discovery of anti-PSGL-1 composition (e.g., monoclonal antibodies and antigen-binding fragments thereof), that regulates myeloid cell inflammatory phenotypes, such as suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells, including polarization, activation, and / or function, and methods of using such anti-PSGL-1 compositions for therapeutic, diagnostic, prognostic, and screening purposes. For example, in one aspect, a monoclonal antibody, or antigen-binding fragment thereof, that binds myeloid cells expressing PSGL-1 polypeptide and increases an inflammatory phenotype of the myeloid cells, optionally in which the myeloid cells comprise suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells, is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 857,169, filed June 4, 2019, U.S. Provisional Application No. 62 / 867,569, filed June 27, 2019, U.S. Provisional Application No. 62 / 947,948, filed December 13, 2019, and U.S. Provisional Application No. 63 / 032,214, filed May 29, 2020, the entire contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Monocytes and macrophages are types of phagocytes, cells that protect the body by engulfing harmful foreign particles, bacteria, and dead or dying cells. In addition to monocytes and macrophages, phagocytes include neutrophils, dendritic cells, and mast cells.

[0003] Macrophages are classically known as large white blood cells that patrol the body, engulfing and digesting cellular debris and foreign particles such as pathogens, microorganisms, and cancer cells through a process known as phagocytosis. Furthermore, macrophages, including tissue macrophages and circulating monocyte-derived macrophages, are important mediators of both the innate and adaptive immune systems.

[0004] Macrophage phenotypes depend on activation via classical or alternative pathways (see, e.g., Classen et al. (2009) Methods Mol. Biol. 531:29-43). Classically activated macrophages are activated by interferon gamma (IFNγ) or lipopolysaccharide (LPS) and exhibit an M1 phenotype. This pro-inflammatory phenotype is associated with increased inflammation and stimulation of the immune system. Alternatively, activated macrophages are activated by cytokines such as IL-4, IL-10, and IL-13 and exhibit an M2 phenotype. This anti-inflammatory phenotype is associated with reduced immune responses, increased wound healing, increased tissue repair, and embryonic development.

[0005] Under non-pathological conditions, the immune system contains a balanced population of immunostimulatory and immunoregulatory macrophages. Disruption of this balance can lead to various disease states. In some cancers, for example, tumors secrete immune factors (e.g., cytokines and interleukins) that polarize macrophage populations in favor of an anti-inflammatory, pro-tumorigenic M2 phenotype, which activates wound healing pathways, promotes new blood vessel growth (i.e., angiogenesis), and provides nutrients and growth signals to tumors. These M2 macrophages are referred to as tumor-associated macrophages (TAMs) or tumor-infiltrating macrophages. TAMs in the tumor microenvironment are key regulators of cancer progression and metastasis (Pollard (2004) Nat. Rev. Cancer 4:71-78). Small molecules and monoclonal antibodies designed to inhibit macrophage gene targets (e.g., CSF1R and CCR2) are being investigated as regulators of macrophage phenotype, such as by modulating the balance between pro-tumorigenic macrophages (e.g., TAMs) and pro-inflammatory macrophages, which can inhibit tumorigenesis. Therapies that modulate the recruitment, polarization, activation, and / or function of monocytes and macrophages to regulate the balance of macrophage populations are referred to as macrophage immunotherapy. Despite advances in the field of macrophage biology, there remains a need for new targets (e.g., genes and / or gene products) for modulating the inflammatory phenotype of macrophages and agents for use in macrophage immunotherapy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Classen et al.(2009)Methods Mol.Biol.531:29-43 [Non-patent document 2] Pollard (2004) Nat. Rev. Cancer 4:71-78 Summary of the Invention [Means for solving the problem]

[0007] The present invention is based, at least in part, on the discovery of anti-PSGL-1 compositions and methods for modulating myeloid cell inflammatory phenotypes and their uses for therapeutic, diagnostic, prognostic, and screening purposes, etc. For example, it has been determined herein that PSGL-1 expression increases upon activation in M2 macrophages, and that anti-PSGL-1 antibodies, including antigen-binding fragments thereof, can be used to increase the myeloid cell inflammatory phenotype.

[0008] For example, in one aspect, a monoclonal antibody, or antigen-binding fragment thereof, is provided that binds to myeloid cells that express a PSGL-1 polypeptide and increases the inflammatory phenotype of the myeloid cells, optionally the myeloid cells including suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0009] Numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, a monoclonal antibody, or antigen-binding fragment thereof, is provided that: a) after contact with the monoclonal antibody or antigen-binding fragment thereof, i) increased expression and / or secretion of cluster of differentiation 80 (CD80), CD86, MHCII, MHC1, interleukin-1 beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); ii) increased expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1; , TGFb, and / or IL-10; iii) increased secretion of at least one cytokine or chemokine selected from the group consisting of IL-1β, TNF-α, IL-12, IL-18, GM-CSF, CCL3, CCL4, and IL-23; iv) an increased ratio of IL-1β, IL-6, and / or TNF-α expression to IL-10 expression; v) increased CD8+ cytotoxic T cell activation; vi) increased mobilization of CD8+ cytotoxic T cell activation; vii) increased and / or xiii) increasing the inflammatory phenotype of myeloid cells by resulting in one or more of: CD4+ helper T cell activity; viii) increased recruitment of CD4+ helper T cell activity; ix) increased NK cell activity; x) increased recruitment of NK cells; xi) increased neutrophil activity; xii) increased macrophage and / or dendritic cell activity; and / or xiii) increased spindle-shaped morphology, flattened appearance, and / or number of dendrites as assessed by microscopy; b) selectively binding to a human PSGL-1 polypeptide at least 1.1-fold greater than a polypeptide selected from the group consisting of human complement C4 protein, human sulfotyrosinylated C4 peptide, human fibrinogen protein, human sulfotyrosinylated fibrinogen peptide, human sulfotyrosinylated CCK peptide, human sulfotyrosinylated CCR2b peptide, human sulfotyrosinylated D6 peptide, wherein the polypeptide is expressed on a cell or in vitro; c) optionally, as measured by ELISA or biolayer interferometry assay.d) binds to human PSGL-1 polypeptide with a kD of about 0.00001 nanomolar (nM) to 1000 nM; d) binds to the N-terminal peptide sequence QATEYEYLDYDFLPETEPPEM of human PSGL-1 polypeptide; e) binds to one or more sulfotyrosine residues of sulfotyrosinylated human PSGL-1 polypeptide; f) cross-reacts with cynomolgus monkey PSGL-1 polypeptide; g) competes or cross-competes with an antibody listed in Table 2 or 3 that binds to PSGL-1 polypeptide or an antigen-binding fragment thereof; h) The antibody or antibody composition of the present invention may be selected from the group consisting of: a antibody that competes with, inhibits, or blocks the binding of PSGL-1 to a PSGL-1 ligand, and optionally the PSGL-1 ligand is VISTA; i) is available as a monoclonal antibody deposited with the ATCC as described herein; j) does not activate unstimulated monocytes; k) has no ADCC activity against PSGL-1-expressing cells; l) has no CDC activity against PSGL-1-expressing cells; m) is capable of binding to and / or endocytosis by PSGL-1-expressing cells; n) not killing PSGL-1 expressing cells upon localization; n) not conjugated to another therapeutic moiety, optionally where the other therapeutic moiety is a cytotoxic agent; o) not activating or inducing T cell apoptosis; p) binding to an epitope comprising residues 45-55 of human PSGL-1, optionally where the binding epitope is a conformational epitope or a linear epitope; q) binding an epitope C-terminal to residues 42-62 of human PSGL-1, optionally where the epitope is a conformational epitope or a linear epitope; r) binding to one or more residues 45, 46, 49, 50, 51, 52, 53, and 55 of human PSGL-1, optionally wherein the residues are selected from the group consisting of the epitope residues listed in Table 13, and optionally wherein the binding epitope is a conformational epitope or a linear epitope; s) binding to one, two, or three of human PSGL-1,or three sulfotyrosinylated residues of human PSGL-1, wherein the sulfotyrosinylated residues are selected from the group consisting of positions 46, 48, and 51, and optionally the binding epitope is a conformational epitope or a linear epitope; t) binding to sulfotyrosinylated human PSGL-1, wherein the ratio of the binding affinity of the mAb to sulfotyrosinylated human PSGL-1 compared to the binding affinity of the mAb to a sulfotyrosinylated protein that is not PSGL-1 is greater than the binding affinity of the mAb to PSG6, PSG3, and / or SELK1 mAb to sulfotyrosinylated human PSGL-1 compared to the binding affinity of the mAb to a sulfotyrosinylated protein that is not PSGL-1. The mAb has one or more of the following characteristics: u) binds to sulfotyrosinylated human PSGL-1, wherein the binding affinity of the mAb to sulfotyrosinylated human PSGL-1 is at least 10% or greater than the affinity of the mAb to a sulfotyrosinylated protein that is not PSGL-1, and optionally the sulfotyrosinylated protein that is not PSGL-1 is C4 alpha chain, complement C4, fibrinogen gamma, fibrinogen, CCK, CC42b, and / or D6; and / or v) has anti-tumor activity in vivo. In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, comprises a) a heavy chain CDR sequence having at least about 90% identity to a heavy chain CDR sequence selected from the group consisting of the sequences listed in Table 2, and / or b) a light chain CDR sequence having at least about 90% identity to a light chain CDR sequence selected from the group consisting of the sequences listed in Table 2. In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, comprises a) a heavy chain sequence having at least about 90% identity to a heavy chain sequence selected from the group consisting of the heavy chain sequences listed in Table 2;and / or b) a light chain sequence having at least about 90% identity to a light chain sequence selected from the group consisting of the light chain sequences listed in Table 2. In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, comprises a) a heavy chain CDR sequence selected from the group consisting of the heavy chain sequences listed in Table 2, and / or b) a light chain CDR sequence selected from the group consisting of the light chain sequences listed in Table 2. In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, comprises a) a heavy chain sequence selected from the group consisting of the heavy chain sequences listed in Table 2, and / or b) a light chain sequence selected from the group consisting of the light chain sequences listed in Table 2. In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is chimeric, humanized, murine, or human. In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, and / or comprises an Fc domain. In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, Fde, sdFv, single-domain antibody (dAb), and bispecific antibody fragment. In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, comprises an immunoglobulin constant domain selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In yet another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, comprises a constant domain derived from a human immunoglobulin. In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, is conjugated to an agent, optionally, the agent being selected from the group consisting of a binding protein, an enzyme, a drug, a chemotherapeutic agent, a biological agent, a toxin, a radionuclide, an immunomodulator, a detectable moiety, and a tag.

[0010] In another aspect, a pharmaceutical composition is provided comprising a therapeutically effective amount of at least one monoclonal antibody encompassed by the present invention, or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or excipient.

[0011] As noted above, numerous embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the pharmaceutically acceptable carrier or excipient is selected from the group consisting of a diluent, a solubilizer, an emulsifier, a preservative, and an adjuvant. In another embodiment, the pharmaceutical composition has less than about 20 EU endotoxin / mg protein. In yet another embodiment, the pharmaceutical composition has less than about 1 EU endotoxin / mg protein.

[0012] In yet another aspect, an isolated nucleic acid molecule is provided that i) hybridizes under stringent conditions to the complement of a nucleic acid encoding an immunoglobulin heavy and / or light chain polypeptide of a monoclonal antibody, or antigen-binding fragment thereof, encompassed by the invention; ii) has a sequence having at least about 90% identity over its entire length to a nucleic acid encoding an immunoglobulin heavy and / or light chain polypeptide of a monoclonal antibody, or antigen-binding fragment thereof, encompassed by the invention; or iii) encodes an immunoglobulin heavy and / or light chain polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2.

[0013] In yet another aspect, there are provided isolated immunoglobulin heavy and / or light chain polypeptides encoded by the nucleic acids encompassed by the invention.

[0014] In another aspect, there is provided a vector comprising an isolated nucleic acid encompassed by the invention, optionally wherein the vector is an expression vector.

[0015] In yet another aspect, host cells are provided that comprise an isolated nucleic acid encompassed by the invention. In some embodiments, host cells are provided that a) express a monoclonal antibody encompassed by the invention, or an antigen-binding fragment thereof, b) comprise an immunoglobulin heavy chain and / or light chain polypeptide encompassed by the invention, c) comprise a vector encompassed by the invention, and / or d) are accessible as the monoclonal antibody deposited under the ATCC deposit accession number(s) set forth herein.

[0016] In yet another aspect, there is provided a device or kit comprising at least one monoclonal antibody, or antigen-binding fragment thereof, encompassed by the present invention, optionally comprising a label for detecting the at least one monoclonal antibody, or antigen-binding fragment thereof, or a complex comprising the monoclonal antibody, or antigen-binding fragment thereof.

[0017] In another aspect, a device or kit is provided that includes a pharmaceutical composition, isolated nucleic acid molecule, isolated immunoglobulin heavy and / or light chain polypeptide, vector, and / or host cell encompassed by the invention.

[0018] In yet another aspect, a method for producing at least one monoclonal antibody, or antigen-binding fragment thereof, encompassed by the present invention is provided, comprising the steps of: (i) culturing a transformed host cell that has been transformed with nucleic acid comprising a sequence encoding at least one monoclonal antibody, or antigen-binding fragment thereof, under conditions suitable to allow expression of the monoclonal antibody, or antigen-binding fragment thereof; and (ii) recovering the expressed monoclonal antibody, or antigen-binding fragment thereof.

[0019] In yet another aspect, a method for detecting the presence or level of a PSGL-1 polypeptide is provided, comprising obtaining a sample and detecting the polypeptide in the sample by using at least one monoclonal antibody, or antigen-binding fragment thereof, encompassed by the present invention. In one embodiment, the at least one monoclonal antibody, or antigen-binding fragment thereof, forms a complex with the PSGL-1 polypeptide, and the complex is detected using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunochemical assay, Western blot, mass spectrometry, a nuclear magnetic resonance assay, or an intracellular flow assay.

[0020] In another aspect, provided is a method of generating bone marrow cells having an increased inflammatory phenotype after contact with an agent encompassed by the present invention, comprising contacting bone marrow cells with an effective amount of the agent, optionally wherein the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0021] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, bone marrow cells having an increased inflammatory phenotype, after contact with a monoclonal antibody, or antigen-binding fragment thereof, exhibit a) increased expression and / or secretion of cluster of differentiation 80 (CD80), CD86, MHCII, MHC1, interleukin-1 beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), b) decreased expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, TGFβ, and / or IL-10, c) decreased expression and / or secretion of IL-1β, TNF-α, IL-12, IL-18, GM-CSF, CCL3, CCL4, and IL-2. 3), d) an increased ratio of IL-1β, IL-6, and / or TNF-α expression to IL-10 expression, e) increased CD8+ cytotoxic T cell activation, f) increased recruitment of CD8+ cytotoxic T cell activation, g) increased CD4+ helper T cell activity, h) increased recruitment of CD4+ helper T cell activity, i) increased NK cell activity, j) increased NK cell recruitment, k) increased neutrophil activity, l) increased macrophage and / or dendritic cell activity, and / or m) increased spindle-shaped morphology, flattened appearance, and / or number of dendrites when assessed by microscopy. In another embodiment, bone marrow cells contacted with the monoclonal antibody, or antigen-binding fragment thereof, are included in a population of cells, and the monoclonal antibody, or antigen-binding fragment thereof, increases the number of type 1 and / or M1 macrophages and / or decreases the number of type 2 and / or M2 macrophages in the population of cells. In yet another embodiment, bone marrow cells contacted with the monoclonal antibody, or antigen-binding fragment thereof, are included in a population of cells, and the monoclonal antibody, or antigen-binding fragment thereof, increases the ratio of i) to ii) in the population of cells, where i) are type 1 and / or M1 macrophages and ii) are type 2 and / or M2 macrophages.In yet another embodiment, the macrophages comprise type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells. In another embodiment, the bone marrow cells are contacted in vitro or ex vivo. In yet another embodiment, the bone marrow cells are primary bone marrow cells. In yet another embodiment, the bone marrow cells are purified and / or cultured prior to contacting with the agent. In another embodiment, the bone marrow cells are contacted in vivo (e.g., by systemic, peritumoral, or intratumoral administration of the agent). In yet another embodiment, the bone marrow cells are contacted within a tissue microenvironment. In yet another embodiment, the method further comprises contacting the bone marrow cells with at least one immunotherapeutic agent that modulates a proinflammatory phenotype, optionally wherein the immunotherapeutic agent comprises an immune checkpoint inhibitor, an immunostimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus.

[0022] In yet another aspect, compositions are provided comprising bone marrow cells produced according to the methods encompassed by the present invention, optionally wherein the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0023] In yet another aspect, a method is provided for increasing the inflammatory phenotype of bone marrow cells in a subject after contact with an agent encompassed by the present invention, comprising administering to the subject an effective amount of the agent.

[0024] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, bone marrow cells having an increased inflammatory phenotype, after contact with an agent, exhibit a) increased expression and / or secretion of cluster of differentiation 80 (CD80), CD86, MHCII, MHC1, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), b) decreased expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, and / or IL-10, c) decreased expression and / or secretion of IL-1β, TNF-α, IL-1, and / or IL-1. and / or j) increased fusiform morphology, flattened appearance, and / or number of dendrites as assessed by microscopy. In another embodiment, the one or more agents increase the number of type 1 and / or M1 macrophages, decrease the number of type 2 and / or M2 macrophages, and / or increase the ratio of i) to ii), wherein i) are type 1 and / or M1 macrophages and ii) are type 2 and / or M2 macrophages in the subject. In yet another embodiment, the number and / or activity of cytotoxic CD8+ T cells in the subject is increased after administration of the agent. In yet another embodiment, the bone marrow cells comprise type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells. In yet another embodiment, the agent is administered in vivo by systemic, peritumoral, or intratumoral administration of the agent.In yet another embodiment, the agent contacts the bone marrow cells within the tissue microenvironment. In yet another embodiment, the method further comprises contacting the bone marrow cells with at least one immunotherapeutic agent that modulates an inflammatory phenotype, optionally the immunotherapeutic agent comprising an immune checkpoint inhibitor, an immunostimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus.

[0025] In another aspect, a method of increasing inflammation in a subject is provided, comprising administering to the subject bone marrow cells contacted with an effective amount of a drug encompassed by the present invention, optionally wherein the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0026] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the bone marrow cells comprise type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells. In another embodiment, the bone marrow cells are genetically engineered, autologous, syngeneic, or allogeneic relative to the subject's bone marrow cells. In yet another embodiment, the agent is administered systemically, peritumorally, or intratumorally.

[0027] In yet another aspect, there is provided a method of sensitizing cancer cells in a subject to cytotoxic CD8+ T cell-mediated killing and / or immune checkpoint therapy, comprising administering to the subject a therapeutically effective amount of an agent encompassed by the present invention.

[0028] In yet another aspect, there is provided a method of sensitizing cancer cells in a subject suffering from cancer to cytotoxic CD8+ T cell-mediated killing and / or immune checkpoint therapy, comprising administering to the subject monocytes and / or macrophage cells that have been contacted with a therapeutically effective amount of an agent encompassed by the present invention, optionally wherein the myeloid cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0029] As noted above, numerous embodiments are further provided that can be applied to any aspect of the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the bone marrow cells comprise type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells. In another embodiment, the bone marrow cells are genetically engineered, autologous, syngeneic, or allogeneic relative to the subject's bone marrow cells. In yet another embodiment, the agent is administered systemically, peritumorally, or intratumorally. In yet another embodiment, the method further comprises treating cancer in the subject by administering at least one immunotherapy to the subject, where the immunotherapy optionally comprises an immune checkpoint inhibitor, an immunostimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus. In another embodiment, the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4. In yet another embodiment, the immune checkpoint is PD-1. In yet another embodiment, the method further comprises treating cancer in the subject by administering to the subject an additional therapeutic agent or regimen for treating the cancer, where optionally the additional therapeutic agent or regimen is selected from the group consisting of chimeric antigen receptor, chemotherapy, radiation, targeted therapy, and surgery. In yet another embodiment, the agent reduces the number of proliferating cells in the cancer and / or reduces the volume or size of a tumor comprising the cancer cells. In yet another embodiment, the agent increases the amount and / or activity of CD8+ T cells infiltrating a tumor comprising cancer cells. In yet another embodiment, the agent a) increases the amount and / or activity of M1 macrophages infiltrating a tumor comprising cancer cells, and / or b) reduces the amount and / or activity of M2 macrophages infiltrating a tumor comprising cancer cells. In another embodiment, the method further comprises administering to the subject at least one additional therapy or regimen for treating the cancer. In yet another embodiment, the therapy is prior to, concurrent with, or subsequent to administration of the agent.

[0030] In another aspect, provided is a method for identifying a bone marrow cell capable of increasing its inflammatory phenotype by modulating at least one target, the method comprising: a) determining the amount and / or activity of at least one target listed in Table 1 from a bone marrow cell using an agent, wherein the agent is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof; b) determining the amount and / or activity of the at least one target in a control using the agent; and c) comparing the amount and / or activity of the at least one target detected in steps a) and b), wherein a presence or increase in the amount and / or activity of at least one target listed in Table 1 in the bone marrow cell compared to the control amount and / or activity of the at least one target indicates that the bone marrow cell is capable of increasing its inflammatory phenotype by modulating the at least one target; and optionally, the bone marrow cell comprises a suppressor myeloid cell, a monocyte, a macrophage, a neutrophil, and / or a dendritic cell.

[0031] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the method further comprises contacting, recommending, prescribing, or administering the cells with an agent that modulates at least one target listed in Table 1. In another embodiment, if it is determined that the subject would not benefit from increasing the inflammatory phenotype by modulating at least one target (e.g., immunotherapy), the method further comprises contacting, recommending, prescribing, or administering the cells with a cancer therapy other than an agent that modulates at least one target listed in Table 1. In yet another embodiment, the method further comprises contacting the cells with and / or administering at least one additional agent that increases the immune response. In yet another embodiment, the additional agent is selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and / or hormone therapy. In another embodiment, the control is from a member of the same species to which the subject belongs. In yet another embodiment, the control is a sample comprising cells. In yet another embodiment, the subject is afflicted with cancer. In another embodiment, the control is a cancer sample from the subject. In yet another embodiment, the control is a non-cancerous sample from the subject.

[0032] In yet another aspect, there is provided a method for predicting clinical outcome of a subject afflicted with cancer, comprising: a) determining the amount and / or activity of at least one target listed in Table 1 from bone marrow cells from the subject using an agent, wherein the agent is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof; b) determining the amount and / or activity of the at least one target from a control subject with a poor clinical outcome using the agent; and c) comparing the amount and / or activity of the at least one target in the subject sample and a sample from the control subject, wherein a presence or increase in the amount and / or activity of at least one target listed in Table 1 from bone marrow cells from the subject compared to the amount and / or activity in the control indicates that the subject will not have a poor clinical outcome, and optionally the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0033] In yet another aspect, a method for monitoring the inflammatory phenotype of bone marrow cells in a subject, comprising: a) detecting in a first subject sample at a first time point the amount and / or activity of at least one target listed in Table 1 from bone marrow cells from the subject using an agent, wherein the agent is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof; b) repeating step a) using a subsequent sample comprising bone marrow cells obtained at a subsequent time point; and c) comparing the amount or activity of at least one target listed in Table 1 detected in steps a) and b). Methods are provided wherein an absence or reduction in the amount and / or activity of at least one target listed in Table 1 from bone marrow cells from the subsequent sample compared to the amount and / or activity from bone marrow cells indicates that the subject's bone marrow cells have an upregulated inflammatory phenotype, or a presence or increase in the amount and / or activity of at least one target listed in Table 1 from bone marrow cells from the subsequent sample compared to the amount and / or activity from bone marrow cells from the first sample indicates that the subject's bone marrow cells have a downregulated inflammatory phenotype, optionally wherein the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0034] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the first and / or at least one subsequent sample comprises bone marrow cells that have been cultured in vitro. In another embodiment, the first and / or at least one subsequent sample comprises bone marrow cells that have not been cultured in vitro. In yet another embodiment, the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from the subject. In another embodiment, the sample comprises blood, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject.

[0035] In another aspect, a method for evaluating the effectiveness of a test agent for increasing the inflammatory phenotype of bone marrow cells in a subject is provided, the method comprising: a) detecting in a subject sample comprising bone marrow cells at a first time point: i) detecting the amount or activity of at least one target listed in Table 1 in or on the bone marrow cells using an agent, wherein the agent is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof, and / or ii) detecting the inflammatory phenotype of the bone marrow cells; b) repeating step a) for at least one subsequent time point after the bone marrow cells have been contacted with the test agent; and c) comparing the values ​​of i) and / or ii) detected in steps a) and b), wherein a lack of, or a reduction in, the amount and / or activity of at least one target listed in Table 1 in the sample at the first time point, and / or an increase in ii) in the subsequent sample, indicates that the test agent increases the inflammatory phenotype of bone marrow cells in the subject.

[0036] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, bone marrow cells contacted with an agent are included in a population of cells, and the agent increases the number of type 1 and / or M1 macrophages within the population of cells. In another embodiment, bone marrow cells contacted with an agent are included in a population of cells, and the agent reduces the number of type 2 and / or M2 macrophages within the population of cells. In yet another embodiment, the bone marrow cells are contacted in vitro or ex vivo. In yet another embodiment, the bone marrow cells are primary monocytes and / or primary macrophages. In another embodiment, the bone marrow cells are purified and / or cultured prior to contacting with the agent. In yet another embodiment, the bone marrow cells are contacted in vivo. In yet another embodiment, the bone marrow cells are contacted in vivo by systemic, peritumoral, or intratumoral administration of the agent. In another embodiment, the bone marrow cells are contacted within a tissue microenvironment. In yet another embodiment, the method further comprises contacting the bone marrow cells with at least one immunotherapeutic agent that modulates the inflammatory phenotype, optionally the immunotherapeutic agent comprising an immune checkpoint inhibitor, an immunostimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus. In yet another embodiment, the subject is a mammal (e.g., a non-human animal model or a human).

[0037] In yet another aspect, a method of evaluating the effectiveness of a test agent for treating cancer in a subject comprises: a) detecting in a subject sample comprising bone marrow cells at a first time point: i) detecting the amount and / or activity of at least one target listed in Table 1 in or on the bone marrow cells using an agent, wherein the agent is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof; and / or ii) detecting an inflammatory phenotype of the bone marrow cells; and b) performing step a) during at least one subsequent time point after administration of the agent. and c) comparing the values ​​of i) and / or ii) detected in steps a) and b), wherein an absence or reduction in the amount and / or activity of at least one target listed in Table 1 in or on the bone marrow cells of the subject sample at a first time point, and / or an increase in ii) in or on the bone marrow cells of the subject sample at a subsequent time point, compared to the amount and / or activity in or on the bone marrow cells of the subject sample at a first time point, indicates that the test agent treats cancer in the subject, and optionally the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0038] As noted above, numerous embodiments are further provided that can be applied to any aspect of the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the subject has undergone treatment for cancer between the first time point and the subsequent time point, has completed the treatment, and / or is in remission. In another embodiment, the first and / or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples. In yet another embodiment, the first and / or at least one subsequent sample is obtained from a non-human animal model of cancer. In yet another embodiment, the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from the subject. In another embodiment, the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject.

[0039] In another aspect, a method for screening a test agent that sensitizes cancer cells to cytotoxic T cell-mediated killing and / or immune checkpoint therapy comprises: a) contacting cancer cells with cytotoxic T cell and / or immune checkpoint therapy in the presence of bone marrow cells contacted with a test agent, wherein the test agent modulates the amount and / or activity of at least one target listed in Table 1 in or on the bone marrow cells as determined using the agent, and the agent is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof; and b) contacting the cancer cells with cytotoxic T cell and / or immune checkpoint therapy in the presence of bone marrow cells contacted with the test agent. a) contacting the cancer cells with cytotoxic T cell and / or immune checkpoint therapy in the presence of control bone marrow cells not contacted with the test agent; and c) identifying a test agent that sensitizes the cancer cells to cytotoxic T cell mediated killing and / or immune checkpoint therapy by identifying an agent that increases the efficacy of cytotoxic T cell mediated killing and / or immune checkpoint therapy in a) compared to b), wherein optionally the bone marrow cells comprise suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0040] As noted above, numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the contacting step occurs in vivo, ex vivo, or in vitro. For example, in one embodiment, the method further comprises determining i) a decrease in the number of proliferating cells in the cancer and / or ii) a decrease in the volume or size of a tumor comprising the cancer cells. In yet another embodiment, the method further comprises determining i) an increase in the number of CD8+ T cells and / or ii) an increase in the number of type 1 and / or M1 macrophages infiltrating the tumor comprising the cancer cells. In yet another embodiment, the method further comprises determining responsiveness to a test agent that modulates at least one target listed in Table 1, as measured by at least one criterion selected from the group consisting of clinical benefit rate, survival to death, pathological complete response, a semi-quantitative measure of pathological response, clinical complete response, clinical partial response, clinical stable disease, relapse-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST criteria. In another embodiment, the method further comprises contacting the cancer cells with at least one additional cancer therapeutic agent or regimen.

[0041] As noted above, numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, myeloid cells having a modulated inflammatory phenotype exhibit a) modulated expression of cluster of differentiation 80 (CD80), CD86, MHCII, MHC1, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), b) modulated expression of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, and / or IL-10, c) modulated expression of IL-1β, TNF-α, IL-12, IL-18, and IL-23. a) modulated secretion of at least one cytokine selected from the group consisting of: d) modulated ratio of IL-1β, IL-6, and / or TNF-α expression to IL-10 expression; e) modulated CD8+ cytotoxic T cell activation; f) modulated CD4+ helper T cell activity; g) modulated NK cell activity; h) modulated neutrophil activity; i) modulated macrophage and / or dendritic cell activity; and / or j) modulated spindle-shaped morphology, flattened appearance, and / or number of dendrites as assessed by microscopy. In another embodiment, the cells and / or bone marrow cells comprise type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, and optionally, the cells and / or bone marrow cells express or are determined to express PSGL-1.In yet another embodiment, the human PSGL-1 polypeptide has the amino acid sequence of SEQ ID NO: 2, the cynomolgus PSGL-1 polypeptide has the amino acid sequence of SEQ ID NO: 17, the human sulfotyrosinylated C4 peptide has the amino acid sequence of NEDY(SO3)EDY(SO3)EY(SO3)DELPAKDDGGK, the human sulfotyrosinylated fibrinogen peptide has the amino acid sequence of (EHPAETEY(SO3)DSLY(SO3)PEDDLGGK), the human sulfotyrosinylated CCK peptide has the amino acid sequence of SHRISDRDY(SO3)MGWMDFGGK, the human sulfotyrosinylated CCR2b peptide has the sequence of TTFFDY(SO3)DY(SO3)GAPSHGGK, and / or the human sulfotyrosinylated D6 peptide has the sequence of ENSSFYY(SO3)Y(SO3)DY(SO3)LDEVAFGGK. In yet another embodiment, the cancer is a solid tumor infiltrated with macrophages, wherein the infiltrating macrophages represent at least about 5% of the mass, volume, and / or number of cells in the tumor or tumor microenvironment, and / or the cancer is selected from the group consisting of mesothelioma, kidney renal clear cell carcinoma, glioblastoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, breast invasive carcinoma, acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, brain low-grade glioma, breast invasive carcinoma, cervical squamous cell carcinoma and adenocarcinoma, cholangiocarcinoma, The cancer is selected from the group consisting of colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe kidney, renal clear cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma of the liver, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, mesothelioma, ovarian serous and cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma, paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid carcinoma, uterine carcinosarcoma, endometrial carcinoma, and uveal melanoma. In another embodiment, the bone marrow cells comprise type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, optionally, the bone marrow cells are TAMs and / or M2 macrophages.In yet another embodiment, the macrophages express or are determined to express PSGL-1. In yet another embodiment, the bone marrow cells are primary bone marrow cells. In another embodiment, the bone marrow cells are contained within a tissue microenvironment. In yet another embodiment, the bone marrow cells are contained within a human tumor model or an animal model of cancer. In yet another embodiment, the subject is a mammal. In another embodiment, the mammal is a human (e.g., a human suffering from cancer).

[0042] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0043] [Figure 1] We show that PSGL-1 expression is predominant in human myeloid cells with a restricted subset of T cells expressing PSGL-1. [Figure 2] We show that TAMs, which represent the majority of cells in ascites fluid samples obtained from gynecological cancers (e.g., M2 TAMs, which express CD16 and CD163), also highly express PSGL-1 protein on their cell surface. [Figure 3] We show that TAMs (e.g., CD11b+ / CD14+ macrophages) obtained from breast tumors dissociated into single-cell suspensions and immunophenotyped via flow cytometry highly express PSGL-1 protein on their cell surface. [Figure 4] Figure 1 shows the rank distribution of macrophage-infiltrated tumors across cancer types in a large public dataset of human cancers (TCGA, The Cancer Genome Atlas, 2017 edition, processed and distributed by OmicSoft / Qiagen) based on their expression of PSGL-1, with highest PSGL-1 expression at the top. [Figure 5-1]We demonstrate the validation of anti-PSGL-1 antibodies in macrophage functional assays. We demonstrate that anti-PSGL-1 antibodies modulate macrophage inflammatory phenotypes in M2-inclined conditions after PSGL-1 inhibition in primary human macrophages, including an increase in M1 proinflammatory cytokines. [Figure 5-2] (continuation) [Figure 6-1] 1 shows the results of a Staphylococcal enterotoxin B (SEB) assay experiment. [Figure 6-2] (continuation) [Figure 6-3] (continuation) [Figure 6-4] (continuation) [Figure 6-5] (continuation) [Figure 7-1] Representative exemplary anti-PSGL-1 mAbs are shown not to activate or induce apoptosis in T cells. [Figure 7-2] (Continued) Ibid. [Figure 8] 1 shows the results of an ex vivo tumor model experiment. [Figure 9] Shown are the results of anti-PSGL-1 antibodies against macrophage inflammatory activation signatures (eg, increased secretion of TNFα and IL-1β) averaged across all tumors analyzed. [Figure 10] Shown are the results of anti-PSGL-1 antibodies against chemokine signatures (e.g., increased secretion of CCL3, CCL4, CCL5, CXCL9, and CXCL10) averaged across all tumors analyzed. [Figure 11] The results of anti-PSGL-1 antibodies against T cell activation signatures (eg, increased secretion of IFNγ and IL-2) averaged across all tumors analyzed are shown. [Figure 12] The results of anti-PSGL1 antibody on increased secretion of TNFα and / or IL-1β in individual tumors are shown. [Figure 13] The results of anti-PSGL-1 antibodies on increased secretion of CCL3, CCL4, CCL5, CXCL9, and / or CXCL10 in individual tumors are shown. [Figure 14] The results of anti-PSGL-1 antibody on increased secretion of IFNγ and / or IL-2 in individual tumors are shown. [Figure 15]

[0033] Figure 1 shows the results of binding characteristics of anti-PSGL-1 antibodies. Binding was determined by ELISA using plate-immobilized proteins and peptides or by flow cytometry. ND, not determined; N / A, not applicable; NB, no binding. [Figure 16] Results are shown for anti-PSGL-1 antibody binding to unmodified, scrambled, and sulfotyrosine-biotin N-terminal PSGL-1 (residues 42-62) peptides. Binding was determined by ELISA using plate-immobilized peptides. ND, not determined; N / A, not applicable; NB, no binding. [Figure 17] 1 shows the amino acid sequences, including sulfated tyrosine, of human sulfotyrosine protein and sulfotyrosine biotin peptide used to characterize anti-PSGL-1 antibodies. [Figure 18] Figure 1 shows the results of anti-PSGL-1 antibody binding to sulfotyrosine human protein and sulfotyrosine peptides. Binding was determined by ELISA using plate-immobilized protein and peptide. ND, not determined. [Figure 19] A comparison of the primary amino acid sequences for representative mature human and cynomolgus monkey PSGL-1 (ECD) is shown. Alignments were generated using known human (Uniprot: Q14242) and cynomolgus monkey (NCBI: XP_005572207.1) PSGL-1 sequences. [Figure 20] Figure 1 shows the results of anti-N-terminal PSGL-1 mAb binding to plate-immobilized PSGL-1(ECD)-hFc1 and biotinylated sulfotyrosine and unmodified PSGL-142-62 peptides. PSGL-1 protein was coated directly, while the biotin peptide was bound to wells pre-coated with streptavidin. [Figure 21-1]The results of anti-N-terminal PSGL-1 mAb binding to plate-immobilized biotinylated alanine-substituted PSGL-142-62 peptide are shown. The bar graph shows the results of mAb binding to the streptavidin-immobilized peptide shown at concentrations approximately 10-fold higher than the EC50 value. MAb binding curves for the alanine-substituted PSGL-142-62 peptide confirmed the importance of specific residues for mAb binding. The heat map summarizes the results of three test and two control (italic) mAbs binding to the wild-type and mutant peptides. The darkest color indicates the weakest mAb binding. The data for the heat map were obtained using a single mAb concentration test, as shown in the bar graph. [Figure 21-2] (continuation) [Figure 22] Figure 1 shows the results of anti-N-terminal PSGL-1 mAb binding to plate-immobilized biotin PSGL-149-56 and PSGL-142-62 peptides. The antibody binding curves show that mAb 18F02 and two control mAbs bind similarly to short and long peptides. In contrast, mAb 16L15 had essentially no binding to the shorter peptides up to the highest antibody concentration tested (10 nM). [Figure 23] The sulfotyrosine motif contained in PSGL-1 and several other sulfotyrosine-containing proteins is shown. [Figure 24] Figure 1 shows control anti-sulfotyrosine N-terminal PSGL-1 mAb cross-reactivity with plate-immobilized sulfotyrosine containing human complement C4 and gamma-fibrinogen proteins. Antibody binding curves show anti-PSGL-1 mAbs, PSG6 (U.S. Patent Publication No. 2007 / 0160601) and SELK1 (Swers et al. (2006) Biochem. Bio. Phys. Res. Commun. 350:508-513), binding to PSGL-1(ECD)-hFc1, complement C4, and gamma-fibrinogen. PSG1 (U.S. Patent Publication No. 2007 / 0154472) mAb is a pan-sulfotyrosine-reactive antibody. [Figure 25]Figure 1 shows the cross-reactivity of anti-sulfotyrosine PSGL-1 mAbs with sulfotyrosine motif-containing proteins and peptides. The bar graph shows 50 nM mAb binding to plate-immobilized sulfotyrosine proteins or peptides. Antibody binding curves show the dose-dependent binding of mAbs 20I15, PSG6, and SELK1 to plate-immobilized complement C4 and gamma-fibrinogen proteins. [Figure 26] Figure 1 shows the results of anti-sulfotyrosine N-terminal PSGL-1 mAb binding to plate-immobilized PSGL-1(ECD)-hFc1 and biotin-sulfotyrosine and unmodified PSGL-142-62. PSGL-1 protein was coated directly, and the biotinylated peptide was bound to wells pre-coated with streptavidin. [Figure 27] Figure 1 shows the results of anti-sulfotyrosine PSGL-1 mAb binding to plate-immobilized biotin-unmodified and sulfotyrosine PSGL-1(42-62). Representative antibody binding curves are shown for the test anti-PSGL-1 mAbs 3D07 and 20I15, as well as the control anti-PSGL-1 mAbs PSG3, PSG6, and SELK1. A commercially available pan-sY-reactive mAb, sulfo-1c-A2 (Millipore, Sigma), confirmed that each modified peptide contained similar amounts of sulfotyrosine. Heat maps were generated from the apparent EC50 values ​​determined from the antibody binding curves. The darkest colors in the heat maps indicate the weakest mAb binding. [Figure 28-1] Figure 1 shows the results of overlapping peptide epitope mapping of anti-PSGL-1 mAbs. The bar graph shows the results of 10 nM mAb binding to the indicated streptavidin-immobilized overlapping peptides. The antibody binding curve confirmed dose-dependent binding of the mAb to the peptides. [Figure 28-2] (continuation) [Figure 29]Figure 1 shows the binding results of anti-PSGL-1 mAbs 18F17 and 19J23 to full-length and C-terminally truncated PSGL-1 (ECD) proteins. The antibody binding curves show that 18F17 bound to PSGL-1 recombinant protein containing the full-length extracellular domain (amino acids 42-320; PSGL-1(ECD)-HIS) and lacking residues 306-320 (PSGL-1(ECD)-hFc1, R&D Systems, catalog no. 3345-PS). In contrast, anti-PSGL-1 mAb 19J23 bound only to proteins containing the full-length ECD. [Figure 30] Antibody binding and epitope mapping data for anti-PSGL-1 mAbs that recognize epitopes outside the N-terminus of PSGL-1 are shown. The PSGL-1 peptide or protein fragment bound by the mAb is shown in parentheses. The epitope of anti-PSGL-1 mAb 15A7 has been reported to lie between D115 and P126 (Johnson et al. (1997) J. Infect. Dis. 176:1215-1224). DETAILED DESCRIPTION OF THE INVENTION

[0044] For any figure showing bar histograms, curves, or other data associated with a legend, the bars, curves, or other data displayed from left to right for each display correspond directly to boxes from top to bottom in the legend.

[0045] The present invention is based, at least in part, on the discovery of anti-PSGL-1 compositions (e.g., monoclonal antibodies) that modulate myeloid cell inflammatory phenotype, including polarization, activation, and / or function. Accordingly, the present invention provides anti-PSGL-1 compositions and methods and uses, including, but not limited to, modulating myeloid cell inflammatory phenotype for therapy, diagnosis, prognosis, and screening.

[0046] I. Definition In some embodiments, the term "about" includes values ​​within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the measured value, or any range therebetween (e.g., plus or minus 2% to 6%). In some embodiments, the term "about" refers to the inherent variation in a method, assay, or error in a measured value, such as the variation that exists between experiments.

[0047] The term "activating receptor" includes immune cell receptors that bind antigens, complexed antigens (e.g., in the context of major histocompatibility complex (MHC) polypeptides), or antibodies. Such activating receptors include T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, Fc receptors, and other ITAM-containing receptors. For example, T cell receptors are present on T cells and associated with CD3 polypeptides. T cell receptors are stimulated by antigens (and polyclonal T cell activation reagents) in the context of MHC polypeptides. T cell activation via TCRs results in many changes, including, for example, protein phosphorylation, membrane lipid changes, ion flux, cyclic nucleotide modifications, RNA transcription changes, protein synthesis changes, and cell volume changes. Similarly, T cell activation of macrophages via activating receptors such as cytokine receptors or pathogen-associated molecular pattern (PAMP) receptors results in changes such as protein phosphorylation, modifications to surface receptor phenotype, protein synthesis and release, and morphological changes.

[0048] The term "activity," when used in reference to a polypeptide, includes activities inherent in the structure of the protein. For example, with respect to a myeloid cell protein, the term "activity" includes the ability to modulate the inflammatory phenotype of the myeloid cell protein by modulating natural binding protein binding or cell signaling of the cell (e.g., by binding to a natural receptor or ligand on an immune cell).

[0049] The term "administering" refers to the actual physical introduction of an agent into or (optionally) onto a desired biological target, such as a host and / or subject. A composition can be administered (e.g., "contacted") to a cell in vitro or in vivo. A composition can be administered to a subject in vivo via an appropriate route of administration. Any and all methods of introducing a composition into a host are contemplated according to the present invention. The present method is not dependent on any particular means of introduction, and should not be construed as such. Introduction means are well known to those skilled in the art and are exemplified herein. The term includes any route of administration that enables an agent to perform its intended function. Examples of administration routes for physical treatment that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. Injection can be a bolus injection or a continuous infusion. Depending on the route of administration, the agent can be coated with or placed within a selected material to protect it from natural conditions that may adversely affect its ability to perform its intended function. The agent can be administered alone or in combination with a pharmaceutically acceptable carrier. Agents can also be administered as prodrugs that are converted in vivo to their active form.

[0050] The term "agent" refers to a compound, a supramolecular complex, a material, and / or a combination or mixture thereof. A compound (e.g., a molecule) can be represented by a chemical formula, a chemical structure, or a sequence. Representative, non-limiting examples of agents include, for example, antibodies, small molecules, polypeptides, polynucleotides (e.g., RNAi agents, siRNA agents, miRNA, piRNA, mRNA, antisense polynucleotides, aptamers, etc.), lipids, and polysaccharides. Generally, agents can be obtained using any suitable method known in the art. In some embodiments, an agent can be a "therapeutic agent" for use in treating a disease or disorder (e.g., cancer) in a subject (e.g., a human).

[0051] The term "agonist" refers to an agent that binds to a target(s) (e.g., a receptor) and activates or increases the biological activity of the target(s). For example, an "agonist" antibody is an antibody that activates or increases the biological activity of the antigen(s) to which it binds.

[0052] The term "altered amount" or "altered level" encompasses an increase or decrease in the copy number (e.g., germline and / or somatic) of a biomarker nucleic acid, or an increase or decrease in the expression level in a sample of interest, compared to the copy number or expression level in a control sample. The term "altered amount" of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g., a cancer sample, compared to the corresponding protein level in a normal and / or control sample. Furthermore, an altered amount of a biomarker protein can be determined by detecting post-translational modifications, such as the methylation status of the marker, which can affect the expression or activity of the biomarker protein. In some embodiments, "altered amount" refers to the presence or absence of a biomarker, as the reference standard can be the absence or presence of the biomarker, respectively. The absence or presence of a biomarker can be determined according to the sensitivity threshold of a given assay used to measure the biomarker.

[0053] An amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker if the amount of the biomarker is higher or lower, respectively, than the normal level by more than the standard error of the assay utilized to assess the amount, preferably at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. Alternatively, the amount of a biomarker in a subject can be considered "significantly" higher or lower than normal if the amount is at least about 2-fold, preferably at least about 3-fold, 4-fold, or 5-fold higher or lower, respectively, than the normal amount of the biomarker. Such "significance" can also apply to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell proliferation, etc.

[0054] The term "altered expression level" of a biomarker refers to the expression level or copy number of the biomarker in a test sample, e.g., a sample from a patient afflicted with cancer, that is greater than or less than the standard error of the assay utilized to assess expression or copy number, and is preferably at least 2-fold, more preferably 3-fold, 4-fold, 5-fold, or 10-fold or more, the expression level or copy number of the biomarker in a control sample (e.g., a sample from a healthy subject without the relevant disease), preferably the average expression level or copy number of the biomarker in several control samples. In some embodiments, the level of a biomarker refers to the level of the biomarker itself, the level of a modified biomarker (e.g., a phosphorylated biomarker), or the level of a biomarker compared to another measured variable, such as a control (e.g., a phosphorylated biomarker compared to a non-phosphorylated biomarker). The term "expression" encompasses the process by which a nucleic acid (e.g., DNA) is transcribed to produce RNA and can further refer to the process by which the RNA transcript is processed and translated into a polypeptide. The sum of the expression of a nucleic acid and its polypeptide counterpart, if present, contributes to the amount of a biomarker, such as one or more targets listed in Table 1.

[0055] The term "altered activity" of a biomarker refers to the activity of the biomarker, which is increased or decreased in a disease state, e.g., a cancer sample, or a treatment state, compared to the activity of the biomarker in a normal control sample. Altered activity of a biomarker can result, for example, from altered expression of the biomarker, altered protein levels of the biomarker, altered structure of the biomarker, or, for example, from altered interaction with other proteins involved in the same or different pathways as the biomarker, or altered interaction with transcriptional activators or inhibitors.

[0056] The term "altered structure" of a biomarker refers to the presence of a mutation or allelic variant in a biomarker nucleic acid or protein, e.g., a mutation affects the expression or activity of the biomarker nucleic acid or protein compared to a normal or wild-type gene or protein. For example, mutations include, but are not limited to, substitutions, deletions, or additional mutations. Mutations can be present in coding or non-coding regions of a biomarker nucleic acid.

[0057] The term "altered subcellular localization" of a biomarker refers to the mislocalization of a biomarker within a cell, as compared to its normal localization within a cell, e.g., a healthy and / or wild-type cell. An indication of normal localization of a marker can be determined through analysis of art-known subcellular localization motifs encompassed by the biomarker polypeptide.

[0058] The terms "antagonist" or "blocking" refer to an agent that binds to a target(s) (e.g., a receptor) and inhibits or reduces the biological activity of the target(s). For example, an "antagonist" antibody is one that significantly inhibits or reduces the biological activity of the antigen(s) to which it binds.

[0059] Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments, fusion proteins, and derivatives of all of the foregoing, which fragments and derivatives retain at least an antigen-binding site. Antibody derivatives can include proteins or chemical moieties conjugated to an antibody.

[0060] The term "biomarker" refers to a gene or gene product that is a target for modulating one or more phenotypes of interest, such as a phenotype of interest in myeloid cells. In this context, the term "biomarker" is synonymous with "target." However, in some embodiments, the term further encompasses measurable entities of a target that have been determined to be indicative of a desired output, such as one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for modulating an inflammatory phenotype, a cancerous condition, etc.). In still other embodiments, the term further encompasses compositions that modulate a gene or gene product, including anti-gene product antibodies and antigen-binding fragments thereof. Thus, biomarkers can include, but are not limited to, nucleic acids (e.g., genomic and / or transcribed nucleic acids), proteins, and antibodies (and antigen-binding fragments thereof), particularly those listed in Table 1.

[0061] The terms "cancer" or "tumor" or "hyperproliferative" refer to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, invasive or metastatic potential, rapid growth, and certain characteristic morphological features. In some embodiments, such cells exhibit such characteristics partially or completely due to the expression and activity of immune checkpoint proteins such as PD-1, PD-L1, PD-L2, and / or CTLA-4.

[0062] Cancer cells are often in the form of tumors, but such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers. Cancers include, but are not limited to, various cancers, carcinomas including those of the bladder (including advanced and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small cell and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testis, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic cancer), esophagus, stomach, gallbladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of the lymphoid system, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkitt's lymphoma; acute and chronic myeloid leukemia Hematopoietic tumors of the myeloid lineage, including leukemia, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer cancer), bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, head and neck cancer, gastric cancer, germ cell tumors, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, childhood sarcoma, nasal and sinonasal natural killer, neoplasms, plasma cell neoplasms; myelodysplastic syndromes; neuroblastoma; testicular germ cell tumors, intraocular melanoma, myelodysplastic syndromes; myelodysplastic / myeloproliferative disorders, synovial sarcoma, chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), multiple myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, mastocytosis and conditions related to mastocytosis, and any metastases thereof.Additionally, disorders include mastocytoses such as urticaria pigmentosa, diffuse cutaneous mastocytosis, and solitary mastocytoma in humans, as well as canine mastocytoma and some rare subtypes such as bullous erythroderma and distant telangiectatic mastocytoma, mastocytoma with associated hematological disorders, e.g., myeloproliferative or myelodysplastic syndromes, or acute leukemia, myeloproliferative disorders associated with mastocytosis, mast cell leukemia, in addition to other cancers. Other cancers also include carcinomas of the bladder, urothelial carcinoma, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, testis, especially testicular seminoma, and skin, including squamous cell carcinoma; gastrointestinal stromal tumors ("GIST"); hematopoietic tumors of the lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkitt's lymphoma; hematopoietic tumors of the myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia; fibrosarcoma and The range of disorders includes, but is not limited to, tumors of mesenchymal origin, including rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetracarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, teratoma, chemotherapy-resistant nonseminomatous germ cell tumor, Kaposi's sarcoma, and any metastases thereof.Other non-limiting examples of cancer types amenable to methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chondroma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bone cancer, brain tumor, lung cancer (including lung adenocarcinoma), small cell lung cancer, and ovarian cancer. Alveolar lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In some embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in various other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated. In some embodiments, the cancer is selected from the group consisting of (advanced) non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, (advanced) urothelial bladder cancer, (advanced) renal cell carcinoma (RCC), microsatellite instability high carcinoma, classical Hodgkin lymphoma, (advanced) gastric cancer, (advanced) cervical cancer, primary mediastinal B-cell lymphoma, (advanced) hepatocellular carcinoma, and (advanced) Merkel cell carcinoma.

[0063] The term "classifying" includes "associating" or "categorizing" a sample with a disease state. In certain cases, "classification" is based on statistical evidence, empirical evidence, or both. In certain embodiments, classification methods and systems use a so-called training set of samples with known disease states. Once established, the training data set serves as a basis, model, or template against which features of unknown samples are compared in order to classify the unknown disease state of the sample. In certain cases, classifying a sample is analogous to diagnosing the disease state of the sample. In certain other cases, classifying a sample is analogous to distinguishing one disease state of a sample from another disease state.

[0064] The term "coding region" refers to the region of a nucleotide sequence that contains codons that are translated into amino acid residues, while the term "non-coding region" refers to the region of a nucleotide sequence that is not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0065] The term "compete" with respect to an antibody or antigen-binding fragment thereof refers to a situation in which a first antibody or antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding portion thereof, such that binding of the first antibody to 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. Alternatively, binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this need not be the case. That is, a first antibody may inhibit binding of a second antibody to its epitope without inhibiting binding of the second antibody to its respective epitope. However, antibodies are said to "cross-compete" with each other for binding of their respective epitope(s) if each antibody detectably inhibits binding of the other antibody to its cognate epitope or ligand to the same, greater, or lesser extent. Both competing and cross-competing antibodies, and antigen-binding fragments thereof, are encompassed by the present invention (e.g., antibodies and antigen-binding fragments described herein that compete or cross-compete with other antibodies and antigen-binding fragments described herein and / or known in the art). 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 of skill in the art will understand, based on the disclosure provided herein and the skill of one of ordinary skill in the art, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0066] The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can base pair with a residue in a second nucleic acid strand that is antiparallel to the first strand if the residue is guanine. A first region of nucleic acid is complementary to a second region of the same or different nucleic acid if at least one nucleotide residue in the first region can base pair with a residue in the second region when the two regions are arranged in an antiparallel fashion. Preferably, the first region comprises a first portion, and the second region comprises a second portion, such that when the first and second portions are arranged in an antiparallel fashion, at least about 50%, preferably at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or more of the nucleotide residues of the first portion can base pair with the nucleotide residues of the second portion. More preferably, all nucleotide residues of the first portion can base pair with the nucleotide residues of the second portion. In some embodiments, complementary polynucleotides can be "sufficiently complementary" or "sufficiently complementary," i.e., have sufficient complementarity to maintain a duplex and / or have the desired activity. For example, in the case of an RNAi agent, such complementarity is sufficient between the agent and the target mRNA to partially or completely prevent translation of the mRNA. For example, an siRNA having a "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the siRNA has a sequence sufficient to induce destruction of the target mRNA by the RNAi machinery or process.

[0067] The term "substantially complementary" refers to the complementarity in the base-paired double-stranded region between two nucleic acids, not in any single-stranded region, such as a terminal overhang or a gap region between two double-stranded regions. Complementarity does not need to be perfect, and any number of base pair mismatches are possible. In some embodiments, when two sequences are referred to herein as "substantially complementary," it means that the sequences are sufficiently complementary to each other to hybridize under selected reaction conditions. Thus, a substantially complementary sequence can refer to a sequence having at least 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60 percent or more base pair complementarity in the double-stranded region, or any number in between.

[0068] As used herein, the terms "cotherapy" and "combination therapy" refer to the administration of two or more therapeutic agents, e.g., a combination of a modulator of more than one target listed in Table 1, at least one modulator of at least one target listed in Table 1, and an additional therapeutic agent, such as immune checkpoint therapy, a combination of more than one modulator of one or more targets listed in Table 1, and combinations thereof. The different agents comprising the combination therapy can be administered simultaneously with, before, or after the administration of one or more other agents. The combination therapy aims to provide a beneficial (additive or synergistic) effect from the combination of these therapeutic agents. The administration of the combination of these therapeutic agents can be carried out over a defined period of time (usually minutes, hours, days, or weeks, depending on the combination selected). In combination therapy, the combined therapeutic agents can be applied sequentially or by substantially simultaneous application.

[0069] The term "control" refers to any reference standard suitable for providing a comparison with the expression product in a test sample. In one embodiment, controlling involves obtaining a "control sample" in which expression product levels are detected and compared to expression product levels from the test sample. Such a control sample can include any suitable sample, including, but not limited to, a sample from a subject, such as a subject with bone marrow cells and / or a control cancer patient with a known outcome (which can be an archived sample or a measurement of a previous sample), normal tissue or cells isolated from a subject, such as a normal patient or a cancer patient, cultured primary cells / tissues isolated from a subject, such as a normal subject or a cancer patient, adjacent normal cells / tissues obtained from the same organ or body location of a cancer patient, a tissue or cell sample isolated from a normal subject, or primary cells / tissues obtained from an archive. In another preferred embodiment, the control can include a reference standard expression product level from any suitable source, including, but not limited to, a housekeeping gene, a range of expression product levels from normal tissue (or other previously analyzed control sample), a range of expression product levels previously determined within a test sample from a group of patients, or in a set of patients with a particular outcome (e.g., 1-, 2-, 3-, 4-year survival, etc.), or receiving a particular treatment (e.g., standard of care for cancer therapy). It will be understood by those skilled in the art that such control sample and reference standard expression product levels can be used in combination as controls in the methods encompassed by the present invention. In one embodiment, the control can include a normal or non-cancerous cell / tissue sample. In another preferred embodiment, the control can include expression levels for a set of patients, such as a set of cancer patients, or a set of cancer patients receiving a particular treatment, or a set of patients with one outcome versus another. In the former case, the particular expression product level for each patient can be assigned a percentile level of expression or expressed as being higher or lower than the mean or average of the reference standard expression level. In another preferred embodiment, the controls may include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancer.In another embodiment, a control can also include a measured value, e.g., the average level of expression of a particular gene in a population compared to the expression level of a housekeeping gene in the same population. Such a population can include normal subjects, cancer patients who have not received any treatment (i.e., untreated), cancer patients receiving standard treatment, or patients with benign cancer. In another preferred embodiment, a control includes a ratio conversion of expression product levels, including, but not limited to, determining the ratio of expression product levels of two genes in a test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining the expression product levels of two or more genes in a test sample and determining the difference in expression product levels in any suitable control; or determining the expression product levels of two or more genes in a test sample, normalizing their expression to the expression of housekeeping genes in the test sample, and comparing it to any suitable control. In a particularly preferred embodiment, a control includes a control sample of the same lineage and / or type as the test sample. In another embodiment, a control can include expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment, a control expression product level is established, and higher or lower levels of expression product, e.g., compared to a particular percentile, are used as the basis for predicting outcome. In another preferred embodiment, the control expression product level is established using expression product levels from cancer control patients with known outcomes, and the expression product level from the test sample is compared to the control expression product level as the basis for predicting outcome. The methods encompassed by the present invention are not limited to the use of a particular cutoff point when comparing the level of expression product in the test sample with the control.

[0070] The "copy number" of a biomarker nucleic acid refers to the number of DNA sequences in a cell (e.g., germline and / or somatic cell) that encode a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. However, copy number can be increased by gene amplification or duplication, or decreased by deletion. For example, a germline copy number alteration includes an alteration at one or more genomic loci that are not accounted for by the copy number of the normal complement of germline copies in a control (e.g., the normal copy number of germline DNA for the same species as that from which the particular germline DNA and corresponding copy number were determined). A somatic copy number alteration includes an alteration at one or more genomic loci that are not accounted for by the copy number in the germline DNA of a control (e.g., the copy number of somatic DNA and corresponding germline DNA for the same subject from which the copy number was determined).

[0071] The term "costimulation" used in reference to activated immune cells includes the ability of a costimulatory polypeptide to provide a second, non-activating receptor-mediated signal ("costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion in, for example, a T cell that has received a T cell receptor-mediated signal. Immune cells that have received a cell receptor-mediated signal, for example, via an activating receptor, are referred to herein as "activated immune cells."

[0072] The term "costimulatory receptor" includes receptors that transmit costimulatory signals to immune cells, such as CD28. As used herein, the term "inhibitory receptor" includes receptors that transmit negative signals to immune cells (e.g., PD-1, CTLA-4, etc.). Inhibitory signals transmitted by inhibitory receptors can occur even when costimulatory receptors (e.g., CD28) are not present on immune cells and are therefore not simply a function of competition between the inhibitory receptor and the costimulatory receptor for binding of costimulatory polypeptides (Fallarino et al. (1998) J. Exp. Med. 188:205). Transmission of inhibitory signals to immune cells can result in unresponsiveness or anergy or programmed cell death in immune cells. Preferably, transmission of inhibitory signals operates via a mechanism that does not involve apoptosis. As used herein, the term "apoptosis" includes programmed cell death, which can be characterized using techniques known in the art. Apoptotic cell death can be characterized by, for example, cell shrinkage, membrane blebbing, and chromatin condensation leading to cell fragmentation. Cells undergoing apoptosis also show characteristic patterns of internucleosomal DNA cleavage. Depending on the form of the polypeptide that binds to the receptor, the signal can be transmitted (for example, by a multivalent form of an inhibitory receptor ligand), or the signal can be inhibited (for example, by a soluble monovalent form of an inhibitory receptor ligand), for example, by competing with the activated form of the ligand for binding to one or more natural binding partners. However, there are cases where soluble polypeptides can be stimulatory. The effect of a modulator can be easily demonstrated using routine screening assays such as those described herein.

[0073] The term "cytokine" refers to a substance secreted by certain cells of the immune system that has a biological effect on other cells. Cytokines can be a variety of substances, such as interferons, interleukins, and growth factors.

[0074] The term "determining a suitable therapeutic regimen for a subject" is understood to mean determining a subject's therapeutic regimen (i.e., a single therapy or a combination of different therapies used to prevent and / or treat cancer in a subject) that is initiated, modified, and / or terminated based on, or essentially based on, or at least in part based on, the results of a biomarker-mediated analysis encompassed by the present invention. One example is determining whether to provide targeted therapy for cancer, to provide therapy using an agent encompassed by the present invention that modulates one or more biomarkers. Another example is initiating adjuvant therapy after surgery aimed at reducing the risk of recurrence. Yet another example is modifying the dosage of a particular chemotherapy. In addition to the results of an analysis according to the present invention, the decision can be based on the personal characteristics of the subject being treated. In most cases, the actual decision on a suitable therapeutic regimen for a subject will be performed by the attending physician or doctor.

[0075] The terms "endotoxin-free" or "substantially endotoxin-free" refer to compositions, solvents, and / or containers containing at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects in a subject), and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, usually gram-negative bacteria, but can also be found in gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), found in the outer membrane of various gram-negative bacteria, and represent a central pathogenic feature of these bacteria's ability to cause disease. Small amounts of endotoxin in humans can result in fever, a drop in blood pressure, activation of inflammation and coagulation, and other adverse physiological effects.

[0076] Therefore, in pharmaceutical manufacturing, it is often desirable to remove most or all traces of endotoxin from formulations and / or drug containers, as even small amounts can have adverse effects in humans. Because temperatures above 300°C are typically required to degrade most endotoxins, depyrogenation ovens can be used for this purpose. For example, based on primary packaging materials such as syringes or vials, a glass temperature of 250°C in combination with a 30-minute hold time is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxins are contemplated, including, for example, chromatography and filtration techniques, as described herein and known in the art. Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus amoeba lysate assay, which utilizes blood from horseshoe crabs, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / ml, or any range therebetween, including 0.05-10 EU / ml. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU / ml.

[0077] The term "epitope" refers to a determinant or site on an antigen to which an antigen-binding protein (e.g., an immunoglobulin, antibody, or antigen-binding fragment) binds. Epitopes of protein antigens can be either linear or conformational. A linear epitope refers to an epitope formed from a continuous linear sequence of linked amino acids. Linear epitopes of protein antigens are typically retained upon exposure to chemical denaturants (e.g., acids, bases, solvents, cross-linking reagents, chaotropic agents, disulfide bond-reducing agents) or physical denaturants (e.g., heat, radiation, or mechanical shear or stress). In contrast, a conformational epitope refers to an epitope formed from non-contiguous amino acids juxtaposed by tertiary folding of a polypeptide. Conformational epitopes are typically lost upon treatment with denaturants. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids in a unique spatial conformation. In some embodiments, an epitope includes fewer than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids in a unique spatial conformation. Generally, an antibody, or antigen-binding fragment thereof, specific for a particular target molecule preferentially recognizes and binds to a particular epitope on the target molecule within a complex mixture of proteins and / or macromolecules. In some embodiments, an epitope does not include all amino acids of the extracellular domain of a biomarker protein.

[0078] The term "expression signature" or "signature" refers to a group of one or more expressed biomarkers that indicate a state of interest. For example, the genes, proteins, etc. that make up this signature may be expressed in a particular cell lineage, stage of differentiation, or during a particular biological response. Biomarkers can reflect biological aspects of the tumor in which they are expressed, such as the inflammatory state of the cells, the cell of origin of the cancer, the nature of non-malignant cells in a biopsy, and the oncogenic mechanisms responsible for the cancer. Expression data and gene expression levels can be stored in a computer-readable medium, such as a computer-readable medium used in conjunction with a microarray or chip reading device. Such expression data can be manipulated to generate an expression signature.

[0079] The terms "immobilized" or "attached" refer to covalent or non-covalent association with a substrate such that the substrate can be rinsed with a fluid (e.g., standard citrate saline, pH 7.4) without a substantial portion of the molecules dissociating from the substrate.

[0080] The term "gene" encompasses a nucleotide (e.g., DNA) sequence that encodes a functional molecule (e.g., RNA, protein, etc.). A gene generally comprises two complementary strands of nucleotides (i.e., dsDNA), a coding strand and a non-coding strand. When referring to DNA transcription, the coding strand is the DNA strand whose base sequence corresponds to the base sequence of the generated RNA transcript (except that thymine is replaced by uracil). The coding strand contains codons, while the non-coding strand contains anticodons. During transcription, RNA Pol II binds to the non-coding strand, reads the anticodon, and transcribes the sequence to synthesize an RNA transcript containing complementary bases. In some embodiments, the gene sequence (i.e., DNA sequence) listed is the sequence of the coding strand.

[0081] A "function-conservative variant" is one in which a given amino acid residue in a protein or enzyme has been altered without altering the overall conformation and function of the polypeptide, including, but not limited to, replacing the amino acid with one that has similar properties (e.g., polarity, hydrogen-bonding potential, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those designated as conserved may differ between proteins, and as a result, the percent similarity of protein or amino acid sequence between any two proteins with similar functions may vary, e.g., 70%-99% when similarity is determined according to an alignment scheme such as the clustering method based on the MEGALIGN algorithm. In some embodiments, a "function-conservative variant" also includes a polypeptide having at least 80%, 81%, 82%, 83%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid identity as determined by BLAST or FASTA algorithms, and has the same or substantially similar properties or functions as the native or parent protein to which it is compared.

[0082] The term "gene product" (also referred to herein as "gene expression product" or "expression product") encompasses products resulting from expression of a gene, such as a nucleic acid (e.g., mRNA) transcribed from the gene, and a polypeptide or protein resulting from translation of such an mRNA. It will be understood that certain gene products may undergo processing or modification, for example, within the cell. For example, an mRNA transcript may be spliced, polyadenylated, etc. before translation, and / or a polypeptide may undergo co- or post-translational processing, such as removal of a secretory signal sequence, removal of an organelle targeting sequence, or modifications such as phosphorylation, glycosylation, methylation, fatty acylation, etc. The term "gene product" encompasses such processed or modified forms. Genomic mRNA and polypeptide sequences for various species, including humans, are readily known in the art and are available in publicly accessible databases, such as those available at the National Center for Biotechnology Information (ncbi.nih.gov) or the Universal Protein Resource (uniprot.org). Other databases include, for example, GenBank, RefSeq, Gene, UniProtKB / SwissProt, UniProtKB / Trembl, etc. Generally, the sequence of the NCBI reference sequence database can be used as the gene product sequence for the gene of interest. It will be understood that multiple alleles of a gene may exist among individuals of the same species. Multiple isoforms of a particular protein may exist as a result of alternative RNA splicing or editing. Generally, when aspects of the present disclosure relate to a gene or gene product, unless otherwise specified, embodiments relating to allelic variants or isoforms are encompassed, if applicable. Certain embodiments can be directed to specific sequence(s), for example, specific allele(s) or isoform(s).

[0083] The term "producing" encompasses any manner in which a desired result is achieved, such as by direct or indirect action. For example, cells having a modulated phenotype described herein can be produced by a direct action, such as by contact with at least one agent that modulates one or more biomarkers described herein, and / or by an indirect action, such as by propagating cells having the desired physical, genetic, and / or phenotypic attributes.

[0084] The term "glycosylation pattern" refers to the pattern of carbohydrate units that are covalently attached to a protein, more particularly to an immunoglobulin protein. The glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to the glycosylation pattern that occurs naturally in antibodies produced by the species of non-human transgenic animal, and one skilled in the art will recognize that the glycosylation pattern of the heterologous antibody is more similar to the glycosylation pattern in the species of non-human transgenic animal than to the species from which the transgenic CH gene was derived.

[0085] The terms "high," "low," "intermediate," and "negative," in reference to cellular biomarker expression, refer to the amount of biomarker expressed relative to cellular expression of the biomarker by one or more reference cells. Biomarker expression can be determined according to any method described herein, including, but not limited to, analysis of cellular levels, activity, structure, etc. of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or polypeptides. In one embodiment, the terms refer to a defined percentage of a population of cells that express the biomarker at the highest, intermediate, or lowest levels, respectively. Such percentages can be defined as the top 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15% or more of a population of cells highly or weakly expressing a biomarker, or any range therebetween. The term "low" excludes cells that do not detectably express a biomarker because their biomarker expression is "negative." The term "intermediate" includes cells that express a biomarker, but at a lower level than a population that expresses it at a "high" level. In another embodiment, the term may, or alternatively, refer to a cell population of biomarker expression identified by a qualitative or statistical plot area. For example, cell populations sorted using flow cytometry can be determined based on biomarker expression levels by identifying distinct plots based on detectable local analysis, such as based on mean fluorescence intensity, etc., according to methods well known in the art. Such plot regions can be refined according to number, shape, overlap, etc., based on methods well known in the art for the biomarkers of interest. In yet another embodiment, terminology can also be determined according to the presence or absence of expression of additional biomarkers.

[0086] The term "substantially identical" refers to a nucleic acid or amino acid sequence that shares at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second nucleic acid or amino acid sequence when optimally aligned, e.g., using the methods described below. "Substantial identity" can be used to refer to various types and lengths of sequences, including full-length sequences, functional domains, coding and / or regulatory sequences, exons, introns, promoters, and genomic sequences. The percent sequence identity between two polypeptide or nucleic acid sequences can be determined using, for example, the BLAST program (Basic Local Alignment Search Tool; Altschul et al., J. Am. Chem. Soc. 1999, 1999; 1999; 1999; 2000). Alignment can be determined in a variety of ways within the skill of the art using publicly available computer software, such as BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Furthermore, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the length of the sequences being compared. For purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is understood to be equivalent to a uracil nucleotide. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine.

[0087] The term "immune cell" refers to a cell capable of directly or indirectly participating in an immune response. Immune cells include, but are not limited to, T cells, B cells, antigen-presenting cells, dendritic cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, eosinophils, basophils, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, peripheral blood mononuclear cells, cytotoxic T cells, tumor-infiltrating lymphocytes (TILs), and the like. "Antigen-presenting cells" (APCs) are cells capable of activating T cells, including, but not limited to, monocytes / macrophages, B cells, and dendritic cells (DCs). The terms "dendritic cell" or "DC" refer to any member of a diverse population of morphologically similar cell types found in lymphoid and non-lymphoid tissues. These cells are characterized by their distinctive morphology and high levels of surface MHC class II expression. DCs can be isolated from many tissue sources. DCs have a high ability to sensitize MHC-restricted T cells and are highly effective at presenting antigens to T cells in situ. Antigens can be self-antigens expressed during T cell development and tolerance, as well as foreign antigens present during normal immune processes. The term "neutrophil" generally refers to a white blood cell that constitutes part of the innate immune system. Neutrophils typically have a segmented nucleus containing approximately two to five lobes. Neutrophils frequently migrate to the site of injury within minutes of trauma. They function by releasing cytotoxic compounds, including oxidants, proteases, and cytokines, at the site of injury or infection. The term "activated DC" refers to a DC that has been pulsed with antigen and is capable of activating immune cells. The term "NK cell" has its common meaning in the art and refers to a natural killer (NK) cell. Those skilled in the art can easily identify NK cells, for example, by determining the expression of certain phenotypic markers (e.g., CD56), and determine their function based on, for example, their ability to express different cytokines or to induce cytotoxicity. The term "B cells" refers to immune cells derived from the bone marrow and / or spleen. B cells can develop into plasma cells that produce antibodies.The term "T cells" refers to thymus-derived immune cells involved in various cellular immune responses, including CD8+ T cells and CD4+ T cells. Conventional T cells, also known as Tconv or Teff, have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) and amplify immune responses thanks to their expression of one or more T cell receptors. Tconv or Teff are generally defined as any T cell population that is not a Treg, and include, for example, naive T cells, activated T cells, memory T cells, resting Tconv, or Tconv differentiated into Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-regulatory T cells (Treg). In some embodiments, Teff is a CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T cells (lymphocytes). As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tconv" is CD4. + Naive T cells differentiate in the bone marrow and successfully undergo positive and negative central selection processes in the thymus, but have not yet been activated by exposure to antigen. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, and CD69, and the absence of memory markers such as CD45RO. Naive T cells are therefore considered quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO 2010 / 101870). The presence and activity of such cells are undesirable in the context of suppressing immune responses. Unlike Tregs, T cells are not anergic and can proliferate in response to antigen-driven T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637). In tumors, exhausted cells can exhibit characteristics of anergy.

[0088] The term "immune disorder" includes immune diseases, conditions, conditions, and predispositions, including, but not limited to, cancer, chronic inflammatory diseases and disorders (including, for example, Crohn's disease, inflammatory bowel disease, reactive arthritis, and Lyme disease), insulin-dependent diabetes mellitus, organ-specific autoimmunity (including, for example, multiple sclerosis, Hashimoto's thyroiditis, autoimmune uveitis, and Graves' disease), contact dermatitis, psoriasis, transplant rejection, graft-versus-host disease, sarcoidosis, atopic diseases (including, for example, asthma and allergies, including, but not limited to, gastrointestinal allergies such as allergic rhinitis and food allergies), eosinophilia, conjunctivitis, glomerulonephritis, systemic lupus erythematosus, scleroderma, helminthiasis (including, for example, leishmaniasis), and certain viral infections (including, for example, HIV and bacterial infections such as tuberculosis and leprosy), and certain pathogen susceptibilities such as malaria.

[0089] The term "immune response" refers to a defensive response developed by the body against "foreign" targets, such as bacteria, viruses, and pathogens, and against targets that do not necessarily originate outside the body, including, but not limited to, defensive responses directed against substances naturally occurring in the body (e.g., autoimmunity directed against self-antigens) or against transformed (e.g., cancer) cells. In particular, an immune response is the activation and / or action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced either by these cells or the liver, including antibodies (humoral response), cytokines, and complement, that result in the selective targeting, binding, damage, destruction, and / or elimination of pathogens invading the vertebrate body, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. The anti-cancer immune response refers to the immune surveillance mechanism by which the body recognizes abnormal tumor cells and initiates both the innate and adaptive immune system to eliminate the dangerous cancer cells.

[0090] The term "immunomodulator" refers to a substance, agent, signaling pathway, or component thereof that modulates the immune response. The terms "regulate," "modify," or "regulate," with respect to an immune response, refer to any alteration in the cells of the immune system or the activity of such cells. Such regulation includes stimulation or suppression of the immune system (or distinct parts thereof), which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which can enhance function in the cancer microenvironment.

[0091] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to generate an immune response against a tumor or cancer in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0092] The terms "inhibition" or "downregulation" include, for example, the reduction, limitation, or blocking of a particular action, function, or interaction. In some embodiments, cancer is "inhibited" if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is further "inhibited" if the recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited when it is reduced compared to a reference state, such as a control, such as a wild-type state. Such inhibition or deficiency can be induced, such as by application of an agent at a particular time and / or location, or can be constitutive, such as by inherited mutation. Such inhibition or deficiency can also be partial or complete (e.g., essentially no measurable activity compared to a reference state, such as a control, such as a wild-type state). In some embodiments, essentially complete inhibition or deficiency is referred to as "blocked." In one embodiment, the term refers to a decrease in the level of a given output or parameter by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or less than the amount in a corresponding control. A decreased level of a given output or parameter may, but need not, mean the absolute absence of the output or parameter. The present invention does not require, and is not limited to, methods that completely eliminate the output or parameter. A given output or parameter can be determined using methods well known in the art, including, but not limited to, immunohistochemistry, molecular biology, cell biology, clinical, and biochemical assays, as discussed herein and in the Examples. The terms "promotion" and "upregulation" have opposite meanings.

[0093] The term "inhibitory signal" refers to a signal transmitted through an inhibitory receptor of a polypeptide (e.g., CTLA4, PD-1, etc.) on an immune cell. Such a signal antagonizes a signal through an activating receptor (e.g., through a TCR, CD3, BCR, TMIGD2, or Fc polypeptide) and can result in, for example, inhibition of second messenger production, inhibition of proliferation, inhibition of effector function in the immune cell, e.g., decreased phagocytosis, decreased antibody production, decreased cytotoxicity, failure of the immune cell to produce mediators (e.g., cytokines (e.g., IL-2) and / or mediators of the allergic response), or the development of anergy.

[0094] The innate immune system is a nonspecific immune system that includes cells (e.g., natural killer cells, mast cells, eosinophils, basophils, and phagocytes, including macrophages, neutrophils, and dendritic cells) and mechanisms that protect the host from infection by other organisms. The innate immune response can initiate cytokine production and activation of the complement cascade and adaptive immune response. The adaptive immune system is a specialized immune system that is required for and participates in the activation of highly specialized systemic cells and processes, such as antigen presentation by antigen-presenting cells, activation of antigen-specific T cells, and cytotoxic effects.

[0095] The term "interaction," when referring to the interaction between two molecules, refers to physical contact (e.g., binding) between the molecules. Generally, such an interaction results in the activity (producing a biological effect) of one or both of the molecules. The activity can be the direct activity of one or both of the molecules (e.g., signal transduction). Alternatively, one or both molecules in the interaction can be prevented from binding to their ligand, and thus remain inactive with respect to ligand binding activity (e.g., bind to their ligand and induce or inhibit costimulation). Inhibiting such an interaction results in the disruption of the activity of one or more molecules involved in the interaction. Strengthening such an interaction prolongs or increases the probability of such physical contact, thereby prolonging or increasing the probability of such activity.

[0096] "Isolated protein" refers to a protein that is isolated from a cell or produced by recombinant DNA techniques, and that is substantially free of other proteins, cellular material, separation medium, and culture medium, or, if chemically synthesized, chemical precursors or other chemicals. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein is derived, or, if chemically synthesized, is substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of biomarker polypeptides or fragments thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of biomarker proteins or fragments thereof having less than about 30% (by dry weight) non-biomarker protein (also referred to herein as "contaminating protein"), more preferably less than about 20% non-biomarker protein, even more preferably less than about 10% non-biomarker protein, and most preferably less than about 5% non-biomarker protein. When an antibody, polypeptide, peptide, or fusion protein or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0097] The term "isotype" refers to the antibody class (e.g., IgM, IgG1, IgG2C, etc.) that is encoded by heavy chain constant region genes.

[0098] "K DThe term "antibody-antigen interaction" is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. The binding affinity of the disclosed antibodies of the invention can be measured or determined by standard antibody-antigen assays, e.g., competition assays, saturation assays, or standard immunoassays such as ELISA or RIA. In some embodiments, the K of an antibody or antigen-binding fragment thereof described herein for a biomarker of interest, such as one or more biomarkers listed in Table 1, is D can be about 0.002 to about 200 nM. In some embodiments, the binding affinity is any of about 250 nM, 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 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, or about 2 pM or less. In some embodiments, the binding affinity is less than about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 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 or less, or any range therebetween, such as from about 5 nM to about 35 nM.

[0099] "kd" or "k オフ The term "off-rate constant" refers to the off-rate constant for dissociation of an antibody from an antibody / antigen complex. The Kd value is a numerical value of the fraction of the complex that decays or dissociates per second and is expressed in units of seconds. -1 It is expressed as:

[0100] "ka" or "k オンThe term "on rate constant" refers to the on rate constant for the association of an antibody with an antigen. The value of Ka is the number of antibody / antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and is expressed in units of M -1 seconds -1 It is expressed as:

[0101] The term "microenvironment" generally refers to a localized region within a tissue region of interest, e.g., the "tumor microenvironment." The term "tumor microenvironment" or "TME" refers to the surrounding microenvironment that constantly interacts with tumor cells, helping to enable crosstalk between tumor cells and their environment. The tumor microenvironment may include the tumor's cellular environment, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix. The tumor environment may include tumor cells or malignant cells that are supported and influenced by the tumor microenvironment to ensure their growth and survival. The tumor microenvironment also includes tumor-infiltrating immune cells, such as lymphocytes and myeloid cells, which can stimulate or inhibit anti-tumor immune responses, as well as stromal cells, such as tumor-associated fibroblasts and endothelial cells, which contribute to the structural integrity of the tumor. Stromal cells include cells that compose tumor-associated blood vessels, such as endothelial cells and pericytes, which contribute to their structural integrity (fibroblasts), as well as tumor-associated macrophages (TAMs) and infiltrating immune cells, including monocytes, neutrophils (PMNs), dendritic cells (DCs), T and B cells, mast cells, and natural killer (NK) cells. While stromal cells constitute the majority of tumor cellularity, the predominant cell type in solid tumors is the macrophage.

[0102] The term "modulate" and its grammatical equivalents refer to either an increase or a decrease (eg, silencing), in other words, an upregulation or a downregulation.

[0103] A "normal" expression level of a biomarker is the expression level of the biomarker in cells of a subject, e.g., a human patient, who is not afflicted with cancer.

[0104] "Overexpression" or "significantly high levels of expression" of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay used to assess expression, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times greater than the expression activity or level of the biomarker in a control sample (e.g., a sample from a healthy subject not having the biomarker-associated disease), preferably the average expression level of the biomarker in several control samples. A "significantly lower level of expression" of a biomarker refers to an expression level in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times lower than the expression level of the biomarker in a control sample (e.g., a sample from a healthy subject not having a biomarker-associated disease), preferably the average expression level of the biomarker in several control samples.

[0105] Such "significance" levels may also be applied to any other measured parameter described herein for expression, inhibition, cytotoxicity, cell proliferation, and the like.

[0106] The term "peripheral blood cell subtype" refers to cell types commonly found in peripheral blood, including, but not limited to, eosinophils, neutrophils, T cells, monocytes, macrophages, NK cells, granulocytes, and B cells.

[0107] The term "polypeptide fragment" or "fragment," when used in reference to a reference polypeptide, refers to a polypeptide that lacks amino acid residues compared to the reference polypeptide itself, but in which the remaining amino acid sequence is typically identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino terminus, internally, or at the carboxyl terminus of the reference polypeptide, or alternatively, both. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, at least 14 amino acids in length, at least 20, 30, 40, or 50 amino acids in length, at least 75 amino acids in length, or at least 100, 150, 200, 300, 500, or more amino acids in length. They may be, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1200, 1220 The ranges can be and / or include lengths of 0, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060, 1080, 1100, 1120, 1140, 1160, 1180, 1200, 1220, 1240, 1260, 1280, 1300, 1320, 1340 or more. Alternatively, they can be less than and / or exclude such ranges, as long as they are shorter than the length of the full-length polypeptide.

[0108] The term "predetermined" biomarker amount and / or activity measurement(s) can refer, by way of example only, to a biomarker amount and / or activity measurement used to evaluate a subject who may be selected for a particular treatment, assess response to a treatment, such as a modulator of one or more of the one or more biomarkers described herein, and / or evaluate a disease state. The predetermined biomarker amount and / or activity measurement(s) can be determined in a population of patients, regardless of whether they have cancer. The predetermined biomarker amount and / or activity measurement(s) can be a single number equally applicable to all patients, or the predetermined biomarker amount and / or activity measurement(s) can vary according to particular subpopulations of patients. A subject's age, weight, height, and other factors can affect an individual's predetermined biomarker amount and / or activity measurement(s). Furthermore, the predetermined biomarker amount and / or activity can be determined individually for each subject. In one embodiment, the amounts determined and / or compared in the methods described herein are based on absolute measurements. In another embodiment, the amounts determined and / or compared in the methods described herein are based on relative measurements such as ratios (e.g., cellular or serum biomarker ratios normalized to the expression of a housekeeping or other generally constant biomarker). The predetermined biomarker amount and / or activity measurement(s) can be any suitable standard. For example, the predetermined biomarker amount and / or activity measurement(s) can be obtained from the same or a different human being for whom the patient selection is being evaluated. In one embodiment, the predetermined biomarker amount and / or activity measurement(s) can be obtained from a previous evaluation of the same patient. In such a manner, the progress of the patient selection can be monitored over time. Furthermore, when the subject is a human, the control can be obtained from the evaluation of another human or multiple humans, e.g., a selected group of humans.In such a manner, the degree of selection of the human being whose selection is being evaluated can be compared with that of suitable other humans, e.g., other humans in a similar situation to the human of interest, e.g., those suffering from a similar or the same condition(s), and / or humans of the same ethnic group.

[0109] The term "predictive" includes the use of biomarker nucleic acid and / or protein status, e.g., tumor over- or underactivity, onset, expression, growth, remission, recurrence, or resistance, before, during, or after treatment, to determine a desirable likelihood. Such predictive uses of biomarkers include, for example, (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, single molecule sequencing, e.g., those described in the art at least in J. Biotechnol., 86:289-301, or qPCR), overexpression or underexpression of biomarker nucleic acids (e.g., by ISH, Northern blot, or qPCR), increased or decreased biomarker protein (e.g., by IHC), or increased or decreased expression of biomarker proteins (e.g., by IHC), e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, %, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more increase or decrease in activity; (2) its absolute or relatively modulated presence or absence in biological samples, such as samples including tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow from human subjects with cancer; or (3) its absolute or relatively modulated presence or absence in clinical subsets of patients with cancer (e.g., those that respond to or develop resistance to particular modulators of T cell-mediated cytotoxicity alone or in combination with immunotherapy).

[0110] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who is not a subject but who is at risk of or susceptible to developing the disease, disorder, or condition.

[0111] The term "probe" refers to any molecule capable of selectively binding to a specifically intended target molecule, e.g., a nucleotide transcript or protein encoded by or corresponding to a biomarker nucleic acid. Probes can be synthesized by one skilled in the art or derived from appropriate biological preparations. For the purpose of detecting the target molecule, probes can be specifically designed to be labeled as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0112] The term "prognosis" includes a prediction of the expected course and outcome of cancer, or the likelihood of recovery from disease. In some embodiments, the use of statistical algorithms provides a prognosis for cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from disease.

[0113] The term "ratio" refers to a relationship between two numbers (e.g., a score, a sum, etc.). However, ratios can be expressed in a particular order (e.g., a vs. b or a:b), and one of ordinary skill in the art will recognize that while trend observations and correlations based on ratios can be reversed, the underlying relationship between the numbers can be expressed in any order without losing the significance of the underlying relationship.

[0114] The term "rearrangement" refers to a V segment that is essentially a complete V H and V L"Germline configuration" refers to a configuration of a heavy or light chain immunoglobulin locus in which the V segment is immediately adjacent to a DJ or J segment in a conformation encoding the respective domains. Rearranged immunoglobulin loci can be identified by comparison with germline DNA, and rearranged loci will have at least one recombined heptamer / nonamer homology element. In contrast, the term "unrearranged" or "germline configuration" with respect to V segments refers to a configuration in which the V segment has not rearranged to be immediately adjacent to a D or J segment.

[0115] The term "receptor" refers to a naturally occurring molecule or complex of molecules that is typically present on the surface of cells of a target organ, tissue, or cell type.

[0116] The terms "cancer response," "response to immunotherapy," or "response to a modulator of T-cell-mediated cytotoxicity / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to a cancer mediator, such as a modulator of T-cell-mediated cytotoxicity, and a change in tumor mass and / or volume after the initiation of neoadjuvant or adjuvant therapy, such as immunotherapy. The term "neoadjuvant therapy" refers to treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormonal therapy. Hyperproliferative disorder response can be assessed, for example, for efficacy or in the neoadjuvant or adjuvant setting, and tumor size after systemic intervention can be compared to the initial size and dimensions measured by CT, PET, mammogram, ultrasound, or palpation. Response can also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response can be recorded quantitatively, such as by percent change in tumor volume, or qualitatively, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of hyperproliferative disorder response can be performed early after the initiation of neoadjuvant or adjuvant therapy, e.g., hours, days, weeks, or preferably months. A typical endpoint for response assessment is the end of neoadjuvant chemotherapy or surgical removal of residual tumor cells and / or tumor bed, which is usually 3 months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the proportion of patients in complete remission (CR), the number of patients in partial remission (PR) at least 6 months after the end of treatment, and the number of patients with stable disease (SD). A shorthand notation for this formula is CBR = CR + PR + SD over 6 months.In some embodiments, the CBR for a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating response to cancer treatment relate to "survival," including survival until death, also known as overall survival (death may be unrelated to either the cause or tumor), "recurrence-free survival" (the term "recurrence" includes both local and distant recurrence), metastasis-free survival, and disease-free survival (the term "disease" includes cancer and related diseases). The length of survival can be calculated by referencing a defined starting point (e.g., diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a specific cancer treatment regimen can be administered to a population of subjects, and the outcome can be correlated with biomarker measurements determined before any cancer treatment was administered. The outcome measurement can be a pathological response to treatment given in a neoadjuvant setting. Alternatively, outcome indicators such as overall survival and disease-free survival can be monitored over a period of time for subjects after cancer treatment for which biomarker measurements are readily available. In certain embodiments, the administered dose is a standard dose known in the art for cancer therapeutic agents. The duration for which subjects are monitored can vary. For example, subjects can be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds that correlate with cancer treatment outcomes can be determined using methods well known in the art, such as those described in the Examples section.

[0117] As indicated, the term can also refer to improved prognosis, for example, as reflected by an increased time to recurrence, which is the time to censoring the first recurrence of a second primary cancer as a first event or death without evidence of recurrence or an increase in overall survival, which is the time from treatment to death from any cause.Responding or having a response means that there is a beneficial endpoint achieved when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated when exposed to a stimulus.It will be understood that assessing the likelihood that a tumor or subject will respond favorably is equivalent to assessing the likelihood that a tumor or subject will not respond favorably (i.e., showing a lack of response or not responding).

[0118] The term "resistance" (i.e., no response or reduced or limited response to therapeutic treatment) refers to acquired or natural resistance of a cancer sample or mammal to cancer treatment, such as a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more reduction in response to cancer treatment, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range therebetween (including borderlines). The reduced response can be measured by comparing the same cancer sample or mammal with that before resistance was acquired, or by comparing with a different cancer sample or mammal known not to be resistant to therapeutic treatment. Typical acquired resistance to chemotherapy is referred to as "multidrug resistance." Multidrug resistance can be mediated by P-glycoprotein or other mechanisms, or can occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of a skilled artisan, and can be measured, for example, by cell proliferation and cell death assays, described herein as "sensitizing." In some embodiments, the term "reverse resistance" refers to the use of a second agent in combination with a primary cancer treatment (e.g., chemotherapy or radiation therapy) that can produce a significant reduction in tumor volume at a statistically significant level (e.g., p<0.05) compared to the tumor volume of an untreated tumor in situations where the primary cancer treatment (e.g., chemotherapy or radiation therapy) alone fails to produce a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor. This generally applies to tumor volume measurements performed when an untreated tumor is growing exponentially.

[0119] The term "sample" used to detect or determine the presence or level of at least one biomarker typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., those described in the definition of "bodily fluid" above), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgically resected tissue. In certain cases, methods encompassed by the present invention further include obtaining a sample from an individual prior to detecting or determining the presence or level of at least one marker in the sample.

[0120] The term "sensitization" refers to modifying cancer or tumor cells in a manner that allows for more effective treatment of the associated cancer with cancer therapies (e.g., anti-immune checkpoint agents, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not affected to the extent that they are unduly damaged by the treatment. Increased or decreased sensitivity to therapeutic treatment can be measured using cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Sensitivity or resistance is measured according to methods known in the art for the particular treatment and methods described below, including, but not limited to, those described infra (Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90). Sensitivity or resistance can also be measured in animals by measuring tumor size reduction over a period of time, e.g., 6 months in humans or 4-6 weeks in mice. A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range therebetween (inclusive), compared to the therapeutic sensitivity or resistance in the absence of such composition or method. Determining sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician.It should be understood that any of the methods described herein for enhancing the effectiveness of cancer treatment are equally applicable to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to cancer treatment.

[0121] The terms "selective modulator" or "selective modulation" as applied to a biologically active agent refer to the ability of the agent to modulate a target, such as a cell population, signaling activity, etc., relative to a non-specific cell population, signaling activity, etc., either directly or through an interaction with the target. For example, an agent that selectively inhibits an interaction between a protein and one native binding partner over another interaction between the protein and another binding partner, and / or such interaction(s) on a cell population of interest, may exhibit at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 2x (fold), 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, ...x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 1 5x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 105x, 110x, 120x, 125x, 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 600x, 700x, 800x, 900x, 1000x , 1500x, 2000x, 2500x, 3000x, 3500x, 4000x, 4500x, 5000x, 5500x, 6000x, 6500x, 7000x, 7500x, 8000x, 8500x, 9000x, 9500x, 10000x, or more, or any range therebetween (inclusive). Such metrics are typically expressed as the relative amount of agent required to reduce the interaction / activity by half. Such metrics apply to other selectivity configurations, such as the binding of nucleic acid molecules to one or more target sequences.

[0122] More generally, the term "selective" refers to a preferential action or function. The term "selective" can be quantified in terms of a preferential effect in a particular subject of interest compared to other targets. For example, the measured variable (e.g., modulation of biomarker expression in desired cells versus other cells, enrichment and / or depletion of desired cells versus other cells, etc.) can be increased by 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold The fold increase can be 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, or any range therebetween (inclusive) (e.g., 50% to 16-fold), differing in intended versus unintended or undesired targets. The same fold analysis can be used to ascertain the magnitude of the effect in a given tissue, cell population, measured variable, and / or measured effect, e.g., cell proportion, hyperproliferative cell growth rate or volume, cell growth rate, cell number, etc.

[0123] In contrast, the term "specific" refers to an exclusive action or function. For example, specific modulation of the interaction between a protein and one binding partner refers to the exclusive modulation of that interaction, and does not refer to significant modulation of the interaction between the protein and another binding partner. In another example, the specific binding of an antibody to a predetermined antigen refers to the antibody's ability to bind to the antigen of interest without binding to other antigens. Typically, antibodies have a specificity of approximately 1 x 10 -7 Less than M, e.g., approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11M, or even lower affinity (K) as determined using an appropriate assay, such as using surface plasmon resonance (SPR) technology in a BIACORE® assay instrument, using the antigen of interest as the analyte and the antibody as the ligand. D The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0124] Methods for determining cross-reactivity include standard binding assays as described herein, such as using surface plasmon resonance (SPR) analysis, flow cytometry analysis, and the like.

[0125] The term "small molecule" is a term of the art and includes molecules less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, a small molecule does not contain exclusively peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptide compound. The term is intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of the chemical structure in question, unless otherwise specified.

[0126] The term "subject" refers to an animal, vertebrate, mammal, or human, particularly one to which an agent is administered, a sample is obtained, or a procedure is performed, e.g., for experimental, diagnostic, and / or therapeutic purposes. In some embodiments, the subject is a mammal, e.g., a human, a non-human primate, a rodent (e.g., a mouse or rat), a livestock animal (e.g., a cow, sheep, cat, dog, and horse), or other animals such as llamas and camels. In some embodiments, the subject is a human. In some embodiments, the subject is a human subject with cancer. The term "subject" is interchangeable with "patient."

[0127] The term "survival" includes all of the following: survival until death, also known as overall survival (death may be either related to the cause or to the tumor), "recurrence-free survival" (the term recurrence includes both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease includes cancer and related diseases). The length of survival can be calculated by reference to a defined starting point (e.g., diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0128] The term "synergistic effect" refers to the combined effect of two or more agents (e.g., modulators of biomarkers listed in Table 1 and immunotherapy combination therapy) that is greater than the sum of the individual effects of the cancer agents / therapies alone.

[0129] The term "target" refers to a gene or gene product that is modulated, inhibited, or silenced by an agent, composition, and / or formulation described herein. Target genes or gene products include wild-type and mutant forms. A non-limiting, representative list of targets encompassed by the present invention is provided in Table 1. Similarly, the terms "target," "target(s)," or "targeting" used as a verb refers to modulating the activity of a target gene or gene product. Targeting refers to up-regulating or down-regulating the activity of a target gene or gene product.

[0130] The term "therapeutic effect" encompasses a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desired physical or mental development and conditions in animals or humans. A prophylactic effect encompassed by the term includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0131] The term "effective amount" or "effective dose" of an agent (including compositions and / or formulations comprising such an agent) refers to an amount sufficient to achieve a desired biological and / or pharmacological effect when delivered to a cell or organism, for example, according to a selected administration form, route, and / or schedule. As will be understood by those skilled in the art, the absolute amount of a particular agent or composition that is effective may vary depending on factors such as the desired biological or pharmacological endpoint, the agent being delivered, the target tissue, etc. Those skilled in the art will further understand that, in various embodiments, an "effective amount" may be contacted with a cell or administered to a subject in a single dose or through the use of multiple doses. The term "effective amount" may be a "therapeutically effective amount."

[0132] The term "therapeutically effective amount" refers to that amount of an agent effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the therapeutic index. Compositions that exhibit large therapeutic indices are preferred. In some embodiments, the LD 50 The lethal dose (ED) can be measured, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more for the drug compared to when the drug is not administered. 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured and can be, for example, increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more for the drug compared to not administering the drug. Similarly, the IC 50The concentration (i.e., the concentration that achieves half-maximal cytotoxicity or cytostatic effect against cancer cells) can be measured and can, for example, be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more for the agent compared to not administering the agent. In some embodiments, cancer cell growth in the assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% reduction in solid malignancies can be achieved.

[0133] More generally, "EC 50 " refers to the concentration of an agent, such as an antibody or antigen-binding fragment thereof, that induces a response that is 50% of the maximal response, such as between the maximal response and the baseline response in an in vitro and / or in vivo assay.

[0134] The terms "tolerance" or "unresponsiveness" include the refractoriness of cells, such as immune cells, to stimuli, e.g., stimulation via activating receptors or cytokines. Unresponsiveness can result, for example, from exposure to immunosuppressants or high doses of antigen. Several independent methods can induce tolerance. One mechanism is called "anergy," which is defined as a state in which cells persist in vivo as unresponsive cells rather than differentiating into cells with effector function. Such unresponsiveness is generally antigen-specific and persists even after exposure to the tolerizing antigen has ceased. For example, anergy in T cells is characterized by a lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even in the presence of a costimulatory polypeptide) results in a failure to produce cytokines and therefore a failure to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, measured by ELISA or proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by multimers of AP1 sequences found within heterologous promoters or enhancers under the control of the 5' IL-2 gene enhancer (Kang et al. (1992) Science 257:1134). Another mechanism is termed "exhaustion." T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections and cancers. It is defined by defective effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector or memory T cells.

[0135] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analog of such RNA or cDNA) that is complementary to or homologous to all or a portion of the mature mRNA produced by transcription of a biomarker nucleic acid and normal post-transcriptional processing (e.g., splicing), if present, an RNA transcript, or reverse transcription of the RNA transcript.

[0136] The term "treatment" refers to the therapeutic management or amelioration of a desired condition (e.g., a disease or disorder). Treatment may include, but is not limited to, the administration of an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed in an attempt to alter the course of a disease in a manner beneficial to the subject (the term is used to refer to a disease, disorder, syndrome, or undesirable condition that warrants, or potentially warrants, treatment). The effect of treatment may include reversing, alleviating, reducing the severity of, delaying the onset of, curing, inhibiting progression of, and / or reducing the likelihood of occurrence or recurrence of a disease or one or more symptoms or signs of a disease. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or temporarily alleviating the condition, and achieving remission or improving prognosis. A therapeutic agent may be administered to a subject who has a disease or who is at increased risk of developing a disease compared to members of the general population. In some embodiments, a therapeutic agent can be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent can be administered, for example, to reduce the likelihood of overt disease recurrence. A therapeutic agent can be administered prophylactically, i.e., before the onset of any symptoms or the manifestation of the disease. "Prophylactic treatment" refers to providing medical and / or surgical management to a subject who has not developed a disease or does not subsequently show evidence of the disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity of the disease if it does occur. A subject can be identified as being at risk of developing a disease (e.g., at an increased risk compared to the general population or having risk factors that increase the likelihood of developing a disease).

[0137] The term "unresponsiveness" includes the refraction of cancer cells to treatment or the refraction of therapeutic cells, such as immune cells, to stimulation, e.g., stimulation via an activating receptor or cytokine. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen. As used herein, the terms "anergy" or "tolerance" include unresponsiveness to activating receptor-mediated stimulation. Such unresponsiveness is generally antigen-specific and persists even after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by a lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even in the presence of a costimulatory polypeptide) results in failure to produce cytokines and therefore failure to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can be observed by the lack of IL-2 production by T lymphocytes measured by ELISA or proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by multimers of AP1 sequences found within heterologous promoters or enhancers under the control of the 5' IL-2 gene enhancer (Kang et al. (1992) Science 257:1134).

[0138] The term "vaccine" refers to a composition for generating immunity for the prevention and / or treatment of disease.

[0139] Furthermore, there is a known and definite correspondence between the amino acid sequence of a particular protein, as defined by the genetic code (below), and the nucleotide sequence that can encode that protein. Similarly, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid, as defined by the genetic code, and the amino acid sequence encoded by that nucleic acid. [Table A]

[0140] An important and well-known feature of the genetic code is its redundancy, which allows more than one coding nucleotide triplet to be used for most amino acids used to make proteins (as shown above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others). Furthermore, occasionally, methylated variants of purines or pyrimidines can be found within a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0141] In view of the above, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to derive a polypeptide amino acid sequence by translating the DNA or RNA into an amino acid sequence using the genetic code. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence capable of encoding the polypeptide can be deduced from the genetic code (which, due to its redundancy, generates multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0142] II. Monocytes and macrophages Monocytes are myeloid-derived immune effector cells that circulate in the blood, bone marrow, and spleen and undergo limited proliferation under steady-state conditions. The term "myeloid cells" can refer to granulocyte or monocyte precursor cells in the bone marrow or spinal cord, or their similarity to those found in the bone marrow or spinal cord. The myeloid cell lineage includes circulating monocytic cells in peripheral blood and the cell populations they become after maturation, differentiation, and / or activation. These populations include non-terminally differentiated myeloid cells, myeloid-derived suppressor cells, and differentiated macrophages. Differentiated macrophages include non-polarized and polarized macrophages, resting and activated macrophages. Without limitation, the myeloid lineage also includes granulocyte precursors, polymorphonuclear-derived suppressor cells, differentiated polymorphonuclear leukocytes, neutrophils, granulocytes, basophils, eosinophils, monocytes, macrophages, microglia, myeloid-derived suppressor cells, dendritic cells, and erythrocytes. Monocytes are found within peripheral blood mononuclear cells (PBMCs), which also contain other hematopoietic and immune cells such as B cells, T cells, and NK cells. Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes have two primary functions in the immune system: (1) they can exit the bloodstream to recruit resident macrophages and dendritic cells (DCs) under normal conditions, and (2) they rapidly migrate to sites of infection in tissues, where they can divide and differentiate into macrophages and inflammatory dendritic cells to elicit immune responses in response to inflammatory signals. Monocytes are typically identified by their large, bilobed nuclei in stained smears. Monocytes also express chemokine receptors and pathogen-recognition receptors, which mediate migration from the blood to tissues during infection. They produce inflammatory cytokines and phagocytes. In some embodiments, bone marrow cells of interest are identified according to CD11b+ and / or CD14+ expression.

[0143] As described in more detail below, monocytes can differentiate into macrophages. Monocytes can also differentiate into dendritic cells, such as through the action of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). In general, the term "monocyte" encompasses undifferentiated monocytes and cell types differentiated therefrom, including macrophages and dendritic cells. In some embodiments, the term "monocyte" can refer to undifferentiated monocytes.

[0144] Macrophages are key immune effectors and regulators of inflammatory and innate immune responses. Macrophages are heterogeneous, tissue-resident, terminally differentiated, innate myeloid cells with remarkable plasticity, capable of altering physiological function in response to local cues from the microenvironment and capable of assuming a spectrum of functional demands ranging from host defense to tissue homeostasis (Ginhoux et al. (2016) Nat. Immunol. 17:34-40). Macrophages are present in nearly every tissue in the body. They are derived either from tissue-resident macrophages, e.g., liver-resident Kupffer cells, or from circulating monocytic precursors (i.e., monocytes), primarily from bone marrow and splenic reservoirs, and migrate to tissues either at steady state or in response to inflammatory or other stimulatory cues. For example, monocytes can be recruited from the blood to tissues to recruit tissue-specific macrophages in bone, alveoli (lungs), the central nervous system, connective tissue, gastrointestinal tract, liver, spleen, and peritoneum.

[0145] The term "tissue-resident macrophages" refers to a heterogeneous population of immune cells that perform tissue-specific and / or microanatomical niche-specific functions, such as tissue immune surveillance, response to infection and resolution of inflammation, and intrinsic homeostatic functions. Tissue-resident macrophages originate in the embryonic yolk sac and mature in specific tissues of the developing fetus, where they acquire tissue-specific roles and alter their gene expression profiles. Local proliferation of tissue-resident macrophages that maintain colony-forming capacity can directly give rise to a population of mature macrophages within the tissue. Local proliferation of tissue-resident macrophages that maintain colony-forming capacity can directly give rise to a population of mature macrophages within the tissue. Tissue-resident macrophages can also be identified and named according to the tissue they occupy. For example, adipose tissue macrophages occupy adipose tissue, Kupffer cells occupy liver tissue, sinus histiocytes occupy lymph nodes, alveolar macrophages (dust cells) occupy alveoli, Langerhans cells occupy skin and mucosal tissue, giant cell-connected histiocytes occupy connective tissue, microglia occupy central nervous system (CNS) tissue, Hofbauer cells occupy placental tissue, intraglomerular mesangial cells occupy kidney tissue, osteoclasts occupy bone tissue, epithelioid cells occupy granulomas, red pulp macrophages (sinusoidal lining cells) occupy the red pulp of splenic tissue, peritoneal macrophages occupy peritoneal tissue, lysosomal cells occupy Peyer's patch tissue, and pancreatic macrophages occupy pancreatic tissue.

[0146] In addition to host defense against infectious pathogens and other inflammatory responses, macrophages can perform various homeostatic functions, including, but not limited to, development, wound healing, and tissue repair, as well as regulating immune responses. Macrophages were first recognized as phagocytic cells in the body that defend against infection through phagocytosis and are an essential component of innate immunity. In response to pathogens and other inflammatory stimuli, activated macrophages can engulf infecting bacteria and other microorganisms, stimulating inflammation and releasing a cocktail of proinflammatory molecules into these intracellular microorganisms. After phagocytosing pathogens, macrophages present pathogenic antigens to T cells, further activating the adaptive immune response for protection. Exemplary proinflammatory molecules include cytokines IL-1β, IL-6, and TNF-α, chemokines MCP-1, CXC-5, and CXC-6, and CD40L.

[0147] In addition to contributing to the host's defense against infection, macrophages also play an important homeostatic role independent of their involvement in the immune response. Macrophages are large phagocytes that eliminate red blood cells, allowing released materials such as iron and hemoglobin to be recycled for reuse by the host. This process of elimination is a critical metabolic contribution without which the host cannot survive.

[0148] Macrophages are also involved in the clearance of cellular debris generated during tissue remodeling, rapidly and efficiently eliminating apoptotic cells. Macrophages are thought to contribute to steady-state tissue homeostasis through the clearance of apoptotic cells. These homeostatic clearance processes are generally mediated by surface receptors on macrophages, including scavenger receptors, phosphatidylserine receptors, thrombospondin receptors, integrins, and complement receptors. These receptors that mediate phagocytosis either fail to signal inducing cytokine gene transcription or actively produce inhibitory signals and / or cytokines. The homeostatic function of macrophages is independent of other immune cells.

[0149] Macrophages can also clear cellular debris / necrotic cells resulting from trauma or other cellular damage. Macrophages detect endogenous danger signals present within necrotic cellular debris via Toll-like receptors (TLRs), intracellular pattern recognition receptors, and interleukin-1 receptors (IL-1Rs), most of which transduce signals via the adaptor molecule myeloid differentiation primary response gene 88 (MyD88). Clearance of cellular debris can significantly alter macrophage physiology. Macrophages clearing necrotic debris can undergo dramatic changes in physiology, including altered surface protein expression and production of cytokines and proinflammatory mediators. Alterations in macrophage surface protein expression in response to these stimuli may be used to identify biochemical markers specific to these altered cells.

[0150] Macrophages play a key role in maintaining homeostasis in many tissues, including white adipose tissue, brown adipose tissue, the liver, and the pancreas. Tissue macrophages can rapidly respond to changes in tissue conditions by releasing cell signaling molecules that trigger a cascade of changes that adapt tissue cells. For example, macrophages in adipose tissue regulate the generation of new adipocytes in response to dietary changes (e.g., macrophages in white adipose tissue) or exposure to cold (e.g., macrophages in brown adipose tissue). Macrophages in the liver, known as Kupffer cells, regulate the breakdown of glucose and lipids in response to dietary changes. Macrophages in the pancreas can regulate insulin production in response to a high-fat diet.

[0151] Macrophages can also contribute to wound healing and tissue repair. For example, in response to signals derived from damaged tissues and cells, macrophages can be activated and induce tissue repair responses to repair damaged tissues (Minutti et al. (2017) Science 356:1076-1080).

[0152] During embryonic development, macrophages also play an important role in tissue remodeling and organ development. For example, resident macrophages actively shape the developing blood vessels in the neonatal mouse heart (Leid et al. (2016) Circ. Res. 118:1498-1511). Microglia in the brain can produce growth factors that guide neurons and blood vessels in the developing brain during embryonic development. Similarly, CD95L, a protein produced by macrophages, binds to the CD95 receptor on the surface of neurons, promoting the development of blood vessels in the brain of mouse embryos and promoting neuronal and vascular development (Chen et al. (2017) Cell Rep. 19:1378-1393). Without the ligand, neurons branch less frequently, and the resulting adult brain exhibits less electrical activity. Monocyte-derived cells known as osteoclasts are involved in bone development; mice lacking these cells develop dense, sclerotic bones, a rare condition known as osteopetrosis. Macrophages also regulate mammary gland development and aid in early postnatal retinal development (Wynn et al. (2013) Nature 496:445-455).

[0153] As mentioned above, macrophages regulate the immune system. In addition to presenting antigens to T cells, macrophages can provide immunosuppressive / inhibitory signals to immune cells under some conditions. For example, in the testes, macrophages help create an environment that protects sperm from attack by the immune system. Tissue-resident macrophages within the testes produce immunosuppressive molecules that prevent immune cell responses to sperm (Mossadegh-Keller et al. (2017) J. Med. 214:10.1084 / jem.20170829).

[0154] The plasticity of macrophages in response to different environmental signals and consistent with their functional requirements has resulted in a range of macrophage activation states, including two extremes of a continuum: "classically activated" M1 and "alternatively activated" M2 macrophages.

[0155] The term "activated" refers to a state of myeloid cells that have been stimulated sufficiently to induce detectable cell proliferation and / or to exert effector functions such as induced cytokine expression and secretion, phagocytosis, cell signaling, antigen processing and presentation, target cell killing, and pro-inflammatory functions.

[0156] The term "M1 macrophage" or "classically activated macrophage" refers to macrophages with a proinflammatory phenotype. The term "macrophage activation" (also called "classical activation") was introduced by Mackaness in the 1960s in the context of infection to describe the antigen-dependent but nonspecific enhanced bactericidal activity of macrophages against BCG (bacillus Calmette-Guerin) and Listeria upon secondary exposure to pathogens (Mackaness (1962) J. Exp. Med. 116:381-406). This enhancement was later associated with Th1 responses and IFN-γ production by antigen-activated immune cells (Nathan et al. (1983) J. Exp. Med. 158:670-689) and extended to cytotoxic and antitumor properties (Pace et al. (1983) Proc. Natl. Acad. Sci. USA 80:3782-3786, Celada et al. (1984) J. Exp. Med. 160:55-74). Therefore, macrophage functions that enhance inflammation through cytokine secretion, antigen presentation, phagocytosis, cell-cell interactions, migration, etc., are considered proinflammatory. In vitro and in vivo assays can measure different endpoints: common in vitro measurements include proinflammatory cell stimulation measured by proliferation, migration, proinflammatory Th1 cytokine / chemokine secretion, and / or migration, while common in vivo measurements further include analysis of pathogen combating, immediate responders to tissue damage, other cell activators, and migration inducers. Proinflammatory antigen presentation can be assessed both in vitro and in vivo. Lipopolysaccharide (LPS), certain Toll-like receptor (TLR) agonists, the Th1 cytokine interferon gamma (IFNγ) (e.g., produced by NK cells in response to stress and infection, as well as by T helper cells with sustained production), and TNF polarize macrophages along the M1 pathway.Activated M1 macrophages phagocytose and destroy microorganisms, eliminate damaged cells (such as tumor cells and apoptotic cells), present antigens to T cells to increase adaptive immune responses, produce high levels of proinflammatory cytokines (e.g., IL-1, IL-6, and IL-23), reactive oxygen species (ROS), and nitric oxide (NO), and activate other immune and non-immune cells. M1 macrophages, characterized by the expression of inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and production of the Th1-associated cytokine IL-12, are well adapted to promote potent immune responses. M1 macrophage metabolism is characterized by enhanced aerobic glycolysis, glucose conversion to lactate, increased flux through the pentose phosphate pathway (PPP), fatty acid synthesis, and a truncated tricarboxylic acid (TCA) cycle leading to the accumulation of succinate and citrate.

[0157] "Type 1" or "M1-like" myeloid cells are myeloid cells that can contribute to a proinflammatory response characterized by at least one of generating an inflammatory stimulus by secreting at least one proinflammatory cytokine, expressing at least one cell surface activation molecule / ligand for an activation molecule on its surface, recruiting / directing / interacting with at least one other cell (including other macrophages and / or T cells) to stimulate a proinflammatory response, presenting antigen in a proinflammatory context, migrating to a site that allows the initiation of a proinflammatory response, or initiating expression of at least one gene predicted to lead to a proinflammatory function. In some embodiments, the term includes activating cytotoxic CD8+ T cells, mediating increased sensitivity of cancer cells to immunotherapy, such as immune checkpoint therapy, and / or mediating the reversal of cancer cell resistance. In certain embodiments, such modulation of the pro-inflammatory state is determined by, but is not limited to, a) increased expression and / or secretion of cluster of differentiation 80 (CD80), CD86, MHCII, MHC1, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); b) decreased expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, TGFβ, and / or IL-10; c) decreased expression and / or secretion of at least one protein selected from the group consisting of IL-1β, TNF-α, IL-12, IL-18, GM-CSF, CCL3, CCL4, and IL-23. The increased secretion of one cytokine or chemokine, d) an increased ratio of IL-1β, IL-6, and / or TNF-α expression to IL-10 expression, e) increased CD8+ cytotoxic T cell activation, f) increased recruitment of CD8+ cytotoxic T cell activation, g) increased CD4+ helper T cell activity, h) increased recruitment of CD4+ helper T cell activity, i) increased NK cell activity, j) increased recruitment of NK cells, k) increased neutrophil activity, l) increased macrophage activity, and / or m) increased spindle-shaped morphology, flattened appearance, and / or number of dendrites as assessed by microscopy.

[0158] In cells that are already pro-inflammatory, an increase in the inflammatory phenotype points to a further pro-inflammatory state.

[0159] In contrast, the term "M2 macrophages" refers to macrophages with an anti-inflammatory phenotype. Th2 and tumor-derived cytokines, such as IL-4, IL-10, IL-13, transforming growth factor beta (TGF-β), and prostaglandin E2 (PGE2), can promote M2 polarization. The metabolic profile of M2 macrophages is defined by reduced OXPHOS, FAO, and glycolysis, and the PPP. The discovery that the mannose receptor is selectively upregulated by Th2 IL-4 and IL-13 in murine macrophages, inducing enhanced endocytic clearance of mannosylated ligands, increasing major histocompatibility complex (MHC) class II antigen expression, and reducing proinflammatory cytokine secretion follows from Stein, Doyle, and colleagues' proposal that IL-4 and IL-13 induce an alternative activation phenotype that is distinct from IFN-γ activation but far from being inactivated (Martinez and Gordon (2014) F1000 Prime Reports 6:13). In vitro and in vivo definitions / assays can measure different endpoints: common in vitro endpoints include proliferation, migration, anti-inflammatory cell stimulation measured by anti-inflammatory Th2 cytokine / chemokine secretion and / or migration, while common in vivo M2 endpoints further include analyzing pathogen fighting, delayed tissue damage / profibrotic responses, Th2 polarization of other cells, migration inducers, etc. Tolerogenic antigen presentation can be assessed both in vitro and in vivo.

[0160] "Type 2" or "M2-like" myeloid cells are myeloid cells that can contribute to an anti-inflammatory response characterized by at least one of generating an anti-inflammatory stimulus by secreting at least one anti-inflammatory cytokine, expressing at least one cell surface inhibitory molecule / ligand for an inhibitory molecule on their surface, recruiting / directing / interacting with at least one other cell to stimulate an anti-inflammatory response, presenting antigen in a tolerogenic context, migrating to a site that allows for the initiation of a tolerogenic response, or initiating the expression of at least one gene predicted to lead to a tolerogenic / anti-inflammatory function. In certain embodiments, such modulation toward a pro-inflammatory state can be measured by several well-known methods, including, but not limited to, the inverse of the type 1 pro-inflammatory state measurement described above.

[0161] A cell having an "increased inflammatory phenotype" is a cell that, following modulation of at least one biomarker encompassed by the present invention (e.g., at least one target listed in Table 1), e.g., contact with an agent that modulates at least one biomarker encompassed by the present invention (e.g., at least one target listed in Table 1), has a greater pro-inflammatory response capability associated with a) an increase in one or more of the type 1 enumerated criteria, and / or b) a decrease in one or more of the type 2 enumerated criteria.

[0162] A cell having a "reduced inflammatory phenotype" is a cell that, following modulation of at least one biomarker encompassed by the present invention (e.g., at least one target listed in Table 1), e.g., contact with an agent that modulates at least one biomarker encompassed by the present invention (e.g., at least one target listed in Table 1), has a greater anti-inflammatory response capability associated with a) a reduction in one or more of the type 1 enumerated criteria, and / or b) an increase in one or more of the type 2 enumerated criteria.

[0163] Thus, macrophages can adopt a continuum of alternatively activated states with phenotypes between type 1 and type 2 states (see, e.g., Biswas et al. (2010) Nat. Immunol. 11:889-896; Mosser and Edwards (2008) Nat. Rev. Immunol. 8:958-969; Mantovani et al. (2009) Hum. Immunol. 70:325-330), and such increased or decreased inflammatory phenotypes can be determined as described above.

[0164] As used herein, the term "alternatively activated macrophages" or "alternatively activated state" refers to essentially all types of macrophage populations other than classically activated M1 proinflammatory macrophages. Originally, the alternatively activated state was designated exclusively for M2 anti-inflammatory macrophages. The term has been expanded to include all other alternatively activated states of macrophages that have dramatic differences in biochemistry, physiology, and function.

[0165] For example, one type of alternatively activated macrophage is one involved in wound healing. In response to innate and adaptive signals released during tissue injury (e.g., surgical wounds), such as IL-4 produced by basophils and mast cells, tissue-resident macrophages can be activated to promote wound healing. Instead of producing high levels of proinflammatory cytokines, wound-healing macrophages secrete large amounts of extracellular matrix components, such as chitinase and chitinase-like proteins YM1 / CHI3L3, YM2, AMCase, and stabilin, all of which exhibit carbohydrate- and matrix-binding activity and are involved in tissue repair.

[0166] Another example of alternatively activated macrophages includes regulatory macrophages, which can be induced by innate and adaptive immune responses. Regulatory macrophages can contribute to immunoregulatory functions. For example, macrophages can respond to hormones (e.g., glucocorticoids) from the hypothalamic-pituitary-adrenal (HPA) axis to inhibit host defense and inflammatory functions, such as inhibiting the transcription of proinflammatory cytokines. Regulatory macrophages can produce the regulatory cytokine TGF-β to dampen immune responses under certain conditions, such as in the late stages of adaptive immune responses. Many regulatory macrophages express high levels of costimulatory molecules (e.g., CD80 and CD86) and can therefore enhance antigen presentation to T cells.

[0167] Many stimuli / cues can induce regulatory macrophage polarization, including, but not limited to, TLR agonists and immune complexes, apoptotic cells, IL-10, prostaglandins, GPC ligands, adenosine, dopamine, histamine, sphingosine 1-phosphate, melanocortin, vasoactive intestinal peptide, and Siglec-9. Some pathogens, such as parasites, viruses, and bacteria, specifically induce the differentiation of regulatory macrophages, resulting in defective pathogen killing and promoting the survival and spread of infecting microorganisms.

[0168] Regulatory macrophages share several common characteristics. For example, they require two stimuli to induce anti-inflammatory effects. Differences between regulatory macrophage subpopulations induced by different cues / stimuli have also been observed, reflecting their heterogeneity.

[0169] Regulatory macrophages are also a heterogeneous population of macrophages, including various subpopulations found in metabolism, development, and homeostasis. In one example, a subpopulation of alternatively activated macrophages is immunoregulatory macrophages, which have inherent immunoregulatory properties that can be induced in the presence of M-CSF / GM-CSF, CD16 ligand (such as immunoglobulin), and IFN-γ (PCT Application Publication No. WO 2017 / 153607).

[0170] Macrophages in tissues can change their activation state in vivo over time. This dynamic reflects the constant influx of macrophages migrating into tissues, the dynamic shift of activated macrophages, and macrophages returning to a resting state. In some conditions, different signals in the environment can induce macrophages into a mixture of different activation states. For example, in chronic wounds, macrophages over time may include pro-inflammatory activated subpopulations, macrophages that promote wound healing, and some macrophages that exhibit pro-resolving activities. Under non-pathological conditions, the immune system contains a balanced population of immunostimulatory and immunoregulatory macrophages. In some disease states, this balance is disrupted, and this imbalance leads to many clinical conditions.

[0171] The apparent plasticity of macrophages also results in unstable responses to environmental cues they encounter in disease states. Macrophages can repolarize in response to various disease states and display distinct characteristics. One example is macrophages that are attracted to and filtered from peripheral blood monocytes into tumor tissue, often referred to as "tumor-associated macrophages" ("TAMs") or "tumor-infiltrating macrophages" ("TIMs"). Tumor-associated macrophages are the most abundant inflammatory cells within tumors, and a significant correlation has been found between high TAM density and poor prognosis in most cancers (Zhang et al. (2012) PloS One 7:e50946.10.1371 / journal.pone.0050946).

[0172] TAMs are a mixed population of both M1-like pro-inflammatory and M2-like anti-inflammatory subpopulations. In the early stages of neoplasia, classically activated macrophages with a pro-inflammatory phenotype reside in normoxic tumor regions and are thought to contribute to the early eradication of transformed tumor cells. However, as tumors grow and progress, the majority of TAMs in later-stage tumors are M2-like regulatory macrophages present in hypoxic regions of the tumor. This phenotypic change in macrophages is significantly influenced by tumor microenvironment stimuli, such as the tumor extracellular matrix, the anoxic environment, and cytokines secreted by tumor cells. M2-like TAMs exhibit a hybrid activation state between wound-healing and regulatory macrophages and display various unique characteristics, including high levels of IL-10 but little or no IL-12 production, defective TNF production, suppression of antigen-presenting cells, and contribution to tumor angiogenesis.

[0173] Generally, TAMs are characterized by an M2 phenotype and suppress M1 macrophage-mediated inflammation through the production of IL-10 and IL-1β. Therefore, TAMs promote tumor growth and metastasis through the activation of wound healing (i.e., anti-inflammatory) pathways that provide nutrients and growth signals for proliferation and invasion and promote the creation of new blood vessels (i.e., angiogenesis). Furthermore, TAMs contribute to an immunosuppressive tumor microenvironment by secreting anti-inflammatory signals that prevent other elements of the immune system from recognizing and attacking tumors. TAMs have been reported to play a critical role in promoting cancer growth, proliferation, and metastasis in many types of cancer (e.g., breast cancer, astrocytoma, head and neck squamous cell carcinoma, type II papillary renal cell carcinoma, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, glioma, classical Hodgkin's lymphoma, ovarian cancer, and colorectal cancer). Generally, cancers characterized by a large population of TAMs are associated with poor disease prognosis.

[0174] Their diverse functions and activation states can have dangerous consequences if not properly controlled: for example, classically activated macrophages can cause damage to host tissues, predispose surrounding tissues, and affect glucose metabolism when overactivated.

[0175] In many disease states, the balanced dynamics of macrophage activation state are disrupted, resulting in an imbalance that leads to disease. For example, tumors are rich in macrophages. Macrophages are found in 75 percent of cancers. Aggressive cancers are often associated with higher infiltration of macrophages and other immune cells. In most malignant tumors, TAMs exert several tumor-promoting functions, including promoting cancer cell survival, proliferation, invasion, extravasation, and metastasis; stimulating angiogenesis; remodeling the extracellular matrix; and suppressing antitumor immunity (Qian and Pollard, 2010, Cell, 141(1):39-51). They may also produce growth-promoting molecules such as ornithine, VEGF, EGF, and TGF-β.

[0176] TAMs stimulate tumor growth and survival in response to CSF1 and IL4 / IL13 encountered in the tumor microenvironment. TAMs can also remodel the tumor microenvironment through the expression of proteases such as MMPs, cathepsins, and uPA, and matrix-remodeling enzymes (e.g., lysyl oxidase and SPARC).

[0177] TAMs play a key role in tumor angiogenesis, which regulates the dramatic increase in blood vessels in tumor tissues necessary for tumor progression. These angiogenic TAMs express the angiopoietin receptor TIE2 and secrete many angiogenic molecules, including members of the VEGF family, TNFα, IL1β, IL8, PDGF, and FGF.

[0178] Diverse macrophage subpopulations execute these distinct tumor-promoting functions. These TAMs exhibit distinct phenotypes depending on the degree of macrophage infiltration and tumor type. For example, detailed profiling of human hepatocellular carcinoma (HCC) cells reveals various macrophage subtypes defined by anatomical location and tumor-promoting and tumor-antitumor properties. M2-like macrophages have been shown to be the primary source of tumor-promoting functions for TAMs. M2-like TAMs have been shown to affect the efficacy of anticancer treatments, contribute to therapy resistance, and mediate tumor recurrence after conventional cancer treatment.

[0179] III. Targets and Biomarkers Useful for Modulating Myeloid Cell Inflammatory Phenotype The present invention encompasses biomarkers such as PSGL-1 that are useful for modulating the inflammatory phenotype of myeloid cells and the corresponding immune response (eg, to augment anti-cancer macrophage immunotherapy).

[0180] Downregulation of PSGL-1 is associated with an increased inflammatory phenotype (e.g., type 1 phenotype), and upregulation is associated with and results in a decreased inflammatory phenotype (e.g., type 2 phenotype).

[0181] Nucleic acid and amino acid sequence information for the loci and biomarkers encompassed by the present invention (e.g., the biomarkers listed in Table 1) is well known in the art and readily available in publicly available databases such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences from publicly available sequence databases are provided below.

[0182] As discussed further below, agents that modulate the expression, translation, degradation, amount, subcellular localization, and other activities of the biomarkers encompassed by the present invention in myeloid cells are useful not only to modulate the inflammatory phenotype of these cells, but also to modulate the immune responses mediated by these cells.

[0183] Although a number of representative orthologs to human sequences are provided below, in some embodiments, human biomarkers (including their regulators and modulators) are preferred. For some biomarkers, the immune responses mediated by such biomarkers in humans are believed to be particularly useful given the differences between the human immune system and the immune systems of other vertebrates.

[0184] The term "PSGL-1" or "SELPLG" refers to selectin P ligand, a glycoprotein expressed as a dimer on the cell surface of myeloid cells and on a subset of activated T cells. It functions as a high-affinity counterreceptor for the cell adhesion molecules P-, E-, and L-selectin expressed on myeloid cells and stimulated T lymphocytes. Thus, the PSGL-1 protein plays an important role in leukocyte trafficking during inflammation by tethering leukocytes to activated platelet- or endothelial-expressed selectins. The PSGL-1 protein possesses two post-translational modifications: tyrosine sulfation and the addition of sialyl Lewis x tetrasaccharide (sLex) to its O-linked glycan for its high-affinity binding activity. In addition to its adhesive function, PSGL-1 has been shown to inhibit T cell function independently of select binding (Tinoco et al. (2017) Trends Immunol. 38:323-335). Furthermore, PSGL-1 antagonists have been developed that have been shown to specifically block the differentiation of cytotoxic T cells or induce apoptosis of both T cells and NK cells (U.S. Patent Application Publication Nos. 2002 / 0058034 and 2003 / 0049252). Despite the description of PSGL-1 expression on macrophages, the ability to modulate the inflammatory phenotype of macrophages, such as driving the phenotype from M2-like macrophages to M1-like macrophages, is believed to have not been previously described. Indeed, loss of PSGL-1 on macrophages has been proposed to increase susceptibility to colorectal cancer in an in vivo model (Li et al. (2017) Mol. Cancer Res. 15:467-477). Aberrant expression of PSGL-1 and polymorphisms in PSGL-1 have been associated with defects in innate and adaptive immune responses. PSGL-1 is an SLe(x)-type proteoglycan that mediates the rapid mobilization of leukocytes on the vascular surface during the early stages of inflammation through high-affinity, calcium-dependent interactions with E-, P-, and L-selectin. PSGL-1 is important for early leukocyte sequestration. PSGL-1 also binds to VISTA polypeptides, particularly at acidic pH (e.g., pH 6.0) (see, e.g., PCT Publication No. WO2018 / 132476).In some embodiments, the PSGL-1 gene, located on human chromosome 12q, consists of three exons. Orthologs are known from chimpanzee, rhesus monkey, dog, cow, mouse, and rat. PSGL-1. tm2Rpmc (Miner et al. (2008) Blood 112:2035-2045), PSGL-1 tm1Fur (Yang et al. (1999) J Exp Med 190:1769-1782), and PSGL-1 tm1Rpmc (Xia et al. (2002) J Clin Invest 109:939-950). In some embodiments, the human PSGL-1 protein has 412 amino acids and / or a molecular weight of 43,201 Da. In some embodiments, the PSGL-1 protein contains an RNase E / G family domain and / or can act as a receptor for enterovirus 71 during microbial infection. Known binding partners of PSGL-1 include, for example, P-, E-, and L-selectin, SNX20, MSN, and SYK.

[0185] The term "PSGL-1" is intended to encompass fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human PSGL-1 cDNA and human PSGL-1 protein sequences are known in the art and publicly available from the National Center for Biotechnology Information (NCBI) (see, e.g., ncbi.nlm.nih.gov / gene / 6404). For example, at least two distinct human PSGL-1 isoforms are known. Human PSGL-1 isoform 1 (NP_001193538.1) can be encoded by transcript variant 1 (NM_001206609.1), which is the longer transcript. Human PSGL-1 isoform 2 (NP_002997.2) can be encoded by transcript variant 2 (NM_003006.4), which differs from variant 1 in its 5' UTR, lacks a portion of the 5' coding region, and initiates translation at a downstream start codon. The encoded isoform 2 has a shorter N-terminus compared to isoform 1. Nucleic acid and polypeptide sequences of PSGL-1 orthologues in organisms other than humans are known, such as chimpanzee PSGL-1 (XM_016924121.2 and XP_016779610.1), rhesus monkey PSGL-1 (XM_015152715.1 and XP_015008201.1, and XM_015152716.1 and XP_015008202.1), dog PSGL-1 (NM_0012 These include bovine PSGL-1 (NM_001037628.2 and NP_001032717.2, and NM_001271160.1 and NP_001258089.1), mouse PSGL-1 (NM_009151.3 and NP_033177.3), and rat PSGL-1 (NM_001013230.1 and NP_001013248.1). Representative sequences of PSGL-1 orthologs are shown in Table 1 below.

[0186] Suitable anti-PSGL-1 antibodies for detecting PSGL-1 protein are well known in the art, including, for example, antibodies GTX19793, GTX54688, and GTX34468 (GeneTex, Irvine, CA), antibodies sc-365506 and sc-398402 (Santa Cruz Biotechnology), antibodies MAB9961, MAB996, NBP2-53344, and AF3345 (Novus Biologicals, Littleton, CO), antibodies ab68143, ab66882, antibody ab110096 (AbCam, Cambridge, MA), antibodies catalog numbers TA349432 and TA338245 (Origene, Rockville, MD), etc. Furthermore, reagents for detecting PSGL-1 expression are well known. Several clinical tests for PSGL-1 are available in the NIH Genetic Testing Registry (GTR®) (e.g., GTR test ID: GTR000532965.2, provided by Fulgent Clinical Diagnostics Lab, Temple City, CA). In addition, several siRNA, shRNA, CRISPR constructs for reducing PSGL-1 expression are listed in the commercial product list of the companies referenced above, such as siRNA product #SR321732, shRNA product #TL309563, TR309563, TG309563, TF309563, TL309563V, and CRISPR product #KN206507 from Origene Technologies (Rockville, MD), CRISPR gRNA product (sc-401534) from Applied Biological Materials (K6134408) and Santa Cruz, and RNAi product (catalog number sc-36323 and sc-42833) from Santa Cruz. Note that this term can also be used to refer to any combination of the features described herein for PSGL-1 molecules. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc. can be used to describe the PSGL-1 molecules encompassed by the present invention. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0187] IV. Antibodies and Antigen-Binding Fragments Thereof The inflammatory phenotype of myeloid cells can be modulated by modulating the amount and / or activity of certain biomarkers (e.g., at least one target listed in Table 1), and such inflammatory phenotype modulation also modulates the immune response.

[0188] The present invention provides antibodies, and antigen-binding fragments thereof, that modulate the targets listed in Table 1. Such compositions are useful for upregulating or downregulating the monocyte and / or macrophage inflammatory phenotype, thereby upregulating or downregulating, respectively, the immune response. Such compositions are also useful for detecting the amount and / or activity of the targets listed in Table 1, and are therefore useful for diagnosing, prognosing, and screening agents whose effects are mediated by such targets.

[0189] Representative exemplary, non-limiting antibodies are shown in Table 2 below. [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

[0190] a. Compositions of antibodies and antigen-binding fragments thereof In general, the antibodies and antigen-binding fragments thereof encompassed by the present invention are characterized by their ability to bind to myeloid cells that express PSGL-1 polypeptide and increase the inflammatory phenotype of the myeloid cells.

[0191] Antibodies (e.g., isolated monoclonal antibodies) directed against PSGL-1, as well as antigen-binding fragments thereof, are provided. In some embodiments, the mAbs have been deposited with the American Type Culture Collection (ATCC) under the terms of the Budapest Treaty, as further described below.

[0192] Because it is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity / affinity of an antibody to an antigen, antibodies encompassed by the present invention, such as those described in Table 2, preferably comprise heavy and light chain CDR3s of a variable region encompassed by the present invention (e.g., comprising a sequence in Table 2, or a portion thereof). The antibody may further comprise a CDR2 of a variable region encompassed by the present invention (e.g., comprising a sequence in Table 2, or a portion thereof). The antibody may further comprise a CDR1 of a variable region encompassed by the present invention (e.g., comprising a sequence in Table 2, or a portion thereof). In other embodiments, the antibody may comprise any combination of CDRs. In some embodiments, CDR1, CDR2, and / or CDR3 may be selected from within the same heavy or light chain sequence encompassed by the present invention (e.g., comprising a sequence in Table 2, or a portion thereof). In other embodiments, CDR1, CDR2, and / or CDR3 may be selected from within the same heavy and light chain sequence pair encompassed by the present invention (eg, including a sequence in Table 2, or a portion thereof).

[0193] The CDR1, CDR2, and / or CDR3 regions of the above-described antibodies and antigen-binding fragments thereof may comprise the exact amino acid sequence(s) as those of the variable regions encompassed by the invention disclosed herein (e.g., including the sequences in Table 2, or portions thereof). However, one of skill in the art will understand that some deviations from the exact CDR sequences may be possible while still retaining the ability of the antibody to effectively bind to PSGL-1 (e.g., conservative sequence modifications). Thus, in another embodiment, an engineered antibody may be composed of one or more CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or more CDRs encompassed by the invention (e.g., including the sequences in Table 2, or portions thereof).

[0194] Structural features of known non-human or human antibodies (e.g., murine or non-rodent anti-human PSGL-1 antibodies) can be used to generate structurally related human anti-human PSGL-1 antibodies that retain at least one functional property of the antibodies encompassed by the present invention, such as binding of PSGL-1. Another functional property includes inhibiting binding of the original known non-human or human antibody in a competitive ELISA assay.

[0195] In some embodiments, antibodies capable of binding to human PSGL-1, and antigen-binding fragments thereof, are provided, comprising a heavy chain whose variable domains include at least CDRs having sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the group of heavy chain variable domain CDRs presented in Table 2.

[0196] Also provided are antibodies capable of binding to human PSGL-1, and antigen-binding fragments thereof, comprising a light chain whose variable domains include at least CDRs having sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the group of light chain variable domain CDRs presented in Table 2.

[0197] Also provided are antibodies capable of binding to human PSGL-1, and antigen-binding fragments thereof, wherein the variable domains comprise at least a heavy chain comprising a CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the group of heavy chain variable domain CDRs presented in Table 2, and at least a light chain comprising a CDR having a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the group of light chain variable domain CDRs presented in Table 2.

[0198] Those skilled in the art will note that such percentage homology may be equivalent to or, alternatively, achieved by introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, e.g., conservative substitutions, within a given CDR of interest.

[0199] The antibodies and antigen-binding fragments thereof encompassed by the present invention comprise a heavy chain whose variable domain comprises at least a CDR having a sequence selected from the group consisting of the heavy chain variable domain CDRs presented in Table 2, and a light chain whose variable domain comprises at least a CDR having a sequence selected from the group consisting of the light chain variable domain CDRs presented in Table 2.

[0200] Such antibodies and antigen-binding fragments thereof may comprise a light chain whose variable domain comprises at least a CDR having a sequence selected from the group consisting of CDR-L1, CDR-L2, and CDR-L3 described herein, and / or a heavy chain whose variable domain comprises at least a CDR having a sequence selected from the group consisting of CDR-H1, CDR-H2, and CDR-H3 described herein. In some embodiments, antibodies and antigen-binding fragments thereof capable of binding to human PSGL-1 comprise or consist of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 described herein.

[0201] The heavy chain variable domain of the antibodies and antigen-binding fragments thereof encompassed by the present invention may comprise or consist of a vH amino acid sequence shown in Table 2, and / or the light chain variable domain of the antibodies and antigen-binding fragments thereof encompassed by the present invention may comprise or consist of a vκ amino acid sequence shown in Table 2.

[0202] Antibodies and antigen-binding fragments thereof encompassed by the present invention can be produced and modified by any technique known in the art. For example, such antibodies and antigen-binding fragments thereof can be murine or non-rodent antibodies. Similarly, such antibodies and antigen-binding fragments thereof can be chimeric, preferably chimeric mouse / human antibodies. In some embodiments, antibodies and antigen-binding fragments thereof are humanized antibodies, such that the variable domains comprise human acceptor framework regions and, optionally, human constant domains, and non-human donor CDRs, such as the murine or non-rodent CDRs described above.

[0203] In other embodiments, the immunoglobulin heavy and / or light chains according to the invention comprise or consist of a vH or vκ variable domain sequence, respectively, provided in Table 2.

[0204] The invention further provides polypeptides having a sequence selected from the group consisting of a vH variable domain, a vκ variable domain, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences described herein. The antibodies, immunoglobulins, and polypeptides of the invention may be used in isolated (e.g., purified) form or may be contained in a vector, such as a membrane or lipid vesicle (e.g., liposome).

[0205] Several modifications, fragments, etc. are further contemplated.

[0206] The term "antibody" or "Ab" is used broadly and specifically includes, but is not limited to, whole antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact, trispecific, or more highly multispecific antibodies), naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE), and recombinant antibodies, antibody fragments, bispecific antibodies, antibody variants, and antibody-derived binding domains that are part of or associated with other peptides. Antibodies are primarily amino acid-based molecules but can also include one or more modifications (including, but not limited to, the addition of sugar moieties, fluorescent moieties, chemical tags, etc.). In some cases, antibodies can include non-amino acid-based molecules. Antibodies encompassed by the present invention can be naturally occurring or bioengineered.

[0207] Antibodies and antigen-binding fragments thereof may be isolated. As used herein, the term "isolated antibody" is intended to refer to an antibody composition (e.g., having a desired antigen specificity) that is substantially free of other antibodies (e.g., those with different antigen specificities) (e.g., an isolated antibody that binds to PSGL-1 and is substantially free of antibodies that do not bind to PSGL-1). However, in some embodiments, an isolated antibody that specifically binds to PSGL-1 may, however, have cross-reactivity with other proteins of interest, such as those from different family members, species, etc. For example, in some embodiments, the antibody maintains specific binding affinity for at least two species, such as humans and other animals, e.g., non-rodents, or other mammalian or non-mammalian species. However, in some embodiments, the antibody maintains a higher or indeed specific affinity and / or selectivity for human PSGL-1. As noted above, such differential or cross-linking may be, for example, about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.5 fold, 3.0 fold, 3.5 fold, 4.0 fold, 4.5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 35 fold, The fold difference compared to the control can be measured as 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, or more, or any range therebetween, such as about 1.5x to about 100x different compared to the control. Additionally, isolated antibodies are typically substantially free of other cellular material and / or chemicals. In one embodiment, a combination of "isolated" monoclonal antibodies with different specificities for human PSGL-1 are combined in a well-defined composition.

[0208] In some embodiments, an antibody or antigen-binding fragment thereof may comprise heavy and light variable domains and an Fc region. The term "Fc region" is generally used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human PSGL-1 IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. Native-sequence Fc regions suitable for use in antibodies encompassed by the present invention include human IgG1, IgG2 (such as IgG2A and IgG2B), IgG3, and IgG4.

[0209] The term "native antibody" refers to a heterotetrameric glycoprotein, usually about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes (e.g., IgG, IgA, IgE, IgM). Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain aligned with the variable domain of the heavy chain. The remaining constant domains of the two heavy chains of an antibody comprise the fragment crystallizable (Fc) region of the antibody.

[0210] The Fc region in the tail region of an antibody interacts with cell surface receptors called Fc receptors and several proteins of the complement system. Generally, the terms "Fc receptor" or "FcR" describe receptors that bind to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Furthermore, a preferred FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0211] The term "light chain" refers to a component of antibodies from any vertebrate species that are assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of the constant domain. Depending on the amino acid sequence of the constant domain of their heavy chain, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The CL of an antibody, such as a human or human chimeric antibody, can be any region belonging to an Ig class, such as the kappa or lambda class.

[0212] The term "variable domain" refers to specific antibody domains on both the heavy and light chains of an antibody that vary significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. For example, the term "VH" refers to a "heavy chain variable domain" and the term "VL" refers to a "light chain variable domain." Variable domains are composed of hypervariable regions. The term "hypervariable region" refers to the regions within a variable domain that contain amino acid residues involved in antigen binding. These regions are hypervariable in sequence and / or form structurally defined loops. The amino acids present within the hypervariable regions determine the structure of the complementarity-determining regions (CDRs), which are part of the antigen-binding site of the antibody. Generally, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity among these six HVRs, and H3 in particular is thought to play a unique role in conferring excellent specificity to antibodies (see, e.g., Xu et al. (2000) Immunity 13, 37-45; Johnson and Wu (2003) Meth. Mol. Biol. 248:1-25). The term "CDR" refers to the region of an antibody that contains the structure complementary to its target antigen or epitope.

[0213] The remaining portions of the variable domain that do not interact with the antigen are referred to as framework (FW) regions. The antigen-binding site (also known as the antigen-binding site or paratope) contains the amino acid residues necessary for interacting with a specific antigen. The exact residues that make up the antigen-binding site are usually elucidated by co-crystallography with the bound antigen, although computational evaluation based on comparison with other antibodies can also be used (Strohl, WR Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54). The residues that make up the CDRs can be determined using, but are not limited to, the methods described by Kabat (Wu et al. (1970) JEM 132:211-250; Kabat et al. (1992), "Sequences of Proteins of Immunological Interest," 5 thEdition,USDepartment of Health and Human Services;Johnson et al.(2000)Nucl.Acids Res.28:214-218), Chothia(Chothia and Lesk(1987)J.Mol.Biol.196:901;Chothia et al.(1989)Nature 342:877;Al-Lazikani et al. (1997) J. Mol. Biol. 273:927-948), Lefranc (Lefranc et al. (1995) Immunome Res. 1:3), Honegger (Honegger and Pluckthun (2001) J. Mol. Biol. 309:657-670), and MacCallum (MacCallum et al. (1996) J. Mol. Biol. 262:732). Accordingly, CDR definitions according to these systems may vary in length and boundary regions with respect to the adjacent framework regions. See, e.g., Kabat, Chothia, and / or MacCallum et al. (Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th Edition, USDapartment of Health and Human Services, 1992; Chothia et al. (1987) J. Mol. Biol. 196, 901; and MacCallum et al., J. Mol. Biol. (1996) 262, 732, each of which is incorporated by reference in its entirety).

[0214] Each VH domain and VL domain has three CDRs. The VL CDRs are referred to herein as CDR-L1, CDR-L2, and CDR-L3, in the order in which they occur when moving from N- to C-terminus along the variable domain polypeptide. The VH CDRs are referred to herein as CDR-H1, CDR-H2, and CDR-H3, in the order in which they occur when moving from N- to C-terminus along the variable domain polypeptide. With the exception of CDR-H3, each CDR adheres to a canonical structure, which contains amino acid sequences that can vary greatly in sequence and length between antibodies, conferring diverse three-dimensional structures to the antigen-binding domain (Nikoloudis et al. (2014) Peer J. 2:e456). In some cases, CDR-H3 can be analyzed across a panel of related antibodies to assess antibody diversity. Methods for determining CDR sequences are readily known in the art and can be applied to known antibody sequences (Strohl, WR Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54).

[0215] Antibodies and antigen-binding fragments thereof described herein include, but are not limited to, those containing CDRs defined according to Chothia CDRs, Kabat CDRs, AbM, CDR contact region, and / or conformational definitions. Determining CDR regions is within the skill of the art. It is understood that in some embodiments, a CDR may be a combination of Kabat and Chothia CDRs (also referred to as a "combined CDR" or "extended CDR"). In some embodiments, a CDR is a Kabat CDR. In other embodiments, a CDR is a Chothia CDR. In some embodiments, a CDR is an extended CDR, which refers to all amino acid residues identified according to the Kabat and Chothia nomenclature. Thus, in some embodiments having more than one CDR, one or more CDRs may be either a Kabat, Chothia, extended CDR, or a combination thereof.

[0216] In some embodiments, antibody fragments and variants can comprise any portion of an intact antibody. The terms "antibody fragment" and "antibody variant" also include any synthetic or genetically engineered protein / polypeptide that acts like an antibody by binding to a specific antigen to form a complex. In some embodiments, antibody fragments and variants comprise the antigen-binding region from an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments; Fd, diabodies; intracellular antibodies, linear antibodies; single-chain antibody molecules, e.g., single-chain variable fragments (scFv); and multispecific antibodies formed from antibody fragments. Regardless of structure, an antibody fragment or variant binds to the same antigen recognized by the parent full-length antibody.

[0217] Antibody fragments generated by limited proteolysis of wild-type antibodies are referred to as proteolytic antibody fragments. These include, but are not limited to, Fab fragments, Fab' fragments, and F(ab')2 fragments. Papain digestion of antibodies generates two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. A residual "Fc" fragment is further generated, the name reflecting its ability to readily crystallize. Pepsin or ficin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen. Generally, an F(ab')2 fragment contains two "arms," ​​each of which contains a variable region that specifically binds to a common antigen. Two Fab' molecules are linked by interchain disulfide bonds at the hinge region of the heavy chains, and the Fab' molecules can be directed against the same (bivalent) or different (bispecific) epitopes. As used herein, a "Fab" fragment contains a single anti-binding domain that includes the Fab and an additional portion of the heavy chain through the hinge region. Compounds and / or compositions encompassed by the present invention can contain one or more of these fragments.

[0218] The term "Fv" refers to an antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. Fv fragments can be generated by proteolytic cleavage, but most are unstable. Recombinant methods known in the art for generating stable Fv fragments typically involve inserting a flexible linker between the light- and heavy-chain variable domains (to form single-chain Fvs (scFvs)) or introducing disulfide bridges between the heavy and light-chain variable domains (Strohl, W. R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia, PA. 2012. Ch. 3, p. 46-47).

[0219] The term "single-chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains, which are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker allows the scFv to form the desired structure for antigen binding. In some embodiments, the VH and VL domains can be linked by a peptide of 10-30 amino acid residues. In some embodiments, scFvs are utilized in conjunction with phage display, yeast display, or other display methods, where they are expressed in association with a surface member (e.g., a phage coat protein) and can be used to identify high-affinity peptides against a specific antigen. In some embodiments, the term "single-chain antibody" can further include, but is not limited to, disulfide-linked Fvs (dsFvs), in which two single-chain antibodies (each of which can be directed against a different epitope) are linked together by a disulfide bond. Using molecular genetics, two scFvs can be engineered in tandem on a single polypeptide separated by a linker domain, termed a "tandem scFv" (tascFv). Constructing a tascFv using the genes of two different scFvs results in a "bispecific single-chain variable fragment" (bis-scFv) (Nelson (2010) Mabs 2:77-83). Maxibodies (bivalent scFvs fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG) can also be included.

[0220] In some embodiments, the antibody can comprise a modified Fc region. By way of non-limiting example, the modified Fc region can be produced by the methods described in U.S. Patent Publication No. US 2015-0065690, or can be any of a range of.

[0221] Antibodies and antigen-binding fragments encompassed by the present invention may be "recombinant," a term that includes antibodies and antigen-binding fragments thereof that are prepared, expressed, produced, or isolated by recombinant means, such as, for example, (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., antibodies isolated from transfectomas, (c) antibodies isolated from recombination, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus modifying the V and constant regions of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0222] The term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as, for example, (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., antibodies isolated from transfectomas, (c) antibodies isolated from recombination, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus modifying the V and constant regions of the recombinant antibody. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0223] The term "polyclonal antibody" includes antibodies generated in an immunogenic response to a protein with many epitopes. Thus, a polyclonal antibody composition (e.g., serum) contains a variety of different antibodies directed against the same and different epitopes within the protein. Methods for generating polyclonal antibodies are readily known in the art (see, e.g., Cooper et al., Section III of Chapter 11: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pp. 11-37 to 11-41).

[0224] In contrast, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and / or bind the same particular epitope on the antigen, except for possible variants that may arise during the generation of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0225] The term "antibody variant" refers to a modified antibody (with respect to a natural or starting antibody) or a biomolecule that resembles the natural or starting antibody in structure and / or function, including some differences in amino acid sequence, composition, or structure compared to the natural or starting antibody (e.g., an antibody mimetic). Antibody variants can have altered amino acid sequence, composition, or structure compared to the natural antibody. Antibody variants include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments. For example, variant constant chain regions are contemplated, such as a mutant IgG4 with a substitution at Ser228, e.g., S228P.

[0226] In some embodiments, antibodies encompassed by the present invention can include antibody fusion proteins. As used herein, the term "antibody fusion protein" refers to a recombinantly produced antigen-binding molecule that links two or more of the same or different natural antibodies, single-chain antibodies, or antibody fragment segments with the same or different specificities. The valency of the fusion protein refers to the total number of binding arms or sites that the fusion protein has for an antigen or epitope, i.e., monovalent, bivalent, trivalent, or multivalent. The multivalency of an antibody fusion protein means that it can utilize multiple interactions in binding to an antigen, thus increasing the avidity of antigen binding. The specificity refers to the number of different antigens or epitopes that the antibody fusion protein can bind, i.e., monospecific, bispecific, trispecific, multispecific, etc. Using these definitions, for example, a natural antibody such as an IgG is bivalent because it has two binding arms, but monospecific because it binds to a single antigen. Monospecific, multivalent fusion proteins have more than one binding site for an epitope, but only bind to the same epitope on the same antigen, e.g., bispecific antibodies, which have two binding sites reactive with the same antigen. Fusion proteins can contain multivalent or multispecific combinations of different antibody components, or multiple copies of the same antibody component. Fusion proteins can further contain a therapeutic agent. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion proteins") and toxins ("antibody-toxin fusion proteins"). One preferred toxin comprises a ribonuclease (RNase), preferably a recombinant RNase.

[0227] In some embodiments, antibodies encompassed by the present invention can include multispecific antibodies. As used herein, the term "multispecific antibody" refers to an antibody that binds to multiple epitopes. As used herein, the term "multibody" or "multispecific antibody" refers to an antibody in which two or more variable regions bind different epitopes. The epitopes can be on the same or different targets. In one embodiment, multispecific antibodies can be generated and optimized by the methods described in PCT Publication No. WO2011 / 109726 and U.S. Patent Publication No. 2015-0252119. These antibodies can bind to multiple antigens with high specificity and high affinity. In some embodiments, multispecific antibodies are bispecific antibodies. As used herein, the term "bispecific antibody" refers to an antibody capable of binding to two different epitopes on the same or different antigens. In one aspect, bispecific antibodies are capable of binding to two different antigens. Such antibodies typically comprise antigen-binding regions from at least two different antibodies. For example, bispecific monoclonal antibodies (BsMAbs, BsAbs) are artificial proteins composed of fragments of two different monoclonal antibodies, allowing BsAbs to bind to two different types of antigens. Bispecific antibodies can include any of those described in Riethmuller (2012) Cancer Immun. 12:12-18, Marvin et al. (2005) Acta Pharmacol. Sinica 26:649-658, and Schaefer et al. (2011) Proc. Natl. Acad. Sci. USA 108:11187-11192. A new generation of BsMAbs, called "trifunctional bispecific" antibodies, has been developed. These are composed of two heavy chains and two light chains, one each from two different antibodies, with two Fab regions (arms) directed against two antigens and an Fc region (foot) containing two heavy chains and forming a third binding site.

[0228] In some embodiments, compositions encompassed by the present invention can include anti-peptide antibodies. As used herein, the term "anti-peptide antibodies" refers to "monospecific antibodies" generated in a humoral response to short (usually 5-20 amino acids) immunogenic polypeptides corresponding to several (preferably one) isolated epitopes of the protein from which they are derived (e.g., a target protein encompassed by the present invention). Multiple anti-peptide antibodies include a variety of different antibodies directed against specific portions of the protein, i.e., amino acid sequences containing at least one, and preferably only one, epitope. Methods for generating polyclonal antibodies are readily known in the art (see, e.g., Cooper et al., Section III of Chapter 11: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pp. 11-42 to 11-46).

[0229] In some embodiments, antibodies encompassed by the present invention can include bispecific antibodies. As used herein, the term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites. Bispecific antibodies comprise a heavy chain variable domain, VH, connected to a light chain variable domain, VL, in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be paired with complementary domains on another chain to generate two antigen-binding sites. Bispecific antibodies are described in further detail, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0230] In some embodiments, antibodies encompassed by the present invention can include intracellular antibodies. The term "intracellular antibody" refers to a form of antibody that is not secreted from the cell in which it is produced, but instead targets one or more intracellular proteins. Intracellular antibodies are a well-known type of antigen-binding molecule that has the characteristics of an antibody but can be expressed intracellularly to bind to and / or inhibit a desired intracellular target (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies to target (e.g., inhibit) intracellular moieties are well known in the art, such as the use of single-chain antibodies (scFv), modification of immunoglobulin VL domains for hyperstability, modification of antibodies to resist reduced intracellular environments, and generation of fusion proteins to enhance intracellular stability and / or modulate intracellular localization. Intracellular antibodies can also be introduced into and expressed in one or more cells, tissues, or organs of a multicellular organism, for example, for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (see, e.g., at least PCT Publication Nos. WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960; U.S. Patent No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0231] Intrabodies can be used to affect numerous cellular processes, including, but not limited to, intracellular trafficking, transcription, translation, metabolic processes, growth signaling, and cell division. In some embodiments, methods encompassed by the present invention can include intracellular antibody-based therapy. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein can be incorporated into one or more constructs for intracellular antibody-based therapy. For example, intracellular antibodies can target one or more glycosylated intracellular proteins or modulate the interaction between one or more glycosylated intracellular proteins and alternative proteins. Intracellular expression of intracellular antibodies in different compartments of mammalian cells allows for the blocking or modulation of the function of endogenous molecules (Biocca et al. (1990) EMBO J. 9:101-108; Colby et al. (2004) Proc. Natl. Acad. Sci. USA101:17616-17621). Intrabodies can alter protein folding, protein-protein, protein-DNA, and protein-RNA interactions, and protein modifications. They can induce phenotypic knockouts and act as neutralizing agents by directly binding to target antigens, altering their intracellular trafficking, or inhibiting their association with binding partners. Because of their high specificity and affinity for target antigens, intrabodies have the advantage of blocking specific binding interactions of specific target molecules while sparing others. Intrabodies can be developed using sequences from donor antibodies. Intrabodies are often recombinantly expressed as single-domain fragments, such as isolated VH and VL domains, or as intracellular single-chain variable fragment (scFv) antibodies. For example, intrabodies are often expressed as a single polypeptide to form a single-chain antibody containing the variable domains of the heavy and light chains connected by a flexible linker polypeptide. Intrabodies typically lack disulfide bonds and can regulate the expression or activity of target genes through their specific binding activity. Single-chain intracellular antibodies are often expressed from recombinant nucleic acid molecules and engineered to be retained intracellularly (eg, in the cytoplasm, endoplasmic reticulum, or periplasm). Intracellular antibodies are, for example, Marasco et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893, Chen et al. (1994) Hum. Gene Ther.5:595-601, Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936, Maciejewski et al. (1995) Nat. Med.1:667-673, Marasco (1995) Immunotech.1:1-19, Mhashilkar et al. (1995) EMBO J.14:542-1451, Chen et al. (1996) Hum. Gene Therap.7:1515-1525, Marasco (1997) Gene Ther.4:11-15, London and Marasco (1997) Annu.Rev.Microbiol.51:257-283, Cohen et al. (1998) Oncogene 17:2445-2456, Proba et al. (1998) J. Mol. Biol. 275:245-253, Cohen et al. (1998) Oncogene 17:2445-2456, Hassanzadeh et al. (1998) FEBS Lett.437:81-86, Richardson et al. (1998) Gene Ther.5:635-644, Ohage and Steipe (1999) J.Mol.Biol.291:1119-1128, Ohage et al. (1999) J.Mol.Biol.291:1129-1134, Wirtz and Steipe(1999)Protein Sci. 8:2245-2250, Zhu et al. (1999) J. Immunol. Methods 231:207-222, Arafat et al. (2000) Cancer Gene Ther. 7:1250-1256, der Maur et al. (2002) J. Biol. Chem. 277:45075-45085, Mhashilkar et al. (2002) Gene Ther. 9:307-319, and Wheeler et al. (2003) FASEB J. 17:1733-1735).

[0232] In some embodiments, antibodies encompassed by the present invention can include chimeric antibodies. As used herein, the term "chimeric antibody" refers to recombinant antibodies in which a portion of the heavy and light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855). For example, chimeric antibodies of interest herein can include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences.

[0233] In some embodiments, antibodies encompassed by the present invention are composite antibodies. As used herein, the term "composite antibody" refers to an antibody having a variable region comprising germline or non-germline immunoglobulin sequences from two or more unrelated variable regions. Additionally, the term "composite, human antibody" refers to an antibody having a constant region derived from human germline or non-germline immunoglobulin sequences and a variable region comprising human germline or non-germline sequences from two or more unrelated human variable regions. Composite, human antibodies are useful as active ingredients in therapeutic agents according to the present invention due to the reduced antigenicity of composite, human antibodies in the human body.

[0234] In some embodiments, antibodies encompassed by the present invention may include xenogenous antibodies. The term "xenogenous antibody" is defined with respect to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence that corresponds to that found in an organism that is not the transgenic non-human animal, and is generally from a species other than that of the transgenic non-human animal.

[0235] In some embodiments, antibodies encompassed by the present invention may be humanized antibodies. As used herein, the term "humanized antibody" refers to a chimeric antibody containing minimal portions derived from one or more non-human (e.g., murine) antibody sources and remainders derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region from the recipient antibody are replaced by residues from a hypervariable region from an antibody of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In one embodiment, the antibody may be a humanized full-length antibody. Humanized antibodies can be generated using protein engineering techniques (e.g., Gussow and Seemann (1991) Meth. Enzymol. 203:99-121). As a non-limiting example, antibodies can be humanized using the methods taught in U.S. Patent Publication No. 2013 / 0303399. As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0236] As used herein, a humanized mouse is a mouse carrying functional human genes, cells, tissues, and / or organs. Humanized mice are commonly used as small animal models in biological and medical research for human therapeutics. Nude mice and severe combined immunodeficiency (SCID) mice may be used for this purpose. NCG, NOG, and NSG mice can be used to engraft human cells and tissues more efficiently than other models. Such humanized mouse models can be used to model the human immune system in healthy and pathological scenarios, allowing for the evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

[0237] In some embodiments, antibodies encompassed by the present invention can include cysteine-modified antibodies. In "cysteine-modified antibodies," a cysteine ​​amino acid is inserted or substituted into the surface of the antibody by genetic engineering and is used, for example, to bind the antibody to another molecule via a disulfide bridge. Cysteine ​​substitution or insertion into antibodies has been described (see, e.g., U.S. Pat. No. 5,219,996). A method for introducing cysteine ​​residues into the constant region of an IgG antibody for use in site-specific antibody conjugation is described in Stimmel et al. (2000) J. Biol. Chem. 275:330445-30450).

[0238] In some embodiments, antibody variants encompassed by the present invention may be antibody mimetics. As used herein, the term "antibody mimetic" refers to any molecule that mimics the function or effect of an antibody and binds specifically and with high affinity to its molecular target. In some embodiments, antibody mimetics may be monobodies designed to incorporate a fibronectin type III domain (Fn3) as a protein scaffold (see U.S. Patent Nos. 6,673,901 and 6,348,584). In some embodiments, antibody mimetics may include any of those readily known in the art, including, but not limited to, affibody molecules, affilins, affitins, anticalins, avimers, centyrins, DARPINS™, finomers, and Kunitz domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.

[0239] In some embodiments, antibodies encompassed by the present invention can comprise a single antigen-binding domain. These molecules are very small, with molecular weights approximately one-tenth that observed for full-sized mAbs. Additional antibodies can include "nanobodies," which are derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, lacking light chains (see, e.g., Nelson (2010) Mabs 2:77-83).

[0240] In some embodiments, antibodies encompassed by the present invention can be "miniaturized." One example of miniaturized mAbs is small modular immunopharmaceuticals (SMIPs). These monovalent or bivalent molecules are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant "effector" domains, all connected by a linker domain (see, e.g., Nelson (2010) Mabs 2:77-83). Such molecules are believed to retain the immune effector functions conferred by constant domains while offering the advantage of increased tissue or tumor penetration required by fragments. Another example of a miniaturized antibody is called a "unibody," in which the hinge region is removed from an IgG4 molecule. While IgG4 molecules are unstable and can interconvert light-heavy chain heterodimers, deleting the hinge region completely prevents heavy-heavy chain pairing, leaving a highly specific monovalent light / heavy chain heterodimer while retaining the Fc region to ensure in vivo stability and half-life. This configuration may minimize the risk of immune activation or increased oncogenicity because IgG4 interacts poorly with FcRs and monovalent unibodies cannot promote the formation of intracellular signaling complexes (see, e.g., Nelson (2010) Mabs 2:77-83).

[0241] In some embodiments, antibody variants encompassed by the present invention may be single domain antibodies (sdAbs, or nanobodies). As used herein, the terms "sdAb" or "nanobody" refer to antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies, they are capable of selectively binding to a specific antigen. In one aspect, an sdAb may be a "camelid Ig" or "camelid VHH." As used herein, the term "camelid Ig" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolté et al. (2007) FASEB J. 21:3490-3498). A "heavy chain antibody" or "camelid antibody" refers to an antibody that comprises two VH domains and no light chains (Hamers-Casterman et al. (1993) Nature 363:446-448 (1993); Sheriff et al. (1996) Nat. Struct. Biol. 3:733-736; Riechmann et al. (1999) J. Immunol. Meth. 231:25-38; PCT Publication Nos. WO1 994 / 04678 and WO1994 / 025591; and U.S. Patent No. 6,005,079). In another aspect, the sdAb may be an "immunoglobulin novel antigen receptor" (IgNAR). The term "immunoglobulin novel antigen receptor" refers to a class of antibodies from the shark immune repertoire consisting of a homodimer of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and possess highly stable and efficient binding properties. Their inherent stability can be attributed to both (i) the underlying Ig scaffold, which displays a significant number of charged and hydrophilic surface-exposed residues compared to traditional antibody VH and VL domains found in murine antibodies, and (ii) stabilizing structural features in the complementarity-determining region (CDR) loops, including interloop disulfide bridges and interloop hydrogen bonding patterns. Other miniaturized antibody fragments can include "complementarity-determining region peptides" or "CDR peptides."CDR peptides (also known as "minimal recognition units") are peptides corresponding to a single complementarity-determining region (CDR) and can be prepared by constructing a gene encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, e.g., Larrick et al. (1991) Methods Enzymol. 2:106).

[0242] Other variants comprising antigen-binding fragments of antibodies include, but are not limited to, disulfide-linked Fvs (sdFvs), VFvs, and VFvs. L , V H , camelid Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3)2), bispecific single chain Fv (Bis-scFv), IgG delta CH2, scFv-Fc, (scFv)2-Fc, affibody, peptide aptamer, avimer, or nanobody, or other antigen-binding subsequence of an intact immunoglobulin.

[0243] In some embodiments, antibodies encompassed by the present invention may be those described in U.S. Patent No. 5,091,513. Such antibodies may comprise one or more sequences of amino acids that constitute a region that behaves as a biosynthetic antibody combining site (BABS). These sites include: 1) noncovalently or disulfide-linked synthetic VH and VL dimers; 2) VH-VL or VL-VH single chains in which the VH and VL are linked by a polypeptide linker; or 3) individual VH or VL domains. A binding domain comprises linked CDR and FR regions, which may be derived from separate immunoglobulins. Biosynthetic antibodies may also contain other polypeptide sequences that function, for example, as enzymes, toxins, binding sites, or attachment sites for immobilization media or radioactive atoms. Methods for producing biosynthetic antibodies, for designing BABS with any specificity that can be elicited by in vivo antibody generation, and for producing analogs thereof are disclosed.

[0244] In some embodiments, antibodies encompassed by the present invention may be antibodies having antibody acceptor frameworks as taught in U.S. Patent No. 8,399,625. Such antibody acceptor frameworks may be particularly suitable for accepting CDRs from an antibody of interest.

[0245] In one embodiment, the antibody can be a conditionally active biological protein.Antibodies can be used to generate conditionally active biological proteins that are reversibly or irreversibly inactivated under normal wild-type physiological conditions, and uses of such conditionally active biological proteins are provided.Such methods and conditionally active proteins are taught, for example, in PCT Publication Nos. WO2015 / 175375 and WO2016 / 036916, and U.S. Patent Publication No. 2014 / 0378660.

[0246] In some embodiments, antibodies encompassed by the present invention are therapeutic antibodies. As used herein, the term "therapeutic antibody" refers to an antibody that is effective in treating a disease or disorder in a mammal having or predisposed to the disease or disorder. The antibody may be a cell-permeable antibody, a neutralizing antibody, an agonist antibody, a partial agonist, an inverse agonist, a partial antagonist, or an antagonist antibody.

[0247] In some embodiments, antibodies encompassed by the present invention may be naked antibodies. As used herein, the term "naked antibody" refers to an intact antibody molecule that does not contain further modifications, such as conjugation with a chelate for binding to a toxin or radionuclide. The Fc portion of a naked antibody provides effector functions such as complement fixation and ADCC (antibody-dependent cellular cytotoxicity), mechanisms that can result in cell lysis (see, e.g., Markrides (1998) Pharmacol. Rev. 50:59-87).

[0248] It is well known that antibodies can cause depletion of cells that extracellularly harbor the antigen specifically recognized by the antibody. This depletion can be mediated through at least three mechanisms: antibody-mediated cytotoxicity (ADCC), complement-dependent lysis, and direct anti-tumor inhibition of tumor growth via signals provided through the antigen targeted by the antibody.

[0249] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies that bind to their cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al. (1997), can be performed.

[0250] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted antibodies that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to and subsequently kill antigen-bearing target cells. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, can be performed. As is well known in the art, the Fc portion can be engineered to provide a desired interaction, or lack thereof, with an Fc receptor.

[0251] Fc receptors are found on many cells involved in the immune response. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). Among the human FcRs identified to date are those that recognize IgG (termed FcγR), IgE (FcεR1), IgA (Fcα), and polymerized IgM / A (FcμαR). FcRs are found on the following cell types: FcεRI (mast cells), FcεR.II (many leukocytes), FcαR (neutrophils), and FcμαR (glandular epithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9:185-86). The extensively studied FcγRs are central to cellular immune defense and are responsible for stimulating the release of mediators of inflammation and hydrolytic enzymes involved in the pathogenesis of autoimmune diseases (Unkeless, JC et al. (1988) Annu. Rev. Immunol. 6:251-81). FcγRs provide a critical link between effector cells and Ig-secreting lymphocytes on macrophages / monocytes, polymorphonuclear leukocytes, and natural killer (NK) cells, as FcγRs confer elements of specific recognition mediated by IgG. Human leukocytes possess at least three distinct receptors for IgG: hFcγ RI (found on monocytes / macrophages), hFcγ RII (monocytes, neutrophils, eosinophils, platelets, and occasionally B cells and the K562 cell line), and Fcγ III (NK cells, neutrophils, eosinophils, and macrophages).

[0252] In some embodiments, antibodies encompassed by the present invention can be conjugated to one or more detectable labels for the purpose of detection by methods well known in the art. The label can be a radioisotope, a fluorescent compound, a chemiluminescent compound, an enzyme, or an enzyme cofactor, or any other label readily known in the art. In some embodiments, an antibody that binds to a desired target (also referred to herein as a "primary antibody") can be detected by binding of a secondary antibody (also referred to herein as a "secondary antibody") that is unlabeled but specifically binds to the primary antibody. According to such methods, the secondary antibody can contain a detectable label.

[0253] In some embodiments, enzymes that can be attached to antibodies can include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase, and glucose oxidase (GOx). Fluorescent compounds can include, but are not limited to, ethidium bromide. Examples of conjugates that can be used to label antibodies include fluorescein and its derivatives (e.g., FITC), cyanine and its derivatives (e.g., indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), rhodamine, Oregon Green, eosin, Texas Red, Nile Red, Nile Blue, cresyl violet, oxazine 170, proflavine, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphine, phthalocyanine, bilirubin, allophycocyanin (APC), green fluorescent protein (GFP) and its variants (e.g., yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and cyan fluorescent protein (CFP)), ALEXIFLOUR® compound (Thermo Fisher Scientific, Waltham, MA), and quantum dots. Other conjugates that can be used to label antibodies include biotin, avidin, and streptavidin.

[0254] For example, conjugates of antibodies or other proteins encompassed by the present invention with heterologous agents can be made using a variety of bifunctional protein coupling agents, including, but not limited to, N-succinimidyl(2-pyridyldithio)propionate (SPDP), succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6 diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for attaching radionucleotides to antibodies (WO 94 / 11026).

[0255] In another aspect, the invention features antibodies that specifically bind to a biomarker of interest conjugated to a therapeutic moiety, such as a cytotoxin, a drug, and / or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxic or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or congeners thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin)), bleomycin, mithramycin, and anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine and vinblastine). Antibodies encompassed by the present invention may be conjugated with radioisotopes, eg, radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating related disorders such as cancer.

[0256] Conjugated anti-biomarker antibodies can be used diagnostically or prognostically to monitor polypeptide levels in tissues as part of a clinical trial procedure, e.g., to determine the effectiveness of a given treatment regimen or to select patients most likely to respond to immunotherapy. For example, cells can be permeabilized in a flow cytometry assay so that antibodies that bind to the biomarker of interest target its recognized intracellular epitope and binding can be detected by analyzing the signal emitted by the bound molecule. Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin (PE); examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and 125 I, 131 I, 35 S, or 3 Examples of radioactive substances include H. As used herein, the term "labeled" with respect to an antibody is intended to encompass direct labeling of the antibody by conjugating (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the antibody, as well as indirect labeling of the antibody by reactivity with a detectable substance.

[0257] The antibody conjugates encompassed by the present invention can be used to modify a given biological response. The therapeutic moiety should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins include, for example, enzymatically active toxins, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, or active fragments thereof; or biological response modifiers, such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other cytokines or growth factors.

[0258] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic See "Use Of Radiolabeled Antibodies In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0259] In some embodiments, the conjugate can be made using a "cleavable linker" that facilitates release of the cytotoxic agent or growth inhibitory agent in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (see, e.g., U.S. Pat. No. 5,208,020) can be used. Alternatively, a fusion protein comprising an antibody and a cytotoxic agent or growth inhibitory agent can be made by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding the two portions of the conjugate adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0260] In some embodiments, the present invention encompasses antibody-drug conjugate (ADC) agents. An ADC is a conjugate of an antibody and another moiety, such that the drug has the targeting capability conferred by the antibody and an additional effect conferred by the moiety. For example, a cytotoxic agent can be tethered to an antibody or its antigen-binding fragment, which targets the drug to cells of interest that contribute to disease progression (e.g., tumor progression) and releases its toxic payload into the cells upon internalization. As noted above, different effects can be achieved based on the conjugated moiety.

[0261] In some embodiments, further modifications and changes can be made in the structure of antibodies (and antigen-binding fragments thereof) and the DNA sequences encoding them to obtain functional molecules that still encode antibodies and polypeptides with desired characteristics. For example, certain amino acids can be substituted by other amino acids in the protein structure without appreciable loss of activity. Because the interaction capabilities and properties of a protein determine its biological functional activity, certain amino acid substitutions can be made in the protein sequence, and of course, its DNA coding sequence, to nevertheless obtain proteins with similar properties. Thus, it is contemplated that various changes can be made in the antibody sequences of the present invention, or the corresponding DNA sequences encoding the polypeptides, without appreciable loss of their biological activity.

[0262] In one embodiment, amino acid changes can be achieved by altering codons in a DNA sequence to encode conservative substitutions based on the conservation of the genetic code. Specifically, as defined by the genetic code (shown below), there is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein. Similarly, as defined by the genetic code, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid (see genetic code chart above).

[0263] As mentioned above, an important and well-known feature of the genetic code is its redundancy, which allows more than one coding nucleotide triplet to be used for most amino acids used to make proteins (as shown above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others). Furthermore, occasionally, methylated variants of purines or pyrimidines can be found within a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0264] When making changes to the amino acid sequence of a polypeptide, the hydrophobic-hydrophilic index of the amino acids may be considered. The importance of the hydrophobic-hydrophilic index amino acid index in imparting an interactive biological function to a protein is generally understood in the art. The relative hydrophobic-hydrophilic index characteristics of amino acids contribute to the resulting protein's secondary structure and are allowed to define the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophobic-hydrophilic index based on their hydrophobic and charge characteristics, which are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (<RTI 3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0265] It is known in the art that a particular amino acid may be substituted by other amino acids having a similar hydrophobic-hydrophilic index or score, still resulting in a protein having a similar biological activity, i.e., still obtaining a biologically functionally equivalent protein.

[0266] Thus, as outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various foregoing characteristics are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0267] Another type of amino acid modification of the antibodies of the invention may be useful to alter the original glycosylation pattern of the antibody, for example, to increase stability. By "modifying" is meant deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically N-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 and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically attaching glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell with glycosylation capabilities for N- or O-linked glycosylation. Depending on the conjugation mode used, the sugar(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO 87 / 05330.

[0268] Similarly, removal of any carbohydrate moieties present on an antibody can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposing the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described in Sojahr H. et al. (1987) and Edge, A S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of various endo- and exoglycosidases as described in Thotakura, N R. et al. (1987).

[0269] Other modifications may involve the formation of immunoconjugates. For example, in one type of covalent modification, antibodies or proteins can be covalently bound to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0270] b.Antibody engineering As noted above, techniques that can be used to produce antibodies and antibody fragments, such as Fabs and scFvs, are well known in the art and include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, Miersch et al. (2012) Methods 57:486-498, Chao et al. (2006) Nat. Protoc. 1:755-768), Huston et al. (1991) Methods Enzymol. 203:46-88, Shu et al. (1993) Proc. Natl. Acad. Sci. USA 90:7995-7999, and Skerra et al. (1988) Science 240:1038-1041). Representative examples of engineered antibodies are described herein, including those with altered residues amenable to chemical modification, as well as those with useful sequence modifications (e.g., CDR sequences that more closely resemble human germline sequences). Such antibody variants are encompassed by the present invention.

[0271] After isolation or selection of target antigen-specific antibodies, the antibody sequences can be used for recombinant production and / or optimization of such antibodies. When antibody fragments are isolated from a display library, for example, as described in detail below, coding regions from the isolated fragments can be used to generate whole antibodies, including human antibodies or any other desired target-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. If desired, IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4) can be synthesized for further testing and / or product development from the variable domain fragments generated or selected according to the methods described herein. Such antibodies can be produced by inserting one or more segments of a cDNA encoding the desired amino acid sequence into an expression vector suitable for IgG production. The expression vector can include a mammalian expression vector suitable for IgG expression in mammalian cells. Mammalian expression of IgG can be performed to ensure that the produced antibodies contain modifications characteristic of mammalian proteins (e.g., glycosylation) and / or to ensure that the antibody preparation lacks endotoxins and / or other contaminants that may be present in protein preparations from bacterial expression systems.

[0272] In some embodiments, affinity maturation is performed. The term "affinity maturation" refers to a method of producing antibodies with increased affinity for a predetermined target through successive rounds of mutation and selection of cDNA sequences encoding the antibody or antibody fragment. In some cases, this process is performed in vitro. To accomplish this, amplification of variable domain sequences (in some cases limited to CDR-encoding sequences) can be performed using error-prone PCR to generate millions of copies containing mutations, including, but not limited to, point mutations, local mutations, insertion mutations, and deletion mutations. As used herein, the term "point mutation" refers to a nucleic acid mutation in which one nucleotide in a nucleotide sequence is changed to a different nucleotide. As used herein, the term "local mutation" refers to a nucleic acid mutation in which two or more consecutive nucleotides are changed to different nucleotides. As used herein, the term "insertion mutation" refers to a nucleic acid mutation in which one or more nucleotides are inserted into a nucleotide sequence. As used herein, the term "deletion mutation" refers to a nucleic acid mutation in which one or more nucleotides are removed from a nucleotide sequence. Insertion or deletion mutations involve the complete replacement of the entire codon or the change of one codon to another by modifying one or two nucleotides in the start codon.

[0273] Mutagenesis can be performed on the cDNA sequences encoding the CDRs to generate millions of variants with specific mutations in the CDR regions of the heavy and light chains. In another approach, random mutations are introduced only at the CDR residues most likely to improve affinity. These newly generated mutagenic libraries can be used in an iterative process of screening for clones encoding antibody fragments with ever-higher affinity for the target peptide. Successive rounds of mutation and selection promote the synthesis of clones with progressively higher affinities (see, e.g., Chao et al. (2006) Nat. Protoc. 1:755-768).

[0274] Affinity-matured clones can be selected based on affinity as determined by binding assays (e.g., FACS, ELISA, surface plasmon resonance, etc.). Selected clones can then be converted to IgG and further tested for affinity and functional activity. In some cases, the goal of affinity optimization is to increase affinity by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1,000-fold or more compared to the affinity of the original antibody. If the optimized affinity is lower than desired, the process can be repeated.

[0275] In some embodiments, it is useful to generate chimeric and / or humanized antibodies. For some uses, including, for example, in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal immunoglobulin and a human immunoglobulin constant region. Methods for generating chimeric antibodies are known in the art (e.g., Morrison (1985) Science 229:1202-1207; Gillies et al. al. (1989) J. Immunol. Meth. 125:191-202, and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397).

[0276] Humanized antibodies are antibody molecules from non-human species that bind to a desired target and have one or more complementarity-determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions are replaced with corresponding residues from the CDRs and framework regions of the donor antibody to alter, preferably improve, target binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions of CDR and framework residues to identify framework residues important for target binding and identifying unusual framework residues at specific positions by sequence comparison (see, e.g., U.S. Patent Nos. 5,693,762 and 5,585,089; Riechmann et al. (1988) Nature 332:323-327).

[0277] Antibodies can be prepared by, for example, CDR grafting (e.g., EP Patent Publication No. 239,400; PCT Publication No. WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering, or resurfacing (e.g., EP Patent Publication No. 592,106; EP Patent Publication No. 519,596; Padlan (1991) Mol. Immunol. 28:489-498; Studnicka et al. (1994) Protein Eng. 7:805-814; Roguska et al. (1994) Protein Eng. 7:805-814). Humanization can be accomplished using a variety of techniques known in the art, including nucleic acid sequencing (see, e.g., U.S. Pat. No. 5,565,332), nucleic acid sequencing (see, e.g., U.S. Pat. No. 5,565,332), and chain recombination (see, e.g., U.S. Pat. No. 5,565,332).

[0278] Fully human antibodies are particularly desirable for therapeutic therapy of human patients to avoid or reduce immune responses to foreign proteins. Human antibodies can be produced by a variety of methods known in the art, including the antibody display methods described above, using antibody libraries derived from human immunoglobulin sequences (see, e.g., U.S. Pat. Nos. 4,444,887 and 4,716,111, and PCT Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741). Human antibodies can also be produced using transgenic mice incapable of expressing functional endogenous immunoglobulins but capable of expressing human immunoglobulin polynucleotides. For example, human heavy and light chain immunoglobulin polynucleotide complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain polynucleotides. The mouse heavy and light chain immunoglobulin polynucleotides can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in the usual manner with a selected immunogen (e.g., a target antigen). Using such techniques, it is possible to produce useful human IgG, IgA, IgM, IgD, and IgE antibodies.As noted above, methods for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, are well known in the art (see, e.g., PCT Publication Nos. WO 98 / 24893, WO 92 / 01047, WO 96 / 34096, and WO 96 / 33735, and U.S. Pat. No. 5,413,993). (See also Nos. 23, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, 5,939,598, 6,075,181, and 6,114,598).

[0279] Once an antibody molecule encompassed by the present invention is produced by an animal, a cell line, chemically synthesized, or recombinantly expressed, it can be purified (i.e., isolated) by any method readily known in the art for the purification of immunoglobulin or polypeptide molecules, for example, by chromatography (e.g., by ion exchange, affinity, particularly affinity for a specific target, Protein A, and size exclusion chromatography), centrifugation, differential solubility, or other standard techniques for the purification of proteins. Furthermore, antibodies encompassed by the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or readily known in the art to facilitate purification.

[0280] According to the present invention, antibodies that specifically bind to antigens can be in solution or bound to a substrate. In some embodiments, the antibodies are coupled to cellulose nanobeads and confined to one or more detection regions of a substrate of a detection device.

[0281] c. Antibody generation The antibodies and antigen-binding fragments thereof encompassed by the present invention may be naturally occurring or artificially generated by any method known in the art, such as conventional hybridoma technology, recombinant technology, mutation or optimization of known antibodies, selection from antibody or antibody fragment libraries, and monoclonal antibodies (mAbs) produced by immunization. The generation of antibodies, whether monoclonal or polyclonal, is well known in the art. Techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane, "Using Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1999; and "Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry," Woodhead Publishing, 2012.

[0282] The antibodies described herein, as well as variants and / or fragments thereof, can be generated using recombinant polynucleotides. In one embodiment, the polynucleotide has a modular design for encoding at least one of the antibodies, fragments, or variants thereof. As non-limiting examples, the polynucleotide construct can encode any of the following designs: (1) an antibody heavy chain; (2) an antibody light chain; (3) an antibody heavy and light chain; (4) a heavy and light chain separated by a linker; (5) a VH1, CH1, CH2, CH3 domain, a linker, and a light chain; or (6) a VH1, CH1, CH2, CH3 domain, a VL region, and a light chain. Any of these designs can include optional linkers between any of the domains and / or regions. Polynucleotides encompassed by the present invention can be engineered to produce any of the standard classes of immunoglobulins using any of the antibodies or components thereof described herein as starting molecules.

[0283] Methods of antibody development typically rely on the use of target molecules for selection, immunization, and / or validation of antibody affinity and / or specificity. In some embodiments, antibodies can be prepared through immunization of a host with one or more target antigens that act as immunogens to elicit an immunological response, using well-established methods well known to those skilled in the art.

[0284] d. Antibody Characterization and Efficacy Antibodies, and antibody-binding fragments thereof, encompassed by the present invention can be characterized by one or more characteristics selected from the group consisting of structure, isotype, binding (e.g., affinity and specificity), conjugation, glycosylation, and other distinguishing characteristics.

[0285] Such antibodies encompassed by the present invention can be of any animal origin, including birds and mammals. Preferably, such antibodies are of human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken origin. Antibodies encompassed by the present invention can be monospecific or multispecific. Multispecific antibodies may be specific for different epitopes of a peptide encompassed by the invention, or may be specific for both a peptide encompassed by the invention and a heterologous epitope, such as a heterologous peptide or solid support material (see, e.g., PCT Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al. (1991) J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al. (1992) J. Immunol. 148:1547-1553). For example, antibodies can be raised against peptides containing repeating units of peptide sequences encompassed by the present invention, or against peptides containing two or more peptide sequences encompassed by the present invention, or combinations thereof. As a non-limiting example, the heterobivalent ligand (HBL) system has been designed to competitively inhibit antigen binding to IgE antibodies bound to mast cells, thereby inhibiting mast cell degranulation (Handlogten et al. (2011) Chem. Biol. 18:1179-1188).

[0286] The characteristics of an antibody can be determined against a standard under normal physiological conditions, either in vitro or in vivo. Measurements can be performed relative to the presence or absence of the antibody. Such measurement methods include Western blots, enzyme-linked immunosorbent assays (ELISAs), activity assays, reporter assays, luciferase assays, polymerase chain reaction (PCR) arrays, gene arrays, real-time reverse transcriptase (RT) PCR, and other standard measurements in tissues or body fluids, such as serum or blood.

[0287] Antibodies can bind to or interact with any number of locations on or along a target protein. Contemplated antibody target sites include any and all possible sites on a target protein. Antibodies can be selected for their ability to bind (reversibly or irreversibly) to one or more epitopes on a particular target. Epitopes on a target include, but are not limited to, one or more features, regions, domains, chemical groups, functional groups, or moieties. Such epitopes can be composed of one or more atoms, groups of atoms, atomic structures, molecular structures, cyclic structures, hydrophobic structures, hydrophilic structures, sugars, lipids, amino acids, peptides, glycopeptides, nucleic acid molecules, or any other antigenic structure.

[0288] Methods for epitope mapping are well known in the art and include, but are not limited to, structural, functional, and computational methods. X-ray crystallography is a known structural approach, and the crystal structure of a bound antibody-antigen pair allows for highly accurate determination of important interactions between individual amino acids, both side chain and main chain atoms, in both the antigen's epitope and the antibody's paratope. Amino acids within 4 angstroms of each other are generally considered to be contact residues. The methodology typically involves purification of the antibody and antigen, complex formation and purification, followed by successive rounds of crystallization screening and optimization to obtain diffraction-quality crystals. Structural solutions are often obtained after X-ray crystallography at synchrotron sources. Other structural methods for epitope mapping include, but are not limited to, hydrogen-deuterium exchange coupled to mass spectrometry, cross-linking mass spectrometry, and nuclear magnetic resonance (NMR) (Epitope Mapping Protocols Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996); Abbott et al. (2014) Immunol. 142:526-535).

[0289] Functional methods for epitope mapping are also well known in the art and typically involve assessing or quantifying antibody binding to whole proteins, protein fragments, or peptides. Functional methods for epitope mapping may be used, for example, to identify linear or conformational epitopes and / or to infer when two or more different antibodies bind to the same or similar epitopes. Functional methods for epitope mapping include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides from a biomarker of interest are tested for reactivity with anti-biomarker antibodies, such as those described herein. Other functional methods for epitope mapping include array-based oligopeptide scanning (alternatively known as "overlapping peptide scanning" or "pepscan analysis"), site-directed mutagenesis (e.g., alanine scanning mutagenesis), and high-throughput mutagenesis mapping (e.g., shotgun mutagenesis mapping).

[0290] Several types of competitive binding assays are known, including, but not limited to, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (Stahli et al. (1983) Meth. Enzymol. 9:242), solid-phase direct biotin-avidin EIAs (Kirkland et al. (1986) J. Immunol. 137:3614), solid-phase direct label assays or solid-phase direct label sandwich assays (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), and I 125These include solid-phase direct-label RIA (Morel et al. (1988) Mol. Immunol. 25:7), solid-phase direct biotin-avidin EIA (Cheung et al. (1990) Virol. 176:546), and direct-label RIA (Moldenhauer et al. (1990) Scand. J. Immunol. 32:77). Typically, such assays involve the use of purified antigen bound to a solid surface or cells, and either 1) an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein, or 2) a labeled test antigen-binding protein and an unlabeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope that is sufficiently close to the epitope bound by the reference antigen-binding protein to create steric hindrance. Further details regarding methods for determining competitive binding are provided in the Examples herein. Typically, when a competing antigen-binding protein is present in excess (e.g., about 1-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold excess), it inhibits or blocks the specific binding of the reference antigen-binding protein to a common antigen by at least about 40-45%, about 45-50%, about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70-75%, or about 75% or more. In some cases, binding is inhibited by at least about 80-85%, about 85-90%, about 90-95%, about 95-97%, or greater than about 97%.

[0291] The effects of the agents described herein, such as antibodies, antigen-binding fragments thereof, and cells, can be evaluated using reagents, methods, and assays well known to those skilled in the art, particularly in light of the Examples. In some embodiments, a control is used for comparison, such as those described in the definitions above. For example, an assay can include contacting a biomarker target, such as on a cell or substrate, with an agent of interest, determining a desired measurement (e.g., amount, activity, cytokine production, cell proliferation, cell death, etc.), and comparing the measurement to a measurement from a reference or control, e.g., a measurement resulting from contact with a control agent, such as a control antibody or antigen-binding fragment thereof that does not specifically bind to the antigen of interest. Any known measurement or assay can be used, particularly those presented in the Examples, such as conventional cytokine production determination assays, cell activation assays, cell proliferation assays, cell death assays, cell migration assays, cell signaling assays, etc.

[0292] Also, as noted in the definition above, a "significant" modulation of a desired measurement may be numerically quantified, such as being above a particular number (e.g., percentage), below a particular number (e.g., percentage), or within a particular range of numbers (e.g., percentage range). Representative, non-limiting examples of quantitative measurements include affinity (K D ), k d , k a , an increase or decrease in the percentage of biomarker expression, an increase or decrease in the percentage of cells (e.g., desired cells, non-desired cells, the ratio of desired cells to non-desired cells, the ratio of desired cells to total cells, the ratio of non-desired cells to total cells, etc., at one time point or compared at different time points, etc.).

[0293] V. Nucleic Acids, Vectors, and Cells, Including Host Cells A further object of the present invention relates to nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof (and fragments thereof), as well as polypeptides, vectors, and cells, including host cells, described herein.

[0294] Nucleic acid drugs One aspect encompassed by the present invention involves the use of nucleic acid molecules. Nucleic acid molecules can be deoxyribonucleic acid (DNA) molecules (e.g., cDNA, genomic DNA, etc.), ribonucleic acid (RNA) molecules (e.g., mRNA, long non-coding RNA, small RNA species, etc.), DNA / RNA hybrids, and DNA or RNA analogs generated using nucleotide analogs. RNA agents can include RNAi (RNA interference) agents (e.g., small interfering RNA (siRNA)), single-stranded RNA (ssRNA) molecules (e.g., antisense oligonucleotides), or double-stranded RNA (dsRNA) molecules. dsRNA molecules comprise a first strand and a second strand, the second strand being substantially complementary to the first strand, and the first and second strands forming at least one double-stranded duplex region. dsRNA molecules can be blunt-ended or have at least one terminal overhang. When used as agents that bind to target nucleic acid sequences, nucleic acid agents encompassed by the present invention can hybridize to any region of the target sequence, such as a genomic sequence and / or mRNA sequence, including, but not limited to, an enhancer region, a promoter region, a transcriptional start and / or stop region, a splice site, a coding region, a 3' untranslated region (3'-UTR), a 5' untranslated region (5'-UTR), a 5' cap, a 3' polyadenylyl tail, or any combination thereof.

[0295] An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule does not contain sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, an isolated nucleic acid molecule can contain approximately 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB, or 0.1 kB of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized.

[0296] Nucleic acid molecules encompassed by the invention can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or part of such nucleic acid sequences, nucleic acid molecules encompassed by the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012).

[0297] Nucleic acid molecules encompassed by the present invention can be...

Claims

[Claim 1] The invention described in the specification.

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