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

Abstract

The present invention is based, in part, on the discovery of anti-PSGL-1 compositions (e.g., monoclonal antibodies and antigen-binding fragments thereof) that modulate the inflammatory phenotype of myeloid cells, such as suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells, including their 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 is provided that binds to myeloid cells that express a PSGL-1 polypeptide and increases the inflammatory phenotype of the myeloid cells, optionally including suppressor myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 857,169, filed on June 4, 2019; U.S. Provisional Application No. 62 / 867,569, filed on June 27, 2019; U.S. Provisional Application No. 62 / 947,948, filed on December 13, 2019; and U.S. Provisional Application No. 63 / 032,214, filed on May 29, 2020, the entire contents of each of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Monocytes and macrophages are a type of phagocytic cell that protects the body by taking in harmful foreign particles, bacteria, and dead or dying cells. In addition to monocytes and macrophages, phagocytic cells include neutrophils, dendritic cells, and mast cells.

[0003] Macrophages are classically known as large white blood cells that patrol the body through a process known as phagocytosis and engulf and digest cellular debris and foreign substances such as pathogens, microorganisms, and cancer cells. Additionally, macrophages, including tissue macrophages and macrophages derived from circulating monocytes, are important mediators of both the innate and adaptive immune systems.

[0004] The phenotype of macrophages depends 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 response, increased wound healing, increased tissue repair, and embryogenesis.

[0005] Under non-pathological conditions, a balanced population of immunostimulatory and immunomodulatory macrophages exists in the immune system. Disruption of the balance can lead to various disease states. In some cancers, for example, tumors secrete immune factors (such as cytokines and interleukins) that polarize the macrophage population to support an anti-inflammatory, tumor-promoting M2 phenotype, which activates wound healing pathways, promotes the growth of new blood vessels (i.e., angiogenesis), and provides nutrients and growth signals to the tumor. These M2 macrophages are referred to as tumor-associated macrophages (TAMs) or tumor-infiltrating macrophages. TAMs in the tumor microenvironment are important 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 (such as CSF1R and CCR2) have been studied as regulators of macrophage phenotypes, such as by modulating the balance of tumor-promoting macrophages (e.g., TAMs) and inflammation-inducing macrophages that can inhibit tumor formation. Therapies that regulate the mobilization, polarization, activation, and / or function of monocytes and macrophages to modulate the balance of the macrophage population are referred to as macrophage immunotherapy. Despite advances in the field of macrophage biology, there remains a need for new targets (such as genes and / or gene products) to regulate the inflammatory phenotype of macrophages and the agents used in macrophage immunotherapy.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0007] The present invention is based, at least in part, on the discovery of anti-PSGL-1 compositions and methods for modulating the myeloid cell inflammatory phenotype, and their use for purposes such as treatment, diagnosis, prognosis, and screening. For example, herein, it has been determined that PSGL-1 expression increases upon activation in M2 macrophages, and that the inflammatory phenotype of myeloid cells can be increased using anti-PSGL-1 antibodies comprising antigen-binding fragments thereof.

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

[0009] A number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiments described herein. For example, in one embodiment, a monoclonal antibody, or an antigen-binding fragment thereof, after contact with a) a monoclonal antibody, or an antigen-binding fragment thereof, results in i) increased expression and / or secretion of surface antigen classification 80 (CD80), CD86, MHCII, MHCI, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), ii) decreased 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 the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, v) increased CD8+ cytotoxic T cell activation, vi) increased recruitment of CD8+ cytotoxic T cell activation, vii) increased 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, flatness of appearance, and / or number of dendritic protrusions when evaluated by microscopy, thereby increasing the inflammatory phenotype of bone marrow cells, b) selectively binds to a human PSGL-1 polypeptide that is at least 1.1-fold greater than a polypeptide selected from the group consisting of human complement C4 protein, human sulfotyrosinated C4 peptide, human fibrinogen protein, human sulfotyrosinated fibrinogen peptide, human sulfotyrosinated CCK peptide, human sulfotyrosinated CCR2b peptide, and human sulfotyrosinated D6 peptide, wherein the polypeptide is expressed on a cell or in vitro, the binding, c) optionally, when measured by ELISA or a biolayer interferometry assay,Binding to human PSGL-1 polypeptide at a KD of approximately 0.00001 nanomolar (nM) to 1000 nM, d) binding to the N-terminal peptide sequence QATEYEYLDYDFLPETEPPEM of human PSGL-1 polypeptide, e) binding to one or more sulfotyrosine residues of sulfotyrosinylated human PSGL-1 polypeptide, f) cross-reacting with cynomolgus PSGL-1 polypeptide, g) competing or cross-competing with an antibody that binds to a PSGL-1 polypeptide or an antigen-binding fragment thereof listed in Table 2 or 3, h) competing with, inhibiting, or blocking the binding of PSGL-1 to its PSGL-1 ligand, optionally, where the PSGL-1 ligand is VISTA, competing with, inhibiting, or blocking, i) being obtainable as a monoclonal antibody deposited with ATCC described herein, j) not activating unstimulated monocytes, k) not having ADCC activity against PSGL-1-expressing cells, l) not having CDC activity against PSGL-1-expressing cells, m) not killing PSGL-1-expressing cells upon binding to and / or internalization by PSGL-1-expressing cells, n) not being conjugated to another therapeutic moiety, optionally, where the other therapeutic moiety is a cytotoxic agent, not being conjugated, o) not activating or inducing T cell apoptosis, p) binding to an epitope comprising residues 45 to 55 of human PSGL-1, optionally, where the binding epitope is a conformational epitope or a linear epitope, binding, q) binding the C-terminus of the epitope to residues 42 to 62 of human PSGL-1, optionally, where the epitope comprises residues 56 to 62, residues 42 to 121, or residues 105 to 125 of human PSGL-1, and further optionally, where the binding epitope is a conformational epitope or a linear epitope, binding, r) binding to one or more of residues 45, 46, 49, 50, 51, 52, 53, and 55 of human PSGL-1, optionally, where the residues are selected from the group consisting of epitope residues listed in Table 13, and further optionally, where the binding epitope is a conformational epitope or a linear epitope, binding, s) one, two,or binding to three sulfotyrosinated residues, wherein the sulfotyrosinated residues of human PSGL-1 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 sulfotyrosinated human PSGL-1, wherein the ratio of the binding affinity of the mAb for sulfotyrosinated human PSGL-1 to the binding affinity of the mAb for a sulfotyrosinated protein that is not PSGL-1 is higher than the ratio of the binding affinity of PSG6, PSG3, and / or SELK1 mAb for sulfotyrosinated human PSGL-1 to the binding affinity of PSG6, PSG3, and / or SELK1 mAb for a sulfotyrosinated protein that is not PSGL-1, and optionally, the sulfotyrosinated protein that is not PSGL-1 is C4 alpha chain, complement C4, fibrinogen gamma, fibrinogen, CCK, CC42b, and / or D6; u) binding to sulfotyrosinated human PSGL-1, wherein the binding affinity of the mAb for sulfotyrosinated human PSGL-1 is at least 10% or more compared to the affinity of the mAb for a sulfotyrosinated protein that is not PSGL-1, and optionally, the sulfotyrosinated protein that is not PSGL-1 is C4 alpha chain, complement C4, fibrinogen gamma, fibrinogen, CCK, CC42b, and / or D6; and / or v) having antitumor activity in vivo, and having one or more of the above characteristics. In another embodiment, the monoclonal antibody, or an 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 an 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 fragments. 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 complexed with a drug, and optionally, the drug is selected from the group consisting of a binding protein, an enzyme, a drug, a chemotherapeutic agent, a biological agent, a toxin, a radionuclide, an immunomodulatory agent, a detectable moiety, and a tag.,

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

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

[0012] In yet another aspect, an isolated nucleic acid molecule is provided that 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 present invention, or has a sequence having at least about 90% identity over its full 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 present invention, or 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, an isolated immunoglobulin heavy and / or light chain polypeptide encoded by a nucleic acid encompassed by the present invention is provided.

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

[0015] In yet another aspect, a host cell comprising an isolated nucleic acid encompassed by the present invention is provided. In some embodiments, the host cell expresses a monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof, contains an immunoglobulin heavy chain and / or light chain polypeptide encompassed by the present invention, contains a vector encompassed by the present invention, and / or is accessible as a monoclonal antibody deposited under the ATCC deposit accession numbers described herein.

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

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

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

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

[0020] In another aspect, a method for generating myeloid cells having an increased inflammatory phenotype after contact with an agent encompassed by the present invention is provided, which includes contacting myeloid cells with an effective amount of the agent, and optionally, the myeloid cells include suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0021] As described above, a number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiments described herein. For example, in one embodiment, bone marrow cells having an increased inflammatory phenotype, after contact with a monoclonal antibody or an antigen-binding fragment thereof, exhibit a) increased expression and / or secretion of surface antigen classification 80 (CD80), CD86, MHCII, MHCI, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), b) reduced expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, TGFb, and / or IL-10, c) 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, d) increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, 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 and / or dendritic cell activity, and / or m) one or more of increased spindle-shaped morphology, flatness of appearance, and / or number of dendritic protrusions when evaluated by microscopy. In another embodiment, bone marrow cells contacted with a monoclonal antibody or an antigen-binding fragment thereof are included within a population of cells, and the monoclonal antibody or an antigen-binding fragment thereof increases the number of type 1 and / or M1 macrophages and / or reduces the number of type 2 and / or M2 macrophages within the population of cells. In yet another embodiment, bone marrow cells contacted with a monoclonal antibody or an antigen-binding fragment thereof are included within a population of cells, and the monoclonal antibody or an antigen-binding fragment thereof increases the ratio of i) to ii), where i) within the population of cells is type 1 and / or M1 macrophages and ii) is type 2 and / or M2 macrophages.In yet another embodiment, the macrophages include 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 contact 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 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 wherein the immunotherapeutic agent comprises an immune checkpoint inhibitor, an immune-stimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus.

[0022] In yet another aspect, a composition comprising bone marrow cells generated according to the methods encompassed by the present invention is provided, optionally wherein the bone marrow cells comprise suppressive bone marrow cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

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

[0024] As described 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 embodiments 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 surface antigen classification 80 (CD80), CD86, MHCII, MHCI, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); b) reduced expression and / or secretion of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, and / or IL-10; c) increased secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, IL-12, IL-18, and IL-23; d) an increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10; e) increased CD8+ cytotoxic T cell activation; f) increased CD4+ helper T cell activity; g) increased NK cell activity; h) increased neutrophil activity; i) increased macrophage and / or dendritic cell activity; and / or j) one or more of an increased spindle-shaped morphology, flatness of appearance, and / or number of dendritic protrusions when evaluated by microscopy. In another embodiment, one or more agents increase the number of type 1 and / or M1 macrophages, reduce the number of type 2 and / or M2 macrophages, and / or increase the ratio of i) to ii), where i) is type 1 and / or M1 macrophages and ii) is type 2 and / or M2 macrophages in a subject. In yet another embodiment, the number and / or activity of cytotoxic CD8+ T cells in a subject increases after administration of the agent. In yet another embodiment, the bone marrow cells include type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAM), 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 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, and optionally, the immunotherapeutic agent comprises an immune checkpoint inhibitor, an immune stimulating 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, comprising administering to the subject bone marrow cells contacted with an effective amount of an agent encompassed by the present invention, optionally, the bone marrow cells comprising suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells, is provided.

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

[0027] In yet another aspect, 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, is provided.

[0028] In yet another aspect, 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 monocyte cells and / or macrophage cells contacted with a therapeutically effective amount of an agent encompassed by the present invention, optionally, wherein the myeloid cells comprise suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells, is provided.

[0029] As described above, a number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiments described herein. For example, in one embodiment, the bone marrow cells include 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 compared 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 to the subject at least one immunotherapy, optionally, the immunotherapy including immune checkpoint inhibitors, immune-stimulating agonists, inflammatory agents, cells, cancer vaccines, and / or viruses. 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 cancer, optionally, the additional therapeutic agent or regimen being selected from the group consisting of chimeric antigen receptors, chemotherapy, radiation, targeted therapy, and surgery. In yet another embodiment, the agent reduces the number of proliferating cells in cancer and / or reduces the volume or size of a tumor containing cancer cells. In yet another embodiment, the agent increases the amount and / or activity of CD8+ T cells infiltrating a tumor containing cancer cells. In yet another embodiment, the agent a) increases the amount and / or activity of M1 macrophages infiltrating a tumor containing cancer cells and / or b) reduces the amount and / or activity of M2 macrophages infiltrating a tumor containing cancer cells. In another embodiment, the method further comprises administering to the subject at least one additional therapy or regimen for treating cancer. In yet another embodiment, the therapy is before, simultaneous with, or after the administration of the agent.

[0030] In another aspect, a method of identifying myeloid cells capable of increasing their inflammatory phenotype by modulating at least one target, comprising: a) determining the amount and / or activity of at least one target listed in Table 1 from myeloid cells 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 at least one target in a control using the agent; and c) comparing the amount and / or activity of at least one target detected in steps a) and b), wherein the presence or increase in the amount and / or activity of at least one target listed in Table 1 in myeloid cells, compared to the control amount and / or activity of the at least one target, indicates that the myeloid cells are capable of increasing their inflammatory phenotype by modulating at least one target, and optionally, the myeloid cells include suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells, a method is provided.

[0031] As described 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 embodiments described herein. For example, in one embodiment, the method further comprises contacting, recommending, prescribing, or administering a cell with an agent that modulates at least one target listed in Table 1. In another embodiment, the method comprises contacting, recommending, prescribing, or administering a cell with a cancer therapy other than an agent that modulates at least one target listed in Table 1 when it is determined that the subject would not benefit from increasing an inflammatory phenotype by modulating at least one target (e.g., immunotherapy). In yet another embodiment, the method further comprises contacting and / or administering a cell with at least one additional agent that increases an immune response. In yet another embodiment, the additional agent is selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and / or hormonal 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 containing cells. In yet another embodiment, the subject has cancer. In another embodiment, the control is a cancer sample from the subject. In yet another embodiment, the control is a non-cancer sample from the subject.

[0032] In yet another aspect, a method for predicting the clinical outcome of a subject having 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 at least one target from a control having a poor clinical outcome using the agent; and c) comparing the amount and / or activity of at least one target in the subject sample and the sample from the control subject, wherein the presence or increase in the amount and / or activity of at least one target listed in Table 1 from the bone marrow cells from the subject, as 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 suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0033] In yet another aspect, a method for monitoring an 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; c) comparing the amount or activity of at least one target listed in Table 1 detected in steps a) and b), wherein a lack 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 as compared to the amount and / or activity from bone marrow cells from the first sample indicates that the subject's bone marrow cells have an upregulated inflammatory phenotype, or the 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 as 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, and optionally, the bone marrow cells comprise suppressive bone marrow cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0034] As described above, a number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiments described herein. For example, in one embodiment, the first and / or at least one subsequent sample comprises bone marrow cells cultured in vitro. In another embodiment, the first and / or at least one subsequent sample comprises bone marrow cells that are not cultured in vitro. In yet another embodiment, the first and / or at least one subsequent sample is part of a single sample or 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, comprising: a) detecting in a subject sample containing bone marrow cells at a first time point, i) detecting the amount or activity of at least one target listed in Table 1 that is within or on the bone marrow cells using an agent, wherein the agent is at least one monoclonal antibody included in 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) during 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 or reduction in the amount and / or activity of at least one target listed in Table 1, compared to the amount and / or activity 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 described 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 embodiments described herein. For example, in one embodiment, bone marrow cells contacted with a drug are included within a population of cells, and the drug increases the number of type 1 and / or M1 macrophages within the population of cells. In another embodiment, bone marrow cells contacted with a drug are included within a population of cells, and the drug 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 drug. 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 drug. 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 an inflammatory phenotype, optionally wherein the immunotherapeutic agent comprises an immune checkpoint inhibitor, an immune stimulatory 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 for evaluating the effectiveness of a test agent for treating cancer in a subject, comprising: a) detecting in a subject sample containing bone marrow cells at a first time point, i) detecting the amount and / or activity of at least one target listed in Table 1 that is within 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; b) repeating 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 a lack or reduction in the amount and / or activity of at least one target listed in Table 1, compared to the amount and / or activity within or on the bone marrow cells of the subject sample at the first time point, and / or an increase in ii) within or on the bone marrow cells of the subject sample at a subsequent time point, indicates that the test agent treats cancer in the subject, and optionally, the bone marrow cells include suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0038] As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the subject has received treatment for cancer, completed treatment, and / or is in remission between the first time point and a subsequent time point. 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 part of a single sample or a pooled sample obtained from the subject. In another embodiment, the sample includes 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, comprising: a) contacting cancer cells with cytotoxic T cells and / or immune checkpoint therapy in the presence of bone marrow cells that have been contacted with the test agent, wherein the test agent regulates the amount and / or activity of at least one target listed in Table 1 in or on the bone marrow cells as determined using a drug, and the drug is at least one monoclonal antibody encompassed by the present invention, or an antigen-binding fragment thereof; b) contacting cancer cells with cytotoxic T cells and / or immune checkpoint therapy in the presence of control bone marrow cells that have not been contacted with the test agent; and c) identifying a drug that increases the effectiveness of cytotoxic T cell-mediated killing and / or immune checkpoint therapy in a) compared to b), thereby identifying a test agent that sensitizes cancer cells to cytotoxic T cell-mediated killing and / or immune checkpoint therapy, optionally wherein the bone marrow cells comprise suppressive myeloid cells, monocytes, macrophages, neutrophils, and / or dendritic cells.

[0040] As described above, a number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiments 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 cancer and / or ii) a decrease in the volume or size of a tumor comprising 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 a tumor comprising cancer cells. In yet another embodiment, the method is measured by at least one criterion selected from the group consisting of clinical benefit rate, survival time to death, pathologic complete response, semi-quantitative measurement of pathologic response, clinical complete remission, clinical partial remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST criteria, and further comprises determining responsiveness to a test agent that modulates at least one target listed in Table 1. In another embodiment, the method further comprises contacting the cancer cells with at least one additional cancer therapeutic agent or regimen.

[0041] As described above, a number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiments described herein. For example, in one embodiment, bone marrow cells having a regulated inflammatory phenotype exhibit: a) regulated expression of surface antigen classification 80 (CD80), CD86, MHCII, MHCI, interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); b) regulated expression of CD206, CD163, CD16, CD53, VSIG4, PSGL-1, and / or IL-10; c) regulated secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, IL-12, IL-18, and IL-23; d) regulated ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10; e) regulated CD8+ cytotoxic T cell activation; f) regulated CD4+ helper T cell activity; g) regulated NK cell activity; h) regulated neutrophil activity; i) regulated macrophage and / or dendritic cell activity; and / or j) exhibit one or more of a regulated spindle shape, flatness of appearance, and / or number of dendritic processes when evaluated by microscopy. In another embodiment, the cells and / or bone marrow cells include type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAM), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, and optionally, the cells and / or bone marrow cells are determined to express or express PSGL-1.In yet another embodiment, the human PSGL-1 polypeptide has the amino acid sequence of SEQ ID NO: 2, the cynomolgus monkey PSGL-1 polypeptide has the amino acid sequence of SEQ ID NO: 17, the human sulfotyrosinated C4 peptide has the amino acid sequence of NEDY(SO3)EDY(SO3)EY(SO3)DELPAKDDGGK, the human sulfotyrosinated fibrinogen peptide has the amino acid sequence of (EHPAETEY(SO3)DSLY(SO3)PEDDLGGK), the human sulfotyrosinated CCK peptide has the amino acid sequence of SHRISDRDY(SO3)MGWMDFGGK, the human sulfotyrosinated CCR2b peptide has the sequence of TTFFDY(SO3)DY(SO3)GAPSHGGK, and / or the human sulfotyrosinated D6 peptide has the sequence of ENSSFYY(SO3)Y(SO3)DY(SO3)LDEVAFGGK. In yet another embodiment, the cancer is a solid tumor infiltrated by macrophages, 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, 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 cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, chromophobe kidney, renal clear cell carcinoma, renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma, paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, thymoma, thyroid carcinoma, uterine carcinosarcoma, endometrial carcinoma of the uterine corpus, and uveal melanoma. In another embodiment, the bone marrow cells include type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAM), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, optionally, the bone marrow cells are TAM and / or M2 macrophages.In yet another embodiment, the macrophage expresses, or is determined to express, PSGL-1. In yet another embodiment, the bone marrow cell is a primary bone marrow cell. In another embodiment, the bone marrow cell is contained within a tissue microenvironment. In yet another embodiment, the bone marrow cell is 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 drawings executed in at least one color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee(s).

Brief Description of the Drawings

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BRIEF DESCRIPTION OF THE INVENTION

[0044] For any figure showing a bar histogram, curve, or other data associated with a legend, the bars, curves, or other data shown left to right for each display directly correspond to the boxes from top to bottom of 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 a myeloid cell inflammatory phenotype, including at least in part polarization, activation, and / or function. Accordingly, the present invention provides anti-PSGL-1 compositions, as well as methods and uses including, but not limited to, modulation of a myeloid cell inflammatory phenotype for treatment, diagnosis, prognosis, and screening.

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

[0047] The term “activating receptor” includes immune cell receptors that bind to an antigen, a complexed antigen (e.g., in the context of a major histocompatibility complex (MHC) polypeptide), or an antibody. 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, the T cell receptor is present on T cells and is associated with CD3 polypeptides. The T cell receptor is stimulated by an antigen (and by polyclonal T cell activation reagents) in the context of an MHC polypeptide. T cell activation via the TCR results in many changes, such as protein phosphorylation, membrane lipid changes, ion fluxes, cyclic nucleotide modifications, RNA transcription changes, protein synthesis changes, and cell volume changes. Similar to T cell activation of macrophages via activating receptors such as cytokine receptors or pathogen-associated molecular pattern (PAMP) receptors, it results in changes such as protein phosphorylation, modifications to the surface receptor phenotype, protein synthesis and release, and morphological changes.

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

[0049] The term "administer" relates to the actual physical introduction of an agent into, or (where appropriate) onto, a target biological subject such as a host and / or subject. The composition can be administered (e.g., "contacted") to cells in vitro or in vivo. The composition can be administered to a subject in vivo via a suitable route of administration. Any and all methods of introducing the composition into a host are contemplated according to the present invention. The method is not dependent on, and should not be construed as being dependent on, any particular means of introduction. Means of introduction are well known to those of skill in the art and are also exemplified herein. The term includes routes of administration that enable the agent to perform the function intended by the agent. Examples of routes of administration for the treatment of the body that can be used can include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be a bolus injection or a continuous infusion. Depending on the route of administration, the agent can be coated with, or disposed within, a selected material to protect the agent from natural conditions that can have an adverse effect on its ability to perform its intended function. The agent can be administered alone or in combination with a pharmaceutically acceptable carrier. The agent can also be administered as a prodrug that is converted to its active form in vivo.

[0050] The term "agent" refers to a compound, supramolecular complex, material, and / or combinations or mixtures thereof. A compound (e.g., a molecule) can be represented by a chemical formula, chemical structure, or sequence. Representative non-limiting examples of agents can include, for example, antibodies, small molecules, polypeptides, polynucleotides (e.g., RNAi agents, siRNA agents, miRNA, piRNA, mRNA, antisense polynucleotides, aptamers, etc.), lipids, and polysaccharides. In general, an agent can be obtained using any suitable method known in the art. In some embodiments, the 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 terms "altered amount" or "altered level" include an increase or decrease in the copy number of a biomarker nucleic acid (e.g., germline and / or somatic) 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. Further, the altered amount of a biomarker protein can be determined by detecting post-translational modifications such as the methylation state of the marker, and can affect the expression or activity of the biomarker protein. In some embodiments, "altered amount" refers to the presence or absence of a biomarker, since 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 threshold of sensitivity of a given assay used to measure the biomarker.

[0053] When the amount of a biomarker is greater or less, respectively, than the normal level due to the amount being greater than the standard error of the assay utilized to assess the amount, the amount of the biomarker in the subject is "significantly" higher or lower than the normal amount of the biomarker, 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 than that amount. Alternatively, the amount of the biomarker in the subject can be considered "significantly" higher or lower than the normal amount when 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, 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 a biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, which is greater or less than the standard error of the assay used to evaluate the expression or copy number, and is at least 2-fold, more preferably 3-fold, 4-fold, 5-fold, or 10-fold or more of the expression level or copy number of the biomarker in a control sample (e.g., a sample from a healthy subject without the related 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 the 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 may further refer to the process by which the RNA transcript is processed and translated into a polypeptide. The total expression of a nucleic acid and its polypeptide counterpart, if present, contributes to the amount of a biomarker such as one or more of the 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. The altered activity of a biomarker can result, for example, from an altered expression of the biomarker, an altered protein level of the biomarker, an altered structure of the biomarker, or an altered interaction of the biomarker with other proteins involved in the same or a different pathway, e.g., or an altered interaction with a transcriptional activator or inhibitor.

[0056] The term "modified structure" of a biomarker refers to the presence of a mutation or allelic variant within the biomarker nucleic acid or protein, where, for example, the mutation affects the expression or activity of the biomarker nucleic acid or protein as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to, substitution, deletion, or addition mutations. Mutations can be present within the coding or non-coding regions of the biomarker nucleic acid.

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

[0058] The term "antagonist" or "blocking" refers 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 an antibody 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 antibodies in their naturally occurring forms (e.g., IgG, IgA, IgM, IgE) and single-chain antibodies, chimeric and humanized antibodies, and recombinant antibodies such as multispecific antibodies, as well as all fragments, fusion proteins, and derivatives of the foregoing, where the fragments and derivatives have at least an antigenic binding site. Antibody derivatives can include a protein or chemical moiety conjugated to the 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 desired phenotype in a bone marrow cell. In this context, the term "biomarker" is synonymous with "target". However, in some embodiments, the term further encompasses a measurable entity of a target that has been determined to indicate an output of interest, such as one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for modulating an inflammatory phenotype, a cancer state, 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 nucleic acids and / or transcribed nucleic acids), proteins, and antibodies (and antigen-binding fragments thereof), particularly those described in Table 1.

[0061] The term "cancer" or "tumor" or "hyperproliferative" refers to the presence of cells having characteristics typical of cells that cause cancer, such as unregulated growth, immortality, the ability to invade or metastasize, rapid growth, and certain characteristic morphological features. In some embodiments, such cells partially or fully exhibit such characteristics 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 often take the form of tumors, but such cells can exist alone within an animal or can 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, 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, urogenital, lymphatic, rectum, larynx, pancreas (including exocrine pancreatic cancer), esophagus, stomach, gallbladder, neck, thyroid, and skin (including squamous cell carcinoma); cancer tumors including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkitt lymphoma; lymphocytic hematopoietic tumors including acute and chronic myelogenous leukemia, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors including melanoma, xeroderma pigmentosum, corneal acanthoma, seminoma, thyroid follicular 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, renal cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, neck cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, head and neck cancer, gastric cancer, germ cell tumor, bone cancer, bone tumor, adult malignant fibrous histiocytoma of bone; pediatric malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, nasal and paranasal natural killer, neoplasm, plasma cell neoplasm; myelodysplastic syndrome; neuroblastoma; testicular germ cell tumor, intraocular melanoma, myelodysplastic syndrome; myelodysplastic / myeloproliferative disease, synovial sarcoma, chronic myelogenous leukemia, acute lymphoblastic leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), multiple myeloma, acute myelogenous leukemia, chronic lymphocytic leukemia, mastocytosis and symptoms associated with mastocytosis, and any metastases thereof are included.Furthermore, disorders include, in addition to other cancers, mastocytosis in humans such as urticaria pigmentosa, diffuse cutaneous mastocytosis, solitary mastocytoma, as well as mastocytoma in dogs, and several rare subtypes such as vesicular erythema and telangiectatic mastocytoma, mastocytoma with associated hematological disorders, for example myeloproliferative or myelodysplastic syndromes, or acute leukemia, myeloproliferative disorders associated with mastocytosis, mast cell leukemia, etc. Other cancers also include carcinomas including skin including bladder, urothelial cancer, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, testis, particularly testicular seminoma, and squamous cell carcinoma; gastrointestinal stromal tumors ("GIST"); lymphoid hematopoietic tumors including leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, and Burkitt lymphoma; myeloid hematopoietic tumors including acute and chronic myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; other tumors including melanoma, seminoma, teratocarcinoma, 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, corneal acanthoma, seminoma, thyroid follicular carcinoma, teratoma, chemotherapy-resistant nonseminomatous germ cell tumors, Kaposi sarcoma, and any metastases thereof, but are not limited to these disorders.Other non-limiting examples of cancer types applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chondroma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial sarcoma, 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, 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 myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemias (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is essentially epithelial and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney 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. The epithelial cancer can be characterized in various other ways including, but not limited to, serous, endometroid, 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) kidney cancer (RCC), high microsatellite instability cancer, 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 disease state with a sample. In certain instances, "classification" is based on statistical evidence, empirical evidence, or both. In certain embodiments, methods and systems for classifying using a so-called training set of samples have a known disease state. Once established, the training dataset functions as a basis, model, or template against which the characteristics of an unknown sample are compared to classify the unknown disease state of the sample. In certain instances, classifying a sample is similar to diagnosing the disease state of the sample. In certain other instances, classifying a sample is similar to distinguishing the disease state of the sample from another disease state.

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

[0065] The term "competes with" with respect to an antibody or an antigen-binding fragment thereof means that a first antibody or an antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or its antigen-binding moiety such that the binding of the first antibody to its cognate epitope is detectably decreased in the presence of the second antibody as compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, but this is not necessary. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its epitope. However, antibodies are said to "cross-compete" with each other with respect to the binding of each epitope( s) if each antibody inhibits the binding of the other antibody to its cognate epitope or ligand, detectably, to the same extent, to a greater extent, or to a lesser extent. Both competing and cross-competing antibodies, as well as their antigen-binding fragments, are encompassed by the present invention (e.g., the 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 changes, or binding to a common epitope or a portion thereof), one of ordinary 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 when 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 when the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if at least one nucleotide residue of the first region can base pair with a residue of the second region when the two regions are arranged in an antiparallel manner. Preferably, the first region includes a first portion and the second region includes a second portion such that when the first and second portions are arranged in an antiparallel manner, 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 of the nucleotide residues of the first portion can base pair with the nucleotide residues of the second portion. In some embodiments, the complementary polynucleotide can be "sufficiently complementary" or have "sufficient complementarity", i.e., sufficient complementarity to maintain a double strand and / or the desired activity. For example, in the case of an RNAi agent, such complementarity is the complementarity between the agent and the target mRNA that is sufficient to partially or completely prevent translation of the mRNA. For example, an siRNA having a "sequence sufficiently complementary to the 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 mechanism or process.

[0067] The term "substantially complementary" refers to complementarity in the base pair double-stranded region between two nucleic acids, and not in any single-stranded regions such as terminal overhangs or gap regions between two double-stranded regions. Complementarity need not be perfect, and there may be any number of base pair mismatches. 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 the selected reaction conditions. Thus, substantially complementary sequences can refer to sequences having at least 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60 percent or more, or any number of base pair complementarity therebetween, in the double-stranded region.

[0068] As used herein, the terms "combination therapy" and "combined therapy" refer to the administration of two or more therapeutic agents, such as a combination of more than one modulator of a target listed in Table 1, at least one modulator of at least one target listed in Table 1, and additional therapeutic agents 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 that make up the combination therapy can be administered simultaneously, before, or after the administration of one or more other agents. The combination therapy is intended 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 (usually, depending on the combination selected, for a few minutes, hours, days, or weeks). In combination therapy, the combination therapeutic agents can be applied by applying them continuously or substantially simultaneously.

[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, the control includes obtaining a "control sample" in which the expression product level is detected and compared with the expression product level from the test sample. Such control samples can include, but are not limited to, normal tissues or cells isolated from subjects such as subjects with bone marrow cells and / or control cancer patients with known results (which can be measurements of stored samples or previous samples), normal patients or cancer patients, cultured primary cells / tissues isolated from subjects such as normal subjects or cancer patients, adjacent normal cells / tissues obtained from the same organ or body location of a cancer patient, tissue or cell samples isolated from a normal subject, or samples from subjects such as primary cells / tissues obtained from a storage location, and any suitable sample. In another preferred embodiment, the control can include reference standard expression product levels from any suitable source, including but not limited to, housekeeping genes, expression product level ranges from normal tissues (or other previously analyzed control samples), within test samples from a group of patients, or expression product level ranges previously determined for a set of patients with a specific outcome (e.g., survival at 1 year, 2 years, 3 years, 4 years, etc.) or a specific treatment (e.g., standard treatment for cancer therapy). Those skilled in the art will understand that such control samples 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 normal or non-cancerous cell / tissue samples. 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 specific treatment, or a set of patients with one outcome versus another outcome. In previous cases, the specific expression product level for each patient can be represented as assigned to a percentile level of expression, or as higher or lower than the average or mean of the reference standard expression level. In another preferred embodiment, the control can include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancer.In another embodiment, the control can also include the average level of expression of a particular gene in a population, e.g., 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 who have received standard treatment, or patients with benign cancer. In another preferred embodiment, the control includes, but is not limited to, a ratio transformation of the expression product levels, which includes determining the ratio of the 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 difference in the expression product levels of two or more genes in a test sample and determining the difference in the expression product levels in any suitable control, determining the expression product levels of two or more genes in a test sample, normalizing their expression to the expression of a housekeeping gene in the test sample, and comparing it to any suitable control. In a particularly preferred embodiment, the control includes a control sample of the same strain and / or type as the test sample. In another embodiment, the 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, control expression product levels are established and, for example, higher or lower levels of expression products compared to a particular percentile are used as a basis for predicting outcome. In another preferred embodiment, the control expression product levels are established using expression product levels from cancer control patients with known outcomes, and the expression product levels from the test sample are compared to the control expression product levels as a basis for predicting outcome. The methods encompassed by the present invention are not limited to the use of a specific cut-off point when comparing the level of the expression product in the test sample to 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 cells) that encode a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. However, the copy number can be increased by gene amplification or duplication, or decreased by deletion. For example, changes in germline copy number include changes 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 of the same species for which a particular germline DNA and corresponding copy number have been determined). Changes in somatic copy number include changes 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 germline DNA of the same subject for which somatic DNA and corresponding copy number have been determined).

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

[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.). The inhibitory signals transmitted by inhibitory receptors can occur even when costimulatory receptors (such as CD28) are not present on immune cells and thus are not simply a function of competition between inhibitory receptors and costimulatory receptors for binding of costimulatory polypeptides (Fallarino et al. (1998) J. Exp. Med. 188:205). Transmission of inhibitory signals to immune cells can lead to unresponsiveness or anergy or programmed cell death in immune cells. Preferably, transmission of inhibitory signals operates via a mechanism not involving apoptosis. As used herein, the term "apoptosis" includes programmed cell death that can be characterized using techniques known in the art. Apoptotic cell death can be characterized, for example, by chromatin condensation leading to cell shrinkage, membrane blebbing, and cell fragmentation. Cells undergoing apoptosis also exhibit a characteristic pattern of internucleosomal DNA fragmentation. Depending on the form of the polypeptide that binds to the receptor, a signal can be transmitted (e.g., by the multivalent form of an inhibitory receptor ligand), or a signal can be inhibited (e.g., by the soluble monovalent form of an inhibitory receptor ligand) by competing with the activated form of the ligand for binding to one or more natural binding partners. However, there are examples where soluble polypeptides can be stimulatory. The effects of the modulators can be readily demonstrated using routine screening assays as described herein.

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

[0074] The term "determining a treatment regimen suitable for a subject" is interpreted to mean the determination of a treatment regimen (i.e., a single therapy or a combination of different therapies used for the prevention and / or treatment of cancer in a subject) of 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 biomarker-mediated analysis encompassed by the present invention. One example is determining whether to provide a targeted therapy for cancer in order 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 for the purpose of reducing the risk of recurrence. Yet another example is modifying the dosage of a particular chemotherapy. In addition to the results of the analysis according to the present invention, the determination can be based on the individual characteristics of the subject being treated. In most cases, the actual determination of a treatment regimen suitable for a subject will be made by the attending physician or a doctor.

[0075] The term "endotoxin-free" or "substantially endotoxin-free" refers to a composition, solvent, and / or container that contains a maximum trace amount (e.g., an amount that has no clinically harmful physiological effects on a subject) of endotoxin, and preferably an undetectable amount of endotoxin. Endotoxins are toxins associated with certain bacteria, usually gram-negative bacteria, but may 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 membranes of various gram-negative bacteria, which represent a central pathogenic characteristic of the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can cause fever, decreased blood pressure, activation of inflammation and coagulation, and other harmful physiological effects.

[0076] Therefore, in the manufacture of pharmaceuticals, since even small amounts can have an adverse effect in humans, it is often desirable to remove most or all of the trace amounts of endotoxin from the formulation and / or the drug container. Since temperatures above 300°C are usually required to decompose most endotoxins, a pyrogen removal oven can be used for this purpose. For example, based on primary packaging materials such as syringes or vials, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to reduce the endotoxin level by 3 logs. Other methods of removing endotoxin, such as chromatography and filtration methods, as described herein and known in the art, are contemplated. Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus amebocyte lysate assay, which utilizes blood from horseshoe crabs, is a very sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the Limulus lysate by a powerful enzyme cascade that amplifies this reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). For substantially endotoxin-free, the endotoxin level 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, for example, 0.05 - 10 EU / ml. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1 - 10 EU.

[0077] The term "epitope" refers to a determinant or site on an antigen to which an antigen-binding protein (e.g., an immunoglobulin, an antibody, or an antigen-binding fragment) binds. Epitopes of protein antigens can be either linear epitopes or conformational epitopes. 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 the tertiary folding of a polypeptide. Conformational epitopes are typically lost upon treatment with denaturants. Epitopes typically contain 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 contains less 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 specific for a particular target molecule, or an antigen-binding fragment thereof, 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 of the 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 can be expressed during a particular cell lineage, stage of differentiation, or particular biological response. Biomarkers can reflect the biological aspects of the tumors in which they are expressed, such as the inflammatory state of the cells, the cells of origin of the cancer, the nature of non-malignant cells in a biopsy, and the carcinogenic mechanisms that cause cancer. Expression data and gene expression levels can be stored in a computer-readable medium, such as a computer-readable medium used in combination 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 associating covalently or non-covalently with a substrate, and such substrate can be rinsed with a fluid (e.g., standard citrate saline, pH 7.4) without substantial dissociation of the molecule from the substrate.

[0080] The term "gene" encompasses a nucleotide (e.g., DNA) sequence that encodes a molecule with a function (e.g., RNA, protein, etc.). A gene generally includes two complementary nucleotide strands (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 anti-codons. During transcription, RNA Pol II binds to the non-coding strand, reads the anti-codons, and transcribes their sequence to synthesize several RNA transcripts with complementary bases. In some embodiments, the recited gene sequence (i.e., DNA sequence) is the sequence of the coding strand.

[0081] A "functionally conserved variant" is one in which a given amino acid residue in a protein or enzyme has been changed without altering the overall three-dimensional structure and function of the polypeptide, including but not limited to replacement of the amino acid with one having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those shown to be conserved may vary between proteins, and as a result, the percent similarity of the protein or amino acid sequences between any two proteins having similar functions may vary, e.g., it may be between 70% and 99% when similarity is determined according to an alignment scheme such as the Clustal method based on the MEGALIGN algorithm. In some embodiments, a "functionally conserved 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 the BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parental protein to which it is being compared.

[0082] The term "gene product" (also referred to herein as "gene expression product" or "expression product") encompasses products resulting from the expression of a gene, such as nucleic acids (e.g., mRNA) transcribed from the gene, and polypeptides or proteins resulting from the translation of such mRNA. It will be understood that a particular gene product can, for example, be processed or modified in a cell. For example, an mRNA transcript can be spliced, polyadenylated, etc. before translation, and / or a polypeptide can undergo co-translational or post-translational processing such as removal of a secretion signal sequence, removal of an organelle targeting sequence, or modification such as phosphorylation, glycosylation, methylation, fatty acylation, etc. The term "gene product" encompasses such processed or modified forms. Genomic mRNA and polypeptide sequences of various species including humans are well 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, sequences in the NCBI reference sequence database can be used as gene product sequences for the gene of interest. It will be understood that multiple alleles of a gene can exist among individuals of the same species. Multiple isoforms of a particular protein can 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 related to allelic variants or isoforms are included where applicable. Particular embodiments can be directed to particular sequences (s), e.g., particular alleles (s) or isoforms (s).

[0083] The term "generate" encompasses any manner by which a desired result is achieved, such as directly or indirectly. For example, a cell having a regulated phenotype described herein can be generated by direct action, such as by contact with at least one agent that regulates one or more biomarkers described herein, and / or by indirect action, such as by growing a cell having the desired physical, genetic, and / or phenotypic attributes.

[0084] The term "glycosylation pattern" is a pattern of carbohydrate units that are covalently bound to a protein, more particularly an immunoglobulin protein. The glycosylation pattern of a xenogeneic 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, in which case one of ordinary skill in the art will recognize that the glycosylation pattern of the xenogeneic antibody is more similar to the glycosylation pattern in the species of non-human transgenic animal than to the glycosylation pattern in the species from which the CH gene of the transgene is derived.

[0085] The terms "high," "low," "intermediate," and "negative" as related to cellular biomarker expression refer to the amount of biomarker expressed as compared to the 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 the cellular level, activity, structure, etc. of one or more biomarker genomic nucleic acids, ribonucleic acids, and / or polypeptides. In one embodiment, the terms refer to defined percentages of populations of cells that express the biomarker at the highest, intermediate, or lowest levels, respectively. Such percentages can be defined as the upper 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, or any range therebetween, of populations of cells that highly or weakly express the biomarker. The term "low" excludes cells that do not detectably express the biomarker, since biomarker expression is "negative." The term "intermediate" includes cells that express the biomarker but at a level lower than the population that expresses it at the "high" level. In another embodiment, the terms may alternatively or additionally refer to populations of cells of biomarker expression identified by qualitative or statistical plot regions. For example, populations of cells sorted using flow cytometry can be determined based on biomarker expression levels by identifying distinct plots based on detectable sub-analyses, such as based on mean fluorescence intensity, 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 biomarker of interest. In yet another embodiment, the terms 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 using, for example, the methods described below. "Substantial identity" can be used to refer to sequences of various types and lengths, such as full-length sequences, functional domains, coding and / or control sequences, exons, introns, promoters, and genomic sequences. The percent sequence identity between two polypeptide or nucleic acid sequences can be determined in a variety of ways within the skill in the art, using, for example, publicly available computer software such as the BLAST program (Basic Local Alignment Search Tool; (Altschul et al. (1995) J. Mol. Biol. 215:403-410), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Further, one of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the length of the sequences being compared. For purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, it is understood that thymine nucleotides are equivalent to uracil nucleotides. 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 cells that can be directly or indirectly involved in the 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 (TIL), and the like. An "antigen-presenting cell" (APC) is a cell that can activate T cells and includes, but is not limited to, monocytes / macrophages, B cells, and dendritic cells (DC). The term "dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or 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 very 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 white blood cells that form part of the innate immune system. Neutrophils usually have a segmented nucleus containing about 2-5 lobes. Neutrophils migrate frequently to the site of injury within minutes after trauma. Neutrophils 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 an antigen and is capable of activating immune cells. The term "NK cell" has its general meaning in the art and refers to natural killer (NK) cells. One of ordinary skill in the art can readily identify NK cells, for example, by determining the expression of certain phenotypic markers (e.g., CD56), and can identify their function based on, for example, their ability to express different types of cytokines or their ability to induce cytotoxicity. The term "B cell" 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 cell" 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, autorecognition, etc.) and increase the immune response due to the expression of one or more T cell receptors. Tconv or Teff is generally defined as any T cell population that is not a Treg, and includes, for example, naive T cells, activated T cells, memory T cells, resting Tconv, or Tconv differentiated into the Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-regulatory T cells (Treg). In some embodiments, Teff is CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17, etc.) and CD8+ cytotoxic T cells (lymphocytes). As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tconv" is CD4. + A T cell that has differentiated in the bone marrow and successfully undergone the processes of positive and negative central selection in the thymus, but has not yet been activated by exposure to an antigen. Naive Tconv is generally characterized by surface expression of L-selectin (CD62L), absence of activation markers such as CD25, CD44, CD69, etc., and absence of memory markers such as CD45RO. Thus, naive Tconv is thought to be in a resting, non-dividing state and requires interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO2010 / 101870). The presence and activity of such cells are undesirable in the context of suppressing the immune response. Unlike Treg, Tconv is not anergic and can proliferate in response to activation of the antigenic T cell receptor (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:625-637). In tumors, exhausted cells may exhibit characteristics of anergy.

[0088] The term "immune disorder" includes immune diseases, conditions, states, and susceptibilities, 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, organ-specific autoimmunity (including, for example, multiple sclerosis, Hashimoto's thyroiditis, autoimmune uveitis, and Graves' disease), contact dermatitis, psoriasis, graft rejection, graft-versus-host disease, sarcoidosis, atopic diseases (including allergies such as asthma and gastrointestinal allergies such as allergic rhinitis and food allergies, but not limited thereto), eosinophilia, conjunctivitis, glomerulonephritis, systemic lupus erythematosus, scleroderma, helminthiasis (including, for example, leishmaniasis), and specific viral infections (including, for example, HIV and bacterial infections such as tuberculosis and Hansen's disease) and specific pathogen susceptibilities such as malaria.

[0089] The term "immune response" means a defensive response developed by the body directed against "foreign substances" such as bacteria, viruses, and pathogens, and not necessarily occurring outside the body, including but not limited to defensive responses directed against substances naturally present in the body (such as autoimmunity directed against self-antigens) or transformed (such as cancer) cells. In particular, an immune response is the activation and / or action of cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies (humoral response), cytokines, and complement) produced by any of these cells or the liver, resulting in selective targeting, binding, damage, destruction, and / or elimination of pathogens that invade the body of a vertebrate, pathogen-infected cells or tissues, cancerous or other abnormal cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An 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 systems to eliminate dangerous cancer cells.

[0090] The term "immune modulator" refers to a substance, agent, signaling pathway, or a component thereof that modulates an immune response. The terms "control," "modify," or "modulate" with respect to an immune response refer to any alteration in the cells of the immune system or the activity of such cells. Such control includes stimulation or suppression of the immune system (or a distinct part thereof), which can manifest 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 can occur within the immune system. Both inhibitory and stimulatory immune modulators have been identified, some of which can enhance their function in the tumor 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 subject's tumor or cancer. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0092] The terms "inhibit" or "downregulate" 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, further, cancer is "inhibited" if recurrence or metastasis of the cancer is decreased, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited if it is reduced as compared to a reference state, such as a control in a wild-type state. Such inhibition or deficiency can be induced, for example, by the application of an agent at a particular time and / or location, or can be constitutive, such as by a genetic mutation. Such inhibition or deficiency can also be partial or complete (e.g., essentially no measurable activity as compared to a reference state, such as a control in a wild-type state). In some embodiments, an essentially complete inhibition or deficiency is referred to as "blocked." In one embodiment, the term refers to reducing 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 of the corresponding control. The reduced level of a given output or parameter can, but need not necessarily, mean an absolute absence of the output or parameter. The present invention does not require, and is not limited to, methods that completely eliminate an 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 "promote" or "upregulate" have the opposite meaning.

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

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

[0095] The term "interaction", when referring to an interaction between two molecules, refers to a physical contact (e.g., binding) between the molecules with each other. Generally, such an interaction results in the activation (generating a biological effect) of one or both of the molecules. The activation can be a direct activation of one or both of the molecules (e.g., signal transduction). Alternatively, one or both of the molecules involved in the interaction can prevent binding to its ligand, and thus can remain inactive with respect to ligand binding activity (e.g., bind to its ligand and induce or inhibit co-stimulation). Inhibiting such an interaction results in the disruption of the activity of one or more of the molecules involved in the interaction. Enhancing such an interaction extends or increases the possibility of the physical contact and extends or increases the possibility of the activity.

[0096] "Isolated protein" refers to a protein that is isolated from cells or produced by recombinant DNA technology and substantially free of other proteins, cellular substances, separation media, and media, or, when chemically synthesized, chemical precursors or other chemical substances. An "isolated" or "purified" protein or a biologically active portion thereof is substantially free of cellular substances or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein is derived, or, when chemically synthesized, substantially free of chemical precursors or other chemical substances. The term "substantially free of cellular substances" includes preparations of the biomarker polypeptide or fragments thereof, where the protein is separated from the cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular substances" includes preparations of the biomarker protein 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., the culture medium represents less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%.

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

[0098] "K DThe term " " is intended to refer to the dissociation equilibrium constant of a specific antibody-antigen interaction. The binding affinity of the antibodies of the present invention disclosed can be measured or determined by standard antibody-antigen assays, such as competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA. In some embodiments, the K D of an antibody or antigen-binding fragment thereof described herein for a biomarker of interest, such as one or more of the biomarkers listed in Table 1, can be from 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, about 2 pM or less. In some embodiments, the binding affinity is lower than any of 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 ten pM, about 5 pM, or about 2 pM or less, or is in any range therebetween, such as from about 5 nM to about 35 nM.

[0099] The term "kd" or "k オフ " refers to the off-rate constant for the dissociation of the antibody from the antibody / antigen complex. The value of Kd is a numerical value of the fraction of the complex that decays or dissociates per second, and the unit is seconds -1 and is represented by.

[0100] The term "ka" or "k オンThe term "Ka" 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 (1 M) solution of antibody and antigen, and is expressed in units of M -1 seconds -1 and is expressed as

[0101] The term "microenvironment" generally refers to a local region within a tissue area of interest and can refer to, for example, the "tumor microenvironment". The term "tumor microenvironment" or "TME" refers to the surrounding microenvironment that constantly interacts with tumor cells and helps enable crosstalk between tumor cells and their environment. The tumor microenvironment can include the cellular environment of the tumor, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix. The tumor environment can include tumor cells or malignant cells that are supported and influenced by the tumor microenvironment to ensure growth and survival. The tumor microenvironment also includes tumor-infiltrating immune cells such as lymphocytes and bone marrow cells, which can stimulate or inhibit stromal cells such as tumor-associated fibroblasts and endothelial cells that contribute to the structural integrity of the tumor, as well as the anti-tumor immune response. Stromal cells include cells that make up the blood vessels associated with the tumor, such as endothelial cells and pericytes, cells that contribute to structural integrity (fibroblasts), as well as tumor-associated macrophages (TAM), and infiltrating immune cells including monocytes, polymorphonuclear neutrophils (PMN), dendritic cells (DC), T and B cells, mast cells, and natural killer (NK) cells. Stromal cells make up the majority of tumor cellularity, but the major cell type in solid tumors is macrophages.

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

[0103] The "normal" expression level of a biomarker is the expression level of the biomarker in cells of a subject not suffering from cancer, e.g., a human patient. <000>

[0104] "Overexpression" or "significantly high level of expression" of a biomarker refers to the expression level in a test sample that is greater than the standard error of the assay used to evaluate the expression, and is at least 10% less than the expression activity or level of the biomarker in a control sample (e.g., a sample from a healthy subject without a biomarker-related disease), preferably, 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 times higher. "Significantly low level of expression" of a biomarker refers to the expression level in a test sample that is at least 10% less than the expression level of the biomarker in a control sample (e.g., a sample from a healthy subject without a biomarker-related disease), preferably, 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 times lower.

[0105] Such a "significance" level may also apply to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell proliferation, etc.

[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 with reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted compared to the reference polypeptide itself, but the remaining amino acid sequence is usually identical to the corresponding positions within the reference polypeptide. Such deletions can occur at the amino terminus, internally, or at the carboxyl terminus of the reference polypeptide, or alternatively at 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 can 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, 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 in length and / or contain, provided that they are shorter than the length of the full-length polypeptide. Alternatively, they can be not longer than and / or exclude such ranges, provided that they are shorter than the length of the full-length polypeptide.

[0108] The term "pre-determined" biomarker amount and / or activity measurement(s) can, by way of example only, be the biomarker amount and / or activity measurement used to evaluate a subject who can be selected for a particular treatment, evaluate the response to treatment such as one or more modulators of one or more of the biomarkers described herein, and / or evaluate a disease state. The pre-determined biomarker amount and / or activity measurement(s) can be determined in a population of patients, regardless of the presence or absence of cancer. The pre-determined biomarker amount and / or activity measurement(s) can be a single value equally applicable to all patients, or the pre-determined biomarker amount and / or activity measurement(s) can vary according to a particular sub-population of patients. The age, weight, height, and other factors of a subject can affect the pre-determined biomarker amount and / or activity measurement(s) of an individual. Further, the amount and / or activity of a pre-determined biomarker can be determined individually for each subject. In one embodiment, the amount determined and / or compared by the methods described herein is based on an absolute measurement. In another embodiment, the amount determined and / or compared by the methods described herein is based on a relative measurement value such as a ratio (e.g., a cell ratio or serum biomarker normalized to the expression of a housekeeping or other generally constant biomarker). The pre-determined biomarker amount and / or activity measurement(s) can be any suitable standard. For example, the pre-determined biomarker amount and / or activity measurement(s) can be obtained from the same or different humans in whom the selection of the patient is being evaluated. In one embodiment, the pre-determined biomarker amount and / or activity measurement(s) can be obtained from a previous evaluation of the same patient. In such a manner, the progression of the selection of the patient can be monitored over time. Further, the control can be obtained from the evaluation of another human or multiple humans, e.g., a group of selected humans, when the subject is a human.In such a manner, the degree of selection of a human being whose selection is being evaluated can be compared to that of other suitable human beings, such as other human beings in a similar situation to the human being of interest, such as those suffering from a similar or the same condition(s), and / or human beings of the same ethnic group.

[0109] The term "predictive" includes the use of biomarker nucleic acids and / or protein states, such as the over- or under-activity, occurrence, expression, growth, remission, recurrence, or resistance of a tumor before, during, or after treatment to determine the desired likelihood. Such predictive use of a biomarker can be determined, for example, by (1) an increase or decrease in copy number (e.g., by FISH, FISH plus SKY, single molecule sequencing, such as those described in at least J. Biotechnol., 86:289-301 in the art, or by qPCR), overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern blot, or qPCR), an increase or decrease in a biomarker protein (e.g., by IHC), or an increase or decrease in activity of greater than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than that of a cancer type or cancer sample of an assayed human being, (2) the absolute or relatively regulated presence or absence thereof in a biological sample, such as a sample containing tissue, whole blood, serum, plasma, buccal swab, saliva, cerebrospinal fluid, urine, feces, or bone marrow from a human subject suffering from cancer, (3) the absolute or relatively regulated presence or absence thereof in a clinical subset of patients having cancer (e.g., those responding, either alone or in combination with immunotherapy, to a specific modifier of T cell-mediated cytotoxicity, or those expressing resistance thereto).

[0110] The terms "prevent", "preventing", "prevention", "preventive treatment", etc. are not the subject, but refer to reducing the likelihood of developing a disease, disorder, or condition in a subject at risk of developing, or prone to developing, the disease, disorder, or condition.

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

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

[0113] The term "ratio" refers to the relationship between two numerical values (e.g., scores, totals, etc.). However, a ratio can be expressed in a particular order (e.g., a to b or a:b), and one of ordinary skill in the art can reverse the observation of trends and correlations based on the ratio, but will recognize that the underlying relationship between the numbers can be expressed in any order without losing the significance of the underlying relationship.

[0114] The term "rearrangement" means that the V segment is an essentially complete V H and V LIn the three-dimensional structure encoding each domain, it refers to the configuration of the heavy or light chain immunoglobulin locus that is directly adjacent to the D-J or J segment. The rearranged immunoglobulin locus can be identified by comparison with germline DNA, and the rearranged locus will have at least one recombination heptamer / nonamer homology element. In contrast, the term "unrearranged" or "germline configuration" with respect to the V segment refers to a configuration in which the V segment is not rearranged to be directly adjacent to the D or J segment.

[0115] The term "receptor" refers to a naturally occurring molecule or complex of molecules that is generally 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 modulators of T cell-mediated cytotoxicity / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to cancer mediators such as modulators of T cell-mediated cytotoxicity, as well as changes in tumor mass and / or volume related to immunotherapy, preferably after the initiation of neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to a treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy. The hyperproliferative disorder response can be evaluated, for example, for effectiveness or in the context of neoadjuvant or adjuvant, and the size of the tumor after systemic intervention can be compared to the initial size and dimensions measured by CT, PET, mammogram, ultrasound, or palpation. The response can also be evaluated by tumor caliper measurement or pathological examination after biopsy or surgical resection. The response can be recorded in a quantitative manner such as the rate of change of tumor volume, or in a qualitative manner such as "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. The evaluation of the hyperproliferative disorder response can be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, after several hours, days, weeks, or preferably after several months. A typical endpoint for response evaluation is at the end of neoadjuvant chemotherapy or at the time of surgical removal of residual tumor cells and / or the tumor bed. This is usually 3 months after the initiation of neoadjuvant therapy. In some embodiments, the clinical effectiveness 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 sum of 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 simplified notation of 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 the response to cancer treatment are related to "survival time", which is also known as overall survival time (the survival time until death, which may not be related to either the cause or the tumor), "recurrence-free survival time" (the term recurrence includes both local and distant recurrences), survival time without metastasis, and disease-free survival time (the term disease includes cancer and related diseases). The length of survival can be calculated by referring to defined starting points (e.g., at the time of diagnosis or treatment initiation) and end points (e.g., death, recurrence, or metastasis). Further, the criteria for treatment effectiveness can be extended to include response to chemotherapy, probability of survival time, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine appropriate thresholds, a particular cancer treatment regimen can be administered to a population of subjects, and the outcomes can be correlated with biomarker measurements determined prior to any cancer treatment. The measurement of outcomes can be the pathological response to treatment given in the neoadjuvant setting. Alternatively, outcome metrics such as overall survival time and disease-free survival time can be monitored over a period of time for subjects after cancer treatment where the biomarker measurements are known. In certain embodiments, the dosage administered is a standard dosage known in the art for the cancer treatment agent. The period during which the subject is monitored can vary. For example, the subject 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 correlated with the outcomes of cancer treatment can be determined using methods well known in the art, such as those described in the Examples section.

[0117] As shown, the term may refer to an improvement in prognosis, for example, as reflected by an increase in the period to recurrence, which is the period until review of the first recurrence for a first event or a second primary cancer as death without evidence of recurrence or extension of overall survival, which is the period from treatment until 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, reduced, or attenuated when exposed to the stimulus. It will be understood that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., show a lack of response or not respond).

[0118] The term "resistance" refers to acquired or natural resistance of a cancer sample or mammalian cancer treatment to cancer treatment, i.e., not responding to a therapeutic treatment, or having a reduced or limited response, where the response to cancer treatment decreases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range in between (including the boundary values). The decrease in response can be measured by comparing the same cancer sample or mammal before the acquisition of resistance, or by comparing with a different cancer sample or mammal that is known to be non-resistant to the therapeutic treatment. A typical acquired resistance to chemotherapy is referred to as "multi-drug resistance". Multi-drug resistance can be mediated by P-glycoprotein or by other mechanisms, or can occur when a mammal is infected with multi-drug resistant microorganisms or a combination of microorganisms. The determination of resistance to a therapeutic treatment is conventional in the art and within the skill of the artisan, and can be measured, for example, by the cell proliferation assays and cell death assays described herein as "sensitizing". In some embodiments, the term "reversing resistance" means that the use of a second agent in combination with a primary cancer treatment (e.g., chemotherapy or radiation therapy) results in a significant reduction in tumor volume at a level of statistical significance (e.g., p < 0.05) compared to the tumor volume of an untreated tumor in a situation where the primary cancer treatment (e.g., chemotherapy or radiation therapy) alone cannot produce a statistically significant reduction in tumor volume compared to the tumor volume of the untreated tumor. This generally applies to tumor volume measurements taken when untreated tumors are growing logarithmically and rhythmically.

[0119] The term "sample" used to detect or determine the presence or level of at least one biomarker typically includes brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears, and any other body fluid (e.g., those described in the definition of "body fluid" above), or tissue samples such as small intestine, colon samples, or surgically excised tissue (e.g., biopsy). In certain instances, the 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" means modifying cancer cells or tumor cells in a manner that enables more effective treatment of the associated cancer by cancer therapy (e.g., anti-immune checkpoint, chemotherapy, and / or radiotherapy). In some embodiments, normal cells are not affected to the extent that they are overly damaged by the treatment. The increased or decreased sensitivity to a therapeutic treatment is measured according to known methods in the art for specific treatments and the methods described below, including but not limited to 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 G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993:415-432, 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 for humans and 4-6 weeks for mice. A composition or method is sensitized to a therapeutic treatment if the increased treatment sensitivity or decreased resistance is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range therebetween (including the boundary values), compared to the treatment sensitivity or resistance in the absence of such composition or method. Determination of sensitivity or resistance to a therapeutic treatment is conventional in the art and within the skill of an ordinary skilled clinician.It should be understood that any method described herein for enhancing the effectiveness of cancer treatment is equally applicable as a method for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to cancer treatment.

[0121] The term "selective modulator" or "selective modulation" as applied to a biologically active agent refers to the ability of the agent to modulate a target such as a cell population, signaling activity, etc., as compared 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 the interaction between a protein and one natural binding partner over another interaction between the protein and another binding partner, and / or over such interaction(s) with a target cell population of interest, inhibits the interaction 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%, 2× (times), 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 55×, 60×, 65×, 70×, 75×, 80×, 85×, 90×, 95×, 100×, 105×, 110×, 120×, 125×, 150×, 200×, 250×, 300×, 350×, 400×, 450×, 500×, 600×, 700×, 800×, 900×, 1000×, 1500×, 2000×, 2500×, 3000×, 3500×, 4000×, 4500×, 5000×, 5500×, 6000×, 6500×, 7000×, 7500×, 8000×, 8500×, 9000×, 9500×, 10000×, or more, or any range therebetween (including the boundary values). Such a measure is typically expressed as the relative amount of the agent required to reduce the interaction / activity by half. Such a measure is applicable to other selective arrangements such as the binding of a nucleic acid molecule 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 object of interest compared to other targets. For example, a measured variable (e.g., regulation of biomarker expression in a desired cell versus other cells, enrichment and / or depletion of a desired cell versus other cells, etc.) can be 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, 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 in between (including the boundary values) (e.g., 50% to 16-fold) and can be different in the target of interest compared to an unintended or undesired target. The same fold analysis can be used to confirm the degree of effect in a given tissue, cell population, measured variable, and / or measured effect, etc., such as in cell ratio, 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 regulation of the interaction between a protein and one binding partner refers to exclusive regulation of that interaction and does not refer to significant regulation of the interaction between the protein and another binding partner. In another example, specific binding of an antibody to a predetermined antigen refers to the ability of the antibody to bind to the antigen of interest without binding to other antigens. Typically, the antibody has a concentration of less than approximately 1×10 -7 M, for example, approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11When determined using an appropriate assay, such as using surface plasmon resonance (SPR) technology in a BIACORE® assay instrument that uses M, or an antigen of interest as the analyte and an antibody as the ligand, binding occurs with an even lower affinity (K 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 in the art and includes molecules having a molecular weight of less than about 1000 or less than about 500. In one embodiment, the small molecule exclusively contains no peptide bonds. In another embodiment, the 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 of interest, unless otherwise specified.

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

[0127] The term "survival period" includes all of the survival period until death, also known as overall survival period (the death may not be related to either the cause or the tumor), "recurrence-free survival period" (the term recurrence includes both local and distant recurrences), survival period without metastasis, and disease-free survival period (the term disease includes cancer and related diseases). The length of the survival can be calculated by referring to a defined starting point (e.g., the time of diagnosis or treatment initiation) and an end point (e.g., death, recurrence, or metastasis). Further, the criteria for the effectiveness of treatment can be extended to include response to chemotherapy, probability of survival period, 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., combination therapies of regulators of biomarkers described in Table 1 and immunotherapy) that is greater than the sum of the individual effects of the cancer agent / therapy alone.

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

[0130] The term "therapeutic effect" encompasses local or systemic effects in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term means any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in animals or humans or for the enhancement of a desired physical or mental development and condition. Preventive effects included within the term are 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 containing such 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 dosage form, route, and / or schedule. As will be understood by those of skill in the art, the absolute amount of a particular agent or composition that is effective can vary depending upon factors such as the desired biological or pharmacological endpoint, the agent being delivered, the target tissue, etc. Those of skill in the art will further understand that in various embodiments, an "effective amount" can be contacted with a cell or administered to a subject as a single dose or through the use of multiple doses. The term "effective amount" can be a "therapeutically effective amount".

[0132] The term "therapeutically effective amount" refers to an 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. The toxicity and therapeutic efficacy of the subject compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD 50 and ED 50 . Compositions showing a large therapeutic index are preferred. In some embodiments, LD 50 (lethal dose) can be measured, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more less for the agent compared to when the agent is not administered. Similarly, ED 50 (i.e., the concentration to achieve maximum half inhibition of symptoms) can be measured, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more greater for the agent compared to when the agent is not administered. Further similarly, IC 50(i.e., the concentration that achieves up to half the maximum cytotoxicity or cell growth inhibitory effect against cancer cells) can be measured, for example, compared to the case where no drug is administered, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increase for the drug. 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 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% reduction can be achieved in solid malignant tumors.

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

[0134] The terms "tolerance" or "unresponsiveness" include the refractivity of cells such as immune cells to stimuli, such as stimuli via activating receptors or cytokines. Unresponsiveness can occur, for example, due to exposure to immunosuppressive agents or high doses of antigen. Several independent methods can induce tolerance. One mechanism is termed "anergy" and is defined as a state in which cells survive in vivo as unresponsive cells rather than differentiating into cells with effector functions. Such unresponsiveness is generally antigen-specific and persists after exposure to the tolerizing antigen has ended. For example, anergy in T cells is characterized by, for example, a lack of cytokine production such as IL-2. T cell anergy occurs when T cells are exposed to antigen and receive the first signal (T cell receptor or CD-3 mediated signal) in the absence of the second signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of co-stimulatory polypeptides) fails to produce cytokines and thus fails to proliferate. However, anergic T cells can proliferate when cultured with cytokines such as 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 using indicator cell lines in a proliferation assay. Alternatively, reporter gene constructs can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by multimers of the AP1 sequence that can be 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 impaired effector function, distinct from functional effector or memory T cells, persistent expression of inhibitory receptors, and transcriptional states.

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

[0136] The term "treatment" refers to the therapeutic management or improvement of a target condition (e.g., a disease or disorder). Treatment can 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 as an attempt to modify the course of a disease in a manner that is beneficial to the subject (this term is used to indicate a disease, disorder, syndrome, or undesirable condition that justifies or potentially justifies treatment). The effects of treatment can include reversal, alleviation, reduction in severity, delay in onset, cure, inhibition of progression, and / or reduction in the likelihood of occurrence or recurrence of a disease or one or more symptoms or signs of the disease. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, recovery or primary alleviation of the medical condition, and remission or improvement of the prognosis. A therapeutic agent can be administered to a subject who has a disease or is at high 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 exhibits evidence of the disease. An 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 a disease. "Prophylactic treatment" refers to providing medical and / or surgical management to a subject who has not developed a disease or does not sequentially exhibit evidence of a disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity if the disease does occur. A subject can be identified as being at risk of developing a disease (e.g., an increased risk compared to the general population or having a risk factor that increases the likelihood of developing the disease).

[0137] The term "non-responsiveness" includes refractivity of cancer cells to treatment, or of therapeutic cells such as immune cells to stimuli, e.g., stimuli via activating receptors or cytokines. Non-responsiveness can occur, for example, due to exposure to immunosuppressive agents or exposure to high doses of antigen. As used herein, the terms "anergy" or "tolerance" include unresponsiveness to activation of receptor-mediated stimuli. Such unresponsiveness is generally antigen-specific and persists after exposure to the tolerizing antigen has ended. For example, (in contrast to non-responsiveness) anergy in T cells is characterized by lack of cytokine production, e.g., lack of IL-2. T cell anergy occurs when a T cell is exposed to an antigen and receives a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure of the cell to the same antigen (even if the re-exposure occurs in the presence of a co-stimulatory polypeptide) fails to produce cytokines and thus fails to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2, etc.). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes measured by ELISA or using an indicator cell line in a proliferation assay. Alternatively, a reporter gene construct can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by multimers of the AP1 sequence that can be found within a heterologous promoter or enhancer 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 a disease.

[0139] Furthermore, as defined by the genetic code (shown below), there is a known and unambiguous correspondence between the amino acid sequence of a particular protein and the nucleotide sequence capable of encoding the protein. Similarly, as defined by the genetic code, there is a known and unambiguous correspondence between the nucleotide sequence of a particular nucleic acid 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, by which more than one coding nucleotide triplet can be used for most of the amino acids used in protein production (shown above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered to be functionally equivalent because the same amino acid sequence is produced in all organisms (although some organisms may translate some sequences more efficiently than others). Additionally, 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, a polypeptide amino acid sequence can be derived by using the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) and 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 that can encode the polypeptide can be deduced from the genetic code (generating multiple nucleic acid sequences for any given amino acid sequence due to its redundancy). Thus, the description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include the description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, the description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include the description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0142] II. Monocytes and Macrophages Monocytes are immune effector cells derived from the bone marrow, circulate in the blood, bone marrow, and spleen, and have restricted proliferation in the steady state. The term "bone marrow cells" can refer to granulocyte or monocyte progenitor cells in the bone marrow or spinal cord, or those with similarities to what is found in the bone marrow or spinal cord. The myeloid cell lineage includes circulating monocyte cells in peripheral blood, as well as cell populations that they become after maturation, differentiation, and / or activation. These populations include non-terminally differentiated bone marrow cells, bone marrow-derived suppressor cells, and differentiated macrophages. Differentiated macrophages include non-polarized and polarized macrophages, resting and activated macrophages. Without limitation, the myeloid system also includes granulocyte precursors, polymorphonuclear-derived suppressor cells, differentiated polymorphonuclear leukocytes, neutrophils, granulocytes, basophils, eosinophils, monocytes, macrophages, microglia, bone marrow-derived suppressor cells, dendritic cells, and erythrocytes. Monocytes are found within peripheral blood mononuclear cells (PBMCs), which also include 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 progenitor cells called monoblasts. Monocytes have two main functions in the immune system: (1) they can leave the bloodstream and replenish resident macrophages and dendritic cells (DCs) under normal conditions, and (2) they can rapidly migrate to sites of infection within tissues and divide / differentiate into macrophages and inflammatory dendritic cells to induce an immune response in response to inflammatory signals. Monocytes are usually identified by a large bilobed nucleus in stained smear specimens. Monocytes also express chemokine receptors and pathogen recognition receptors that mediate their migration from the blood to tissues during infection. They produce inflammatory cytokines and phagocytic cells. In some embodiments, the bone marrow cells of interest are identified according to CD11b+ expression and / or CD14+ expression.

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

[0144] Macrophages are important immune effectors and regulators of inflammation and innate immune responses. Macrophages are heterogeneous, tissue-resident, terminally differentiated, innate myeloid cells with significant plasticity, capable of changing their physiological functions in response to local cues from the microenvironment, and can assume a spectrum of functional requirements from host defense to tissue homeostasis (Ginhoux et al. (2016) Nat. Immunol. 17:34-40). Macrophages are present in almost all tissues in the body. They are either tissue-resident macrophages, such as Kupffer cells that reside in the liver, or derived from circulating monocyte precursors (i.e., monocytes), mainly from the bone marrow and spleen reservoirs, and migrate to tissues in the steady state or in response to inflammation or other stimulatory cues. For example, monocytes can be mobilized from the blood to tissues to replenish tissue-specific macrophages in bone, alveoli (lungs), 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 homeostatic functions. Tissue-resident macrophages develop in the embryonic yolk sac, mature in specific tissues of the developing fetus, acquire tissue-specific roles there, and change their gene expression profiles. Local proliferation of tissue-resident macrophages that maintain colony-forming ability can directly give rise to a population of mature macrophages within the tissue. Local proliferation of macrophages present in tissues that maintain colony-forming ability may directly give rise to a population of mature macrophages within the tissue. Tissue-resident macrophages can also be identified and named according to the tissues 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 tissues, histiocytes that give rise to giant cells occupy connective tissue, microglia occupy central nervous system (CNS) tissue, Hofbauer cells occupy placental tissue, mesangial cells within glomeruli occupy kidney tissue, osteoclasts occupy bone tissue, epithelioid cells occupy granulomas, red pulp macrophages (sinusoidal lining cells) occupy the red pulp of spleen tissue, peritoneal macrophages occupy peritoneal tissue, lysomac cells occupy Peyer's patch tissue, and pancreatic macrophages occupy pancreatic tissue.

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

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

[0148] Macrophages are also involved in the elimination of cell debris generated during tissue remodeling and rapidly and efficiently eliminate cells that have undergone apoptosis. Macrophages are thought to be involved in maintaining tissue homeostasis through the elimination of apoptotic cells. These homeostatic elimination 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 induce signaling that transcribes cytokine genes or actively produce inhibitory signals and / or cytokines. The homeostatic functions of macrophages are independent of other immune cells.

[0149] Macrophages can also eliminate cell debris / necrotic cells resulting from trauma or other damage to cells. Macrophages detect endogenous danger signals present within the fragments of necrotic cells via Toll-like receptors (TLRs), intracellular pattern recognition receptors, and interleukin-1 receptors (IL-1R), and most of them transmit signals via the adapter molecule myeloid differentiation primary response gene 88 (MyD88). The elimination of cell debris can significantly modify the physiological functions of macrophages. Macrophages that eliminate necrosis can cause dramatic changes in physiological functions, including alterations in the expression of surface proteins and the production of cytokines and pro-inflammatory mediators. The modification of macrophage surface protein expression in response to these stimuli may be used to identify biochemical markers specific to these altered cells.

[0150] Macrophages have important functions in maintaining the homeostasis of many tissues such as white adipose tissue, brown adipose tissue, liver, and pancreas. Tissue macrophages can respond rapidly to changes in tissue status by releasing cell signaling molecules that trigger a cascade of changes to adapt tissue cells. For example, macrophages in adipose tissue can regulate the generation of new adipocytes in response to changes in diet (e.g., macrophages in white adipose tissue) or exposure to low temperature (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 changes in diet. 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, macrophages can be activated in response to signals derived from damaged tissues and cells and induce a tissue repair response to repair the damaged tissue (Minutti et al. (2017) Science 356:1076-1080).

[0152] During embryonic development, macrophages also play important roles in tissue remodeling and organ development. For example, resident macrophages actively form the development of blood vessels in the heart of neonatal mice (Leid et al. (2016) Circ.Res. 118:1498 - 1511). Microglia in the brain can generate growth factors that guide neurons and blood vessels during brain development during embryonic development. Similarly, CD95L, a protein produced by macrophages, binds to the CD95 receptor on the surface of neurons, develops blood vessels in the brain of mouse embryos, and promotes the development of neurons and blood vessels (Chen et al. (2017) Cell Rep. 19:1378 - 1393). Without the ligand, the branching frequency of neurons is reduced, and the resulting adult brain shows less electrical activity. Monocyte-derived cells known as osteoclasts are involved in bone development, and mice lacking these cells develop dense and hardened bone, a rare condition known as marble bone disease. Macrophages also regulate breast development and assist in the early postnatal development of the retina (Wynn et al. (2013) Nature 496:445 - 455).

[0153] As described above, macrophages control the immune system. In addition to antigen presentation to T cells, macrophages can provide immunosuppressive / suppressive signals to immune cells under some conditions. For example, in the testis, macrophages help create an environment that prevents sperm from being attacked by the immune system. Resident macrophages in the tissues within the testis produce immunosuppressive molecules that prevent the immune cell response to sperm (Mossadegh-Keller et al. (2017) J.Med. 214:10.1084 / jem.20170829).

[0154] In response to different environmental signals, the plasticity of macrophages to match their functional requirements has led to a series of macrophage activation states, including two extreme ends of a continuum, namely "classically activated" M1 and "alternatively activated" M2 macrophages.

[0155] The term "activation" refers to the state of myeloid cells that have been sufficiently stimulated and / or induced to exert effector functions such as the expression and secretion of cytokines, phagocytosis, cell signaling, antigen processing and presentation, killing of target cells, and inflammation-inducing functions, which are stimulated to induce detectable cell proliferation.

[0156] The term "M1 macrophage" or "classically activated macrophage" refers to macrophages with an inflammatory phenotype. The term "macrophage activation" (also referred to as "classical activation") was introduced by Mackaness in the context of infection in the 1960s (Mackaness (1962) J. Exp. Med. 116:381-406) to account for the antigen-dependent but nonspecific enhanced bactericidal activity of macrophages against BCG (bacillus Calmette-Guerin) and Listeria upon secondary exposure to pathogens. The enhancement was later associated with the Th1 response 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. U.S.A. 80:3782-3786, Celada et al. (1984) J. Exp. Med. 160:55-74). Thus, macrophage functions that enhance inflammation through cytokine secretion, antigen presentation, phagocytosis, cell-cell interactions, migration, etc. are considered pro-inflammatory. In vitro and in vivo assays can measure different endpoints: common in vitro measurements include proliferation, migration, pro-inflammatory cell stimulation measured by the secretion of pro-inflammatory Th1 cytokines / chemokines and / or migration, while common in vivo measurements further include the analysis of battles against pathogens, immediate responders to tissue damage, other cell activators, migration-inducing substances, etc. In both in vitro and in vivo settings, pro-inflammatory antigen presentation can be evaluated. Lipopolysaccharide (LPS), specific Toll-like receptor (TLR) agonists, the Th1 cytokine interferon gamma (IFNγ) (e.g., IFNγ produced by NK cells in response to stress and infection, as well as T helper cells with persistent 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 the adaptive immune response, produce high levels of pro-inflammatory 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 the production of IL-12, a Th1-related cytokine, are well adapted to promote a strong immune response. The metabolism of M1 macrophages is characterized by enhanced aerobic glycolysis, conversion of glucose to lactate, increased flux through the pentose phosphate pathway (PPP), fatty acid synthesis, and a shortened tricarboxylic acid (TCA) cycle leading to the accumulation of succinate and citrate.

[0157] "Type 1" or "M1-like" myeloid cells contribute to an inflammatory response by at least one of the following: generating an inflammatory stimulus by secreting at least one pro-inflammatory 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 an inflammatory response; presenting an antigen in an inflammatory context; migrating to a site enabling the initiation of an inflammatory response, or initiating the expression of at least one gene predicted to lead to an inflammatory function. In some embodiments, the term includes mediating the activation of cytotoxic CD8+ T cells, increasing the sensitivity of cancer cells to immunotherapies such as immune checkpoint therapies, and / or reversing the resistance of cancer cells. In certain embodiments, such modulation to an inflammatory state can be measured in several well-known ways, including, but not limited to: a) increased expression and / or secretion of surface antigen classification 80 (CD80), CD86, MHCII, MHCI, 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, TGFb, and / or IL-10; c) 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; d) increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10; 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, flatness of appearance, and / or number of dendritic processes when evaluated by microscopy.

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

[0159] In contrast, the term "M2 macrophage" 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), prostaglandin E2 (PGE2), etc., can promote M2 polarization. The metabolic profile of M2 macrophages is defined by reduced OXPHOS, FAO, glycolysis, and PPP. The finding that the mannose receptor is selectively enhanced by Th2 IL-4 and IL-13 in mouse macrophages, induces high endocytosis of mannosylated ligands, increases the expression of major histocompatibility complex (MHC) class II antigens, and decreases the secretion of inflammatory cytokines led to the proposal by Stein, Doyle, and colleagues that IL-4 and IL-13 induce an alternative activation phenotype that is quite different from but far from inactivation of IFN-γ activation (Martinez and Gordon (2014) F1000 Prime Reports 6:13). In in vitro and in vivo definitions / assays, different endpoints can be measured: common in vitro endpoints include proliferation, migration, anti-inflammatory Th2 cytokine / chemokine secretion, and / or anti-inflammatory cell stimulation measured by migration, while common in vivo M2 endpoints further include analysis of the fight against pathogens, the response to delay / facilitation of tissue damage / fibrosis, Th2 polarization of other cells, chemoattractants, etc. In both in vitro and in vivo, tolerogenic antigen presentation can be evaluated.

[0160] "Type 2" or "M2-like" myeloid cells can contribute to an anti-inflammatory response by producing 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 its surface, mobilizing / instructing / interacting with at least one other cell to stimulate an anti-inflammatory response, presenting an antigen under tolerogenic circumstances, migrating to a site that enables the initiation of a tolerogenic response, or initiating the expression of at least one gene expected to lead to tolerogenic / anti-inflammatory functions. In certain embodiments, such modulation of the pro-inflammatory state can be measured by several well-known methods including, but not limited to, the opposite of the type 1 pro-inflammatory state measurements described above.

[0161] Cells having an "increased inflammatory phenotype" are cells having a higher pro-inflammatory response capacity associated with, for example, an increase in one or more of the type 1 listed criteria and / or a decrease in one or more of the type 2 listed criteria after modulation of at least one biomarker (e.g., at least one target listed in Table 1) encompassed by the present invention, such as by contact with an agent that modulates at least one biomarker (e.g., at least one target listed in Table 1) encompassed by the present invention.

[0162] Cells having a "decreased inflammatory phenotype" are cells having a higher anti-inflammatory response capacity associated with, for example, a decrease in one or more of the type 1 listed criteria and / or an increase in one or more of the type 2 listed criteria after modulation of at least one biomarker (e.g., at least one target listed in Table 1) encompassed by the present invention, such as by contact with an agent that modulates at least one biomarker (e.g., at least one target listed in Table 1) encompassed by the present invention.

[0163] Thus, macrophages can adopt a continuum of alternatively activated states with phenotypes between the M1 and M2 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 terms "alternatively activated macrophage" or "alternatively activated state" refer to essentially all types of macrophage populations other than classically activated M1 pro-inflammatory macrophages. Originally, the alternatively activated state was designated only for M2 anti-inflammatory macrophages. The terms are extended 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 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. Wound-healing macrophages secrete large amounts of extracellular matrix components, such as chitinases and chitinase-like proteins YM1 / CHI3L3, YM2, AMCase, and stabilin, instead of producing high levels of pro-inflammatory cytokines, all of which exhibit carbohydrate and matrix-binding activities and are involved in tissue repair.

[0166] Another example of alternatively activated macrophages includes regulatory macrophages that can be induced by innate and adaptive immune responses. Regulatory macrophages can contribute to immune regulatory functions. For example, macrophages can assume a state in which inflammatory functions such as host defense and the transcription of pro-inflammatory cytokines are inhibited in response to hormones (e.g., glucocorticoids) from the hypothalamic-pituitary-adrenal (HPA) axis. Regulatory macrophages can produce the regulatory cytokine TGF-β and can dampen the immune response under certain conditions, for example, at the late stages of the adaptive immune response. Many regulatory macrophages express high levels of co-stimulatory molecules (e.g., CD80 and CD86) and can thus enhance antigen presentation to T cells.

[0167] Many stimuli / cues can induce the polarization of regulatory macrophages. Cues include, but are not limited to, combinations of TLR agonists and immune complexes, apoptotic cells, IL-10, prostaglandins, GPcR 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, leading to incomplete killing of the pathogen and promoting the survival and spread of the infected microbe.

[0168] Regulatory macrophages share some common features. For example, regulatory macrophages require two stimuli to induce an anti-inflammatory effect. Differences are also observed between subpopulations of regulatory macrophages induced by different cues / stimuli, reflecting their heterogeneity.

[0169] Controlling macrophages are also a heterogeneous population of macrophages, including various subpopulations found in metabolism, development, and maintenance of homeostasis. In one example, a subpopulation of alternatively activated macrophages are immunoregulatory macrophages with unique immunoregulatory properties that can be induced in the presence of M-CSF / GM-CSF, CD16 ligand (such as immunoglobulins), and IFN-γ (PCT Application Publication No. WO2017 / 153607).

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

[0171] The apparent plasticity of macrophages also results in an unstable response to environmental cues they receive in disease states. Macrophages can re-polarize in response to various disease states and exhibit distinct characteristics. One example is macrophages attracted from peripheral blood monocytes to tumor tissue and filtered, which are often referred to as "tumor-associated macrophages" ("TAM") or "tumor-infiltrating macrophages" ("TIM"). Tumor-associated macrophages are the most abundant inflammatory cells within tumors, and a significant correlation was found between high TAM density and poor prognosis of 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 are present 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 late-stage tumors are M2-like regulatory macrophages that are 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 hypoxic environment, and cytokines secreted by tumor cells. M2-like TAMs exhibit a hybrid activation state of wound-healing macrophages and regulatory macrophages, produce high levels of IL-10 but little or no IL-12, show incomplete TNF production, suppress antigen-presenting cells, and exhibit various unique characteristics including contributions to tumor angiogenesis.

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

[0174] Diverse functions and activation states can lead to dangerous consequences if not properly regulated. For example, classically activated macrophages can cause damage to host tissues, predispose surrounding tissues, and affect glucose metabolism when over-activated.

[0175] In many disease states, the balanced dynamics of macrophage activation states are disrupted, and the imbalance causes 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 anti-tumor immunity (Qian and Pollard, 2010, Cell, 141(1):39-51). They may also produce growth-promoting molecules such as ornithine, VEGF, EGF, TGF-β, etc.

[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, cathepsin, uPA, and matrix remodeling enzymes (e.g., lysyl oxidase and SPARC).

[0177] TAMs play an important role in tumor angiogenesis that regulates a dramatic increase in tumor tissue blood vessels necessary for the transition of tumors to a malignant state. These angiogenic TAMs express the angiopoietin receptor TIE2 and secrete many angiogenic molecules such as members of the VEGF family, TNFα, IL1β, IL8, PDGF, and FGF.

[0178] The diversity of macrophage subpopulations executes these individual tumor-promoting functions. These TAMs have different phenotypes depending on the degree of macrophage infiltration and the type of tumor. For example, detailed profiling of human hepatocellular carcinoma shows various macrophage subtypes defined from the perspectives of anatomical location, tumor-promoting characteristics, and anti-tumor characteristics. M2-like macrophages have been shown to be a major source of the tumor-promoting functions of TAMs. M2-like TAMs have been shown to affect the efficacy of anti-cancer treatment, contribute to treatment resistance, and mediate tumor recurrence after conventional cancer treatment.

[0179] III. Targets and Biomarkers Useful for Modulating Myeloid Cell Inflammatory Phenotypes The present invention encompasses biomarkers such as PSGL-1 useful for modulating the inflammatory phenotype of myeloid cells, as well as corresponding immune responses (e.g., to increase anti-cancer macrophage immunotherapy).

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

[0181] Nucleic acid and amino acid sequence information of the loci and biomarkers (e.g., the biomarkers described in Table 1) encompassed by the present invention are well-known in the art and are readily available in publicly accessible databases such as the National Center for Biotechnology Information (NCBI) of the United States. Exemplary nucleic acid and amino acid sequences derived from publicly accessible sequence databases are provided below.

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

[0183] A number of representative orthologs to the human sequences are provided below, but in some embodiments, human biomarkers (including its modulators and regulators) are preferred. For some biomarkers, the immune response mediated by such biomarkers in humans is considered to be particularly useful considering 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 that is expressed as a dimer on the cell surface of myeloid cells and on subsets of activated T cells. It functions as a high-affinity counter-receptor 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 platelets or endothelium-expressed selectins. The PSGL-1 protein has two post-translational modifications, tyrosine sulfation and addition of the sialyl Lewis x tetrasaccharide (sLex) to its O-linked glycans for its high-affinity binding activity. In addition to its adhesion function, PSGL-1 has been shown to play a role in inhibiting T cell function independently of the selection of binding function (Tinoco et al. (2017) Trends Immunol. 38:323-335). Furthermore, PSGL-1 antagonists have been developed that specifically block the differentiation of cytotoxic T cells or induce apoptosis in 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 using anti-PSGL-1 antagonists, has not been previously described. Indeed, deletion of PSGL-1 on macrophages has been proposed to increase susceptibility to colorectal cancer in in vivo models (Li et al. (2017) Mol. Cancer Res. 15:467-477). Aberrant expression and polymorphisms in PSGL-1 are associated with deficiencies in natural and acquired immune responses. PSGL-1 is an SLe(x)-type proteoglycan that mediates the rapid recruitment of leukocytes on the vascular surface at the initial stages of inflammation through high-affinity calcium-dependent interactions with E-, P-, and L-selectin. PSGL-1 is important for initial leukocyte capture. PSGL-1 also binds to the VISTA polypeptide, particularly at acidic pH (such as pH 6.0) (see, for example, PCT Publication No. WO2018 / 132476).In some embodiments, the PSGL-1 gene located on human chromosome 12q consists of three exons. Orthologs from chimpanzee, rhesus monkey, dog, cow, mouse, and rat are known. 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) knockout mouse lines exist. In some embodiments, the human PSGL-1 protein has 412 amino acids and / or a molecular weight of 43201 Da. In some embodiments, the PSGL-1 protein contains a ribonuclease 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 include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human PSGL-1 cDNA and human PSGL-1 protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI) (e.g., see ncbi.nlm.nih.gov / gene / 6404). For example, at least two different 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 has a different 5'UTR compared to variant 1, 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 orthologs in organisms other than human are well known and include, for example, 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_001242719.1 and NP_001229648.1), 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] Anti-PSGL-1 antibodies suitable for detecting PSGL-1 protein are well-known in the art and include, 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. Further, reagents for detecting PSGL-1 expression are well-known. Multiple 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)). Additionally, multiple siRNAs, shRNAs, and CRISPR constructs for reducing PSGL-1 expression are described in the commercial product lists of the companies referenced above, such as siRNA product #SR321732, shRNA products #TL309563, TR309563, TG309563, TF309563, TL309563V, and CRISPR product #KN206507 from Origene Technologies (Rockville, MD), CRISPR gRNA products (sc-401534) from Applied Biological Materials (K6134408) and Santa Cruz, and RNAi products (catalog numbers sc-36323 and sc-42833) from Santa Cruz. Note that this term can be further used to refer to any combination of the features described herein with respect to the PSGL-1 molecule. 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 Their Antigen-Binding Fragments The inflammatory phenotype of bone marrow cells can be regulated by modulating the amount and / or activity of specific biomarkers (e.g., at least one target listed in Table 1), and such regulation of the inflammatory phenotype also regulates the immune response.

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

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

Table 2-1

Table 2-2

Table 2-3

[0190] a. Composition of an antibody and its antigen-binding fragment Generally, the antibodies and their antigen-binding fragments included in the present invention are characterized by their ability to bind to myeloid cells expressing the PSGL-1 polypeptide and to increase the inflammatory phenotype of myeloid cells.

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

[0192] Since it is well known in the art that the antibody heavy and light chain CDR3 domains play particularly important roles in the binding specificity / affinity of an antibody for an antigen, the antibodies encompassed by the present invention, e.g., the antibodies described in Table 2, preferably comprise the heavy and light chain CDR3s of the variable regions (e.g., the sequences of Table 2, or a portion thereof) encompassed by the present invention. The antibody may further comprise the CDR2 of the variable region encompassed by the present invention (e.g., the sequences of Table 2, or a portion thereof). The antibody may further comprise the CDR1 of the variable region encompassed by the present invention (e.g., the sequences of 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 (e.g., the sequences of Table 2, or a portion thereof) encompassed by the present invention. In other embodiments, CDR1, CDR2, and / or CDR3 may be selected from within the same heavy and light chain sequence pair (e.g., the sequences of Table 2, or a portion thereof) encompassed by the present invention.

[0193] The CDR1, CDR2, and / or CDR3 regions of the above-mentioned antibodies and their antigen-binding fragments may contain the exact amino acid sequence(s) as those of the variable regions encompassed by the present invention disclosed herein (e.g., the sequences in Table 2, or a part thereof). However, those skilled in the art will understand that some deviation 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, the engineered antibody may be composed of one or more CDRs (e.g., including the sequences in Table 2, or a part thereof) that are 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 present invention.

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

[0195] In some embodiments, antibodies and their antigen-binding fragments that can bind to human PSGL-1 are provided, wherein the variable domain comprises a heavy chain that includes a CDR having a sequence that is 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] Similarly, provided are antibodies and antigen-binding fragments thereof that can bind to human PSGL-1, comprising a light chain that comprises a CDR having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the group of light chain variable domain CDRs presented in Table 2.

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

[0198] One of ordinary skill in the art will note that such percentage homologies are equal to or alternatively can be achieved by introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, such as 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 that comprises a CDR having a sequence selected from the group consisting of at least the heavy chain variable domain CDRs presented in Table 2, and a light chain that comprises a CDR having a sequence selected from the group consisting of at least the light chain variable domain CDRs shown in Table 2.

[0200] Such antibodies, and antigen-binding fragments thereof, may include a light chain whose variable domain includes a CDR having a sequence selected from the group consisting of at least CDR-L1, CDR-L2, and CDR-L3 described herein, and / or a heavy chain whose variable domain includes a CDR having a sequence selected from the group consisting of at least CDR-H1, CDR-H2, and CDR-H3 described herein. In some embodiments, antibodies and antigen-binding fragments thereof that can bind to human PSGL-1 include 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 include or consist of the 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 include or consist of the vκ amino acid sequence shown in Table 2.

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

[0203] In other embodiments, the immunoglobulin heavy and / or light chains according to the present invention each include or consist of the vH or vκ variable domain sequences provided in Table 2.

[0204] The present invention further provides a polypeptide having a sequence selected from the group consisting of the vH variable domain, 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 present invention can be used in isolated (e.g., purified) form or can be included in vectors such as membranes or lipid vesicles (e.g., liposomes).

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

[0206] The term "antibody" or "Ab" is used in a broad sense and includes, but is not limited to, whole antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies, trispecific, or higher multispecific antibodies), antibodies in their naturally occurring form (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 related to 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. The antibodies encompassed by the present invention can occur naturally or can be produced by biotechnology.

[0207] Antibodies, and antigen-binding fragments thereof, can be isolated. As used herein, the term "isolated antibody" is intended to refer to an antibody composition (such as one having a desired antigen specificity) that is substantially free of other antibodies (such as those having different antigen specificities). For example, 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 can 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-rodent animals, or other mammalian or non-mammalian species. However, in some embodiments, the antibody maintains a higher or actually specific affinity and / or selectivity for human PSGL-1. As described above, such differential or cross-binding can be measured as a fold difference compared to a control, such as, 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, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, or more, or any range therebetween, such as about 1.5-fold to about 100-fold different compared to a control. In addition, an isolated antibody typically is substantially free of other cellular materials and / or chemicals. In one embodiment, a combination of "isolated" monoclonal antibodies having different specificities for human PSGL-1 are combined in a defined composition.

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

[0209] The term "native antibody" refers to a heterotetrameric glycoprotein, usually of 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 between the heavy chains of different immunoglobulin isotypes (e.g., IgG, IgA, IgE, IgM). Each heavy and light chain also has regularly spaced intra-chain disulfide cross-links. 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 the other end, and the constant domain of the light chain aligns with the first constant domain of the heavy chain, while the variable domain of the light chain aligns with the variable domain of the heavy chain. The remaining constant domains of the heavy chains of the two heavy chains of the antibody constitute the fragment crystallizable (Fc) region of the antibody.

[0210] In the tail region of an antibody, the Fc region interacts with several proteins of the cell surface receptor called Fc receptor and the complement system. Generally, the term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Further, preferred FcRs bind to IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Among the FcγRII receptors, there are FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which mainly have similar amino acid sequences that differ in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory 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 to be identified in the future, are encompassed by the term "FcR" herein.

[0211] The term "light chain" refers to a component of an antibody from any vertebrate species that has been assigned to one of two distinct types, designated kappa and lambda, based on the amino acid sequence of the constant domain. Depending on the amino acid sequence of their heavy chain constant domains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The CL of an antibody, such as a human or humanized antibody, can be any region belonging to an Ig, 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 differ greatly in sequence between antibodies and are used in the binding and specificity of each particular antibody for its specific antigen. For example, the term "VH" refers to the "heavy chain variable domain" and the term "VL" refers to the "light chain variable domain". The variable domain is composed of hypervariable regions. The term "hypervariable region" refers to a region within the variable domain that contains amino acid residues involved in antigen binding. These regions are highly variable 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, an antibody contains six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the highest diversity among these six HVRs, and in particular, H3 is thought to play a unique role in conferring excellent specificity to the antibody (see, for example, Xu et al. (2000) Immunity 13, 37-45; Johnson and Wu (2003) Meth. Mol. Biol. 248:1-25). The term "CDR" refers to a region of an antibody that contains a structure complementary to its target antigen or epitope.

[0213] Other parts of the variable domain that do not interact with the antigen are referred to as the framework (FW) regions. The antigen-binding site (also known as the antigen-binding site or paratope) contains the amino acid residues necessary to interact 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 evaluations based on comparisons with other antibodies can also be used (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54). The determination of the residues that make up the CDRs includes, but is not limited to, Kabat (Wu et al. (1970) JEM 132:211-250; Kabat et al. (1992), “Sequences of Proteins of Immunological Interest,” 5 thThe use of numbering schemes, including those taught by the Edition, U.S. Department 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) can be included. Thus, the CDR definitions by these systems can differ in length and boundary regions with respect to the adjacent framework regions. See, for example, Kabat, Chothia, and / or MacCallum et al. (Kabat et al., “Sequences of Proteins of Immunological Interest,” 5 th Edition, U.S. Department 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 of the VH domain and the VL domain has three CDRs. The VL CDRs are referred to herein as CDR-L1, CDR-L2, and CDR-L3 in the order that occurs when moving from the N-terminus to the 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 that occurs when moving from the N-terminus to the C-terminus along the variable domain polypeptide. Except for CDR-H3, each CDR supports a canonical structure, which includes amino acid sequences that can vary greatly in sequence and length between antibodies, resulting in diverse three-dimensional structures in the antigen-binding domain (Nikoloudis et al. (2014) Peer J. 2: e456). In some cases, CDR-H3 can be analyzed among a panel of related antibodies to evaluate antibody diversity. Various methods for determining CDR sequences are well known in the art and can be applied to known antibody sequences (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54).

[0215] The antibodies and antigen-binding fragments thereof described herein include, but are not limited to, those that include CDRs defined according to Chothia CDR, Kabat CDR, AbM, CDR contact regions, and / or conformational definitions. Determination of the CDR regions is within the scope of the art. It is understood that in some embodiments, the CDRs can be a combination of Kabat and Chothia CDRs (also referred to as "combined CR" or "extended CDR"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In some embodiments, the CDRs are extended CDRs that refer to all amino acid residues identified according to the Kabat and Chothia nomenclatures. Thus, in some embodiments having more than one CDR, one or more of the CDRs can be any of Kabat, Chothia, extended CDR, or combinations thereof.

[0216] In some embodiments, the antibody fragments and variants can comprise any portion of the 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, the antibody fragments and variants comprise the antigen-binding region from the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; Fd, bispecific antibodies; 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, the antibody fragment or variant binds to the same antigen recognized by the parental full-length antibody.

[0217] Antibody fragments generated by limited proteolysis of a wild-type antibody 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 an antibody generates two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site. A residual "Fc" fragment is also generated, the name of which reflects its ability to readily crystallize. Treatment with pepsin or ficin yields an F(ab')2 fragment that has two antigen-binding sites and can cross-link antigens. Generally, an F(ab')2 fragment contains two "arms", each of which contains a variable region that is directed towards and specifically binds to a common antigen. The two Fab' molecules are joined by an interchain disulfide bond in the hinge region of the heavy chain, and the Fab' molecules can be directed towards the same (bivalent) or different (bispecific) epitopes. As used herein, a "Fab" fragment comprises a single antigen-binding domain that includes the Fab and an additional portion of the heavy chain through the hinge region. The compounds and / or compositions encompassed by the present invention can comprise one or more of these fragments.

[0218] The term "Fv" refers to antibody fragments that contain the complete antigen recognition and antigen-binding site. These regions consist of a dimer of one heavy-chain variable domain and one light-chain variable domain in a 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-chain variable domain and the heavy-chain variable domain (to form a single-chain Fv (scFv)), or by introducing a disulfide bridge between the heavy and light-chain variable domains (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p46-47).

[0219] The term "single-chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains, which domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables the scFv to form a structure desirable for antigen binding. In some embodiments, the VH and VL domains can be linked by a peptide of 10 to 30 amino acid residues. In some embodiments, the scFv is utilized in combination with phage display, yeast display, or other display methods, which are expressed in association with surface members (e.g., phage coat proteins) and can be used for the identification of high-affinity peptides for a particular antigen. In some embodiments, the term "single-chain antibody" can further include, but is not limited to, a disulfide-linked Fv (dsFv) in which two single-chain antibodies (each of which can be directed to a different epitope) are linked together by a disulfide bond. Using molecular genetics, two scFvs can be engineered in tandem as a single polypeptide separated by a linker domain and are referred to as "tandem scFv" (tascFv). Construction of 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 include a modified Fc region. By way of non-limiting example, the modified Fc region can be made by the methods described in U.S. Patent Publication No. US 2015-0065690 or can be within any range.

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

[0222] The term "recombinant human antibody" includes, for example, (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) that are transgenic for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express an antibody, e.g., antibodies isolated from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) any other antibodies prepared, expressed, produced, or isolated by any other means including splicing of human immunoglobulin gene sequences to 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, if transgenic animals are used for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the V H and V L regions are related to and derived from human germline V H and V L sequences but may not naturally occur within the human antibody germline repertoire in vivo.

[0223] The term "polyclonal antibody" includes antibodies produced in an immunogenic response to a protein having multiple epitopes. Thus, a composition of polyclonal antibodies (e.g., serum) contains a variety of different antibodies that target the same and different epitopes within the protein. Methods for generating polyclonal antibodies are well 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 substantially homogeneous population of cells (or clones), i.e., the individual antibodies that make up the population are identical, except for variants that may arise during the production of the monoclonal antibody, and / or bind to the same specific epitope of the antigen, and such variants generally occur in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies include, in particular, "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived 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 the corresponding sequence in an antibody derived 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 (in relation to a native or starting antibody) or a biomolecule that is similar to the native or starting antibody in structure and / or function and includes some differences in amino acid sequence, composition, or structure when compared to the native or starting antibody (e.g., antibody mimetic). Antibody variants can have their amino acid sequence, composition, or structure modified compared to the native antibody. Antibody variants include, but are not limited to, antibodies with modified 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, mutant constant chain regions such as mutant IgG4 with a substitution at Ser228 like S228P are contemplated.

[0226] In some embodiments, the 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 in which two or more of the same or different natural antibodies, single-chain antibodies, or antibody fragment segments, having the same or different specificities, are linked. The valency of the fusion protein indicates the total number of binding arms or sites that the fusion protein has for an antigen or epitope, i.e., monovalent, divalent, trivalent, or multivalent. The multivalency of an antibody fusion protein means that multiple interactions can be utilized in binding to an antigen, and thus enhances the binding strength to the antigen. Specificity indicates the number of different antigens or epitopes to which an antibody fusion protein can bind, i.e., monospecific, bispecific, trispecific, multispecific, etc. Using these definitions, for example, a natural antibody such as IgG is divalent because it has two binding arms, but is monospecific because it binds to one antigen. A monospecific multivalent fusion protein has more than one binding site for an epitope, but binds only to the same epitope on the same antigen, e.g., a bispecific antibody having two binding sites that react with the same antigen. The fusion protein can include a multivalent or multispecific combination of different antibody components, or multiple copies of the same antibody component. The fusion protein can further include a therapeutic agent. Examples of suitable therapeutic agents for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion proteins") and toxins ("antibody-toxin fusion proteins"). One preferred toxin includes ribonuclease (RNase), preferably recombinant RNase.

[0227] In some embodiments, the 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 to different epitopes. The epitopes can be on the same or different targets. In one embodiment, the 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, the 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, the bispecific antibody is capable of binding to two different antigens. Such antibodies typically include antigen-binding regions from at least two different antibodies. For example, bispecific monoclonal antibodies (BsMAb, BsAb) are artificial proteins composed of fragments of two different monoclonal antibodies, so BsAb can 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. U.S.A. 108:11187-11192. A new generation of BsMAb, referred to as "trifunctional bispecific" antibodies, has been developed. These are composed of two heavy chains and two light chains, one each from two different antibodies, with the two Fab regions (arms) directed against two antigens, and the Fc region (foot) containing the two heavy chains, forming a third binding site.

[0228] In some embodiments, the compositions encompassed by the present invention can include anti-peptide antibodies. As used herein, the term "anti-peptide antibody" refers to a "monospecific antibody" generated in a humoral response against a short (usually 5 to 20 amino acids) immunogenic polypeptide corresponding to some (preferably one) isolated epitope of the protein from which it is derived (e.g., the target protein encompassed by the present invention). Multiple anti-peptide antibodies include various different antibodies directed to a particular portion of the protein, i.e., an amino acid sequence containing at least one, preferably only one, epitope. Methods for generating polyclonal antibodies are well known in the art (see, for example, 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, the 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. A bispecific antibody includes a heavy-chain variable domain VH connected to a light-chain variable domain VL within 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 the complementary domains on another chain to generate two antigen-binding sites. Bispecific antibodies are further described, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448.

[0230] In some embodiments, the 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 type of well-known antigen-binding molecule that have the characteristics of antibodies and can be expressed intracellularly to bind and / or inhibit a target intracellular molecule of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies to target (e.g., inhibit) intracellular compartments are well known in the art, such as the use of single-chain antibodies (scFv), modification of immunoglobulin VL domains for enhanced stability, modification of antibodies to resist degradation in the intracellular environment, and generation of fusion proteins to enhance intracellular stability and / or regulate intracellular localization. Intracellular antibodies can also be introduced and expressed in one or more cells, tissues, or organs of a multicellular organism (e.g., as gene therapy) for, e.g., prophylactic and / or therapeutic purposes (see, e.g., at least PCT Publications WO08 / 020079, WO94 / 02610, WO95 / 22618, and WO03 / 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] Intracellular antibodies can be used to affect a number of cellular processes including, but not limited to, intracellular trafficking, transcription, translation, metabolic processes, proliferation signaling, and cell division. In some embodiments, the methods encompassed by the present invention can include intracellular antibody-based therapies. 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 therapies. For example, an intracellular antibody can target one or more glycosylated intracellular proteins or can modulate the interaction between one or more glycosylated intracellular proteins and alternative proteins. Intracellular expression of intracellular antibodies in different compartments of mammalian cells enables 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. U.S.A.101: 17616 - 17621). Intracellular antibodies can modify protein folding, protein - protein, protein - DNA, protein - RNA interactions, and protein modifications. They can act as neutralizing agents by inducing phenotypic knockout, by directly binding to the target antigen, by altering its intracellular transport, or by inhibiting its association with binding partners. Intracellular antibodies have the advantage of blocking specific binding interactions of a particular target molecule while sparing others, due to their high specificity and affinity for the target antigen. Sequences from donor antibodies can be used to develop intracellular antibodies. Intracellular antibodies 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, intracellular antibodies are often expressed as a single polypeptide, forming a single - chain antibody that contains the variable domains of the heavy and light chains linked by a flexible linker polypeptide. Intracellular antibodies usually lack disulfide bonds and it is possible to regulate the expression or activity of the target gene through their specific binding activity. Single - chain intracellular antibodies are often expressed from recombinant nucleic acid molecules and engineered to be retained intracellularly (e.g., retained within the cytoplasm, endoplasmic reticulum, or periplasm). Intracellular antibodies are described, for example, in Marasco et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:7889 - 7893, Chen et al. (1994) Hum. Gene Ther. 5:595 - 601, Chen et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:5932 - 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, Rondon and Marasco (1997) Annu. Rev. Microbiol.It can be generated using methods well-known in the art, such as those disclosed and outlined in 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, the antibodies encompassed by the present invention can include chimeric antibodies. As used herein, the term "chimeric antibody" refers to a recombinant antibody in which a portion of the heavy and light chains is identical or homologous to the corresponding sequences within an antibody derived 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 the corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, insofar as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855). For example, chimeric antibodies of interest herein can include "primatized" antibodies that include variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences.

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

[0234] In some embodiments, the antibodies encompassed by the present invention may include heterologous antibodies. The term "heterologous antibody" is defined with respect to transgenic non-human organisms that produce such antibodies. This term refers to antibodies having amino acid sequences or coding nucleic acid sequences corresponding to those found in organisms that are not transgenic non-human animals, and generally are from species other than those of transgenic non-human animals.

[0235] In some embodiments, the antibodies encompassed by the present invention may be humanized antibodies. As used herein, the term "humanized antibody" refers to chimeric antibodies that include minimal portions from one or more non-human (e.g., murine) antibody sources and the remainder derived from one or more human immunoglobulin sources. In most parts, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable regions from the recipient's antibody are replaced by residues from the hypervariable regions of an antibody of a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) having the desired specificity, affinity, and / or potency. In one embodiment, the antibody can 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, an antibody can be humanized using the methods taught in U.S. Patent Publication No. 2013 / 0303399. The term "humanized antibody" as used herein also includes antibodies in which CDR sequences derived from the germline of another mammalian species such as a mouse are transplanted into a human framework sequence.

[0236] As used herein, a humanized mouse is a mouse that carries functional human genes, cells, tissues, and / or organs. Humanized mice are commonly used as small animal models in biological and medical research for human therapeutic agents. For this purpose, nude mice and severe combined immunodeficiency (SCID) mice may be used. NCG mice, NOG mice, 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 both healthy and pathological scenarios and can enable the evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

[0237] In some embodiments, the antibodies encompassed by the present invention can include cysteine-modified antibodies. In a "cysteine-modified antibody", a cysteine amino acid is inserted or substituted on 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 of antibodies has been described (see, for example, U.S. Patent No. 5,219,996). Methods for introducing cysteine residues into the constant region of IgG antibodies for use in site-specific conjugation of antibodies are described in Stimmel et al. (2000) J. Biol. Chem. 275: 330445-30450).

[0238] In some embodiments, the antibody variants encompassed by the present invention can 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 their molecular targets. In some embodiments, the antibody mimetic can be a monobody designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold (see U.S. Pat. Nos. 6,673,901 and 6,348,584). In some embodiments, the antibody mimetic can include, but is not limited to, any of those known in the art, such as affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINS™, finomers, and knottins, and domain peptides. In other embodiments, the antibody mimetic can include one or more non-peptide regions.

[0239] In some embodiments, the antibodies encompassed by the present invention can include a single antigen-binding domain. These molecules are very small, with a molecular weight that is about one-tenth of the molecular weight observed for full-size mAbs. Further antibodies can include "nanobodies" derived from the antigen-binding variable heavy chain region (VHH) of heavy chain antibodies found in camels and llamas that lack a light chain (see, e.g., Nelson (2010) Mabs 2:77-83).

[0240] In some embodiments, the antibodies encompassed by the present invention can be "miniaturized." An example of mAb miniaturization is the small modular immunopharmaceutical (SMIP). These monovalent or bivalent molecules are recombinant single-chain molecules that contain one VL, one VH antigen-binding domain, and one or two constant "effector" domains, all connected by linker domains (see, e.g., Nelson (2010) Mabs 2:77-83). Such molecules are thought to provide the advantage of increased tissue or tumor penetration required by the fragment while retaining the immune effector function provided by the constant domain. Another example of a miniaturized antibody is termed a "unibody," in which the hinge region has been removed from the IgG4 molecule. IgG4 molecules are unstable and can exchange their heavy and light chain heterodimers with each other, but deletion of the hinge region completely prevents heavy chain-heavy chain pairing and leaves a very specific monovalent light chain / heavy chain heterodimer while retaining the Fc region to ensure stability and half-life in vivo. IgG4 has insufficient interaction with FcR, and this configuration can minimize the risk of immune activation or increased carcinogenicity because the monovalent unibody cannot promote the formation of intracellular signaling complexes (see, e.g., Nelson (2010) Mabs 2:77-83).

[0241] In some embodiments, the antibody variants encompassed by the present invention can 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, it is capable of selectively binding to a specific antigen. In one aspect, the sdAb can 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-No lte et al (2007) FASEB J. 21: 3490-3498). "Heavy chain antibody" or "camelid antibody" refers to an antibody that contains two VH domains and no light chain (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 No. WO1 994 / 04678, and No. WO1994 / 025591, and U.S. Patent No. 6,005,079). In another aspect, the sdAb can be an "immunoglobulin new antigen receptor" (IgNAR). The term "immunoglobulin new antigen receptor" refers to a class of antibodies from the shark immune repertoire consisting of a homodimer of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains. IgNAR represents some of the smallest known immunoglobulin-based protein scaffolds, is highly stable, and possesses efficient binding properties. The inherent stability can result from both (i) the underlying Ig scaffold presenting a significant number of charged and hydrophilic surface-exposed residues compared to the conventional antibody VH and VL domains found in murine antibodies, and (ii) the stabilization of structural features in the complementarity-determining region (CDR) loops, including inter-loop disulfide bridges and patterns of inter-loop hydrogen bonds. Other miniaturized antibody fragments can include "complementarity-determining region peptides" or "CDR peptides".A CDR peptide (also known as the "minimal recognition unit") is a peptide corresponding to a single complementarity-determining region (CDR) and can be prepared by constructing a gene encoding the CDR of the antibody of interest. Such a gene is prepared by, for example, using polymerase chain reaction to synthesize the variable region from the RNA of antibody-producing cells (see, for example, Larrick et al (1991) Methods Enzymol. 2:106).

[0242] Other variants containing the antigen-binding fragment of the antibody include, but are not limited to, disulfide-linked Fv (sdFv), V L , V H , camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), tribody (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 subsequences of intact immunoglobulins may be included.

[0243] In some embodiments, the antibodies encompassed by the present invention can be the antibodies described in U.S. Patent No. 5,091,513. Such antibodies can include one or more sequences of amino acids that constitute a region that behaves as a biosynthetic antibody-binding site (BABS). These sites include 1) non-covalently or disulfide-bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains in which VH and VL are linked by a polypeptide linker, or 3) individual VH or VL domains. The binding domain includes the linked CDR and FR regions, which can be derived from separate immunoglobulins. The biosynthetic antibody can also include, for example, other polypeptide sequences that function as enzymes, toxins, binding sites, or attachment sites to an immobilization medium or radioactive atom. Methods for producing biosynthetic antibodies, methods for designing BABS having any specificity that can be induced by in vivo generation of antibodies, and methods for producing analogs thereof are disclosed.

[0244]

[0244] In some embodiments, the antibodies encompassed by the present invention can be antibodies having an antibody acceptor framework as taught in U.S. Patent No. 8,399,625. Such an antibody acceptor framework can be particularly suitable for accepting CDRs from the 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 physiological conditions of the wild type, and the use of such conditionally active biological proteins is provided. Such methods and conditionally active proteins are taught, for example, in PCT Publications WO2015 / 175375 and WO2016 / 036916, and U.S. Patent Publication 2014 / 0378660.

[0246] In some embodiments, the antibodies encompassed by the present invention are therapeutic antibodies. As used herein, the term "therapeutic antibody" means an antibody that is effective in treating a disease or disorder in a mammal having or at risk of having the disease or disorder. The antibody can 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]

[0245] In some embodiments, the antibodies encompassed by the present invention can be naked antibodies. As used herein, the term "naked antibody" refers to an intact antibody molecule that does not include further modifications, such as a complex with a chelate for binding to a toxin or a radionuclide. The Fc portion of a naked antibody provides effector functions such as complement binding and ADCC (antibody-dependent cell cytotoxicity), and acts on 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 carry the antigen specifically recognized by the antibody. This depletion can be mediated through at least three mechanisms: antibody-dependent cell-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 antigens. To evaluate complement activation, for example, a CDC assay such as that described by 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 target cells bearing the antigen and subsequently kill the target cells. To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in U.S. Patent 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 effect the desired interaction with the Fc receptor or its absence.

[0251] Fc receptors are found on many cells involved in the immune response. Fc receptors (FcR) are cell surface receptors for the Fc portion of immunoglobulin polypeptides (Ig). Among the human FcRs that have been identified to date, some recognize IgG (referred to as FcγR), IgE (FcεR1), IgA (Fcα), and polymeric IgM / A (FcμαR). FcRs are found in 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 widely studied FcγR is central to cellular immune defense and plays a role in stimulating inflammation and the release of mediators of hydrolytic enzymes involved in the etiology of autoimmune diseases (Unkeless, J.C. et al. (1988) Annu. Rev. Immunol. 6:251-81). FcγR provides an important link between effector cells and lymphocytes that secrete Ig by conferring an element of specific recognition mediated by IgG. Human leukocytes have at least three different receptors for IgG: hFcγRI (found on monocytes / macrophages), hFcγRII (monocytes, neutrophils, eosinophils, platelets, in some cases B cells, and the K562 cell line), and FcγIII (NK cells, neutrophils, eosinophils, and macrophages).

[0252] In some embodiments, the antibodies encompassed by the present invention can be complexed with 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 known in the art. In some embodiments, an antibody that binds to a desired target (also referred to herein as a "primary antibody") is unlabeled but can be detected by the binding of a secondary antibody (referred to herein as a "secondary antibody") that 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 attach 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, fluorescein and its derivatives (e.g., FITC), cyanine and its derivatives (e.g., indocyanine, 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, porphyrin, 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), ALEXAFLUOR® compounds (Thermo Fisher Scientific, Waltham, MA), as well as quantum dots. Other complexes that can be used to label antibodies include biotin, avidin, and streptavidin.

[0254] For example, the conjugation of an antibody or other protein encompassed by the present invention with a heterologous agent can be prepared using various 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-azide 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-isothiocyanatobenzylmethyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding a radioactive nucleotide to an antibody (WO94 / 11026).

[0255] In another aspect, the invention features an antibody that specifically binds to a target biomarker and is conjugated to a therapeutic moiety such as a cytotoxin, drug, and / or radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." Cytotoxic or cytocidal agents include any agent that is detrimental to cells (e.g., kills). Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogs or homologs. 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, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin)), bleomycin, mitramycin, and anthramycin (AMC), and mitotic inhibitors (e.g., vincristine and vinblastine). The antibodies encompassed by the invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate a cytotoxic radiopharmaceutical for treating related disorders such as cancer.

[0256] Composite anti-biomarker antibodies can be used diagnostically or prognostically as part of clinical trial procedures, for example, to determine the effectiveness of a given treatment regimen or to select patients most likely to respond to immunotherapy, by monitoring polypeptide levels in tissues. For example, cells can be permeabilized in a flow cytometry assay so that an antibody that binds to a biomarker of interest targets its recognized intracellular epitope and binding can be detected by analyzing the signal emitted from the binding molecule. Detection can be facilitated by conjugating (i.e., physically linking) the antibody with a detectable substance. Examples of detectable substances include various enzymes, avidin-biotin families, 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 avidin-biotin family complexes including streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances including umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin (PE); examples of luminescent substances including luminol; examples of bioluminescent substances including luciferase, luciferin, and aequorin, and 125 I, 131 I, 35 S, or 3 examples of radioactive substances including H are included. As used herein, the term "labeled" with respect to an antibody is intended to include both direct labeling of the antibody by conjugating (i.e., physically linking) the antibody with a detectable substance such as a radioactive agent or fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)), and 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 being limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the 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, see, for example, 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 Prospective Of The Therapeutic Use Of Radiolabeled Antibody 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 promotes the release of a cytotoxic or growth-inhibiting agent in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker, or a disulfide-containing linker (see, e.g., U.S. Patent No. 5,208,020) can be used. Alternatively, a fusion protein comprising an antibody and a cytotoxic or growth-inhibiting agent can be made by recombinant techniques or peptide synthesis. The length of the DNA can include regions encoding linker peptides that do not disrupt the desired properties of the conjugate, and regions encoding each of the two portions of the conjugate that are adjacent to or separated from each other.

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

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

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

[0263] As noted 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 of the amino acids used in protein production (shown above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered to be functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although some organisms may translate some sequences more efficiently than others). Additionally, occasionally, methylated variants of purines or pyrimidines may 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 conferring an interactive biological function on 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 another amino acid having a similar hydrophobic-hydrophilic index or score, still resulting in a protein having 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, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various characteristics described above are well known to those skilled 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 present invention can be useful, for example, for modifying the original glycosylation pattern of the antibody in order to increase its stability. "Modifying" means deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites not present in the antibody. Glycosylation of antibodies is usually N-linked. "N-linked" refers to the binding of a 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 other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of a glycosylation site to an antibody is conveniently achieved by changing the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). Another type of covalent modification involves chemically or enzymatically attaching a glycoside to the antibody. These procedures are advantageous in that they do not require the production of the antibody in a host cell having glycosylation capacity for N- or O-linked glycosylation. Depending on the conjugation mode used, the sugar(s) can be linked 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 WO87 / 05330.

[0268] Similarly, removal of any carbohydrate moieties present on the 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 cleavage of most or all of the sugars except the bound sugars (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 the antibody can be accomplished by use of various endoglycosidases and exoglycosidases as described in Thotakura, N R. et al. (1987).

[0269] Other modifications can involve the formation of immune complexes. For example, in certain types of covalent modification, an antibody or protein can be covalently attached to one of various 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 described above, techniques that can be used to produce antibodies and antibody fragments such as Fab and scFv are well known in the art and include those described in U.S. Patent 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. U.S.A. 90:7995-7999, and Skerra et al. (1988) Science 240:1038-1041). Representative examples of genetically engineered antibodies are described herein, such as those in which residues capable of chemical modification have been modified, as well as those having useful sequence modifications (e.g., CDR sequences that are more similar to the human germ line). Such antibody variants are encompassed by the present invention.

[0271] After isolation or selection of a target antigen-specific antibody, the antibody sequence can be used for recombinant production and / or optimization of such antibody. When isolating antibody fragments from a display library, for example, as described in detail below, the coding region from the isolated fragment can be used to generate an entire antibody, including a human antibody or any other desired target-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. Optionally, IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4) can be synthesized for further testing and / or product development from variable domain fragments generated or selected according to the methods described herein. Such antibodies can be produced by inserting one or more segments of 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 antibody contains modifications characteristic of mammalian proteins (e.g., glycosylation), and / or to ensure that the antibody preparation is free of 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 an antibody by enhancing the affinity for a given target through successive rounds of mutation and selection of the cDNA sequences encoding the antibody or antibody fragment. In some cases, this process is performed in vitro. To achieve this, amplification of the variable domain sequences (which in some cases is limited to the 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 within 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 contiguous 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 include complete replacement of an entire codon, or a change from one codon to another, by modifying one or two nucleotides of the start codon.

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

[0274] Affinity matured clones can be selected based on affinity determined by a binding assay (e.g., FACS, ELISA, surface plasmon resonance, etc.). Next, the selected clones can be converted to IgG and further tested for affinity and functional activity. In some cases, the goal of affinity optimization is to enhance the 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, this process can be repeated.

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

[0276] A humanized antibody is an antibody molecule from a non-human species that binds to a desired target and has one or more complementarity-determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule. In many cases, framework residues within the human framework regions are replaced with the corresponding residues from the CDRs and framework regions of the donor antibody to modify, preferably improve, target binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions of the CDR and framework residues to identify framework residues important for target binding, as well as by sequence comparison to identify unusual framework residues at specific positions (see, for example, U.S. Patent Nos. 5,693,762 and 5,585,089; Riechmann et al. (1988) Nature 332:323-327).

[0277] Antibodies can be humanized using a variety of techniques known in the art, including, for example, grafting of CDRs (see, for example, EP Patent Publication No. 239,400; PCT Publication No. WO91 / 09967; U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (see, for example, 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) Proc. Natl. Acad. Sci. U.S.A. 91:969-973), and chain shuffling (see, for example, U.S. Patent No. 5,565,332).

[0278] Fully human antibodies are particularly desired for the therapeutic treatment of human patients in order to avoid or reduce immune responses to foreign proteins. Human antibodies can be made by a variety of methods known in the art including the above-described antibody display methods using antibody libraries derived from human immunoglobulin sequences (see, e.g., U.S. Pat. Nos. 4,444,887 and 4,716,111, and PCT Publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, WO91 / 10741). Human antibodies can also be made using transgenic mice that are unable to express functional endogenous immunoglobulins but are able to express human immunoglobulin polynucleotides. For example, human heavy and light chain immunoglobulin polynucleotide complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain polynucleotides, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin polynucleotides can be rendered non-functional separately or simultaneously from the introduction of the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are grown and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal 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 indicated 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, for example, PCT Publications WO98 / 24893, WO92 / 01047, WO96 / 34096, and WO96 / 33735, as well as U.S. Patents Nos. 5,413,923, 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] When the antibody molecules encompassed by the present invention are produced by an animal, cell line, chemically synthesized, or recombinantly expressed, they can be purified (i.e., isolated) by any method 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, by differences in solubility, or by other standard techniques for protein purification. Further, the antibodies or fragments thereof encompassed by the present invention can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0280] According to the present invention, an antibody that specifically binds to an antigen can be present in solution or can be bound to a substrate. In some embodiments, the antibody is bound to cellulose nanobeads and confined to one or more detection regions of the substrate of a detection device.

[0281] c. Antibody production The antibodies and antigen-binding fragments thereof included in the present invention can be produced artificially through any method known in the art, such as being naturally occurring or being produced by conventional hybridoma technology, recombinant technology, mutation or optimization of known antibodies, selection from antibody libraries or antibody fragment libraries, and monoclonal antibodies (mAbs) produced by immunization. Whether monoclonal or polyclonal, the production of antibodies 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, as well as variants and / or fragments thereof, described herein can be generated using recombinant polynucleotides. In one embodiment, the polynucleotide has a modular design for encoding at least one of an antibody, a fragment, or a variant thereof. By way of non-limiting example, the polynucleotide construct can encode any one of (1) the heavy chain of an antibody, (2) the light chain of an antibody, (3) the heavy and light chains of an antibody, (4) the heavy and light chains separated by a linker, (5) the VH1, CH1, CH2, CH3 domains, a linker, and a light chain, or (6) the VH1, CH1, CH2, CH3 domains, the VL region, and a light chain. Any of these designs can include optional linkers between any domains and / or regions. The polynucleotides encompassed by the present invention can be engineered to produce any standard class of immunoglobulin using any of the antibodies or components thereof described herein as starting molecules.

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

[0284] d. Characterization and efficacy of antibodies The antibodies encompassed by the present invention, and antibody-binding fragments thereof, can be characterized by one or more characteristics selected from the group consisting of structure, isotype, binding (e.g., affinity and specificity), complexation, 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. The antibodies encompassed by the present invention can be monospecific or multispecific. Multispecific antibodies can be specific for different epitopes of the peptides encompassed by the present invention, or can be specific for both the peptides encompassed by the present invention and heterologous epitopes such as heterologous peptides or solid support substances (see, for example, PCT Publications WO93 / 17715, WO92 / 08802, WO91 / 00360, and WO92 / 05793, Tutt et al. (1991) J. Immunol. 147:60-69, U.S. Patents 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 produced against peptides containing repeating units of the 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, a heterobivalent ligand (HBL) system has been designed that competitively inhibits the binding of an antigen to an IgE antibody bound to a mast cell, thereby inhibiting mast cell degranulation (Handlogten et al. (2011) Chem. Biol. 18:1179-1188).

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

[0287] Antibodies can bind or interact with any number of positions on or along a target protein. The intended antibody target sites include any and all possible sites on the 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 of 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 a very accurate determination of the important interactions between individual amino acids from both side chains and backbone atoms in both the epitope of the antigen and the paratope of the antibody. Amino acids within 4 angstroms of each other are generally considered to be contacting residues. The methodology typically involves purification of the antibody and antigen, formation and purification of the complex, followed by successive rounds of crystallization screening and optimization to obtain crystals of diffraction quality. Structure solution is obtained after frequent X-ray crystallography at a synchrotron source. 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, G.E. 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 the evaluation or quantification of 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, and overlapping or contiguous peptides from the 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] Multiple types of competitive binding assays are known, including the following non-limiting examples: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (Stahli et al. (1983) Meth. Enzymol. 9:242), solid-phase direct biotin-avidin EIA (Kirkland et al. (1986) J. Immunol. 137:3614), solid-phase direct labeled assay or solid-phase direct labeled sandwich assay (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), I 125Solid-phase direct-labeled RIAs using labels (Morel et al. (1988) Mol. Immunol. 25:7), solid-phase direct biotin-avidin EIAs (Cheung et al. (1990) Virol. 176:546), and direct-labeled RIAs (Moldenhauer et al. (1990) Scand. J. Immunol. 32:77) are included. Typically, such assays involve the use of a purified antigen bound to a solid surface or cell, 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 cell in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive 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 such that steric hindrance occurs. Further details regarding methods for determining competitive binding are provided in the examples herein. Usually, when the competitive 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 its 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, the binding is inhibited by at least about 80 - 85%, about 85 - 90%, about 90 - 95%, about 95 - 97%, or about 97% or more.

[0291] The effects of the agents described herein, such as antibodies, their antigen-binding fragments, cells, etc., can be evaluated using reagents, methods, and assays well known to those of ordinary skill in the art, particularly in view of the examples. In some embodiments, a control is used for comparison, such as those described in the above definitions. For example, an assay may involve contacting a biomarker target, such as on a cell or substrate, with the agent of interest, determining a desired measurement (e.g., quantity, activity, cytokine production, cell proliferation, cell death, etc.), and comparing the measurement to a measurement from a reference or control, such as a control agent like a control antibody or its antigen-binding fragment 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 described in the above definitions, a "significant" modulation of a desired measurement can be numerically quantified, such as being above a particular numerical value (e.g., percentage), below a particular numerical value (e.g., percentage), or within a particular numerical range (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, undesired cells, ratio of desired cells to undesired cells, ratio of desired cells to total cells, ratio of undesired cells to total cells, etc., compared at a certain point in time or at different points in time, etc.).

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

[0294] a. Nucleic Acid Agents One aspect included in the present invention involves the use of nucleic acid molecules. The 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 analogs of DNA or RNA generated using nucleotide analogs. RNA agents can include RNA interference (RNAi) agents (e.g., small interfering RNA (siRNA)), single-stranded RNA (ssRNA) molecules (e.g., antisense oligonucleotides), or double-stranded RNA (dsRNA) molecules. The dsRNA molecule includes a first strand and a second strand, the second strand is substantially complementary to the first strand, and the first strand and the second strand form at least one double-stranded duplex region. The dsRNA molecule can have blunt ends or at least on...

Claims

1. A monoclonal antibody that binds to a PSGL-1 polypeptide, or an antigen-binding fragment thereof, comprising a heavy-chain CDRH1 having residues 26 to 32 (GYTFTTY) of SEQ ID NO: 134, a CDRH2 having residues 52 to 57 (NTYSGV) of SEQ ID NO: 134, and a CDRH3 having residues 99 to 108 (HYYGSHYFDY) of SEQ ID NO: 134, and a light-chain CDRL1 having residues 24 to 40 (KSSQSLSSSNQKNYLA) of SEQ ID NO: 135, a CDRL2 having residues 56 to 62 (FASTRES) of SEQ ID NO: 135, and a CDRL3 having residues 95 to 103 (QQHYFSPLT) of SEQ ID NO: 135, said monoclonal antibody or antigen-binding fragment thereof.

2. The monoclonal antibody according to claim 1, or an antigen-binding fragment thereof, which binds to a PSGL-1 polypeptide on bone marrow cells.

3. Said monoclonal antibody, or an antigen-binding fragment thereof, is a) after contact with said monoclonal antibody, or an antigen-binding fragment thereof, i) increased expression and / or secretion of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), ii) reduced expression and / or secretion of IL-10, iii) increased secretion of at least one cytokine or chemokine selected from the group consisting of IL-1β, TNF-α, IL-12, GM-CSF, CCL3, and CCL4, iv) an increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, v) increased CD8+ cytotoxic T cell activation, vi) increased recruitment of CD8+ cytotoxic T cell activation, vii) increased CD4+ helper T cell activity, viii) increased recruitment of CD4+ helper T cell activity, ix) increased macrophage activity, and / or x) increasing the inflammatory phenotype of said bone marrow cells by resulting in one or more of increased spindle shape, flatness of appearance, and / or number of dendritic processes as evaluated by microscopy, b) cross-reacting with a cynomolgus monkey PSGL-1 polypeptide, c) having no ADCC activity against PSGL-1-expressing cells, d) having no CDC activity against PSGL-1-expressing cells, e) not killing PSGL-1-expressing cells upon binding to and / or internalization by PSGL-1-expressing cells; f) not being conjugated with another therapeutic moiety; g) not activating or inducing T cell apoptosis; h) binding the epitope C-terminus to residues 42-62 of human PSGL-1; i) increasing inflammation in tumors and / or having anti-tumor activity in vivo; j) being chimeric, humanized, murine, or human; k) being detectably labeled, comprising an effector domain, and / or comprising an Fc domain; l) being selected from the group consisting of Fv, F(av), F(ab')2, Fab', dsFv, scFv, sc(Fv)2, Fde, sdFv, and bispecific antibody fragments; m) comprising an immunoglobulin constant domain selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM; n) comprising a constant domain derived from a human immunoglobulin, and / or o) being conjugated with a drug. The monoclonal antibody according to claim 2, or an antigen-binding fragment thereof, having one or more of the characteristics of

4. The monoclonal antibody, or an antigen-binding fragment thereof, wherein it comprises a heavy chain variable domain sequence having at least 90% identity with SEQ ID NO: 134, and / or it comprises a light chain variable domain sequence having at least 90% identity with SEQ ID NO: 135, the monoclonal antibody according to claim 1 or 2, or an antigen-binding fragment thereof.

5. The monoclonal antibody, or an antigen-binding fragment thereof, wherein it comprises the heavy chain variable domain sequence of SEQ ID NO: 134, and / or it comprises the light chain variable domain sequence of SEQ ID NO: 135, the monoclonal antibody according to claim 1 or 2, or an antigen-binding fragment thereof.

6. a) the myeloid cells having an increased inflammatory phenotype A) increased expression of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); B) reduced expression of IL-10; C) increased secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, and IL-12; D) an increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10. E) Increased CD8+ cytotoxic T cell activation, F) Increased CD4+ helper T cell activity, G) Increased macrophage activity, and / or H) Increased spindle shape, flatness of appearance, and / or number of dendrites when evaluated by microscopy showing one or more of, b) said bone marrow cells comprising 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, c) said bone marrow cells expressing PSGL-1 or determined to express PSGL-1, d) said bone marrow cells being primary bone marrow cells, and / or e) said bone marrow cells being contained within the tissue microenvironment, The monoclonal antibody according to any one of claims 2 to 5, or an antigen-binding fragment thereof, having one or more of the characteristics.

7. A pharmaceutical composition comprising a therapeutically effective amount of at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or excipient.

8. An isolated nucleic acid molecule encoding an immunoglobulin heavy chain polypeptide comprising the heavy chain variable domain of SEQ ID NO: 134 and a light chain polypeptide comprising the light chain variable domain of SEQ ID NO:

135.

9. An isolated immunoglobulin heavy chain and light chain polypeptide encoded by the nucleic acid according to claim 8.

10. A vector comprising the isolated nucleic acid according to claim 8.

11. The vector according to claim 10, which is an expression vector.

12. A host cell comprising the isolated nucleic acid according to claim 8.

13. a) expressing the monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, b) comprising the immunoglobulin heavy chain and light chain polypeptides according to claim 9, and / or c) comprising the vector according to claim 10 or 11, The host cell according to claim 12.

14. A device or kit comprising at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof.

15. The device or kit according to claim 14, comprising the at least one monoclonal antibody, or a label for detecting an antigen-binding fragment thereof, or a complex comprising the monoclonal antibody or an antigen-binding fragment thereof.

16. A device or kit comprising a pharmaceutical composition, an isolated nucleic acid molecule, an isolated immunoglobulin heavy and light chain polypeptide, a vector, and / or a host cell according to any one of claims 7 to 13.

17. A method for producing at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, comprising: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding the at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, under conditions suitable for allowing expression of the monoclonal antibody or an antigen-binding fragment thereof; and (ii) recovering the expressed monoclonal antibody or an antigen-binding fragment thereof.

18. A method for detecting the presence or level of a PSGL-1 polypeptide, comprising detecting the polypeptide in a sample obtained by use of at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof.

19. The method according to claim 18, wherein the at least one monoclonal antibody, or an antigen-binding fragment thereof, forms a complex with the PSGL-1 polypeptide, and the complex is detected in a form using an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical assay, Western blot, mass spectrometry assay, nuclear magnetic resonance assay, or intracellular flow assay.

20. A method for generating myeloid cells having an increased inflammatory phenotype after contact with the monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, the pharmaceutical composition according to claim 7, the isolated nucleic acid molecule according to claim 8, the isolated immunoglobulin heavy and light chain polypeptides according to claim 9, the vector according to claim 10 or 11, and / or the host cell according to claim 12 or 13, the method comprising contacting myeloid cells with an effective amount of said monoclonal antibody, or an antigen-binding fragment thereof, said pharmaceutical composition, said isolated nucleic acid molecule, said isolated immunoglobulin heavy and light chain polypeptides, said vector, and / or host cell, wherein said myeloid cells are not contacted in humans.

21. After contact of the myeloid cells having an increased inflammatory phenotype with the monoclonal antibody, or an antigen-binding fragment thereof, a) increased expression and / or secretion of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), b) reduced expression and / or secretion of IL-10, c) increased secretion of at least one cytokine or chemokine selected from the group consisting of IL-1β, TNF-α, IL-12, GM-CSF, CCL3, and CCL4, d) increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, 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 macrophage activity, and / or j) showing one or more of increased spindle-shaped morphology, flatness of appearance, and / or number of dendritic processes when evaluated by microscopy, the method according to claim 20.

22. a) The myeloid cells contacted with the monoclonal antibody, or an antigen-binding fragment thereof, are contained within a population of cells, and the monoclonal antibody, or an antigen-binding fragment thereof, i) increases the number of type 1 and / or M1 macrophages within the population of cells, and / or ii) reduces the number of type 2 and / or M2 macrophages. b) the myeloid cells contacted with the monoclonal antibody or an antigen-binding fragment thereof are contained within a population of cells, and the monoclonal antibody or an antigen-binding fragment thereof increases the ratio of i) to ii), wherein i) within the population of cells is a type 1 and / or M1 macrophage, and ii) is a type 2 and / or M2 macrophage; c) the myeloid cells include 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; d) the myeloid cells are contacted in vitro or ex vivo; e) the myeloid cells are primary myeloid cells; f) the myeloid cells are purified and / or cultured prior to contact with the monoclonal antibody or an antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell; g) the myeloid cells are contacted in a non-human animal model and / or; h) the method further comprises contacting the myeloid cells with at least one immunotherapeutic agent that increases the inflammatory phenotype; The method according to claim 20 or 21.

23. The monoclonal antibody or an antigen-binding fragment thereof according to claim 2, wherein the myeloid cells include suppressive myeloid cells, monocytes, and / or macrophages.

24. A composition comprising the monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, the pharmaceutical composition according to claim 7, the isolated nucleic acid molecule according to claim 8, the isolated immunoglobulin heavy and light chain polypeptides according to claim 9, the vector according to claim 10 or 11, and / or the host cell according to claim 12 or 13, for increasing the inflammatory phenotype of myeloid cells in a subject, after contact with the monoclonal antibody or an antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell.

25. after contact of said bone marrow cells having said increased inflammatory phenotype with said monoclonal antibody, or antigen-binding fragment thereof, said pharmaceutical composition, said isolated nucleic acid molecule, said isolated immunoglobulin heavy and light chain polypeptides, said vector, and / or host cell, a) increased expression and / or secretion of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), b) reduced expression and / or secretion of IL-10, c) increased secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, and IL-12, d) increased ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, e) increased CD8+ cytotoxic T cell activation, f) increased CD4+ helper T cell activity, g) increased macrophage activity, and / or h) the composition according to claim 24, showing one or more of increased spindle shape, flatness of appearance, and / or number of dendritic processes when evaluated by microscopy. **Claim 26** a) said monoclonal antibody, or antigen-binding fragment thereof, said pharmaceutical composition, said isolated nucleic acid molecule, said isolated immunoglobulin heavy and light chain polypeptides, said vector, and / or host cell increases the number of type 1 and / or M1 macrophages, reduces the number of type 2 and / or M2 macrophages, and / or increases the ratio of i) to ii), wherein i) in said subject is type 1 and / or M1 macrophages and ii) is type 2 and / or M2 macrophages, b) the number and / or activity of cytotoxic CD8+ T cells in said subject increases after administration of said composition, c) said bone marrow cells include type 1 macrophages, M1 macrophages, type 2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAM), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, d) said composition is administered in vivo by systemic, peritumoral, or intratumoral administration of said composition, and / or e) said bone marrow cells are further contacted with at least one immunotherapeutic agent that increases said inflammatory phenotype, The composition according to claim 24 or 25.

27. The composition according to claim 26, wherein the monoclonal antibody, or an antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell are contacted with the myeloid cells in the tissue microenvironment.

28. The composition according to claim 26 or 27, wherein the immunotherapeutic agent comprises an immune checkpoint inhibitor, an immune stimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus.

29. A composition for increasing inflammation in a subject, comprising myeloid cells contacted with a monoclonal antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 6, the pharmaceutical composition according to claim 7, the isolated nucleic acid molecule according to claim 8, the isolated immunoglobulin heavy and light chain polypeptides according to claim 9, the vector according to claim 10 or 11, and / or the host cell according to claim 12 or 13.

30. a) the myeloid cells comprise suppressive myeloid cells, monocytes, and / or macrophages; b) the myeloid cells comprise M1 macrophages, type 1 macrophages, M2 macrophages, type 2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells; c) the myeloid cells are genetically engineered, autologous, syngeneic, or allogeneic compared to the myeloid cells of the subject, and / or d) the monoclonal antibody, or an antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell are administered systemically, peritumorally, or intratumorally; e) the myeloid cells having a regulated inflammatory phenotype have A) regulated expression of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α); B) regulated expression of IL-10; C) regulated secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, and IL-12; D) a regulated ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10. E) Modulated CD8+ cytotoxic T cell activation, F) Modulated CD4+ helper T cell activity, G) Modulated macrophage activity, and / or H) Modulated spindle shape, flatness of appearance, and / or number of dendrites when evaluated by microscopy showing one or more of: b) said bone marrow cells express PSGL-1 or are determined to express PSGL-1, c) said bone marrow cells are primary bone marrow cells, d) said bone marrow cells are contained within a tissue microenvironment, and / or e) said subject is a mammal or a human, The composition according to claim 29.

31. A composition for use in a method of sensitizing cancer cells in a subject to cytotoxic CD8+ T cell-mediated killing and / or immune checkpoint therapy, comprising the monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, the pharmaceutical composition according to claim 7, the isolated nucleic acid molecule according to claim 8, the isolated immunoglobulin heavy and light chain polypeptides according to claim 9, the vector according to claim 10 or 11, and / or the host cell according to claim 12 or 13, wherein the method comprises administering the composition to the subject.

32. a) the composition is administered systemically, peritumorally, or intratumorally, b) the method further comprises treating the cancer in the subject by administering to the subject at least one immunotherapy, c) the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4, d) the immune checkpoint is PD-1, e) the method further comprises treating the cancer in the subject by administering to the subject an additional therapeutic agent or regimen for treating cancer, f) the monoclonal antibody, or an antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell reduce the number of proliferating cells in the cancer and / or reduce the volume or size of the tumor comprising the cancer cells. g) the monoclonal antibody, or antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell increases the amount and / or activity of CD8+ T cells infiltrating the tumor comprising the cancer cells, h) the monoclonal antibody, or antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell increases i) the amount and / or activity of M1 macrophages infiltrating the tumor comprising the cancer cells and / or ii) reduces the amount and / or activity of M2 macrophages infiltrating the tumor comprising the cancer cells, i) the myeloid cells having a regulated inflammatory phenotype, A) regulated expression of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), B) regulated expression of IL-10, C) regulated secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, and IL-12, D) regulated ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, E) regulated CD8+ cytotoxic T cell activation, F) regulated CD4+ helper T cell activity, G) regulated macrophage activity, and / or H) regulated spindle shape, flat appearance, and / or number of dendritic processes when evaluated by microscopy, exhibiting one or more of: j) the cells, and / or myeloid cells comprise M1 macrophages, M1 macrophages, M2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, k) the cancer is a solid tumor infiltrated by macrophages, the infiltrating macrophages represent at least 5% of the cell mass, volume, and / or number in the tumor or tumor microenvironment, and / or the cancer is selected from the group consisting of renal tumors, ovarian tumors, endometrial tumors, lung tumors, retinal tumors, uterine tumors, l) the myeloid cells express PSGL-1 or are determined to express PSGL-1, m) the bone marrow cells are primary bone marrow cells, n) the bone marrow cells are contained within a tissue microenvironment, and / or o) the subject is a mammal or a human, The composition according to claim 31.

33. A composition for use in a method of sensitizing cancer cells in a subject suffering from cancer to cytotoxic CD8+ T cell-mediated killing and / or immune checkpoint therapy, the monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, the pharmaceutical composition according to claim 7, the isolated nucleic acid molecule according to claim 8, the isolated immunoglobulin heavy and light chain polypeptides according to claim 9, the vector according to claim 10 or 11, and / or a composition comprising bone marrow cells contacted with the host cell according to claim 12 or 13, the method comprising administering the composition to the subject.

34. a) the bone marrow cells comprise suppressive myeloid cells, monocytes, and / or macrophages, b) the bone marrow cells comprise M1 macrophages, M1 macrophages, M2 macrophages, M2 macrophages, M2c macrophages, M2d macrophages, tumor-associated macrophages (TAMs), CD11b+ cells, CD14+ cells, and / or CD11b+ / CD14+ cells, c) the bone marrow cells are genetically engineered, autologous, syngeneic, or allogeneic compared to the subject's bone marrow cells, and / or d) the monoclonal antibody, or an antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell are administered systemically, peritumorally, or intratumorally, e) the method further comprises treating the cancer in the subject by administering to the subject at least one immunotherapy, f) the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4, g) the immune checkpoint is PD-1, h) the method further comprises treating the cancer in the subject by administering to the subject an additional therapeutic agent or regimen for treating cancer. i) the monoclonal antibody, or antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor comprising the cancer cells, j) the monoclonal antibody, or antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell increases the amount and / or activity of CD8+ T cells infiltrating the tumor comprising the cancer cells, k) the monoclonal antibody, or antigen-binding fragment thereof, the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and light chain polypeptides, the vector, and / or the host cell increases i) the amount and / or activity of M1 macrophages infiltrating the tumor comprising the cancer cells and / or ii) reduces the amount and / or activity of M2 macrophages infiltrating the tumor comprising the cancer cells, l) the myeloid cells having a regulated inflammatory phenotype, A) regulated expression of interleukin 1-beta (IL-1β), IL-6, CCL3, CCL4, CXCL10, CXCL9, GM-CSF, and / or tumor necrosis factor alpha (TNF-α), B) regulated expression of IL-10, C) regulated secretion of at least one cytokine selected from the group consisting of IL-1β, TNF-α, and IL-12, D) a regulated ratio of the expression of IL-1β, IL-6, and / or TNF-α to the expression of IL-10, E) regulated CD8+ cytotoxic T cell activation, F) regulated CD4+ helper T cell activity, G) regulated macrophage activity, and / or H) a regulated spindle shape, flatness of appearance, and / or number of dendritic processes when evaluated by microscopy, exhibiting one or more of the above, m) the cancer is a solid tumor infiltrated by macrophages, the infiltrating macrophages represent at least 5% of the cell mass, volume, and / or number in the tumor or tumor microenvironment, and / or the cancer is selected from the group consisting of renal tumors, ovarian tumors, endometrial tumors, lung tumors, retinal tumors, uterine tumors, n) the myeloid cells express PSGL-1 or are determined to express PSGL-1, o) the bone marrow cells are primary bone marrow cells, p) the bone marrow cells are contained within a tissue microenvironment, and / or q) the subject is a mammal or a human, The composition according to claim 33.

35. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, or the composition according to any one of claims 29 to 34, wherein the bone marrow cells express PSGL-1 or it is determined that they express PSGL-1.

36. The composition according to any one of claims 34, wherein the immunotherapy comprises an immune checkpoint inhibitor, an immune stimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus.

37. The composition according to any one of claims 32, 34, and 36, wherein the additional therapeutic agent or regimen is selected from the group consisting of a chimeric antigen receptor, chemotherapy, radiotherapy, targeted therapy, and surgery.

38. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, or the composition according to any one of claims 29 to 37, wherein the bone marrow cells are TAM and / or M2 macrophages.

39. The composition according to any one of claims 29 to 38, wherein the human is suffering from cancer or an immunological disorder.

40. An in vitro or ex vivo method for obtaining, as an indicator for identifying bone marrow cells capable of increasing the inflammatory phenotype thereof by regulating the amount and / or activity of at least one target selected from the amino acid sequences of SEQ ID NOs: 8, 10, 12, 14, 16, and 18 to 28 from bone marrow cells, a) determining the amount and / or activity of the at least one target from the bone marrow cells using an agent, wherein the agent is at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, said determining, b) determining the amount and / or activity of the at least one target in a control using the agent, c) comparing the amount and / or activity of the at least one target detected in steps a) and b), The presence or increase in the amount and / or activity of the at least one target in the bone marrow cells, compared to the control amount and / or activity of the at least one target, indicates that the bone marrow cells can increase their inflammatory phenotype by increasing the at least one target, the method.

41. a) the cells are contacted, recommended, prescribed, or administered with an agent that increases the at least one target, the agent being at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof; b) if it is determined that the subject from which the cells are derived does not benefit from increasing the inflammatory phenotype by increasing the at least one target, the cells are contacted, recommended, prescribed, or administered with a therapy other than an agent that increases the at least one target, the agent being at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof; c) the therapy is a cancer therapy; d) the cells are contacted with and / or administered at least one additional agent that increases or decreases the immune response, the at least one additional agent being at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof; e) the control is from a member of the same species from which the cells are derived; f) the control is a sample containing cells; g) the cells are derived from a subject suffering from cancer; h) the control is a sample from the subject from which the cells are derived, and / or i) the control is a non-cancer sample from the subject from which the cells are derived, The method according to claim 40.

42. An in vitro or ex vivo method for obtaining the amount and / or activity of at least one target comprising an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16 and 18 to 28 from bone marrow cells from a subject suffering from cancer or an immunological disorder, as an indicator for predicting the clinical outcome of the subject, a) determining the amount and / or activity of at least one target from bone marrow cells from the subject using an agent, the agent being at least one monoclonal antibody according to any one of claims 1 to 6, or an antigen-binding fragment thereof, the determining; b) determining the amount and / or activity of said at least one target from a control having an adverse clinical outcome using said agent; c) comparing the amount and / or activity of said at least one target in said test sample from the subject and said control, wherein the presence or increase in the amount and / or activity of said at least one target from said bone marrow cells from the subject, as compared to the amount and / or activity in the control, indicates that the subject does not have an adverse clinical outcome, said method.

43. An in vitro or ex vivo method of obtaining the amount and / or activity of at least one target comprising an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16 and 18-28 from bone marrow cells from a subject as an indicator for monitoring the inflammatory phenotype of bone marrow cells in said subject, a) detecting the amount and / or activity of said at least one target from bone marrow cells from the subject using an agent at a first time point with a first test sample from the subject, wherein said agent is at least one monoclonal antibody according to any one of claims 1-6, or an antigen-binding fragment thereof, said detecting; b) repeating step a) using a subsequent sample containing bone marrow cells obtained at a subsequent time point; c) comparing the amount and / or activity of said at least one target detected in steps a) and b), wherein the absence or reduction in the amount and / or activity of said at least one target from said bone marrow cells from the subsequent sample, as compared to the amount and / or activity from said bone marrow cells from the first test sample, indicates that the bone marrow cells of the subject have an upregulated inflammatory phenotype, or wherein the presence or increase in the amount and / or activity of said at least one target from said bone marrow cells from the subsequent sample, as compared to the amount and / or activity from said bone marrow cells from the first test sample, indicates that the bone marrow cells of the subject have a downregulated inflammatory phenotype, said method.

44. i) The amount and / or activity of at least one target comprising an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, and 18-28, present in or on the bone marrow cells, and / or ii) The inflammatory phenotype of the bone marrow cells, as an indicator for evaluating the effectiveness of a test agent for increasing the inflammatory phenotype of bone marrow cells in a subject, an in vitro or ex vivo method of obtaining, a) Detecting in a test sample containing bone marrow cells at a first time point, i) Detecting the amount and / or activity of the at least one target present in or on the bone marrow cells using a drug, wherein the drug is at least one monoclonal antibody according to any one of claims 1-6, or an antigen-binding fragment thereof, said detecting, and / or ii) Detecting the inflammatory phenotype of the bone marrow cells, said detecting, b) Repeating step a) during at least one subsequent time point after the bone marrow cells have been contacted with the test agent, c) Comparing the values of i) and / or ii) detected in steps a) and b), the absence or reduction of the amount and / or activity of the at least one target, compared to the amount and / or activity in the sample at the first time point, and / or the increase in ii) in the subsequent sample, indicates that the test agent increases the inflammatory phenotype of the bone marrow cells in the subject, comprising the method.

45. a) The bone marrow cells contacted with the drug are contained within a cell population, and the drug increases the number of type 1 and / or M1 macrophages within the cell population, b) The bone marrow cells contacted with the drug are contained within a cell population, and the drug reduces the number of type 2 and / or M2 macrophages within the cell population, c) The bone marrow cells are contacted in vitro or ex vivo, d) The bone marrow cells are primary bone marrow cells, e) The bone marrow cells are purified and / or cultured before being contacted with the drug, h) The bone marrow cells are contacted within a tissue microenvironment, i) The method further comprises contacting the bone marrow cells with at least one immunotherapeutic agent that increases the inflammatory phenotype, and / or j) The subject is a mammal, The method according to claim 44.

46. The method according to claim 22 or 45, wherein the immunotherapeutic agent comprises an immune checkpoint inhibitor, an immune stimulatory agonist, an inflammatory agent, a cell, a cancer vaccine, and / or a virus.

47. The method according to claim 45 or 46, wherein the mammal is a non-human animal model or a human.

48. i) the amount and / or activity of at least one target comprising an amino acid sequence selected from SEQ ID NO: 8, 10, 12, 14, 16 and 18-28, within or on bone marrow cells, and / or ii) an in vitro or ex vivo method of obtaining the inflammatory phenotype of the bone marrow cells as an indicator for evaluating the effectiveness of a test agent for treating cancer or an immunological disorder in a subject, a) detecting in a subject sample containing bone marrow cells at a first time point, i) detecting the amount and / or activity of the at least one target within or on the bone marrow cells using an agent, wherein the agent is at least one monoclonal antibody according to any one of claims 1-6, or an antigen-binding fragment thereof, said detecting, and / or ii) detecting the inflammatory phenotype of the bone marrow cells, said detecting, b) repeating step a) between at least one subsequent time point after administration of the agent, c) comparing the values of i) and / or ii) detected in steps a) and b), wherein the presence, or increase, of the amount and / or activity of the at least one target, or the increase of ii) within or on the bone marrow cells of the subject sample at the subsequent time point, compared to the amount and / or activity within or on the bone marrow cells of the subject sample at the first time point, indicates that the test agent treats the cancer in the subject, or the absence, or reduction, of the amount and / or activity of the at least one target, or the reduction of ii) within or on the bone marrow cells of the subject sample at the subsequent time point, compared to the amount and / or activity within or on the bone marrow cells of the subject sample at the first time point, indicates that the test agent treats the cancer in the subject, the method comprising.

49. a) between the first time point and the subsequent time point, the subject has received treatment for the cancer or the immunological disorder, has completed the treatment, and / or is in remission; b) the first and / or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples; c) the first and / or at least one subsequent sample is obtained from a non-human animal model of the cancer; d) the first and / or at least one subsequent sample is part of a single sample or a pooled sample obtained from the subject, and / or e) the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject. The method according to claim 48.

50. An in vitro or ex vivo method for screening a test agent that sensitizes cancer cells to cytotoxic T cell-mediated killing and / or immune checkpoint therapy, comprising: a) contacting cancer cells with cytotoxic T cells and / or immune checkpoint therapy in the presence of bone marrow cells contacted with the test agent, wherein the test agent increases the amount and / or activity of at least one target comprising an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, and 18-28 in or on the bone marrow cells when determined using a drug, and the drug is at least one monoclonal antibody according to any one of claims 1-6, or an antigen-binding fragment thereof, said contacting; b) contacting cancer cells with cytotoxic T cells and / or immune checkpoint therapy in the presence of control bone marrow cells not contacted with the test agent; c) identifying a test agent that sensitizes cancer cells to cytotoxic T cell-mediated killing and / or immune checkpoint therapy by identifying a drug that increases the effectiveness of cytotoxic T cell-mediated killing and / or immune checkpoint therapy in a) compared to b). The method as described above.

51. The method according to any one of claims 20 and 40-50, wherein the bone marrow cells comprise suppressive bone marrow cells, monocytes, and / or macrophages.

52. a) further comprising 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. b) further comprising 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 said cancer cells, c) further comprising determining the responsiveness of said at least one target increasing test agent, measured by at least one criterion selected from the group consisting of clinical benefit rate, survival time to death, pathological complete response, semi-quantitative measurement of pathological response, clinical complete remission, clinical partial remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST criteria, and / or d) further comprising contacting said cancer cells with at least one additional cancer therapeutic agent or regimen, The method according to claim 50 or 51.

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