Combination therapy of immune checkpoint inhibitor and extracellular matrix component binder and method of use thereof

Abstract

A clinical combination therapy comprising one or more pharmaceutical compositions for treating various cancers. The one or more pharmaceutical compositions include at least a first composition of an immune checkpoint inhibitor and a second composition that interacts with an extracellular matrix component of the tumor microenvironment. The first composition may, for example, include pembrolizumab, and the second composition may include an LAIR-2Fc fusion protein, such as NC410. The compositions follow a dosing and timing regimen.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of combination therapies for the treatment of cancer. [Background technology]

[0002] The importance of normal immune surveillance in controlling the growth of neoplastic transformation has been known for several decades (Disis, ML, "Immune regulation of cancer", J Clin Oncol., 28: 4531-8 (2010)). Accumulating evidence indicates a correlation between tumor-infiltrating lymphocytes in cancer tissue and favorable prognosis in various malignancies. Specifically, the presence of CD8+ T cells and the ratio of CD8+ effector T cells to FoxP3+ regulatory T cells (T-reg) correlate with improved prognosis and long-term survival in solid malignancies such as ovarian, colorectal, and pancreatic cancer; hepatocellular carcinoma; malignant melanoma; and renal cell carcinoma. Tumor-infiltrating lymphocytes can be expanded ex vivo and reinfused to induce durable tumor regression in cancers such as melanoma (Dudley, ME, et al., "Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma", J Clin Oncol., 23: 2346-57 (2005); Hunder, NN, et al., "Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1", N Engl J Med., 358:2698-703 (2008)).

[0003] Therapeutic trials in mouse models have shown that administration of antibodies that block the PD-1 / PD-L1 interaction, either as monotherapy or in combination with other therapies, promotes the infiltration of tumor-specific CD8+ T cells and ultimately leads to tumor rejection (Strome, SE, et al., "B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma", Cancer Res., 63:6501-5 (2003); Blank, C., et al., "PD-L1 / B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells", Cancer Res., 64:1140-5 (2004); Hirano, F., et al., "Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity", Cancer Res., 65:1089-96 (2005); Curran, MA, et al., “PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors”, Proc Natl Acad Sci USA, 107:4275-80 (2010);Pilon-Thomas, S., et al., “Blockade of programmed death ligand 1 enhances the therapeutic efficacy of combination immunotherapy against melanoma”, J Immunol., 184:3442-9 (2010);Weber, J., “Immune checkpoint proteins: a new therapeutic paradigm for cancer--preclinical background: CTLA-4 and PD-1 blockade”, Semin Oncol., 37:430-9 (2010);Spranger, S., et al., “Mechanism of tumor rejection with doublets of CTLA-4, PD-1 / PD-L1, or IDO blockade involves IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment”, J Immunother Cancer., 2:3 (2014)). Anti-mouse PD-1 or PD-L1 antibodies have demonstrated anti-tumor responses in models of squamous cell carcinoma, pancreatic cancer, melanoma, acute myeloid leukemia, and colorectal cancer (Strome, SE, et al., "B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma", Cancer Res., 63:6501-5 (2003); Nomi, 2007; Zhang, 2009; Curran, MA, et al., "PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors", Proc Natl Acad Sci USA, 107:4275-80 (2010); Pilon-Thomas, S., et al., "Blockade of programmed death ligand 1 enhances the therapeutic efficacy of combination immunotherapy against melanoma", J Cancer Res., 107:4275-80 (2010)). Immunol., 184:3442-9 (2010)). These studies observed tumor infiltration by CD8+ T cells and increased expression of IFN-γ, granzyme B, and perforin, suggesting that the mechanism underlying the antitumor activity of PD-1 checkpoint blockade involves local infiltration and activation of effector T cell function in vivo (Curran, MA, et al., “PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors”, Proc Natl Acad Sci USA, 107:4275-80 (2010)). Experiments have confirmed the in vivo efficacy of anti-mouse PD-1 antibodies in syngenic mouse tumor models, both as monotherapy and in combination with chemotherapy. However, immune checkpoint inhibitors (ICIs), such as anti-PD-1 antibodies, may be limited due to components within the extracellular matrix (ECM) of the tumor microenvironment (TME) that can bind to ICIs.

[0004] There remains a need in the art to improve the function of immune checkpoint inhibitors in treating, reducing, and killing tumors.

[0005] Incorporation by Reference All of the foregoing applications and all documents cited therein or cited during prosecution thereof ("Application Citation Documents"), and all documents cited or referenced in the Application Citation Documents, and all documents cited or referenced herein ("Herein Citation Documents"), and all documents cited or referenced in the Herein Citation Documents, together with manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or described in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of this invention.

[0006] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0007] It should be understood that this summary is not an extensive overview of the disclosure. This summary is illustrative and not limiting, and is not intended to identify key or critical elements of the disclosure or to delineate its scope. Its sole purpose is to describe and illustrate certain concepts of the disclosure as a prelude to the more complete and extensive detailed description that follows.

[0008] The present disclosure relates to a combination therapy comprising at least two pharmaceutical compositions. This combination therapy substantially treats or alleviates the symptoms of diseases, including but not limited to cancer. Cancers include but are not limited to colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, or head and neck cancer.

[0009] The first pharmaceutical composition comprises an immune checkpoint inhibitor (ICI), such as an ICI that targets the programmed cell death protein 1 (PD-1) pathway. Such ICIs include, but are not limited to, pembrolizumab. The first pharmaceutical composition can be administered at an approximate dosage of about 400 mg on the first day of a repeated 42-day cycle. Alternatively, pembrolizumab can be administered at a dose of 200 mg every 21 days.

[0010] The second pharmaceutical composition comprises a protein configured to bind to one or more components of the extracellular matrix (ECM) of the tumor microenvironment (TME), such as collagen or C1q. Such proteins include, but are not limited to, LAIR-2 protein, LAIR-2 functional fragments, LAIR-2 variants, and LAIR-2 fusion proteins (e.g., NC410). The LAIR-2 protein or functional fragment or variant may comprise at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 5. The second pharmaceutical composition can be administered on days 1, 15, and 29 of a repeated 42-day cycle. Potential doses of the second pharmaceutical composition include about 15 mg, about 30 mg, about 60 mg, about 100 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, and about 400 mg. The second pharmaceutical composition can be administered on a variety of schedules and for a variety of lengths of time, which can be determined by considering routine factors and routine experimentation known to those skilled in the art. In one embodiment of the present invention, the composition is administered at about 15 mg, about 30 mg, about 60 mg, about 100 mg, or about 200 mg on days 1, 15, and 29 of a repeated 42-day cycle. In another embodiment, the second pharmaceutical composition can be administered at about 100 mg once weekly of a repeated 42-day cycle.

[0011] Features and components in the following figures are shown to emphasize the general principles of the present disclosure. Throughout the figures, corresponding features and components may be designated by corresponding reference characters for consistency and clarity. [Brief explanation of the drawings]

[0012] [Figure 1] We demonstrate that in a spontaneous Müllerian inhibitory factor type II receptor (MISIIR) transgenic model of ovarian cancer, LAIR-1 is expressed on CD11c+CD11b+ suppressor dendritic cells (DCs) at primary and metastatic sites of disease, but not on CD103+ stimulatory DCs. [Figure 2]We show that NC410 binds to tumor-associated ligands (collagen and C1q) and blocks LAIR-1 inhibition, promoting adaptive (T cell) and innate (dendritic cell) immune responses and activating macrophages, ultimately leading to tumor cell killing. [Figure 3] 1 shows the dose escalation study design according to the present disclosure. [Figure 4] 1 displays exemplary dosing and timing regimens according to the present disclosure. [Figure 5] 1 shows a dosage rule matrix according to the present disclosure. [Figure 6] Analysis of five representative samples of gastric adenocarcinoma (STAD) analyzed for LAIR-2-Fc (NC410) binding by H&E staining and by immunostaining of LAIR-1, CD45, CD3, and CD163 positive cells is shown. [Figure 7] 1 shows the Simon two-stage design dosing rules of the present disclosure. [Figure 8] Collagen staining by trichome staining (left) and LAIR-1 staining within immune cells by IHC in multiple tumor types (right) are shown. [Figure 9] Displays a line graph showing that the combination of NC410 and anti-PD-L1 results in synergistic and reproducible tumor killing in a mouse model. NC410, 200ug Q4D for 5 doses ± PD-L1, 100ug Q7D for 2 doses. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure may be understood more readily by reference to the following detailed description, examples, figures, and claims, as well as their preceding and following explanations. However, before the present compositions and / or methods are disclosed and described, it is to be understood that, unless otherwise specified, the disclosure is not limited to the particular compositions and / or methods disclosed, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0014] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any compositions, methods, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned are incorporated herein by reference in their entirety.

[0015] Unless otherwise defined, all composition percentage values ​​used herein are given in terms of weight percent.

[0016] In the context of describing the presently claimed invention (particularly in the context of the claims), the terms "a," "an," "the," and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated in the specification or clearly contradicted by context.

[0017] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, unless otherwise stated herein, and each separate value is incorporated into the specification as if it were individually set forth herein.

[0018] The use of the term "about" is intended to describe any value above or below the stated value within approximately + / - 10%. In other embodiments, values ​​may range anywhere above or below the stated value within approximately + / - 5%. In other embodiments, values ​​may range anywhere above or below the stated value within approximately + / - 2%. In other embodiments, values ​​may range anywhere above or below the stated value within approximately + / - 1%. The above ranges are intended to be made clear by context and are not further limited. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples provided herein, or exemplary language (e.g., "etc."), is intended merely to better clarify the invention and does not impose limitations on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0019] As used herein, "administration," as applied to a human, primate, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous ligand, reagent, placebo, small molecule, pharmaceutical, therapeutic agent, diagnostic agent, or composition with a subject, cell, tissue, organ, or biological fluid, etc. "Administration" can refer to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of a cell encompasses contacting a reagent with a cell, as well as contacting a reagent with a fluid, which is in contact with the cell. "Administration" also encompasses in vitro and ex vivo treatment of a cell, for example, with a reagent, diagnostic, binding composition, or with another cell.

[0020] "Agonist" includes a molecule, combination of molecules, complex, or combination of reagents that stimulates a receptor due to the interaction of a ligand and receptor. For example, an agonist of granulocyte-macrophage colony-stimulating factor (GM-CSF) can include GM-CSF, a mutein or derivative of GM-CSF, a peptidomimetic of GM-CSF, a small molecule that mimics the biological function of GM-CSF, or an antibody that stimulates the GM-CSF receptor.

[0021] As used herein, an "analog" or "derivative" with respect to a peptide, polypeptide, or protein refers to another peptide, polypeptide, or protein that has a similar or identical function as the original peptide, polypeptide, or protein, but does not necessarily contain a similar or identical amino acid sequence or structure as the original peptide, polypeptide, or protein. An analog preferably satisfies at least one of the following: (a) a proteinaceous agent having an amino acid sequence at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the original amino acid sequence; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the original amino acid sequence; and (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding the original amino acid sequence.

[0022] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule having a "variable region" antigen recognition site. The term "variable region" is intended to distinguish such domains of an immunoglobulin from domains broadly shared by antibodies (such as the antibody Fc domain). The variable region includes a "hypervariable region," the residues of which are responsible for antigen binding. The hypervariable region includes amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., typically approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and approximately residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.The term antibody includes monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Pat. No. 6,005,079), single-chain Fvs (scFvs) (see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315) and other antibodies. (1994), single-chain antibodies, disulfide-linked Fvs (sdFvs), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-anti-Id antibodies directed against antibodies). Specifically, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0023] As used herein, "antigen-presenting cells" (APCs) are cells of the immune system used to present antigens to T cells. APCs include dendritic cells, monocytes, macrophages, marginal zone Kupffer cells, microglia, Langerhans cells, T cells, and B cells. Dendritic cells develop in at least two lineages. The first lineage includes pre-DC1, myeloid DC1, and mature DC1. The second lineage includes CD34 + CD45RA - Early progenitor pluripotent cells, CD34 + CD45RA + cells, CD34 + CD45RA + CD4 + IL-3Rα +Pro-DC2 cells, CD4 + CD11c - These include plasmacytoid pre-DC2 cells, lymphoid human DC2 plasmacytoid-derived DC2, and mature DC2.

[0024] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity-determining regions ("CDRs") and, optionally, framework residues comprising the antibody's "variable region" antigen recognition site, and that exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab'), Fv, single-chain (ScFv), and mutants, naturally occurring variants thereof, as well as fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor, or a receptor ligand, etc.).

[0025] As used herein, an "attenuated gene" includes a gene that mediates virulence, pathology, or pathogenicity to a host, growth in a host, or survival in a host, where the gene has been mutated to reduce, reduce, or eliminate the virulence, pathology, or pathogenicity. This reduction or elimination can be assessed by comparing the pathogenicity or toxicity mediated by the mutated gene to that mediated by the non-mutated (or parent) gene. A "mutated gene" includes deletions, point mutations, and frameshift mutations in the regulatory region of the gene, the coding region of the gene, the non-coding region of the gene, or any combination thereof.

[0026] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from the abnormal, uncontrolled growth of cells. As used herein, cancer explicitly includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and that exhibit phenotypic traits distinct from non-cancerous cells, such as colony formation in a three-dimensional substrate such as soft agar, or the formation of a tubular network or web-like matrix in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells do not form colonies in soft agar, but rather form distinct, spherical structures in a three-dimensional basement membrane or extracellular matrix preparation.

[0027] As used herein, a "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0028] As used herein, the term "chimeric receptor" is defined as a cell surface receptor that comprises an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic costimulatory signaling domain in a combination that does not naturally occur together on a single protein. This specifically includes receptors in which the extracellular and cytoplasmic domains do not naturally occur together on a single receptor protein. Furthermore, chimeric receptors differ from TCRs expressed on native T cell lymphocytes.

[0029] As used herein, "costimulatory" signals encompass positive costimulatory signals (e.g., signals that result in an enhanced activity) and negative costimulatory signals (e.g., signals that result in an inhibited activity).

[0030] Throughout this specification, unless the context requires otherwise, the word "comprise," "comprises," or "comprising" should be understood to mean the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, "include," "has," "contain," and "comprise" are used synonymously.

[0031] "Consisting of" means inclusive of, and limited to, everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0032] "Consisting essentially of" means inclusive of any elements listed after the phrase, and limits other elements to elements that do not interfere with or contribute to the activity or function specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that the other elements are not optional, and that the other elements may or may not be present depending on whether they affect the activity or function of the listed elements.

[0033] The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target as a parent or reference antibody, but differs in amino acid sequence from the parent or reference antibody or antigen-binding fragment thereof by containing one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the parent or reference antibody or antigen-binding fragment thereof. In some embodiments, such derivatives have substantially the same immunospecificity and / or properties as the parent or reference antibody or antigen-binding fragment thereof, or the same immunospecificity and properties. The amino acid substitutions or additions in such derivatives can include naturally occurring (i.e., DNA-encoded) amino acid residues or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, chimeric or humanized variants, as well as variants in which the CH1, hinge, CH2, CH3, or CH4 regions have been altered to form, for example, antibodies with variant Fc regions with enhanced or diminished effector or binding properties.

[0034] As used herein, "effective amount" includes, but is not limited to, an amount (e.g., an amount of a protein, polypeptide, fragment thereof, etc.) that can ameliorate, relieve, alleviate, prevent, or diagnose a symptom or sign of a disease state or disorder. Unless otherwise indicated explicitly or contextually, an "effective amount" is not limited to a minimum amount sufficient to ameliorate a condition.

[0035] References throughout this specification to "an embodiment," "one embodiment," "an embodiment," "a certain embodiment," "related embodiments," "certain embodiments," "additional embodiments," or "further embodiments," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0036] The term "endogenous concentration" refers to the level at which a molecule is naturally expressed (i.e., in the absence of an expression vector or recombinant promoter) by a cell (which may be a normal cell, a cancer cell, or an infected cell).

[0037] As used herein, "epitope" refers to an antigenic determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0038] As used herein, "extracellular fluid" includes serum, plasma, blood, interstitial fluid, cerebrospinal fluid, secretions, lymph, bile, sweat, feces, and urine. "Extracellular fluid" can also include colloids or suspensions, such as whole blood or clotted blood.

[0039] As used herein, a "fragment" in the context of a polypeptide includes a peptide or polypeptide that comprises an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of the larger polypeptide.

[0040] As used herein, the term "humanized antibody" refers to an immunoglobulin containing a human framework region and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." Constant regions are not necessarily present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-99%, or about 95% or more identical. Thus, all portions of a humanized immunoglobulin, except for the CDRs as the case may be, are substantially identical to corresponding portions of a native human immunoglobulin sequence. A humanized antibody is an antibody containing a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody does not include conventional chimeric antibodies, for example, because the entire variable region of a chimeric antibody is non-human.

[0041] As used herein, "immune cell" refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0042] As used herein, "immune checkpoint" refers to an inhibitory pathway of the immune system that is involved in maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage. Immune checkpoints are controlled by immune checkpoint proteins.

[0043] As used herein, an "immune checkpoint protein" is a protein, typically a receptor (e.g., CTLA4 or PD-1) or a ligand (e.g., PD-L1), that regulates or modulates the extent of an immune response. Immune checkpoint proteins can be inhibitory or stimulatory. In particular, immune checkpoint proteins are inhibitory to the activation of an immune response. Thus, inhibition of an inhibitory immune checkpoint protein acts to stimulate or activate an immune response, such as T cell activation and proliferation.

[0044] As used herein, "immune checkpoint inhibitor" or "immune checkpoint suppressor," or "immune checkpoint blocker," refers to an agent that binds to and blocks the activity of an inhibitory immune checkpoint protein. The inhibition can be competitive or noncompetitive, and it can be steric or allosteric. When the immune checkpoint protein is an immunostimulatory protein, the immune checkpoint inhibitor acts to promote the activity of the immunostimulatory protein by inhibiting it, such as by binding to and activating the stimulatory immune checkpoint protein, or by interfering with, such as by binding to or inactivating, an inhibitor of the stimulatory immune checkpoint protein. An example of an immune checkpoint inhibitor is an anti-immune checkpoint protein antibody.

[0045] The "target" of an immune checkpoint inhibitor is the immune checkpoint protein that the immune checkpoint inhibitor or immune checkpoint suppressor binds to and blocks its activity. Typically, immune checkpoint inhibitors specifically bind to their targets. For example, the target of an exemplary anti-CTLA4 antibody called ipilimumab is CTLA4.

[0046] An "immunogenic agent" or "immunogen" is capable of inducing an immunological response against itself when administered to a mammal, optionally in combination with an adjuvant.

[0047] As used herein, the terms "immunological," "immunological," or "immune" response refer to the development in a recipient patient of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response to a peptide. Such a response can be an active response induced by administration of an immunogen, or a passive response induced by administration of antibodies or primed T cells. A cellular immune response is elicited by presentation of a polypeptide epitope in association with class I or class II MHC molecules and results in antigen-specific CD4 + T helper cells and / or CD8 + Activates cytotoxic T cells. This response may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, and activation or recruitment of neutrophils or other innate immune components. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0048] As used herein, a molecule is said to be capable of "immunospecifically binding" to a second molecule if such binding exhibits the specificity and affinity of an antibody for its cognate antigen. An antibody is said to be capable of immunospecifically binding to a target region or structure ("epitope") of an antigen if the binding involves an antigen recognition site on an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind other antigens with lower affinity, but may not bind completely unrelated antigens, if the other antigens share some sequence or conformational similarity recognized by the antigen recognition site as determined, for example, by immunoassays, BIACORE® assays, or other assays known in the art. However, in some embodiments, antibodies (and their antigen-binding fragments) do not cross-react with other antigens. Antibodies can also bind other molecules in a non-immunospecific manner, such as FcR receptors, by binding domains in other regions / domains of the molecule not involved in the antigen recognition site, such as the Fc region.

[0049] As used herein, the terms "individual," "host," "subject," "participant," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents such as mice and rats, and other laboratory animals. Thus, the methods and compositions described herein are applicable to both human and veterinary diseases. In certain embodiments, the subject is a "patient," such as a living human receiving medical treatment for a disease or condition. This includes those without a clearly identified illness who are being investigated for signs of pathology.

[0050] As used herein, "inflammatory molecules" refers to molecules that result in an inflammatory response, including, but not limited to, cytokines and metalloproteases, including, but not limited to, IL-1β, TNF-α, TGF-beta, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

[0051] As used herein, "ligand" refers to a small molecule, peptide, polypeptide, or membrane-associated or membrane-bound molecule that is an agonist or antagonist of a receptor. "Ligand" also encompasses binding agents that are neither agonists nor antagonists and do not have agonist or antagonist properties. By convention, if a ligand is membrane-bound on a first cell, the receptor typically occurs on a second cell. The second cell may have the same ID (same name) as the first cell or a different ID (different name). A ligand or receptor may be entirely intracellular; that is, it may reside in the cytosol, nucleus, or some other intracellular compartment. A ligand or receptor may change location, for example, from an intracellular compartment to the outer surface of the plasma membrane. A complex of a ligand and a receptor is called a "ligand-receptor complex." When a ligand and a receptor are involved in a signaling pathway, the ligand occurs upstream of the signaling pathway, and the receptor occurs downstream of the signaling pathway.

[0052] As used herein, the term "isolated" means material that is substantially or essentially free from components that normally accompany it in its natural state. In certain embodiments, the terms "obtained" or "derived" are used synonymously with "isolated."

[0053] As used herein, "managing" or "controlling" one or more symptoms or effects of a disease or condition (e.g., cancer) refers to using the compositions or methods contemplated herein to better control tumor activity and clinical signs associated with cancer in a subject in need thereof, thereby improving the animal's quality of life.

[0054] As used herein, the term "modulate" refers to the ability to alter an effect, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating but not abolishing) or can completely abolish (e.g., neutralize) such activity. Modulation can include receptor internalization after antibody binding or reducing receptor expression on target cells. Agonistic modulation can enhance, otherwise increase, or potentiate an activity (e.g., signal transduction). In still further embodiments, such modulation can alter the nature of the interaction between a ligand and its cognate receptor, so as to alter the nature of the induced signal transduction. For example, a molecule, by binding to a ligand or receptor, can alter the ability of such molecule to bind to other ligands or receptors, thereby altering their overall activity. In some embodiments, such modulation results in at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or at least a 100-fold change in such activity.

[0055] As used herein, the terms "percent sequence identity" and "% sequence identity" refer to the percentage of sequence similarity found by comparing or aligning two or more amino acid or nucleic acid sequences. Percent identity can be determined by directly comparing the sequence information between two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Algorithms for calculating percent identity include the Smith-Waterman homology search algorithm (see, e.g., Kann and Goldstein (2002) Proteins 48:367-376; Arslan, et al. (2001) Bioinformatics 17:327-337). As non-limiting examples, the percent sequence identity can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and any percentage therebetween.

[0056] As used herein, "peptide" refers to a short sequence of amino acids, where the amino acids are linked to one another by peptide bonds. Peptides can occur free or can be bound to another moiety, such as a polymer, lipid, oligo- or polysaccharide, and / or polypeptide. Even when the peptide is incorporated into a polypeptide chain, the term "peptide" may still be used to specifically refer to a short sequence of amino acids. A "peptide" can be linked to another moiety via a peptide bond or some other type of bond. A peptide is at least two amino acids long, with the maximum length being a function of convention or context.

[0057] The term "percent sequence identity (%)" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity.Alignment for determining percent sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared, can be determined by known methods.

[0058] For purposes of this specification, the % sequence identity between a given nucleotide or amino acid sequence C and a given nucleic acid sequence D (which may also be expressed as a given sequence C having or containing a certain % sequence identity to given sequence D or to given sequence D) is calculated as follows: 100 x fraction X / Y, where W is the number of nucleotides or amino acids scored as identical matches in the alignment of C and D by a sequence alignment program, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, then the % sequence identity of C to D will not equal the % sequence identity of D to C.

[0059] As used herein, "pharmaceutically acceptable excipients," "pharmaceutically acceptable carriers," or "diagnostically acceptable excipients" include, but are not limited to, sterile distilled water, physiological saline, phosphate buffer solution, amino acid-based buffer, or bicarbonate buffer. The excipient selected and the amount of excipient used will depend on the mode of administration. Administration includes injection, infusion, or a combination thereof. Additional pharmaceutically acceptable carriers include, but are not limited to, suitable carriers or diluents commonly used in the pharmaceutical arts, including aqueous or organic solvents or solvent mixtures. These organic solvents are described, for example, in Remington Pharmaceutical Sciences, 21 st Edition (2005). Other solvents and / or additives that may be used in the topical compositions include, but are not limited to, PEG esters of carboxylic and dicarboxylic acids and PEG esters of fatty acids, glycerol esters including triacetin, caprylic / capric triglyceride (Miglyol 812®), and the like; glycerol ethers including glycerol formal; propylene glycol dicaprylate / dicaprate (Miglyol 840®), lauryl lactylate, triacetin, diisopropyl adipate (DIPA, also known as Ceraphile 230), diisobutyl adipate, dimethyl isosorbide (DMI), acetyl tributyl citrate, oleic acid; carboxylic acid esters including diacid esters, ketones including acetone, methyl isobutyl ketone (MIK), methyl ethyl ketone, and the like; acetonitrile, C1-C 12Alcohols (including benzyl alcohol, methanol, ethyl alcohol, isopropanol, and butanol); aromatic ethers such as anisole; amides such as dimethylacetamide, monomethylacetamide, and dimethylformamide; dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, glycol carbonates (including but not limited to propylene carbonate and butylene carbonate); C1-C2 of carboxylic acids including 2-pyrrolidone, N-methylpyrrolidone, butyl or octyl acetate, and benzyl acetate. 12 Alkyl esters of dicarboxylic acids C1-C 12 These include, but are not limited to, alkyl esters; aryl esters such as benzyl benzoate, ethyl benzoate; and diethyl phthalate, or a mixture of at least two of these solvents.

[0060] As used herein, the term "polypeptide" refers to an amino acid chain of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. A polypeptide can be "exogenous," meaning "heterologous," i.e., foreign to the host cell utilized, such as, for example, a human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. The sequences are written from left to right in the amino- to carboxy-terminal direction. According to standard nomenclature, amino acid residue sequences are designated by either the three-letter or one-letter code shown below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0061] As used herein, "prevent," and similar terms such as "prevented," "preventing," and the like, refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of one or more symptoms or other effects of a disease or condition disclosed herein (such as a disease or condition such as colon cancer, head and neck cancer, gastrointestinal cancer, gastroesophageal cancer, and other known diseases and conditions). For example, in embodiments relating to the treatment of cancer in a subject, "prevent," and similar terms such as "prevented," "preventing," and the like, refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence of clinical disease and symptoms associated with cancer. This approach also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms.

[0062] As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disorder or disease before any symptoms of such disorder or disease are detected. A "prophylactically effective" amount is the amount of a prophylactic agent (e.g., a protein, polypeptide, fragment thereof, etc.) sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of a prophylactic agent that provides a prophylactic benefit in preventing disease. Typically, but not necessarily, a prophylactically effective amount may be less than a therapeutically effective amount, as it is a prophylactic dose used in subjects before or at an early stage of disease.

[0063] As used herein, "protein" generally refers to the sequence of amino acids comprising a polypeptide chain. Protein may also refer to the three-dimensional structure of a polypeptide. A "denatured protein" refers to a partially denatured polypeptide that has some residual three-dimensional structure, or alternatively, has an essentially random three-dimensional structure, as in a fully denatured protein. Polypeptide variants may be generated by glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal or leader sequence processing, covalent and non-covalently bound cofactors, oxidation variants, and the like.

[0064] As used herein, "recombinant," when used in reference to a nucleic acid, cell, animal, virus, plasmid, vector, etc., refers to the introduction of exogenous non-native nucleic acid, the modification of a naturally occurring nucleic acid, or the modification derived in whole or in part from a recombinant nucleic acid, cell, virus, plasmid, or vector. Recombinant protein refers to a protein derived from a recombinant nucleic acid, virus, plasmid, vector, etc.

[0065] As used herein, "sample" refers to a sample from a human, animal, placebo, or research sample, such as a cell, tissue, organ, bodily fluid, gas, aerosol, slurry, colloid, or coagulated material. A "sample" may be tested in vivo (i.e., without removal from a human or animal) or in vitro. A sample may be examined after processing, such as by histological methods. A "sample" also refers to cells comprising a bodily fluid or tissue sample, or cells separated from a bodily fluid or tissue sample. A "sample" may also refer to a cell, tissue, organ, or bodily fluid that is freshly removed from a human or animal, or a cell, tissue, organ, or bodily fluid that has been processed or stored.

[0066] "Specifically" or "selectively" binding, when referring to a ligand / receptor, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pair (e.g., cytokine for cytokine receptor), refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under specified conditions, a particular ligand will bind to a specific receptor and will not bind in significant amounts to other proteins present in a sample. Specific binding can also mean, for example, that a binding compound, nucleic acid ligand, antibody, or binding composition derived from an antigen-binding portion of an antibody of a contemplated method binds to its target with an affinity that is often at least 25% higher, more often at least 50% higher, most often at least 100% (2-fold) higher, usually at least 10-fold higher, more usually at least 20-fold higher, and most usually at least 100-fold higher than that of other binding compounds.

[0067] The term "substantially" when used in the context of a binding effect or a demonstrated effect is intended to indicate that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule can substantially inhibit the activity of a ligand or receptor if the degree of blockage is physiologically or therapeutically relevant (e.g., if such degree is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or properties as another molecule if such immunospecificity and properties are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical. As used herein, the term "therapeutically effective amount" is defined as the amount of a reagent or pharmaceutical composition sufficient to induce a desired immune response specific to the encoded heterologous antigen and benefit a patient (e.g., resulting in the reduction, prevention, or amelioration of the symptoms of the condition being treated). If the agent or pharmaceutical composition includes a diagnostic agent, a "diagnostically effective amount" is defined as an amount sufficient to produce a signal, image, or other diagnostic parameter. The effective amount of a pharmaceutical preparation varies depending on factors such as the individual's degree of susceptibility, the individual's age, sex, and weight, and the individual's specific response (US Pat. No. 5,888,530).

[0068] As used herein, "treat," "treating," "treatment," and "therapeutic use" (with respect to a condition or disease) are approaches for obtaining beneficial or desired results, preferably including clinical results. For purposes of this disclosure, beneficial or desired results with respect to a disease include, but are not limited to, one or more of: ameliorating symptoms associated with the disease, curing the disease, reducing the severity of the disease, delaying the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of a person suffering from the disease, and / or prolonging survival. Similarly, for purposes of this disclosure, beneficial or desired results with respect to a condition include, but are not limited to, one or more of: ameliorating the condition, curing the condition, reducing the severity of the condition, delaying the progression of the condition, alleviating one or more symptoms associated with the condition, improving the quality of life of a person suffering from the condition, and / or prolonging survival.

[0069] As used herein, "tumor microenvironment" or "TME" refers to the normal cells, molecules, fibroblasts, immune cells, and blood vessels that surround and nourish tumor cells. The tumor microenvironment also includes proteins produced by all cells present in the tumor that support the growth of cancer cells.

[0070] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more alterations (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0071] Modifications and variations can be made to the structure of the polypeptides of the present disclosure and still obtain molecules with similar properties (e.g., conservative amino acid substitutions) to the polypeptides. For example, certain amino acids can be substituted for other amino acids within the sequence without significant loss of activity. Because the biological functional activity of a polypeptide is defined by its ability to interact and its properties, certain amino acid sequences can be substituted into the polypeptide sequence and still obtain a polypeptide with similar properties.

[0072] When making such changes, the hydropathic index of the amino acid can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art. It is known that a particular amino acid can be substituted with another amino acid having a similar hydropathic index or score while still resulting in a polypeptide having similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are as follows: 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), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0073] The relative hydropathic properties of amino acids are believed to determine the secondary structure of the resulting polypeptide, which in turn defines the polypeptide's interactions with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that substitution of an amino acid with another amino acid having a similar hydropathic index can result in a functionally equivalent polypeptide. Among such changes, substitution of amino acids with hydropathic indices within ±2 is preferred, with substitutions within ±1 being particularly preferred, and substitutions within ±0.5 being even more particularly preferred.

[0074] Similar amino acid substitutions can also be made on the basis of hydrophilicity, particularly when the resulting biologically functional equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±1), glutamic acid (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), proline (-0.5±1), threonine (-0.4), alanine (-0.5), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), tryptophan (-3.4). It is understood that an amino acid can be substituted with another having a similar hydrophilicity value and still obtain a biologically equivalent, and particularly an immunologically equivalent, polypeptide. Among such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0075] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg:Lys), (Asn:Gln, His), (Asp:Glu, Cys, Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn, Gln), (Ile:Leu, Val), (Leu:Ile, Val), (Lys:Arg), (Met:Leu, Tyr), (Ser:Thr), (Thr:Ser), (Tip:Tyr), (Tyr:Trp, Phe), and (Val:Ile, Leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of the above polypeptides. In particular, embodiments of the polypeptides may include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the subject polypeptides.

[0076] II. Composition The present disclosure relates to a combination of one or more pharmaceutical compositions. The one or more pharmaceutical compositions may be combined in a single administration, as described herein, or may be administered separately according to various dosing and timing regimens. The combination can be administered to a subject in need thereof, where the subject has an advanced, unresectable and / or metastatic immune checkpoint inhibitor (ICI)-refractory solid tumor, or an ICI-naive, microsatellite-stable / microsatellite-unstable (MSS / MSI)-low solid tumor. The combination can be administered to a subject in need thereof, where one composition of the combination is an ICI (e.g., an anti-PD-1 therapy), and a second composition of the combination exhibits affinity for binding components of the extracellular matrix (ECM) within the tumor microenvironment (TME). The second composition may bind to a component of the ECM, allowing the ICI of the first composition to more efficiently target the tumor. This combination then more efficiently treats, reduces, or kills cancer-related tumors. The composition exhibiting affinity for binding components of the ECM may bind to collagen. The combination can include, but is not limited to, a first pharmaceutical composition containing pembrolizumab and a second pharmaceutical composition containing LAIR-2 or an LAIR-2Fc fusion protein. The second pharmaceutical composition may specifically contain NC410, an LAIR-2Fc fusion protein. The combination may also include additional compositions, including, but not limited to, one or more of an LAIR-2IgG1 fusion protein, LAIR-1, an LAIR-1Fc fusion protein, a humanized monoclonal antibody (e.g., a humanized monoclonal antibody against PD-1 (IgG4)), a collagen-derived product (e.g., C4G, Pro-C3, Pro-C6, etc.), any combination thereof, and other such components known in the art.

[0077] A. Immune Checkpoint Pathway The present disclosure relates to a combination of one or more compositions configured to effectively inhibit the programmed cell death protein 1 (PD-1) pathway. Inhibiting the PD-1 pathway within the tumor microenvironment (TME) using an immune checkpoint inhibitor (e.g., pembrolizumab) in combination with a protein configured to interact with components of the extracellular matrix (e.g., a LAIR protein) promotes immune cell activation, coupled with extracellular matrix (ECM) remodeling, promoting further T cell infiltration into the TME, providing a new and improved therapeutic approach for patients with ICI-refractory advanced metastatic solid tumors, regardless of MSI status or MSS or MSI-poor advanced unresectable and / or metastatic solid tumors. The PD-1 receptor-ligand interaction is a major pathway that tumors hijack to suppress immune control. Under healthy conditions, the normal function of PD-1, expressed on the cell surface of activated T cells, is to downregulate unwanted or excessive immune responses, including autoimmune responses. PD-1 (encoded by the gene PDCD1) is a member of the immunoglobulin (Ig) superfamily related to cluster of differentiation 28 (CD28) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and has been shown to negatively regulate antigen receptor signaling by binding to its ligands (PD-L1 and / or PD-L2) (Okazaki, T., et al., "PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine", Proc Natl Acad Sci USA, 98:13866-71 (2001); Greenwald, RJ, et al., "The B7 family revisited", Annu Rev Immunol., 23:515-48 (2005)).

[0078] The sequence of human PDCD1 is known in the art. For example, the consensus sequence for PDCD1 is as follows: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1, UniProt Accession No. Q15116, incorporated by reference in its entirety).

[0079] The structure of mouse PD-1 has been elucidated (Zhang, 2004). PD-1 and its family members are type I transmembrane glycoproteins containing an Ig variable (IgV) domain responsible for ligand binding and a cytoplasmic tail responsible for binding to signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs: an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif.Following T cell stimulation, PD-1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to an immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, resulting in the dephosphorylation of effector molecules involved in the CD3 T cell signaling cascade, such as CD3 zeta (CD3ζ), protein kinase C theta (PKCθ), and zeta chain-associated protein kinase (ZAP70) (Okazaki, T., et al., "PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine", Proc Natl Acad Sci USA, 98:13866-71 (2001); Chemnitz, JM, et al., "SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor "PD-1 ligation prevents T cell activation," J Immunol., 173:945-54 (2004); Sheppard, KA, et al., "PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70 / CD3zeta signalosome and downstream signaling to PKCtheta.", FEBS Lett., 574:37-41 (2004); Riley, JL, "PD-1 signaling in primary T cells," Immunol Rev., 229:114-25 (2009)). The mechanism by which PD-1 down-regulates T cell responses is similar to but distinct from that of CTLA-4.Because both molecules regulate overlapping signaling proteins (Parry, RV, et al., "CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms", Mol Cell Biol., 25:9543-53 (2005); Francisco, LM, et al., "The PD-1 pathway in tolerance and autoimmunity", Immunol Rev., 236:219-42 (2010)), the PD-1 / PD-L1 pathway is an attractive target in the tumor microenvironment (TME) and an effective approach for cancer therapy.

[0080] B. LAIR Proteins The present disclosure relates to compositions comprising LAIR proteins. The LAIR proteins may comprise the full-length amino acid sequence of LAIR-1 or LAIR-2 proteins, or fragments or variants thereof, or fusion proteins thereof (including, but not limited to, LAIR-1Fc fusion proteins or LAIR-2Fc fusion proteins). The LAIR-2Fc fusion protein may comprise NC410. The LAIR protein compositions can be administered to a subject in need thereof in combination with one or more compositions of pembrolizumab. The LAIR protein compositions can be administered simultaneously with one or more compositions or separately. The LAIR protein compositions can be administered according to various dosing and timing regimens described herein.

[0081] Inhibitory LAIR-1 signaling may prevent autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroid disease, and atherosclerosis, as well as contact hypersensitivity (Sun et al., 2014). Meanwhile, overexpression of LAIR-2 may promote autoimmunity through decoy binding of LAIR-1 ligands. LAIR-2 binding of LAIR-1 ligands essentially reduces cell surface cross-linking of LAIR-1, limiting the inhibitory signaling pathways that lead to hyperreactive immune function. Conversely, it has been hypothesized that elevated levels of LAIR-2 may promote antitumor immunity through the same mechanism.

[0082] The presence of a collagen-rich extracellular matrix (ECM) is increasingly recognized as an important determinant of tumor response to ICI therapy. Collagen can be secreted within the TME by cancer-associated fibroblasts (CAFs), cancer cells, and macrophages. LAIR-1-expressing cells localized in the tumor microenvironment can be specifically suppressed through collagen cross-linking of LAIR-1 and subsequent inhibitory signaling. Interestingly, both collagen and C1q have been shown to limit or alter LAIR-1-mediated differentiation and activation of antigen-presenting cells (monocytes / macrophages / DCs). Studies have shown that T cell cross-linking of LAIR-1 on NK cells can inhibit their proliferation and function. Collagen-dense ECM, which acts as a physical barrier against immune cell infiltration into tumors, has been shown to suppress anti-tumor immunity and bind to PD-1 / PD-L1-resistant tumors (Peng, DH, et al., “Collagen promotes anti-PD-1 / PD-L1 resistance in cancer through LAIR1-dependent CD8(+) T cell exhaustion”, Nat Commun., 11:4520 (2020)).

[0083] Dysregulation of the ECM in the TME supports tumor progression, leads to immune dysfunction, and provides a target for cancer therapy. Leukocyte-associated immunoglobulin-like receptor (LAIR)-1 and LAIR-2 are members of the leukocyte receptor complex (LRC) on human chromosome 19 (Lebbink, RJ, et al., "The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen / LAIR-1 inhibitory immune interaction", J Immunol., 180:1662-9 (2008); Lebbink, RJ, et al., "Identification of multiple potent binding sites for human leukocyte-associated Ig-like receptor LAIR on collagens II and III", Matrix Biol., 28:202-10 (2009); Olde Nordkamp, ​​MJ, et al., "Enhanced secretion of leukocyte-associated immunoglobulin-like receptor 2 (LAIR-2) and soluble LAIR-1 in rheumatoid arthritis: LAIR-2 is a more efficient antagonist of the LAIR-1-collagen inhibitory interaction than is soluble;Olde Nordkamp, ​​MJ, et al., “Leukocyte-associated Ig-like receptor-1 is a novel inhibitory receptor for surfactant protein D”, J Leukoc Biol, 96:105-11 (2014)).LAIR-1 is a well-described co-inhibitory receptor expressed on several subsets of immune cells and functions to limit immune responses (Afshar-Kharghan, V., “The role of the complement system in cancer”, J Clin Invest., 127:780-9 (2017); Pearce, OMT, et al., “Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers”, Cancer Discov., 8:304-19 (2018)). In some cancers, LAIR-1 expression has been observed to be associated with suppressive immune cell populations. As shown in Figure 1, in both mouse and human ovarian cancer, stimulatory, but not suppressive, dendritic cell (DC) subpopulations and suppressive macrophages express LAIR-1, indicating that inhibition of LAIR-1 in ovarian cancer should reverse immune suppression (Flies, DB, et al., “Immune checkpoint blockade reveals the stimulatory capacity of tumor-associated CD103(+) dendritic cells in late-stage ovarian cancer”, Oncoimmunology., 5:e1185583 (2016)).

[0084] i.LAIR-1 The sequence of human LAIR-1 is known in the art. For example, the consensus sequence for LAIR-1a (isoform 1) is: MSPHPTALLGLVLCLAQTIHT QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLYILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 2, UniProtKB-Q6GTX8(LAIR1_HUMAN)) is.

[0085] Amino acids 1-21 are the signal sequence, amino acids 22-165 (underlined) are the extracellular domain, amino acids 166-186 are the transmembrane domain, and amino acids 187-287 are the cytoplasmic domain. Amino acids 29-117 form the Ig-like C2 domain. Amino acids 249-254 and 279-284 form ITIM motifs 1 and 2, respectively. LAIR-1b (also known as isoform 2) lacks amino acids 122-138 compared to SEQ ID NO:2. LAIR-1c (also known as isoform 3) lacks amino acids 23-23 and 122-138 compared to SEQ ID NO:2. LAIR-1d (also known as isoform 4) lacks amino acids 210-287 compared to SEQ ID NO:2.

[0086] As mentioned above, the extracellular domain of human LAIR-1 is QEEDLPR PSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLE LLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLY (SEQ ID NO: 3), or a fragment thereof, for example, the Ig-like C2 domain (underlined amino acids 8-96 of SEQ ID NO: 3), or the region surrounded by the disulfide-bond-forming cysteines between amino acids 49-101 of SEQ ID NO: 2 (amino acids 28-80 of SEQ ID NO: 3, shown in italics).

[0087] Known variants and mutations of LAIR-1 include E63D, Y251F, and Y251F relative to SEQ ID NO: 2. Evidence indicates that Y215F reduces tyrosine phosphorylation, abolishes binding to PTPN6 and CSK, completely abolishes inhibitory activity, and also abolishes phosphorylation and calcium mobilization inhibition when bound to F-281 (Xu, et al., J. Biol. Chem. 275:17440-17446 (2000), Verbrugge, et al., Int. Immunol., 15:1349-1358 (2003), Verbrugge, et al., Eur. J. Immunol., 36:190-198 (2006)). Y281F exhibits reduced tyrosine phosphorylation and loss of binding to PTPN6, as well as partial inhibition of cytotoxic activity.

[0088] Studies have demonstrated that components of the ECM function as ligands for the collagen-binding inhibitory receptor LAIR-1. All collagens are composed of three polypeptide chains characterized by a repeating Gly-X-X' sequence, where X is often proline and X' is often 4-R-hydroxyproline (Hyp,O) (Brondijk, Blood, 115(7):1364-1373 (2010)). The Gly-X-X' triplet is an almost unique feature of collagen, allowing it to form the characteristic triple-helical collagen structure.

[0089] LAIR-1 ligands include several collagens and collagen domain-containing ligands, including complement component C1q, mannan-binding lectin (MBL), and surfactant protein D (SP-D) (Lebbink, RJ, et al., "The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen / LAIR-1 inhibitory immune interaction", J Immunol., 180:1662-9 (2008); Lebbink, RJ, et al., "Identification of multiple potent binding sites for human leukocyte-associated Ig-like receptor LAIR on collagens II and III", Matrix Biol., 28:202-10 (2009); Olde Nordkamp, ​​MJ, et al., "Enhanced secretion of leukocyte-associated immunoglobulin-like receptor 2 (LAIR-2) and soluble LAIR-1 in rheumatoid arthritis: LAIR-2"). is a more efficient antagonist of the LAIR-1-collagen inhibitory interaction than is solubl;Olde Nordkamp, ​​MJ, et al., “Leukocyte-associated Ig-like receptor-1 is a novel inhibitory receptor for surfactant protein D”, J Leukoc Biol, 96:105-11 (2014)).

[0090] In cancer, it has been hypothesized that LAIR-1 expression on some subsets of leukocytes prevents optimal immune responses by limiting both innate and adaptive immune functions. LAIR-1 functions to suppress antitumor immunity through inhibition of stimulatory signaling pathways. Specifically, LAIR-1 is a checkpoint and adhesion receptor on T cells that limits T cell activation and increases adhesion to collagen (Meyaard, L., "The inhibitory collagen receptor LAIR-1 (CD305)", J Leukoc Biol., 83:799-803 (2008)).

[0091] In addition to its role in T cell function, LAIR-1 is also expressed on NK cells, monocytes, macrophages, dendritic cells, and neutrophils, where it functions to limit immune responses. Furthermore, LAIR-1 expression has been shown to be associated with suppressive DC and macrophage subpopulations in both mouse and human ovarian cancer (Flies, DB, et al., "Immune checkpoint blockade reveals the stimulatory capacity of tumor-associated CD103(+) dendritic cells in late-stage ovarian cancer," Oncoimmunology., 5:e1185583 (2016)). Blocking LAIR-1 in cancer should reduce the inhibitory mechanisms that redirect myeloid cells toward promoting stimulatory responses, including T cell responses, against tumors. Overall, these data suggest that targeting the LAIR-1 pathway in cancer patients may be a rational approach to enhance antitumor immunity.

[0092] Certain tumors with high collagen deposition have been shown to confer either intrinsic or acquired resistance to PD-1 / PD-L1 blockade due to alternative immunosuppressive pathways and a reduction in total intratumoral CD8+ T cells (Peng, DH, et al., "Collagen promotes anti-PD-1 / PD-L1 resistance in cancer through LAIR1-dependent CD8(+) T cell exhaustion", Nat Commun., 11:4520 (2020)). This phenomenon of collagen-induced CD8+ T cell immunosuppression is due to enhanced collagen production, driven in part by overexpression of LAIR-1 on immune cells and increased TGF-β signaling upon treatment with PD-1 / PD-L1 blockade.

[0093] ii. LAIR-2 LAIR-2 is a soluble homolog of LAIR-1 that binds to collagen and competes with collagen for LAIR-1 binding, functioning as a natural decoy to promote immune function. LAIR-2 can block the functional interaction of LAIR-1 with its ligands, resulting in improved immune function in multiple immune cell subsets. Because LAIR-2 binds to collagen with higher affinity than LAIR-1, overexpression of LAIR-2 leads to blockade of LAIR-1 signaling, sensitizing resistant tumors to PD-1 blockade and significantly reducing tumor growth and metastasis.

[0094] LAIR-2 is a secreted protein that shares 77.5% homology with the transmembrane protein LAIR-1 in the Ig-like C2 domain of its extracellular region. It functions as a natural endogenous secretory decoy of LAIR-1, which is primarily produced by activated T cells (Meyaard, L., "The inhibitory collagen receptor LAIR-1 (CD305)", J Leukoc Biol., 83:799-803 (2008)). LAIR-2 can block the functional interaction of LAIR-1 with its ligand, resulting in improved immune function in multiple immune cell subsets, as shown in Figure 2. Of note is the observation that dysregulation of LAIR-1 ligands leads to the overproduction of collagen and complement C1q, as well as other forms of collagen, which can exert potent inhibitory effects in the TME (Afshar-Kharghan, V., "The role of the complement system in cancer", J Clin Invest., 127:780-9 (2017); Pearce, OMT, et al., "Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers", Cancer Discov., 8:304-19 (2018)). Therefore, disrupting the interaction of LAIR-1 ligands with LAIR-1 and utilizing LAIR-2-based therapies to counteract the inhibitory effects of the TME are novel approaches to cancer treatment.

[0095] The sequence of human LAIR-2 is known in the art. For example, the consensus sequence for LAIR-2a (isoform 1) is: MSPHLTALLGLVLCLAQTIHT QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAP (SEQ ID NO: 4, UniProtKB-Q6ISS4(LAIR2_HUMAN)) is.

[0096] Amino acids 1-21 are a signal sequence, and amino acids 22-152 (underlined) are the leukocyte-associated immunoglobulin-like receptor 2 domain. Amino acids 29-117 form the Ig-like C2 domain. LAIR-2b (also known as isoform 2) lacks amino acids 122-138 compared to SEQ ID NO: 4. As introduced above, the leukocyte-associated immunoglobulin-like receptor 2 domain of human LAIR-2 is: QEGALPR PSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSSEGNAGLYRCLYYKPPGWSEHSDFLE LLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAP (SEQ ID NO: 5), or a fragment thereof, for example, the Ig-like C2 domain (underlined amino acids 8-96 of SEQ ID NO: 5), or the region surrounded by the disulfide-bond-forming cysteines between amino acids 49-101 of SEQ ID NO: 1 (amino acids 28-80 of SEQ ID NO: 5, shown in italics).

[0097] Known variants and mutations of LAIR-2 include G78S, H87R, and F115Y compared to SEQ ID NO:4.

[0098] Human LAIR-2 has been shown to bind to collagen and SP-D with higher affinity than LAIR-1 (Meyaard, L., "The inhibitory collagen receptor LAIR-1 (CD305)", J. Leukoc. Biol., 83:799-803 (2008)). Dr. Linde Meyaard demonstrated that LAIR-2 also binds to C1q and mannose-binding lectin (MBL), both of which contain collagen-like domains (Olde Nordkamp et al., J. Innate Immun., 2014, 6(3):284-92). This finding supports evidence by Son et al. that LAIR-2 binds to C1q (Son et al., 2012, Proc. Natl. Acad. Sci. USA 109:E3160-3167). While collagen and C1q are ubiquitously expressed, SP-D is primarily restricted to mucosal surfaces (pulmonary alveolar surface and gastrointestinal tract), where it functions as a first line of innate defense against pathogens (Herias et al., 2007, Mol. Immunol. 44:3324-3332).

[0099] An exemplary alignment of the human LAIR-1 and human LAIR-2 extracellular domains is shown below: JPEG2025537248000002.jpg38159Query is sequence number 2 and Sbjct is sequence number 4.

[0100] iii.NC410 NC410 is a dimeric form of the LAIR-2 protein fused to the human Fc domain of the immunoglobulin (Ig) subtype IgG1. As shown in Figure 2, NC410 targets tumor collagen to reverse LAIR-1-mediated immunosuppression and induces ECM remodeling to promote immune cell infiltration and function in the TME.

[0101] Preclinical studies using NC410 in mouse models (HT-29, P815) have shown that it promotes T cell proliferation (both CD4+ and CD8+ cells) and increases IFN-γ and granzyme B production, resulting in a dose-dependent increase in antitumor efficacy. Because tumor-associated collagen induces CD8+ T cell exhaustion via LAIR-1-SHP-1 signaling, overexpression of LAIR-2, which inhibits LAIR-1 binding to collagen, reduced tumor growth in a lung tumor model. Furthermore, combining LAIR-2 overexpression with anti-PD-1 treatment significantly reduced proliferation and metastasis within one week of treatment, an effect that persisted throughout the treatment period (Peng, DH, et al., “Collagen promotes anti-PD-1 / PD-L1 resistance in cancer through LAIR1-dependent CD8(+) T cell exhaustion”, Nat Commun., 11:4520 (2020)). Furthermore, the combination of NC410 with anti-PD-1 inhibitors has consistently been shown to reduce tumor burden.

[0102] NC410 has been tested in various in vitro and in vivo systems to support its use as an investigational drug in oncology. These studies have demonstrated mechanistic studies and enhanced immune activity in tumor models. NC410 binds with high affinity to LAIR-1 ligands, including collagen, C1q, MBL, and SP-D, blocking the interaction of LAIR-1 with its ligands. NC410 reverses the inhibitory effects of collagen on lipopolysaccharide (LPS)-induced NFκB and interferon signaling, key signaling pathways leading to immune cell activation. NC410 promotes the activation and differentiation of primary monocytes into a stimulatory macrophage phenotype. NC410 dose-dependently enhances human T cell proliferation and activation, which correlates with antitumor efficacy in murine P815 and human HT29 tumor models and the production of chemokines such as CXCL10, CXCL11, and CXCL12. NC410 promotes T cell-dependent cytokine and chemokine production in the tumor microenvironment, which correlates with tumor control and promotes tumor remodeling as evidenced by altered levels of collagen degradation products.

[0103] A. Protein and Polypeptide Compositions The ECM-binding agent and ICI can be a protein, polypeptide, or fusion protein. For example, the ECM-binding agent and ICI can be an isolated or recombinant LAIR-2 or pembrolizumab protein or polypeptide, or a functional fragment, variant, or fusion protein thereof, as described above.

[0104] The protein or polypeptide, or its functional fragment, variant, or fusion protein, can be an agonist or antagonist. For example, in some embodiments, the LAIR-2 antagonist is an LAIR-1 or LAIR-2 polypeptide, or a fragment or fusion protein thereof, that binds to a ligand of LAIR-2. The polypeptide can be a soluble fragment, such as the extracellular domain of LAIR-2, or a functional fragment thereof, or a fusion protein thereof. In some embodiments, a soluble ligand of LAIR-2 can function as an agonist that increases signal transduction via LAIR-2.

[0105] The activity (i.e., agonist or antagonist) of an LAIR-2 protein or polypeptide, or any fragment, variant, or fusion protein thereof, can be determined using functional assays known in the art, including those discussed below. Typically, the assay involves determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through the LAIR-2 receptor. In some embodiments, the assay involves determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) an immune response (i.e., costimulation or co-inhibition) associated with LAIR-2. Typically, the assay involves determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through LAIR-2. In some embodiments, the assay involves determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof reduces (i.e., is an agonist) or increases (i.e., is an antagonist) an immune response that is negatively regulated by LAIR-2. In some embodiments, the assay involves determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., antagonizes) apoptosis and differentiation of acute myeloid leukemia and acute lymphoblastic leukemia cells, resulting in a decrease in the self-renewal capacity of AML and ALL stem cells.

[0106] Nucleic acid and polypeptide sequences of LAIR-1 and LAIR-2 are known in the art, and exemplary protein and peptide sequences are provided above. These sequences can be used by those skilled in the art to prepare any LAIR-1 or LAIR-2 protein or polypeptide, or any fragment, variant, or fusion protein thereof, as discussed in more detail below. Generally, LAIR-1 and LAIR-2 proteins, polypeptides, fragments, variants, and fusions thereof are expressed from nucleic acids containing sequences encoding a signal sequence. Typically, the signal sequence is cleaved from the immature polypeptide, generating a mature polypeptide lacking the signal sequence. The signal sequence can be replaced with the signal sequence of another polypeptide using standard molecular biology techniques, which can affect the expression level, secretion, solubility, or other properties of the polypeptide. Both LAIR-1 and LAIR-2 are disclosed with and without a signal sequence. It is understood that in some cases, the mature protein known or described in the art, i.e., the protein sequence without the signal sequence, is the putative mature protein. During normal cellular expression, the signal sequence can be removed by cellular peptidases to yield the mature protein, the sequence of which can be determined or confirmed using methods known in the art.

[0107] i. Fragment As used herein, a fragment of LAIR-1 or LAIR-2 refers to any subset of a polypeptide that is at least one amino acid shorter than the full-length protein. Useful fragments include those that retain the ability to bind to their natural ligand(s). A polypeptide that is a fragment of any full-length LAIR-1 or LAIR-2 has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or more than 100% of the ability to bind to the natural ligand, respectively, compared to the full-length protein.

[0108] Fragments of LAIR-1 and LAIR-2 include cell-free fragments. Cell-free polypeptides can be fragments of full-length transmembrane polypeptides that can be shed, secreted, or otherwise extracted from producing cells. Cell-free fragments of polypeptides include part or all of the extracellular domain of the polypeptide and may lack part or all of the intracellular and / or transmembrane domains of the full-length protein. In one embodiment, the polypeptide fragment includes the entire extracellular domain of the full-length protein. In other embodiments, cell-free fragments of polypeptides include fragments of the extracellular domain that retain the biological activity of the full-length protein. The extracellular domain can include 1, 2, 3, 4, or 5 consecutive amino acids from the transmembrane domain and / or 1, 2, 3, 4, or 5 consecutive amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5, or more amino acids removed from the C-terminus, N-terminus, or both. In some embodiments, the extracellular domain is the only functional domain of the fragment (e.g., the ligand-binding domain).

[0109] ii. Variants Variants of LAIR-1 and LAIR-2, as well as fragments thereof, are also provided. In some embodiments, the variants are at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any one of SEQ ID NOs: 2-5. Useful variants include those that increase biological activity or increase the half-life or stability of the protein, as demonstrated by any of the assays described herein. LAIR-1 or LAIR-2 proteins and polypeptides, as well as fragments, variants, and fusion proteins thereof, can be engineered to increase biological activity. For example, in some embodiments, an LAIR-2 polypeptide, protein, or fragment, variant, or fusion thereof is modified with at least one amino acid substitution, deletion, or insertion that increases its function.

[0110] Other variants are those that have been engineered to selectively bind to one or more types of LAIR-1 and / or LAIR-2 ligands relative to other LAIR-1 and / or LAIR-2 ligands. For example, variants can be engineered to preferentially bind to one or more of collagen, SP-D, C1q, or MBL, or a specific combination thereof. Preferential binding refers to binding of one type of ligand to another type of ligand that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0111] Still other variants can be engineered to have reduced binding to one ligand relative to another, and these variants can be used in combination with variants with stronger binding properties to modulate the immune response with moderate effect.

[0112] In still other embodiments, variants can be engineered to exhibit reduced binding to one or more collagen-binding sites compared to other embodiments. As discussed in Brondijk, et al., Blood, 18(115):1364-73 (2010), LAIR-1 residue mutations can have different effects on binding to different collagen ligands. For example, the R59A, E61A, R65A, and E111A mutants significantly reduced adhesion to immobilized collagens I, III, and IV, although the magnitude of the effect varied depending on the type of collagen tested. Furthermore, the R62A and N69A mutants exhibited some reduction in adhesion. In some embodiments, variants are mutated at one or more of R59, E61, R62, E63, R65, S66, Y68, N69, I102, R100, W109, E111, Q112, and Y115 relative to SEQ ID NO: 2. In some embodiments, the variant is mutated at one or more of R59, E61, R65, E111, R62A, and N69A. In certain embodiments, the mutation(s) is a substitution with alanine.

[0113] Finally, variant polypeptides can be engineered to have a longer half-life compared to the wild-type. These variants are usually modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. Variants can be modified to adjust the effect of receptor affinity on the half-life of proteins, polypeptides, fragments, or fusions thereof at serum and endosomal pH.

[0114] iii. Fusion proteins The fusion polypeptide has a first fusion partner comprising all or part of the polypeptide LAIR-1 or LAIR-2 fused to a second polypeptide either directly or via a linker peptide sequence fused to the second polypeptide. The fusion protein optionally includes a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker domain may be a separate domain or, alternatively, may be included within one of the other domains of the fusion protein (the first polypeptide or the second polypeptide). Similarly, the domain that functions to dimerize or multimerize the fusion protein may be a separate domain or, alternatively, may be included within one of the other domains of the fusion protein (the first polypeptide, the second polypeptide, or the peptide / polypeptide linker domain). In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0115] The fusion proteins disclosed herein are represented by Formula I: N-R1-R2-R3-C wherein "N" represents the N-terminus of the fusion protein and "C" represents the C-terminus of the fusion protein. In some embodiments, "R1" is an LAIR-1 or LAIR-2 polypeptide or protein, or a fragment or variant thereof, "R2" is an optional peptide / polypeptide linker domain, and "R3" is a second polypeptide. Alternatively, R3 may be an LAIR-1 or LAIR-2 polypeptide or protein, or a fragment or variant thereof, and R1 may be a second polypeptide. In some embodiments, the LAIR-1 or LAIR-2 polypeptide is an extracellular domain or a fragment thereof, such as an Ig-like C2 domain, or a region surrounded by disulfide bond-forming cysteines as described above.

[0116] Dimerization or multimerization can occur between two or more fusion proteins via dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers formed can be homodimers / homomultimers or heterodimers / heteromultimers.

[0117] In some embodiments, the fusion protein comprises the extracellular domain of LAIR-1 or LAIR-2, or a fragment or variant thereof, fused to an IgFc region. Recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain of the extracellular domain, or a fragment or variant thereof, to the Fc region of human IgG1, IgG2, IgG3, or IgG4, or mouse IgG2a, or other suitable Ig domain, as previously described (Chapoval, et al., Methods Mol. Med., 45:247-255 (2000)).

[0118] iv. Exemplary Fusion Proteins An exemplary fusion protein is provided below. The signal sequence is double-underlined, the LAIR-2 extracellular domain is single-underlined, and the Ig domain is italicized. The signal sequence is usually removed in the mature protein. Additionally, signal peptides from other polypeptides or organisms can be used (e.g., substituted) to enhance secretion of the fusion protein from the host during production.

[0119] In some embodiments, the human LAIR2-hIg fusion protein (hIgG1) (hLAIR2.hG1), with or without a signal sequence, MEWSWVFLFFLSVTTGVHSQEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 6), has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the amino acid sequence of

[0120] SEQ ID NO:6 without the signal sequence is QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAP DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 7) is.

[0121] The human LAIR2-hIg fusion protein (hIgG1) (hLAIR2.hG1) functions as a decoy for the LAIR-1 ligand, thereby acting as an antagonist of LAIR-1 signaling and can be used to treat cancer or infectious diseases.

[0122] In some embodiments, the human LAIR2.mIg fusion protein (mIgG2a) is MEWSWVFLFFLSVTTGVHSQEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAPEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG (SEQ ID NO: 8), has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the amino acid sequence of

[0123] SEQ ID NO:8 without the signal sequence is

[0124] QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAP EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG (SEQ ID NO: 9). The human LAIR2.mIg fusion protein (mIgG2a) can be used to generate antagonistic anti-LAIR2 (e.g., mAbs, or fragments thereof) that can be used to treat autoimmune diseases.

[0125] IV. Immune Checkpoint Inhibitors A. PD-1 antagonists In some embodiments, LAIR-2-Fc is co-administered with a PD-1 receptor antagonist. Programmed death-1 (PD-1) is a member of the CD28 family of receptors that, when induced on T cells, exerts a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the intensity and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated by reference in their entireties, and include compounds or agents that bind to and block the PD-1 ligand, preventing or inhibiting binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor, thereby preventing inhibitory signaling through the PD-1 receptor.

[0126] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without inducing inhibitory signaling and also binds to a ligand of the PD-1 receptor, reducing or preventing the ligand from inducing signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and cause transmission of inhibitory signals, fewer cells will be attenuated by the negative signals delivered by PD-1 signaling, and a more robust immune response may be achieved.

[0127] PD-1 signaling is thought to be driven by binding of PD-1 ligands (such as B7-H1 or B7-DC) to peptide antigens presented by the major histocompatibility complex (MHC) in close proximity (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0128] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or prevents PD-1 receptor signaling by binding to a ligand of PD-1 or by binding to PD-1 itself, particularly when co-ligation of PD-1 and TCR does not follow such binding, thereby leading to inhibitory signaling through the PD-1 receptor.

[0129] Other PD-1 antagonists contemplated by the methods of the present invention include antibodies that bind to PD-1 or a ligand of PD-1, and other antibodies.

[0130] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications, the contents of each of which are incorporated herein in their entirety: PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196), PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168), PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708), PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771), PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286), PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875), PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174), PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874), PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533), Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0131] Specific examples of anti-PD-1 antibodies include those described in Kosak, US 20070166281 (published July 19, 2007) at par. 42, i.e., human anti-PD-1 antibodies, which in some embodiments are administered at a dose of 3 mg / kg.

[0132] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following publications: PCT / US06 / 022423 (WO / 2006 / 133396, published December 14, 2006) PCT / US07 / 088851 (WO / 2008 / 083174, published July 10, 2008) US2006 / 0110383 (released May 25, 2006) A specific example of an anti-B7-H1 antibody is the described antibody, human anti-B7-H1 antibody (WO / 2007 / 005874, published Jan. 11, 2007).

[0133] Additional anti-PD-1 and anti-B7-H1 antibodies are disclosed in 2014 / 0044738, which is specifically incorporated by reference in its entirety.

[0134] For anti-B7-DC antibodies, see 7,411,051, 7,052,694, 7,390,888, and U.S. Published Application No. 2006 / 0099203.

[0135] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides (including homologs and variants thereof), active fragments of any of the foregoing, and fusion proteins incorporating any of the foregoing. In some embodiments, the fusion protein comprises a soluble portion of B7-DC linked to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0136] The PD-1 antagonist can also be a fragment of mammalian B7-H1, e.g., from a primate, e.g., mouse, or human, that binds to and blocks PD-1 but does not cause inhibitory signaling through PD-1. The fragment can also be part of a fusion protein, e.g., an Ig fusion protein.

[0137] Other useful polypeptide PD-1 antagonists include those that bind to the ligand of the PD-1 receptor. These include the PD-1 receptor protein, or a soluble fragment thereof, which can bind to a PD-1 ligand, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signaling. B7-H1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of the PD-1 protein containing mutations, such as the A99L mutation, that increase binding to the natural ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or a soluble fragment thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signaling, is also useful.

[0138] PD-1 and B7-H1 antisense nucleic acids (both DNA and RNA) and siRNA molecules can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 in T cells and the production of T cell ligands, such as B7-H1, PD-L1, and / or PD-L2. For example, siRNA (e.g., approximately 21 nucleotides in length, specific for the gene encoding PD-1 or the gene encoding the PD-1 ligand; these oligonucleotides are readily available commercially) complexed with a carrier such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009)) is readily taken up by cells expressing PD-1 and its ligand, reducing the expression of these receptors and ligands, thereby reducing inhibitory signaling in T cells and activating them.

[0139] B. Pembrolizumab Several monoclonal antibodies that inhibit the interaction of PD-1 with one or both of its ligands, PD-L1 and PD-L2, have been approved for the treatment of cancer. Pembrolizumab is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) with high binding specificity to the programmed cell death 1 (PD-1) receptor and inhibits its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor-blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an intravenous (IV) immunotherapy for advanced malignancies. Keytruda® (pembrolizumab) is indicated for the treatment of patients with various indications.

[0140] The present disclosure relates to compositions comprising immune checkpoint inhibitors (ICIs). As a non-limiting example, pembrolizumab is an ICI known to inhibit the PD-1 pathway. The pembrolizumab composition can be administered to a subject in need thereof in combination with one or more compositions capable of binding to components of the extracellular matrix (ECM) of the tumor microenvironment (TME), such as collagen. As a non-limiting example, an LAIR-2Fc fusion protein (e.g., NC410) can bind to collagen. The pembrolizumab composition can be administered simultaneously with one or more compositions or separately. The pembrolizumab composition can be administered according to various dosing and timing regimens described herein.

[0141] Over the past decade, several other checkpoint inhibitors have also attracted significant attention in cancer treatment due to their durability of response and improved survival rates. Several favorable factors have emerged as predictors of response to ICIs, including, but not limited to, the presence and activation status of tumor-infiltrating T cells, PD-L1 expression, or high tumor mutation burden (TMB). The presence of multiple neoantigens derived from highly mutated tumors leads to an increase in tumor-infiltrating T cells, which is favorable for ICI response. Similarly, mismatch repair-deficient (dMMR) tumors have a 10- to 100-fold increase in somatic mutations compared to pMMR (mismatch repair-deficient) tumors. Both dMMR colorectal cancer (CRC) and dMMR non-CRC tumors showed excellent responses to pembrolizumab, whereas pMMR CRC did not (Le, DT, et al., “PD-1 Blockade in Tumors with Mismatch-Repair Deficiency”, N Engl J Med., 372:2509-20 (2015)). Subsequently, the FDA approved pembrolizumab for unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) solid tumors that have progressed after prior therapy and have no satisfactory alternative treatment options. It is hypothesized that this favorable response to ICIs is due to the abundance of neoantigens associated with immune cell infiltration in the TME. In contrast, responses to ICIs in patients with MSS / MSI-L / pMMR tumors, who have a low mutational burden and lack an “immune-competent” TME, have not met expectations. Therefore, there remains an unmet medical need to explore new strategies to enhance the response to ICIs in MSS / MSI-L tumors.

[0142] Furthermore, only 30% of patients with certain types of advanced cancer benefit from ICI monotherapy or combination therapy with chemotherapy or other agents, and the majority lose response due to various resistance mechanisms, including the emergence of T-regs, myeloid-derived suppressor cells (MDSCs), M2 macrophages, TGF-β-driven collagens that promote anti-PD-1 / PD-L1 resistance, and LAIR-1-mediated immunosuppression, including prevention of CD8+ and CD4+ T cell proliferation and activation.

[0143] V. Therapeutic composition A. Dosage Regimen The following compositions should be understood as exemplary compositions related to the present disclosure, and as such are not intended to limit the scope of the present disclosure.

[0144] The compositions described herein can be administered to a subject in need thereof, either alone or in combination with pharmaceutically acceptable excipients and / or carriers, in an amount sufficient to induce an appropriate anti-tumor response. Administration can include injection, infusion, other methods disclosed herein, and other methods known in the art. Administration can include, but is not limited to, intravenous, intramuscular, subcutaneous, and the like. Responses include, but are not limited to, specific immune responses, non-specific immune responses, both specific and non-specific responses, innate immune responses, primary immune responses, adaptive immune responses, secondary immune responses, memory immune responses, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.

[0145] The present invention provides a method for providing anti-tumor immunity to a mammal by administering an effective amount of a combination therapy to the mammal. The combination therapy described herein includes a first component of an immune checkpoint inhibitor (ICI), and a second component of the combination therapy exhibits affinity for binding components of the extracellular matrix (ECM) in the tumor microenvironment (TME). The effective amount of the combination therapy can be determined by one skilled in the art, taking into account individual differences in the patient's (i.e., subject's) age, weight, tumor size, extent of infection or metastasis, and pathological condition. In general, it can be stated that the components of the combination therapy can be administered simultaneously or separately according to the same or different dosing and timing regimens described herein. The combination therapy may also be administered multiple times at these dosages. The combination therapy can be administered using injection techniques commonly known in immunotherapy (Rosenberg, et al., New Eng. J. of Med., 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by a medical professional by monitoring the patient's symptoms and adjusting treatment accordingly. The effective amount for a particular patient may vary depending on factors such as the condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects. Guidance regarding treatment and diagnostic methods is available (Maynard, et al., Interpharm Press, 1996; Dent, Urch Publ., 2001).

[0146] The effective amount of the composition described herein can be given in a single dose, but is not limited to a single dose. Thus, the administration of the composition can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In the method of the present invention, when multiple administrations are performed, the administrations can be performed at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes, or at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, etc. In relation to time, the term "about" refers to any time interval within plus or minus 30 minutes. Administrations can also be spaced apart by time intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The present disclosure is not limited to administration intervals that are evenly spaced in time and encompasses non-equally spaced administrations, such as, by way of non-limiting example, a priming schedule consisting of administrations every 1 day, 4 days, 7 days, and 25 days. In such embodiments, the various compositions can be administered using different dosing and interval regimens. In such embodiments, a first composition can be administered in one or more doses spaced apart by a certain time interval, while a second composition can be administered in a different number of doses spaced apart by different time intervals. In such embodiments, the first and second compositions can be different in composition.

[0147] The compositions of the present invention can be administered in doses or dosages, each dose comprising about 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, etc. The compositions of the present invention can be administered in doses or dosages, with each dose depending on the subject's body weight. As a non-limiting example, a dose or dosage can be administered at about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12 mg / kg, etc. The various compositions disclosed herein can be administered in different dosages. As a non-limiting example, a first composition can be administered at one dosage and a second composition can be administered at a different dosage.

[0148] For the compositions disclosed herein, dosing schedules such as once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, and once every six weeks are available. Dosing schedules include, for example, dosing for a total period of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months. In some examples, two different dosing schedules may have a similar benefit-risk profile. As a non-limiting example, a dosing schedule of 400 mg every six weeks may have a similar benefit-risk profile as a subject receiving a dosing schedule of 200 mg every three weeks.

[0149] Cycles of the above-mentioned dosing schedules are provided. The cycles can be repeated, for example, approximately every 7 days, approximately every 14 days, approximately every 21 days, approximately every 28 days, approximately every 35 days, approximately every 42 days, approximately every 49 days, approximately every 56 days, approximately every 63 days, approximately every 70 days, etc. Non-dosing intervals can occur between cycles, where the intervals can be, for example, about 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 70 days, etc. The dosing schedules of the present disclosure can be related to cycles. The dosing schedules of the compositions disclosed herein can be designed so that doses are given on specific days of the cycle. As a non-limiting example, doses can be administered on days 1, 15, and 29 of a repeated 42-day cycle. As a non-limiting example, the cycle can be repeated until the subject shows adverse side effects to the dose. The cycle can further be repeated until the subject is sufficiently cured of the disease. In this context, the term "about" means plus or minus 1 day, plus or minus 2 days, plus or minus 3 days, plus or minus 4 days, plus or minus 5 days, plus or minus 6 days, or plus or minus 7 days.

[0150] Methods for co-administration with additional therapeutic agents are well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA).

[0151] An effective amount of a therapeutic agent is an amount that reduces or alleviates symptoms, usually by at least 10%, more usually by at least 20%, most usually by at least 30%, typically by at least 40%, more typically by at least 50%, most typically by at least 60%, often by at least 70%, more often by at least 80%, most often by at least 90%, conveniently by at least 95%, more conveniently by at least 99%, and most conveniently by at least 99.9%.

[0152] The dose or dosage of the compositions disclosed herein may be modified. Dosage and timing regimens may be modified according to factors known in the art. As a non-limiting example, administration of a dosage of NC410 in combination with pembrolizumab may be delayed until any observed toxicity resolves. If the participant does not have a medical condition or other circumstances that make them unsuitable for further treatment, administration may be resumed.

[0153] In some embodiments, administration of a composition disclosed herein should not occur after a certain time has elapsed. As a non-limiting example, administration of NC410 should typically not be delayed more than 28 days between consecutive doses, although this may vary. If a dose of NC410 or any other composition disclosed herein is omitted, the subject in need thereof may maintain their original treatment schedule and administer NC410 or any other composition disclosed herein at the next scheduled dose.

[0154] Formulations of the therapeutic agent can be prepared for storage by mixing with physiologically acceptable carriers, excipients, or stabilizers, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension.

[0155] While certain embodiments have been disclosed in the foregoing specification, it will be appreciated that many modifications and other embodiments to which the present disclosure pertains will come to mind to one skilled in the art having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is therefore understood that the present disclosure is not limited to the particular embodiments disclosed above, but that many modifications and other embodiments are intended to be included within the scope of any claims that can recite the disclosed subject matter.

[0156] B. Oral Formulations In embodiments, the combination therapy and related compositions are formulated for oral delivery. Oral delivery may include a single solid or liquid dosage form, as described below, containing multiple compositions (e.g., one composition of immune checkpoint inhibitor and a second composition of extracellular matrix binding component), or one or more separate solid or liquid dosage forms. Dosage forms formulated for oral administration may be administered according to the dosing and timing regimens described herein. Oral solid dosage forms are generally described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co., Easton Pa. 18042). Solid dosage forms include tablets, capsules, pills, troches, or lozenges, cachets, pellets, powders, or granules, or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, or liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed substances. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which is incorporated herein by reference. The composition may be prepared in liquid form or in dry powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the composition. Liposomal encapsulation may be used, and liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.T. Rhodes Chapter 10, 1979. Generally, the formulation includes a peptide (or a chemically modified form thereof) and an inactive ingredient that protects the peptide in the stomach environment and releases the biologically active substance in the intestine.

[0157] Drugs can be chemically modified to allow for effective oral delivery of derivatives. Generally, the intended chemical modification is to attach at least one moiety to the component molecule itself, which allows for uptake from the stomach or intestine into the bloodstream, or direct uptake into the intestinal mucosa. It is also desirable to increase the overall stability of the component(s) and increase their circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical applications. Other components that can be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-tioxocane (see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs, Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4:185-189).

[0158] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain adjuvants such as inert diluents, wetting agents, emulsifying agents, suspending agents, and other ingredients including sweetening, flavoring, and perfuming agents.

[0159] Controlled-release oral formulations may be desirable. The drug can be incorporated into an inert matrix, such as a gum, that allows release by diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Another form of controlled release is based on the Oros Therapeutic System (Alza Corp.), in which the drug is encased in a semipermeable membrane, allowing water to enter through a single small opening and push the drug out by osmotic effects.

[0160] For oral formulations, the release location can be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. In some embodiments, release avoids the deleterious effects of the gastric environment by protecting the drug (or derivative) or by releasing the drug (or derivative) beyond the gastric environment into the intestine. To ensure complete gastric resistance, a coating that is impermeable to at least a pH of 5.0 is essential. Examples of more common inactive ingredients used as enteric coatings include cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may also be used as mixed films.

[0161] C. Manufacturing method i. Antibody production method Antibodies can be produced in cell culture, phage, or in a variety of animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Thus, in one embodiment, the antibody is a mammalian antibody. Phage technology can be used to isolate the original antibody or to generate variants with altered specificity or binding characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting a host cell with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect DNA sequences expressing at least one VL region and one VH region into the host cell. Examples describing recombinant means for antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, most recent edition).

[0162] The disclosed antibodies can be modified by recombinant means to further enhance the effectiveness of the antibodies in mediating the desired function. Therefore, it is within the scope of the present invention that antibodies can be modified by substitution using recombinant means. Typically, the substitutions are conservative. For example, at least one amino acid in the antibody constant region can be replaced with a different residue. See, for example, U.S. Pat. Nos. 5,624,821, 6,194,551, WO9958572, and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such modifications are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Antibodies are often labeled by covalent or noncovalent attachment of a substance that produces a detectable signal. A wide variety of labeling and conjugation techniques are known and are widely reported in both the scientific and patent literature. These antibodies can be screened for binding to LAIR-1, LAIR-2, or pembrolizumab proteins, polypeptides, or fusion proteins. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0163] For example, suitable antibodies with desired biological activity can be identified using in vitro assays, including, but not limited to, in vivo assays such as proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and tumor growth inhibition. The antibodies provided herein may also be useful for diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind to a specific antigen without inhibiting the antigen's receptor binding or biological activity. As neutralizing antibodies, they may be useful in competitive binding assays.

[0164] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulins (i.e., intact antibodies) of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domain differs in sequence from antibody to antibody and is responsible for the binding and specificity of each antibody for a particular antigen. However, variability is not typically evenly distributed throughout the variable domain of an antibody. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). Natural heavy and light chain variable domains each consist of four FR regions, primarily adopting a beta-sheet configuration and connected by three CDRs, which form loops that connect to and, in some cases, form part of the beta-sheet structure. The CDRs within each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0165] Also disclosed are biologically active antibody fragments, which include insertions, deletions, substitutions, or other selected modifications of particular regions or particular amino acid residues, whether or not linked to other sequences, but which do not significantly alter or impair the activity of the fragment compared to the unaltered antibody or antibody fragment.

[0166] Techniques can also be adapted for the production of single-chain antibodies specific to antigenic peptides. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be created by fusing heavy and light chain variable domains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of the other variable domain via a 15-25 amino acid peptide or linker without significantly impairing antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in the appropriate conformation.

[0167] Bivalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by generating a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. ScFvs are sometimes designed with a linker peptide (approximately five amino acids) that is too short for the two variable regions to fold together, forcing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than their scFv counterparts, meaning they have much higher affinity for their targets. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies.

[0168] Monoclonal antibodies are obtained from substantially homogeneous antibody populations, i.e., the individual antibodies within the population are identical except for naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, so long as the desired antagonistic activity is exhibited, as well as fragments of such antibodies.

[0169] Monoclonal antibodies can be produced using any procedure that produces monoclonal antibodies. In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0170] Antibodies can also be produced using recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

[0171] Methods for producing antibodies using protein chemistry are also known in the art. One method for producing proteins, including antibodies, is to link two or more peptides or polypeptides using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art will readily understand that peptides or polypeptides corresponding to antibodies can be synthesized, for example, by standard chemical reactions. For example, a peptide or polypeptide can be synthesized but not cleaved from its synthesis resin, whereas another fragment of the antibody can be synthesized and then cleaved from the resin, thereby exposing a functionally blocked terminal group on the other fragment. These two fragments can be covalently linked at their carboxyl and amino termini via peptide bonds by a peptide condensation reaction to form an antibody or fragment thereof. Alternatively, the peptides or polypeptides can be independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides can be linked to form antibodies or antigen-binding fragments thereof through similar peptide condensation reactions.

[0172] For example, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to generate larger peptide fragments, polypeptides, or entire protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct larger peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-α-thioester with another unprotected peptide segment containing an amino-terminal Cys residue, yielding a thioester-linked intermediate as the initial conjugation product. Without changing the reaction conditions, this intermediate undergoes a simultaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.

[0173] ii. Protein production method The disclosed proteins, polypeptides, fragments, variants, and fusions thereof can be produced using conventional techniques known in the art. Isolated fusion proteins can be obtained, for example, by chemical synthesis or by recombinant production in a host cell. To recombinantly produce a protein, polypeptide, fragment, variant, or fusion thereof, a nucleic acid comprising a nucleotide sequence encoding the protein, polypeptide, fragment, variant, or fusion thereof can be used to transform, transduce, or transfect bacterial or eukaryotic host cells (e.g., insect, yeast, or mammalian cells). Generally, a nucleic acid construct comprises a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, fragment, variant, or fusion thereof. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product but instead affect the expression of nucleic acid sequences to which they are operably linked.

[0174] Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art and include, for example, strains of E. coli such as BL-21, and cultured mammalian cells such as CHO cells.

[0175] In eukaryotic host cells, several viral-based expression systems can be utilized to express fusion proteins. Viral-based expression systems are well known in the art and include, but are not limited to, baculovirus, SV40, retrovirus, or vaccinia-based viral vectors.

[0176] Mammalian cell lines stably expressing proteins, polypeptides, fragments, variants, or fusions thereof can be produced using expression vectors containing appropriate regulatory elements and selectable markers. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expressing proteins, polypeptides, fragments, variants, or fusions thereof in Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVECs), among others. Additional suitable expression systems include the GS Gene Expression System™ available from Lonza Group Ltd.

[0177] After introducing the expression vector by electroporation, lipofection, calcium phosphate, calcium chloride co-precipitation, DEAE-dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by metabolic selection or antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured so that the polypeptide of interest is expressed, and the polypeptide can be recovered, for example, from the cell culture supernatant or from lysed cells. Alternatively, proteins, polypeptides, fragments, variants, or fusions thereof can be produced by (a) ligating the amplified sequence into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) in vitro transcription and translation using wheat germ extract or rabbit reticulocyte lysate.

[0178] Proteins, polypeptides, fragments, variants, or fusions thereof can be isolated using chromatographic methods, such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. In some embodiments, proteins, polypeptides, fragments, variants, or fusions thereof can be engineered to contain additional domains containing amino acid sequences that enable the polypeptide to be captured on an affinity matrix. For example, Fc-fusion polypeptides in cell culture supernatants or cytoplasmic extracts can be isolated using a Protein A column. Additionally, tags such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted at any position within the polypeptide, including either the carboxyl or amino terminus. Other fusions that may be useful include enzymes that aid in polypeptide detection, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides. Fusion proteins can further be engineered to contain a secretion signal that enables the protein, polypeptide, fragment, variant, or fusion thereof to be secreted by the cell in which it is produced (if a secretion signal is not already present). The secreted protein, polypeptide, fragment, variant, or fusion thereof can then be conveniently isolated from the cell culture medium.

[0179] iii. Methods for Producing Isolated Nucleic Acid Molecules Isolated nucleic acid molecules can be produced by standard techniques, including, but not limited to, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) can be used to obtain isolated nucleic acids encoding variant polypeptides. PCR is a technique for enzymatically amplifying target nucleic acids. Typically, sequence information from the ends of the target region or beyond can be used to design oligonucleotide primers identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA and RNA, including sequences derived from total genomic DNA or total cellular RNA. Primers are typically 14-40 nucleotides in length, but can range from 10 nucleotides to several hundred nucleotides in length. General PCR techniques are described, for example, in *PCR Primer: A Laboratory Manual*, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When RNA is used as the template source, a complementary DNA (cDNA) strand can be synthesized using reverse transcriptase. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification can also be used to obtain isolated nucleic acids. See, e.g., Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

[0180] Isolated nucleic acids can be chemically synthesized as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated 3' to 5' DNA synthesis). For example, one or more long oligonucleotide pairs (e.g., more than 100 nucleotides) containing the desired sequence can be synthesized, each pair containing a short complementary segment (e.g., about 15 nucleotides) so that a duplex forms when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in one double-stranded nucleic acid molecule per oligonucleotide pair, which can then be ligated into a vector. Isolated nucleic acids can also be obtained by mutagenesis. Protein-encoding nucleic acids can be mutated using standard techniques, such as oligonucleotide-directed mutagenesis and / or PCR-mediated site-directed mutagenesis. See Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.

[0181] D. Assays and Antibody Screening The production of LAIR-2Fc fusion proteins ("LAIR-2-Fc") for cancer therapy avoids the need for the development and screening of LAIR-1 mAbs. Because LAIR-2 has a higher affinity than LAIR-1, in some embodiments, LAIR-2-Fc is chosen over LAIR-1Fc fusion proteins ("LAIR-1-Fc") as a therapeutic treatment. In some embodiments, LAIR-1-Fc can be utilized in mouse preclinical models because LAIR-2 is not present in mice.

[0182] a. LAIR-2-Fc assay 1. Confirmation of the ability to bind to multiple forms of collagen, SP-D, C1q, and MBL by ELISA. 2. Confirmation of the ability of LAIR-2-Fc to inhibit the binding of multiple collagens, SP-D, and C1q to LAIR-1. This can be tested by 1) an ELISA competition assay and 2) flow cytometry using LAIR-1-transfected cells incubated in the presence of titrated amounts of LAIR-2-Fc and fluorescently labeled LAIR ligand. 3. Analysis of the binding affinity of LAIR-2-Fc to its ligands compared to LAIR-1. 4. Functional assays to confirm that LAIR-2-Fc prevents signaling by LAIR-1-expressing cells. Reporter cells may be utilized for these assays, or primary LAIR-1+ cells are another option.

[0183] E. How to use Current evidence illustrates the inhibitory role of the LAIR-1 cell surface receptor, suggesting that LAIR-2 indirectly antagonizes LAIR-1 function by binding to the same ligand as LAIR-1, thus essentially functioning as a decoy receptor. The tumor microenvironment is often enriched in extracellular matrix proteins (ECM), including the LAIR-1 ligand collagen (Rygiel et al., 2011, Mol. Immunol. 49:402-406). Therefore, LAIR-1-expressing cells localized in the tumor microenvironment may be particularly suppressed through LAIR-1 collagen cross-linking and subsequent inhibitory signaling. Increased LAIR-1 expression and signaling have been shown to inhibit the proliferation, differentiation, and function of several immune cell subsets, and thus are thought to suppress antitumor immunity, particularly in tumor microenvironments with high levels of the LAIR-1 ligands collagen, C1q, and SP-D.

[0184] Both collagen and C1q have been shown to limit or alter LAIR-1-mediated differentiation and activation of antigen-presenting cells (monocytes, macrophages, and dendritic cells (DCs)). LAIR-1 is also known to be expressed on NK and T cells, but at much lower levels than on APCs. However, studies have shown that cross-linking of LAIR-1 on NK and T cells can inhibit their proliferation and function. Therefore, reducing LAIR-1 cross-linking is thought to enhance immune responses to cancer and infectious diseases. Increasing levels of LAIR-2 is thought to promote antitumor immunity through the same mechanism. Therefore, soluble LAIR-1 and LAIR-2, including LAIR-1 and LAIR-2 polypeptides and LAIR-1 and LAIR-2 fusion proteins, can be used to treat human diseases. For example, LAIR-2Fc protein can be used in cancer immunotherapy to enhance immune function by preventing ligand binding to LAIR-1. This strategy is particularly promising because LAIR-2 binds to its ligand with higher affinity than LAIR-1.

[0185] Alternatively, LAIR-1-mediated signaling in AML cancer cells expressing high levels of LAIR-1 inhibits apoptosis and differentiation through the intrinsic LAIR-1-SHP-1-CAMK1-CREB pathway, maintaining the self-renewal capacity, or "stemness," of AML cells (Kang et al., 2015, Nat. Cell Biol. 17:665-677). In these cancers, reduced LAIR-1 signaling leads to AML cell death. Therefore, blocking (i.e., antagonizing) LAIR-1 signaling in leukemia may be a therapeutic approach to eradicate leukemia. Therefore, blocking LAIR-1 signaling with LAIR-1 monoclonal antibodies or soluble LAIR-1 and soluble LAIR-2 (including LAIR-1 and LAIR-2 polypeptides and LAIR-1 and LAIR-2 fusion proteins) may be used to treat leukemia by directly inhibiting cancer cell survival and promoting antitumor immune responses.

[0186] Conversely, reduced expression or function of LAIR-1 is associated with several autoimmune conditions, while overexpression of LAIR-2 can promote autoimmunity through decoy binding of LAIR-1 ligands. LAIR-2 binding of LAIR-1 ligands essentially reduces cell surface cross-linking of LAIR-1, limiting inhibitory signaling pathways that lead to overreactive immune function. Therefore, increasing LAIR-1 cross-linking is thought to reduce excessive or inappropriate immune responses, for example, in cases of autoimmune disease or inflammation. For example, blocking LAIR-2 with mAbs can be used to treat autoimmune diseases because it increases ligand binding to LAIR-1 and thus downregulates the immune response. Targeting LAIR-2 is particularly effective in diseases where there is an imbalance between cell surface LAIR-1 and soluble LAIR-2 expression, as shown in rheumatoid arthritis (Lebbink et al., 2008, J. Immunol 180:1662-1669).

[0187] Exemplary methods are described in further detail below.

[0188] i. Treatment strategies Methods for inducing or enhancing an immune response in a subject are provided. Typically, the methods involve administering to the subject an effective amount of an immunomodulatory agent or cells primed ex vivo with an immunomodulatory agent. The immune response can be, for example, a primary immune response to an antigen, or an improvement in effector cell function, such as increasing antigen-specific proliferation of T cells, enhancing cytokine production by T cells, stimulating differentiation, or a combination thereof. In some embodiments, the agent can increase the development of naive T cells into Th1, Th17, Th22, or other cells that secrete, or cause other cells to secrete, proinflammatory molecules, including, but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In some embodiments, the agent can reduce or inhibit the activity of Tregs, reduce the production of cytokines such as IL-10 from Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the proportion of Tregs within an immune cell population, or reduce the survival of Tregs. The immunomodulatory agent can be administered to a subject in need thereof in an amount effective to overcome T cell exhaustion and / or T cell anergy. Overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.

[0189] This method can be used in vivo or ex vivo for therapeutic purposes to stimulate an immune response. Thus, in some embodiments, the agent or a nucleic acid encoding the agent is administered directly to a subject. Thus, in some embodiments, the agent or a nucleic acid encoding the agent is contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to a subject (e.g., adoptive transfer). Generally, the disclosed immunomodulatory agents can be used to treat subjects suffering from or predisposed to any disease or disorder to which the subject's immune system mounts an immune response. These agents can enable a more robust immune response. The disclosed compositions are useful for stimulating or enhancing T cell-mediated immune responses.

[0190] Immunomodulatory agents utilized to increase immune responses typically reduce LAIR-1 expression, ligand binding, cross-linking, negative signaling, or a combination thereof. For example, the agent may be an LAIR-1 antagonist, such as an antagonist (blocking) anti-LAIR-1 antibody or an antigen-binding fragment thereof. In some embodiments, the antagonist binds to the LAIR-1 collagen-binding domain (see, e.g., Brondijk, et al., Blood, 18(115):1364-73 (2010), and Zhou, et al., Blood, 127(5):529-537 (2016), and supplementary information thereto, which are specifically incorporated by reference in their entireties). In some embodiments, the LAIR-1 antagonist, such as a function-blocking antibody or functional fragment thereof, specifically binds to an epitope comprising one or more of R59, E61, R62, E63, R65, S66, Y68, N69, I102, R100, W109, E111, Q112, and Y115 of LAIR-1 (e.g., relative to SEQ ID NO: 1). The agent can also be an LAIR-1 polypeptide, such as a soluble polypeptide, or a fusion protein thereof that can function as a decoy receptor for one or more LAIR-1 ligands. The agent can also be LAIR-2, or a functional fragment or fusion protein thereof, that can function as a decoy receptor for one or more LAIR-1 ligands.

[0191] For example, in some embodiments, an effective amount of an LAIR-2 fusion protein, e.g., LAIR-2-Fc, is administered to a subject with cancer or an infectious disease. Treating a patient with LAIR-2-Fc reduces cross-linking of LAIR-1 receptors, subsequently reducing inhibitory signaling in LAIR-1+ cells and improving immune function. In particular, in tumor microenvironments where LAIR-1 / 2 ligand expression is highly expressed, increasing the ratio of soluble LAIR-2 levels relative to cell surface LAIR-1 favors enhanced anti-tumor immunity.

[0192] Tumor microenvironments with immune infiltrates that express high levels of both collagen, SP-D, and / or C1q and high levels of LAIR-1 are ideal for the disclosed immunotherapies (e.g., LAIR-2-Fc immunotherapy). Ovarian cancer has high levels of collagen, but it is unclear whether it also has high levels of SP-D and C1q. On the other hand, lung and GI cancers may have high levels of both collagen and SP-D and therefore may be cancers that can be targeted with LAIR-2-Fc. In other embodiments, soluble LAIR-2, soluble LAIR-1, or an LAIR-1 fusion protein (e.g., LAIR-1-Fc) is utilized. In some embodiments, LAIR-2-based molecules may be selected because LAIR-2 binds to its ligand with higher affinity than LAIR-1.

[0193] Blockade of LAIR-1, e.g., the use of function-blocking anti-LAIR-1 antibodies, can be an alternative or complement to soluble LAIR-1 and LAIR-2 polypeptides and fusion proteins. For example, in some embodiments, an LAIR-1 blocker is combined with a decoy receptor, such as soluble LAIR-1 or LAIR-2, or a fusion protein thereof. Combination treatments (e.g., LAIR-2-Fc and LAIR-1 blockade) can be complementary.

[0194] In some embodiments, immune response-stimulating therapy (e.g., in the treatment of cancer or infectious diseases) involves depletion of LAIR-1+ cells. LAIR-1 is highly expressed in mouse and human ovarian cancer ascites. LAIR-1 upregulation is restricted to immunoregulatory macrophages and F4 / 80+ DCs, both of which co-express high levels of PD-L1. Therefore, targeted depletion of LAIR-1-expressing cells should improve the overall state of the tumor microenvironment by removing immunoregulatory populations. Although LAIR-1 expression has not been observed in other cell subsets in ovarian cancer, because LAIR-1 is broadly inhibitory, depletion of other LAIR-1+ cells should also have the effect of reducing immune inhibition and improving anti-tumor immunity. LAIR-1 has also been shown to be expressed on the surface of acute myeloid leukemia tumors and is essential for acute myeloid leukemia development (Kang et al., Nature Cell Biology, Vol. 17, No. 5, 2015; pp. 665-679). Therefore, reducing hematopoietic ("blood") cancers with mAbs that reduce LAIR-1 should have the direct effect of reducing or eradicating LAIR-1-positive cancers.

[0195] ii. Cancer treatment The disclosed compositions and methods can be used to treat cancer. Generally, these agents are used to stimulate or enhance an immune response against cancer in a subject by administering to the subject an amount of an immunomodulatory agent that reduces LAIR-1 expression, ligand binding, cross-linking, negative signaling, or a combination thereof in combination with an immune checkpoint inhibitor (ICI). Administration includes injection, infusion, and other such known administration methods according to the dosage and timing regimens disclosed herein. This method can alleviate one or more symptoms of cancer.

[0196] The immune system has been demonstrated to be a defense against cancer initiation and growth. Regulation of the immune response is governed by cell surface interactions that direct immune cell function along specific pathways, including activation or inhibition against cancer cells. LAIR-1 is an inhibitory receptor on the surface of several immune cell (leukocyte) subsets, preventing optimal immune responses. Meanwhile, LAIR-2 is a soluble homolog that functions as a decoy to block LAIR-1-mediated inhibition. Pembrolizumab is an ICI that interacts with the PD-1 pathway.

[0197] In one embodiment, the LAIR-2Fc fusion protein promotes immune responses in vitro and in vivo. In another embodiment, LAIR-2Fc reduces tumor growth and promotes survival. In yet another embodiment, LAIR-2Fc promotes anti-PD-1 immunotherapy in combination with an ICI (e.g., pembrolizumab). The data provided herein demonstrate that LAIR-1 mAb has in vitro activity in human T cell and myeloid cell lines and exhibits specific agonist and antagonist activity for specific mAb clones. These findings demonstrate the potential modulation of the LAIR-1 pathway by LAIR-2Fc or LAIR-1 mAb for immunotherapeutic intervention in cancer and other diseases.

[0198] In another embodiment, LAIR-2Fc increases the responsiveness of primary human T cells to TCR stimulation. In another embodiment, LAIR-2Fc increases antigen-specific T cell responses in vivo.

[0199] Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through a variety of mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, limitless expressive potential, and persistent angiogenesis. The term "cancer cell" is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor that remains localized. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and metastasized to distant sites. In still other embodiments, cancer is associated with a specific cancer antigen (e.g., pan-cancer antigen (KS1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0200] The methods and compositions disclosed herein are useful for the treatment or prevention of various cancers or other abnormal proliferative disorders, including, but not limited to, carcinomas, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, squamous cell carcinoma; lymphoid hematopoietic tumors, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkett's lymphoma; acute and chronic Hematopoietic tumors of the myeloid lineage, including myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, 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, subcutaneous pigmented macules, hyperkeratosis gracilis, seminoma, thyroid follicular carcinoma, and teratocarcinoma.

[0201] Cancers caused by abnormalities in apoptosis can also be treated by the methods and compositions of the present disclosure. Such cancers include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovaries, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In certain embodiments, malignant tumors or dysproliferative changes (e.g., metaplasia and dysplasia) or hyperproliferative disorders in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus are treated or prevented by the methods and compositions. In other specific embodiments, sarcoma, melanoma, or leukemia are treated or prevented by the methods and compositions.

[0202] The disclosed compositions and methods are particularly useful for treating cancers associated with cells expressing abnormally high levels of LAIR-1, high levels of LAIR-1 ligand, low levels of LAIR-2, or a combination thereof.

[0203] Specific cancers and related diseases that can be treated or prevented by the methods and compositions disclosed herein include acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, and myelodysplastic syndromes), chronic leukemia (including chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia); polycythemia vera; lymphomas, such as Hodgkin's disease or non-Hodgkin's disease lymphomas (including, for example, diffuse anaplastic lymphoma kinase (ALK)-negative large B-cell lymphoma (DLBCL)). ;Diffuse anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML);Multiple myeloma including smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma;Waldenstrom's macroglobulinemia;Monoclonal gammopathy of undetermined significance;Benign monoclonal gammopathy;Heavy chain disease;Osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, and vascular Bone and connective tissue sarcomas, such as sarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors, including but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary cerebral lymphoma; breast cancer (including but not limited to adenocarcinoma, lobular carcinoma, intraductal carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, papillary carcinoma, Paget's disease, and inflammatory breast cancer), adrenal carcinoma (including but not limited to pheochromocytoma and adrenocortical carcinoma), thyroid cancer (including but not limited to papillary or follicular carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma); pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, carcinoid or islet cell tumors, pituitary carcinoma, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, diabetes insipidus; eye cancer, including but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, ciliary body melanoma, and retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma;vulvar cancer (including but not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease); cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial cancer and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancer (including squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, uterine cancer ... cancer of the liver, including but not limited to hepatocellular carcinoma and hepatoblastoma; cancer of the gallbladder, including but not limited to adenocarcinoma, papillary carcinoma, nodular carcinoma, and diffuse carcinoma; gastric cancer, including but not limited to adenocarcinoma, neoplasia (polypoid), ulcerative colitis, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, including but not limited to adenocarcinoma, papillary carcinoma, nodular carcinoma, and diffuse carcinoma Lung cancer, including but not limited to cholangiocarcinoma, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancer (including but not limited to germinoma, seminoma, undifferentiated carcinoma, typical carcinoma, spermatocytic carcinoma, non-seminoma, embryonal carcinoma, teratoma carcinoma, and choriocarcinoma (yolk sac tumor)), prostate cancer (including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma); penile cancer; oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma salivary gland carcinoma, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal carcinoma, including but not limited to squamous cell carcinoma and verrucous carcinoma; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, and acral lentigo melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal nephroma, fibrosarcoma, transitional cell carcinoma (of the renal pelvis and / or uterus); Wilms' tumor;Included are, but are not limited to, bladder cancer, including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for reviews, see Fishman et al., 1985, Medicine, 2nd Ed., J.B. Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America).

[0204] It should be emphasized that the above-described aspects are merely possible examples presented for a clear understanding of the principles of the present invention. The process descriptions and blocks in the flow diagrams should be understood as representing modules, segments, or code portions containing one or more executable instructions for implementing specific logical functions or steps in the process. Alternative implementations, including those in the art of the present disclosure, in which no functions are included or performed at all, in reverse order from that shown or described, or substantially simultaneously or in reverse order, are also included, depending on the functionality involved, as would be understood by one skilled in the art of the present disclosure. Many variations and modifications can be made to the above-described aspect(s) without substantially departing from the spirit and principles of the present disclosure. Furthermore, the scope of the present disclosure is intended to include any and all combinations and subcombinations of all elements, features, and aspects discussed above. All such modifications and variations are intended to be included herein within the scope of the present disclosure, and all possible claims to individual aspects or combinations of elements or steps are intended to be supported by the present disclosure. [Example]

[0205] Example 1. Safety and tolerability study of NC410 in combination with pembrolizumab.

[0206] method: Safety and tolerability will be assessed by monitoring the frequency, duration, and severity of adverse events (AEs). Toxicity assessment will be performed using NCICTCAE v5.0. This study will be divided into two phases, Phase 1b and Phase 2, as described below. Phase 1b of this study will enroll patients with advanced unresectable and / or metastatic solid tumors (e.g., colorectal cancer, gastric cancer including the gastroesophageal junction, esophageal cancer, ovarian cancer, and head and neck cancer) with or without prior immune checkpoint inhibitor (ICI) therapy or microsatellite stability / microsatellite instability (MSS / MSI), as shown in Figure 3. Participants will include both men and women aged 18 years or older. Participants must provide written informed consent and have adequate organ function.

[0207] Participants must have measurable disease based on RECIST v1.1 and consent to non-target lesion biopsies before and during treatment. RECIST v1.1 was adapted to take into account the unique tumor response characteristics seen with immunotherapy (Chiou, VL, et al., "Pseudoprogression and Immune-Related Response in Solid Tumors," J Clin Oncol., 33:3541-3 (2015)). Immunotherapeutic agents may confer antitumor efficacy by enhancing endogenous cancer-specific immune responses. Response patterns seen with such approaches may exceed the typical response time course seen with cytotoxic agents, revealing clinical responses after an initial increase in tumor burden or even the appearance of new lesions. Standard RECIST may not provide accurate response assessments for immunotherapeutic agents. Immunotherapy RECIST (iRECIST) is an adaptation of RECIST v1.1, as described below, to account for the unique tumor responses seen with immunotherapy. After site identification of progressive disease, iRECIST is used by site investigators to assess progression of tumor response and make treatment decisions. Archival tissue may be submitted in lieu of a fresh pre-treatment biopsy. In-treatment biopsies will be fresh biopsies. If a participant is scheduled to undergo a tumor biopsy for the purpose of this study and it is subsequently determined that tumor tissue cannot be safely obtained, the participant may still be enrolled in the study. Participants with certain serious medical conditions (in addition to a cancer diagnosis) will be excluded from study participation.

[0208] The recommended phase 2 dose (RP2D) of NC410 (LAIR-2-Fc) in combination with standard-dose pembrolizumab will be defined during a phase 1b safety and tolerability study in participants with advanced unresectable and / or metastatic ICI-refractory solid tumors (regardless of MSI status) or ICI-naive MSS / MSI-low solid tumors.

[0209] All eligible participants will receive a unique participant number, assigned through non-random allocation, with the first four digits serving as the site number after signing an Informed Consent Form (ICF). Participant numbering is controlled by the site. Investigative sites must complete all applicable CRFs for participants who consent to the study, even if the participant is not receiving treatment with the investigational drug (screening failure). Dose level and cohort assignment will be made at the time of participant enrollment and as directed by the sponsor or designee. Dose level and cohort assignment will be maintained in the CRF according to CRF completion guidelines and will be managed by the sponsor / CRO.

[0210] Participants will receive pembrolizumab on day 1 of each 42-day cycle, followed by NC410. Additional doses of NC410 will be administered on days 15 and 29, or weekly, of each 42-day cycle, as shown in Figure 4 and described in Table 1 below.

[0211] [Table 1]

[0212] *Varies by dose level.

[0213] The study will be conducted in two phases. Phase 1b will involve dose escalation of NC410 to determine the optimal dosing schedule and the RP2D in combination with standard-dose pembrolizumab. Phase 2 will involve dose expansion to evaluate the recommended Phase 2 dose (RP2D) of NC410 in combination with standard-dose pembrolizumab. Participants will remain in the study until disease progression, unacceptable adverse events (AEs), intercurrent illnesses that prevent continued treatment, the investigator's decision to withdraw the participant, the participant's withdrawal of consent, the participant's pregnancy, non-compliance with study treatment or procedural requirements, the participant receives 18 treatments with pembrolizumab (approximately two years), or an administrative reason requiring treatment discontinuation occurs.

[0214] Participants who discontinue for reasons other than disease progression will be followed for post-treatment disease status until disease progression, initiation of non-study cancer treatment, withdrawal of consent, or loss to follow-up. All participants will be followed for overall survival (OS) until death, withdrawal of consent, or end of study. After completion of treatment, each participant will be followed for 30 days for AE monitoring. Serious adverse events (SAEs) and clinically significant events (ECIs) will be collected for 90 days after the end of treatment or 30 days after the end of treatment if the participant begins a new anti-cancer therapy, whichever comes first.

[0215] In Phase 1b, a modified toxicity probability interval (mTPI) (Ji, Y., et al., “Modified toxicity probability interval design: a safer and more reliable method than the 3 + 3 design for practical phase I trials”, J Clin Oncol., 31:1785-91 (2013)) with a target dose-limiting toxicity (DLT) rate of approximately 30% will be applied for dose escalation and confirmation to determine the recommended phase 2 dose (RP2D) of NC410 in combination with pembrolizumab.

[0216] The Phase 1b portion of the study will evaluate five dose levels of NC410. NC410 is in a frozen liquid form formulated for iv infusion. The drug is provided in concentrations of 1 mg / mL (16 mg / vial) and 20 mg / mL (320 mg / vial).

[0217] Predetermined dose levels of NC410 (in combination with a fixed dose of 400 mg pembrolizumab) will be considered on an individual basis. The dose levels are as follows: Dose level-1 (escalating dose): 15 mg Dose level 1 (starting dose): 30 mg Dose level 2: 60 mg Dose level 3: 100 mg Dose level 4: 200 mg The starting dose is 30 mg of NC410 (dose level 1). If the starting dose of NC410 is deemed intolerable in combination with pembrolizumab, reduced doses of NC410 are available. All dose escalation and deescalation decisions will be based on the occurrence of dose-limiting toxicities (DLTs) at a given dose during the first 42 days (i.e., cycle 1) (also referred to as the DLT observation period) and will be made jointly by the investigator and sponsor. All DLTs will be assessed by the investigator using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCICTCAE) v5.0. The pembrolizumab dose will be maintained constant at 400 mg every 6 weeks at each NC410 dose level and in each cohort through phase 2.

[0218] On day 1 of each 42-day cycle, pembrolizumab 400 mg will be administered by intravenous (iv) infusion over a minimum of 30 minutes, followed by NC410 by i.v. infusion over a minimum of 30 minutes, with an interval of 30-60 minutes between each study treatment. However, due to potential site-specific variations in infusion pumps, a time window of -5 to +10 minutes will be allowed (i.e., infusion time will be 30 minutes (-5 minutes / +10 minutes)).

[0219] NC410 is also administered as a standalone solution or in combination with pembrolizumab at 15 mg, 30 mg, 60 mg, 100 mg, or 200 mg on days 1, 15, and 29, or weekly in each 42-day cycle. NC410 can also be administered as a standalone solution or in combination with pembrolizumab at 100 mg weekly. Participants will remain in the study until disease progression, unacceptable AEs, intercurrent illnesses that prevent continued treatment, the investigator's decision to withdraw the participant, the participant's withdrawal of consent, the participant's pregnancy, noncompliance with study treatment or procedural requirements, the participant receives 18 treatments with pembrolizumab (approximately 2 years), or an administrative reason requiring discontinuation of treatment occurs.

[0220] In Figure 5, the number of participants who received treatment is shown in the columns, and the number of participants who experienced a DLT is shown in the rows. Dosing decisions shown in Figure 5 include escalating to the next higher dose (E), maintaining the current dose (S), de-escalating to the next lower dose (D), and de-escalating to a lower dose and not retesting this dose (i.e., unacceptable toxicity dose, DU). Pending safety considerations and new data, alternative dose levels or dosing schedules may be considered.

[0221] As a non-limiting example, if three participants have been enrolled, the dose escalation rules may proceed as follows: if zero of the first three participants at a given dose level develop a DLT, the next available participant may be enrolled in the next level cohort and the dose may be escalated, with no further expansion. If one of the first three participants at a given dose level develops a DLT, no more than three additional participants should be enrolled at this dose level until additional DLT data are available. If all three additional participants (i.e., four of six participants) experience a DLT, the dose is deemed unacceptably toxic. If two of the first three participants at a given dose level develop a DLT, the dose is tapered to a lower dose level cohort. If three of the first three participants at a given dose level develop a DLT, the dose is deemed unacceptably toxic (i.e., the dose is reduced and will not be escalated to that dose again). The same principles apply whether three, four, five, or six participants are enrolled in the same dose cohort, according to Figure 5.

[0222] Based on the mTPI design, the number of participants enrolled at a dose but not yet fully evaluable for DLT assessment may not exceed the number of remaining participants at risk of developing a DLT before the dose is deemed unacceptably toxic (labeled DU in Figure 5). To determine how many more participants can be enrolled at a dose level, one can count the diagonal (down and right) steps from the current cell to the first cell marked DU. A total of 3 to 14 participants may be enrolled at a given dose level.

[0223] Dose escalation and confirmation will end after 10 evaluable participants have been treated with one of the selected doses. The pooled-adjacent-violator algorithm (Ji, Y., et al., "Modified toxicity probability interval design: a safer and more reliable method than the 3 + 3 design for practical phase I trials," J Clin Oncol., 31:1785-91 (2013)) will be used to estimate dose-specific DLT rates.

[0224] The dose with the estimated DLT rate closest to 30% may be treated as the preliminary maximum tolerated dose (MTD). Before selecting the dose to proceed to Phase 2, the totality of data is considered, and an escalation schedule may be adjusted based on pharmacokinetic (PK), pharmacodynamic (PD), and safety data obtained throughout the study to determine the RP2D.

[0225] If a participant is not evaluable during the DLT observation period for any reason, they may be replaced by the next available participant if the escalation or de-escalation rules are not met. The dose level cohort determined as the RP2D will be expanded until at least 10 participants have been dosed and observed during the 42-day DLT observation period before moving on to Phase 2 dose expansion.

[0226] Example 2. Clinical trial of NC410 in combination with pembrolizumab.

[0227] method: The participant selection parameters used in the Phase 1b study (i.e., safety and tolerability study) described above, as well as the parameters applied to participant withdrawal from the study, are also applicable to the Phase 2 study (i.e., clinical trial) described below. To further identify tumor types potentially suitable for NC410 therapy, immunohistochemistry (IHC) studies were performed. These IHC studies were also based on the criteria of previous detailed preclinical analyses of tumor types. The following criteria were used to select tumor types for IHC studies: LAIR-1 expression in the TME, a CD163M2-like macrophage marker assessed for relative increase in cancer tissue compared with normal tissue, and analysis of several LAIR ligands assessed for overexpression in the TME compared with normal tissue. To evaluate these markers, IHC analyses were performed, including hematoxylin and eosin (H&E) and trichrome staining, LAIR-1 expression, LAIR-2-Fc (NC410) binding, and immune cell infiltration. An example of the analysis results for gastric adenocarcinoma (STAD) is shown in Figure 6. For LAIR-2-Fc binding, brown staining indicates LAIR-2Fc binding, and blue staining is a hematoxylin counterstain, indicating no LAIR-2Fc binding. LAIR-2Fc-positive (green) and -negative (red) areas were quantified as shown in the associated pie chart. For immune cell quantification, five regions of interest (ROIs) with a diameter of 600 μm were randomly selected in the LAIR-2Fc-positive area. One of the ROIs was enlarged to show LAIR-1 staining. The graph shows immune cell quantification. The cell numbers of LAIR-1+, CD45+, CD3+, and CD163+ cells within the five ROIs were quantified and calculated as × 103 / mm2.

[0228] In Phase 2 of the study, the clinical benefit of the recommended Phase 2 dose (RP2D) of NC410 obtained in Phase 1b in combination with standard-dose pembrolizumab will be further evaluated in participants with advanced unresectable and / or metastatic solid tumors in various cohorts outlined below, based on a history of immune checkpoint inhibitor (ICI) refractory or treatment-naive status (as shown in Figure 7). Assessment of anti-tumor activity will be used to evaluate the clinical benefit of NC410 in combination with pembrolizumab and to confirm preclinical studies showing that NC410 in combination with anti-PD-L1 results in synergistic and reproducible tumor killing in mouse models (Figure 9).

[0229] Factors to be evaluated include (but are not limited to) objective response rate (ORR), disease control rate (DCR), duration of response (DoR), progression-free survival (PFS) (based on RECIST v1.1 as assessed by the investigator), and overall survival (OS). Pharmacokinetic (PK) evaluation of serum NC410 concentrations and evaluation of combination treatments on pharmacokinetic / pharmacodynamic (PK / PD) profiles will be observed.

[0230] This study will enroll patients with ICI-refractory solid tumors in Cohort 1 and ICI-naive solid tumors in Cohorts 2a, 2b, and 2c, as follows: Prior to entry into these cohorts, patients must have confirmed microsatellite stable (MSS) or microsatellite instability low (MSI-L) status (either from previous results or during screening). The cohorts are defined as follows: Cohort 1: ICI-refractory solid tumors (colorectal cancer microsatellite instability-high (CRC MSI-H), gastric cancer including the gastroesophageal (GE) junction, esophageal cancer, endometrial cancer, and head and neck (H&N) cancer).

[0231] Cohort 2a: ICI-naive MSS or MSI-L CRC Cohort 2b: ICI-naive MSS or MSI-L gastric cancer involving the GE junction Cohort 2c: ICI-naive MSS or MSI-L ovarian cancer

Claims

1. A combination therapy comprising at least two or more pharmaceutical compositions administered to a subject in need thereof, wherein the pharmaceutical compositions comprise: a. a first pharmaceutical composition comprising an immune checkpoint inhibitor (ICI); b. a second pharmaceutical composition comprising a protein configured to bind to one or more components of the extracellular matrix (ECM) of the tumor microenvironment (TME); The combination therapy comprising:

2. The combination therapy of claim 1 , wherein the subject in need thereof has cancer.

3. 10. The combination therapy of claim 1, wherein the subject in need thereof has colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, or head and neck cancer.

4. 2. The combination therapy of claim 1, wherein the immune checkpoint inhibitor inhibits the programmed cell death protein 1 (PD-1) pathway.

5. 2. The combination therapy of claim 1, wherein the immune checkpoint inhibitor is pembrolizumab.

6. 10. The combination therapy of claim 1, wherein the subject in need thereof receives a dose of the first pharmaceutical composition on the first day of a repeating 42-day cycle.

7. 7. The combination therapy of claim 6, wherein the dose is about 400 mg of the first pharmaceutical composition.

8. The combination therapy of claim 1 , wherein the protein binds to collagen or C1q.

9. 2. The combination therapy of claim 1, wherein the protein is a LAIR-2 fusion protein having a nucleic acid sequence of at least 95%, or 100%, sequence identity to SEQ ID NO: 7, or a functional fragment or variant thereof.

10. 10. The combination therapy of claim 1, wherein the subject in need thereof receives doses of the second pharmaceutical composition on days 1, 15, and 29 of a repeating 42-day cycle.

11. 11. The combination therapy of claim 10, wherein the dose is about 15 mg, 30 mg, 60 mg, 100 mg, or 200 mg of the second pharmaceutical composition.

12. 10. The combination therapy of claim 1, wherein the subject in need thereof receives a dose of the second pharmaceutical composition once a week for a repeating 42-day cycle.

13. 13. The combination therapy of claim 12, wherein the dose is about 100 mg of the second pharmaceutical composition.

14. 10. A method of treating cancer in a patient comprising administering the combination therapy of claim 1 to a subject in need thereof, wherein the cancer is colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, or head and neck cancer.

15. 15. The method of claim 14, wherein the subject in need of the combination therapy receives a dose of the first pharmaceutical composition on the first day of a repeating 42-day cycle.

16. 15. The method of claim 14, wherein the subject in need of the combination therapy receives doses of the second pharmaceutical composition on days 1, 15, and 29 of a repeating 42-day cycle.

17. 17. The method of claim 16, wherein the dose is about 15 mg, 30 mg, 60 mg, 100 mg, or 200 mg of the second pharmaceutical composition.

18. 15. The method of claim 14, wherein the subject in need of the combination therapy receives doses of the second pharmaceutical composition once a week for a repeating 42-day cycle.

19. 19. The method of claim 18, wherein the dose is about 100 mg of the second pharmaceutical composition.

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