Immune checkpoint inhibitor and extracellular matrix component binding agent combination therapies and methods of use thereof

Abstract

A clinical combination therapy including one or more pharmaceutical compositions to treat various cancers. The one or more pharmaceutical compositions comprise at least a first composition of an immune checkpoint inhibitor and a second composition that interacts with a component of an extracellular matrix of a tumor microenvironment. As one example, the first composition may comprise palbolizumab, and the second composition may comprise a LAIR-2 Fc fusion protein, such as NC410. The compositions follow the dosing and time regimen.

Description

Technical Field

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

[0002] The importance of intact immunosurveillance function in controlling the outgrowth of neoplastic transformation has been known for decades (Disis, M.L., “Immune regulation of cancer”, J Clin Oncol., 28:4531 - 8 (2010)). Increasing 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 / FoxP3+ regulatory T cells (T-reg) are associated with improved prognosis and long-term survival in solid malignancies such as ovarian cancer, colorectal cancer, and pancreatic cancer; hepatocellular carcinoma; malignant melanoma; and renal cell carcinoma. Tumor-infiltrating lymphocytes can be expanded ex vivo and re-infused to induce durable objective tumor responses in cancers such as melanoma (Dudley, M.E. 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, N.N. 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 studies in mouse models have shown that the administration of antibodies that block the PD-1 / PD-L1 interaction, either as a single therapy or in combination with other treatment modalities, enhances the infiltration of tumor-specific CD8+ T cells and ultimately leads to tumor rejection (Strome, S.E. 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, M.A. 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 U.S.A., 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 restored 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 anti-mouse PD-L1 antibodies have shown anti-tumor responses in squamous cell carcinoma, pancreatic cancer, melanoma, acute myeloid leukemia, and colorectal cancer models (Strome, S.E 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, M.A. 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 U.S.A., 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)). In such studies, tumor infiltration of CD8+ T cells and increased expression of IFN-γ, granzyme B, and perforin were observed, suggesting a potential mechanism of anti-tumor activity of PD-1 checkpoint inhibition involving local infiltration and in vivo activation of effector T cell function (Curran, M.A.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 U.S.A., 107:4275-80 (2010)). Experiments have demonstrated the in vivo efficacy of anti-mouse PD-1 antibody as a single therapy and in combination with chemotherapy in syngeneic mouse tumor models. However, since components within the extracellular matrix (ECM) of the tumor microenvironment (TME) may bind to immune checkpoint inhibitors (ICIs), ICIs such as anti-PD-1 antibodies may be limited.

[0004] There is still a need in the art to improve the role of immune checkpoint inhibitors in tumor treatment, reduction, and killing.

[0005] Incorporated by reference

[0006] Any of the foregoing applications and all documents cited therein or cited during their examination ("application cited documents") and all documents cited or referenced in the application cited documents, all documents cited or referenced herein ("herein cited documents") and all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets of any products mentioned in any document mentioned herein or incorporated herein by reference, are hereby incorporated herein by reference and may be used to practice the present invention.

[0007] The citation or identification of any document in this application does not admit that such document is prior art to the present invention. Summary of the Invention

[0008] It should be understood that this Summary of the Invention is not an exhaustive review of the present disclosure. This Summary of the Invention is exemplary and not restrictive, and it is neither intended to identify the key or important elements of the present disclosure nor to limit its scope. The sole purpose of this Summary of the Invention is to explain and illustrate certain concepts of the present disclosure as a preamble to the following complete and comprehensive Detailed Description.

[0009] The present disclosure relates to a combination therapy comprising at least two pharmaceutical compositions. The combination therapy significantly treats or reduces the symptoms of diseases including but not limited to cancer. Cancer includes but is not limited to colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, or head and neck cancer.

[0010] The first pharmaceutical composition comprises an immune checkpoint inhibitor (ICI), such as an ICI targeting 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 dose 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.

[0011] 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 for a variable duration according to a variable schedule, as can be determined by considering common factors and routine experiments 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 weekly at about 100 mg according to a repeated 42-day cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The features and components of the following drawings are shown to emphasize the general principles of the disclosure. For consistency and clarity, corresponding features and components throughout the drawings may be represented by matching reference characters.

[0013] Figure 1 A transgenic spontaneous model of Müllerian inhibiting substance type II receptor (MISIIR) of ovarian cancer is shown, where LAIR-1 is expressed on CD11c+CD11b+ inhibitory dendritic cells (DCs) at the primary and metastatic sites of the disease. LAIR-1 is not expressed on CD103+ stimulatory DCs.

[0014] Figure 2Shows NC410 binding to tumor - associated ligands (collagen and C1q) to block LAIR - 1 inhibition and promote adaptive (T - cell) and innate (dendritic cell) immune responses, as well as activate macrophages, ultimately leading to tumor cell killing.

[0015] Figure 3 Shows a dose - escalation study design according to the present disclosure.

[0016] Figure 4 Shows an exemplary dosing and timing regimen according to the present disclosure.

[0017] Figure 5 Shows a dose - exploration rule matrix according to the present disclosure.

[0018] Figure 6 Shows the analysis of five representative gastric adenocarcinoma (STAD) samples, analysis of LAIR - 2 - Fc (NC410) binding by H&E staining, and analysis by immunostaining of LAIR - 1, CD45, CD3, and CD163 - positive cells.

[0019] Figure 7 Shows a dose - exploration rule according to the Simon 2 - stage design of the present disclosure.

[0020] Figure 8 Shows collagen staining by triple - staining (left) and LAIR - 1 staining in immune cells by IHC (right) in multiple tumor types.

[0021] Figure 9 Shows a line graph demonstrating that the combination of NC410 and anti - PD - L1 produces synergistic and reproducible tumor killing in a murine model. 200 ug Q4D of NC410 (5 doses) ± 100 ug Q7D of PD - L1 (2 doses). Detailed Description

[0022] The present disclosure can be more readily understood by reference to the following detailed description, examples, drawings, and claims, as well as the description before and after them. However, prior to disclosing and describing the compositions and / or methods, it is to be understood that the present disclosure is not limited to the specific compositions and / or methods disclosed (unless otherwise specified) and can, of course, vary. It is also to be understood that the terms used herein are for the purpose of describing particular aspects only and are not intended to be limiting.

[0023] I. Definitions

[0024] 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. Although any compositions, methods, and materials similar to 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.

[0025] Unless otherwise defined, all percentage values of components used herein are given by weight percentage.

[0026] In the context of describing the claimed invention (especially in the context of the claims), the terms "a", "an", "the", and similar references should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0027] Unless otherwise specified herein, the recitation of value ranges herein is merely intended to be a shorthand method of individually referring to each separate value falling within the stated range, and each separate value is incorporated into the specification as if it were individually described herein.

[0028] The use of the term "about" is intended to describe values that are within about + / - 10% above or below the stated value; in other embodiments, the range of the value can be within about + / - 5% above or below the stated value; in other embodiments, the range of the value can be within about + / - 2% above or below the stated value; in other embodiments, the range of the value can be within about + / - 1% above or below the stated value. The foregoing ranges are intended to be made clear by the context and do not imply further limitations. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not impose a limitation on the scope of the invention, unless otherwise required. The language in this specification should not be construed as indicating any non-claimed element as essential for the practice of the invention.

[0029] As used herein, "administer" means to bring an exogenous ligand, reagent, placebo, small molecule, medicament, therapeutic agent, diagnostic agent, or composition into contact with a subject, cell, tissue, organ, or biological fluid when applied to a human, primate, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ, or biological fluid. "Administer" can refer to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of cells encompasses bringing a reagent into contact with the cells, as well as bringing a reagent into contact with a fluid, where the fluid contacts the cells. "Administer" also includes treatment of cells in vitro and ex vivo by a reagent, diagnostic agent, binding composition, or by another cell.

[0030] "Agonist", when referring to a ligand and a receptor, includes a molecule, combination of molecules, complex, or combination of reagents that stimulates the receptor. For example, a granulocyte-macrophage colony-stimulating factor (GM-CSF) agonist can encompass GM-CSF, a mutant protein 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.

[0031] As used herein, an "analogue" or "derivative" of a peptide, polypeptide, or protein refers to another peptide, polypeptide, or protein having a similar or identical function to the original peptide, polypeptide, or protein, but not necessarily containing an amino acid sequence or structure similar or identical to the original peptide, polypeptide, or protein. An analogue preferably satisfies at least one of the following: (a) a protein agent having an amino acid sequence having 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% identity to the original amino acid sequence; (b) a protein agent encoded by a nucleotide sequence that hybridizes to the nucleotide sequence encoding the original amino acid sequence under stringent conditions; and (c) a protein agent encoded by a nucleotide sequence having 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% identity to the nucleotide sequence encoding the original amino acid sequence.

[0032] As used herein, the term "antibody" is intended to mean an immunoglobulin molecule having an antigen recognition site in the "variable region". The term "variable region" is intended to distinguish such domains of the immunoglobulin from domains that are widely shared by antibodies (such as the antibody Fc domain). The variable region includes "hypervariable regions" whose residues are responsible for antigen binding. The hypervariable regions contain amino acid residues from "complementary determining regions" or "CDRs" (i.e., approximately residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) in the light chain variable domain and approximately residues 27 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) in 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 those residues from "hypervariable loops" (i.e., residues 26 - 32 (L1), 50 - 52 (L2), and 91 - 96 (L3) in the light chain variable domain and residues 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) in 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 defined herein. The term antibody includes monoclonal antibodies, multispecific 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. Patent No. 6,005,079), single-chain Fv (scFv) (see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds. Springer-Verlag, New York, pp. 269 - 315 (1994)), single-chain antibodies, disulfide-linked Fv (sdFv), intracellular antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., 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.

[0033] As used herein, an "antigen-presenting cell" (APC) is a cell of the immune system that presents antigen to T cells. APCs include dendritic cells, monocytes, macrophages, marginal zone Kupffer cells, microglia, Langerhans cells, T cells, and B cells. Dendritic cells exist in at least two lineages. The first lineage encompasses pre-DC1, myeloid DC1, and mature DC1. The second lineage encompasses CD34 + CD45RA - early multipotent progenitors, CD34 + CD45RA + cells, CD34 + CD45RA + CD4 + IL-3Rα + pre-DC2 cells, CD4 + CD11c - plasmacytoid pre-DC2 cells, lymphoid human DC2 plasmacytoid-derived DC2, and mature DC2.

[0034] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the complementarity-determining regions ("CDRs") of the antibody and optionally framework residues that include the antigen recognition site of the "variable region" of the antibody and exhibit the ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')2, Fv, single-chain (ScFv) and its mutants, naturally occurring variants, and fusion proteins that contain the antigen recognition site of the antibody "variable region" and a heterologous protein (e.g., a toxin, antigen recognition site of a different antigen, an enzyme, a receptor, or a receptor ligand, etc.).

[0035] As used herein, "attenuated gene" encompasses a gene that mediates toxicity, pathology, or virulence to a host, growth in a host, or survival in a host, wherein the gene is mutated in a manner that reduces, decreases, or eliminates toxicity, pathology, or virulence. Reduction or elimination can be evaluated by comparing the virulence or toxicity mediated by the mutated gene with that mediated by the non-mutated (or parental) gene. "Mutated gene" encompasses 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.

[0036] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from the abnormal, uncontrolled growth of cells. As used herein, cancer specifically includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells having the potential to metastasize to distal sites and exhibiting phenotypic characteristics (e.g., forming colonies in a three-dimensional matrix such as soft agar or forming a tubular network or reticular matrix in a three-dimensional basement membrane or extracellular matrix preparation) that are different from those of non-cancer cells. Non-cancer cells do not form colonies in soft agar but form distinct spherical structures in a three-dimensional basement membrane or extracellular matrix preparation.

[0037] As used herein, a "chimeric antibody" refers to a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as an antibody having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0038] As used herein, the term "chimeric receptor" is defined as a cell-surface receptor that comprises in combination an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic co-stimulatory signaling domain, where the respective domains do 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. Additionally, chimeric receptors are distinct from TCRs expressed in native T cell lymphocytes.

[0039] As used herein, "co-stimulatory" signals include positive co-stimulatory signals (e.g., signals that result in enhanced activity) and negative co-stimulatory signals (e.g., signals that result in inhibitory activity).

[0040] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" or "comprising" will be understood to mean including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements. In specific embodiments, the terms "include", "have", "contain" and "comprise" are used synonymously.

[0041] "Consisting of" is intended to include but not be limited to all of the content following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory and that no other elements may be present.

[0042] "Consisting essentially of" is intended to include any elements listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or function specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that no other elements are optional and may or may not be present, depending on whether they affect the activity or function of the listed elements.

[0043] The term "derivative" refers to an antibody or an antigen-binding fragment thereof that immunospecifically binds to the same target as the parental or reference antibody, but whose amino acid sequence differs from that of the parental or reference antibody or its antigen-binding fragment by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parental or reference antibody or its antigen-binding fragment. In some embodiments, such derivatives will have substantially the same immunospecificity and / or characteristics, or the same immunospecificity and characteristics, as the parental or reference antibody or its antigen-binding fragment. Amino acid substitutions or additions in such derivatives may include naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, chimeric or humanized variants, as well as variants having altered CH1, hinge, CH2, CH3 or CH4 regions, thereby forming, for example, antibodies having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

[0044] As used herein, "effective amount" encompasses, but is not limited to, an amount (e.g., an amount of a protein, polypeptide, fragment thereof, etc.) that can ameliorate, reverse, mitigate, prevent or diagnose symptoms or signs of a medical disorder or condition. Unless otherwise expressly stated or the context otherwise requires, an "effective amount" is not limited to the minimal amount sufficient to improve the situation.

[0045] References throughout this specification to "an embodiment", "one embodiment", "an embodiment", "a particular embodiment", "a related embodiment", "a certain embodiment", "another embodiment" or "a further embodiment" or combinations thereof mean that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases throughout this specification need not all refer to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures or characteristics may be combined in any suitable manner.

[0046] 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 cell may be a normal cell, a cancer cell or an infected cell.

[0047] As used herein, "epitope" refers to an antigenic determinant capable of specifically binding to an antibody. Epitopes are usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that in the presence of a denaturing solvent, binding to the former is lost, while binding to the latter is not.

[0048] As used herein, "extracellular fluid" encompasses serum, plasma, blood, interstitial fluid, cerebrospinal fluid, secretions, lymph, bile, sweat, feces, and urine. "Extracellular fluid" can include colloids or suspensions such as whole blood or clotted blood.

[0049] As used herein, "fragment" in the context of a polypeptide includes a peptide or polypeptide that contains 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 a larger polypeptide.

[0050] As used herein, the term "humanized antibody" refers to an immunoglobulin that includes human framework regions and one or more CDRs from a non-human (usually murine 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 required to be present, but if they are present, they should be substantially the same as human immunoglobulin constant regions, i.e., at least about 85 - 99% or about 95% or greater identity. Thus, all parts of the humanized immunoglobulin (possibly except for the CDRs) are substantially the same as the corresponding parts of the native human immunoglobulin sequence. A humanized antibody is an antibody that includes humanized light chain and humanized heavy chain immunoglobulins. For example, humanized antibodies do not encompass typical chimeric antibodies because, for example, the entire variable region of a chimeric antibody is non-human.

[0051] 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.

[0052] As used herein, "immune checkpoint" refers to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. Immune checkpoints are regulated by immune checkpoint proteins.

[0053] As used herein, "immune checkpoint protein" is a protein that is 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. Specifically, immune checkpoint proteins are inhibitory to the activation of an immune response. Thus, inhibition of inhibitory immune checkpoint proteins is used to stimulate or activate an immune response, such as T cell activation and proliferation.

[0054] As used herein, "immune checkpoint inhibitor" or "immune checkpoint inhibiting agent" or "immune checkpoint blocker" refers to an agent that binds to an inhibitory immune checkpoint protein and blocks its activity. The inhibition can be competitive inhibition or non-competitive inhibition, which can be steric inhibition or allosteric inhibition. In the case where the immune checkpoint protein is an immune stimulatory protein, an immune checkpoint inhibitor is used to promote the activity of the immune stimulatory protein, such as by binding and activating the stimulatory immune checkpoint protein or by inhibiting via interference (such as by binding or inactivating) an inhibitor of the stimulatory immune checkpoint protein. Examples of immune checkpoint inhibitors are anti-immune checkpoint protein antibodies.

[0055] The "target" of an immune checkpoint inhibitor is the immune checkpoint protein to which the immune checkpoint inhibitor or immune checkpoint inhibitory agent binds to block its activity. Typically, an immune checkpoint inhibitor binds specifically to the target. For example, the target of an exemplary anti-CTLA4 antibody (designated ipilimumab) is CTLA4.

[0056] An "immunogenic agent" or "immunogen" is capable of inducing an immune response against itself when administered to a mammal (optionally in combination with an adjuvant).

[0057] As used herein, the terms "immune," "immunological," or "immunal" response are beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) responses elicited in a patient against a peptide. Such responses can be active responses induced by administration of an immunogen, or passive responses induced by administration of an antibody or primed T cells. Cellular immune responses are caused by the presentation of polypeptide epitopes associated with class I or class II MHC molecules, thereby activating antigen-specific CD4 + T helper cells and / or CD8 +Cytotoxic T cells. The response may also involve activated monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, activated or recruited neutrophils, or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by a proliferation assay (CD4 + T cells) or a CTL (cytotoxic T lymphocyte) assay. The relative contributions of humoral and cellular responses to the protective or therapeutic effects of an immunogen can be distinguished by separately isolating antibodies and T cells from immunized syngeneic animals and measuring the protective or therapeutic effects in a second subject.

[0058] As used herein, a molecule is considered to be able to "immunologically specifically bind" to a second molecule if such binding exhibits the specificity and affinity of an antibody for its cognate antigen. An antibody is considered to be able to immunologically specifically bind to a target region or conformation ("epitope") of an antigen if such binding involves the antigen recognition site of an immunoglobulin molecule. If other antigens have some sequence or conformational similarity that is recognized by the antigen recognition site (as determined, for example, by an immunoassay, assay, or other assays known in the art), an antibody that immunologically specifically binds to a specific antigen may bind to other antigens with a lower affinity, but will not bind to completely unrelated antigens. However, in some embodiments, an antibody (and its antigen-binding fragment) will not cross-react with other antigens. An antibody can also bind to other molecules in a non-immunologically specific manner, such as binding to an FcR receptor, which is achieved by a binding domain in another region / domain of the molecule that does not involve the antigen recognition site (such as the Fc region).

[0059] As used herein, the terms "individual", "host", "subject", "participant", and "patient" are used interchangeably herein and refer to a mammal, including but not limited to a human, a rodent (such as a mouse and a rat), 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 who is receiving medical care for a disease or disorder. This includes humans who do not have an apparent disease but are undergoing examination for pathological signs.

[0060] As used herein, an "inflammatory molecule" refers to a molecule that causes an inflammatory response, including but not limited to cytokines and metalloproteinases, such as including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMP.

[0061] As used herein, "ligand" refers to a small molecule, peptide, polypeptide, or membrane - associated or membrane - bound molecule, i.e., an agonist or antagonist of a receptor. "Ligand" also encompasses binders that are not agonists or antagonists and do not possess agonist or antagonist properties. By convention, when a ligand binds to a membrane on a first cell, the receptor is typically present on a second cell. The second cell may have the same identity (same name) as the first cell or a different identity (different name). A ligand or receptor can be entirely intracellular, i.e., it may be located in the cytoplasm, nucleus, or some other intracellular compartment. A ligand or receptor may change its location, such as from an intracellular compartment to the outer surface of the plasma membrane. The complex of a ligand and a receptor is referred to as a "ligand - receptor complex". When a ligand and a receptor are involved in a signal transduction pathway, the ligand is located upstream in the signal transduction pathway, while the receptor is located downstream in the signal transduction pathway.

[0062] As used herein, the term "isolated" means that a substance is substantially or essentially free of components that normally accompany it in its native state. In a specific embodiment, the terms "obtained" or "derived" are used synonymously with isolated.

[0063] As used herein, "managing" or "controlling" one or more symptoms or effects of a disease or disorder (e.g., cancer) means using a composition or method contemplated herein to improve the quality of life of an animal by better controlling tumor activity and cancer - related clinical symptoms in a subject in need thereof.

[0064] As used herein, the term "modulate" relates to the ability to alter an action, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuate but not eliminate), or it can completely abolish such activity (e.g., neutralize). Modulation can include internalization of a receptor following antibody binding or a decrease in receptor expression on a target cell. Agonistic modulation can enhance or otherwise increase or augment an activity (e.g., signal transduction). In yet another embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor, and thereby alter the nature of the signal transduction elicited. For example, by binding to a ligand or receptor, a molecule can change the ability of such molecule to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments, such modulation will provide at least a 10% change, at least a 50% change, or at least a 2 - fold, 5 - fold, 10 - fold, or at least 100 - fold change in measurable immune system activity.

[0065] 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. The 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. The algorithm used to calculate percent identity is 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 a non-limiting example, 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.

[0066] As used herein, "peptide" refers to a short sequence of amino acids, wherein the amino acids are linked to each other by peptide bonds. A peptide can exist free or in combination with another moiety, such as a macromolecule, lipid, oligosaccharide or polysaccharide and / or polypeptide. When a peptide is incorporated into a polypeptide chain, the term "peptide" can still be used to specifically refer to the short sequence of amino acids. A "peptide" can be linked to another moiety by a peptide bond or some other type of linkage. The length of a peptide is at least two amino acids, where the maximum length depends on convention or context.

[0067] 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 introducing gaps, if necessary, to achieve the maximum percent sequence identity. The alignment for the purposes of determining percent sequence identity can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. The appropriate parameters for measuring the alignment can be determined by known methods, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0068] For the purposes of this disclosure, the percent sequence identity of a given nucleotide or amino acid sequence C with, to, or against a given nucleic acid sequence D (which can alternatively be stated as a given sequence C having or containing a certain percent sequence identity with, to, or against a given sequence D) is calculated as follows:

[0069] 100 multiplied by the fraction W / Z,

[0070] Where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program when aligning C and D, and where Z is the total number of nucleotides or amino acids in D. It is understood that when the length of sequence C is not equal to the length of sequence D, the percent sequence identity of C to D will not be equal to the percent sequence identity of D to C.

[0071] As used herein, "pharmaceutically acceptable excipient", "pharmaceutically acceptable carrier", or "diagnostically acceptable excipient" includes, but is not limited to, sterile distilled water, saline, phosphate buffered solutions, amino acid-based buffers, or bicarbonate buffered solutions. The excipient selected and the amount of excipient used 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 art of formulation, including aqueous or organic solvents or solvent mixtures. These organic solvents may be found, for example, in Remington Pharmaceutical Sciences, 21st Edition (2005). Other solvents and / or additives useful for topical compositions include, but are not limited to, PEG ethers and PEG esters including, but not limited to, PEG esters of carboxylic and dicarboxylic acids and PEG esters of fatty acids, glycerides including triacetin, caprylic / capric triglyceride (Miglyol ) and the like; glycerol ethers including glycerol formal; propylene glycol dicaprylate / dicaprate (Miglyol ), lauryl lactate, triacetin, diisopropyl adipate (DIPA, also known as CERAPHYL 230), diisobutyl adipate, dimethyl isosorbide (DMI), tributyl acetyl citrate, oleic acid; carboxylic acid esters including diesters; ketones including acetone, methyl isobutyl ketone (MIK), methyl ethyl ketone, and the like; acetonitrile, C1-C 12 alcohols, including benzyl alcohol, methanol, ethanol, isopropyl alcohol, and butanol; aromatic ethers, such as anisole; amides including dimethylacetamide, monomethylacetamide, and dimethylformamide; dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, glycol carbonates, including, but not limited to, propylene carbonate and butylene carbonate; 2-pyrrolidone, N-methylpyrrolidone, C1-C 12 alkyl esters of carboxylic acids including butyl acetate or octyl acetate and benzyl acetate; C1-C 12 alkyl esters of dicarboxylic acids; aryl esters, including benzyl benzoate, ethyl benzoate, and the like; and diethyl phthalate, or a mixture of at least two of these solvents.

[0072] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, whether or not modified (e.g., phosphorylated or glycosylated). The term polypeptide includes proteins and fragments thereof. Polypeptides can be "exogenous", meaning that they are "heterologous", i.e., foreign to the host cell utilized, such as a human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein in the form of amino acid residue sequences. Those sequences are written from left to right in the direction from the amino to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or one-letter codes, as follows: 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).

[0073] As used herein, "prevent" and like terms such as "prevented", "preventing", etc. refer to a method of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of one or more symptoms or other effects of a disease or disorder disclosed herein, such as colorectal cancer, head and neck cancer, gastrointestinal cancer, gastroesophageal cancer, and other known diseases and disorders. For example, in embodiments involving treating cancer in a subject, "prevent" and like terms such as "prevented", "preventing", etc. refer to a method for preventing, inhibiting, or reducing the likelihood of the occurrence of clinical signs and symptoms associated with cancer. It also refers to delaying the onset or recurrence of a disease or disorder, or delaying the onset or recurrence of symptoms.

[0074] As used herein, the term "preventive agent" refers to an agent that can be used to prevent a disorder or disease before any symptoms of the disorder or disease are detected. A "preventively effective" amount is the amount of a preventive agent (e.g., a protein, polypeptide, fragment thereof, etc.) sufficient to mediate such protection. A preventively effective amount can also refer to the amount of a preventive agent that provides a preventive benefit in preventing a disease. Generally but not necessarily, since preventive doses are used in a subject before or at an early stage of a disease, a preventively effective amount may be lower than a therapeutically effective amount.

[0075] As used herein, "protein" generally refers to the sequence of amino acids that make up a polypeptide chain. A protein can 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 refers to a substantially random three-dimensional structure, as in the case of a fully denatured protein. Polypeptide variants can be generated by glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal sequence or leader sequence processing, covalently and non-covalently bound cofactors, oxidative variants, and the like.

[0076] As used herein, "recombinant," when used in reference to nucleic acids, cells, animals, viruses, plasmids, vectors, etc., denotes a modification made by introducing exogenous, non-native nucleic acids, altering native nucleic acids, or by being derived in whole or in part from recombinant nucleic acids, cells, viruses, plasmids, or vectors. A recombinant protein is a protein that is derived from recombinant nucleic acids, viruses, plasmids, vectors, etc.

[0077] As used herein, "sample" refers to a sample from a human, animal, placebo, or study sample, such as cells, tissues, organs, fluids, gases, aerosols, slurries, colloids, or coagulates. A "sample" can be tested in vivo (i.e., without removal from the human or animal), or it can be tested in vitro. A sample can be tested after processing, such as by histological methods. A "sample" also refers to the cells that make up a fluid or tissue sample, or cells isolated from a fluid or tissue sample. A "sample" can also refer to cells, tissues, organs, or fluids freshly collected from a human or animal, or cells, tissues, organs, or fluids that have been processed or stored.

[0078] When referring to a ligand / receptor, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pair (e.g., a cytokine and a cytokine receptor), "specific" or "selective" binding indicates a binding reaction that identifies the presence of a protein in a heterogeneous protein population and other biological agents. Thus, under specified conditions, a designated ligand binds to a specific receptor and does not bind extensively to other proteins present in the sample. Specific binding can also mean, for example, that the binding affinity of the binding compound, nucleic acid ligand, antibody, or antigen-binding site derived from an antibody of the method under consideration for its target is generally at least 25% greater, more typically at least 50% greater, most typically at least 100% (2-fold) greater, often at least 10-fold greater, more often at least 20-fold greater, and most often at least 100-fold greater than its affinity for any other binding compound.

[0079] As used in the context of the role of binding or presentation, the term "substantially" is intended to mean that the observed role is physiologically relevant or therapeutically relevant. Thus, for example, if the degree of blockade 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), the molecule is capable of substantially blocking the activity of a ligand or receptor. Similarly, if such immune specificity and characteristics are greater than 60% identity, greater than 70% identity, greater than 75% identity, greater than 80% identity, greater than 85% identity, greater than 90% identity, greater than 95% identity or greater than 97% identity, the molecule is considered to have substantially the same immune specificity and / or characteristics as another molecule.

[0080] 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 for an encoded heterologous antigen to benefit a patient (e.g., result in a reduction, prevention or amelioration of the symptoms of the disorder being treated). When the agent or pharmaceutical composition contains a diagnostic agent, a "diagnostically effective amount" is defined as the amount sufficient to generate a signal, image or other diagnostic parameter. The effective amount of a pharmaceutical formulation will vary depending on factors such as the susceptibility of the individual, the age, sex and weight of the individual, and the individual's unique response (U.S. 5,888,530).

[0081] As used herein, "treat", "treating", "treatment" and "therapeutic use" (with respect to a disorder or disease) are a method for obtaining a beneficial or desired result, including and preferably a clinical result. For the purposes of this disclosure, beneficial or desired results with respect to a disease include, but are not limited to, one or more of the following: improvement of a condition associated with the disease, cure of the disease, alleviation of the severity of the disease, retardation of the progression of the disease, remission of one or more symptoms associated with the disease, improvement of the quality of life of an individual suffering from the disease and / or prolongation of survival. Similarly, for the purposes of this disclosure, beneficial or desired results with respect to a disorder include, but are not limited to, one or more of the following: improvement of the disorder, cure of the disorder, alleviation of the severity of the disorder, retardation of the progression of the disorder, remission of one or more symptoms associated with the disorder, improvement of the quality of life of an individual suffering from the disorder and / or prolongation of survival.

[0082] 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 the cells present in the tumor that support the growth of cancer cells.

[0083] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential characteristics. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited, such that the sequences of the reference polypeptide and the variant are overall very similar and identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by one or more modifications (e.g., substitutions, additions, and / or deletions). The amino acid residues substituted or inserted may or may not be amino acid residues encoded by the genetic code. Variants of a polypeptide can be naturally occurring, such as allelic variants, or they can be variants not known to occur naturally.

[0084] The structure of the polypeptides of the present disclosure can be modified and altered and still obtain molecules having similar characteristics to the polypeptides (e.g., conservative amino acid substitutions). For example, certain amino acids can substitute for other amino acids in the sequence without significant loss of activity. Since the interaction ability and properties of a polypeptide define the biological functional activity of this polypeptide, certain amino acid sequence substitutions can be made in the polypeptide sequence, yet still obtain a polypeptide having the same characteristics.

[0085] When making such changes, the hydropathic index of the amino acids can be considered. In the art, there is a general recognition of the importance of the amino acid hydropathic index in conferring the biological function of polypeptide interactions. It is known that certain amino acids can substitute for other amino acids having similar hydropathic indices or scores and still produce polypeptides having similar biological activities. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0086] The relative hydropathic character of the amino acids is believed to determine the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, substitutions of amino acids with hydropathic indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0087] Amino acid-like substitutions can also be made according to hydrophilicity, especially when the resulting biologically equivalent polypeptides or peptides are 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 should be understood that an amino acid can substitute for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent and specifically immunologically equivalent polypeptide. In such changes, substitutions of amino acids having hydrophilicity values within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0088] As described 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 taking into account the various foregoing characteristics are well known to those skilled 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). Accordingly, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides as described above. Specifically, embodiments of the polypeptide can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the polypeptide of interest.

[0089] II. Compositions

[0090] The present disclosure relates to combinations of one or more pharmaceutical compositions. The one or more pharmaceutical compositions can be combined for administration in a single dose or administered separately according to multiple dosing and timing regimens, as described herein. The combination can be administered to a subject in need thereof, wherein the subject is experiencing advanced unresectable and / or metastatic immune checkpoint inhibitor (ICI)-refractory solid tumors or microsatellite stable / microsatellite unstable (MSS / MSI) low solid tumors that have not been treated with ICI. The combination can be administered to a subject in need thereof, wherein one composition in the combination is an ICI (e.g., anti-PD-1 therapy), and a second composition in the combination exhibits an affinity for binding components of the extracellular matrix (ECM) in the tumor microenvironment (TME). The second composition can bind to components of the ECM, thereby enabling the ICI of the first composition to more effectively target the tumor. The combination then more effectively treats, reduces, or kills tumors associated with cancer. A composition that exhibits an avidity for binding components of the ECM can bind to collagen. The combination can include, but is not limited to, a first pharmaceutical composition comprising pembrolizumab and a second pharmaceutical composition comprising LAIR-2 or an LAIR-2Fc fusion protein. The second pharmaceutical composition can specifically comprise NC410, which is an LAIR-2Fc fusion protein. The combination can include additional compositions, including but not limited to one or more compositions selected from the following: LAIR-2IgG1 fusion protein, LAIR-1, LAIR-1Fc fusion protein, humanized monoclonal antibodies (such as a humanized monoclonal antibody against PD-1 (IgG4)), collagen-derived products (e.g., C4G, Pro-C3, Pro-C6, etc.), any combination thereof, and other such components known in the art.

[0091] A. Immune Checkpoint Pathways

[0092] The present disclosure relates to combinations of one or more compositions that are configured to effectively inhibit the Programmed Cell Death Protein 1 (PD-1) pathway. Inhibiting the PD-1 pathway in the tumor microenvironment (TME) using immune checkpoint inhibitors (such as pembrolizumab) in combination with proteins that interact with extracellular matrix components (such as LAIR proteins) should promote immune cell activation along with extracellular matrix (ECM) remodeling and further promote T cell infiltration into the TME, thus providing a novel and improved treatment method for participants with ICI-refractory advanced metastatic solid tumors, regardless of MSI status or MSS or MSI-low advanced unresectable and / or metastatic solid tumors. PD-1 receptor-ligand interaction is the main pathway hijacked by tumors 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 after engagement with 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 U.S.A., 98:13866-71 (2001); Greenwald, R.J. et al., “The B7 family revisited”, Annu Rev Immunol., 23:515-48 (2005)).

[0093] The sequence of human PDCD1 is known in the art. For example, the consensus sequence of PDCD1 is: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO:1, UniProt accession number Q15116, which is incorporated by reference in its entirety).

[0094] The structure of murine PD-1 has been resolved (Zhang, 2004). PD-1 and its family members are type I transmembrane glycoproteins that contain an Ig variable (IgV) domain responsible for ligand binding and a cytoplasmic tail responsible for binding signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs—the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif. Upon T cell stimulation, PD-1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, resulting in the dephosphorylation of effector molecules such as CD3 zeta (CD3ζ), protein kinase C-theta (PKCθ), and zeta chain-associated protein kinase (ZAP70), which are involved in the CD3 T cell signaling cascade (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 U.S.A., 98:13866-71 (2001); Chemnitz, J.M. 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 ligation prevents T cell activation”, J Immunol., 173:945-54 (2004); Sheppard, K.A. 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, J.L., “PD-1 signaling in primary T cells”, Immunol Rev., 229:114-25 (2009)).The mechanism by which PD-1 downregulates T cell responses is similar but distinct from that of CTLA-4, as both molecules regulate an overlapping set of signaling proteins (Parry, R.V. et al., “CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms”, Mol Cell Biol., 25:9543-53 (2005); Francisco, L.M. et al., “The PD-1 pathway in tolerance and autoimmunity”, Immunol Rev., 236:219-42 (2010)). Thus, the PD-1 / PD-L1 pathway is an attractive target in the tumor microenvironment (TME) and is an effective approach for cancer therapy.

[0095] B. LAIR proteins

[0096] The present disclosure relates to compositions comprising LAIR proteins. The LAIR proteins can include the amino acid sequences of full-length 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 can include NC410. The compositions of LAIR proteins can be administered to a subject in need thereof in combination with a composition of one or more pembrolizumab. The compositions of LAIR proteins can be administered simultaneously or separately from a composition of one or more. The compositions of LAIR proteins can be administered according to a variety of dosing and time regimens as described herein.

[0097] LAIR-1 inhibitory signaling may prevent autoimmune diseases such as lupus, rheumatoid arthritis, autoimmune thyroid disease, and atherosclerosis, etc., as well as contact hypersensitivity (Sun et al., 2014). At the same time, overexpression of LAIR-2 may promote autoimmunity through decoy binding of LAIR-1 ligands. The LAIR-2 binding of LAIR-1 ligands can essentially reduce the cell surface crosslinking of LAIR-1, thereby defining an inhibitory signaling pathway that leads to hyperreactive immune function. Conversely, it is hypothesized that increasing LAIR-2 levels may promote anti-tumor immunity through the same mechanism.

[0098] The presence of a collagen-dense extracellular matrix (ECM) is increasingly recognized as a key determinant of tumor response to ICI therapy. Collagen can be secreted by cancer-associated fibroblasts (CAFs), cancer cells, and macrophages in the tumor microenvironment (TME). Cells expressing LAIR-1 localized in the tumor microenvironment can be specifically inhibited through LAIR-1 collagen crosslinking and subsequent inhibitory signaling. Interestingly, both collagen and C1q have been shown to limit or alter antigen-presenting cell (monocyte / macrophage / DC) differentiation and activation through LAIR-1. Studies have shown that crosslinking LAIR-1 on NK cells and T cells inhibits proliferation and function. The collagen-dense ECM can act as a physical barrier preventing immune cell infiltration into tumors and has been shown to inhibit anti-tumor immunity and be associated with PD-1 / PD-L1-resistant tumors (Peng, D.H. et al., “Collagen promotes anti-PD-1 / PD-L1 resistance in cancer through LAIR1-dependent CD8(+)T cell exhaustion”, Nat Commun., 11:4520 (2020)).

[0099] The ECM abnormality in the TME supports tumor progression, leads to immune dysfunction, and provides targets for cancer therapies. Leukocyte-Associated Immunoglobulin-like Receptor (LAIR)-1 and LAIR-2 are members of the Leukocyte Receptor Complex (LRC) on human chromosome 19 (Le bbink, R.J. 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, R.J. 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, M.J. 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, M.J. 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 is used to delineate the immune response (Afshar-Kharghan, V., “The role of the complement system in cancer”, J Clin Invest., 127:780-9 (2017); Pearce, O.M.T. et al., “Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers”, Cancer Discov., 8:304-19 (2018)). It has been observed that LAIR-1 expression is associated with inhibitory immune cell populations in some cancers. For example. Figure 1 As shown, in both murine and human ovarian cancer, inhibitory but not stimulatory dendritic cell (DC) subsets and inhibitory macrophages express LAIR-1, suggesting that blocking LAIR-1 in ovarian cancer should reverse immune suppression (Flies, D.B. et al., “Immune checkpoint blockade reveals the stimulatory capacity of tumor-associated CD103(+) dendritic cells in late-stage ovarian cancer”, Oncoimmunology., 5:e1185583 (2016)).

[0100] i. LAIR-1

[0101] The sequence of human LAIR-1 is known in the art. For example, the consensus sequence of LAIR-1a (isoform 1) is MSPHPTALLGLVLCLAQTIHT QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSES EARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQG LKAEHLY ILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO:2, UniProtKB-Q6GTX8(LAIR1_HUMAN)).

[0102] 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 isotype 2) is a deletion of amino acids 122 - 138 relative to SEQ ID NO:2. LAIR - 1c (also known as isotype 3) is a deletion of amino acids 23 - 23 and 122 - 138 relative to SEQ ID NO:2. LAIR - 1d (also known as isotype 4) is a deletion of amino acids 210 - 287 relative to SEQ ID NO:2.

[0103] As introduced above, the extracellular domain of human LAIR - 1 can be QEEDLPR PSISAEPGTVIPLGSHVTFVCRGP VGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLE LLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLY (SEQ ID NO:3),

[0104] or a fragment thereof. For example, the Ig - like C2 - domain (underlined amino acids 8 - 96 of SEQ ID NO:3), or the region framed by the cysteines that form the disulfide bond between amino acids 49 - 101 of SEQ ID NO:2 (amino acids 28 - 80 of SEQ ID NO:3, italicized).

[0105] Known variants and mutants of LAIR - 1 relative to SEQ ID NO:2 include E63D, Y251F, and Y251F. Evidence shows that when associated with F - 281, Y215F reduces tyrosine phosphorylation and loses binding to PTPN6 and CSK, as well as completely loses inhibitory activity, and loses phosphorylation and inhibition of calcium mobilization (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 shows reduced tyrosine phosphorylation and loss of binding to PTPN6, as well as partial inhibition of cytotoxic activity.

[0106] Studies have shown that components of the ECM serve as ligands for the collagen-binding inhibitory receptor LAIR-1. All collagens consist of three polypeptide chains, characterized by a repeating Gly-X-X’ sequence, where X is typically proline and X' is typically 4-R-hydroxyproline (Hyp, O) (Brondijk, Blood, 115(7):1364-1373(2010)). The GPO triplet is an almost exclusive feature of collagen and allows for the formation of the characteristic triple-helical collagen structure.

[0107] The LAIR-1 ligands include several types of collagens, as well as ligands with collagen domains, including complement component C1q, mannose-binding lectin (MBL), and surfactant protein D (SP-D) (Lebbink, R.J. 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, R.J. 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, M.J. 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, M.J. et al., “Leukocyte-associated Ig-like receptor-1 is a novel inhibitory receptor for surfactant protein D”, J Leukoc Biol, 96:105-11 (2014)).

[0108] In cancer, it is hypothesized that LAIR-1 expression on several leukocyte subsets prevents optimal immune responses by restricting both innate and adaptive immune functions. LAIR-1 inhibits anti-tumor immunity by suppressing stimulatory signaling pathways. Specifically, LAIR-1 is a checkpoint and adhesion receptor on T cells that restricts T cell activation and increases adhesion to collagen (Meyaard, L., “The inhibitory collagen receptor LAIR-1(CD305)”, JLeukoc Biol., 83:799-803(2008)).

[0109] In addition to its role in T cell function, LAIR-1 is also expressed on NK, monocytes, macrophages, dendritic cells, and neutrophils and serves to demarcate the immune response. Furthermore, LAIR-1 expression has been shown to be associated with inhibitory DC and macrophage subsets in both murine and human ovarian cancer (Flies, D.B. et al., “Immune checkpoint blockade reveals the stimulatory capacity of tumor-associated CD103(+) dendritic cells in late-stage ovarian cancer”, Oncoimmunology., 5:e1185583(2016)). Blockade of LAIR-1 in cancer should reduce inhibitory mechanisms, thereby redirecting myeloid cells to promote stimulatory responses against the tumor, including T cell responses. Overall, these data suggest that targeting the LAIR-1 pathway in cancer patients may be a rational approach to enhancing anti-tumor immunity.

[0110] It has been shown that certain tumors with high collagen deposition lead to intrinsic or acquired resistance to PD-1 / PD-L1 blockade due to alternative immunosuppressive pathways and reduced total CD8+ T cells within the tumor (Peng, D.H. 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 collagen-induced CD8+ T cell immunosuppression phenomenon is driven by overexpression of LAIR-1 on immune cells and enhanced collagen production driven by increased TGF-β signaling following partial treatment with PD-1 / PD-L1 blockade.

[0111] ii. LAIR-2

[0112] LAIR-2 is a soluble homolog of LAIR-1 that binds to LAIR-1 and outcompetes LAIR-1's binding to collagen and serves as a natural decoy to boost immune function. LAIR-2 is capable of blocking the functional interaction of LAIR-1 with ligands, resulting in improved immune function in multiple immune cell subsets. Given that LAIR-2 has a higher affinity for collagen than LAIR-1, overexpression of LAIR-2 leads to blockade of LAIR-1 signaling, rendering resistant tumors sensitive to PD-1 blockade and significantly reducing tumor growth and metastasis.

[0113] LAIR-2 is a secreted protein whose Ig-like C2 domain in the extracellular region has 77.5% homology with the transmembrane protein LAIR-1 and serves as a natural, endogenous, secreted decoy for LAIR-1 produced mainly by activated T cells (Meyaard, L., “The inhibitory collagen receptor LAIR-1(CD305)”, J Leukoc Biol., 83:799-803(2008)). LAIR-2 is capable of blocking the functional interaction of LAIR-1 with ligands, thereby resulting in improved immune function on multiple immune cell subsets, as Figure 2 shown. Of note is the following observation: dysregulation of LAIR-1 ligands leads to overproduction of collagen and complement C1q and altered forms of collagen, which may have a strong inhibitory effect on the TME (Afshar-Kharghan, V., “The role of the complement systemin cancer”, JClinInvest., 127:780-9(2017); Pearce, O.M.T. et al., “Deconstruction of a MetastaticTumor Microenvironment Reveals a Common Matrix Response in Human Cancers”, Cancer Discov., 8:304-19(2018)). Thus, disrupting the interaction between LAIR-1 ligands and LAIR-1 and interrupting the inhibitory effect on the TME (using LAIR-2-based therapies) is a novel cancer treatment approach.

[0114] The sequence of human LAIR-2 is known in the art. For example, the consensus sequence of LAIR-2a (isoform 1) is MSPHLTALLGLVLCLAQTIHT QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSES EARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAP (SEQ IDNO:4, UniProtKB-Q6ISS4(LAIR2_HUMAN)).

[0115] Amino acids 1 - 21 are the 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 isotype 2) is a deletion of amino acids 122 - 138 relative to SEQ ID NO:4. As introduced above, the leukocyte - associated immunoglobulin - like receptor 2 domain of human LAIR - 2 can be QEGALPR PSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHID SVSEGNAGLYRCLYYKPPGWSEHSDFLE 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 framed by cysteines that form disulfide bonds between amino acids 49 - 101 of SEQ ID NO:1 (amino acids 28 - 80 of SEQ ID NO:5, in italics).

[0116] Known variants and mutants of LAIR - 2 relative to SEQ ID NO:4 include G78S, H87R, and F115Y.

[0117] It has been shown that LAIR - 2 in the human body has a higher binding affinity for collagen and SP - D than LAIR - 1 (Meyaard, L., “The inhibitory collagen receptor LAIR - 1(CD305)”, J Leukoc Biol., 83:799 - 803(2008), J.Leukoc.Biol.83:799 - 803). Dr. Linde Meyaard has demonstrated that LAIR - 2 also binds 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 confirmed the evidence of Son et al. that LAIR - 2 binds C1q (Son et al., 2012, Proc.Natl.Acad.Sci.USA 109:E3160 - 3167). Although collagen and C1q are ubiquitously expressed, SP - D is mainly restricted to mucosal surfaces (alveolar surface and gastrointestinal tract), where it functions as the first - line innate defense against pathogens (Herias et al., 2007, Mol.Immunol.44:3324 - 3332).

[0118] An exemplary alignment of the extracellular domains of human LAIR - 1 and human LAIR - 2 is shown below:

[0119]

[0120] The Query is SEQ ID NO:2 and the Sbjct is SEQ ID NO:4.

[0121] III. NC410

[0122] NC410 is a dimeric form of the LAIR-2 protein fused to the human Fc domain of the immunoglobulin (Ig) subtype IgG1. NC410 targets tumor collagen to reverse LAIR-1-mediated immunosuppression and to induce ECM remodeling to promote infiltration and function of immune cells in the TME, as Figure 2 shown.

[0123] Preclinical studies with NC410 in mouse models (HT-29, P815) have shown enhanced T cell expansion (both CD4+ and CD8+ cells), increased production of IFN-γ and granzyme B, and an anti-tumor effect in a dose-dependent manner. Since tumor-associated collagen induces CD8+ T cell exhaustion through LAIR-1-SHP-1 signaling, overexpression of LAIR-2 inhibits the binding of LAIR-1 to collagen, resulting in reduced tumor growth in a lung tumor model. In addition, when LAIR-2 overexpression is combined with anti-PD-1 therapy, growth and metastasis are significantly reduced within 1 week of treatment and persist throughout the treatment (Peng, D.H. et al., “Collagen promotes anti-PD-1 / PD-L1 resistance in cancer through LAIR1-dependent CD8(+)T cell exhaustion”, Nat Commun., 11:4520 (2020)). In addition, it has been demonstrated that NC410 together with an anti-PD-1 inhibitor continuously reduces the tumor burden.

[0124] NC410 has been studied in a variety of in vitro and in vivo systems to support its use as an oncology research agent. These studies have demonstrated enhanced immune activity in mechanistic studies and tumor models. NC410 binds with high affinity to LAIR-1 ligands (including collagen, C1q, MBL, and SP-D) and blocks the interaction of LAIR-1 with its ligands. NC410 reverses the inhibitory effect of collagen on lipopolysaccharide (LPS)-induced NFκB and interferon signaling (important signaling pathways leading to immune cell activation). NC410 promotes primary monocyte activation and differentiation into a stimulatory macrophage phenotype. NC410 enhances human T cell expansion and activation in a dose-dependent manner, which is associated with antitumor efficacy and the production of chemokines such as CXCL10, CXCL11, and CXCL12 in murine P815 and human HT29 tumor models. NC410 promotes the production of T cell-dependent cytokines and chemokines in the tumor microenvironment, which is associated with tumor control and promotes tumor remodeling, as demonstrated by changes in the levels of collagen degradation products.

[0125] A. Protein and polypeptide compositions

[0126] The ECM binder and ICI can be a protein, polypeptide, or fusion protein. For example, the ECM binder and ICI can be an isolated or recombinant protein or polypeptide of LAIR-2 or pembrolizumab as described above, or a functional fragment, variant, or fusion protein thereof.

[0127] The protein or polypeptide or a functional fragment, variant, or fusion protein thereof can be an agonist or antagonist. For example, in some embodiments, an antagonist of LAIR-2 is an LAIR-1 or LAIR-2 polypeptide or a fragment or fusion protein thereof that binds to the ligand of LAIR-2. The polypeptide can be a soluble fragment, such as the extracellular domain of LAIR-2, or a functional fragment, or a fusion protein thereof. In some embodiments, a soluble ligand of LAIR-2 can be used as an agonist to increase signal transduction through LAIR-2.

[0128] The activity (i.e., agonist or antagonist) of a protein or polypeptide of LAIR-2, or any fragment, variant, or fusion protein thereof, can be determined using functional assays known in the art and includes the assays discussed below. Generally, the assays include determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signal transduction through the LAIR-2 receptor. In some embodiments, the assays include determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) the immune response associated with LAIR-2 (i.e., costimulation or coinhibition). Generally, the assays include determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signal transduction through the LAIR-2. In some embodiments, the assays include determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof decreases (i.e., agonist) or increases (i.e., antagonist) the immune response negatively regulated by LAIR-2. In some embodiments, the assays include determining whether the protein, polypeptide, or fragment, variant, or fusion protein thereof increases (i.e., antagonist) apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells, thereby resulting in a reduced self-renewal capacity of AML and ALL stem cells.

[0129] The 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. As discussed in more detail below, one of ordinary skill in the art can use these sequences to prepare any protein or polypeptide of LAIR-1 or LAIR-2, or any fragment, variant, or fusion protein thereof. Generally, the proteins, polypeptides, fragments, variants, and fusions of LAIR-1 and LAIR-2 are expressed from nucleic acids comprising a sequence encoding a signal sequence. The signal sequence is generally excised from the immature polypeptide to produce 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 to affect the expression level, secretion, solubility, or other properties of the polypeptide. LAIR-1 and LAIR-2 with and without signal sequences are disclosed. It should be understood that in some cases, such as the mature proteins known or described in the art, i.e., the protein sequences without the signal sequence, are presumed to be mature proteins. During normal cellular expression, the signal sequence can be removed by cellular peptidases to produce the mature protein. The sequence of the mature protein can be determined or confirmed using methods known in the art.

[0130] i. Fragment

[0131] As used herein, a fragment of LAIR-1 or LAIR-2 refers to any subset of polypeptides that are at least one amino acid shorter than the full-length protein. Useful fragments include those that retain the ability to bind to one or more of their native ligands. Compared to the full-length protein, a polypeptide, i.e., a fragment of any full-length LAIR-1 or LAIR-2, typically has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or even more than 100% of the ability to bind its native ligand.

[0132] Fragments of LAIR-1 and LAIR-2 include cell-free fragments. A cell-free polypeptide can be a fragment of a full-length, transmembrane polypeptide that can be shed, secreted, or otherwise extracted from the producing cell. A cell-free fragment of a polypeptide can include some or all of the extracellular domain of the polypeptide and lack some or all of the intracellular and / or transmembrane domains of the full-length protein. In one embodiment, a polypeptide fragment includes the entire extracellular domain of the full-length protein. In other embodiments, a cell-free fragment of a polypeptide includes a fragment of the extracellular domain that retains 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).

[0133] ii. Variants

[0134] Also provided are variants of LAIR-1 and LAIR-2 and fragments thereof. In some embodiments, a variant has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 2-5. Useful variants include those that increase biological activity (as specified by any assay described herein) or increase the protein half-life or stability. The proteins and polypeptides of LAIR-1 or LAIR-2 and their fragments, variants, and fusion proteins can be engineered to increase biological activity. For example, in some embodiments, a LAIR-2 polypeptide, protein, or fragment, variant, or fusion thereof has been modified to have at least one amino acid substitution, deletion, or insertion that increases its function.

[0135] Other variants are those engineered to selectively bind to one or more types of LAIR-1 and / or LAIR-2 ligands rather than other LAIR-1 and / or LAIR-2 ligands. For example, variants can be engineered to preferentially bind to one or more collagens, SP-D, C1q, or MBL or specific combinations thereof. Preferential binding means that the binding to one type of ligand is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more greater than the binding to another type of ligand.

[0136] There are also other variants that can be engineered to have reduced binding to one ligand compared to another ligand. These variants can be used in combination with variants having stronger binding properties to modulate the immune response with a moderate effect.

[0137] In still other embodiments, variants can be engineered to have reduced binding to one or more collagen binding sites relative to others. As discussed by Brondijk et al., Blood, 18(115):1364-73 (2010), mutations to LAIR-1 residues can have different effects on binding to different collagen ligands. For example, adhesion to immobilized collagens I, III, and IV was significantly reduced in the R59A, E61A, R65A, and E111A mutants, although the magnitude of the effect depended on the type of collagen tested. In addition, adhesion was reduced for the mutants R62A and N69A. In some embodiments, the variant is 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 a specific embodiment, one or more of the mutations are substitutions with alanine.

[0138] Finally, variant polypeptides can be engineered to have an increased half-life relative to the wild type. These variants are typically modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds to resist enzymatic degradation. There are various modifications known in the art for achieving this. Variants can be modified to adjust the effect of affinity for the receptor on the half-life of the protein, polypeptide, fragment, or fusion thereof at serum and endosomal pH.

[0139] iii. Fusion proteins

[0140] The fusion polypeptide has a first fusion partner comprising all or part of the polypeptide LAIR-1 or LAIR-2, which is fused to a second polypeptide directly or via a linker peptide sequence fused to the second polypeptide. The fusion protein optionally contains a domain for dimerizing or multimerizing two or more fusion proteins. The peptide / polypeptide linker domain can be a separate domain or alternatively can be included within one of the other domains (the first polypeptide or the second polypeptide) of the fusion protein. Similarly, the domain for dimerizing or multimerizing the fusion protein can be a separate domain or alternatively can be included within one of the other domains (the first polypeptide, the second polypeptide, or the peptide / polypeptide linker domain) of the fusion protein. In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0141] The fusion proteins disclosed herein have the formula I:

[0142] N-R1-R2-R3-C

[0143] 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 a polypeptide or protein of LAIR-1 or Liar-2, or a fragment or variant thereof, "R2" is an optional peptide / polypeptide linker domain, and "R3" is a second polypeptide. Alternatively, R3 can be a polypeptide or protein of LAIR-1 or LAIR-2, or a fragment or variant thereof, and R1 can 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 framed by cysteines forming disulfide bonds as discussed above.

[0144] Dimerization or multimerization can occur between two or more fusion proteins through the dimerization or multimerization domain. Alternatively, dimerization or multimerization of the fusion protein can occur by chemical crosslinking. The dimers or multimers formed can be homodimeric / homomultimeric or heterodimeric / heteromultimeric.

[0145] In some embodiments, the fusion protein comprises the extracellular domain of LAIR-1 or LAIR-2, or a fragment or variant thereof, fused to the Ig Fc region. The recombinant Ig fusion protein can be prepared by fusing the coding region of the extracellular domain or a fragment or variant thereof to the Fc region of human IgG1, IgG2, IgG3, or IgG4 or murine IgG2a or other suitable Ig domains, as described previously (Chapoval et al., Methods Mol. Med., 45:247-255 (2000)).

[0146] iv. Exemplary fusion proteins

[0147] Exemplary fusion proteins are provided below. The signal sequence is indicated by double underlining, the LAIR-2 extracellular domain is indicated by single underlining, and the Ig domain is indicated in italics. The signal sequence is typically removed in the mature protein. Additionally, a signal peptide from another polypeptide or organism can be used (e.g., substituted) to enhance secretion of the fusion protein from the host during manufacture.

[0148] In some embodiments, the human LAIR2-hIg fusion protein (hIgG1) (hLAIR2.hG1) has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with the following amino acid sequence:

[0149]

[0150] This sequence may or may not have a signal sequence.

[0151] SEQ ID NO:6 without the signal sequence is

[0152]

[0153] The human LAIR2-hIg fusion protein (hIgG1) (hLAIR2.hG1) can act as an antagonist of LAIR-1 signaling by serving as a decoy for the LAIR-1 ligand and can be used to treat cancer or infectious diseases.

[0154] In some embodiments, the human LAIR2.mIg fusion protein (mIgG2a) has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with the following amino acid sequence:

[0155] This sequence may or may not have a signal sequence.

[0156] SEQ ID NO:8 without the signal sequence is

[0157]

[0158] The human LAIR2.mIg fusion protein (mIgG2a) can be used to generate antagonistic anti-LAIR2 (e.g., mAb or its fragment), which can be used to treat autoimmune diseases.

[0159] IV. Immune checkpoint inhibitors

[0160] A. PD-1 antagonists

[0161] 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 receptor family that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands, B7-H1 or B7-DC, induces an inhibitory response that reduces the intensity and / or duration of T cell proliferation and / or T cell responses. Suitable PD-1 antagonists are described in U.S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416 (which are hereby expressly incorporated by reference in their entirety), and include compounds or agents that bind to and block the ligand of PD-1 to interfere with or inhibit ligand binding to the PD-1 receptor, or compounds or agents that directly bind and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[0162] In some embodiments, the PD-1 receptor antagonist directly binds to the PD-1 receptor without triggering inhibitory signal transduction and also binds to the ligand of the PD-1 receptor to reduce or inhibit ligand-triggered signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligand that binds to the PD-1 receptor and triggers inhibitory signal transduction, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction, and a more robust immune response can be achieved.

[0163] PD-1 signaling is believed to be driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) that is in close proximity to the peptide presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. U.S.A, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent the co-ligation of PD-1 and the TCR on the T cell membrane are also useful PD-1 antagonists.

[0164] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or an antibody that reduces or interferes with PD-1 receptor signal transduction by binding to the ligand of PD-1 or PD-1 itself, particularly when the co-ligation of PD-1 and the TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.

[0165] Other PD-1 antagonists contemplated by the methods of the invention include antibodies that bind to PD-1 or a PD-1 ligand and other antibodies.

[0166] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications (the contents of which are hereby incorporated by reference in their entirety):

[0167] PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196)

[0168] PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168)

[0169] PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708)

[0170] PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771)

[0171] PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286)

[0172] PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875)

[0173] PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174)

[0174] PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874)

[0175] PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533)

[0176] Berger et al., Clin. Cancer Res., 14:3044 - 3051 (2008).

[0177] A specific example of an anti - PD - 1 antibody is the antibody described in Kosak, US20070166281 (published on July 19, 2007), paragraph 42), i.e., a human anti - PD - 1 antibody, which is administered at a dose of 3 mg / kg in some embodiments.

[0178] Exemplary anti - B7 - H1 antibodies include, but are not limited to, those described in the following publications:

[0179] PCT / US06 / 022423 (WO / 2006 / 133396, published on December 14, 2006)

[0180] PCT / US07 / 088851 (WO / 2008 / 083174, published on July 10, 2008)

[0181] US2006 / 0110383 (published on May 25, 2006)

[0182] A specific example of an anti-B7-H1 antibody is the antibody described (WO / 2007 / 005874, published on January 11, 2007), namely a human anti-B7-H1 antibody.

[0183] Additional anti-PD-1 and anti-B7-H1 antibodies are disclosed in 2014 / 0044738, which is hereby incorporated by reference in its entirety.

[0184] For anti-B7-DC antibodies, see 7,411,051, 7,052,694, 7,390,888 and US Published Application No. 2006 / 0099203.

[0185] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, and active fragments of any of the foregoing, and fusion proteins incorporating any of these substances. In some embodiments, the fusion protein comprises the soluble portion of B7-DC coupled 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.

[0186] A PD-1 antagonist can also be a fragment of mammalian B7-H1, such as from a mouse or a primate (such as a human), wherein the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragment can also be part of a fusion protein (such as an Ig fusion protein).

[0187] Other useful polypeptide PD-1 antagonists include those that bind to ligands of the PD-1 receptor. These include the PD-1 receptor protein or soluble fragments 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 signal transduction. B7-H1 has also been shown to bind the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that contains mutations that increase binding to the native ligand, such as the A99L mutation (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof are also useful, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction.

[0188] Antisense nucleic acids (both DNA and RNA) and siRNA molecules against PD-1 and B7-H1 can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands such as B7-H1, PD-L1, and / or PD-L2. For example, siRNA complexed with a carrier such as polyethyleneimine (e.g., about 21 nucleotides in length, specific for the gene encoding PD-1 or the gene encoding a PD-1 ligand, and the oligonucleotide can be readily commercially available) (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244 (2009)) is easily taken up by cells expressing PD-1 and PD-1 ligands and reduces the expression of these receptors and ligands to achieve a reduction in inhibitory signal transduction in T cells, thereby activating T cells.

[0189] B. Pembrolizumab

[0190] Several monoclonal antibodies that inhibit the interaction between PD-1 and 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) that binds with high specificity to the programmed cell death 1 (PD-1) receptor, thereby inhibiting 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 for PD-1 and potent receptor-blocking activity. Pembrolizumab has acceptable preclinical safety characteristics and is currently in clinical development as an intravenous (IV) immunotherapy for advanced malignancies. (Pembrolizumab) is indicated for the treatment of patients with a variety of indications.

[0191] 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. Compositions of pembrolizumab can be administered in combination with one or more compositions that can bind components of the extracellular matrix (ECM) of the tumor microenvironment (TME) such as collagen. As a non-limiting example, the LAIR-2Fc fusion protein (e.g., NC410) can bind collagen. Compositions of pembrolizumab can be administered simultaneously or separately with one or more compositions. Compositions of pembrolizumab can be administered according to a variety of dosing and timing regimens as described herein.

[0192] Over the past decade, several other checkpoint inhibitors have gained widespread attention in cancer treatment due to improved response durability and survival benefits. A number of favorable factors have emerged as predictors of response to ICI, including but not limited to the presence and activation status of tumor-infiltrating T cells, expression of PD-L1, or high tumor mutational burden (TMB). The presence of multiple neoantigens derived from highly mutated tumors leads to increased tumor-infiltrating T cells that favor ICI response. Similarly, somatic mutations in mismatch repair-deficient (dMMR) tumors are increased 10- to 100-fold compared to pMMR (mismatch repair proficient) tumors. dMMR colorectal cancer (CRC) and dMMR non-CRC tumors have an excellent response to pembrolizumab, while pMMR CRC has no response to pembrolizumab (Le, D.T. 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 treatment and have no satisfactory alternative treatment options. It is hypothesized that this favorable response to ICI is due to the presence of a large number of neoantigens in the TME and the accompanying immune cell infiltration. In contrast, patients with MSS / MSI-L / pMMR tumors with low mutational burden and a lack of an “immunocompetent” TME have a disappointing response to ICI. Therefore, there is a high unmet medical need to explore new strategies to enhance the response to ICI in MSS / MSI-L tumors.

[0193] In addition, only 30% of patients with certain types of advanced cancer can benefit from ICI monotherapy or in combination with chemotherapy or other agents, and most patients lose response due to multiple resistance mechanisms, including but not limited to the emergence of T-regs, myeloid-derived suppressor cells (MDSCs), M2 macrophages, TGF-β-driven collagen that promotes anti-PD-1 / PD-L1 resistance, and immune suppression mediated by LAIR-1, including preventing the proliferation and activation of CD8+ and CD4+ T cells.

[0194] V. Therapeutic Compositions

[0195] A. Administration Regimen

[0196] The following compositions should be understood as exemplary compositions relevant to the present disclosure. These are not intended to limit the scope of the present disclosure.

[0197] The compositions described herein can be administered, alone or in combination with a pharmaceutically acceptable excipient and / or carrier, to a subject in need thereof 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 includes, but is not limited to, intravenous, intramuscular, subcutaneous, etc. Responses can include, but are not limited to, specific immune responses, non-specific immune responses, specific and non-specific responses, innate responses, primary immune responses, adaptive immunity, secondary immune responses, memory immune responses, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.

[0198] The present invention provides a method for providing anti-tumor immunity in a mammal by administering an effective amount of a combination therapy to the mammal. As described herein, the combination therapy includes a first composition of an immune checkpoint inhibitor (ICI), and the second composition of the combination therapy exhibits an affinity for an extracellular matrix (ECM) binding component in the tumor microenvironment (TME). One of ordinary skill in the art determines the effective amount of the combination therapy by considering differences in the individual with respect to age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (i.e., subject). Generally speaking, it can be noted that the compositions of the combination therapy can be administered to the subject simultaneously or separately according to the same or different dosing and timing regimens, as described herein. The combination therapy can also be administered in multiple doses at these dosages. The combination therapy can be administered by using infusion techniques well known in immunotherapy (Rosenberg et al., New Eng. J. of Med., 1988). One of ordinary skill in the medical art can readily determine the optimal dose and treatment regimen for a particular patient by monitoring the signs of the patient's disease and adjusting the treatment accordingly. For a particular patient, the effective amount may vary depending on factors such as the disorder being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. Guidance for treatment and diagnostic methods is available (Maynard et al., Interpharm Press, 1996; Dent, Urch Publ., 2001).

[0199] An effective amount of the compositions described herein can be administered in one dose, but is not limited to one dose. Thus, administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more administrations of the composition. When there is more than one administration in the method, the administrations can be separated by time intervals of one minute, two minutes, three minutes, four minutes, five minutes, six minutes, seven minutes, eight minutes, nine minutes, ten minutes or more, separated by intervals of about one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, ten hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, etc. In the context of hours, the term "about" means plus or minus any time interval within 30 minutes. The administrations can also be separated by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten 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 equally spaced dosing intervals in time, but encompasses non-equally spaced doses, such as a priming schedule consisting of administrations on 1 day, 4 days, 7 days and 25 days, which provides only non-limiting examples. In such aspects, various dosing and interval regimens can be used to administer the various compositions. In such aspects, the first composition can be administered in one or more doses separated by a certain time interval, while the second composition can be administered in a different number of doses separated by a different time interval. In such aspects, the first composition and the second composition can differ in composition.

[0200] The compositions of the present invention can be administered in one dose or multiple doses, where each dose comprises 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 one dose or multiple doses, where each dose depends on the body weight of the subject. As a non-limiting example, one or more doses 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 doses. As a non-limiting example, the first composition can be administered in one dose, while the second composition can be administered in another composition.

[0201] For the compositions disclosed herein, available dosing schedules are, for example, 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, once every six weeks, etc. The dosing schedule encompasses a total period of administration of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, and twelve months. In some instances, it can be seen that two different dosing schedules have similar benefit-risk profiles. As a non-limiting example, a dosing schedule of 400 mg once every six weeks may have a similar benefit-risk profile to a subject who experiences a dosing schedule of 200 mg once every three weeks.

[0202] The periods of the above dosing schedules are provided. The period can, for example, repeat approximately every 7 days; every 14 days; every 21 days; every 28 days; every 35 days; every 42 days; every 49 days; every 56 days; every 63 days; every 70 days; etc. A non-dosing interval can occur between one period and the next, where the interval can be approximately, for example, 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 a period. A dosing schedule can be designed for the compositions disclosed herein such that a dose is administered on certain days of a period. As a non-limiting example, a dose can be administered on days 1, 15, and 29 of a repeating 42-day period. As a non-limiting example, the period can be repeated until the subject exhibits an adverse side effect to the dose. The period can alternatively be repeated until the subject's disease is adequately cured. In this context, the term "about" means ± one day, ± two days, ± three days, ± four days, ± five days, ± six days, or ± seven days.

[0203] Methods for co - administration with other 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).

[0204] An effective amount of a therapeutic agent is often to reduce or alleviate symptoms by at least 10%, more often at least 20%, most often at least 30%, usually at least 40%, more usually at least 50%, most usually at least 60%, often at least 70%, more often at least 80% and most often at least 90%, routinely at least 95%, more routinely at least 99% and most routinely at least 99.9%.

[0205] The administration of doses or dosages of the compositions disclosed herein may vary. The dosing and timing regimens can be altered according to factors known in the art. As a non - limiting example, the administration of each dose of NC410 in combination with pembrolizumab can be delayed to allow any observed toxicity to subside. If there is no medical condition or other circumstance that renders the participant unfit for further treatment, the administration can be resumed.

[0206] In some instances, the administration of the compositions disclosed herein should not occur after a certain time has passed. As a non - limiting example, the administration of NC410 should generally not be delayed by more than 28 days between consecutive doses, although this may vary. If a dose of NC410 or any other composition disclosed herein is missed, the subject in need can maintain the original treatment schedule, where NC410 or any other composition disclosed herein is administered at the next scheduled dose.

[0207] Formulations of the therapeutic agent can be prepared for storage by mixing with a physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, slurry, aqueous solution, or suspension.

[0208] Although several aspects have been disclosed in the foregoing specification, those skilled in the art will appreciate that many modifications and other aspects related to the present disclosure can be conceived in light of the foregoing description and the teachings presented in the related drawings. Accordingly, it is to be understood that the present disclosure is not limited to the specific aspects disclosed above, and that many modifications and other aspects are intended to be included within the scope of any claims in which the disclosed subject matter may be recited.

[0209] B. Formulations for Oral Administration

[0210] In embodiments, the combination therapy and related compositions are formulated for oral delivery. Oral delivery can include the following single solid or liquid dosage forms that contain more than one composition (e.g., one composition of an immune checkpoint inhibitor and a second composition of an extracellular matrix binding component) or one or more solid or liquid dosage forms in which the compositions are separated. Dosage forms formulated for oral administration can be administered according to the dosing and timing regimens described herein. Oral solid dosage forms are generally described in Remington's Pharmaceutical Sciences, 18th Edition 1990 (Mack Publishing Co., Easton Pa. 18042), Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders or granules, or particulate preparations incorporating the material into polymeric compounds such as polylactic acid, polyglycolic acid, etc. or incorporated into liposomes. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosure. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton, Pa. 18042), pages 1435 - 1712, which is incorporated herein by reference. The compositions can be prepared in liquid form or can be in dry powder (e.g., lyophilized) form. Liposome or proteoid encapsulation can be used to formulate the compositions. Liposome encapsulation can be used and the liposomes can be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K., Modern Pharmaceutics, edited by G.S. Banker and C.T. Rhodes, Chapter 10, 1979. Generally, the formulations will contain a peptide (or a chemically modified form thereof) and inert ingredients that protect the peptide in the gastric environment and release the bioactive material in the intestine.

[0211] The agent can be chemically modified such that oral delivery of the derivative is effective. Generally, the chemical modifications considered are attaching at least one moiety to the component molecule itself, where the moiety permits absorption from the stomach or intestine into the bloodstream, or directly into the intestinal mucosa. It is also desirable to enhance the overall stability of one or more components and increase the circulation time in the body. PEGylation is an exemplary chemical modification for drug use. Other moieties that can be used include: copolymers of propylene glycol, ethylene glycol, and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-trioxocane [see, for example, Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Edited by Hocenberg and Roberts (Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark et al. (1982) J. Appl. Biochem. 4:185-189].

[0212] Another embodiment provides a liquid dosage form for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components, including inert diluents; adjuvants such as wetting agents, emulsifying agents, and suspending agents; and sweetening, flavoring, and aromatic agents.

[0213] Controlled-release oral formulations may be desirable. The agent can be incorporated into an inert matrix that permits release by diffusion or leaching mechanisms, such as gums. Slowly degradable matrices can also be incorporated into the formulation. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), which involves enclosing the drug in a semipermeable membrane that permits water to enter and push out the drug through a single small opening due to osmosis.

[0214] For oral formulations, the release location can be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. In some embodiments, the release will avoid the harmful effects of the gastric environment, either by a protective agent (or derivative) or by releasing the agent (or derivative) outside the gastric environment (such as in the intestine). To ensure complete gastric resistance, a coating that is impermeable at least at pH 5.0 is crucial. Examples of more common inert ingredients used as enteric coatings are: cellulose acetate phthalate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D TM 、Aquateric TM, cellulose acetate phthalate (CAP), Eudragit L TM , Eudragit S TM and Shellac TM . These coatings can be used as mixed films.

[0215] C. Manufacturing methods

[0216] i. Methods for preparing antibodies

[0217] Antibodies can be generated in cell cultures, phages, or various 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 primary antibodies or generate variants with altered specificity or affinity characteristics. Such techniques are routine and well-known in the art. In one embodiment, the antibody is produced by recombinant methods known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect the host cells with the DNA sequence expressing at least one VL region and one VH region. Exemplary descriptions of recombinant methods 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, latest version).

[0218] The disclosed antibodies can be modified by recombinant methods to increase the efficacy of the antibody in mediating desired functions. Accordingly, modification of the antibody by substitution using recombinant methods is also within the scope of the invention. Typically, the substitution will be a conservative substitution. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and Angal et al., Mol. Immunol. 30:105-08 (1993). Modifications of amino acids include deletion, addition, and substitution of amino acids. In some cases, such changes are made to reduce undesired activities, such as complement-dependent cytotoxicity. Typically, antibodies are labeled by covalently or non-covalently linking a substance that provides a detectable signal. A variety of labeling and conjugation techniques are known and widely reported in both the scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, or fusion proteins of LAIR-1, LAIR-2, or pembrolizumab. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0219] For example, in vitro assays can be used to identify suitable antibodies having desired biological activities, including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays (such as inhibition of tumor growth). The antibodies provided herein can also be used in diagnostic applications. As capture antibodies or non-neutralizing antibodies, their ability to bind to a specific antigen can be screened without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be used in competitive binding assays.

[0220] Antibodies that can be used in the disclosed compositions and methods include intact immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. In an antibody, the sequences of the variable domains are different and are used for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not often evenly distributed throughout the variable domain of the antibody. In both the light and heavy chain variable domains, it is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domain are called frameworks (FRs). The variable domains of native heavy and light chains each contain four FR regions, mostly adopting a β-sheet configuration, connected by three CDRs, forming loops that connect the β-sheet structures and in some cases forming part of the β-sheet structure. The CDRs in each chain are held very close together by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody.

[0221] Also disclosed are bioactive antibody fragments. Whether or not attached to other sequences, the fragments include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0222] This technology can also be applied to generate single-chain antibodies specific for antigenic peptides. Methods for generating single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be created by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstructing the 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 tethered to the N-terminus of the other variable domain via a 15- to 25-amino acid peptide or linker, without significantly disrupting antigen binding or binding specificity. The linker is chosen to allow the heavy and light chains to bind together in their appropriate conformational orientations.

[0223] Diabody 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 regions and two VL regions, thereby producing tandem scFvs. ScFvs can also be designed to have a linker peptide that is too short for the two variable regions to fold together (about five amino acids), thus forcing the scFv to dimerize. This type is called a diabody. Diabodies have been shown to have a dissociation constant that is 1 / 40 of that of the corresponding scFv, meaning that they have a much higher affinity for their target. Even shorter linkers (one or two amino acids) result in the formation of trimers (triabodies or trisomes). Tetrabodies have also been produced. They exhibit an even higher affinity for their target than diabodies.

[0224] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequences of an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequences of an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided that they exhibit the desired antagonistic activity.

[0225] Any method for producing monoclonal antibodies can be used to prepare 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 will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0226] Antibodies can also be prepared by recombinant DNA methods. DNA encoding the disclosed single-chain antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). Antibody or active antibody fragment libraries can also be generated and screened using phage display techniques.

[0227] Methods for preparing antibodies using protein chemistry are also known in the art. One method for generating a protein comprising an antibody is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using Fmoc (9-fluorenylmethoxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry with currently available laboratory equipment. (Applied Biosystems, Inc., Foster City, CA). One of ordinary skill in the art can readily appreciate that, for example, peptides or polypeptides corresponding to antibodies can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin, while another fragment of the antibody can be synthesized and subsequently cleaved from the resin, thereby exposing terminal groups that are functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently linked via peptide bonds at their carboxyl and amino termini, respectively, to form an antibody or a fragment thereof. Alternatively, the peptides or polypeptides are synthesized independently in vivo. Once isolated, these independent peptides or polypeptides can be linked via similar peptide condensation reactions to form an antibody or its antigen-binding fragment.

[0228] For example, enzymatic ligation of cloned or synthetic peptide segments permits relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthesize large peptides or polypeptides from shorter peptide fragments. The method consists of two chemical reactions. The first step is a chemoselective reaction of an unprotected synthetic peptide-α-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without changing the reaction conditions, this intermediate undergoes a spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.

[0229] ii. Methods for producing proteins

[0230] The disclosed proteins, polypeptides, fragments, variants, and their fusions can be made 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 its fusion, a nucleic acid containing the nucleotide sequence encoding the protein, polypeptide, fragment, variant, or its fusion can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). Generally, a nucleic acid construct includes regulatory sequences operably linked to the nucleotide sequence encoding the protein, polypeptide, fragment, variant, or its fusion. Regulatory sequences (also referred to herein as expression control sequences) generally do not encode a gene product but affect the expression of the nucleic acid sequence operably linked to them.

[0231] Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art and include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

[0232] In eukaryotic host cells, a variety of virus-based expression systems can be utilized to express fusion proteins. Virus-based expression systems are well known in the art and include, but are not limited to, virus vectors based on baculovirus, SV40, retrovirus, or vaccinia virus.

[0233] Expression vectors with appropriate control elements and selectable markers can be used to generate mammalian cell lines that stably express a protein, polypeptide, fragment, variant, or its fusion. 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 a protein, polypeptide, fragment, variant, or its fusion in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human umbilical vein endothelial cells (HUVEC). Additional suitable expression systems include the GS Gene Expression System available from Lonza Group Ltd TM .

[0234] After introduction of an expression vector by electroporation, lipofection, calcium phosphate or calcium chloride co - precipitation, DEAE - dextran or other suitable transfection methods, 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 such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, cell culture supernatants or lysed cells. Alternatively, proteins, polypeptides, fragments, variants or fusions thereof can be produced by (a) ligating an amplified sequence to a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies) and (b) performing in vitro transcription and translation using wheat germ extract or rabbit reticulocyte lysate.

[0235] Proteins, polypeptides, their fragments, variants or fusions thereof can be separated using, for example, 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 that contain amino acid sequences that allow the polypeptide to be captured on an affinity matrix. For example, an Fc - fusion polypeptide in cell culture supernatant or cytoplasmic extract can be separated using a protein A column. Additionally, tags such as c - myc, hemagglutinin, polyhistidine or Flag TM (Kodak) etc. can be used to aid in polypeptide purification. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides. Fusion proteins can also be engineered to contain a secretion signal (if one is not already present), which causes the protein, polypeptide, fragment, variant or fusion thereof to be secreted by the cell that produces it. The secreted protein, polypeptide, fragment, variant or fusion thereof can then be conveniently isolated from the cell culture medium.

[0236] iii. Methods for generating isolated nucleic acid molecules

[0237] Isolated nucleic acid molecules can be produced by standard techniques, including but not limited to conventional molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain isolated nucleic acids encoding variant polypeptides. PCR is a technique in which a target nucleic acid is enzymatically amplified. Typically, sequence information from the ends or beyond the region of interest can be used to design oligonucleotide primers that are identical in sequence to the opposite strand of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. The length of the primers is typically from 14 to 40 nucleotides, but can range from 10 nucleotides to several hundred nucleotides. General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, edited by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When RNA is used as the template source, reverse transcriptase can be used to synthesize complementary DNA (cDNA) strands. 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, for example, 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.

[0238] Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., automated DNA synthesis in the 3' to 5' direction using phosphoramidite techniques). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) containing the desired sequence can be synthesized, where each pair contains short segments (e.g., about 15 nucleotides) with complementarity such that a duplex is formed when the oligonucleotide pairs are annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in the production of a single double-stranded nucleic acid molecule from each oligonucleotide pair, which can then be ligated into a vector. Isolated nucleic acids can also be obtained by mutagenesis. Standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis by PCR, can be used to mutate nucleic acids encoding proteins. See Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.

[0239] D. Assays and Antibody Screening

[0240] The generation of LAIR-2Fc fusion protein (“LAIR-2-Fc”) for cancer therapy bypasses the need to develop and screen LAIR-1mAb. Since LAIR-2 has greater affinity than LAIR-1, in some embodiments, LAIR-2-Fc is selected over LAIR-1Fc fusion protein (“LAIR-1-Fc”) for therapeutic treatment. In some embodiments, LAIR-1-Fc can be used in mouse preclinical models as LAIR-2 is absent in mice.

[0241] a. Assays for LAIR-2-Fc

[0242] 1. Confirm the ability to bind multiple forms of collagen SP-D, C1q, and MBL by ELISA.

[0243] 2. Confirm 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) ELISA competition assay, and 2) flow cytometry using LAIR-1 transfected cells incubated in the presence of titrated LAIR-2-Fc and fluorescently labeled LAIR ligand.

[0244] 3. Analyze the binding affinity of LAIR-2-Fc to ligands compared to LAIR-1.

[0245] 4. Functional assays to confirm that LAIR-2-Fc blocks signal transduction by cells expressing LAIR-1. Reporter cells can be used for these assays, or primary LAIR-1+ cells are another option.

[0246] E. Methods of Use

[0247] To date, evidence suggests that the LAIR-1 cell surface receptor has an inhibitory role and that LAIR-2 indirectly antagonizes the function of LAIR-1 by binding to the same ligands as LAIR-1 and thus essentially acts as a decoy receptor. The tumor microenvironment is typically rich in extracellular matrix proteins (ECM), including the LAIR-1 ligand collagen (Rygiel et al., 2011, Mol. Immunol. 49:402-406). Thus, cells expressing LAIR-1 that are localized to the tumor microenvironment can be particularly inhibited through collagen crosslinking of LAIR-1 and subsequent inhibitory signaling. It has been shown that increased LAIR-1 expression and signaling inhibits the proliferation, differentiation, and function of multiple immune cell subsets and is thus thought to inhibit anti-tumor immunity, particularly in tumor microenvironments with high levels of the LAIR-1 ligands collagen C1q and SP-D.

[0248] Both collagen and C1q have been shown to restrict or alter the differentiation and activation of antigen-presenting cells (monocytes / macrophages / dendritic cells (DC)) through LAIR-1. It has also been found that LAIR-1 is also expressed on NK and T cells, but its expression level is much lower than that on APCs. Nevertheless, studies have shown that crosslinking LAIR-1 on NK and T cells can inhibit proliferation and function. Thus, it is believed that reducing LAIR-1 crosslinking can increase the immune response to cancer and infectious diseases. It is believed that increased LAIR-2 levels promote anti-tumor immunity through the same mechanism. Thus, soluble LAIR-1 and soluble 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, the 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 has a higher affinity for binding ligands than LAIR-1.

[0249] Alternatively, signaling through LAIR-1 on AML cancer cells expressing high levels of LAIR-1 maintains the self-renewal capacity or "stemness" of AML cells by inhibiting apoptosis and differentiation through the unique LAIR-1-SHP-1-CAMK1-CREB pathway (Kang et al., 2015, Nat. Cell Biol. 17:665-677). In these cancers, reduced LAIR-1 signaling leads to AML cell death. Thus, blocking (i.e., antagonizing) LAIR-1 signaling on leukemia is considered a treatment to eradicate leukemia. Accordingly, blocking LAIR-1 signaling using 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) can be used to treat leukemia by directly inhibiting cancer cell survival and by promoting an anti-tumor immune response.

[0250] Conversely, reduced LAIR-1 expression or function is associated with multiple autoimmune manifestations, and, simultaneously, overexpression of LAIR-2 may promote autoimmunity through decoy binding of LAIR-1 ligands. LAIR-2 binding to LAIR-1 ligands can essentially reduce cell surface cross-linking of LAIR-1, thereby defining an inhibitory signaling pathway that results in hyperreactive immune function. Thus, it is believed that, for example, in the case of autoimmune diseases or inflammation, increasing LAIR-1 cross-linking can reduce hyperactive or inappropriate immune responses. For example, mAb blocking of LAIR-2 can be utilized to treat autoimmune diseases because this increases ligand binding to LAIR-1, thereby downregulating the immune response. Targeting LAIR-2 is particularly effective for diseases in which there is an imbalance between cell surface LAIR-1 and soluble LAIR-2 expression, as demonstrated in rheumatoid arthritis (Lebbink et al., 2008, J. Immunol 180:1662-1669).

[0251] Exemplary methods are discussed in more detail below.

[0252] i. Treatment strategies

[0253] Methods are provided for inducing or enhancing an immune response in a subject. Generally, the methods include administering to the subject an effective amount of an immunomodulator, or cells primed ex vivo with an immunomodulator. The immune response can be, for example, a primary immune response to an antigen or an increase in effector cell function, such as an increase in antigen-specific proliferation of T cells, enhanced cytokine production by T cells, stimulation of differentiation, or a combination thereof. In some embodiments, the agent can promote the development of naive T cells into Th1, Th17, Th22, or other cells that secrete inflammatory molecules, or cause other cells to secrete inflammatory 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 MMP. In some embodiments, the agent can reduce or inhibit the activity of Tregs, reduce the production of cytokines (such as IL-10) by Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the ratio of Tregs in an immune cell population, or reduce the survival of Tregs. An effective amount of an immunomodulator can be administered to a subject in need thereof 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.

[0254] The methods can be used in vivo or ex vivo as therapeutic applications for stimulating an immune response. Thus, in some embodiments, the agent or a nucleic acid encoding the agent is administered directly to the subject. In some embodiments, the agent or a nucleic acid encoding the agent is contacted ex vivo with cells (such as immune cells), and the treated cells are administered to the subject (such as adoptive transfer). Generally, the disclosed immunomodulators can be used to treat subjects suffering from or susceptible to any disease or disorder in which the subject's immune system mounts an immune response against the disease or disorder. The agent can enable a more robust immune response. The disclosed compositions can be used to stimulate or enhance an immune response involving T cells.

[0255] Immunomodulators for enhancing an immune response are generally those that reduce LAIR-1 expression, ligand binding, crosslinking, negative signaling, or combinations thereof. For example, the agent can be an antagonist of LAIR-1, 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 their supplementary information, which are hereby incorporated by reference in their entirety). In some embodiments, the LAIR-1 antagonist (such as a function-blocking antibody or a functional fragment thereof) specifically binds to an epitope of LAIR-1, including one or more of R59, E61, R62, E63, R65, S66, Y68, N69, I102, R100, W109, E111, Q112, and Y115 (e.g., relative to SEQ ID NO:1). The agent can also be a LAIR-1 polypeptide, such as a soluble polypeptide or a fusion protein thereof, which can serve 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, which can serve as a decoy receptor for one or more LAIR-1 ligands.

[0256] For example, in some embodiments, an effective amount of a LAIR-2 fusion protein (such as LAIR-2-Fc) is administered to a subject suffering from cancer or an infection. Treating a patient with LAIR-2-Fc results in reduced LAIR-1 receptor crosslinking and subsequently reduced inhibitory signaling of LAIR-1+ cells and improved immune function. Increasing the ratio towards soluble LAIR-2 levels, as compared to cell surface LAIR-1, especially in a tumor microenvironment where LAIR-1 / 2 ligands are highly expressed, will be beneficial for enhanced anti-tumor immunity.

[0257] A tumor microenvironment with an immune infiltrate having high levels of both collagen SP-D and / or C1q and expressing high levels of LAIR-1 is ideal for the disclosed immunotherapies (such as LAIR-2-Fc immunotherapy). While ovarian cancer has high levels of collagen, it is not clear whether SP-D and C1q levels are also high. However, lung cancer and gastrointestinal cancers may have high levels of both collagen and SP-D and may thus be cancers targeted with LAIR-2-Fc. In other embodiments, soluble LAIR-2, soluble LAIR-1, or a LAIR-1 fusion protein (e.g., LAIR-1-Fc) is utilized. In some embodiments, LAIR-2-based molecules can be selected because LAIR-2 binds ligands with a higher affinity than LAIR-1.

[0258] LAIR-1 blockade using, for example, a function-blocking anti-LAIR-1 antibody can be an alternative or supplement to soluble LAIR-1 and LAIR-2 polypeptides and fusion proteins. For example, in some embodiments, LAIR-1 blockade is combined with a decoy receptor, such as soluble LAIR-1 or LAIR-2 or a fusion protein thereof. The combination therapy (e.g., LAIR-2-Fc and LAIR-1 blockade) can be complementary.

[0259] In some embodiments, immunostimulatory therapy (e.g., in the treatment of cancer or infection) includes depletion of LAIR-1+ cells. LAIR-1 is highly expressed in murine and human ovarian cancer ascites. The upregulation of LAIR-1 is restricted to immunoregulatory macrophages and F4 / 80+ DCs, both of which co-express high levels of PD-L1. Thus, depletion of cells expressing LAIR-1 will improve the overall condition of the tumor microenvironment by removing the immunoregulatory population. Although LAIR-1 expression on other cell subsets in ovarian cancer has not been observed, depletion of other LAIR-1+ cells also has the effect of reducing immunosuppression and enhancing anti-tumor immunity due to the general inhibitory nature of LAIR-1. It has been shown that LAIR-1 is also expressed on the surface of acute myeloid leukemia cancer and is essential for its development (Kang et al., Nature Cell Biology, Vol. 17, No. 5, 2015; pp. 665-679). Thus, depletion of hematopoietic (“blood”) cancers using LAIR-1-depleting mAb will have a direct effect of reducing or eliminating LAIR-1-positive cancers.

[0260] ii. Treatment of cancer

[0261] The disclosed compositions and methods can be used to treat cancer. Generally, the agent is used to stimulate or enhance the immune response of a subject to cancer by administering to the subject a combination of an immunomodulator that reduces LAIR-1 expression, ligand binding, crosslinking, negative signaling, or a combination thereof, and an immune checkpoint inhibitor (ICI). Administration includes injection, infusion, and other such known administration methods according to the dosing and timing regimens disclosed herein. The method can reduce one or more symptoms of cancer.

[0262] The immune system has been shown to be capable of defending against cancer development and growth. The regulation of the immune response is governed by cell surface interactions that direct immune cells to function along specific pathways, including activation or inhibition of cancer cells. LAIR-1 is an inhibitory receptor on the surface of several subsets of immune cells (white blood cells) that prevents optimal immune responses. LAIR-2 is a soluble homolog that acts as a decoy to block LAIR-1-mediated inhibition. Pembrolizumab is an ICI that interacts with the PD-1 pathway.

[0263] In one embodiment, the LAIR-2Fc fusion protein promotes immune responses in vitro and in vivo. In another embodiment, LAIR-2Fc slows tumor growth and promotes survival. In still another embodiment, LAIR-2Fc promotes anti-PD-1 immunotherapy in combination with ICI (e.g., pembrolizumab). The data provided herein demonstrate that LAIR-1mAb has in vitro activity in human T cells and myeloid cell lines, showing agonist and antagonist activities specific to particular mAb clones. These findings demonstrate the potential LAIR-1 pathway modulation by LAIR-2Fc or LAIR-1mAb for immunotherapeutic intervention in cancer and other diseases.

[0264] 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.

[0265] Although by various mechanisms, cancer cells acquire a characteristic repertoire of functional capabilities during their development. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, unlimited replicative potential, and sustained angiogenesis. The term "cancer cell" is intended 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 spread to distant sites. In still other embodiments, cancer is associated with specific cancer antigens (e.g., pan-cancer antigen (KS1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0266] The methods and compositions disclosed herein can be used to treat or prevent a variety of cancers or other hyperproliferative diseases, including (but not limited to) the following: carcinomas, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer; including squamous cell carcinoma; hematopoietic tumors of the lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of the myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular carcinoma of the thyroid, and teratocarcinoma.

[0267] Cancers caused by abnormal apoptosis can also be treated by the disclosed methods and compositions. Such cancers can include, but are not limited to, follicular lymphoma, cancers with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovary, and premalignant lesions (such as familial adenomatous polyposis and myelodysplastic syndromes). In certain embodiments, malignant tumors or abnormal proliferative changes (such as 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 certain embodiments, sarcomas, melanomas, or leukemias are treated or prevented by the methods and compositions.

[0268] The disclosed compositions and methods are particularly useful for treating cancers associated with cells that express abnormally high levels of LAIR-1, high levels of LAIR-1 ligand, low levels of LAIR-2, or combinations thereof.

[0269] Specific cancers and related disorders that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, leukemia, including but not limited to acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia leukemia, and myelodysplastic syndrome, chronic leukemia such as but not limited to chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphoma such as but not limited to Hodgkin's disease or non-Hodgkin's disease, lymphoma (e.g., 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, such as but not limited to 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; bone and connective tissue sarcomas, such as but not limited to osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including but not limited to glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget’s disease, and inflammatory breast cancer; adrenal cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vasoactive intestinal polypeptide-secreting tumor, somatostatin-secreting tumor, and carcinoid or islet cell tumors;Pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer, including but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and 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 cancer, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, including but not limited to adenocarcinoma, fungating (polypoid), ulcerative, superficially spreading, diffusely 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; bile duct cancer, including but not limited to papillary, nodular, and diffuse; lung cancer, including but not limited to 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 germ cell tumors, seminoma, anaplastic, classic (typical), spermatocytic nonseminomatous carcinoma, embryonal carcinoma, teratocarcinoma, 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 cell carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; throat cancer, 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, superficially spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, renal epithelioma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor;Bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelioma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for a review of such conditions, 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 U.S.A., Inc., United States of America).;

[0270] It should be emphasized that the above aspects are only possible examples of embodiments, presented merely for a clear understanding of the principles of the present disclosure. Any process description or block in a flowchart should be understood to represent a module, segment, or portion of code that includes one or more executable instructions for implementing a specific logical function or step in the process, and may include alternative embodiments that may not include or perform the function at all, which may be executed in an order different from that shown or discussed, including substantially simultaneously or in the reverse order, depending on the functions involved, as would be understood by a reasonably skilled person in the art of the present disclosure. Many variations and modifications can be made to the above one or more aspects without substantially departing from the spirit and principles of the present disclosure. In addition, the scope of the present disclosure is intended to cover any and all combinations and sub-combinations of all the elements, features, and aspects discussed above. All such modifications and variations are intended to be included within the scope of the present disclosure, and all possible claims directed to each aspect or combination of elements or steps are intended to be supported by the present disclosure.

[0271] Example

[0272] Example 1. Safety and Tolerability Study of NC410 in Combination with Pembrolizumab.

[0273] Methods:

[0274] Safety and tolerability will be evaluated by monitoring the frequency, duration, and severity of adverse events (AEs). Toxicity grading will conform to NCI CTCAE v5.0. The study will be divided into two phases, including Phase 1b and Phase 2, as described below. The Phase 1b portion of the study will enroll participants with advanced unresectable and / or metastatic solid tumors (including CRC, gastric cancer (including GE junction cancer), esophageal cancer, ovarian cancer, and head and neck cancer), regardless of prior treatment with immune checkpoint inhibitors (ICI) or microsatellite stable / microsatellite unstable (MSS / MSI) status, as Figure 3 shown. Participants include males and females 18 years of age and older. Participants must provide written informed consent and have adequate organ function.

[0275] Participants must have measurable disease based on RECIST v1.1 and agree to undergo biopsies of non-target lesions before and during treatment. RECIST v1.1 will be adjusted to account for the unique tumor response characteristics seen with immunotherapy (Chiou, V.L. et al., “Pseudoprogression and Immune-Related Response in Solid Tumors”, J Clin Oncol., 33:3541-3 (2015)). Immunotherapeutic agents can produce antitumor effects by enhancing the endogenous cancer-specific immune response. The response patterns observed by such methods may exceed the typical response time course seen with cytotoxic agents and can exhibit a clinical response after an initial increase in tumor burden or even the appearance of new lesions. Standard RECIST may not provide an accurate assessment of response to immunotherapeutic agents. Immune Therapy RECIST (iRECIST) is RECIST 1.1 adjusted as described below to account for the unique tumor responses seen with immunotherapy. After the determination of sites of progressive disease, site investigators will use iRECIST to evaluate tumor response progression and make treatment decisions. Archival tissue may be submitted in lieu of fresh biopsies prior to treatment. Biopsies during treatment will be fresh biopsies. If a participant plans to have a tumor biopsy for the purposes of this study and it is subsequently determined that tumor tissue cannot be safely obtained, the participant may still be enrolled in this study. Participants with certain severe medical conditions (other than the diagnosis of cancer) will be excluded from participating in the trial.

[0276] The recommended Phase 2 dose (RP2D) of NC410 (LAIR-2-Fc) in combination with standard-dose pembrolizumab will be defined during the Phase 1b safety and tolerability study in patients with advanced unresectable and / or metastatic ICI-refractory solid tumors (regardless of MSI status) or untreated MSS / MSI-low solid tumors.

[0277] All eligible participants will be assigned through non-random allocation after signing the informed consent form (ICF) and will be given a unique participant number, with the first 4 digits used as the site number. Participant numbers are managed by each site. Even if a participant is not treated with the study drug (i.e., screening failure), the study site must complete all applicable CRFs for participants who consent to participate in the trial. Dose levels and cohort assignments will be made at the time of participant enrollment and directed by the sponsor or designee. Dose levels and cohort assignments will be maintained within the CRF according to the CRF completion guidelines and managed by the sponsor / CRO.

[0278] Participants will receive pembrolizumab on Day 1 of each 42-day cycle, followed by the administration of NC410. As Figure 4 shown and described in Table 1 below, additional doses of NC410 will be given on Days 15 and 29 of each 42-day cycle or once weekly.

[0279] Table 1. Trial Treatments

[0280]

[0281] Abbreviations: IV = intravenous; Q2W = every 2 weeks; Q6W = every 6 weeks; RP2D = recommended Phase 2 dose.

[0282] * Depends on dose level.

[0283] The study will be conducted in two phases. Phase 1b will include dose escalation of NC410 to determine the optimal dosing schedule and RP2D when combined with standard-dose pembrolizumab. Phase 2 will include dose expansion to evaluate the recommended Phase 2 dose (RP2D) of NC410 when combined with standard-dose pembrolizumab. Participants will continue until progressive disease occurs, unacceptable adverse events (AEs), concurrent diseases that impede further treatment administration, the researcher decides to withdraw the participant, the participant withdraws consent, the participant becomes pregnant, non-compliance with trial treatment or procedural requirements, the participant receives 18 doses (approximately 2 years) of pembrolizumab treatment, or administrative reasons require treatment to be stopped.

[0284] Participants who stop for reasons other than progressive disease will be followed for disease status after treatment until progressive disease, 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 study end. After treatment ends, each participant will be followed for 30 days for AE monitoring. Serious adverse events (SAEs) and events of clinical interest (ECIs) will be collected within 90 days after treatment ends or within 30 days after treatment ends (if the participant starts a new anti-cancer therapy), whichever is earlier.

[0285] In the Phase 1b, a modified Toxicity Probability Interval (mTPI) with an approximately 30% target dose-limiting toxicity (DLT) rate (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 applied for dose escalation and confirmation to determine the recommended Phase 2 dose (RP2D) of NC410 in combination with pembrolizumab.

[0286] During the Phase 1b part of this study, five dose levels of NC410 will be evaluated. NC410 is in a frozen liquid form and is formulated for intravenous infusion. The drug is provided at concentrations of 1 mg / mL (16 mg / vial) and 20 mg / mL (320 mg / vial).

[0287] The pre-determined dose levels of NC410 (in combination with a fixed dose of 400 mg of pembrolizumab) will be explored independently. The dose levels are as follows:

[0288] Dose Level -1 (de-escalating dose): 15 mg

[0289] Dose Level 1 (starting dose): 30 mg

[0290] Dose Level 2: 60 mg

[0291] Dose Level 3: 100 mg

[0292] Dose Level 4: 200 mg

[0293] The starting dose will be 30 mg (dose level 1) of NC410. If the starting dose of NC410 is considered intolerable in combination with pembrolizumab, a reduced dose of NC410 will be available. All dose escalation and de-escalation decisions will be based on the occurrence of dose-limiting toxicity (DLT) at the given dose during the first 42-day period (i.e., the first cycle) (also known as the DLT observation period) and will be made jointly by the investigator and the sponsor. All DLTs will be evaluated by the investigator using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. At each dose level of NC410 and in each cohort up to phase 2, the dose of pembrolizumab will remain constant at 400 mg administered once every 6 weeks.

[0294] 400 mg of pembrolizumab will be administered by intravenous (iv) infusion over at least 30 minutes on day 1 of each 42-day cycle, followed by an iv infusion of NC410 for at least 30 minutes, with a 30- to 60-minute interval between each administration of study treatment. However, a window of -5 minutes to +10 minutes is allowed (i.e., infusion time of 30 minutes (-5 minutes / +10 minutes)) to account for differences in infusion pumps from site to site.

[0295] NC410 will also be administered on days 1, 15, and 29 of each 42-day cycle or once weekly at 15 mg, 30 mg, 60 mg, 100 mg, or 200 mg as a single solution or in combination with pembrolizumab. NC410 may also be administered once weekly at 100 mg as a single solution or in combination with pembrolizumab. Participants will continue until progressive disease occurs, unacceptable AEs, concurrent illnesses that impede further administration of treatment, the investigator decides to withdraw the participant, the participant withdraws consent, the participant becomes pregnant, non-compliance with trial treatment or procedural requirements, the participant receives 18 (approx. 2 years) administrations of pembrolizumab, or for administrative reasons requiring discontinuation of treatment.

[0296] In Figure 5 the number of treated participants is indicated in the columns, and the number of participants experiencing DLT is indicated in the rows. Figure 5 The dosing decisions shown in

[0297] As a non-limiting example, if 3 participants are enrolled, the dose escalation rule can proceed as follows: If none of the first 3 participants at a given dose level experience a DLT, the dose can be escalated and the next available participant enrolled into the next level cohort without further expansion. If 1 of the first 3 participants at a given dose level experiences a DLT, no more than an additional 3 participants should be enrolled at that dose level until additional DLT data are obtained. If all 3 additional participants experience DLT (i.e., 4 out of 6 participants), the dose will be considered to have unacceptable toxicity. If 2 of the first 3 participants at a given dose level experience DLT, the dose will be de-escalated to a lower dose level cohort. If 3 of the first 3 participants at a given dose level experience DLT, the dose will be considered to have unacceptable toxicity (i.e., the dose will be de-escalated and never escalated back to this dose). According to Figure 5 , the same principles will apply regardless of whether 3, 4, 5, or 6 participants are enrolled in the same dose cohort.

[0298] Based on the mTPI design, the number of participants enrolled at a given dose but not yet fully evaluated for DLT assessment may not exceed the number of remaining participants at risk of experiencing DLT before the dose is considered to have unacceptable toxicity (represented as DU in Figure 5 ). To determine how many more participants can be enrolled at a given dose level, the number of steps along the diagonal (down and to the right) from the current cell to the first cell marked DU can be counted. A total of 3 to 14 participants can be enrolled at a given dose level.

[0299] After any one of the doses has been selected for treatment in 10 evaluable participants, dose escalation and confirmation will end. The pool-adjacent-violators-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 the DLT rate at each dose.

[0300] The dose with the DLT rate closest to 30% can be used as the initial maximum tolerated dose (MTD) for treatment. All data will be considered before selecting a dose to enter phase 2, and the escalation schedule can be adjusted based on the pharmacokinetics (PK), pharmacodynamics (PD), and safety data that emerge throughout the study to determine the RP2D.

[0301] If participants within the DLT observation period cannot be evaluated for any reason, they can be replaced with the next available participants if the escalation or de-escalation rules have not been met. The dose level cohort determined to be the RP2D will be expanded until at least 10 participants have been dosed and observed for the duration of the 42-day DLT observation period, and then enter the phase 2 dose expansion.

[0302] Example 2. Clinical study of NC410 in combination with pembrolizumab.

[0303] Methods:

[0304] The selection parameters for study participants used in the above phase 1b (i.e., safety and tolerability study) and the parameters applicable to participants who withdrew from the study also apply to the following phase 2 (i.e., clinical study). Immunohistochemistry (IHC) studies were conducted to further understand the identification of potential tumor types for NC410 therapy. These IHC studies were also based on the criteria of early in-depth non-clinical analysis of tumor types. The selection of tumor types for IHC studies employed the following criteria: LAIR-1 expression in the TME, CD163 M2-like macrophage marker (such as evaluating the relative increase in cancer compared to normal tissue), and the analysis of several LAIR ligands (such as evaluating overexpression in the TME compared to normal tissue). To evaluate these markers, IHC analysis was performed, including hematoxylin and eosin (H&E) and trichrome staining, LAIR-1 expression, LAIR-2-Fc (NC410) binding, and immune cell infiltration. Figure 6 Examples of these results showing the analysis of gastric adenocarcinoma (STAD) are presented. For LAIR-2-Fc binding, brown staining indicates the binding of LAIR-2Fc, and blue staining is the hematoxylin counterstain, indicating no binding of LAIR-2Fc. The LAIR-2Fc positive (green) and negative regions (red) were quantified, as indicated by the associated pie chart. Five regions of interest (ROIs) with a diameter of 600 μm were randomly selected in the LAIR-2Fc positive region for immune cell quantification. One of the ROIs was magnified to show LAIR-1 staining. This figure shows the quantification of immune cells. The cell counts of LAIR-1+, CD45+, CD3+, and CD163+ cells within the 5 ROIs were quantified and calculated as ×103 / mm2.

[0305] The Phase 2 of this study will further evaluate the clinical benefit of the combination of NC410 at the recommended Phase 2 dose (RP2D) obtained from Phase 1b and standard-dose pembrolizumab in participants with advanced unresectable and / or metastatic solid tumors in different cohorts (as shown below based on prior history of immune checkpoint inhibitor (ICI) refractoriness or untreated history) such as Figure 7 shown. Antitumor activity assessment will be used to evaluate the clinical benefit of the combination of NC410 and pembrolizumab and confirm preclinical studies showing that the combination of NC410 and anti-PD-L1 resulted in synergistic and reproducible tumor killing in murine models ( Figure 9 ).

[0306] 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) (as evaluated by the investigator, based on RECIST v1.1), and overall survival (OS). Pharmacokinetic (PK) assessment of NC410 concentration in serum will be observed, as well as assessment of the pharmacokinetic / pharmacodynamic (PK / PD) properties of the combination therapy.

[0307] This study will enroll patients with ICI-refractory solid tumors in Cohort 1 and untreated solid tumors in Cohorts 2a, 2b, and 2c, as described below. Before entry into these cohorts, microsatellite stable (MSS) or microsatellite instability-low (MSI-L) status must be confirmed (either by historical results or during screening). Cohort definitions are as follows:

[0308] Cohort 1: ICI-refractory solid tumors (colorectal cancer microsatellite instability-high (CRC MSI-H), gastric cancer including gastroesophageal (GE) junction cancer, esophageal cancer, endometrial cancer, and head and neck (H&N) cancer).

[0309] Cohort 2a: Untreated MSS or MSI-L CRC

[0310] Cohort 2b: Untreated MSS or MSI-L gastric cancer (including GE junction cancer)

[0311] Cohort 2c: Untreated MSS or MSI-L ovarian cancer.

Claims

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

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

3. The combination therapy according to 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. The combination therapy according to claim 1, wherein the immune checkpoint inhibitor inhibits the programmed cell death protein 1 (PD-1) pathway.

5. The combination therapy according to claim 1, wherein the immune checkpoint inhibitor is pembrolizumab.

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

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

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

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

7.

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

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

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

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

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

15. The method according to claim 14, wherein the subject in need thereof receives a dose of the first pharmaceutical composition on the first day of a repeated 42-day cycle.

16. The method according to claim 14, wherein the subject in need thereof receives a dose of the second pharmaceutical composition on days 1, 15 and 29 of a repeated 42-day cycle.

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

18. The method according to claim 14, wherein the subject in need thereof receives a dose of the second pharmaceutical composition once a week for a repeated 42-day cycle.

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

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