Collagen-localized immunomodulatory molecules and methods thereof

Abstract

The present disclosure provides immunomodulatory fusion proteins comprising a collagen-binding domain operably linked to an immunomodulatory domain. The disclosure also features compositions and methods of using the same, for example, to treat cancer.

1 . An immunomodulatory fusion protein comprising:
 (i) an immunomodulatory domain;   (ii) a collagen-binding domain, wherein the collagen-binding domain specifically binds type I and/or type IV collagen and binds type I collagen with a K D ≤500 nM, and wherein the collagen-binding domain has an isoelectric point pI<10 and a molecular weight (MW) of ≥5 kDa; and   (iii) optionally, a linker,   wherein the immunomodulatory domain is operably linked with or without the linker to the collagen-binding domain.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The immunomodulatory fusion protein of  claim 1 , wherein the collagen-binding domain comprises
 (i) one or more leucine-rich repeats from a human proteoglycan Class II member of the small leucine-rich proteoglycan (SLRP) family comprising lumican, decorin, biglycan, fibromodulin, chondroadherin, asporin, PRELP, osteoadherin/osteomodulin, opticin, osteoglycin/mimecan, podocan, perlecan, and nidogen; or   (ii) a human type I glycoprotein having an Ig-like domain selected from LAIR1, LAIR2, and Glycoprotein IV, or an extracellular portion thereof which binds collagen, or variants thereof.   
     
     
         5 - 11 . (canceled) 
     
     
         12 . The immunomodulatory fusion protein of  claim 4 , wherein the lumican comprises the amino acid sequence as set forth in SEQ ID NO: 107. 
     
     
         13 - 16 . (canceled) 
     
     
         17 . The immunomodulatory fusion protein of  claim 4 , wherein the human type I glycoprotein is LAIR1 or a variant thereof and the collagen-binding domain comprises (j) amino acid residues 22-122 of the amino acid sequence as set forth in SEQ ID NO: 98, (ii) one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98, or (iii) an increased or decreased binding affinity to collagen relative to a collagen binding affinity of a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. 
     
     
         18 - 20 . (canceled) 
     
     
         21 . The immunomodulatory fusion protein of  claim 1 , wherein the immunomodulatory domain comprises a polypeptide that (i) activates, enhances or promotes a response by an immune cell, or (ii) inhibits, reduces or suppresses a response by an immune cell. 
     
     
         22 . (canceled) 
     
     
         23 . The immunomodulatory fusion protein of  claim 21 , wherein the immune cell is a lymphoid cell selected from an innate lymphoid cell, a T cell, a B cell, an NK cell, a monocyte, a neutrophil, a granulocyte, a mast cell, a macrophage, a dendritic cell, or a combination thereof. 
     
     
         24 . (canceled) 
     
     
         25 . The immunomodulatory fusion protein of  claim 21 , wherein the response by the immune cell comprises cytokine production, antibody production, production of antigen-specific immune cells, increased effector function and/or cytotoxicity, or a combination thereof. 
     
     
         26 . The immunomodulatory fusion protein of  claim 1 , wherein the immunomodulatory domain comprises one or more selected from a cytokine, a chemokine, an activating ligand/receptor, an inhibitory ligand/receptor, an agonist antibody, an antagonist antibody, or a combination thereof. 
     
     
         27 . (canceled) 
     
     
         28 . The immunomodulatory fusion protein of  claim 26 , wherein the cytokine is (i) a human gamma common chain receptor interleukin selected from IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-15/IL-15RA, IL-21, or a combination thereof; (ii) a human IL-12 family member selected from IL-12 (p35), IL-12 (p40), IL-12(p35)/IL-12(p40), IL-23, IL-27 IL-35, or a combination thereof, (iii) a human IL-1 family member selected from IL-1, IL-18, IL-33, or a combination thereof, or (iv) TNFα, INFα, IFN-γ, GM-CSF, FLT3L, G-CSF, M-CSF, or a combination thereof. 
     
     
         29 - 34 . (canceled) 
     
     
         35 . The immunomodulatory fusion protein of  claim 26 , wherein the immunomodulatory domain comprises one or more chemokines selected from LIF, MIP-2, MIP-1α, MIP-1β, CXCL1, CXCL9, CXCL10, MCP-1, Eotaxin, RANTES, LIX, CCL3, CCL4, CCL5, Eotaxin or a combination thereof. 
     
     
         36 - 37 . (canceled) 
     
     
         38 . The immunomodulatory fusion protein of  claim 26 , wherein the immunomodulatory domain comprises one or more activating ligands/receptors selected from
 (i) a TNF superfamily ligand selected from TNF-alpha, CD40L, 4-1BBL, OX40, or an antibody or antigen binding fragment thereof selected from an anti-TNFR1 antibody, an anti-TNFR2 antibody, an anti-CD40 antibody, an anti-4-1BB antibody and an anti-OX40 antibody;   (ii) a CD28 receptor superfamily or a B7 ligand family-selected from ICOS ligand, CD80, CD86, or an antibody or antigen binding fragment thereof selected from an anti-ICOS antibody and an anti-CD28 antibody; or   (iii) a T cell receptor comprising an antibody or antigen binding fragment thereof selected from an anti-CD3γ antibody, an anti-CD3δ antibody, an anti-CD3ζ antibody, and an anti-CD3ε antibody.   
     
     
         39 - 44 . (canceled) 
     
     
         45 . The immunomodulatory fusion protein of  claim 26 , wherein the immunomodulatory domain comprises one or more inhibitory ligands/receptors selected from (i) a CD28 receptor superfamily member comprising an antibody or antigen binding fragment thereof selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA4 antibody,
 (ii) a TNF superfamily member comprises an antibody or antigen binding fragment selected from an anti-TIGIT antibody and an anti-BTLA antibody, or (iii) a checkpoint inhibitor member comprising an antibody or antigen binding fragment selected from an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD47 antibody, and an anti-SIRPα antibody.   
     
     
         46 - 49 . (canceled) 
     
     
         50 . The immunomodulatory fusion protein of  claim 1 , wherein the immunomodulatory domain is operably linked to the collagen-binding domain via a linker of sufficient length or mass to reduce adsorption of the immunomodulatory domain onto collagen fibrils, and/or provides sufficient molecular weight to the fusion protein reduce diffusion from a tissue, optionally wherein the linker allows for steric separation of the immunomodulatory domain from collagen fibrils to promote receptor/ligand engagement. 
     
     
         51 - 53 . (canceled) 
     
     
         54 . The immunomodulatory fusion protein of  claim 50 , wherein the linker (i) is a hydrophilic polypeptide comprising “N” amino acids in length, wherein 1-1000, 10-900, 30-800, 40-700, 50-600, 100-500, or 200-400, (ii) is human serum albumin or fragment thereof, or (iii) comprises an Fc domain or a mutant Fc domain with reduced FcR interaction. 
     
     
         55 - 56 . (canceled) 
     
     
         57 . The immunomodulatory fusion protein of  claim 1 , wherein the fusion protein is of sufficient mass to reduce size dependent escape by diffusion or convection upon administration in vivo, optionally wherein the fusion protein is >60 kDa. 
     
     
         58 . (canceled) 
     
     
         59 . The immunomodulatory fusion protein of  claim 50 , wherein the fusion protein binds type I and/or type IV collagen upon administration in vivo, thereby reducing systemic exposure of the immunomodulatory fusion protein. 
     
     
         60 - 146 . (canceled) 
     
     
         147 . A pharmaceutical composition comprising an immunomodulatory fusion protein of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         148 . A nucleic acid comprising a nucleotide sequence encoding an immunomodulatory fusion protein of  claim 1 . 
     
     
         149 . An expression vector comprising the nucleic acid of  claim 148 . 
     
     
         150 . A cell transformed with an expression vector of  claim 149 . 
     
     
         151 . A method for producing an immunomodulatory fusion protein, the method comprising maintaining a cell according to  claim 150  under conditions permitting expression of the immunomodulatory fusion protein. 
     
     
         152 . (canceled) 
     
     
         153 . A method for activating, enhancing or promoting a response by an immune cell in a subject or inhibiting, reducing or suppressing a response by an immune cell in a subject, comprising administering to a subject in need thereof, an effective amount of the pharmaceutical composition of  claim 147 . 
     
     
         154 - 158 . (canceled) 
     
     
         159 . A method for treating cancer, or reducing or inhibiting tumor growth, comprising administering to a subject in need thereof, an effective amount of the pharmaceutical composition of  claim 147 . 
     
     
         160 - 162 . (canceled) 
     
     
         163 . The method of  claim 159 , wherein infiltration of immune cells into a tumor microenvironment is increased after administration of the pharmaceutical composition. 
     
     
         164 - 166 . (canceled) 
     
     
         167 . A kit comprising a container comprising a pharmaceutical composition of  claim 147 , and a package insert comprising instructions for administration of the pharmaceutical composition alone or in combination with another agent, for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof. 
     
     
         168 - 170 . (canceled) 
     
     
         171 . A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof, an effective amount of the pharmaceutical composition of  claim 147 , and an effective amount of a second composition comprising (i) a tumor antigen-targeting antibody, optionally wherein the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen and/or wherein the tumor antigen-targeting antibody specifically binds human HER-2/neu, EGFR, VEGFR, CD20, CD33, CD38 or antigen-binding fragment thereof, (ii) a cancer vaccine, optionally wherein the cancer vaccine is a peptide comprising one or more tumor-associated antigens, or a population of cells immunized in vitro with a tumor antigen and administered to the subject, (iii) an immune checkpoint inhibitor, optionally comprising an antibody or antigen binding fragment thereof which binds PD-1, PD-L1, CTLA-4, LAG3, or TIM3, or (iv) an adoptive cell therapy, optionally comprising an immune effector cell comprising a chimeric antigen receptor (CAR) molecule which binds to a tumor antigen, thereby reducing or inhibiting tumor growth or treating cancer in the subject. 
     
     
         172 - 176 . (canceled) 
     
     
         177 . The method of  claim 171 , wherein the cancer vaccine is an amphiphilic peptide conjugate comprising a tumor-associated antigen, a lipid, and optionally a linker, wherein the amphiphilic peptide conjugate binds albumin under physiological conditions, optionally wherein the cancer vaccine further comprises an adjuvant. 
     
     
         178 - 182 . (canceled) 
     
     
         183 . The method of  claim 171 , wherein the CAR molecule comprises an antigen binding domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and/or a primary signaling domain, wherein the antigen binding domain binds to the tumor antigen associated with the disease, optionally wherein the tumor antigen is selected from CD19, EGFR, Her2/neu, CD30 and BCMA. 
     
     
         184 - 201 . (canceled)