Immunomodulatory fusion protein-metal hydroxide complexes and methods thereof

Abstract

To provide novel immunomodulatory fusion protein-metal hydroxide complexes and methods thereof.SOLUTION: The present disclosure provides immunomodulatory fusion protein-metal hydroxide complexes comprising an immunomodulatory domain adsorbed to a metal hydroxide via ligand exchange. The disclosure also features compositions and methods of using the immunomodulatory fusion protein-metal hydroxide complexes, for example, to treat cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 867,162, filed June 26, 2019. The entire contents of the provisional patent application are incorporated herein by reference.

[0002] Government funding This invention was made with Government support under Grant No. R01 CA174795 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention. [Background technology]

[0003] Immune checkpoint blockade therapies can significantly extend progression-free survival in patients with non-small cell lung cancer, metastatic melanoma, and more (Hodi et al. (2010) 363: 711-723; Ribas et al. (2018) Science 359: 1350-1355). However, these treatment regimens usually only show improvement in a small proportion of patients. Combining these therapies with immune-modulating cytokines such as IL-2 has shown promise in mice (Moynihan, Kelly D., et al. Nature medicine 22: 1402 (2016)), but translating these from preclinical models to clinical studies has been difficult due to dose-limiting toxicity of potent cytokines (Milling, et al. Advanced drug delivery reviews 114: 79-101 (2017)). Thus, there is great interest in limiting toxicity while maintaining efficacy. Alternative routes of administration along with novel mechanisms of in vivo retention may be key to boosting antitumor immune responses in all treated individuals while preventing toxic side effects (Aznar et al. The Journal of Immunology 198:31-39 (2017)). Some proposed in vivo retention strategies include fusing payloads to other high half-life proteins and binding proteins to degradable biomaterials such as hydrogels (Zhu, et al. Cancer cell 27:489-501 (2015); Chao, et al. Nature Biomedical Engineering 2:611 (2018)). However, even these can only extend protein persistence for a short period of time and may rely on multiple therapies for efficacy.

[0004] Thus, there remains a need for novel immunotherapeutic approaches. Summary of the Invention

[0005] The present disclosure is based, at least in part, on the surprising discovery that immunomodulatory domains (e.g., cytokines, anti-immunoreceptor antibodies, anti-tumor associated antigen antibodies, etc.) designed to be conjugated to aluminum hydroxide (alum) have increased antitumor efficacy when delivered by intratumoral injection compared to unconjugated immunomodulatory domains. Alum provides a particulate scaffold known to persist at the site of injection in preclinical models for weeks. Without being bound by theory, alum provides a particulate scaffold that retains the immunomodulatory domain at the site of intratumoral injection, thereby enhancing the persistence of the immunotherapy within the tumor microenvironment while limiting systemic exposure of the immunotherapy below levels that would result in undesirable toxicity. It is well understood that phosphorylated protein antigens exhibit stronger adsorption to alum through ligand exchange. This discovery has led to the development of phosphonated small molecule adjuvants, such as phosphonated TLR agonists, that can be localized at the vaccination site by adsorption to alum. However, the use of adsorption to alum to localize immunomodulatory polypeptides for the treatment of cancer has not been demonstrated. Thus, herein Provided are immunomodulatory fusion proteins that contain hydroxyl substituents (e.g., phosphate groups) for adsorption by ligand exchange with metal hydroxides (e.g., alum) for the purpose of improving tumor retention and antitumor efficacy of the immunomodulatory domain for use in cancer immunotherapy.

[0006] In some embodiments, the present disclosure provides methods for improving the efficacy of immunomodulatory domains, where the immunomodulatory domains are modified to provide tight binding to metal hydroxides (e.g., alum) via one or more phosphoserine residues. In some embodiments, the present disclosure provides methods and compositions comprising an immunomodulatory domain modified with a peptide containing a phosphorylated residue, where the phosphorylated peptide undergoes a ligand exchange reaction with the surface of alum to immobilize the immunomodulatory domain to the metal hydroxide (e.g., alum). As a result of such linkage, the modified immunomodulatory domains have been found to increase binding to alum in vitro compared to unmodified immunomodulatory domains that nonspecifically adsorb to alum. Furthermore, it has been found that modified immunomodulatory domains when adsorbed to alum by ligand exchange persist in tumors for more than 29 days, whereas in the absence of alum, the immunomodulatory domains are cleared from tumors within 3 days. Additionally, improved retention corresponds to improved antitumor efficacy, with modified immunomodulatory domains adsorbed to alum by ligand exchange being found to promote increased survival and tumor clearance in tumor-bearing animals compared to unmodified immunomodulatory domains or immunomodulatory domains adsorbed to alum by nonspecific interactions. Thus, strong binding to metal hydroxides (e.g., alum) mediated by ligand exchange promotes improved tumor retention and antitumor efficacy of immunomodulatory therapies.

[0007] Thus, in some embodiments, the disclosure provides an immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein comprising an immunomodulatory domain, a metal hydroxide-binding peptide comprising at least one target motif of a secretory pathway kinase modified with a phosphate group, and optionally a stabilization domain; and (b) a metal hydroxide (e.g., alum), wherein the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange via the at least one phosphate group of the metal hydroxide-binding peptide, thereby forming the immunomodulatory fusion protein-metal hydroxide complex.

[0008] In some embodiments, the present disclosure provides an immunomodulatory fusion protein-metal hydroxide complex and uses thereof. In some embodiments, the present disclosure provides an immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein, (i) an immunomodulatory domain, (ii) a metal hydroxide-binding peptide comprising at least one kinase targeting motif of a secretory pathway kinase, the kinase comprising at least one phosphorylated amino acid; and (iii) an immunomodulatory fusion protein, optionally including a stabilization domain; and (b) a metal hydroxide; An immunomodulatory fusion protein-metal hydroxide complex is provided in which the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange via at least one phosphorylated amino acid of the metal hydroxide-binding peptide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

[0009] In some embodiments, the immunomodulatory fusion protein comprises a metal hydroxide binding peptide comprising at least one kinase targeting motif, the kinase targeting motif being selected from the group consisting of Fam20C, protein kinase A, cAMP-dependent protein kinase, cyclin-dependent kinase, extracellular regulated kinase-2, casein kinase 1, casein kinase 2, glycogen kinase, and the like. The protein kinase C comprises an amino acid sequence that is phosphorylated by a kinase selected from the group consisting of: insulin synthase kinase-3, calmodulin-dependent protein kinase-2, Abelson murine leukemia virus tyrosine kinase, Rous sarcoma virus tyrosine kinase, insulin receptor tyrosine kinase, protein kinase B, protein kinase D, proviral integration site kinases 1-3, AMP-activated protein kinase, mitogen-activated protein kinase, or NimA-related kinase.

[0010] In some embodiments, the immunomodulatory fusion protein comprises a metal hydroxide binding peptide comprising at least one kinase target motif, wherein the at least one kinase target motif comprises an amino acid sequence that is phosphorylated by Fam20C. In some embodiments, the at least one kinase target motif of the metal hydroxide binding peptide comprises phosphoserine, phosphotyrosine or phosphothreonine. In some embodiments, the at least one kinase target motif of the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SXE, SX-pS, or SXQXXDE, wherein X is any amino acid. In some embodiments, the at least one kinase target motif of the metal hydroxide binding peptide comprises the amino acid sequence SXE, wherein X is any amino acid, wherein the serine is phosphorylated. In some embodiments, X is selected from E, S, V, H, Q and G. In some embodiments, X is E.

[0011] In another aspect, the present disclosure provides an immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein, (i) an immunomodulatory domain, (ii) a metal hydroxide-binding peptide comprising at least one kinase targeting motif of the secretory pathway kinase Fam20C comprising the amino acid sequence SXE; and (iii) an immunomodulatory fusion protein, optionally including a stabilization domain; and (b) a metal hydroxide; An immunomodulatory fusion protein-metal hydroxide complex is provided, in which at least one kinase target motif of the metal hydroxide binding peptide comprises a phosphoserine, and the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange via the at least one phosphoserine of the metal hydroxide binding peptide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

[0012] In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex comprises a metal hydroxide-binding peptide operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain.

[0013] In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex comprises an immunomodulatory domain, a stabilizing domain, and a metal hydroxide-binding peptide, where the stabilizing domain is operably linked, optionally via an amino acid linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either end of the immunomodulatory domain or the stabilizing domain.

[0014] In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex comprises an immunomodulatory domain, a stabilizing domain, and a metal hydroxide-binding peptide, wherein the metal hydroxide-binding peptide is operably linked, optionally via an amino acid linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the stabilizing domain is operably linked, optionally via a linker, to either the metal hydroxide-binding peptide or the immunomodulatory domain.

[0015] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises about 3-6, about 6-15, about 10-25, or about 10-50 amino acids.

[0016] In any of the foregoing or related embodiments, the metal hydroxide binding peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more kinase target motifs comprising phosphorylated amino acids.

[0017] In any of the aforementioned or related aspects, the kinase target motif comprises a phosphorylated amino acid that is phosphoserine.

[0018] In any of the foregoing or related embodiments, the metal hydroxide binding peptide comprises two or more kinase target motifs of a secretory pathway kinase, wherein the two or more kinase target motifs comprise the same or different amino acid sequences, and optionally, the two or more kinase target motifs are separated by a peptide linker.

[0019] In any of the foregoing or related embodiments, at least one kinase target motif comprises the amino acid sequence SXE, where X is any amino acid, and the serine is phosphorylated. In some embodiments, X is selected from E, S, V, H, Q, and G. In some embodiments, X is E.

[0020] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises at least one, two, or three kinase targeting motifs, and optionally, the kinase targeting motifs are contiguous. In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises an amino acid sequence selected from SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, and SEQ ID NO:125.

[0021] In any of the aforementioned or related embodiments, the metal hydroxide-binding peptide comprises the amino acid sequence XXSXX (SEQ ID NO:127) or XXSEEXX (SEQ ID NO:128), where X is any amino acid.

[0022] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide has the amino acid sequence Xaa 1 -Xaa 2 -S-Xaa 3 -E-Xaa 4 -Xaa 5 (SEQ ID NO: 127), 1 is F, M, or G, and Xaa 2 is Q, E, or G, and Xaa 3 are E, S, V, H, Q, and G, and Xaa 4 is Q, S, or G, and Xaa5 is Q, N, or G. In some embodiments, Xaa 3 is E. In some embodiments, Xaa 3 is E and Xaa 1 is F and Xaa 2 is Q. In some embodiments, Xaa 3 is E and Xaa 1 is M and Xaa 2 is E. In some embodiments, Xaa 3 is E and Xaa 1 is G and Xaa 2 In some embodiments, Xaa is G. 3 is E and Xaa 4 is Q and Xaa 5 is Q. In some embodiments, Xaa 3 is E and Xaa 4 is E and Xaa 5 is S. In some embodiments, Xaa 3 is E and Xaa 4 is G and Xaa 5 is G.

[0023] In any of the aforementioned or related embodiments, the metal hydroxide-binding peptide comprises the amino acid sequence FQSEEQQ (SEQ ID NO: 129), MESEESN (SEQ ID NO: 130), or GGSEEGG (SEQ ID NO: 131).

[0024] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide has the amino acid sequence Xaa 1 -Xaa 2 -S-Xaa 3 -E-Xaa 4 -Xaa 5 -[L]-S-Xaa 3 -E-Xaa 6 -Xaa 7 (SEQ ID NO: 133), Xaa 1 is F, M or G, and Xaa 2 is Q, E, or G, and Xaa 3are E, S, V, H, Q, and G, and Xaa 4 is Q, S, or G, and Xaa 5 is Q, N, or G, and Xaa 5 is G and Xaa 6 is G and L is a peptide linker, optionally a G / S linker, optionally GGGS.

[0025] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises a sequence of linked amino acids having the formula [A]x, where A is an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and x is an integer whose value indicates the number of linked amino acid sequences represented by A, and x is 1 to 4.

[0026] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[B], where A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131.

[0027] In any of the foregoing or related embodiments, the metal hydroxide binding peptide has the formula ([A]-[B]): X wherein A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131, and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[B], and x is 1 to 4.

[0028] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[L]-[A], where A is an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

[0029] In any of the foregoing or related embodiments, the metal hydroxide binding peptide has the formula ([A]-[L]-[A]): x wherein A is an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131; x is an integer representing the number of linked amino acid sequences represented by [A]-[L]-[A], where x is 1 to 4; and L is a peptide linker, optionally a G / S linker, optionally GGGS.

[0030] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[L]-[B], where A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

[0031] In any of the foregoing or related embodiments, the metal hydroxide binding peptide has the formula ([A]-[L]-[B]): x wherein A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[L]-[B], where x is 1 to 4, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

[0032] In any of the aforementioned or related aspects, the metal hydroxide binding peptide comprises: The amino acid sequence is selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101.

[0033] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide has the formula [C] x wherein C is an amino acid sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101; and x is an integer with a value indicating the number of linked amino acid sequences represented by C, where x is 1 to 4.

[0034] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide has the formula [C] x -[D] y wherein C and D are the same or different amino acid sequences, C and D are selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101, x is an integer with a value indicating the number of linked amino acid sequences represented by C, y is an integer with a value indicating the number of linked amino acid sequences represented by D, x is 1 to 4, y is 1 to 4, and x and y are the same or different.

[0035] In any of the foregoing or related embodiments, the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 115. In some embodiments, the metal hydroxide binding peptide comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the metal hydroxide binding peptide comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the metal hydroxide binding peptide comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the metal hydroxide binding peptide comprises the amino acid sequence of SEQ ID NO: 115.

[0036] In any of the foregoing or related embodiments, the metal hydroxide-binding peptide comprises about 1-5, 1-10, 1-15, or 1-20 phosphoserine residues, and the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange of the phosphoserine residues.

[0037] In another aspect, the present disclosure provides an immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein, (i) an immunomodulatory domain, optionally linked to a stabilization domain; (ii) an immunomodulatory fusion protein comprising a terminal metal hydroxide binding peptide comprising one or more hydroxyl substituents coupled by a protein reactive moiety, optionally via a linker; (b) a metal hydroxide; An immunomodulatory fusion protein-metal hydroxide complex is provided in which the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange via at least one hydroxyl substituent of the metal hydroxide-binding peptide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

[0038] In some embodiments, the disclosure provides an immunomodulatory fusion protein-metal hydroxide complex, where the protein reactive moiety comprises a sulfhydryl reactive moiety, optionally, the sulfhydryl reactive moiety is a maleimide.

[0039] In some embodiments, the disclosure provides an immunomodulatory fusion protein-metal hydroxide complex, wherein the protein-reactive moiety comprises a sortase recognition motif.

[0040] In any of the foregoing or related embodiments, the metal hydroxide binding peptide is selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, In some embodiments, the hydroxyl substituent comprises at least one hydroxyl group, 19, 20 or more hydroxyl substituents. In some embodiments, the hydroxyl substituent is selected from the group consisting of a fluoride group, a citrate group, a phosphate group, a carbonate group, and a sulfate group, and optionally, the hydroxyl substituent is a phosphate group. In some embodiments, the hydroxyl substituent comprises at least one phosphorylated amino acid residue, and optionally, the phosphorylated amino acid residue is selected from phosphoserine, phosphotyrosine, and phosphothreonine. In some embodiments, the phosphorylated amino acid residue is phosphoserine.

[0041] In any of the foregoing or related embodiments, the metal hydroxide is selected from aluminum hydroxide, aluminum phosphate, calcium hydroxide, calcium phosphate, iron hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, zinc hydroxide, and zirconium hydroxide. In some embodiments, the metal hydroxide is aluminum hydroxide (alum).

[0042] In another aspect, the present disclosure provides an immunomodulatory fusion protein comprising: (a) an immune modulating domain; (b) a metal hydroxide-binding peptide comprising at least one kinase targeting motif of the secretory pathway kinase Fam20C comprising the amino acid sequence SXE; and (c) optionally, a stabilization domain; An immunomodulatory fusion protein is provided, wherein at least one kinase target motif of the metal hydroxide-binding peptide comprises a serine modified with a phosphate group, and the immunomodulatory fusion protein undergoes ligand exchange with alum via at least one phosphoserine of the metal hydroxide-binding peptide, thereby coupling the immunomodulatory fusion protein to alum and forming an immunomodulatory fusion protein-metal hydroxide complex.

[0043] In another aspect, the present disclosure provides an immunomodulatory fusion protein comprising: (a) an immunomodulatory domain, optionally linked to a stabilization domain, and (b) a metal hydroxide-binding peptide comprising one or more phosphorylated amino acids coupled, optionally via a linker, by a protein-reactive moiety; An immunomodulatory fusion protein is provided, wherein the immunomodulatory fusion protein undergoes ligand exchange with alum via at least one hydroxyl substituent of the metal hydroxide-binding peptide, thereby coupling the immunomodulatory fusion protein to the alum and forming an immunomodulatory fusion protein-metal hydroxide complex.

[0044] In some embodiments, the metal hydroxide-binding peptide is coupled to the N-terminus or C-terminus of the immunomodulatory domain by a protein-reactive moiety.

[0045] In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising an immunomodulatory domain, a stabilizing domain, and a metal hydroxide-binding peptide, wherein the stabilizing domain is N-terminal or C-terminal to the immunomodulatory domain, optionally via an amino acid linker, and the metal hydroxide-binding peptide is coupled to the end of the immunomodulatory domain or the stabilizing domain by a protein-reactive moiety.

[0046] In another aspect, the disclosure provides a method for increasing phosphorylation of an immunomodulatory fusion protein, comprising: (a) Immunomodulatory domain, Metal hydroxide binding peptides containing one or more kinase targeting motifs; a nucleotide sequence encoding an immunomodulatory fusion protein, optionally including a stabilization domain; and (b) ER targeting leader sequence, operably linked thereto a kinase domain operably linked thereto contacting the cell with a nucleotide sequence encoding a kinase comprising an anchor peptide; Methods are provided in which a kinase is localized in the secretory pathway and one or more kinase target motifs of the metal hydroxide binding peptide are phosphorylated by the kinase within the secretory pathway, thereby increasing phosphorylation of the immunomodulatory fusion protein.

[0047] In some embodiments of the foregoing methods, the kinase comprises an ER targeting leader sequence that directs the kinase to the secretory pathway, and optionally, the kinase comprises a kinase domain selected from the group consisting of protein kinase A, cAMP-dependent protein kinase, cyclin-dependent kinase, extracellular regulated kinase-2, casein kinase 1, casein kinase 2, glycogen synthase kinase-3, calmodulin-dependent protein kinase-2, Abelson murine leukemia virus tyrosine kinase, Rous sarcoma virus tyrosine kinase, insulin receptor tyrosine kinase, protein kinase B, protein kinase D, proviral integration site kinases 1-3, AMP-activated protein kinase, mitogen-activated protein kinase, or NimA-related kinase.

[0048] In some embodiments of the aforementioned methods, the kinase comprises Fam20C, wherein Fam20C comprises the amino acid sequence set forth in SEQ ID NO:135.

[0049] In some embodiments of the aforementioned methods, the kinase comprises an anchor peptide that inhibits secretion of the kinase, and optionally, the anchor peptide comprises the amino acid sequence KDEL or HDEL.

[0050] In some embodiments of the aforementioned methods, the cell is contacted with an expression vector comprising a nucleic acid encoding an immunomodulatory fusion protein.

[0051] In some embodiments of the aforementioned methods, the cell is contacted with an expression vector comprising a nucleic acid encoding a kinase.

[0052] In some embodiments of the aforementioned methods, the cell is contacted with an expression vector comprising a nucleic acid encoding a kinase and a nucleic acid encoding an immunomodulatory fusion protein.

[0053] In another aspect, the disclosure provides a method for increasing phosphorylation of an immunomodulatory fusion protein comprising an immunomodulatory domain, a metal hydroxide-binding peptide comprising one or more kinase target motifs of the secretory pathway kinase Fam20C, and optionally a stabilization domain, comprising contacting a cell with an expression vector comprising a nucleic acid encoding the immunomodulatory fusion protein and an expression vector comprising a nucleic acid encoding the secretory pathway kinase Fam20C operably linked to an anchor peptide, wherein the secretory pathway kinase Fam20C is localized to the secretory pathway by the anchor peptide, and the one or more kinase target motifs are phosphorylated by Fam20C within the secretory pathway, thereby increasing phosphorylation of the immunomodulatory fusion protein.

[0054] In some embodiments, the method comprises maintaining the cells under conditions that allow for expression of the immunomodulatory fusion protein. In some embodiments, the method further comprises isolating the immunomodulatory fusion protein.

[0055] Another aspect of the present disclosure is the immunomodulatory fusion protein produced by the method of the present disclosure. and wherein the immunomodulatory fusion protein comprises at least one phosphorylated amino acid, and the immunomodulatory fusion protein is adsorbed by ligand exchange with alum via the at least one phosphorylated amino acid, thereby coupling the immunomodulatory fusion protein to the alum and forming an immunomodulatory fusion protein-metal hydroxide complex.

[0056] In any of the foregoing or related aspects, the immunomodulatory domain comprises a polypeptide that activates, enhances, or promotes a response by an immune cell.

[0057] In any of the foregoing or related aspects, the immunomodulatory domain comprises a polypeptide that inhibits, reduces, or suppresses a response by an immune cell.

[0058] In any of the foregoing or related embodiments, the immune cell is a lymphoid cell selected from an innate lymphoid cell, a T cell, a B cell, a NK cell, and combinations thereof.

[0059] In any of the foregoing or related aspects, the immune cells are myeloid cells selected from monocytes, neutrophils, granulocytes, mast cells, macrophages, dendritic cells, and combinations thereof.

[0060] In any of the foregoing or related aspects, the response by the immune cells includes cytokine production, antibody production, production of antigen-specific immune cells, increased effector function and / or cytotoxicity, and combinations thereof.

[0061] In any of the foregoing or related embodiments, the immunomodulatory domain comprises one or more selected from a cytokine, a chemokine, an activating ligand / receptor, an inhibitory ligand / receptor, or a combination thereof.

[0062] In any of the foregoing or related embodiments, the immunomodulatory domain comprises one or more cytokines. In some embodiments, the cytokine is 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, and combinations thereof. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-15 / IL15RA. In some embodiments, the cytokine is 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, and combinations thereof. In some embodiments, the cytokine is a single chain fusion of IL-12(p35) / IL-12(p40). In some embodiments, the cytokine is a human IL-1 family member selected from IL-1, IL-18, IL-33, and combinations thereof. In some embodiments, the cytokine is IL-18. In some embodiments, the cytokine is selected from TNFα, INFα, IFN-γ, GM-CSF, FLT3L, G-CSF, M-CSF, and combinations thereof.

[0063] In any of the above or related embodiments, the immunomodulatory domain comprises one or more chemokines. In some embodiments, the chemokines are selected from LIF, MIP-2, MIP-1α, MIP-1β, CXCL1, CXCL9, CXCL10, MCP-1, eotaxin, RANTES, LIX, and combinations thereof. In some embodiments, the chemokines are selected from CCL3, CCL4, CCL5, eotaxin, and combinations thereof.

[0064] In any of the above or related embodiments, the immunomodulatory domain comprises one or more activating ligands / receptors. In some embodiments, the activating ligands / receptors are selected from the TNF superfamily, the CD28 receptor superfamily, the B7 ligand family, and T cell receptors. In some embodiments, the activating ligands / receptors are TNF superfamily ligands selected from TNF-alpha, CD40L, 4-1BBL, OX40, and combinations thereof. In some embodiments, the activating ligands / receptors are TNF superfamily receptors, and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from anti-TNFR1 antibody, anti-TNFR2 antibody, anti-CD40 antibody, anti-4-1BB antibody, and anti-OX40 antibody. In some embodiments, the activating ligands / receptors are CD28 superfamily members or B7 family members selected from ICOS ligand, CD80, and CD86, and combinations thereof. In some embodiments, the activating ligand / receptor is a CD28 superfamily member and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-ICOS antibody and an anti-CD28 antibody. In some embodiments, the activating ligand / receptor is a T cell receptor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-CD3γ antibody, an anti-CD3δ antibody, an anti-CD3ζ antibody, and a CD3ε antibody.

[0065] In any of the foregoing or related embodiments, the immunomodulatory domain comprises one or more inhibitory ligands / receptors. In some embodiments, the inhibitory ligands / receptors are selected from the CD28 receptor superfamily, the TNF superfamily, and checkpoint inhibitors. In some embodiments, the inhibitory ligands / receptors are CD28 superfamily members, and the immunomodulatory domain comprises 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. In some embodiments, the inhibitory ligands / receptors are TNF superfamily members, and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-TIGIT antibody and an anti-BTLA antibody. In some embodiments, the inhibitory ligands / receptors are TNF superfamily members, and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof that is an anti-TIGIT antibody. In some embodiments, the inhibitory ligand / receptor is a checkpoint inhibitor and the immunomodulatory domain comprises 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.

[0066] In any of the aforementioned or related aspects, the stabilization domain comprises human serum albumin or a fragment thereof.

[0067] In any of the above or related embodiments, the stabilization domain comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

[0068] In any of the foregoing or related embodiments, the immunomodulatory fusion protein-metal hydroxide complex is of sufficient mass to reduce size-dependent diffusion away from the injection site upon administration in vivo.

[0069] In another aspect, the present disclosure provides a pharmaceutical composition comprising an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure and a pharma- ceutically acceptable carrier.In another aspect, the present disclosure provides a pharmaceutical composition comprising an immunomodulatory fusion protein of the present disclosure and a pharma- ceutically acceptable carrier.

[0070] In another aspect, the present disclosure provides a nucleoside encoding an immunomodulatory fusion protein of the present disclosure. In another aspect, the disclosure provides an expression vector comprising a nucleic acid of the disclosure. In another aspect, the disclosure provides a cell transformed with an expression vector of the disclosure.

[0071] In other aspects, the disclosure provides methods for producing an immunomodulatory fusion protein, the methods comprising maintaining cells under conditions that allow for expression of the immunomodulatory fusion protein, hi some aspects, the methods further comprise obtaining the immunomodulatory fusion protein and adsorbing the immunomodulatory fusion protein to a metal hydroxide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

[0072] In another aspect, the present disclosure provides a method for activating, enhancing or promoting an immune cell mediated response in a subject, comprising administering an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need of an immune cell mediated response. In another aspect, the present disclosure provides a method for activating, enhancing or promoting an immune cell mediated response in a subject, comprising administering an effective amount of an immunomodulatory fusion of the present disclosure or a pharmaceutical composition thereof to a subject in need of an immune cell mediated response.

[0073] In another embodiment, the present disclosure provides a method for inhibiting, reducing or suppressing an immune cell-mediated response in a subject, comprising administering an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need of inhibiting, reducing or suppressing an immune cell-mediated response. In another embodiment, the present disclosure provides a method for inhibiting, reducing or suppressing an immune cell-mediated response in a subject, comprising administering an effective amount of an immunomodulatory fusion protein of the present disclosure or a pharmaceutical composition thereof to a subject in need of inhibiting, reducing or suppressing an immune cell-mediated response. In some embodiments, the immune cell is a lymphoid cell selected from innate lymphocytes, T cells, B cells, NK cells, and combinations thereof. In some embodiments, the immune cell is a myeloid cell selected from monocytes, neutrophils, granulocytes, mast cells, macrophages, dendritic cells, and combinations thereof. In some embodiments, the immune cell-mediated response includes cytokine production, antibody production, production of antigen-specific immune cells, increased effector function and / or cytotoxicity, and combinations thereof. In some embodiments, the immune cell response occurs in the tumor microenvironment.

[0074] In another aspect, the present disclosure provides a method for reducing or inhibiting tumor growth comprising administering an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need thereof.In another aspect, the present disclosure provides a method for reducing or inhibiting tumor growth comprising administering an effective amount of an immunomodulatory fusion protein of the present disclosure or a pharmaceutical composition thereof to a subject in need thereof.

[0075] In other embodiments, the present disclosure provides a method for treating cancer in a subject, comprising administering an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need of cancer treatment. In some embodiments, an anti-tumor immune response is induced in the subject after administration of the immunomodulatory fusion protein-metal hydroxide complex or pharmaceutical composition. In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex or pharmaceutical composition is administered intratumorally.

[0076] In other aspects, the present disclosure provides a method for treating cancer in a subject, comprising administering to a subject in need of cancer treatment an effective amount of an immunomodulatory fusion protein of the present disclosure or a pharmaceutical composition of the present disclosure. An immune response is induced in the subject following administration of the immunomodulatory fusion protein or pharmaceutical composition. In some embodiments, the immunomodulatory fusion protein or pharmaceutical composition is administered intratumorally.

[0077] In other aspects, the present disclosure provides a kit comprising a container containing an immune modulatory fusion protein-metal hydroxide complex of the present disclosure and an optional pharma- ceutical composition of the present disclosure, and a package insert containing instructions for administering the fusion protein or pharmaceutical composition, for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

[0078] In other aspects, the present disclosure provides kits for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, comprising a container containing an immunomodulatory fusion protein of the present disclosure and optionally a pharma- ceutical composition of the present disclosure, and a package insert containing instructions for administering the fusion protein or pharmaceutical composition.

[0079] In other aspects, the disclosure provides kits for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, comprising a container containing an immunomodulatory fusion protein-metal hydroxide complex of the disclosure and optionally a pharma- ceutical composition of the disclosure, and a package insert containing instructions for administering the antibody or pharmaceutical composition alone or in combination with another agent.

[0080] In other aspects, the present disclosure provides kits for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, comprising a container containing an immunomodulatory fusion protein of the present disclosure and optionally a pharma- ceutical composition of the present disclosure, and a package insert containing instructions for administering the antibody or pharmaceutical composition alone or in combination with another agent.

[0081] Another aspect of the present disclosure provides the use of an immune modulatory fusion protein-metal hydroxide complex of the present disclosure, and optionally a pharma- ceutical composition of the present disclosure, and an optional pharma- ceutical acceptable carrier, for the manufacture of a medicament for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

[0082] In some embodiments, the present disclosure provides the use of an immunomodulatory fusion protein of the present disclosure, and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition of the present disclosure, for the manufacture of a medicament for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

[0083] In yet another aspect, the disclosure provides an immune modulatory fusion protein-metal hydroxide complex of the disclosure and an optional pharma- ceutically acceptable carrier, or a pharmaceutical composition of the disclosure, in the manufacture of a medicament for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

[0084] In other aspects, the disclosure provides an immunomodulatory fusion protein of the disclosure and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition of the disclosure, in the manufacture of a medicament for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

[0085] Another aspect relates to a pharmaceutical composition comprising an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure and an optional pharma- ceutical acceptable carrier, or a pharmaceutical composition of the present disclosure, for use as a medicament. In some aspects, the present disclosure provides an immunomodulatory fusion of the present disclosure and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition of the present disclosure, for use as a medicament.

[0086] In another embodiment, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of such treatment an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure and an effective amount of a second composition comprising a tumor antigen targeting antibody or antigen-binding fragment thereof, thereby reducing or inhibiting tumor growth or treating cancer in the subject.In another embodiment, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of such treatment an effective amount of an immunomodulatory fusion protein of the present disclosure or a pharmaceutical composition of the present disclosure and an effective amount of a second composition comprising a tumor antigen targeting antibody or antigen-binding fragment thereof, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

[0087] In any of the foregoing or related embodiments, the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen. In some embodiments, the tumor antigen-targeting antibody specifically binds to human HER-2 / neu, EGFR, VEGFR, CD20, CD33, or CD38.

[0088] In another embodiment, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of reducing or inhibiting tumor growth or treating cancer an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of the present disclosures or a pharmaceutical composition of the present disclosure, and an effective amount of a second composition comprising a cancer vaccine, thereby reducing or inhibiting tumor growth or treating cancer in the subject.In another embodiment, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of reducing or inhibiting tumor growth or treating cancer an effective amount of an immunomodulatory fusion protein of any one of the present disclosures or a pharmaceutical composition of the present disclosure, and an effective amount of a second composition comprising a cancer vaccine, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

[0089] In any of the above or related embodiments, the cancer vaccine is a population of cells that are immunized with tumor antigen in vitro and administered to a subject.In some embodiments, the cancer vaccine is a peptide that comprises one or more tumor-associated antigens.In some embodiments, the cancer vaccine is an amphiphilic peptide conjugate that comprises tumor-associated antigen, lipid, and optionally a linker, and the amphiphilic peptide conjugate binds to albumin under physiological conditions.In some embodiments, the cancer vaccine further comprises an adjuvant.

[0090] Yet another aspect of the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of such treatment an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure, and an effective amount of a second composition comprising an immune checkpoint inhibitor, thereby reducing or inhibiting tumor growth in the subject or treating cancer. In another aspect, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of such treatment an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure, and an effective amount of an immune checkpoint inhibitor. The method includes administering to a subject in need thereof a second composition comprising an immune checkpoint inhibitor, thereby reducing or inhibiting tumor growth or treating cancer in a subject.

[0091] In any of the aforementioned or related embodiments, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof that binds to PD-1, PD-L1, CTLA-4, LAG3, or TIM3.

[0092] Another embodiment of the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of such treatment an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of the present disclosure or a pharmaceutical composition of the present disclosure, and an effective amount of a second composition comprising adoptive cell therapy, thereby reducing or inhibiting tumor growth in the subject or treating cancer. In another embodiment, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising administering to a subject in need of such treatment an effective amount of an immunomodulatory fusion protein of the present disclosure or a pharmaceutical composition of the present disclosure, and an effective amount of a second composition comprising adoptive cell therapy, thereby reducing or inhibiting tumor growth in the subject or treating cancer.

[0093] In any of the foregoing or related embodiments, the adoptive cell therapy comprises an immune effector cell comprising a chimeric antigen receptor (CAR) molecule that binds to a tumor antigen. In some embodiments, 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. In some embodiments, the antigen binding domain binds to a tumor antigen associated with a disease. In some embodiments, the tumor antigen is selected from CD19, EGFR, Her2 / neu, CD30, and BCMA. In some embodiments, the immune effector cell is a T cell, such as a CD8+ T cell. In some embodiments, the immune effector cell is a natural killer (NK) cell.

[0094] In any of the foregoing or related embodiments, the immunomodulatory fusion protein-metal hydroxide complex or pharmaceutical composition is administered intratumorally. In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex or pharmaceutical composition and the second composition are administered simultaneously or sequentially.

[0095] In any of the foregoing or related embodiments, the immunomodulatory fusion protein or pharmaceutical composition is administered intratumorally. In some embodiments, the immunomodulatory fusion protein or pharmaceutical composition and the second composition are administered simultaneously or sequentially.

[0096] In any of the foregoing or related embodiments, the methods described herein include administering a plurality of immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or pharmaceutical compositions, wherein the immunomodulatory domains are different. In some embodiments, the immunomodulatory domains are different cytokines. In some embodiments, the plurality of immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or pharmaceutical compositions are formulated together. In some embodiments, the plurality of immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or pharmaceutical compositions are formulated separately. In some embodiments, the plurality of immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or pharmaceutical compositions are administered simultaneously or sequentially. [Brief description of the drawings]

[0097] [Figure 1] A schematic diagram showing a representative plasmid used to generate an alum-binding protein is provided. The illustrated plasmid was used to generate a fusion of mouse serum albumin (MSA) with interleukin-2 (IL2) and the Fam20C targeting motif ABP10 with an N-terminal secretion leader sequence and a C-terminal His tag (MSA-IL2-ABP10, SEQ ID NO: 147). [Figure 2A] 1 provides an image of a denaturing SDS-PAGE gel showing the molecular weights of purified MSA-IL2 and MSA-IL2 protein variants having a C-terminal ABP that is either ABP3 (SEQ ID NO: 91), ABP4 (SEQ ID NO: 93), ABP5 (SEQ ID NO: 95), ABP6 (SEQ ID NO: 97), ABP7 (SEQ ID NO: 99), ABP8 (SEQ ID NO: 101), ABP10 (SEQ ID NO: 103), or ABP11 (SEQ ID NO: 105). [Figure 2B] FIG. 1 provides a line graph showing protein elution peaks measured by size-exclusion fast protein liquid chromatography (FPLC) for purified MSA-IL2 and MSA-IL2 with C-terminal ABP10 (MSA-IL2-ABP10, SEQ ID NO: 146). [Figure 2C] 1 provides an image of a denaturing SDS-PAGE gel showing the molecular weight of purified MSA-IL2 or MSA-IL2-ABP10 following co-transfection with wild-type Fam20C kinase (indicated by the "K" annotation) or inactive Fam20C kinase (indicated by the "IK" annotation). [Figure 3A] FIG. 3C provides line graphs showing the measurement of protein molecular weight by MALDI-MS for purified MSA-IL2 or MSA-IL2-ABP10 after co-transfection with wild-type Fam20C kinase (FIGS. 3C and 3D, respectively) or inactive Fam20C kinase (FIGS. 3A and 3B, respectively). [Figure 3B] FIG. 3C provides line graphs showing the measurement of protein molecular weight by MALDI-MS for purified MSA-IL2 or MSA-IL2-ABP10 after co-transfection with wild-type Fam20C kinase (FIGS. 3C and 3D, respectively) or inactive Fam20C kinase (FIGS. 3A and 3B, respectively). [Figure 3C] FIG. 3C provides line graphs showing the measurement of protein molecular weight by MALDI-MS for purified MSA-IL2 or MSA-IL2-ABP10 after co-transfection with wild-type Fam20C kinase (FIGS. 3C and 3D, respectively) or inactive Fam20C kinase (FIGS. 3A and 3B, respectively). [Figure 3D] FIG. 3C provides line graphs showing the measurement of protein molecular weight by MALDI-MS for purified MSA-IL2 or MSA-IL2-ABP10 after co-transfection with wild-type Fam20C kinase (FIGS. 3C and 3D, respectively) or inactive Fam20C kinase (FIGS. 3A and 3B, respectively). [Figure 4A]Provides images of denaturing SDS-PAGE gels showing the molecular weight (MW) of purified proteins of interest fused to the C-terminal ABP and expressed alone or co-expressed with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). Figure 4A provides a MW comparison of IFNg-IFNg-ABP10 (SEQ ID NO: 162, K and IK), IFNg-IFNg-MSA-ABP10 (SEQ ID NO: 160, K and IK), MSA-IL2-ABP10 (SEQ ID NO: 146, K and IK), and MSA-IL2 (SEQ ID NO: 204, K and IK). Figure 4B provides a MW comparison of MSA-IL2-ABP10 (K and IK), lysozyme, and lysozyme-ABP10 (SEQ ID NO: 156). [Figure 4B] Provides images of denaturing SDS-PAGE gels showing the molecular weight (MW) of purified proteins of interest fused to the C-terminal ABP and expressed alone or co-expressed with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). Figure 4A provides a MW comparison of IFNg-IFNg-ABP10 (SEQ ID NO: 162, K and IK), IFNg-IFNg-MSA-ABP10 (SEQ ID NO: 160, K and IK), MSA-IL2-ABP10 (SEQ ID NO: 146, K and IK), and MSA-IL2 (SEQ ID NO: 204, K and IK). Figure 4B provides a MW comparison of MSA-IL2-ABP10 (K and IK), lysozyme, and lysozyme-ABP10 (SEQ ID NO: 156). [Figure 5A]1 provides a bar graph showing quantification of phosphate content using a malachite green assay of purified MSA-IL2 or MSA-IL2 variants fused to a C-terminal ABP and expressed alone (without Fam20C) or co-expressed with wild-type Fam20C kinase (K). Figure 5A provides quantification of MSA-IL2 alone or fused to ABP3 (SEQ ID NO: 91), ABP4 (SEQ ID NO: 93), ABP5 (SEQ ID NO: 95), ABP6 (SEQ ID NO: 97), ABP7 (SEQ ID NO: 99), ABP8 (SEQ ID NO: 101), ABP10 (SEQ ID NO: 103) or ABP11 (SEQ ID NO: 105), Figure 5B provides quantification of MSA-IL2 alone or fused to ABP8 (MSA-IL2-ABP8, SEQ ID NO: 150) and Figure 5C provides quantification of MSA-IL2 alone, MSA-IL2-ABP10 or MSA-IL2 fused to ABP17 (MSA-IL2-ABP17, SEQ ID NO: 152). [Figure 5B] 1 provides a bar graph showing quantification of phosphate content using a malachite green assay of purified MSA-IL2 or MSA-IL2 variants fused to a C-terminal ABP and expressed alone (without Fam20C) or co-expressed with wild-type Fam20C kinase (K). Figure 5A provides quantification of MSA-IL2 alone or fused to ABP3 (SEQ ID NO: 91), ABP4 (SEQ ID NO: 93), ABP5 (SEQ ID NO: 95), ABP6 (SEQ ID NO: 97), ABP7 (SEQ ID NO: 99), ABP8 (SEQ ID NO: 101), ABP10 (SEQ ID NO: 103) or ABP11 (SEQ ID NO: 105), Figure 5B provides quantification of MSA-IL2 alone or fused to ABP8 (MSA-IL2-ABP8, SEQ ID NO: 150) and Figure 5C provides quantification of MSA-IL2 alone, MSA-IL2-ABP10 or MSA-IL2 fused to ABP17 (MSA-IL2-ABP17, SEQ ID NO: 152). [Figure 5C]1 provides a bar graph showing quantification of phosphate content using a malachite green assay of purified MSA-IL2 or MSA-IL2 variants fused to a C-terminal ABP and expressed alone (without Fam20C) or co-expressed with wild-type Fam20C kinase (K). Figure 5A provides quantification of MSA-IL2 alone or fused to ABP3 (SEQ ID NO: 91), ABP4 (SEQ ID NO: 93), ABP5 (SEQ ID NO: 95), ABP6 (SEQ ID NO: 97), ABP7 (SEQ ID NO: 99), ABP8 (SEQ ID NO: 101), ABP10 (SEQ ID NO: 103) or ABP11 (SEQ ID NO: 105), Figure 5B provides quantification of MSA-IL2 alone or fused to ABP8 (MSA-IL2-ABP8, SEQ ID NO: 150) and Figure 5C provides quantification of MSA-IL2 alone, MSA-IL2-ABP10 or MSA-IL2 fused to ABP17 (MSA-IL2-ABP17, SEQ ID NO: 152). [Figure 6A] 1 provides a bar graph showing quantification of phosphate content using a malachite green assay of purified MSA-IL2 or MSA-IL2-ABP10 proteins after co-expression with either wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). These figures represent data collected on proteins isolated from separate purification runs. ****p<0.0001. [Figure 6B] 1 provides a bar graph showing quantification of phosphate content using a malachite green assay of purified MSA-IL2 or MSA-IL2-ABP10 proteins after co-expression with either wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). These figures represent data collected on proteins isolated from separate purification runs. ****p<0.0001. [Figure 7] 1 provides images of immunoblots measuring serine phosphorylation of purified MSA-IL2 or MSA-IL2-ABP10 following co-expression with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). [Figure 8]1 provides images of immunoblots measuring serine phosphorylation of purified MSA-ABP10 (60 kDa) or MSA-IL2-ABP10 (90 kDa) following co-expression with wild-type Fam20C kinase (lane labeled "K") compared to expression without the kinase. [Figure 9] FIG. 1 is a graph showing protein elution peaks measured by anion exchange chromatography using FPLC for MSA-IL2-ABP10 co-expressed with Fam20C kinase (MSA-IL2-ABP10K) or MSA-IL2-ABP10 expressed without kinase (MSA-IL2-ABP10). [Figure 10] 1 provides a bar graph showing quantification of phosphate content, measured by malachite green assay, of purified MSA-IL2 or IFNg-IFNg-MSA protein alone, or fused to C-terminal ABP10, after co-expression with either wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). [Figure 11A] 1 provides an image of a denaturing SDS-PAGE gel showing the molecular weight (MW) of purified scIL12-ABP10 or scIL12-MSA-ABP10 co-expressed with wild-type Fam20C kinase (K) or without the kinase. [Figure 11B] 11C provides a bar graph showing quantification of phosphate content measured by malachite green assay for purified scIL12-ABP10 or scIL12-MSA-ABP10 co-expressed with wild-type Fam20C kinase (K) or kinase-free (FIG. 11B, ****p<0.0001), or for purified scIL12-ABP10, scIL12-MSA-ABP10, scIL12-ABP17, and scIL12-MSA-ABP17 co-expressed with wild-type Fam20C kinase (K) or kinase-free (FIG. 11C) compared to purified MSA-IL2 and MSA-IL2-ABP10K. [Figure 11C]11C provides a bar graph showing quantification of phosphate content measured by malachite green assay for purified scIL12-ABP10 or scIL12-MSA-ABP10 co-expressed with wild-type Fam20C kinase (K) or kinase-free (FIG. 11B, ****p<0.0001), or for purified scIL12-ABP10, scIL12-MSA-ABP10, scIL12-ABP17, and scIL12-MSA-ABP17 co-expressed with wild-type Fam20C kinase (K) or kinase-free (FIG. 11C) compared to purified MSA-IL2 and MSA-IL2-ABP10K. [Figure 12] 1 provides a bar graph showing quantification of phosphate content measured by malachite green assay for purified MSA-IL2 variants after co-expression with Fam20C kinase (K). MSA-IL2 variants are fused to a C-terminal ABP with a functional phosphorylation motif (ABP10), a C-terminal ABP10 with a modified motif to prevent Fam20C phosphorylation (ABP13), or a C-terminal ABP10 containing a trypsin cleavage site (ABP15). **p<0.01. [Figure 13A] 13A provides line graphs showing quantification of unbound protein in the supernatant of alum and protein mixtures as measured by fluorescence spectroscopy over time. Adsorption to alum was measured for fluorescently labeled MSA-IL2 or MSA-IL2-ABP10 co-expressed with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). The presence of unbound protein in the supernatant was measured for protein:alum mixtures following incubation with 10% mouse serum (MS) in phosphate-free buffer (FIG. 13A) or 10% MS in phosphate-buffered saline (PBS) (FIG. 13B). [Figure 13B]13A provides line graphs showing quantification of unbound protein in the supernatant of alum and protein mixtures as measured by fluorescence spectroscopy over time. Adsorption to alum was measured for fluorescently labeled MSA-IL2 or MSA-IL2-ABP10 co-expressed with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). The presence of unbound protein in the supernatant was measured for protein:alum mixtures following incubation with 10% mouse serum (MS) in phosphate-free buffer (FIG. 13A) or 10% MS in phosphate-buffered saline (PBS) (FIG. 13B). [Figure 14A] Figure 13 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 13A-13B and incubated with 10% FBS in phosphate-free buffer (Figures 14A-C) or 10% FBS in PBS (Figures 14D-F). [Figure 14B] Figure 13 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 13A-13B and incubated with 10% FBS in phosphate-free buffer (Figures 14A-C) or 10% FBS in PBS (Figures 14D-F). [Figure 14C] Figure 13 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 13A-13B and incubated with 10% FBS in phosphate-free buffer (Figures 14A-C) or 10% FBS in PBS (Figures 14D-F). [Figure 14D]Figure 13 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 13A-13B and incubated with 10% FBS in phosphate-free buffer (Figures 14A-C) or 10% FBS in PBS (Figures 14D-F). [Figure 14E] Figure 13 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 13A-13B and incubated with 10% FBS in phosphate-free buffer (Figures 14A-C) or 10% FBS in PBS (Figures 14D-F). [Figure 14F] Figure 13 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 13A-13B and incubated with 10% FBS in phosphate-free buffer (Figures 14A-C) or 10% FBS in PBS (Figures 14D-F). [Figure 15] Provides images of immunoblots used to measure unbound protein in samples of protein alone (column 1) or in the supernatants of protein mixed with alum (columns 2-6). Adsorption to alum was measured for MSA-IL2-ABP10 co-expressed with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK). The presence of unbound protein in the supernatant was measured before the addition of mouse serum (column 2) or at specific time intervals after the addition of mouse serum (columns 3-6 = 0, 1, 2, or 24 h). [Figure 16]1 provides a line graph showing quantification of unbound protein in the supernatant of alum and protein mixtures measured over time by fluorescence spectroscopy after exposure to 10% MS in PBS. Adsorption to alum was measured for fluorescently labeled fusion proteins including MSA-IL2-ABP10 coexpressed with wild-type Fam20C kinase (MSA-IL2-ABP10K), and MSA-IL2-ABP8 coexpressed with wild-type Fam20C kinase (MSA-IL2-ABP8K) or MSA-IL2-ABP8 expressed alone. [Figure 17] 1 provides a line graph showing quantification of alum-bound protein for alum and protein mixtures measured by fluorescence spectroscopy before and after 17 hours of exposure to 20% MS or 40% MS in PBS. Adsorption to alum was measured for fluorescently labeled MSA-IL2-ABP10 expressed alone, or for fluorescently labeled MSA-IL2-ABP10 coexpressed with wild-type Fam20C kinase (MSA-IL2-ABP10K), and for MSA-IL2-ABP17 coexpressed with wild-type Fam20C kinase (MSA-IL2-ABP17K). [Figure 18A] Provides line graphs showing quantification of unbound protein in the supernatant of alum and protein mixtures as measured by fluorescence spectroscopy over time. Adsorption to alum was measured for fluorescently labeled IFNg-IFNg-ABP10 or IFNg-IFNg-MSA-ABP10 after co-expression with Fam20C kinase (K) or inactive Fam20C kinase (IK). The presence of unbound protein in the supernatant was measured for protein:alum mixtures incubated with 10% MS in TBS without phosphate (FIG. 18A) or 10% MS in PBS (FIG. 18B). [Figure 18B]Provides line graphs showing quantification of unbound protein in the supernatant of alum and protein mixtures as measured by fluorescence spectroscopy over time. Adsorption to alum was measured for fluorescently labeled IFNg-IFNg-ABP10 or IFNg-IFNg-MSA-ABP10 after co-expression with Fam20C kinase (K) or inactive Fam20C kinase (IK). The presence of unbound protein in the supernatant was measured for protein:alum mixtures incubated with 10% MS in TBS without phosphate (FIG. 18A) or 10% MS in PBS (FIG. 18B). [Figure 19A] Figure 18 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 18A-18B and incubated with 10% MS in TBS without phosphate (Figures 19A-B) or 10% MS in PBS (Figures 19C-D). [Figure 19B] Figure 18 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 18A-18B and incubated with 10% MS in TBS without phosphate (Figures 19A-B) or 10% MS in PBS (Figures 19C-D). [Figure 19C] Figure 18 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 18A-18B and incubated with 10% MS in TBS without phosphate (Figures 19A-B) or 10% MS in PBS (Figures 19C-D). [Figure 19D]Figure 18 provides line graphs showing quantification of unbound protein by sandwich ELISA in the supernatants of samples containing alum and protein mixtures (solid lines) or protein alone (dashed lines). Adsorption to alum was measured for proteins shown in Figures 18A-18B and incubated with 10% MS in TBS without phosphate (Figures 19A-B) or 10% MS in PBS (Figures 19C-D). [Figure 20A] Bar graphs are provided showing the percentage of protein bound to alum as measured by fluorescence spectroscopy either before serum treatment (FIG. 20A) or after 17 hours of incubation in PBS containing 10% MS (FIG. 20B). Adsorption to alum was measured for purified fusion proteins scIL12-MSA-ABP10, scIL12-ABP10, and MSA-IL2-ABP10, either expressed alone or co-expressed with wild-type Fam20C kinase (K). [Figure 20B] Bar graphs are provided showing the percentage of protein bound to alum as measured by fluorescence spectroscopy either before serum treatment (FIG. 20A) or after 17 hours of incubation in PBS containing 10% MS (FIG. 20B). Adsorption to alum was measured for purified fusion proteins scIL12-MSA-ABP10, scIL12-ABP10, and MSA-IL2-ABP10, either expressed alone or co-expressed with wild-type Fam20C kinase (K). [Figure 21] FIG. 1 provides a line graph showing fluorescence intensity measured over time by an in vivo imaging system (IVIS) for mouse adenocarcinoma (MC38) flank tumors injected intratumorally with alum alone, fluorescent phosphoserine peptide (pSer4-AF647) alone, or alum + pSer4-AF647 complex. [Figure 22]A line graph is provided showing fluorescence intensity measured over time by IVIS for mouse MC38 flank tumors injected with fluorescently labeled IFNg-IFNg-MSA-ABP10 protein co-expressed with wild-type Fam20C kinase (K) or inactive Fam20C kinase (IK) alone (dashed line) or complexed with alum (solid line). [Diagram 23] 1 provides a line graph showing the fluorescence intensity measured by IVIS over time of mouse B16F10-Trp2 knockout tumors injected with fluorescently labeled MSA-IL2-ABP10K either as free protein or complexed with alum, or with fluorescently labeled lumican-MSA-IL2 (MSA-IL2 fused to the C-terminus of the collagen-anchored protein lumican). False color image of post-sacrifice ("post-sac") tumors injected with MSA-IL2-ABP10K complexed with alum, measured by IVIS, shown in the inset. [Figure 24A] 24A-24B provide confocal microscopy images of B16F10 tumor slices prepared from B16F10 tumor-bearing mice administered intratumoral injections of fluorescently labeled MSA-IL2-ABP10K, either free (FIG. 24A) or complexed with alum (FIG. 24B). Tumor isolation was performed 5 days after injection. Images are provided showing the tumor border outlined and the fluorescent MSA-IL2-ABP10K signal detected by confocal microscopy in white. Scale bar: 500 μm. [Figure 24B] 24A-24B provide confocal microscopy images of B16F10 tumor slices prepared from B16F10 tumor-bearing mice administered intratumoral injections of fluorescently labeled MSA-IL2-ABP10K, either free (FIG. 24A) or complexed with alum (FIG. 24B). Tumor isolation was performed 5 days after injection. Images are provided showing the tumor border outlined and the fluorescent MSA-IL2-ABP10K signal detected by confocal microscopy in white. Scale bar: 500 μm. [Figure 24C]Confocal microscopy images of B16F10 tumor slices prepared from B16F10 tumor-bearing mice administered intratumoral injections of fluorescently labeled MSA-IL2-ABP10K complexed with fluorescent alum-pSer4 are provided. Tumor isolation was performed 1 h after injection. Images of tumor slices showing either fluorescent MSA-IL2-ABP10K (left panel) or fluorescent alum-pSer4 (right panel) as detected by confocal microscopy imaging are provided, with tumor borders outlined and signal in grey. Scale bar: 500 μm. [Figure 24D] High-resolution confocal microscopy images of B16F10 tumor slices prepared from B16F10 tumor-bearing mice administered intratumoral injections of fluorescently labeled MSA-IL2-ABP10K complexed with fluorescent alum-pSer4 are provided. Tumor isolation was performed 5 days after injection. Images of tumor slices showing either tumor cells (left panel), fluorescent alum-pSer4 (middle panel), or fluorescent MSA-IL2-ABP10K (right panel) as detected by confocal microscopy imaging are provided, with signals shown in grey. Scale bar: 100 μm. [Figure 25A] Schematics are provided showing protein configurations evaluated for alum binding, including MSA alone, MSA-IL2, or MSA-IL2 fused to ABP10 at the C-terminus or N-terminus and co-expressed with wild-type Fam20C kinase (MSA-IL2-ABP10K and ABP10K-MSA-IL2, respectively). [Figure 25B] Figure 25A provides a line graph showing proliferation of CTLL-2 cells as measured by CellTiter Glo assay after treatment with different IL2 concentrations of protein either administered to cells alone (Figure 25B) or adsorbed to alum (Figure 25C). [Figure 25C] Figure 25A provides a line graph showing proliferation of CTLL-2 cells as measured by CellTiter Glo assay after treatment with different IL2 concentrations of protein either administered to cells alone (Figure 25B) or adsorbed to alum (Figure 25C). [Figure 26A]26A provides line graphs showing proliferation of CTLL-2 cells as measured by CellTiter Glo assay following treatment with alum-binding fusion proteins at different IL2 concentrations, including MSA-IL2-ABP10K or MSA-IL2-ABP8K (FIG. 26A), and MSA-IL2-ABP10K or MSA-IL-ABP17K (FIG. 26B). [Figure 26B] 26A provides line graphs showing proliferation of CTLL-2 cells as measured by CellTiter Glo assay following treatment with alum-binding fusion proteins at different IL2 concentrations, including MSA-IL2-ABP10K or MSA-IL2-ABP8K (FIG. 26A), and MSA-IL2-ABP10K or MSA-IL-ABP17K (FIG. 26B). [Figure 27A] 1 provides a schematic diagram of the fusion proteins of scIL12 and scIL12-MSA to phosphorylated ABP10 (scIL12-ABP10K and scIL12-MSA-ABP10K, respectively). [Figure 27B] 1 provides a bar graph quantifying the bioactivity of scIL12-ABP10K and scIL12-MSA-ABP10K as free proteins or complexed with alum as measured by in vitro activation of IL12 signaling in HEK-Blue IL12 reporter cells. Shown is a comparison with scIL12-MSA as a positive control and MSA as a negative control. [Figure 28A] FIG. 1 provides a schematic showing the treatment schedule for mice inoculated with B16F10 melanoma flank tumors and treated with a single dose of tumor-targeting antibody (TA99) administered by intraperitoneal (i.p.) injection and MSA-IL2 fusion protein administered by intratumoral (i.tu.) injection. [Figure 28B] FIG. 28A provides a line graph showing mouse survival after treatment with TA99 alone or in combination with free MSA-IL2, MSA-IL2 fused to a collagen binding domain (lumican), MSA-IL2 adsorbed to alum, MSA-IL2-ABP10K adsorbed to alum, or ABP10K-MSA-IL2 adsorbed to alum. [Figure 28C] FIG. 28C provides line graphs showing tumor area measured at regular intervals after treatment with TA99 alone (FIG. 28C) or in combination with MSA-IL2 (FIG. 28D), lumican-MSA-IL2 (FIG. 28E), MSA-IL2 adsorbed to alum (FIG. 28F), MSA-IL2-ABP10K adsorbed to alum (FIG. 28G), or ABP10K-MSA-IL2 adsorbed to alum (FIG. 28H). [Figure 28D] FIG. 28C provides line graphs showing tumor area measured at regular intervals after treatment with TA99 alone (FIG. 28C) or in combination with MSA-IL2 (FIG. 28D), lumican-MSA-IL2 (FIG. 28E), MSA-IL2 adsorbed to alum (FIG. 28F), MSA-IL2-ABP10K adsorbed to alum (FIG. 28G), or ABP10K-MSA-IL2 adsorbed to alum (FIG. 28H). [Figure 28E] FIG. 28C provides line graphs showing tumor area measured at regular intervals after treatment with TA99 alone (FIG. 28C) or in combination with MSA-IL2 (FIG. 28D), lumican-MSA-IL2 (FIG. 28E), MSA-IL2 adsorbed to alum (FIG. 28F), MSA-IL2-ABP10K adsorbed to alum (FIG. 28G), or ABP10K-MSA-IL2 adsorbed to alum (FIG. 28H). [Figure 28F] FIG. 28C provides line graphs showing tumor area measured at regular intervals after treatment with TA99 alone (FIG. 28C) or in combination with MSA-IL2 (FIG. 28D), lumican-MSA-IL2 (FIG. 28E), MSA-IL2 adsorbed to alum (FIG. 28F), MSA-IL2-ABP10K adsorbed to alum (FIG. 28G), or ABP10K-MSA-IL2 adsorbed to alum (FIG. 28H). [Figure 28G]FIG. 28C provides line graphs showing tumor area measured at regular intervals after treatment with TA99 alone (FIG. 28C) or in combination with MSA-IL2 (FIG. 28D), lumican-MSA-IL2 (FIG. 28E), MSA-IL2 adsorbed to alum (FIG. 28F), MSA-IL2-ABP10K adsorbed to alum (FIG. 28G), or ABP10K-MSA-IL2 adsorbed to alum (FIG. 28H). [Fig. 28H] FIG. 28C provides line graphs showing tumor area measured at regular intervals after treatment with TA99 alone (FIG. 28C) or in combination with MSA-IL2 (FIG. 28D), lumican-MSA-IL2 (FIG. 28E), MSA-IL2 adsorbed to alum (FIG. 28F), MSA-IL2-ABP10K adsorbed to alum (FIG. 28G), or ABP10K-MSA-IL2 adsorbed to alum (FIG. 28H). [Figure 29A] FIG. 1 provides a schematic showing the treatment schedule for mice inoculated with B16F10 flank tumors and treated with a single dose of TA99 administered by intraperitoneal injection and MSA-IL2 fusion protein administered by intratumoral injection. [Figure 29B] Provides a graph showing mouse survival after treatment with TA99 alone or in combination with free MSA-IL2, MSA-IL2 and alum, free MSA-IL2-ABP10K, MSA-IL2-ABP10K complexed with alum, lumican-MSA-IL2, or alum alone according to FIG. 29A. *=p<0.05, ***=p<0.001, ****=p<0.0001 [Figure 29C] 29A provides a graph quantifying the number of B16F10 reactive spot forming units (SFU) per million splenocytes as measured by IFNγ ELISPOT. Splenocytes were isolated from mice treated according to FIG. 29A on day 12 after tumor inoculation. [Figure 29D]29A provides a graph showing mouse survival following treatment with TA99 in combination with MSA-IL2-ABP10K complexed with alum, MSA-IL2-ABP8K complexed with alum, or alum alone, according to the schedule shown in FIG. 29A. [Figure 30A] Provides a schematic showing the treatment schedule for mice inoculated with B16F10 flank tumors and treated with anti-PD-1 antibody administered by intraperitoneal injection and a single dose of IL12 fusion protein administered by intratumoral injection. The IL12 fusion protein was either scIL12 directly fused to ABP10 (scIL12-ABP10) or scIL12-MSA fused to ABP10 (scIL12-MSA-ABP10). Mice received IL12 fusion protein complexed with alum only, IL12 fusion protein complexed with alum and anti-PD-1 antibody, or free IL12 fusion protein and anti-PD-1 antibody. Control mice received alum by intratumoral injection and anti-PD-1 antibody by intraperitoneal injection. [Figure 30B] Graphs are provided showing mouse survival after treatment with either scIL12-ABP10 fusion protein (FIG. 30B) or scIL12-MSA-ABP10 fusion protein (FIG. 30C) according to FIG. 30A. *=p<0.05, **=p<0.01 [Figure 30C] Graphs are provided showing mouse survival after treatment with either scIL12-ABP10 fusion protein (FIG. 30B) or scIL12-MSA-ABP10 fusion protein (FIG. 30C) according to FIG. 30A. *=p<0.05, **=p<0.01 [Figure 31A]30A provides a graph showing tumor area over time after tumor inoculation in B16F10 tumor-bearing mice administered anti-PD-1 antibody and IL12 fusion protein according to the treatment schedule shown in FIG. 30A. The IL12 fusion protein was either scIL12 fused to ABP10 (scIL12-ABP10) and complexed with alum (FIG. 31A), or scIL12 fused to ABP17 (scIL12-ABP17) and complexed with alum (FIG. 31B). Treatment with scIL12-ABP10 without alum (FIG. 31C) and alum only (FIG. 31D) were used as control groups. [Figure 31B] 30A provides a graph showing tumor area over time after tumor inoculation in B16F10 tumor-bearing mice administered anti-PD-1 antibody and IL12 fusion protein according to the treatment schedule shown in FIG. 30A. The IL12 fusion protein was either scIL12 fused to ABP10 (scIL12-ABP10) and complexed with alum (FIG. 31A), or scIL12 fused to ABP17 (scIL12-ABP17) and complexed with alum (FIG. 31B). Treatment with scIL12-ABP10 without alum (FIG. 31C) and alum only (FIG. 31D) were used as control groups. [Figure 31C] 30A provides a graph showing tumor area over time after tumor inoculation in B16F10 tumor-bearing mice administered anti-PD-1 antibody and IL12 fusion protein according to the treatment schedule shown in FIG. 30A. The IL12 fusion protein was either scIL12 fused to ABP10 (scIL12-ABP10) and complexed with alum (FIG. 31A), or scIL12 fused to ABP17 (scIL12-ABP17) and complexed with alum (FIG. 31B). Treatment with scIL12-ABP10 without alum (FIG. 31C) and alum only (FIG. 31D) were used as control groups. [Figure 31D]30A provides a graph showing tumor area over time after tumor inoculation in B16F10 tumor-bearing mice administered anti-PD-1 antibody and IL12 fusion protein according to the treatment schedule shown in FIG. 30A. The IL12 fusion protein was either scIL12 fused to ABP10 (scIL12-ABP10) and complexed with alum (FIG. 31A), or scIL12 fused to ABP17 (scIL12-ABP17) and complexed with alum (FIG. 31B). Treatment with scIL12-ABP10 without alum (FIG. 31C) and alum only (FIG. 31D) were used as control groups. [Figure 32A] Schematic diagram showing the treatment schedule for mice inoculated with B16F10 flank tumors and treated with anti-PD-1 antibodies administered by intraperitoneal injection, and a single dose of IL12 and / or IL2 fusion proteins administered by intratumoral injection. Mice were administered anti-PD-1 antibodies in combination with (i) free MSA-IL2-ABP10K and free scIL12-MSA-ABP10K, (ii) MSA-IL2-ABP10K and scIL12-MSA-ABP10K, respectively, complexed to alum, or (iii) scIL12-MSA-ABP10K complexed to alum. Control mice received intratumoral injections of alum. [Figure 32B] Graphs are provided showing percent weight change over time normalized to pre-treatment weight (FIG. 32B) and survival of mice after treatment with FIG. 32A (FIG. 32C). **p<0.01; ****p<0.0001. [Figure 32C] Graphs are provided showing percent weight change over time normalized to pre-treatment weight (FIG. 32B) and survival of mice after treatment with FIG. 32A (FIG. 32C). **p<0.01; ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] Immunomodulatory fusion proteins and metal hydroxide complexes The present disclosure provides novel compositions and methods for enhancing immune responses (e.g., cancer-specific immune responses) against tumors resulting from administration of an immunomodulatory fusion protein to a vertebrate. According to the present disclosure, the immune response is enhanced by administering the immunomodulatory fusion protein in a form in which its presentation to the immune response enhances the response. For example, promoting, increasing, or enhancing the period during which the immunomodulatory fusion protein is available at the tumor or tumor-draining lymph nodes, or promoting, increasing, or enhancing the activation of immune cells (e.g., dendritic cells, cytotoxic T cells) against tumor cells. The present disclosure arises from the discovery that immunomodulatory fusion proteins adsorbed to metal hydroxides (e.g., alum) by electrostatic and other secondary forces alone do not remain highly adsorbed to the metal hydroxide (e.g., alum). However, antigens adsorbed to metal hydroxides (e.g., alum) by ligand exchange remain tightly bound and remain at the injection site for extended periods of time following injection into the tumor site, resulting in a strong cancer-specific immune response. Accordingly, provided herein are methods and compositions for generating immune modulating fusion proteins for adsorption via ligand exchange to metal hydroxides (e.g., alum) for use in generating cancer-specific immune responses following administration in vivo.

[0099] Thus, provided herein are methods for increasing phosphorylation of an immunomodulatory fusion protein to increase adsorption from or decrease release from a metal hydroxide (e.g., alum). In some embodiments, the methods of the disclosure include methods for increasing phosphorylation of an immunomodulatory fusion protein to increase adsorption from or decrease release from a metal hydroxide (e.g., alum). Provided is an immunomodulatory fusion protein comprising an immunomodulatory domain, a metal hydroxide binding peptide, and optionally a stabilizing domain, where the metal hydroxide binding peptide comprises one or more kinase target motifs of a secretory pathway kinase. When expressed in a host cell comprising a secretory pathway kinase, the one or more kinase target motifs are phosphorylated to provide an immunomodulatory fusion protein modified during recombinant expression with a phosphate group. The resulting immunomodulatory fusion protein is further contacted with a metal hydroxide (e.g., alum), where the phosphorylated kinase target motif of the metal hydroxide binding peptide allows for adsorption via ligand exchange to the metal hydroxide (e.g., alum), thereby forming an immunomodulatory fusion protein-metal hydroxide complex. Such methods are useful, for example, by allowing for easier modes of production using methods of recombinant protein expression and purification known to those of skill in the art.

[0100] In other embodiments, the method of the disclosure provides a polypeptide comprising an immunomodulatory domain, optionally operably linked thereto, where the stabilizing domain is further crosslinked to a metal hydroxide-binding peptide comprising one or more hydroxyl substituents (e.g., phosphorylated amino acid residues) via a polypeptide reactive moiety. Thus, the method allows for modification of the polypeptide comprising the immunomodulatory domain after recombinant expression of the polypeptide. The resulting immunomodulatory fusion protein is further contacted with a metal hydroxide (e.g., alum), allowing one or more hydroxyl substituents (e.g., phosphorylated amino acid residues) of the metal hydroxide-binding peptide to adsorb to the metal hydroxide (e.g., alum) via ligand exchange, thereby forming an immunomodulatory fusion protein-metal hydroxide complex. Such methods are useful to those skilled in the art, for example, by allowing covalent attachment of a metal hydroxide-binding peptide comprising a non-natural amino acid residue that confers higher affinity binding to the metal hydroxide (e.g., alum).

[0101] metal hydroxide In some embodiments, the present disclosure provides an immunomodulatory fusion protein-metal hydroxide complex, in which the immunomodulatory fusion protein is absorbed to a metal hydroxide. Adsorption to the metal hydroxide occurs by a ligand exchange mechanism, and the displacement results in the formation of an inner sphere surface complex comprising the immunomodulatory fusion protein and the metal hydroxide, whereby the immunomodulatory fusion protein is strongly adsorbed to the adjuvant particle. As used herein, the term "ligand exchange" is defined as the replacement or exchange of a surface hydroxyl with another ligand, in this case an antigen containing a hydroxyl substituent. Methods for measuring adsorption by ligand exchange are known to those skilled in the art. For example, adsorption can be measured by ellipsometry (ELM), surface plasmon resonance (SPR), optical waveguide light mode spectroscopy (OWLS), attenuated total internal reflection infrared spectroscopy (ATR-IR), circular dichroism spectroscopy (CD), total internal reflection infrared spectroscopy (TIRF), and other high-resolution microscopy techniques. In some embodiments, these methods show the spatial arrangement between domains of the immunomodulatory fusion protein.

[0102] As used herein, the term "metal hydroxide" is used to refer to a material that contains at least one hydroxyl group bound to a metal, can adsorb an immunomodulatory fusion protein having a hydroxyl-substituted moiety, and can assist the immunomodulatory fusion protein in eliciting a cancer-specific immune response when delivered to a vertebrate. In some embodiments, the metal hydroxide is selected as being biocompatible with humans and non-humans. A preferred metal hydroxide adjuvant for use according to the present invention is an aluminum-containing metal hydroxide. The term "aluminum-containing metal hydroxide" is defined as a material that contains at least one hydroxyl group bound to aluminum. Examples of aluminum-containing metal hydroxides are aluminum hydroxide and aluminum phosphate.

[0103] Those skilled in the relevant art will understand that the term "aluminum hydroxide" is used in the art to identify crystalline oxyhydroxide compounds. Aluminum hydroxide has only hydroxyl groups on the surface, which are covalently bonded to the aluminum. The term "aluminum phosphate" is used in the art to identify amorphous aluminum hydroxyphosphate. Aluminum phosphate has phosphate and hydroxyl groups on the surface, which are covalently bonded to the aluminum.

[0104] The principles of the present invention are equally applicable to metal hydroxides other than aluminum-containing metal hydroxides. As further alternative, non-limiting examples of metal hydroxides, the present invention contemplates that iron hydroxide or calcium phosphate are suitable for use in accordance with the present invention.

[0105] The present invention also contemplates the use of modified metal hydroxides, such as modified aluminum-containing metal hydroxides, in immunogenic formulations for delivering modified hydroxyl-substituted immunomodulatory fusion proteins. As used herein, the term "modified metal hydroxide" is used to refer to metal hydroxides in which some of the surface hydroxyl groups have been replaced or modified so that the number of surface hydroxyl groups available for ligand exchange is reduced. One exemplary method of modifying metal hydroxide adjuvants according to the present invention is by contacting the metal hydroxide with a phosphate-containing solution for a sufficient time to cause phosphate replacement of the surface hydroxyl groups (also referred to herein as phosphorylation of some of the surface hydroxyl groups). For example, when aluminum hydroxide is exposed to phosphate, the phosphate can replace the surface hydroxyls and form inner sphere surface complexes (covalent bonds) with the surface aluminum, thereby modifying the surface structure of the aluminum hydroxide adjuvant. The surface hydroxyl groups of aluminum phosphate adjuvant can be phosphorylated in a similar manner.

[0106] Metal hydroxide (e.g., alum) can be modified as described to modify the density of hydroxyl groups on the surface of metal hydroxide particles available for ligand exchange. Alternatively, or in addition, modification of metal hydroxide (e.g., alum) in this manner may be desirable for other reasons. For example, the present disclosure contemplates that altering the density of hydroxyl groups on the surface of metal hydroxide (e.g., alum) particles accordingly alters the degree of adsorption or coupling of immunogenic compositions.

[0107] Non-limiting examples of hydroxyl substituents include fluoride, citrate, phosphate, sulfate, and carbonate groups. Aluminum hydroxide has a high affinity for phosphate and can replace surface hydroxyls in ligand exchange reactions. In some embodiments, the metal hydroxide bond contains one or more hydroxyl substituents that contain phosphate groups. Aluminum has an even higher affinity for fluorine. In some embodiments, the metal hydroxide bond contains one or more hydroxyl substituents that contain fluorine groups.

[0108] In some embodiments, the immunomodulatory fusion protein adsorbed by ligand exchange to metal hydroxide is of sufficient mass to prevent size-dependent diffusion from the injection site in tissue (e.g., tumor). Methods for measuring diffusion from tissue are known to those skilled in the art. For example, diffusion can be measured by in vivo imaging or via microscopic examination of tissue sections over time. Exemplary methods are described at least in Schmidt & Wittrup, Mol Canc Ther. 2009' and Wittrup et al., Methods in Enzymol 2012, each of which is incorporated herein by reference in its entirety.

[0109] Kinase targeting motif In some embodiments, the present disclosure provides a method for the preparation of a medicament comprising administering to a subject a medicament comprising administering to said subject a medicament ... The present invention provides an immunomodulatory fusion protein comprising a metal hydroxide-binding peptide that contains ... See, e.g., Kemp, et al., (1975) PNAS 72:3448-3452, Daile, et al., (1975) Nature 257:416-418, and Pearson, et al. (1991) Methods Kinase target motifs are determined using mutational analysis of known kinase substrates, as described by Enzymol. 200:62-81. In another example, a peptide library screen is used to determine kinase target motifs, and the kinase of interest is screened with ATP to identify 10 kinases with only one phosphorylatable residue, as described by Songyang, et al. (1994) Curr Biol 4:973-982. 9A soluble mixture of peptides is added to the soluble mixture of peptides. The kinase reaction is allowed to occur for a short period of time before the phosphorylated peptides are separated from the non-phosphorylated peptides and the mixture is sequenced. Identification of the preferred amino acid at each position is obtained by comparing the abundance of the amino acid at each position in the phosphorylated fraction compared to the starting mixture. In another example, a biotinylated double-oriented peptide library containing Ser and / or Thr residues at a first fixed position and a second fixed amino acid as described by Hutti, et al (2004) Nature Methods 1:27-29. The peptide mixture is incubated with the kinase in a 96-well plate configuration and transferred to an avidin-coated membrane for analysis of phosphorylation by autoradiography. Using such methods, kinase target motifs have been identified for specific cellular kinases such as those listed in Table 1 and further described in the listed references or by Pinna, et al (1996) Biochim Biophys Acta 1314:191-225. [Table 1-1] [Table 1-2]

[0110] In some embodiments, the present disclosure provides an immunomodulatory fusion protein comprising a metal hydroxide-binding peptide that includes one or more kinase targeting motifs of secretory pathway kinases. The secretory pathway refers to the endoplasmic reticulum (ER), the Golgi apparatus, and the vesicles that move between them, as well as the plasma membrane and lysosomal storage compartments. The secretory pathway is the route by which cells secrete proteins into the extracellular environment. Many proteins are synthesized and sorted into the secretory pathway by entering the ER. This occurs during translation, when the ribosomes that synthesize the protein bind to the rough ER and the synthesized protein cotranslationally crosses the ER membrane. Entry into the secretory pathway is directed by an ER targeting leader sequence. The ER targeting leader sequence, which contains a stretch of hydrophobic amino acids, is generally present at the N-terminus of a protein and directs the translocation of the protein into the ER lumen.

[0111] Soluble proteins sorted into the secretory pathway are localized in the lumen of the ER and are subsequently sorted into the lumen of other organelles or secreted from the cell. Proteins destined for secretion are assembled into small transport vesicles and trafficked to the cis-Gogli endoplasmic reticulum. Proteins are either recycled back to the ER or trafficked to the trans-Golgi by cisternal migration. From the trans face of the Golgi, secretory proteins are sorted into transport vesicles for secretion or into secretory vesicles for storage within the cell.

[0112] It is known in the art that intracellular proteins localized in the cytosol or nucleus can be modified with an ER targeting leader sequence to direct the protein to the secretory pathway. Thus, in some embodiments, kinases localized in the cytosol or nucleus, such as those listed in Table 1, are modified with an ER targeting leader sequence to direct the kinase to the secretory pathway, thereby generating a "secretory pathway kinase". Furthermore, polypeptides comprising a C-terminal anchor peptide (e.g., KDEL, e.g., HDEL) are known in the art to have increased retention in the secretory pathway. In some embodiments, the kinase is further modified with an anchor peptide to promote or increase retention in the secretory pathway and / or reduced secretion.

[0113] In some embodiments, an immunomodulatory fusion protein comprising an immunomodulatory domain, a metal hydroxide binding peptide, and optionally a stabilization domain as described herein is made in a transfected host cell using recombinant DNA techniques, wherein the metal hydroxide binding peptide comprises one or more kinase target motifs of a cellular kinase listed in Table 1. In some embodiments, a cell is transfected with a recombinant DNA molecule encoding the immunomodulatory fusion protein and a recombinant DNA molecule encoding a kinase comprising an ER targeting leader sequence, a kinase domain and an anchor peptide derived from a kinase of Table 1, wherein the kinase is localized to the secretory pathway by the ER targeting leader sequence and the anchor peptide, and the one or more kinase target motifs of the metal hydroxide binding peptide are phosphorylated by a kinase in the secretory pathway, thereby increasing phosphorylation of the immunomodulatory fusion protein.

[0114] Most kinases are localized in the nucleus or cytosol of the cell. However, some kinases reside in the secretory pathway and function to phosphorylate proteins destined for secretion. Naturally occurring secretory pathway kinases have been identified and described (Ishikawa, et al (2008) Science 321:401-404; Tagliabracci et al, (2012) Science 336:1150-1153; Tagliabracci, et al (2015) Cell 161:1619-1632; Tagliabracci et al (2013) Trends Biochem Sci 38:121-130). As described, secretory pathway kinases contain an N-terminal ER targeting leader sequence and a C-terminal kinase domain to direct the kinase to the secretory pathway. Additionally, secretory pathway kinases lack predicted transmembrane helices, allowing the kinase domain to orient in the ER and / or Golgi lumen in close proximity to secretory pathway proteins. Naturally occurring human secretory pathway kinases include, but are not limited to, 4-junction box kinase 1, Fam20A, Fam20B, Fam20C (also called Golgi casein kinase), Fam198A, Fam198B, Fam69A, Fam69B, Fam69C, and vertebrate orphan kinase (VLK).

[0115] In some embodiments, the present disclosure provides an immunomodulatory fusion protein comprising a metal hydroxide-binding peptide that comprises two or more kinase target motifs of the secretory pathway kinase Fam20C. Fam20C is a human kinase that has been identified for phosphorylation of over 100 secreted phosphoproteins, including a significant portion of the extracellular phosphoproteome (Tagliabracci, et al (2015) Cell 161:1619-1632). Fam20C phosphorylates casein and is often referred to as Golgi-enriched fraction casein kinase. Additionally, Fam20C phosphorylates serine and has been shown to phosphorylate kinase target motifs containing the amino acid sequences Ser-X-Glu (e.g., SXE), Ser-X-pSer (e.g., SX-pS), and Ser-X-Gln-XX-Asp-Glu-Glu (SXQXXDEE), where X is any amino acid and pS is phosphorylated serine (Mercier, et al (1981) Biochimie, 63:1-17; Mercier et al (1971) Eur J. Biochem. 23:41-51; Lasa-Benito (1996) FEBS Lett. 382:149; Brunati, et al (2000) 3:765, Tagliabraccci, et al (2015) Cell 161:1619-1632; Tagliabraccci, et al (2012) Science 336:1150-1153). For example, a peptide substrate derived from β-casein containing the amino acid sequence [ka] (β28-40, SEQ ID NO:99) contains an SXE kinase target motif and the serine is phosphorylated by Fam20C.

[0116] In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a metal hydroxide binding peptide comprising one or more kinase target motifs of secretory pathway kinase Fam20C, wherein the kinase target motif comprises the amino acid sequence SXE, where X is any amino acid, and the serine is modified with phosphate. In some embodiments, the one or more kinase target motifs of secretory pathway kinase Fam20C comprise the amino acid sequence SXE, where X is E, S, V, or G, and the serine is modified with phosphate. In some embodiments, the one or more kinase target motifs of secretory pathway kinase Fam20C comprise the amino acid sequence SEE.

[0117] In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a metal hydroxide binding peptide comprising one or more kinase targeting motifs of the secretory pathway kinase Fam20C, where the kinase targeting motif comprises the amino acid sequence SXS, where X is any amino acid, and one or both serine residues are modified with phosphate.

[0118] In some embodiments, the metal hydroxide binding peptide comprises at least one, two, or three kinase target motifs. In some embodiments, the kinase target motifs are consecutive.

[0119] Metal hydroxide binding peptides In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a metal hydroxide binding peptide. In some embodiments, the metal hydroxide binding peptide comprises at least one phosphorylated amino acid. In some embodiments, the metal hydroxide binding peptide comprises at least two phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least three phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least four phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least five phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least six phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least seven phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least eight phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least nine phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least ten phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least eleven phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least twelve phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least 13 phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least 14 phosphorylated amino acids. In some embodiments, the metal hydroxide binding peptide comprises at least 15 phosphorylated amino acids.

[0120] In some embodiments, the metal hydroxide binding peptide comprises a phosphorylated amino acid selected from the group consisting of phosphoserine, phosphotyrosine, or phosphothreonine. In some embodiments, the metal hydroxide binding peptide comprises at least one phosphoserine.

[0121] In some embodiments, the metal hydroxide-binding peptide comprises a phosphoserine residue. In some embodiments, the metal hydroxide-binding peptide comprises 1 to 15 consecutive phosphoserine residues.

[0122] In some embodiments, the metal hydroxide binding peptide is about 6-15, about 10-25, about 10-50, about 10-100 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 10 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 15 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 20 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 25 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 30 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 35 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 40 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 45 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 50 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 55 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 60 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 65 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 70 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 75 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 80 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 85 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 90 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 95 amino acids in length. In some embodiments, the metal hydroxide binding peptide is about 100 amino acids in length.

[0123] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise one or more target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises one kinase target motif of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises two kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises three kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises four kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises five kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises six kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises seven kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises eight kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises nine kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises ten kinase target motifs of a secretory pathway kinase. In some embodiments, the metal hydroxide binding peptide comprises 11 kinase target motifs of secretory pathway kinases.In some embodiments, the metal hydroxide binding peptide comprises 12 kinase target motifs of secretory pathway kinases.

[0124] In some embodiments, the metal hydroxide binding peptides of the disclosure comprise two or more kinase target motifs of secretory pathway kinases, where the amino acid sequences of the two or more kinase target motifs are the same. In some embodiments, the metal hydroxide binding peptides of the disclosure comprise two or more kinase target motifs of secretory pathway kinases, where the amino acid sequences of the two or more kinase target motifs are different.

[0125] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise two or more kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptides comprise two kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptides comprise three kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptides comprise four kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptides comprise five kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptides comprise six kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptides comprise seven kinase target motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers. In some embodiments, the metal hydroxide binding peptide comprises eight kinase targeting motifs of secretory pathway kinases that are contiguous with no intervening amino acid linkers.

[0126] In some embodiments, the metal hydroxide binding peptides of the disclosure comprise two or more kinase target motifs of secretory pathway kinases with an intervening amino acid linker, the amino acid linker comprising about 1-5, about 1-10, about 1-15, about 1-20, about 1-25, about 1-30, about 1-35, about 1-40, about 1-45, about 1-50 amino acids. In some embodiments, the metal hydroxide binding peptides comprise two or more kinase target motifs of secretory pathway kinases with an intervening amino acid linker, the amino acid linker comprising about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 amino acid. In some embodiments, the linker comprises a gly-ser polypeptide linker.

[0127] In some embodiments, the metal hydroxide binding peptide has the amino acid sequence ([A]-[L]) x wherein A comprises the amino acid sequence of a kinase targeting motif of a secretory pathway kinase disclosed herein, L comprises the amino acid sequence of an amino acid linker, and x=1-15.

[0128] In some embodiments, the metal hydroxide binding peptides of the disclosure comprise one or more kinase targeting motifs of a secretory pathway kinase, where a first kinase targeting motif (e.g., an N-terminal kinase targeting motif) is located at the N-terminus of the metal hydroxide binding peptide. In some embodiments, the metal hydroxide binding peptides comprise one or more kinase targeting motifs of a secretory pathway kinase, where a first kinase targeting motif (e.g., an N-terminal kinase targeting motif) is separated from the N-terminus by about 1-5, about 1-10, about 5-10, about 5-15, about 10-15, about 10-20, about 10-30, about 10-40, about 10-50 amino acids. In some embodiments, the metal hydroxide binding peptide comprises one or more kinase targeting motifs of secretory pathway kinases, and the first kinase targeting motif (e.g., the N-terminal kinase targeting motif) is separated from the N-terminus by about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1 amino acid. In some embodiments, the metal hydroxide binding peptide comprises one or more kinase targeting motifs of secretory pathway kinases, and the first kinase targeting motif (e.g., the N-terminal kinase targeting motif) is separated from the N-terminus by one amino acid. In some embodiments, the metal hydroxide binding peptide comprises one or more kinase targeting motifs of secretory pathway kinases, and the first kinase targeting motif (e.g., the N-terminal kinase targeting motif) is separated from the N-terminus by two amino acids. In some embodiments, the metal hydroxide binding peptide comprises one or more kinase targeting motifs of a secretory pathway kinase, and a first kinase targeting motif (e.g., an N-terminal kinase targeting motif) is separated from the N-terminus by three amino acids. In some embodiments, the metal hydroxide binding peptide comprises one or more kinase targeting motifs of a secretory pathway kinase, and a first kinase targeting motif (e.g., an N-terminal kinase targeting motif) is separated from the N-terminus by four amino acids.In some embodiments, the metal hydroxide binding peptide comprises one or more kinase targeting motifs of a secretory pathway kinase, wherein a first kinase targeting motif (e.g., an N-terminal kinase targeting motif) is separated from the N-terminus by five amino acids.

[0129] In some embodiments, the metal hydroxide binding peptides of the disclosure comprise one kinase targeting motif of a secretory pathway kinase, where the kinase targeting motif is located at or near the C-terminus of the metal hydroxide binding peptide (e.g., separated by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids). In some embodiments, the metal hydroxide binding peptides comprise two kinase targeting motifs of secretory pathway kinases, where the second kinase targeting motif (e.g., the C-terminal kinase targeting motif) is located at or near the C-terminus of the metal hydroxide binding peptide. The metal hydroxide-binding peptide includes three kinase targeting motifs of a secretory pathway kinase, and the third kinase targeting motif (e.g., a C-terminal kinase targeting motif) is located at or near the C-terminus of the metal hydroxide-binding peptide (e.g., separated by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids). In some embodiments, the metal hydroxide-binding peptide includes three kinase targeting motifs of a secretory pathway kinase, and the third kinase targeting motif (e.g., a C-terminal kinase targeting motif) is located at or near the C-terminus of the metal hydroxide-binding peptide (e.g., separated by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids). In some embodiments, the metal hydroxide binding peptide comprises four kinase targeting motifs of secretory pathway kinases, with a fourth kinase targeting motif (e.g., a C-terminal kinase targeting motif) being located at or near the C-terminus of the metal hydroxide binding peptide (e.g., separated by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids). In some embodiments, the metal hydroxide binding peptide comprises five kinase targeting motifs of secretory pathway kinases, with a fifth kinase targeting motif (e.g., a C-terminal kinase targeting motif) being located at or near the C-terminus of the metal hydroxide binding peptide (e.g., separated by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids). In some embodiments, the metal hydroxide binding peptide comprises six kinase targeting motifs of secretory pathway kinases, with a sixth kinase targeting motif (e.g., a C-terminal kinase targeting motif) being located at or near the C-terminus of the metal hydroxide binding peptide (e.g., separated by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids).

[0130] Exemplary Metal Hydroxide Binding Peptides In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a metal hydroxide binding peptide comprising one or more kinase targeting motifs of the secretory pathway kinase Fam20C, wherein the one or more kinase targeting motifs comprise the sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises two kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises three kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises four kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises five kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises six kinase targeting motifs comprising the amino acid sequence SXE, where X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises seven kinase targeting motifs comprising the amino acid sequence SXE, where X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises eight kinase targeting motifs comprising the amino acid sequence SXE, where X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises nine kinase targeting motifs comprising the amino acid sequence SXE. In some embodiments, the metal hydroxide binding peptide comprises 10 kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises 11 kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different. In some embodiments, the metal hydroxide binding peptide comprises 12 kinase targeting motifs comprising the amino acid sequence SXE, X is any amino acid sequence, the serine is modified with phosphate, and the kinase targeting motifs are the same or different.

[0131] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, where X is any amino acid and the serine is modified with phosphate. In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, where X is selected from the group consisting of E, S, V, H, and Q, and at least one serine is modified with phosphate. In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, where X is E and the serine is modified with phosphate.

[0132] In some embodiments, a metal hydroxide binding peptide of the present disclosure comprises at least one kinase targeting motif comprising the amino acid sequence SXE, wherein the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of XXSXX (SEQ ID NO: 127) or XXSEEXX (SEQ ID NO: 128), where X is any amino acid and at least one serine is modified with phosphate.

[0133] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide comprises the amino acid sequence Xaa 1 -Xaa 2 -S-Xaa 3 -E-Xaa 4 -Xaa 5 (SEQ ID NO: 127), 1 is F, M or G, and Xaa 2 is Q, E, or G, and Xaa 3 are E, S, V, H, Q, and G, and Xaa 4 is Q, S, or G, and Xaa 5 is Q, N, or G, and at least one serine is modified with phosphate. 3 is E. In some embodiments, Xaa 1 is F and Xaa 2 In some embodiments, Xaa is Q. 1 is M and Xaa 2 is E. In some embodiments, Xaa 1 is G and Xaa 2 In some embodiments, Xaa is G. 4 is Q and Xaa 5 In some embodiments, Xaa is Q. 4 is E and Xaa 5 is S. In some embodiments, Xaa 4 is G and Xaa 5 is G.

[0134] In some embodiments, a metal hydroxide binding peptide of the present disclosure comprises at least one kinase target motif comprising the amino acid sequence SXE, wherein the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, wherein X is any amino acid and at least one serine is phosphorylated.

[0135] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase target motif comprising the amino acid sequence SEE, wherein the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and at least one serine is phosphorylated.

[0136] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide comprises the amino acid sequence Xaa 1 -Xaa 2 -S-Xaa 3 -E-Xaa 4 -Xaa 5 -[L]-S-Xaa 3 -E-Xaa 6 -Xaa 7 Contains Xaa 1 is F, M or G, and Xaa 2 is Q, E, or G, and Xaa 3 are E, S, V, H, Q, and G, and Xaa 4 is Q, S, or G, and Xaa 5 is Q, N, or G, and Xaa 5 is G and Xaa 6 is G and L is a peptide linker, optionally a gly-ser polypeptide linker, optionally GGGS. [Table 2]

[0137] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide has the formula [A] xwherein A is an amino acid sequence selected from the group consisting of SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131; and x is an integer with a value indicating the number of linked amino acid sequences represented by A, where x is 1 to 15.

[0138] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[B], where A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131.

[0139] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide has the formula ([A]-[B]): X wherein A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131, and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[B], and x is 1 to 8.

[0140] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, The linker comprises a sequence of linked amino acids comprising the formula [A]-[L]-[A], where A is the same or a different amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and L comprises an amino acid linker such as those described herein. In some embodiments, the linker comprises a gly-ser polypeptide linker. In some embodiments, L comprises the amino acid sequence GGGS.

[0141] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide has the formula ([A]-[L]-[A]) x wherein A is an amino acid sequence selected from the group consisting of SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131, x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[L]-[A], x is 1 to 4, and L comprises an amino acid linker such as those described herein. In some embodiments, the linker comprises a gly-ser polypeptide linker. In some embodiments, L comprises the amino acid sequence GGGS.

[0142] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[L]-[B], where A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and L comprises an amino acid linker such as those described herein, including: In some embodiments, the linker comprises a gly-ser polypeptide linker; In some embodiments, L comprises the amino acid sequence GGGS.

[0143] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase targeting motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide has the formula ([A]-[L]-[B]): xwherein A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[L]-[B], where x is 1 to 4, and L comprises an amino acid linker such as those described herein. In some embodiments, the linker comprises a gly-ser polypeptide linker. In some embodiments, L comprises the amino acid sequence GGGS.

[0144] In some embodiments, the metal hydroxide binding peptide of the present disclosure comprises at least one kinase target motif comprising the amino acid sequence SXE, and the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101, as shown in Table 3. [Table 3]

[0145] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise one or more kinase targeting motifs comprising the amino acid sequence SXE, and the metal hydroxide binding peptide has the formula [C] x wherein C is an amino acid sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101; and x is an integer with a value indicating the number of linked amino acid sequences represented by C, where x is 1 to 4.

[0146] In some embodiments, the formula [C] x wherein C is the amino acid sequence set forth in SEQ ID NO:91 and x is 2. The metal hydroxide binding peptide comprises the amino acid sequence set forth in SEQ ID NO:115.

[0147] In some embodiments, the formula [A]x wherein A is the amino acid sequence set forth in SEQ ID NO:8 and x is 2. The metal hydroxide binding peptide comprises the amino acid sequence set forth in SEQ ID NO:107.

[0148] In some embodiments, the metal hydroxide binding peptides of the present disclosure comprise one or more kinase targeting motifs comprising the amino acid sequence SXE, and the metal hydroxide binding peptide has the formula [C] x -[D] y wherein C and D are the same or different amino acid sequences, C and D are selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101, x is an integer having a value indicating the number of linked amino acid sequences represented by C, y is an integer having a value indicating the number of linked amino acid sequences represented by D, x is 1 to 4, y is 1 to 4, and x and y are the same or different.

[0149] In some embodiments, the formula [C] x -[D] y wherein C is the amino acid sequence set forth in SEQ ID NO:91, D is the amino acid sequence set forth in SEQ ID NO:93, is 1, and y is 1. The metal hydroxide binding peptide comprises the amino acid sequence set forth in SEQ ID NO:103. [Table 4]

[0150] Polypeptide reactive moieties In some embodiments, a polypeptide comprising at least one immunomodulatory domain, and optionally a stabilizing domain, is modified with a polypeptide reactive moiety linked to a metal hydroxide binding peptide, thereby forming an immunomodulatory fusion protein.

[0151] In some embodiments provided by the present disclosure, the polypeptide reactive moiety comprises a reactive or functional group selected from the group consisting of an amine reactive group, a carboxyl to amine reactive group, a sulfhydryl reactive group, an aldehyde or carbonyl reactive group, a hydroxyl reactive group, an azide reactive group, and a photoreactive group.

[0152] In some embodiments, the polypeptide reactive moiety comprises an amine reactive group. Non-limiting examples of amine reactive groups include isothiocyanates, isocyanates, sulfonyl chlorides, aldehydes, carbodiimides, acyl azides, anhydrides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters (NHS esters), imidoesters, epoxides, and fluorophenyl esters. In some embodiments, the polypeptide reactive moiety comprises an amine reactive group selected from the group consisting of N-hydroxysuccinimide esters (NHS esters), sulfo-NHS esters, imidoesters, pentafluorophenyl esters, and hydroxymethylphosphines.

[0153] In some embodiments, the polypeptide reactive moiety comprises a carboxyl to amine reactive group that comprises a carbodiimide. In some embodiments, the carbodiimide is EDC. In other embodiments, the carbodiimide is DCC.

[0154] In some embodiments, the polypeptide reactive moiety comprises a sulfhydryl reactive group. Non-limiting examples of sulfhydryl reactive groups include maleimide, haloacetyl (bromo- or iodo-), pyridyl disulfide, thiosulfonate, and vinyl sulfone. In some embodiments, the polypeptide reactive moiety comprises a sulfhydryl reactive group that includes maleimide.

[0155] In some embodiments, the polypeptide reactive moiety comprises an aldehyde or carbonyl reactive group. Examples of aldehyde or carbonyl reactive groups include, but are not limited to, hydrazides and alkoxyamines.

[0156] In some embodiments, the polypeptide reactive moiety comprises a hydroxyl reactive group. A non-limiting example of a hydroxyl reactive group is an isocyanate.

[0157] In some embodiments, the polypeptide reactive moiety comprises an azide reactive group. A non-limiting example of an azide reactive group is a phosphine.

[0158] In some embodiments, the polypeptide reactive moiety comprises a photoreactive group. Examples of functional groups include, but are not limited to, phenyl azide, orthohydroxyphenyl azide, metahydroxyphenyl azide, tetrafluorophenyl azide, orthonitrophenyl azide, metanitrophenyl azide, diazirine, azidomethylcoumarin, and psoralen.

[0159] In some embodiments, the polypeptide reactive moiety targets and reacts with reactive or functional groups selected from the group consisting of primary amine groups (-NH2), carboxyl groups (-COOH), sulfhydryl groups (-SH), carbonyl groups (-CHO), and azide groups (-N3).

[0160] In some embodiments provided by the present disclosure, the polypeptide reactive moiety can react with one or more reactive or functional groups comprising a polypeptide of interest under conditions that maintain the polypeptide in a folded state (e.g., physiological conditions). In some embodiments, the polypeptide reactive moiety reacts with one or more reactive or functional groups of an antigen, such as the side chain groups of Lys, Cys, Ser, Thr, Tyr, His or Arg amino acid residues of the antigen. The polypeptide reactive moiety can be amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive, or guanidinyl-reactive. Further exemplary reactive or functional groups suitable for polypeptide reactive moieties and methods of using them are described in Hermanson "Bioconjugate Techniques" 3rd Edition, Academic Press, 2013 (incorporated herein by reference in its entirety).

[0161] In some embodiments, the polypeptide reactive moiety comprises a sortase recognition motif, which reacts with terminal amino acid residues (e.g., glycine and / or alanine residues) of an immunomodulatory fusion protein through the catalytic action of a sortase. Methods for using sortase to mediate cross-linking between N- or C-terminal amino acid residues (e.g., glycine and / or alanine residues) of a protein and a sortase recognition motif are known in the art and further described by Theile, et al (2013) Nat Protoc 8:1800-1807 and Guimaraes, et al (2013) Nat.Protoc.8:1787-1799, and references cited therein. Briefly, a peptide (e.g., a metal hydroxide-binding peptide) containing a sortase recognition motif, such as an LPXTG or LPXTA amino acid sequence (where X is any amino acid), is added to a polypeptide of interest (e.g., an immunomodulatory fusion protein comprising an immunomodulatory domain, and optionally a stabilizing domain, further comprising a terminal stretch of glycine and / or alanine residues) modified with a terminal amino acid sequence comprising glycine and / or alanine residues along with a sortase, such as sortase A from Staphylococcus aureas. The sortase cleaves between the threonine and the glycine or alanine residue of the sortase recognition motif, forming a thioester intermediate comprising the peptide (e.g., a metal hydroxide-binding peptide). Nucleophilic attachment by the terminally modified polypeptide of interest (e.g., an immunomodulatory fusion protein comprising an immunomodulatory domain, and optionally a stabilizing domain) results in the formation of a covalent bond between the peptide (e.g., a metal hydroxide-binding peptide) and the terminus of the polypeptide of interest.

[0162] Additionally, in some embodiments, a polypeptide of interest (e.g., an immunomodulatory fusion protein comprising an immunomodulatory domain and, optionally, a stabilizing domain) comprises a sortase recognition motif that reacts via a sortase-mediated reaction to an amino acid linker comprising glycine and / or alanine residues attached to a metal hydroxide-binding peptide. In some embodiments, a polypeptide of interest comprises a terminal sortase recognition motif (e.g., at the N-terminus or C-terminus) that reacts via a sortase-mediated reaction to an amino acid linker comprising glycine and / or alanine residues attached to a metal hydroxide-binding peptide. In an embodiment, the polypeptide of interest comprises an internal loop containing a sortase recognition motif that reacts via a sortase-mediated reaction to an amino acid linker comprising glycine and / or alanine residues attached to a metal hydroxide-binding peptide.

[0163] Linker In some embodiments, a polypeptide comprising at least one immunomodulatory domain and optionally a stabilizing domain is modified with a polypeptide reactive moiety linked to a metal hydroxide binding peptide, thereby forming an immunomodulatory fusion protein. In some embodiments, the metal hydroxide binding peptide is coupled to the polypeptide reactive moiety using a linker.

[0164] In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain and a metal hydroxide-binding peptide, where the metal hydroxide-binding peptide is operably linked to a terminus (e.g., the N-terminus or C-terminus) of the immunomodulatory domain by a polypeptide reactive moiety, optionally via a linker, thereby forming the immunomodulatory fusion protein.

[0165] In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain, a stabilization domain, and a metal hydroxide-binding peptide, where the stabilization domain is operably linked to either the N-terminus or C-terminus of the immunomodulatory domain, optionally via a linker, and the metal hydroxide-binding peptide is operably linked to either end (N-terminus or C-terminus) of the immunomodulatory domain or the stabilization domain by a peptide reactive moiety, optionally via a linker.

[0166] In some embodiments, metal hydroxide-binding peptides, optionally including a polypeptide reactive moiety and / or a linker, are effective to replace a hydroxyl group of a metal hydroxide (e.g., alum), thereby providing at least one hydroxyl substituent (e.g., a phosphate group) that facilitates, increases, or enhances adsorption to the metal hydroxide via ligand exchange.

[0167] For example, in some embodiments, a metal hydroxide-binding peptide comprising 1-15 consecutive phosphoserine residues is attached to a short poly(ethylene glycol) linker and an N-terminal maleimide functional group. In some embodiments, the maleimide functional group at the N-terminus of the metal hydroxide-binding peptide is covalently linked via a thioether linkage to a thiol group on a polypeptide comprising at least one immunomodulatory domain, and optionally a stabilizing domain, thereby forming an immunomodulatory fusion protein. In some embodiments, the immunomodulatory fusion protein is adsorbed to the metal hydroxide via the 1-15 consecutive phosphoserine residues of the metal hydroxide-binding peptide.

[0168] As a further example, in some embodiments, a metal hydroxide-binding peptide comprising 1-15 consecutive phosphoserine residues is attached via a short amino acid linker to an N-terminal sortase recognition tag. In some embodiments, the sortase recognition tag at the N-terminus of the metal hydroxide-binding peptide is cleaved by a sortase-mediated reaction to form an amide bond with a terminal glycine on a polypeptide comprising at least one immunomodulatory domain, and optionally a stabilizing domain, thereby forming an immunomodulatory fusion protein.

[0169] In some embodiments, the linker is a polypeptide linker, an ethylene glycol linker, or an oligonucleotide linker.

[0170] In yet another embodiment, the linker containing the metal hydroxide-binding peptide is The linker is conjugated to the immunomodulatory fusion protein via a functional group and coupled to the DBCO-modified immunomodulatory fusion protein. Preferably, a linker compatible with the present invention is relatively non-immunogenic and will not interfere with the non-covalent binding between the monomeric subunits of the binding protein (e.g., antibody). Exemplary linker domains are disclosed in U.S. Patent No. 6,660,843 (herein incorporated by reference).

[0171] In some embodiments, the linker may be a non-cleavable linker or a cleavable linker. A non-cleavable linker may comprise an amide bond or a phosphate bond, and a cleavable linker may comprise a disulfide bond, an acid-cleavable linkage, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-cleavable linkage.

[0172] Polypeptide Linkers In some embodiments, a polypeptide linker is used to covalently link a polypeptide reactive moiety that includes a sulfhydryl reactive moiety to a metal hydroxide-binding peptide that includes one or more hydroxyl substituents, where the hydroxyl substituents include a phosphate group.

[0173] In some embodiments, the polypeptide linker is synthetic. As used herein, the term "synthetic" with respect to a polypeptide linker includes a peptide (or polypeptide) that includes an amino acid sequence (which may or may not occur naturally) linked to a reactive substance in a linear sequence of amino acids. For example, the polypeptide linker may be a modified form of a naturally occurring polypeptide (e.g., includes mutations such as additions, substitutions, or deletions), or may include a non-naturally occurring polypeptide that includes a first amino acid sequence (which may or may not occur naturally).

[0174] In some embodiments, the polypeptide linker comprises or consists of a Gly-Ser linker. As used herein, the term "Gly-Ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary Gly-Ser linker has the formula (Gly 4 Ser) n wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). In certain embodiments, the Gly-Ser linker comprises an amino acid sequence of 4 Ser) 1 In certain embodiments, the Gly-Ser linker is 4 Ser) 2 In certain embodiments, the Gly-Ser linker is 4 Ser) 3 In certain embodiments, the Gly-Ser linker is 4 Ser) 4 In certain embodiments, the Gly-Ser linker is 4 Ser) 5In certain embodiments, the gly-ser linker can be inserted between two other sequences of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In other embodiments, the Gly-Ser linker is attached to one or both ends of another sequence of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In yet other embodiments, two or more Gly-Ser linkers are incorporated in tandem into the polypeptide linker.

[0175] Other linkers suitable for use in preparing metal hydroxide-binding peptides linked to polypeptide reactive moieties described herein are known in the art, such as the serine-rich linkers disclosed in US 5,525,491, the helix-forming peptide linkers (e.g., A(EAAAK)nA (n=2-5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, and the stable linkers disclosed in Chen et al., Mol Pharm 2011;8:457-65, i.e., the dipeptide linker LE, the thrombin-sensitive disulfide cyclopeptide linker, and the alpha-helix-forming linker LEA(EAAAK). 4 ALEA(EAAAK) 4 It is ALE.

[0176] Other exemplary linkers include the GS linker (i.e., (GS)n), the GGSG linker (i.e., (GGSG)n), the GSAT linker, the SEG linker, and the GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for use in preparing metal hydroxide binding peptides linked to polypeptide reactive moieties can be found using publicly available databases such as the Linker Database (ibi.vu.nl / programs / linkerdbwww). The Linker Database is a database of interdomain linkers of multifunctional enzymes, which serve as potential linkers for novel fusion proteins (e.g., George et al., Protein Synthesis, 1999). (See Engineering 2002;15:871-9).

[0177] It will be understood that variant forms of these exemplary polypeptide linkers can be made by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the polypeptide linker, such that one or more amino acid substitutions, additions or deletions are introduced into the polypeptide linker. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0178] The polypeptide linkers of the present invention are at least one amino acid in length and may be of various lengths. In one embodiment, the polypeptide linkers of the present invention are about 1 to about 50 amino acids in length. When used in this context, the term "about" refers to + / - 2 amino acid residues. Since the length of the linker must be a positive integer, a length of about 1 to about 50 amino acids in length means a length of 1 to 48 to 52 amino acids in length. In another embodiment, the polypeptide linkers of the present invention are about 1 to 5 amino acids in length. In another embodiment, the polypeptide linkers of the present invention are about 5 to 10 amino acids in length. In another embodiment, the polypeptide linkers of the present invention are about 10 to 20 amino acids in length. In another embodiment, the polypeptide linkers of the present invention are about 15 to about 50 amino acids in length.

[0179] In another embodiment, the polypeptide linker of the invention is about 20 to about 45 amino acids in length. In another embodiment, the polypeptide linker of the invention is about 15 to about 25 amino acids in length. In another embodiment, the polypeptide linker of the invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 or more amino acids in length.

[0180] Polypeptide linkers can be introduced into the polypeptide sequence using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transfect host cells to stably produce the produced polypeptide.

[0181] Ethylene Glycol Linker In some embodiments, the linker is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker. Polyethylene glycol or PEG refers to a compound made up of repeating ethylene glycol units. Exemplary "PEG Linkers" include: " includes compounds of the formula: H-(O-CH2-CH2)n-OH, where n is a positive integer (e.g., 1, 10, 20, 50, 100, 200, 300, 400, 500, 600). In some embodiments, the PEG linker is PEG1000. In some embodiments, the PEG linker is PEG2000. In some embodiments, the PEG linker is PEG3000.

[0182] In some embodiments, the metal hydroxide binding peptides linked to polypeptide reactive moieties provided by the present disclosure may include any polyethylene glycol (PEG) linker that joins any protein reactive moiety to any metal hydroxide binding peptide that includes one or more hydroxyl substituents described herein. For example, in some embodiments, a polyethylene glycol (PEG) linker can be used to covalently link a protein reactive moiety that includes a sulfhydryl reactive moiety to a metal hydroxide binding peptide that includes one or more hydroxyl substituents, where the hydroxyl substituents include a phosphate group.

[0183] In some embodiments, the exact number of ethylene glycol (EG) units comprising the metal hydroxide binding peptide linked to the polypeptide reactive moiety can range from about 1 to about 100, about 20 to about 80, about 30 to about 70, or about 40 to about 60 EG units. In some embodiments, the ethylene glycol linker has about 45 to 55 EG units. For example, in one embodiment, the ethylene glycol linker has 45 EG units. For example, in one embodiment, the ethylene glycol linker has 48 EG units.

[0184] Oligonucleotide Linkers In some embodiments, the linker is an oligonucleotide. The linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence or a sequence specifically selected for its molecular or biochemical properties. In some embodiments, the linker comprises a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or one or more analogs thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof.

[0185] In one embodiment, the linker is one or more guanines, for example, 1 to 10 guanines. In some embodiments, the linker in the ABP conjugate may contain 0, 1, or 2 guanines. In some embodiments, the oligonucleotide comprises phosphorothioate intersubunit linkages.

[0186] Immunomodulatory Domain The immunomodulatory fusion protein disclosed herein comprises at least one immunomodulatory domain. In some embodiments, the immunomodulatory fusion protein comprises one, two, three, four, or five immunomodulatory domains. In some embodiments, when there are multiple immunomodulatory domains in the fusion protein, the immunomodulatory domains are the same. In some embodiments, when there are multiple immunomodulatory domains in the fusion protein, the immunomodulatory domains are different.

[0187] In some embodiments, an immunomodulatory fusion protein comprises at least one immunomodulatory domain and a metal hydroxide-binding peptide comprising one or more phosphorylated amino acids, wherein the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either the N-terminus or the C-terminus of the immunomodulatory domain, thereby forming an immunomodulatory fusion protein. Form.

[0188] In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain, a stabilization domain, and a metal hydroxide-binding peptide, where the stabilization domain is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either end of the immunomodulatory domain or the stabilization domain, thereby forming the immunomodulatory fusion protein.

[0189] In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain, a stabilization domain, and a metal hydroxide-binding peptide, where the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the stabilization domain is operably linked, optionally via an amino acid linker, to either end of the metal hydroxide-binding peptide or the immunomodulatory domain, thereby forming the immunomodulatory fusion protein.

[0190] In some embodiments, the immunomodulatory fusion protein comprises at least one immunomodulatory domain and a metal hydroxide-binding peptide, where the metal hydroxide-binding peptide is operably linked to a terminus (e.g., the N-terminus or C-terminus) of the at least one immunomodulatory domain by a polypeptide reactive moiety, optionally via a linker, thereby forming the immunomodulatory fusion protein.

[0191] In some embodiments, the immunomodulatory fusion protein comprises at least one immunomodulatory domain, a stabilization domain, and a metal hydroxide-binding peptide, where the stabilization domain is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the at least one immunomodulatory domain, and the metal hydroxide-binding peptide is operably linked, optionally via a linker, by a peptide-reactive moiety to the remaining terminus (e.g., the N-terminus or C-terminus) of the at least one immunomodulatory domain or to an end of the stabilization domain, thereby forming the immunomodulatory fusion protein.

[0192] In some embodiments, the immunomodulatory domain activates the activity of cells of the immune system. For example, in some embodiments, the immunomodulatory domain is an immune response stimulator, such as, but not limited to, a cytokine, such as an interleukin, a chemokine, a member of the TNF family, an agonist antibody, an immune checkpoint blocker, or a combination thereof. In some embodiments, the immunomodulatory domain enhances the immune response. In some embodiments, the enhanced immune response includes stimulating T cells, stimulating B cells, stimulating dendritic cell responses, or a combination thereof. In some embodiments, the enhanced immune response results in cytokine production, antibody production, antigen-specific immune cell (e.g., CD8+ T cells or CD4+ T cells) production, stimulating type I interferon responses, or a combination thereof.

[0193] In some embodiments, the immunomodulatory domain comprises a polypeptide that activates, enhances or promotes a response by an immune cell. In some embodiments, the immunomodulatory domain comprises a polypeptide that inhibits, reduces or suppresses a response by an immune cell. In some embodiments, the immune cell is a lymphoid cell, including but not limited to T cells, B cells, NK cells and innate lymphocytes. In some embodiments, the immune cell is a myeloid cell, including but not limited to monocytes, neutrophils, macrophages, dendritic cells, mast cells and granulocytes.

[0194] In some embodiments, the immune cell response is determined by cytokine production, antibody production, antigen production, or the like. the production of specific immune cells, or a combination thereof.

[0195] Interleukins In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an interleukin (IL). Interleukins are secreted proteins that bind to their specific receptors and play a role in communication between white blood cells. Interleukins suitable for use as the immunomodulatory domain of the immunomodulatory fusion proteins include, but are not limited to, IL-2, IL-12, IL-15, IL-15 superagonist (IL-15SA), IL-21, IL-6, IL-5, IL-8, IL-7, IL-17, IL-23, IL-18, IL-1, IL-4, IL-3, IL-10, IL-13, and IL-9. In some embodiments, the immunomodulatory domain suitable for use as the immunomodulatory domain comprises an amino acid sequence selected from SEQ ID NOs: 1-5 and 9-24. In some embodiments, the immunomodulatory domain is an IL-2 polypeptide. In some embodiments, the immunomodulatory domain is an IL-12 polypeptide. In some embodiments, the immunomodulatory domain is an IL-15 polypeptide. In some embodiments, the immunomodulatory domain is an IL-15SA polypeptide.

[0196] In some embodiments, the immunomodulatory domain is an interleukin polypeptide that binds to the common gamma chain receptor. Interleukins that bind to the common gamma chain receptor include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-15 / IL-15Rα, and IL-21.

[0197] In some embodiments, the immunomodulatory domain is a polypeptide belonging to the IL-12 family. The IL-12 family includes heterodimeric ligands composed of an α subunit (e.g., IL-12p35, IL-23p19, IL-27p28) and a β subunit (e.g., IL-12p40, IL-23p40 (which is identical to IL-12p40), EBI3) with a helical structure. Exemplary members include IL-12, IL-23, IL-27, and IL-35.

[0198] In some embodiments, the immunomodulatory domain is a polypeptide belonging to the IL-1 superfamily. The interleukin-1 (IL-1) family consists of eleven structurally related family members (IL-1α, IL-1-β, IL-1Ra, IL-18, IL-33, and IL-1F5-IL-1F10), which are the most potent immune system signaling molecules acting through a group of closely related receptors. All IL-1 receptors have a similar mode of activation. Upon binding of a ligand to the primary receptor subunit (i.e., IL-1R1 for IL-1α and β, IL-18R for IL-18, and ST2 for IL-33), a second receptor subunit is recruited (i.e., IL-1RAP for IL-1α and β, IL-18RAP for IL-18, and IL-1RAP for IL-33), which initiates signal transduction through the cytoplasmic Toll / IL-1 receptor (TIR) ​​domain of the juxtaposed receptor subunit. The dimerized TIR domain provides a docking platform for the MYD88 adaptor protein. This leads to the activation of the proinflammatory nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways through the recruitment of other intermediates. Members of the IL-1 family are mainly produced by innate immune cells and act on various cell types during immune responses. Thus, in some embodiments, the immunomodulatory domain is an IL-18 polypeptide.

[0199] Interleukin-2 (IL-2) In some embodiments, the immunomodulatory fusion protein is a member of the IL-2 family. In some embodiments, the member of the IL-2 family is IL-2. Interleukin-2 (IL-2) is a cytokine that induces proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. The biological activity of IL-2 is mediated through a multisubunit IL-2 receptor complex (IL-2R) of three polypeptide subunits that span the cell membrane: p55 (IL-2Rα, alpha subunit, also known as CD25 in humans), p75 (IL-2Rβ, beta subunit, also known as CD122 in humans), and p64 ​​(IL-2Rγ, gamma subunit, also known as CD132 in humans). The response of T cells to IL-2 depends on a variety of factors, including: (1) the concentration of IL-2, (2) the number of IL-2R molecules on the cell surface, and (3) the number of IL-2Rs occupied by IL-2 (i.e., the affinity of the binding interaction between IL-2 and IL-2R (Smith, “Cell Growth Signal Transduction is Quantal” In Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)). The IL-2:IL-2R complex is internalized upon ligand binding, and the various components undergo different sorting. IL-2Rα is recycled to the cell surface, whereas IL-2 associated with the IL-2:IL-2RPγ complex is delivered to lysosomes for degradation. When administered as an intravenous (iv) bolus, IL-2 has rapid systemic clearance (an early clearance phase with a half-life of 12.9 minutes, followed by a slower clearance phase with a half-life of 85 minutes) (Konrad et al., Cancer Res. 50:2009-2017, 1990).

[0200] The outcome of systemic IL-2 administration in cancer patients is far from ideal. Although 15-20% of patients respond objectively to high doses of IL-2, the majority do not, and many suffer from severe and life-threatening side effects, including nausea, confusion, hypotension, and septic shock. The severe toxicity associated with high-dose IL-2 treatment is primarily due to the activity of natural killer (NK) cells. NK cells bind to the intermediate affinity receptor IL-2RPγ c and thus are stimulated by nanomolar concentrations of IL-2, and indeed, during high-dose IL-2 treatment, the serum of patients is produced. By reducing the dose and adjusting the dosing regimen, the serum concentration is reduced, and thus the IL-2RaPγ c Attempts have been made to selectively stimulate cells with IL-2, and such treatments have also been less effective, although with lower toxicity. Given the toxicity issues associated with high-dose IL-2 cancer treatments, many groups have attempted to improve the anticancer effects of IL-2 by simultaneously administering therapeutic antibodies. However, such efforts have been largely unsuccessful and provide no additional or limited clinical benefit compared to IL-2 therapy alone. Thus, novel IL-2 therapies are needed to combat various cancers more effectively.

[0201] In some embodiments, the IL-2 is human recombinant IL-2, such as Proleukin® (aldesleukin). Proleukin® is a human recombinant interleukin-2 product produced in E. coli. Proleukin® differs from native interleukin-2 in that it is a) not glycosylated, b) does not have an N-terminal alanine, and c) has a serine substituting a cysteine ​​at amino acid position 125. Proleukin® exists as biologically active non-covalently bound microaggregates with an average size of 27 recombinant interleukin-2 molecules. Proleukin® (aldesleukin) is administered by intravenous infusion. In some embodiments, the IL-2 is wild-type IL-2 (e.g., human IL-2 in its precursor form, or mature IL-2). In some embodiments, the IL-2 comprises the amino acid sequence set forth in SEQ ID NO:1.

[0202] In certain embodiments, the IL-2 has a reduced IL-2 activity compared to unmodified IL-2. The IL-2Rα variants have been mutated to have altered affinity (e.g., higher affinity) for the IL-2R alpha receptor. Site-directed mutagenesis can be used to isolate IL-2 variants, i.e., IL-2Rα, that exhibit high affinity binding to CD25 compared to wild-type IL-2. Increasing the affinity of IL-2 for IL-2Rα at the cell surface increases receptor occupancy within a limited IL-2 concentration range, increasing the local concentration of IL-2 at the cell surface.

[0203] In some embodiments, the present disclosure features IL-2 variants, which may, but are not necessarily, substantially purified and can function as high affinity CD25 binders. IL-2 is a T cell growth factor that induces proliferation of antigen-activated T cells and stimulation of NK cells. Exemplary IL-2 variants that are high affinity binders include those described in WO2013 / 177187A2, which is incorporated herein by reference in its entirety. Further exemplary IL-2 variants with increased affinity for CD25 are disclosed in US 7,569,215, the contents of which are incorporated herein by reference.

[0204] In some embodiments, the disclosure features IL-2 variants that have reduced binding affinity to CD25 compared to wild-type IL-2. In some embodiments, the IL-2 variants do not bind to CD25.

[0205] In some embodiments, the IL-2 variant comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1 that binds CD25. For example, in some embodiments, the IL-2 variant has at least one mutation (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) that increases affinity for the alpha subunit of the IL-2 receptor compared to wild-type IL-2. It should be understood that the mutations identified in mouse IL-2 may be made in the corresponding residues of full-length human IL-2 (nucleic acid sequence (Accession: NM000586), amino acid sequence (Accession: P60568)), or human IL-2 without the signal peptide. Thus, in some embodiments, the IL-2 is human IL-2. In other embodiments, the IL-2 is a mutant human IL-2.

[0206] In some embodiments, the IL-2 variant is at least or about 50%, at least or about 65%, at least or about 70%, at least or about 80%, at least or about 85%, at least or about 87%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 98%, or at least or about 99% identical in amino acid sequence to wild-type IL-2 (its precursor form or preferably its mature form). Mutations may consist of changes in the number or content of amino acid residues. For example, the IL-2 variant can have a greater or lesser number of amino acid residues than the wild-type IL-2. Alternatively or additionally, the IL-2 variant can contain substitutions of one or more amino acid residues present in wild-type IL-2.

[0207] By way of illustration, a polypeptide comprising an amino acid sequence that is at least 95% identical to a reference amino acid sequence of SEQ ID NO: 1 is a polypeptide comprising a sequence that is identical to the reference sequence, except that it comprises up to five modifications of the reference amino acids of SEQ ID NO: 1. For example, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or several amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the amino (N-) or carboxy (C-) terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually between residues in the reference sequence, or interspersed in one or more contiguous groups in the reference sequence.

[0208] The substituted amino acid residues may be, but are not necessarily, conservative substitutions. This typically includes substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. These mutations may be in amino acid residues that contact IL-2Rα.

[0209] Interleukin-12 (IL-12) In some embodiments, the immunomodulatory fusion protein comprises an IL-12 polypeptide. Interleukin-12 (IL-12) is a proinflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16:495-521 (1998). IL-12 functions primarily as a 70 kDa heterodimeric protein consisting of two disulfide-linked p35 and p40 subunits. The precursor form of the IL-12 p40 subunit (NM_002187; P29460; also called IL-12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2) is 328 amino acids in length, while its mature form is 306 amino acids in length. The precursor form of the IL-12 p35 subunit (NM_000882; P29459; also called IL-12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids long, and its mature form is 197 amino acids long. Id. The genes for the IL-12 p35 and p40 subunits are located on different chromosomes and are regulated independently of each other. Gately, MK et al., Annu Rev Immunol. 16:495-521 (1998). Many different immune cells (e.g., dendritic cells, macrophages, monocytes, neutrophils, and B cells) produce IL-12 upon antigen stimulation. The active IL-12 heterodimer is formed after protein synthesis. Id.

[0210] Due to its ability to activate both NK cells and cytotoxic T cells, IL-12 protein has been investigated as a promising anti-cancer therapeutic since 1994. See Nastala, CL et al., J Immunol 153:1697-1706 (1994). However, despite high expectations, early clinical trials did not produce satisfactory results. Lasek W. et al., Cancer Immunol Immunother 63:419-435, 424 (2014). Repeated administration of IL-12 in most patients led to an adaptive response and a gradual decline in IL-12-induced interferon gamma (IFNγ) levels in the blood. Id. Furthermore, although it was recognized that IL-12-induced anti-cancer activity was primarily mediated by secondary secretion of IFNγ, severe toxicity was caused by IL-12 due to the simultaneous induction of IFNγ together with other cytokines (e.g., TNF-α) or chemokines (IP-10 or MIG). Id.

[0211] In addition to negative feedback and toxicity, the modest efficacy of IL-12 therapy in clinical situations may be caused by the strong immunosuppressive environment in humans. Id. To minimize the toxicity of IFNγ and improve the efficacy of IL-12, scientists have tried various approaches, such as different doses and time protocols of IL-12 therapy. See Sacco, S. et al., Blood 90:4473-4479 (1997); Leonard, JP et al., Blood 90:2541-2548 (1997); Coughlin, CM et al., Cancer Res. 57:2460-2467 (1997); Asselin-Paturel, C. et al., Cancer 91:113-122 (2001); and Saudemont, A. et al., Leukemia 16:1637-1644 (2002). Nevertheless, these approaches have not had a significant impact on patient survival. Kang, WK, et al., Human Gene Therapy 12:671-6 84(2001).

[0212] Membrane-anchored versions of IL-12 have been investigated as a means of reducing the toxicity associated with systemic administration, using retroviral and adenoviral vectors for expression in tumor cells. See Pan, WY. et al., Mol. Ther. 20(5):927-937 (2012). However, the use of viral vectors presents potential health risks, as the underlying virus may act as an oncogene and viral vectors may be immunogenic.

[0213] Thus, in some embodiments, the immune modulating fusion proteins disclosed herein comprise an IL-12 polypeptide. In some embodiments, the IL-12 polypeptide comprises IL-12A (e.g., SEQ ID NO: 3). In some embodiments, the IL-12 polypeptide comprises IL-12B (e.g., SEQ ID NO: 2). In some embodiments, the IL-12 polypeptide comprises both IL-12A and IL-12B.

[0214] In some embodiments, IL-12B is located N-terminal to IL-12A in an IL-12 polypeptide. In some embodiments, IL-12A is located N-terminal to IL-12B in an IL-12 polypeptide. The phrase "located N-terminal to" indicates a location within a polypeptide relative to other sequences of the polypeptide that are related to the N-terminus of the polypeptide. For example, IL-12B being "N-terminal" to IL-12A means that IL-12B is located closer to the N-terminus of the IL-12 polypeptide than IL-12A.

[0215] In some embodiments, the IL-12 polypeptide comprises a single polypeptide chain comprising IL-12B and IL-12A, which are either directly fused to each other or linked to each other by a linker (referred to herein as a "subunit linker"), non-limiting examples of which are disclosed elsewhere herein.

[0216] In some embodiments, the IL-12 polypeptides of the disclosure comprise IL-12A and / or IL-12B, which are variants, functional fragments, or contain substitutions, insertions and / or additions, deletions, and / or covalent modifications relative to the wild-type IL-12A or IL-12B sequence. In some embodiments, amino acid residues located at the carboxy, amino terminal, or internal regions of the IL-12 polypeptide are deleted, thereby providing fragments.

[0217] In some embodiments, the IL-12 polypeptide comprises a substitution variant of the IL-12A and / or IL-12B amino acid sequence, which may include one, two, three, or more than three substitutions. In some embodiments, the substitution variant may include one or more conservative amino acid substitutions. In other embodiments, the variant is an insertion variant. In other embodiments, the variant is a deletion variant.

[0218] As will be appreciated by those of skill in the art, IL-12 protein fragments, functional protein domains, variants, and homologous proteins (orthologs) are also contemplated within the scope of the IL-12 polypeptides of the present disclosure. Non-limiting examples of IL-12 polypeptides suitable for use in the immunomodulatory fusion proteins disclosed herein are set forth in SEQ ID NOs: 2-3.

[0219] In some embodiments, the immunomodulatory fusion protein comprises an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. The fusion protein comprises an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the immune modulating fusion protein comprises an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 2 and 3.

[0220] Interleukin-15 (IL-15) In some embodiments, the immunomodulatory fusion protein comprises an IL-15 polypeptide. IL-15 is a member of the four alpha-helical bundle family of cytokines and plays an important role in the development of an effective immune response. Waldmann, TA, Cancer Immunol. Res. 3:219-227 (2015). IL-15 is essential for proper growth of NK cells and long-term maintenance of memory CD8+ T cells. The IL-15 gene encodes a 162 amino acid preprotein with a 48 amino acid signal peptide, and the mature protein is 114 amino acids in length. Bamford, RN, et al., Proc. Natl. Acad. Sci. USA 93:2897-2902 (1996). See also, for example, GenBank accession numbers NM_000585 for the Homo sapiens IL15 transcript variant 3 mRNA sequence and NP_000576 for the corresponding IL15 isoform 1 preproprotein.

[0221] IL-15 shares certain structural similarities with interleukin-2 (IL-2). Like IL-2, IL-15 signals through the IL-2 receptor beta chain (CD122) and common gamma chain (CD132). However, unlike IL-2, IL-15 cannot effectively bind to CD122 and CD132 by itself. IL-15 must first bind to the IL-15 alpha receptor subunit (IL-15Rα). The IL-15Rα gene encodes a 267 amino acid preprotein with a 30 amino acid signal peptide, and the mature protein is 237 amino acids in length. See, for example, GenBank accession numbers NM_002189 for Homo sapiens IL-15Rα transcript variant 1 mRNA and NP_002180 for Homo sapiens IL-15Rα isoform 1 precursor amino acid sequence.

[0222] Human IL-15Rα is a transmembrane protein that primarily binds IL-15 on the surface of cells such as activated dendritic cells and monocytes. Waldmann, TA, Cancer Immunol. Res. 3:219-227 (2015). The membrane-bound complex of IL-15 / IL-15Rα then presents IL-15 in trans to the CD122 and CD132 subunits. Thus, IL-15Rα is an essential component of IL-15 activity.

[0223] To overcome the short half-life of systemically injected IL-15, precomplexing IL-15 with soluble recombinant IL-15Ra to generate an IL-15 superagonist (IL-15SA) has been shown to enhance the systemic potency of IL-15 by approximately 50-fold, extending the half-life of the cytokine in serum after systemic injection to approximately 20 hours. (Stoklasek et al., J Immunol 177(9):6072, 2006; Dubois et al., J Immunol 180(4):2099, 2008; Rubinstein et.al. Proc Natl Acad Sci USA 103(24):9166, 2006.)

[0224] Thus, in some embodiments, the immunomodulatory domain of the immunomodulatory fusion protein is an IL-15 polypeptide. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the IL-15 polypeptide comprises an IL-15Rα fusion protein comprising IL-15 and IL-15Rα. In some embodiments, the IL-15 superagonist comprises the amino acid sequence set forth in SEQ ID NOs: 4 and 5.

[0225] interferon In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an interferon (IFN). Interferons comprise a family of secreted proteins that are induced in response to certain extracellular stimuli by stimulation of Toll-like receptors (TLRs). In some embodiments, interferons enhance the immune system's antiviral defenses (e.g., antigen presentation). IFNs stimulate genes using signaling molecules via high affinity cell surface receptors. Interferons suitable for use as an immunomodulatory domain in an immunomodulatory fusion protein include, but are not limited to, IFN-gamma and IFN-alpha.

[0226] In some embodiments, the immunomodulatory fusion protein comprises an IFN-gamma polypeptide. IFN-gamma is produced by various immune cells, such as activated T cells and NK cells. IFN-gamma interacts with specific receptors on the cell surface and activates signal transduction pathways that produce immunomodulatory effects. Thus, in some embodiments, the immunomodulatory domain is an IFN-gamma polypeptide. In some embodiments, the IFN-gamma polypeptide comprises the amino acid sequence shown in SEQ ID NO:7.

[0227] In some embodiments, the immunomodulatory fusion protein comprises an IFN-alpha polypeptide. IFN-alpha is produced by B lymphocytes, null lymphocytes, and macrophages, and activates NK cells with antiviral and antitumor activity. Thus, in some embodiments, the immunomodulatory domain is an IFN-alpha polypeptide. In some embodiments, the IFN-alpha polypeptide comprises the amino acid sequence shown in SEQ ID NO:8.

[0228] Immune cell differentiation stimulating factor In some embodiments, the immune-modulating domain suitable for use in the immune-modulating fusion protein of the present disclosure is an immune cell differentiation stimulating factor. In some embodiments, the immune cell differentiation stimulating factor activates intracellular signaling pathways that cause the differentiation, development, and proliferation of hematopoietic progenitor cells into specific subtypes of immune cells. Immune cell differentiation stimulating factors suitable for use in the immune-modulating fusion protein disclosed herein include, but are not limited to, GM-CSF (granulocyte macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor) and FLT3L (FMS-like tyrosine kinase 3 ligand).

[0229] In some embodiments, the immunomodulatory domain is a GM-CSF polypeptide. GM-CSF is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts. In addition to its function of stimulating the growth and differentiation of hematopoietic progenitor cells, GM-CSF exerts a variety of effects on immune cells that express the GM-CSF receptor. In some embodiments, the GM-CSF polypeptide comprises the amino acid sequence shown in SEQ ID NO:27.

[0230] In some embodiments, the immunomodulatory domain is a FLT3L polypeptide. FLT3 is a receptor tyrosine kinase (RTK) expressed by immature hematopoietic progenitor cells. FLT3L is a transmembrane or soluble protein, and is expressed by many cells, including hematopoietic and stromal cells of bone marrow. FLT3L, in combination with other growth factors, stimulates the proliferation and development of various cell types, including myeloid and lymphoid progenitor cells, dendritic cells, and NK cells. In some embodiments, the FLT3L polypeptide comprises the amino acid sequence shown in SEQ ID NO:28.

[0231] In some embodiments, the immunomodulatory domain is a G-CSF polypeptide. In some embodiments, G-CSF regulates the proliferation, differentiation and functional activation of neutrophil granulocytes. In some embodiments, the G-CSF polypeptide comprises the amino acid sequence shown in SEQ ID NO:29.

[0232] Chemokines In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is a chemokine. In some embodiments, a chemokine is a protein that induces directional chemotaxis of responsive cells (e.g., leukocytes). Generally, chemokines are classified into four subfamilies: CXC, CC, (X)C, and CX3C. In CXC chemokines, one amino acid separates the first two cysteines (the "CXC motif"). ELR+CXC chemokines are G protein-coupled seven-transmembrane domain receptors that are ligands for the CXCR1 and / or CXCR2 chemokine receptors and specifically bind to ELR+CXC chemokines. The seven human ELR+CXC chemokines are human Gro-alpha (also known as CXCL1), human Gro-beta (also known as CXCL2), human Gro-gamma (also known as CXCL3), human ENA-78 (also known as CXCL5), human GCP-2 (also known as CXCL6), human NAP-2 (also known as CXCL7), and human IL-8 (also known as CXCL8). All ELR+CXC chemokines bind to the CXCR2 receptor. In addition, some ELR+CXC chemokines bind to both the CXCR1 and CXCR2 receptors (i.e., CXCL6 and CXCL8), both of which contribute to the redundancy of activation pathways. The five murine ELR+CXC chemokines are keratinocyte chemoattractant (KC) (also known as CXCL1), macrophage inflammatory protein-2 (MIP-2) (also known as CXCL2), dendritic cell inflammatory protein-1 (DCIP-1) (also known as CXCL3), lipopolysaccharide-inducible CXC chemokine (LIX) (also known as CXCL5), and neutrophil-activating peptide-2 (NAP-2) (also known as CXCL7).

[0233] Chemokines suitable for use in the immune modulating fusion proteins disclosed herein include, but are not limited to, LIF, M-CSF, MIP-2, MIP-1 beta, KP (CXLC1), MIG (CXCL9), IP-10 (CXCL10), MCP-1, eotaxin, RANTES, LIX and MIP-1 alpha.

[0234] Listed below are amino acid sequences encoding exemplary chemokines suitable for use as immunomodulatory domains in the immunomodulatory fusion proteins disclosed herein. [Table 5]

[0235] Tumor necrosis factor (TNF) superfamily In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is the extracellular domain of a member of the tumor necrosis factor (TNF) superfamily. The ligands and receptors of the tumor necrosis factor superfamily are a series of structurally homologous cell surface proteins that transmit signals by forming trimeric clusters of ligand-receptor complexes. Ligation that activates TNF superfamily receptors can trigger a wide range of pro-immune responses, including proliferation, enhanced effector functions, and the production of chemokines and cytokines. Some ligands, such as Fas, can lead to the induction of apoptosis and are expressed on the surface of immune cells. Additionally, other ligands function as inhibitory receptors that dampen immune responses. In some embodiments, the extracellular domain is derived from TNF-alpha, LIGHT, LT-alpha, LT-beta, BTLA, CD160, CD40L, FasL, CD30L, 4-1BBL, CD27L, OX40L, TWEAK, APRIL, BAFF, RANKL, TRAIL, EDA1, EDA2, or GITRL. The extracellular domain can bind to a receptor for the selected TNF superfamily member, thereby inducing or stimulating an immune response.

[0236] The table below shows the receptors to which the derived extracellular domains correspond. [Table 6]

[0237] CD28 family In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is the extracellular domain of a member of the CD28 family. The CD28 family is a family of inhibitory (PD1, CTLA-4) and activating (CD28, ICOS) receptors that bind members of the B7 family of ligands. CD28 is a costimulatory receptor that provides the second signal required to activate naive T cells (along with ligation of the TCR) and has two natural ligands, CD80 and CD86. CD28 signaling can help increase proliferation, effector function, and anti-apoptotic signaling. CD28 signaling has recently been shown to be necessary for effective PD1 / PDL1 blockade. ICOS (inducible T cell costimulatory molecule) is a closely related surface receptor that is expressed on activated T cells and exhibits similar functions as CD28.

[0238] Thus, in some embodiments, the immunomodulatory domain is the extracellular domain of CD80 (B7-1). In some embodiments, the immunomodulatory domain comprises the amino acid sequence set forth in SEQ ID NO:69.

[0239] Thus, in some embodiments, the immunomodulatory domain is the extracellular domain of CD86 (B7-2), which is capable of binding to CD28. In some embodiments, the immunomodulatory domain comprises the amino acid sequence set forth in SEQ ID NO:70.

[0240] Thus, in some embodiments, the immunomodulatory domain is the extracellular domain of ICOSLG. In some embodiments, the immunomodulatory domain comprises the amino acid sequence set forth in SEQ ID NO:71.

[0241] Agonist antibodies In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an agonist antibody or an antigen-binding fragment thereof. Agonist antibodies activate targets of interest, in contrast to antagonist antibodies that block the function of those targets. In some embodiments, agonist antibodies or antigen-binding fragments thereof bind to immune activation receptors. In some embodiments, immune activation receptors include, but are not limited to, tumor necrosis factor (TNF) receptors, CD28 family members, T cell receptors (TCRs), killer cell Ig-like receptors (KIRs), leukocyte Ig-like receptors (LIRs), CD94 / NKG2 receptors, Fc receptors, signaling lymphocyte activation molecules (SLAMs), and activating Siglec receptors.

[0242] Tumor necrosis factor (TNF) superfamily In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a tumor necrosis factor (TNF) superfamily member receptor. The TNF superfamily is described above. For example, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment that binds to TNFR1, thereby activating the receptor.

[0243] The following table lists TNF superfamily member receptors to which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 7-1] [Table 7-2]

[0244] In some embodiments, the immunomodulatory domain is an anti-4-1BB agonist antibody. In some embodiments, the immunomodulatory domain is an anti-OX40 agonist antibody. In some embodiments, the immunomodulatory domain is a CD40 agonist antibody.

[0245] CD28 receptor superfamily In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a CD28 superfamily receptor. The CD28 superfamily is described above. For example, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment that binds to CD28 and thereby activates the receptor.

[0246] The following table lists CD28 superfamily member receptors to which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 8]

[0247] T cell receptor (TCR) complex In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to the T cell receptor (TCR) complex. The T cell receptor (TCR) is a cell surface receptor responsible for conferring antigen specificity to T cells. Each TCR is specific for a particular peptide presented by either MHC class I (for CD8+ T cells) or MHC class II (for CD4+ T cells). In the case of naive T cells, ligation of the TCR provides the first of two signals required to activate the T cell. TCR ligation in CD8+ T cells results in the death of cells displaying their cognate pMHC (and potentially bystander cells) through the release of soluble factors such as perforin and granzyme B, and the upregulation of apoptosis-inducing ligands such as Fas ligand. In the case of CD4+ helper T cells, ligation of the TCR with its cognate pMHC results in the release of cytokines.

[0248] Thus, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment thereof that binds to the TCR. For example, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment thereof that binds to CD3γ, thereby activating the receptor.

[0249] The following table lists members of the TCR complexes to which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 9]

[0250] Killer cell Ig-like receptors (KIR) In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to killer cell Ig-like receptors (KIRs). Killer cell immunoglobulin-like receptors (KIRs) are a family of receptors that are expressed primarily on NK cells and some subsets of T cells. These receptors are primarily involved in self-recognition (and thus inhibitory function) by binding to MHC class I (HLA-A, HLA-B, and HLA-C) molecules. These receptors are either activating or inhibitory depending on the length of their cytoplasmic tails. Inhibitory receptors have long tails and contain ITIM domains. Activating KIRs have shorter cytoplasmic domains and bind to DAP12 to mediate signal transduction.

[0251] Activating KIRs against which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated are shown in the table below. [Table 10]

[0252] Leukocyte Ig-like receptors (LIRs) In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to leukocyte Ig-like receptors (LIRs). LIR receptors are a type of immune receptor that are mainly expressed on innate immune cells. Their main ligands are MHC class I molecules, and although some have activating functions, they mainly exhibit inhibitory functions. For example, LIRA2 functions as an innate sensor of immunoglobulin fragments cleaved by microbial proteases.

[0253] In some embodiments, the immunomodulatory domain is an agonist antibody or antigen-binding fragment thereof that binds to LIRA2. An antibody capable of detecting the antibody can be generated based on Uniprot ID Q8N149.

[0254] CD94 / NKG2 receptor family In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to the CD94 / NKG2 receptor. CD94 / NKG2 is a heterodimeric C-type lectin receptor expressed on the surface of NK cells and some subsets of CD8 T cells. They bind to HLA-E molecules (non-classical MHC class I molecules) and can transmit both inhibitory and activating signals to NK cells. Inhibitory receptors contain ITIM domains in their cytoplasmic tails, and activating receptors bind DAP12 and DAP10, which contain ITAM domains.

[0255] Activating CD94 / NKG2 receptors to which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated are shown in the table below.

[0256] In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the present disclosure is the extracellular domain of a CD94 / NKG2 ligand. The following table shows the receptors to which the derived extracellular domains correspond: [Table 11]

[0257] Fc receptors In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to an Fc receptor. Fc receptors are immune cell receptors expressed primarily on innate immune cells that bind to the constant region of antibodies and elicit a wide range of functions. Fc receptors are almost exclusively activating receptors (with the exception of FcγRIIB, which transmits inhibitory signals). Ligation of Fc receptors results in the transmission of activation signals that enhance ADCC, phagocytosis, degranulation, and effector functions. It may cause.

[0258] The following table lists Fc receptors to which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 12]

[0259] Signaling Lymphocyte Activation Molecule (SLAM) In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an agonist antibody or an antigen-binding fragment thereof that binds to signaling lymphocyte activation molecule (SLAM) receptors. SLAM receptors are a series of molecules that function as both receptors and ligands. SLAM molecules interact with each other on adjacent cells to transmit activating or inhibitory signals. SLAM molecules contain an immunoreceptor tyrosine-based Swith motif in their cytoplasmic tails, allowing them to bind to both activating and inhibitory signaling molecules within cells.

[0260] The following table lists SLAM receptors against which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated.

[0261] In some embodiments, a suitable immunomodulatory domain for use in an immunomodulatory fusion protein of the present disclosure is the extracellular domain of a SLAM ligand. The following table shows the receptors to which the derived extracellular domains correspond: [Table 13]

[0262] Siglec family receptors In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the present disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a Siglec family receptor. Siglecs are a family of surface receptors found primarily on immune cells that are part of the lectin family (sugar-binding proteins). These receptors bind sialic acid-containing ligands. These receptors function primarily as inhibitory receptors on a wide range of immune cell types, although some (siglecs 14, 15, and 16) act as ITAM-activating receptors. Contains the main.

[0263] Activated Siglec receptors to which agonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated are provided in the table below. [Table 14]

[0264] Antagonist antibodies In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion proteins of the present disclosure is an antagonistic antibody or an antigen-binding fragment thereof. Antagonistic antibodies block the function of their target. In some embodiments, antagonistic antibodies or antigen-binding fragments thereof bind to immunoinhibitory receptors, thereby allowing induction of immune responses. In some embodiments, antagonistic antibodies or antigen-binding fragments thereof bind to immunoinhibitory ligands, thereby allowing induction of immune responses. In some embodiments, immunoinhibitory receptors and ligands include, but are not limited to, CD28 receptors, tumor necrosis factor (TNF) superfamily receptors, Siglec receptors, CD94 / NKG2 receptors, leukocyte Ig-like receptors (LIRs), killer cell Ig-like receptors (KIRs), Fc receptors, adenosine pathway molecules, other checkpoint inhibitors, and LAIR1.

[0265] CD28 molecule In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or its antigen-binding fragment that binds to CD28 molecule.As mentioned above, the CD28 family includes both activating and inhibitory molecules.Therefore, in some embodiments, immune response is induced or stimulated by antagonizing inhibitory molecules.

[0266] The following table lists CD28 molecules to which antagonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 15]

[0267] In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds to PD-1. is an antagonist antibody or antigen-binding fragment thereof that binds to PD-L1. In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds to CTLA-4.

[0268] TNF superfamily molecules In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or an antigen-binding fragment thereof that binds to a TNF superfamily member.As mentioned above, the TNF superfamily includes both activating and inhibitory molecules.Therefore, in some embodiments, immune response is induced or stimulated by antagonizing inhibitory molecules.

[0269] The following table lists TNF superfamily molecules against which antagonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 16]

[0270] Siglec receptors In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or an antigen-binding fragment thereof that binds to Siglec receptor.As mentioned above, the Siglec family includes both activating and inhibitory molecules.Therefore, in some embodiments, immune response is induced or stimulated by antagonizing inhibitory molecules.

[0271] The following table lists Siglec receptors against which antagonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 17]

[0272] CD94 / NKG2 receptor In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds to the CD94 / NKG2 receptor. As described above, the CD94 / NKG2 family includes both activating and inhibitory molecules. Thus, in some embodiments, the immune response is induced or stimulated by antagonizing the inhibitory molecules.

[0273] Thus, in some embodiments, the immunomodulatory domain is an antagonistic antibody or antigen-binding fragment thereof that binds to CD94 / NKG2A. In some embodiments, such an antibody is generated based on UniProt ID P26715.

[0274] In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds to CD94 / NKG2B. In some embodiments, such an antibody is generated based on UniProt ID Q13241.

[0275] Leukocyte Ig-like receptors (LIRs) In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or an antigen-binding fragment thereof that binds to leukocyte Ig-like receptor (LIR).As mentioned above, the LIR family includes both activating and inhibitory molecules.Therefore, in some embodiments, immune response is induced or stimulated by antagonizing inhibitory molecules.

[0276] The following table lists LIRs against which antagonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 18]

[0277] Killer cell Ig-like receptors (KIR) In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or an antigen-binding fragment thereof that binds to killer cell Ig-like receptor (KIR).As mentioned above, the KIR family includes both activating and inhibitory molecules.Therefore, in some embodiments, immune response is induced or stimulated by antagonizing inhibitory molecules.

[0278] The following table lists KIRs against which antagonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 19]

[0279] Fc receptors In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonistic antibody or its antigen-binding fragment that binds to Fc receptor.As mentioned above, the family of Fc receptors includes both activating and inhibitory molecules.Therefore, in some embodiments, immune response is induced or stimulated by antagonizing inhibitory molecules.

[0280] In some embodiments, the inhibitor Fc receptor is FcγRIIB. In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds to FcγRIIB. In some embodiments, such an antibody is generated based on UniProt ID P31994.

[0281] Adenosine pathway molecules In some embodiments, the immunomodulatory domain suitable for use in the immunomodulatory fusion protein of the present disclosure is an antagonist antibody or an antigen-binding fragment thereof that binds to adenosine pathway members.For example, CD39 and CD73 are enzymes expressed on the surface of cells that catalyze the transfection of ATP to adenosine.Extracellular ATP is a dangerous molecule that induces immune response, while adenosine is an immunosuppressant.These molecules contribute to the local immunosuppressive environment by generating adenosine.

[0282] Thus, in some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds to CD39. In some embodiments, such an antibody is generated based on UniProt ID P49961.

[0283] In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds to CD73. In some embodiments, such an antibody is generated based on UniProt ID P21589.

[0284] Other checkpoint inhibitors In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the present disclosure is an antagonist antibody or antigen-binding fragment thereof that binds to an immune checkpoint inhibitor, in some embodiments, an immune response is induced or stimulated by antagonizing such immune checkpoint inhibitor.

[0285] The following table lists immune checkpoint inhibitors against which antagonist antibodies or antigen-binding fragments thereof suitable for use in the immunomodulatory fusion proteins described herein can be generated. [Table 20]

[0286] Stabilization domain In some embodiments, the immunomodulatory fusion protein comprises one or more immunomodulatory domains and a stabilizing domain. In some embodiments, the stabilizing domain comprises a polypeptide that promotes or increases expression of the immunomodulatory fusion protein. In some embodiments, the stabilizing domain promotes or increases expression of the immunomodulatory fusion protein by promoting or maintaining folding of the immunomodulatory fusion protein after expression. In some embodiments, the stabilizing domain promotes or increases expression of the immunomodulatory fusion protein by preventing or reducing aggregation of the immunomodulatory fusion protein after expression. In some embodiments, the stabilizing domain promotes or increases expression of the immunomodulatory fusion protein by preventing or reducing degradation of the immunomodulatory fusion protein after expression.

[0287] In some embodiments, host cells transfected with a recombinant nucleic acid encoding an immunomodulatory fusion protein comprising a stabilization domain have increased expression compared to transfection with a recombinant nucleic acid encoding an immunomodulatory fusion protein lacking a stabilization domain. In some embodiments, expression is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, expression is increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold.

[0288] In some embodiments, the stabilization domain comprises a polypeptide that promotes or increases the stability of the immunomodulatory fusion protein after expression and isolation. Methods for measuring protein stability are known in the art and include differential scanning calorimetry, circular dichroism spectroscopy, thermal shift analysis, mass spectrometry, or activity-based assays.

[0289] Stabilization domains useful in the present disclosure are non-immunogenic protein domains that do not induce an immune response in the treated patient. Exemplary stabilization domains are further described below.

[0290] Serum albumin In some embodiments, the immunomodulatory fusion protein comprises a stabilization domain that is serum albumin or a fragment thereof. Methods of fusing serum albumin to a protein are disclosed, for example, in US2010 / 0144599, US2007 / 0048282, and US2011 / 0020345, which are incorporated herein by reference in their entirety. In some embodiments, the stabilization domain is human serum albumin (HSA), or a variant or fragment thereof, as described, for example, in US5,876,969, WO2011 / 124718, WO2013 / 075066, and WO2011 / 0514789. This is disclosed in.

[0291] Albumins suitable for use in immunomodulatory fusion proteins may be derived from humans, primates, rodents, cows, horses, donkeys, rabbits, goats, sheep, dogs, chickens, or pigs. In some embodiments, the albumin is serum albumin, such as human serum albumin (SEQ ID NO: 88), primate serum albumin (e.g., chimpanzee serum albumin, gorilla serum albumin), rodent serum albumin (e.g., hamster serum albumin, guinea pig serum albumin, mouse serum albumin, and rat serum albumin), bovine serum albumin, horse serum albumin, donkey serum albumin, rabbit serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, chicken serum albumin, and pig serum albumin.

[0292] In some embodiments, albumin, or a variant or fragment thereof, has at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of wild-type HSA as set forth in SEQ ID NO:88.

[0293] In some embodiments, the number of modifications, e.g., substitutions, insertions, or deletions, in the albumin variant is between 1 and 20, such as between 1 and 10, and between 1 and 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications, compared to the corresponding wild-type albumin (e.g., HSA).

[0294] In some embodiments, a fragment of albumin, or a fragment of its variant, is suitable for use in the immunomodulatory fusion protein. Exemplary albumin fragments are disclosed in WO2011 / 124718. In some embodiments, the fragment of albumin (e.g., a fragment of HSA) is at least 20 amino acids long, such as at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids long.

[0295] In some embodiments, the albumin fragment may comprise at least one entire subdomain of albumin. The domains of HSA have been expressed as recombinant proteins (Dockal et al., JBC 1999;274:9303-10), with domain I defined as consisting of amino acids 1-197 of HSA (SEQ ID NO:88), domain II defined as consisting of amino acids 189-385, and domain III defined as consisting of amino acids 381-585. Partial overlap of the domains occurs given the extended α-helical structure (h10-h1) present between domains I and II, and between domains II and III (Peters, 1996, supra, Tables 2-4). HSA also comprises six subdomains (subdomains IA, IB, NA, NB, INA, and NIB). Subdomain IA comprises amino acids 6-105 of SEQ ID NO:88, subdomain IB comprises amino acids 120-177, subdomain NA comprises amino acids 200-291, subdomain NB comprises amino acids 316-369, subdomain INA comprises amino acids 392-491, and subdomain NIB comprises amino acids 512-583 of SEQ ID NO:88.

[0296] In some embodiments, the fragment also contains one or more domains as defined above. In some embodiments, the albumin fragment comprises at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of albumin or a domain of albumin, or a variant or fragment thereof.

[0297] Fc domain In some embodiments, a stabilization domain suitable for use in the immunomodulatory fusion proteins described herein is an Fc domain, in some embodiments, the Fc domain is a component of the agonist or antagonist antibodies described above, and thus a separate Fc domain is not required.

[0298] In certain embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the Fc domain does not comprise a variable region that binds to an antigen. In some embodiments, the Fc domain comprises a variable region that binds to an antigen. Fc domains suitable for the immunomodulatory fusion proteins disclosed herein can be obtained from several different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. The Fc domain of human IgG1 is set forth in SEQ ID NO: 90. However, it is understood that the Fc domain can be derived from an immunoglobulin of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, the Fc domain or a portion thereof can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0299] In some embodiments, the immunomodulatory fusion protein comprises a mutant Fc domain. In some embodiments, the immunomodulatory fusion protein comprises a mutant, IgG1 Fc domain. In some embodiments, the mutant Fc domain comprises one or more mutations in the hinge, CH2, and / or CH3 domain. In some embodiments, the mutant Fc domain comprises a D265A mutation.

[0300] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains, including Fc domain sequences, can be selected lacking specific effector functions and / or with specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be derived from these sequences using art-recognized techniques. The genetic material obtained using any of the aforementioned methods can then be modified or synthesized to obtain polypeptides suitable for use in the methods disclosed herein. Furthermore, it will be understood that the scope of the present disclosure encompasses alleles, variants, and mutations of constant region DNA sequences.

[0301] Fc domain sequences can be cloned, for example, using the polymerase chain reaction and primers selected to amplify the domain of interest. To clone Fc domain sequences from an antibody, mRNA is isolated from hybridomas, spleen, or lymphoid cells, reverse transcribed to DNA, and the antibody genes are amplified by PCR. PCR amplification methods are described in U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, and in, for example, "PCR Protocols: A Guide to Methods and Applications" Innis. et al. eds., Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51; Horton PCR is described in detail in (E. et al. 1993. Methods Enzymol. 217:270). PCR can be initiated by consensus constant region primers or by more specific primers based on published heavy and light chain DNA and amino acid sequences. As mentioned above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of an antibody. In this case, the library may be screened by consensus primers or a larger homologous probe such as a mouse constant region probe. Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509), rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33), antibody leader sequences (Larrick et al. Biochem Biophys Res Commun 1989;160:1250)). Cloning of antibody sequences is further described in US Pat. No. 5,658,570 to Newman et al., filed Jan. 25, 1995, which is incorporated herein by reference.

[0302] In some embodiments, the disclosed immunomodulatory fusion proteins comprise one or more Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains). In certain embodiments, the Fc domains can be of different types. In certain embodiments, at least one Fc domain present in an immunomodulatory fusion protein comprises a hinge domain or a portion thereof. In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH2 domain or a portion thereof. In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH3 domain or a portion thereof. In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH4 domain or a portion thereof. In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain comprising at least one hinge domain or a portion thereof and at least one CH2 domain or a portion thereof (e.g., in a hinge-CH2 orientation). In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in a CH2-CH3 orientation). In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain comprising at least one hinge domain or portion thereof, at least one CH2 domain or portion thereof, and at least one CH3 domain or portion thereof, e.g., in a hinge-CH2-CH3, hinge-CH3-CH2, or CH2-CH3-hinge orientation.

[0303] In certain embodiments, the immunomodulatory fusion protein comprises at least one complete Fc region (e.g., an Fc domain including the hinge, CH2 and CH3 domains, which do not have to be from the same antibody) derived from one or more immunoglobulin heavy chains. In certain embodiments, the immunomodulatory fusion protein comprises at least two complete Fc domains derived from one or more immunoglobulin heavy chains. In certain embodiments, the complete Fc domain is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG1).

[0304] In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain that includes an intact CH3 domain. In certain embodiments, an immunomodulatory fusion protein comprises at least one Fc domain that includes an intact CH2 domain. In certain embodiments, an immunomodulatory fusion protein comprises at least a CH3 domain and at least one The fusion protein comprises at least one Fc domain comprising a hinge region and a CH2 domain. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising a hinge and a CH3 domain. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising a hinge, a CH2 and a CH3 domain. In certain embodiments, the Fc domain is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG1).

[0305] The constant region domains or portions thereof constituting the Fc domain of the immunomodulatory fusion protein may be derived from different immunoglobulin molecules. For example, a polypeptide suitable for use in the immunomodulatory fusion proteins disclosed herein may comprise a CH2 domain or portion thereof derived from an IgG1 molecule and a CH3 region or portion thereof derived from an IgG3 molecule. In some embodiments, the immunomodulatory fusion protein comprises an Fc domain that comprises a hinge domain that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. As described herein, it will be understood by those skilled in the art that the Fc domain may be modified to vary in amino acid sequence from a naturally occurring antibody molecule.

[0306] In certain embodiments, the immunomodulatory fusion protein lacks one or more constant region domains of the complete Fc region, i.e., they are partially or completely deleted. In certain embodiments, the immunomodulatory fusion protein lacks the entire CH2 domain. In certain embodiments, the immunomodulatory fusion protein comprises a CH2 domain-deleted Fc region derived from a vector (e.g., from IDEC Pharmaceuticals (San Diego)) encoding an IgG1 human constant region domain (see, e.g., WO02 / 060955A2 and WO02 / 096948A2). This exemplary vector is designed to delete the CH2 domain and provide a synthetic vector expressing a domain-deleted IgG1 constant region. It should be noted that these exemplary constructs are preferably engineered to fuse the binding CH3 domain directly to the hinge region of the respective Fc domain.

[0307] In other constructs, it may be desirable to provide a peptide spacer between one or more of the constituent Fc domains. For example, a peptide spacer may be placed between the hinge region and the CH2 domain, and / or between the CH2 and CH3 domains. For example, a compatible construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (synthetic or non-synthetic) is joined to the hinge region using a peptide spacer of 1-20, 1-10, or 1-5 amino acids. Such a peptide spacer may be added, for example, to ensure that the regulatory elements of the constant region domain remain free and accessible, or that the hinge region remains flexible. Preferably, any compatible stabilization domain peptides used in the present disclosure will be relatively non-immunogenic and will not interfere with the proper folding of the Fc.

[0308] In certain embodiments, the Fc domain used in the immunomodulatory fusion protein is altered or modified, for example, by amino acid mutation (e.g., addition, deletion, or substitution). As used herein, the term "Fc domain variant" refers to an Fc domain having at least one amino acid modification, such as an amino acid substitution, compared to the wild-type Fc from which the Fc domain is derived. For example, if the Fc domain is derived from a human IgG1 antibody, the variant contains at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position of the human IgG1 Fc region.

[0309] In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the hinge domain or a portion thereof. In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the CH2 domain or a portion thereof. In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the CH3 domain or a portion thereof. In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the CH4 domain or a portion thereof.

[0310] In certain embodiments, the immunomodulatory fusion protein comprises an Fc variant that comprises multiple amino acid substitutions. The immunomodulatory fusion protein may, for example, comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in the Fc domain. Preferably, the amino acid substitutions are spaced apart from each other by at least one amino acid position or more, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions or more. More preferably, the engineered amino acids are spaced apart from each other by at least 5, 10, 15, 20, or 25 amino acid positions or more.

[0311] In some embodiments, the Fc domain contains changes in the region of amino acids 234-238, including the sequence LLGGP at the start of the CH2 domain. In some embodiments, the Fc variants modify Fc-mediated effector functions, particularly ADCC, and / or reduce avidity for Fc receptors. In some aspects, sequence changes closer to the CH2-CH3 junction, such as at positions K322 or P331, may eliminate complement-mediated cytotoxicity and / or modify the avidity of FcR binding. In some embodiments, the Fc domain incorporates changes at residues P238 and P331, e.g., changing the wild-type proline at these positions to a serine. In some embodiments, modifications of the hinge region at one or more of the three hinge cysteines to encode CCC, SCC, SSC, SCS, or SSS at these residues may also affect FcR binding and molecular uniformity, such as by removing unpaired cysteines that may destabilize the folded protein.

[0312] Other amino acid mutations in the Fc domain are contemplated to reduce binding to Fc gamma receptors and Fc gamma receptor subtypes. For example, as described in U.S. Patent No. 6,737,056, issued May 18, 2004 (hereby incorporated by reference in its entirety), the Fc region positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 297, 298, 29 ...8, 299, 290, 292, 293, 294, 295, 296, 297, 298, 299, 290, 292, 293, 294, 295, 296, 297, 298, 299, 290, 292, 293, Mutations at 296, 298, 301, 303, 305, 307, 312, 315, 322, 324, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373, 376, 378, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 can alter binding. The patent reports that changing Pro331 to Ser in IgG3 results in a six-fold decrease in affinity compared to unmutated IgG3, implicating Pro331 in Fc gamma RI binding. In addition, US 5,624,821, issued April 29, 1997 (hereby incorporated by reference in its entirety), discloses amino acid modifications at positions 234, 235, 236, and 237, 297, 318, 320, and 322 as potentially altering receptor binding affinity.

[0313] Further mutations contemplated for use include, for example, those described in U.S. Patent Application Publication No. 2006 / 0235208, published October 19, 2006, which is incorporated by reference in its entirety. Additionally, the mutations described in U.S. Patent Application Publication No. 2006 / 0235208, which is incorporated by reference in its entirety, are contemplated for use. The mutant L234A / L235A is described, for example, in U.S. Patent Application Publication No. 2003 / 0108548, published June 12, 2003, which is incorporated by reference in its entirety. (which is incorporated herein in its entirety). In embodiments, the described modifications are included individually or in combination. In a specific embodiment, the mutation is D265A in human IgG1.

[0314] In certain embodiments, the immunomodulatory fusion protein comprises an Fc variant that comprises amino acid substitutions that modify the antigen-dependent effector function of the polypeptide, particularly ADCC or complement activation, for example, compared to wild-type Fc region.Such an immunomodulatory fusion protein exhibits reduced binding to FcR gamma when compared to wild-type polypeptide, and thus mediates reduced effector function.Fc variants with reduced FcR gamma binding affinity are expected to reduce effector function, and such molecules are also useful for treating conditions in which destruction of target cells is undesirable, for example, when normal cells may express target molecules or when chronic administration of the polypeptide may result in undesirable immune system activation.

[0315] In certain embodiments, the immunomodulatory fusion protein exhibits altered binding to an activating FcγR (e.g., Fcγ1, Fcγlla, or FcγRIIIa). In certain embodiments, the immunomodulatory fusion protein exhibits altered binding affinity to an inhibitory FcγR (e.g., FcγRIIb). Exemplary amino acid substitutions that alter FcR or complement binding activity are disclosed in International PCT Publication No. WO05 / 063815, which is incorporated herein by reference.

[0316] In some embodiments, the immunomodulatory fusion protein comprises an amino acid substitution that alters the glycosylation of the fusion protein. For example, in some embodiments, the Fc domain comprises a mutation that results in reduced glycosylation (e.g., N- or O-linked glycosylation) or comprises an altered glycoform of the wild-type Fc domain (e.g., low-fucose or fucose-free glycans). In certain embodiments, the immunomodulatory fusion protein has an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in WO05 / 018572 and US2007 / 0111281, the contents of which are incorporated herein by reference. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain with an engineered cysteine ​​residue or analog thereof located on a solvent-exposed surface. In certain embodiments, an immunomodulatory fusion protein comprises an Fc domain that comprises at least one engineered free cysteine ​​residue, or analog thereof, that is substantially free of disulfide bonds with a second cysteine ​​residue. Any of the above engineered cysteine ​​residues, or analogs thereof, can then be conjugated to a functional domain (e.g., conjugated to a thiol-reactive heterobifunctional stabilization domain) using art-recognized techniques.

[0317] In certain embodiments, the immunomodulatory fusion protein comprises a genetically fused Fc domain having two or more of its constituent Fc domains independently selected from the Fc domains described herein. In certain embodiments, the Fc domains are the same. In certain embodiments, at least two of the Fc domains are different. For example, the Fc domains may comprise the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In certain embodiments, the Fc domains differ in sequence at one or more amino acid positions. For example, at least two of the Fc domains may differ in sequence at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, about 50 positions, about 50 positions, about 60 positions, about 70 positions, about 80 positions, about 90 positions, about 10 ... They may differ at about 40 positions, or at about 50 positions.

[0318] Exemplary Immunomodulatory Fusion Proteins In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain and a metal hydroxide-binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group, wherein the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain, thereby forming the immunomodulatory fusion protein.

[0319] In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain, a stabilization domain, and a metal hydroxide-binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group, where the stabilization domain is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the metal hydroxide-binding peptide is operably linked, optionally via an amino acid linker, to either end of the immunomodulatory domain or the stabilization domain, thereby forming the immunomodulatory fusion protein.

[0320] In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain, a stabilization domain, and a metal hydroxide-binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group, where the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the stabilization domain is operably linked, optionally via an amino acid linker, to either end of the metal hydroxide-binding peptide or the immunomodulatory domain, thereby forming the immunomodulatory fusion protein.

[0321] IL-2 fusion proteins In some embodiments, the immunomodulatory fusion protein comprises IL-2, serum albumin, and a metal hydroxide binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group. In some embodiments, the IL-2 is operably linked to serum albumin. In some embodiments, the metal hydroxide binding peptide is operably linked to IL-2 or serum albumin.

[0322] In some embodiments, an immunomodulatory fusion protein comprises human IL-2 operably linked to the N-terminus of human serum albumin and further comprises a metal hydroxide binding peptide operably linked to the C-terminus of human serum albumin or the N-terminus of human IL-2. In some embodiments, an immunomodulatory fusion protein comprises human IL-2 operably linked to the C-terminus of human serum albumin and further comprises a metal hydroxide binding peptide operably linked to the N-terminus of human serum albumin or the C-terminus of human IL-2.

[0323] In some embodiments, the immunomodulatory fusion protein comprises human IL-2 operably linked to the human serum albumin sequence set forth in SEQ ID NO: 88. In some embodiments, the immunomodulatory fusion protein further comprises a metal hydroxide binding peptide comprising the four kinase motifs of the secretory pathway kinase Fam20C set forth by SEQ ID NO: 103, wherein the metal hydroxide binding peptide is operably linked to IL-2 or serum albumin.

[0324] IL-12 fusion proteins In some embodiments, the immunomodulatory fusion protein comprises at least one of IL-12, serum albumin, and a phosphate-modified secretory pathway kinase, Fam20C. and a metal hydroxide binding peptide comprising a kinase motif. In some embodiments, IL-12 is operably linked to serum albumin. In some embodiments, the metal hydroxide binding peptide is operably linked to IL-12 or serum albumin.

[0325] In some embodiments, an immunomodulatory fusion protein comprises human IL-12 operably linked to the N-terminus of human serum albumin and further comprises a metal hydroxide binding peptide operably linked to the C-terminus of human serum albumin or the N-terminus of human IL-12. In some embodiments, an immunomodulatory fusion protein comprises human IL-12 operably linked to the C-terminus of human serum albumin and further comprises a metal hydroxide binding peptide operably linked to the N-terminus of human serum albumin or the C-terminus of human IL-12.

[0326] In some embodiments, the immunomodulatory fusion protein comprises human IL-12 operably linked to the human serum albumin sequence set forth in SEQ ID NO: 88. In some embodiments, the immunomodulatory fusion protein further comprises a metal hydroxide binding peptide comprising the four kinase motifs of the secretory pathway kinase Fam20C set forth by SEQ ID NO: 103, wherein the metal hydroxide binding peptide is operably linked to IL-12 or serum albumin.

[0327] IFNg fusion protein In some embodiments, the immunomodulatory fusion protein comprises at least one IFNg, serum albumin, and a metal hydroxide binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group. In some embodiments, the IFNg is operably linked to serum albumin. In some embodiments, the metal hydroxide binding peptide is operably linked to the IFNg or serum albumin.

[0328] In some embodiments, an immunomodulatory fusion protein comprises two operably linked human IFNg polypeptides operably linked to the N-terminus of human serum albumin, and further comprises a metal hydroxide binding peptide operably linked to the C-terminus of human serum albumin or the N-terminus of human IFNg. In some embodiments, an immunomodulatory fusion protein comprises two operably linked human IFNg polypeptides operably linked to the C-terminus of human serum albumin, and further comprises a metal hydroxide binding peptide operably linked to the N-terminus of human serum albumin or the C-terminus of human IFNg.

[0329] In some embodiments, the immunomodulatory fusion protein comprises two operably linked human IFNg polypeptides and further comprises a metal hydroxide-binding peptide operably linked to the C-terminus or N-terminus of the two IFNg polypeptides.

[0330] In some embodiments, the immunomodulatory fusion protein comprises two operably linked human IFNg polypeptides operably linked to a human serum albumin sequence set forth in SEQ ID NO: 88. In some embodiments, the immunomodulatory fusion protein further comprises a metal hydroxide binding peptide comprising the four kinase motifs of the secretory pathway kinase Fam20C set forth by SEQ ID NO: 103, wherein the metal hydroxide binding peptide is operably linked to IFNg or serum albumin.

[0331] In some embodiments, the immunomodulatory fusion protein comprises two operably linked human IFNg polypeptides operably linked to a metal hydroxide binding peptide comprising the four kinase motifs of the secretory pathway kinase Fam20C as set forth by SEQ ID NO: 103, wherein the metal hydroxide binding peptide is operably linked to IFNg or serum albumin. It has been done.

[0332] Antibody Fusion Proteins In some embodiments, the immunomodulatory fusion protein comprises an anti-4-1-BB antibody and a metal hydroxide-binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group, wherein the anti-4-1-BB antibody is operably linked to the metal hydroxide-binding peptide. In some embodiments, the anti-4-1-BB antibody is operably linked to the N-terminus of the metal hydroxide-binding peptide. In some embodiments, the anti-4-1-BB antibody is operably linked to the C-terminus of the metal hydroxide-binding peptide.

[0333] In some embodiments, the immunomodulatory fusion protein comprises an anti-OX40 antibody and a metal hydroxide binding peptide comprising at least one kinase motif of the secretory pathway kinase Fam20C modified with a phosphate group, wherein the anti-OX40 antibody is operably linked to the metal hydroxide binding peptide. In some embodiments, the anti-OX40 antibody is operably linked to the N-terminus of the metal hydroxide binding peptide. In some embodiments, the anti-OPX40 antibody is operably linked to the C-terminus of the metal hydroxide binding peptide.

[0334] Methods for making immunomodulatory fusion protein-metal hydroxide complexes In some embodiments, the polypeptides described herein (e.g., kinases, cytokines, antibodies, stabilizing domains, metal hydroxide binding peptides) are produced in transfected host cells using recombinant DNA technology. To do so, recombinant DNA molecules encoding the polypeptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the sequences encoding the polypeptides can be excised from DNA using suitable restriction enzymes. Alternatively, chemical synthesis techniques such as the phosphoramidate method can be used to synthesize the DNA molecules. These techniques can also be used in combination.

[0335] The method of making polypeptide also includes a vector that can express peptide in a suitable host. The vector includes a DNA molecule that codes for peptide operably linked to a suitable expression control sequence. The method of influencing this functional linkage, either before or after the DNA molecule is inserted into the vector, is well known. The expression control sequence includes promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation.

[0336] The resulting vector, with the DNA molecule thereon, is used to transform an appropriate host, which can be carried out using methods well known in the art.

[0337] Any of a large number of available and well-known host cells may be suitable for use in the methods disclosed herein. The selection of a particular host depends on many factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, rate of transformation, ease of recovery of the peptide, expression characteristics, biological safety, and cost. These factors must be balanced with the understanding that not all hosts are equally effective in expressing a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.), and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

[0338] The transformed host is then cultured and purified. The host cells can be cultured under conventional fermentation conditions so that the desired compound is expressed. Such fermentation conditions are well known in the art. Finally, the peptide is purified from the culture by methods well known in the art.

[0339] Compounds can also be made by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable techniques are well known in the art and are described in, for example, Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; US Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2:257-527. Solid phase synthesis is the preferred technique for making individual peptides because it is the most cost-effective method for making small peptides. Compounds containing derivatized peptides or containing non-peptide groups may be synthesized by well-known organic chemistry techniques.

[0340] Other methods are those of molecular expression / synthesis, generally known to one of ordinary skill in the art.

[0341] The nucleic acid molecules described above can be contained within vectors, for example, that are capable of directing their expression in cells transduced with the vector. Thus, in addition to the polypeptide variants, expression vectors containing nucleic acid molecules encoding the variants and cells transfected with these vectors are among particular embodiments.

[0342] Suitable vectors for use include T7-based vectors for use in bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for use in insect cells (e.g., expression vector pBacPAKS from Clontech, Palo Alto, Calif). In such vectors, the nucleic acid insert encoding the polypeptide of interest can be operably linked to a promoter selected, for example, based on the cell type in which expression is desired. For example, the T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, for higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are named for their ability to direct the expression of nucleic acid molecules in specific tissues or cell types in the body. Those of skill in the art are familiar with the numerous promoters and other regulatory elements that can be used to direct expression of a nucleic acid.

[0343] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication and other genes that encode selectable markers. For example, neomycin resistance (neomycin-resistant r The ) gene confers G418 resistance to cells in which it is expressed, thus allowing for phenotypic selection of transfected cells. One of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental situation.

[0344] Suitable viral vectors for use include, for example, retroviruses, adenoviruses and adeno-associated vectors, herpes viruses, Simian Virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press (Cold Spring Harbor, NY)).

[0345] Prokaryotic or eukaryotic cells that contain and express the nucleic acid molecule encoding the polypeptide variant are also suitable for use. The cell is a transfected cell, i.e., a cell into which a nucleic acid molecule, for example, a nucleic acid molecule encoding a variant polypeptide, is introduced by recombinant DNA technology. The descendants of such cells are also considered suitable for use in the methods disclosed herein.

[0346] The exact components of the expression system are not critical. For example, the polypeptide variants may be produced in prokaryotic hosts such as the bacterium E. coli, or eukaryotic hosts such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, the only important thing is that the components are compatible with each other. A craftsman or ordinary skill can make such a determination. Furthermore, if guidance is needed in selecting an expression system, the skilled artisan may refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, 1999). Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0347] The expressed polypeptides can be purified from the expression system using conventional biochemical procedures and can be used, for example, as therapeutic agents, as described herein.

[0348] In some embodiments, an immunomodulatory fusion protein comprising an immunomodulatory domain, a metal hydroxide binding peptide comprising one or more kinase targeting motifs, and optionally a stabilization domain as described herein, is produced in a transfected host cell using recombinant DNA technology. To do so, a recombinant DNA molecule encoding the polypeptide is prepared. The method further comprises a vector capable of expressing the recombinant DNA molecule encoding the polypeptide. The resulting vector comprising the recombinant DNA molecule is used to transfect a suitable host cell. A method provided by the present disclosure for increasing phosphorylation of an immunomodulatory fusion protein comprises transfecting a cell with a recombinant DNA molecule encoding an immunomodulatory fusion protein and a recombinant DNA molecule encoding a kinase comprising an ER targeting leader sequence, a kinase domain derived from a kinase, and an anchor peptide, wherein the kinase is localized to the secretory pathway by the ER targeting leader sequence and the anchor peptide, and the one or more kinase targeting motifs of the metal hydroxide binding peptide are phosphorylated by a kinase in the secretory pathway, thereby increasing phosphorylation of the immunomodulatory fusion protein. In some embodiments, the kinase is a naturally occurring kinase comprising an ER targeting leader sequence that localizes the kinase to the secretory pathway. In some embodiments, naturally occurring kinases are modified with a terminal (e.g., C-terminal) anchor peptide to increase, promote, or improve localization to the secretory pathway and / or reduce or prevent secretion. In some embodiments, kinases of the disclosure include any kinase domain modified with an ER-targeting leader sequence and an anchor peptide to increase, promote, or improve localization to the secretory pathway and / or reduce or prevent secretion.

[0349] In some embodiments, the recombinant DNA molecule encoding the immunomodulatory fusion protein and the recombinant DNA molecule encoding the kinase comprising the ER targeting leader sequence, the kinase domain, and the anchor peptide used to transfect cells are the same or different.

[0350] Thus, in some embodiments, an immunomodulatory fusion protein prepared according to such methods of the present disclosure comprises an immunomodulatory domain, a metal hydroxide-binding peptide comprising at least one kinase target motif of a secretory pathway kinase comprising a phosphorylated amino acid, and optionally a stabilization domain. In some embodiments, the immunomodulatory fusion protein undergoes ligand exchange with a metal hydroxide (e.g., alum) via the at least one phosphorylated amino acid of the metal hydroxide-binding peptide, thereby coupling the immunomodulatory fusion protein to the metal hydroxide (e.g., alum) to form an immunomodulatory fusion protein-metal hydroxide complex.

[0351] In some embodiments, an immunomodulatory fusion protein comprising an immunomodulatory domain and optionally a stabilizing domain is made in a transfected host cell using recombinant DNA techniques and further coupled to a metal hydroxide-binding peptide comprising at least one hydroxyl substituent, a polypeptide reactive moiety, and optionally a linker, thereby preparing an immunomodulatory fusion protein comprising an immunomodulatory domain, a metal hydroxide-binding peptide, and optionally a stabilizing domain, which undergoes ligand exchange with a metal hydroxide (e.g., alum) via the at least one hydroxyl substituent to form an immunomodulatory fusion protein-metal hydroxide complex.

[0352] In some embodiments, an immunomodulatory fusion protein comprising an immunomodulatory domain and, optionally, a stabilizing domain, is modified to include one or more amino acids not present in its native form (e.g., cysteine) to create or increase the ability of the immunomodulatory fusion protein to react with a polypeptide reactive moiety.

[0353] In some embodiments, a metal hydroxide-binding peptide of the present disclosure comprising at least one hydroxyl substituent (e.g., a phosphorylated amino acid) operably linked, optionally via a linker, to a polypeptide reactive moiety reacts with an immunomodulatory fusion protein comprising an immunomodulatory domain and, optionally, a stabilizing domain, and the polypeptide reactive moiety crosslinks the metal hydroxide-binding peptide, optionally comprising a linker, to the immunomodulatory fusion protein.

[0354] One non-limiting method of achieving this modification known in the art that is particularly well suited to modifying polypeptides is by including an amino acid in the immunomodulatory fusion protein that provides a reactive moiety (e.g., cysteine, -SH), and by further contacting the modified immunomodulatory fusion protein containing the reactive moiety with a polypeptide reactive moiety operably linked to the metal hydroxide-binding peptide, optionally via a linker. Another non-limiting method of achieving this modification known in the art is by including a short sequence of terminal amino acids (e.g., a sequence of glycine or alanine amino acids) in the immunomodulatory fusion protein that enables a reaction catalyzed by a recombinant sortase having a metal hydroxide-binding peptide that contains a polypeptide reactive moiety that is a sortase recognition motif, thereby forming a covalent linkage between the immunomodulatory fusion protein and the metal hydroxide-binding peptide.

[0355] In some embodiments, the present disclosure provides that an immunomodulatory fusion protein that includes one or more hydroxyl-substituted moieties in its native form increases the rate of ligand exchange adsorption or enhances the affinity of the fusion protein to gold. It is contemplated that metal hydroxides may be modified in accordance with the present invention to increase the strength of adsorption of immunomodulatory fusion proteins to metal hydroxides.

[0356] Pharmaceutical Compositions and Modes of Administration In certain embodiments, the present disclosure provides pharmaceutical compositions comprising an immunomodulatory fusion protein-metal hydroxide complex with a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.In certain embodiments, the present disclosure provides pharmaceutical compositions comprising an immunomodulatory fusion protein with a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0357] In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used. In certain embodiments, the formulation materials are for sc and / or IV administration. In certain embodiments, the formulation materials are for local administration, e.g., intratumoral administration. In certain embodiments, the pharmaceutical composition can contain formulation materials to modify, maintain, or preserve, for example, pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration.In certain embodiments, suitable formulation ingredients include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium bisulfite, sodium hydrogen sulfite, etc.); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavors, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salts. These include, but are not limited to, forming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, polysorbates such as tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2; 50 mM NaCl; and / or 10 mM NaOAC, pH 5.2, 9% sucrose.In certain embodiments, optimal pharmaceutical compositions will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the immunomodulatory fusion protein-metal hydroxide complex.

[0358] In some embodiments, formulations comprising an immunomodulatory fusion protein-metal hydroxide complex or an immunomodulatory fusion protein described herein can be administered at temperatures between 4° C. and 3° C. when administered to a subject. It's 7 degrees Celsius.

[0359] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, possibly supplemented with other materials common to compositions for parenteral administration. In certain embodiments, saline comprises isotonic phosphate buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, which may further comprise sorbitol or a suitable substitute therefor. In certain embodiments, the compositions comprising the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein may be prepared by dissolving the selected composition having the desired purity in a lyophilized cake or aqueous solution form, together with any formulation agents (Remington's Pharmaceutical Sciences, supra. Moreover, in certain embodiments, compositions comprising an immunomodulatory fusion protein-metal hydroxide complex or an immunomodulatory fusion protein are formulated as a lyophilizate using appropriate excipients, such as sucrose.

[0360] In certain embodiments, the pharmaceutical composition is selected for parenteral delivery. In certain embodiments, the composition is selected for inhalation or delivery via the digestive tract, such as orally. The preparation of such pharma- ceutically acceptable compositions is within the capabilities of those skilled in the art.

[0361] In certain embodiments, the components of the formulation are present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8.

[0362] In certain embodiments, when parenteral administration is intended, the therapeutic composition is in the form of a pyrogen-free parenterally acceptable aqueous solution containing the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein in a pharma- ceutical acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water in which the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein is formulated as a sterile, isotonic solution that is appropriately preserved. In certain embodiments, the preparation may include formulation of the desired molecule with agents such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes that can provide controlled or sustained release of the product that can be delivered via depot injection. In certain embodiments, hyaluronic acid may also be used, which may have the effect of promoting sustained duration in the circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0363] In certain embodiments, the pharmaceutical composition is formulated for inhalation. In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein is formulated as a dry powder for inhalation. In certain embodiments, the inhalation solution containing the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein is formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes the delivery of chemically modified proteins to the lungs.

[0364] In certain embodiments, it is contemplated that the formulations are administered orally. In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory The fusion protein may be formulated with or without these carriers that are typically used in the formulation of solid dosage forms such as tablets and capsules. In certain embodiments, the capsule is designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent is included to facilitate absorption of the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[0365] In certain embodiments, the pharmaceutical composition comprises an effective amount of the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein in a mixture with a non-toxic excipient suitable for producing tablets. In certain embodiments, the solution is prepared in unit dose form by dissolving the tablet in sterile water or another suitable vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate, or binders such as starch, gelatin or acacia, or lubricants such as magnesium stearate, stearic acid, talc, etc.

[0366] Additional pharmaceutical compositions, including formulations that include immunomodulatory fusion protein-metal hydroxide complexes or immunomodulatory fusion proteins in sustained or controlled delivery formulations, will be apparent to those skilled in the art. In certain embodiments, techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. For example, see PCT Application No. PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained release preparations can include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra), or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained release compositions include liposomes, which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985), EP 036,676, EP 088,046, and EP 143,949.

[0367] The pharmaceutical composition used for in vivo administration is usually sterile.In certain embodiments, this is achieved by filtration through a sterile filtration membrane.In certain embodiments, the composition is lyophilized, sterilization using this method is carried out before or after lyophilization and reconstitution.In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in solution.In certain embodiments, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0368] In certain embodiments, once the pharmaceutical composition has been formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations may be stored in a ready-to-use form, or The formulation may be stored either in a form (eg, lyophilized) that is reconstituted prior to administration.

[0369] In certain embodiments, kits are provided for producing single-dose administration units.In certain embodiments, kits can include both a first container with dry protein and a second container with aqueous formulation.In certain embodiments, kits include single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0370] In certain embodiments, the effective amount of the immunomodulatory fusion protein-metal hydroxide complex or pharmaceutical composition comprising the immunomodulatory fusion protein used for treatment will depend, for example, on the context and purpose of the treatment. Thus, those skilled in the art will understand that the appropriate dosage level for treatment according to a particular embodiment will vary depending in part on the molecule being delivered, the indication for which the immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein is being used, the route of administration, and the size (weight, body surface or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician can titrate the dosage and modify the route of administration to obtain optimal therapeutic effect.

[0371] In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of the immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein in the formulation used. In certain embodiments, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, in certain embodiments, the composition can be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of the tasks routinely performed by those skilled in the art. In certain embodiments, the appropriate dosage can be ascertained by using appropriate dose-response data.

[0372] In certain embodiments, the route of administration of pharmaceutical composition is consistent with known methods, for example, intravenous, intraperitoneal, intracerebral (intracemata), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional route, sustained release system, or by implantation device.In certain embodiments, the composition can be administered by bolus injection, or continuously by infusion, or by implantation device.In certain embodiments, the individual components of combination therapy can be administered by different routes.

[0373] In certain embodiments, the composition can be administered locally by implantation of a membrane, sponge, or another suitable material in which the desired molecule is absorbed or encapsulated. In certain embodiments in which an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, sustained release bolus, or continuous administration. In certain embodiments, it may be desirable to use a pharmaceutical composition comprising an immunomodulatory fusion protein-metal hydroxide complex or an immunomodulatory fusion protein in an ex vivo manner. In such cases, cells, tissues, and / or organs removed from a patient are exposed to a pharmaceutical composition comprising an immunomodulatory fusion protein-metal hydroxide complex or an immunomodulatory fusion protein, and then the cells, tissues, and / or organs are implanted back into the patient.

[0374] In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein is delivered by implanting specific cells that have been genetically engineered, using methods as described herein, to express and secrete the polypeptide. In certain embodiments, such cells can be animal or human cells, including autologous cells. The cells may be native, heterologous, or xenogeneic. In certain embodiments, the cells may be immortalized. In certain embodiments, to reduce the possibility of an immune response, the cells may be encapsulated to avoid infiltration of surrounding tissues. Certain encapsulation materials are typically biocompatible semi-permeable polymer enclosures or membranes that allow release of the protein product embodiment but prevent destruction of the cells by the patient's immune system or other harmful factors from the surrounding tissues.

[0375] Treatment methods The immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, and / or nucleic acids expressing immunomodulatory fusion proteins, or compositions thereof described herein, are useful for treating disorders associated with abnormal apoptotic or differentiation processes (e.g., cell proliferation disorders (e.g., hyperproliferative disorders) or cell differentiation disorders, such as cancer). Non-limiting examples of cancers suitable for treatment by the methods of the present disclosure are described below.

[0376] Examples of cell proliferative and / or differentiative disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders, e.g., leukemia). Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, tumors of the prostate, colon, lung, breast, and liver. Thus, for example, compositions used herein, including immunomodulatory fusion protein-metal hydroxide complexes, can be administered to patients with cancer.

[0377] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative", and "neoplastic" refer to cells capable of autonomous growth (i.e., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic conditions may be classified as pathological (i.e., characterizing or constituting a disease state) or non-pathological (i.e., a deviation from normal but not associated with a disease state). The term is meant to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include the proliferation of cells associated with wound repair.

[0378] The term "cancer" or "neoplasm" is used to refer to malignant tumors of various organ systems, including those affecting the lung, breast, thyroid, lymphatic glands and tissues, gastrointestinal tract, and genitourinary tract, as well as adenocarcinomas, which are generally considered to include malignant tumors such as most colon cancers, renal cell carcinoma, prostate and / or testicular cancers, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.

[0379] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissues, including respiratory, gastrointestinal, genitourinary, testicular, breast, prostate, endocrine, and melanoma. The immunomodulatory fusion protein-metal hydroxide complex, immunomodulatory fusion protein, or compositions thereof, can be used to treat patients who have, are suspected of having, or may be at high risk of developing any carcinoma, including renal or melanoma, or any viral disease. Exemplary carcinomas include carcinomas formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0380] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. In this context, the term "hematopoietic neoplastic disorder" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroblastic lineages, or their precursor cells. Preferably, the disease arises from poorly differentiated acute leukemia (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. in Oncol. / Hemotol. 11:267-97). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease.

[0381] It will be understood by those skilled in the art that the amount of immunomodulatory fusion protein-metal hydroxide complex, immunomodulatory fusion protein or composition thereof sufficient to reduce tumor growth and size, or a therapeutically effective amount, will vary depending on the particular compound or composition selected, as well as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist. The length of time that the compounds used in the methods of the invention are given will vary from individual to individual.

[0382] It will be understood by those skilled in the art that the B16 melanoma model used herein is a generalized model of solid tumors. That is, the efficacy of treatment in this model is predictive of the efficacy of treatment in other non-melanoma solid tumors. See, e.g., Baird et al. (J As described in Immunology 2013;190:469-78;Epub Dec 7,2012), the efficacy of cps, a parasite strain that induces adaptive immune responses in mediating antitumor immunity against B16F10 tumors, was found to be generalizable to other solid tumors, including models of lung carcinoma and ovarian cancer. In another example, the results of a series of studies on VEGF targeting lymphocytes have also shown that the results in B16F10 tumors can be generalized to other tumor types studied (Chinnasamy et al.,JCI 2010;120:3953-68;Chinnasamy et al.,Clin Cancer Res 2012;18:1672-83). In yet another example, immunotherapy involving LAG-3 and PD-1 reduced tumor burden with generalizable results in fibrosarcoma and colon adenocarcinoma cell lines (Woo et al., Cancer Res 2012;72:917-27).

[0383] In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or compositions thereof disclosed herein are used to treat cancer. In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or compositions thereof disclosed herein are used to treat melanoma, leukemia, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, and brain cancer.

[0384] In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or compositions thereof disclosed herein inhibit tumor cell growth and / or proliferation. In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or compositions thereof disclosed herein reduce tumor size. In certain embodiments, the immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins, or compositions thereof disclosed herein inhibit metastasis of primary tumors.

[0385] It will be appreciated by those skilled in the art that references herein to treatment extend to prophylaxis as well as treatment of the cancers and conditions mentioned.

[0386] Combination therapy In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein disclosed herein is used in combination with other therapies. For example, in some embodiments, the immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein is used in combination with another immunotherapy. Exemplary immunotherapies include, but are not limited to, chimeric antigen receptor (CAR) T-cell therapy, tumor-associated antigen targeting antibodies, immune checkpoint inhibitors, and cancer vaccines. In some embodiments, the immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein is used in combination with another immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein with a different immunomodulatory domain.

[0387] Chimeric Antigen Receptor (CAR) Effector Cells In some embodiments, the present disclosure provides immunomodulatory fusion protein-metal hydroxide complexes or immunomodulatory fusion proteins for use or practice in conjunction with chimeric antigen receptor (CAR) effector cell therapy (e.g., CAR T cells).

[0388] Chimeric antigen receptors (CARs) are engineered artificial transmembrane receptors that confer an arbitrary specificity to an immune effector cell (e.g., a T cell, a natural killer cell, or other immune cell) for a ligand that, upon recognition and binding to the ligand, activates the effector cell. Typically, these receptors are used to confer the antigen specificity of a monoclonal antibody to a T cell.

[0389] In some embodiments, CARs comprise three domains: 1) an ectodomain, which typically includes a signal peptide, a ligand or antigen recognition region (e.g., scFv), and a flexible spacer; 2) a transmembrane (TM) domain; and 3) an endodomain, which typically includes one or more intracellular signaling domains (alternatively known as the "activation domain"). The ectodomain of a CAR is extracellular and exposed to the extracellular space, making it accessible for interaction with its cognate ligand. The TM domain allows the CAR to be anchored to the cell membrane of an effector cell. The third endodomain (also known as the "activation domain") aids in the activation of effector cells once the CAR binds to its specific ligand. In some embodiments, effector cell activation includes the induction of cytokine and chemokine production, as well as activation of the cytolytic activity of the cell. In some embodiments, the CAR redirects cytotoxicity against tumor cells.

[0390] In some embodiments, the CAR comprises a ligand or antigen-specific recognition domain (also called a binding domain) that binds to a specific target ligand or antigen. In some embodiments, the binding domain is a single chain antibody variable fragment (scFv), a tethering ligand or an extracellular domain of a co-receptor, fused to a transmembrane domain, which is then linked to a signaling domain. In some embodiments, the signaling domain is derived from CD3zeta or FcRgamma. In some embodiments, the CAR comprises one or more costimulatory domains derived from proteins such as CD28, CD137 (also known as 4-1BB), CD134 (also known as OX40) and CD278 (also known as ICOS).

[0391] Engagement of the antigen-binding domain of the CAR with its target antigen on the surface of the target cell results in the CAR are clustered and an activating stimulus is delivered to the CAR-containing cells. In some embodiments, the main feature of CARs is their ability to redirect immune effector cell specificity, thereby triggering the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, leveraging the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors. Although scFv-based CARs engineered to contain signaling domains from CD3ζ or FcRγ have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to elicit signals that promote T cell survival and expansion in the absence of accompanying costimulatory signals. Emerging CARs containing binding domains, hinges, transmembrane, and signaling domains derived from CD3ζ or FcRγ along with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278) have been shown to more effectively direct antitumor activity and increase cytokine secretion, lytic activity, survival and proliferation of CAR-expressing T cells in vitro in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17:1453-1464; Zhong et al., Molecular Therapy, 2010; 18:413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0392] In some embodiments, chimeric antigen receptor-expressing effector cells (e.g., CAR-T cells) are cells derived from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with any specificity for a ligand. The cells are stimulated or induced by specific binding of the ligand to the CAR to perform at least one effector function (e.g., induction of cytokines) useful for treating the disease or condition of the same patient. The effector cells can be T cells (e.g., cytotoxic T cells or helper T cells). Those skilled in the art will understand that other cell types (e.g., natural killer cells or stem cells) can express CARs, and that chimeric antigen receptor effector cells can include effector cells other than T cells. In some embodiments, an effector cell is a T cell (e.g., a cytotoxic T cell) that, upon contact with or proximity to a target or target cell (e.g., a cancer cell), exerts its effector function (e.g., a cytotoxic T cell response) against the target cell (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).

[0393] Prolonged exposure of T cells to their cognate antigens exhausts effector functions, allowing the persistence of infected or transformed cells. Recently developed strategies to stimulate or activate host effector functions using agents that induce immune checkpoint blockade have been successful in the treatment of several cancers. Emerging evidence suggests that T cell exhaustion may be a significant obstacle in maintaining long-lived antitumor activity by chimeric antigen receptor-expressing T cells (CAR-T cells). In some embodiments, the differentiation state of T cells taken from patients prior to CAR transduction, and the pre-transplant treatments that patients undergo prior to reintroduction of CAR-T cells (e.g., application or elimination of alkylating agents, fludarabine, total body irradiation) can greatly affect the persistence and cytotoxicity of CAR-T cells. In vitro culture conditions that stimulate (via anti-CD3 / CD28 or stimulator cells) and expand (via cytokines such as IL-2) T cell populations can also modify the differentiation state and effector function of CAR-T cells (Ghoneim et al., (2016) Trends in Molecular Medicine 22(12):1000-1011).

[0394] In some embodiments, particularly for the treatment of ALL and / or NHL, suitable CARs target CD19 or CD20. Non-limiting examples include CARs comprising the following structures: (i) anti-CD19 scFv, CD8 H / TM domains, 4-1 (ii) anti-CD19 scFv, CD28 hinge and transmembrane domain, CD28 costimulatory domain and CD3ζ TCR signaling domain, (iii) anti-CD20 scFv, IgG hinge and transmembrane domain, CD28 / 4-1BB costimulatory domain and CD3ζ TCR signaling domain. In some embodiments, CAR effector cells suitable for combination with the combinations and methods disclosed herein target CD19 or CD20, including but not limited to Kymriah™ (tisagenlecleucel, Novartis, formerly CTL019) and Yescarta™ (axicabtadin cilorucel, Kite Pharma).

[0395] Retargeted CAR T cells In some embodiments, a CAR-T therapy suitable for use in combination with an immunomodulatory fusion protein-metal hydroxide complex is a retargeted CAR-T cell. In some embodiments, effector cells (e.g., T cells) modified to express a universal immune receptor, a tag, a switch, or a CAR that binds to an Fc region on an immunoglobulin are suitable for the methods described herein.

[0396] In some embodiments, effector cells (e.g., T cells) are modified to express a universal immune receptor or UnivIR. One type of UnivIR is a biotin-binding immune receptor (BBIR) (see, e.g., U.S. Patent Publication No. US2014 / 0234348(A1), which is incorporated herein by reference in its entirety). Other examples of methods and compositions related to universal chimeric receptors and / or effector cells expressing universal chimeric receptors are described in International Patent Application WO2016 / 123122(A1), WO2017 / 143094(A1), WO2013 / 074916(A1), U.S. Patent Application US2016 / 0348073(A1), which are all incorporated herein by reference in their entirety.

[0397] In some embodiments, effector cells (e.g., T cells) are modified to express a universal and modular anti-tag chimeric antigen receptor (UnicAR). This system allows retargeting of UniCAR-engrafted immune cells to multiple antigens (see, e.g., U.S. Patent Publication US2017 / 0240612(A1), incorporated herein by reference in its entirety; Cartellieri et al., (2016) Blood Cancer Journal 6, e458, incorporated herein by reference in its entirety).

[0398] In some embodiments, effector cells (e.g., T cells) are modified to express a switchable chimeric antigen receptor and a chimeric antigen receptor effector cell (CAR-EC) switch. In this system, the CAR-EC switch has a first region that is bound by the chimeric antigen receptor on the CAR-EC and a second region that binds to a cell surface molecule on the target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell. In some embodiments, the CAR-EC is a T cell, and the CAR-EC switch can function as an "on switch" for CAR-EC activity. The activity can be "off" by reducing or ceasing management of the switch. These CAR-EC switches can be used with the CAR-ECs disclosed herein, as well as existing CAR T cells, for the treatment of diseases or conditions, such as cancer, where the target cell is a malignant cell. Such treatments are sometimes referred to herein as switchable immunotherapy (U.S. Patent Publication US9624276(B2), incorporated herein by reference in its entirety).

[0399] In some embodiments, effector cells (e.g., T cells) are human immunoglobulins. The cells are modified to express a receptor that binds to the Fc portion of Brin (e.g., CD16V-BB-ζ) (Kudo et al., (2014) Cancer Res 74(1):93-103, which is incorporated herein by reference in its entirety).

[0400] In some embodiments, effector cells (e.g., T cells) are modified to express a universal immune receptor (e.g., switchable CAR, sCAR) that binds a peptide neoepitope (PNE). In some embodiments, the peptide neoepitope (PNE) is incorporated into an antibody targeting an antigen at a defined, distinct location (antibody switch). Thus, the specificity of the sCAR-T cell is redirected only to the PNE, which does not occur in the human proteome, allowing for orthogonal interactions between the sCAR-T cell and the antibody switch. In this way, the sCAR-T cell strictly relies on the presence of the antibody switch to be fully activated, thus excluding CAR T cell off-target recognition of endogenous tissues or antigens in the absence of the antibody switch (Arcangeli et al., (2016) Transl Cancer Res 5(Suppl 2):S174-S177, incorporated herein by reference in its entirety). Other examples of switchable CARs are provided by U.S. Patent Application US2016 / 0272718(A1), which is incorporated by reference in its entirety.

[0401] As used herein, the term "tag" encompasses universal immune receptors, tags, switches, or Fc regions of immunoglobulins, as described above. In some embodiments, the effector cells are modified to express a CAR that includes a tag-binding domain. In some embodiments, the CAR is conjugated with fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alcalanine phosphatase, glucose oxidase, or maltose protein.

[0402] Anti-TAG chimeric antigen receptor (AT-CAR) In some embodiments, a CAR-T therapy suitable for use in combination with an immunomodulatory fusion protein-metal hydroxide complex or an immunomodulatory fusion protein is an anti-tag CAR T cells. There are several limitations to the generalized clinical application of CAR cells. For example, there is no single tumor antigen that is universally expressed by all carcinomas, so each scFv of the CAR needs to be specifically engineered to the tumor antigen of interest. In addition, the tumor antigen targeted by the CAR may be downregulated or mutated in response to treatment, resulting in tumor escape.

[0403] Alternatively, universal anti-tag chimeric antigen receptors (AT-CARs) and CAR-T cells have been developed. For example, human T cells have been engineered to express anti-fluorescein isothiocyanate (FITC) CARs (termed anti-FITC-CARs). This platform exploits the high affinity interaction between anti-FITC scFv (on the cell surface) and FITC, and the ability to conjugate FITC molecules (or other tags) to anti-cancer-based monoclonal antibodies such as cetuximab (anti-EGFR), retuximab (anti-CD20), and herceptin (anti-Her2).

[0404] Thus, in some embodiments, effector cells (e.g., T cells) are modified to express a universal anti-tag chimeric antigen receptor (AT-CAR), at least as described in WO2012 / 082841 and US2016 / 0129109(A1), which are incorporated by reference in their entireties. In such AT-CAR systems, T cells recognize and bind to tagged proteins, such as antibodies. For example, in some embodiments, AT-CAR T cells express a tagged protein, such as a FITC-labeled antibody. Which tag-labeled antibody recognizes. In some embodiments, the anti-tumor antigen antibody is conjugated to a tag (e.g., FITC) and administered before, simultaneously with, or after AT-CAR therapy. Anti-tumor antigen antibodies are known to those skilled in the art.

[0405] As shown, the binding specificity of tag-binding domain depends on the identity of the tag that is attached to the protein that is used to bind target cells.For example, in some embodiments of the present disclosure, the tag is FITC, and the tag-binding domain is anti-FITC scFv.Alternatively, in some embodiments of the present disclosure, the tag is biotin or PE (phycoerythrin), and the tag-binding domain is anti-biotin scFv or anti-PE scFv.

[0406] In some embodiments, the proteins of each formulation of tagged proteins are the same or different, and the proteins are antibodies or antigen-binding fragments thereof. In some embodiments, the antibodies or antigen-binding fragments thereof are cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), retuximab (anti-CD20), omalizumab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-Her2), gemtuzumab (anti-CD33), alemtuzumab (anti-CD52), and bevacuzimab (anti-VEGF).

[0407] Thus, in some embodiments, the tagged proteins include FITC-conjugated antibodies, biotin-conjugated antibodies, PE-conjugated antibodies, histidine-conjugated antibodies, and streptavidin-conjugated antibodies, which bind to a TAA or TSA expressed by a target cell. For example, tagged proteins include, but are not limited to, FITC-conjugated cetuximab, FITC-conjugated retuximab, FITC-conjugated Herceptin, biotin-conjugated cetuximab, biotin-conjugated retuximab, biotin-conjugated Herceptin, PE-conjugated cetuximab, PE-conjugated retuximab, PE-conjugated Herceptin, histidine-conjugated cetuximab, histidine-conjugated retuximab, histidine-conjugated Herceptin, streptavidin-conjugated cetuximab, streptavidin-conjugated retuximab, and streptavidin-conjugated Herceptin.

[0408] In some embodiments, the AT-CARs of each population of AT-CAR-expressing T cells are the same or different, and the AT-CARs comprise a tag-binding domain, a transmembrane domain, and an activation domain. In some embodiments, the tag-binding domain is an antibody or an antigen-binding fragment thereof. In some embodiments, the tag-binding domain specifically binds to FITC, biotin, PE, histidine, or streptavidin. In some embodiments, the tag-binding domain is an antigen-binding fragment, and the antigen-binding fragment is a single-chain variable fragment (scFv), such as an scFv, that specifically binds to FITC, biotin, PE, histidine, or streptavidin. In some embodiments, the transmembrane domain is the hinge and transmembrane region of human CD8α chain. In some embodiments, the activation domain comprises one or more of the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), OX40, HVEM, CD3ζ, and FcRε.

[0409] In some embodiments, the tags of each formulation of tagged protein are the same or different, and the tags are selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alcalanine phosphatase, glucose oxidase, and maltose binding protein.

[0410] Tags can be conjugated to proteins using techniques such as chemical coupling and chemical cross-linking agents. Alternatively, polynucleotide vectors can be prepared that code for tagged proteins as fusion proteins. Cell lines can then be engineered to express tagged proteins, which can be isolated from culture medium, purified, and used in the methods disclosed herein.

[0411] In some embodiments, the tagged protein is administered to the subject before, or simultaneously with, or after administration of the AT-CAR-expressing T cells. In some embodiments, the present disclosure provides a method of treating cancer in a subject, comprising: (a) administering a combination of tagged proteins to a subject in need of treatment, where the tagged proteins bind to cancer cells in the subject, and (b) administering a therapeutic population of anti-tag chimeric antigen receptor (AT-CAR)-expressing T cells to the subject, thereby treating the cancer, where the AT-CAR-expressing T cells bind to the tagged protein and induce cancer cell death, thereby treating the cancer.

[0412] Tandem CAR (TanCAR) effector cells In some embodiments, the CAR-T therapy suitable for use in combination with the immunomodulatory fusion protein-metal hydroxide complex or the immunomodulatory fusion protein is a tandem CAR effector cell. It has been observed that using the CAR approach for cancer treatment, tumor heterogeneity and immunoediting can cause deviation from CAR therapy (Grupp et al., New Eng. J. Med (2013) 368: 1509-1518). As an alternative approach, bispecific CARs, known as tandem CARs or TanCARs, have been developed in an attempt to simultaneously target multiple cancer-specific markers. In TanCARs, the extracellular domain contains two antigen binding specificities in tandem joined by a linker. Thus, the two binding specificities (scFvs) are both linked to a single transmembrane moiety. One scFv is juxtaposed to the membrane and the other is in a distal position. An exemplary TanCAR is described by Grada et al. (Mol Ther Nucleic Acids (2013) 2, e105) described a TanCAR containing a CD19-specific scFv followed by a Gly-Ser linker and a HER2-specific scFv. The HER2-scFv was in a membrane-apposed position and the CD19-scFv was in a distal position. TanCAR has been shown to induce distinct T cell reactivity against each of the two tumor-restricted antigens.

[0413] Thus, some embodiments of the present disclosure relate to tandem chimeric antigen receptors that mediate bispecific activation and targeting of T cells. Although the present disclosure refers to the bispecificity of CARs, in some embodiments, CARs can target three, four, or more tumor antigens. Using CAR T cells to target multiple antigens may enhance T cell activation and / or counteract tumor escape due to antigen loss. TanCARs also target multiple expressed antigens, target various tumors using the same cell product with broad specificity, and / or provide a better toxicity profile with weaker signaling CARs that achieve the same results due to multiple specificities.

[0414] In some embodiments, the disclosure provides a TanCAR comprising two targeting domains. In some embodiments, the disclosure provides a multispecific TanCAR comprising three or more targeting domains. In another embodiment, the disclosure provides a first CAR and a second CAR on a cell surface, each CAR comprising an antigen binding domain, the antigen binding domain of the first CAR binds to a first tumor antigen (e.g., CD19, CD20, CD22, HER2) and the antigen binding domain of the second CAR binds to another (different) tumor antigen. TanCARs are disclosed in US2016 / 0303230(A1) and US2017 / 0 No. 340705(A1), which is incorporated herein by reference.

[0415] In some embodiments, the TanCARs of the present disclosure target two or more tumor antigens. Exemplary tumor antigens include one or more of CD19, CD20, CD22, k light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2, ErbB3 / 4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL-13 receptor alpha 2, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CALX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-alpha, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, TEM1, and / or TEM8.

[0416] In some embodiments, the present disclosure provides a bispecific TanCAR that targets CD19 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets CD22 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets HER2 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets IL13R-alpha2 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets VEGF-A and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets Tem8 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets FAP and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets EphA2 and another tumor antigen. In some embodiments, the present disclosure provides bispecific TanCARs that target one or more, two or more, three or more, or four or more of the following tumor antigens: CD19, CD22, HER2, IL13R-alpha2, VEGF-A, Tem8, FAP, or EphA2, and any combination thereof. In some embodiments, the present disclosure provides bispecific TanCARs that target HER2 and IL13R-alpha2. In some embodiments, the present disclosure provides bispecific TanCARs that target CD19 and CD22.

[0417] Methods for generating chimeric antigen receptors and CAR effector cells In some embodiments, the subject's effector cells (e.g., T cells) are genetically modified with a chimeric antigen receptor (Sadelain et al., Cancer Discov. 3:388-398, 2013). For example, effector cells (e.g., T cells) are provided, and a recombinant nucleic acid encoding a chimeric antigen receptor is introduced into the patient-derived effector cells (e.g., T cells) to generate a CAR cell. In some embodiments, effector cells (e.g., T cells) that are not derived from the subject are genetically modified with a chimeric antigen receptor. For example, in some embodiments, the effector cells (e.g., T cells) are allogeneic cells that have been engineered to be used as an "off-the-shelf" adoptive cell therapy, such as the universal chimeric antigen receptor T cells (UCART) developed by Cellectis. UCARTs are allogeneic CAR T cells that have been engineered to be used to treat the maximum number of patients with a particular carcinoma. Non-limiting examples of UCARTs under development by Cellectis include those targeting the tumor antigens CD19, CD123, CD22, CS1, and CD38.

[0418] Any of the nucleic acids or expression vectors disclosed herein can be introduced into effector cells (e.g., T cells) using a variety of different methods known in the art. Non-Limiting Examples of Methods for Introducing Nucleic Acids into Effector Cells (e.g., T Cells) These include lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, phototransfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene guns, magnetofection, viral transfection, and nucleofection. Additionally, CRISPR / Cas9 genome editing technology known in the art can be used to introduce CAR nucleic acids into effector cells (e.g., T cells) and / or introduce other genetic modifications (e.g., as described below) into effector cells (e.g., T cells) to enhance CAR cell activity (see, e.g., US9,890,393; US9,855,297; US2017 / 0175128; US2016 / 0184362; US2016 / 0272999; WO2015 / 161276; WO2014 / 191128; CN106755088; CN106591363; CN106480097; CN106399375; CN104894068 for the use of CRISPR / Cas9 technology in connection with CAR cells).

[0419] Provided herein are methods that can be used to generate any of the cells or compositions described herein, each cell capable of expressing a CAR (e.g., any of the CARs described herein).

[0420] A chimeric antigen receptor (CAR) includes an antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain that includes a cytoplasmic sequence of a CD3ζ sequence sufficient to stimulate a T cell when the antigen-binding domain binds to an antigen, and optionally a cytoplasmic sequence of one or more (e.g., 2, 3, or 4) costimulatory proteins (e.g., one or more cytoplasmic sequences of B7-H3, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD40L, CD80, CD160, CD244, ICOS, LAG3, LFA-1, LIGHT, NKG2C, 4-1BB, OX40, PD-1, PD-L1, TIM3, and a ligand that specifically binds to CD83) that costimulate the T cell when the antigen-binding domain binds to an antigen. In some embodiments, the CAR can further include a linker. Non-limiting aspects and functions of the CAR are described below. Additional aspects of CARs and CAR cells, including exemplary antigen binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described in, e.g., Kakarla et al., Cancer J. 20:151-155, 2014; Srivastava et al., Trends Immunol. 36:494-502, 2015; Nishio et al. al.,Oncoimmunology 4(2):e988098,2015;Ghorashian et al.,Br.J.Haematol.169:463-478,2015;Levine,Cancer Gene Ther.22:79-84,2015;Jensen et al.,Curr.Opin.Immunol.33:9-15,2015;Singh et al.,Cancer Gene Ther.22:95-100,2015;Li et al.,Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1753-1756,2014;Gill et al. al.,Immunol.Rev.263:68-89,2015;Magee et al.,Discov.Med.18:265-271,2014;Gargett et al.,Front.Pharmacol.5:235,2014;Yuan et al.,Zhongguo Xu Yan 22:1137-1141,2014;Pedgram et al.,Cancer J.20:127-133,2014;Eshhar et al.,Cancer J.20:123-126,2014;Ramos et al.,Canc er J.20:112-118,2014;Maus et al.,Blood 123:2625-2635,2014;Jena et al.,Curr.Hematol.Malig.Rep.9:50-56,2014;Maher et al.,Curr.43;Riches:301-Ther. al.,Discov.Med.16:295-302,2013;Cheadle et al.,Immunol.Rev.257:83-90,2014;Davila et al.,Int.J.Hematol.99:361-371,2014;Xu et al.,Cancer Lett.343:172-178,2014;Kochenderfer et al.,Nat.Rev.Clin.Oncol.10:267-276,2013;Hosing et al.,Curr.Hematol.Malig.Rep.8:60-70,2013;Hombach et al.,Curr.Mol.Med.13:1079-1088,2013;Xu et al.,Leuk.Lymphoma 54:255-260,2013;Gilham et al.,Trends Mol.Med.18:377-384,2012;Lipowska-Bhalla et al.,Cancer Immunol.Immunother.61:953-962,20...

Claims

1. 1. An immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein, (i) an immunomodulatory domain, (ii) a metal hydroxide binding peptide comprising at least one kinase targeting motif of a secretory pathway kinase, the kinase comprising at least one phosphorylated amino acid; and (iii) an immunomodulatory fusion protein, optionally including a stabilization domain; and (b) a metal hydroxide; An immunomodulatory fusion protein-metal hydroxide complex, wherein the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange via the at least one phosphorylated amino acid of the metal hydroxide binding peptide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

2. 2. The immunomodulatory fusion protein-metal hydroxide complex of claim 1, wherein said at least one kinase target motif of said metal hydroxide binding peptide comprises an amino acid sequence that is phosphorylated by a kinase selected from the group consisting of Fam20C, protein kinase A, cAMP-dependent protein kinase, cyclin-dependent kinase, extracellular regulated kinase-2, casein kinase 1, casein kinase 2, glycogen synthase kinase-3, calmodulin-dependent protein kinase-2, Abelson murine leukemia virus tyrosine kinase, Rous sarcoma virus tyrosine kinase, insulin receptor tyrosine kinase, protein kinase B, protein kinase D, proviral integration site kinases 1-3, AMP-activated protein kinase, mitogen-activated protein kinase, or NimA-related kinase.

3. 3. The immunomodulatory fusion protein-metal hydroxide complex of claim 2, wherein said at least one kinase target motif of said metal hydroxide binding peptide comprises an amino acid sequence that is phosphorylated by Fam20C.

4. 4. The immunomodulatory fusion protein-metal hydroxide complex of claim 2 or 3, wherein said at least one kinase target motif of said metal hydroxide binding peptide comprises phosphoserine, phosphotyrosine, or phosphothreonine.

5. 5. The immunomodulatory fusion protein-metal hydroxide complex of claim 4, wherein said at least one kinase targeting motif of said metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of S-X-E, S-X-pS, or S-X-Q-X-X-D-E, where X is any amino acid.

6. 1. An immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein, (i) an immunomodulatory domain, (ii) a metal hydroxide binding peptide comprising at least one kinase targeting motif of said secretory pathway kinase Fam20C comprising the amino acid sequence S-X-E; and (iii) an immunomodulatory fusion protein, optionally including a stabilization domain; and (b) a metal hydroxide; An immunomodulatory fusion protein-metal hydroxide complex is provided, wherein the at least one kinase target motif of the metal hydroxide binding peptide comprises a phosphoserine, and the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange via the at least one phosphoserine of the metal hydroxide binding peptide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

7. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 6, wherein the metal hydroxide-binding peptide is operably linked, optionally via a linker, to either the N-terminus or C-terminus of the immunomodulatory domain.

8. 7. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 6, comprising an immunomodulatory domain, a stabilizing domain, and a metal hydroxide binding peptide, wherein the stabilizing domain is operably linked, optionally via an amino acid linker, to either the N-terminus or C-terminus of the immunomodulatory domain, and the metal hydroxide binding peptide is operably linked, optionally via a linker, to either the immunomodulatory domain or the stabilizing domain at its terminus.

9. 7. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 6, comprising an immunomodulatory domain, a stabilizing domain, and a metal hydroxide-binding peptide, wherein the metal hydroxide-binding peptide is operably linked, optionally via an amino acid linker, to either the N-terminus or the C-terminus of the immunomodulatory domain, and the stabilizing domain is operably linked, optionally via a linker, to either terminus of the metal hydroxide-binding peptide or the immunomodulatory domain.

10. 10. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 9, wherein the metal hydroxide-binding peptide comprises about 3-6, about 6-15, about 10-25, or about 10-50 amino acids.

11. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 10, wherein the metal hydroxide binding peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more kinase target motifs comprising phosphorylated amino acids.

12. The immunomodulatory fusion protein-metal hydroxide complex of claim 11, wherein said kinase target motif comprises a phosphorylated amino acid that is phosphoserine.

13. 13. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 12, wherein the metal hydroxide binding peptide comprises two or more kinase target motifs of secretory pathway kinases, the two or more kinase target motifs comprising the same or different amino acid sequences, and optionally the two or more kinase target motifs are separated by a peptide linker.

14. 14. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 13, wherein said at least one kinase target motif comprises the amino acid sequence S-X-E, where X is any amino acid, and serine is phosphorylated.

15. 15. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein said metal hydroxide-binding peptide comprises at least one, two, or three kinase targeting motifs, optionally wherein said kinase targeting motifs are contiguous.

16. The immunomodulatory fusion protein-metal hydroxide complex of claim 14 or 15, wherein X is selected from E, S, V, H, Q, and G.

17. 17. The immunomodulatory fusion protein-metal hydroxide complex of claim 16, wherein X is E.

18. 18. The immunomodulatory fusion protein-metal hydroxide complex of claim 17, wherein the metal hydroxide-binding peptide comprises an amino acid sequence selected from SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, and SEQ ID NO:

125.

19. 15. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein the metal hydroxide binding peptide comprises the amino acid sequence XXSXEXX (SEQ ID NO: 127) or XXSEEXX (SEQ ID NO: 128), where X is any amino acid.

20. The metal hydroxide binding peptide has the amino acid sequence Xaa １ -Xaa ２ -S-Xaa ３ -E-Xaa ４ -Xaa ５ (SEQ ID NO: 127), １ is F, M, or G; ２ is Q, E, or G; ３ is E, S, V, H, Q, and G; ４ is Q, S, or G; ５ is Q, N, or G.

21. Xaa ３ The immunomodulatory fusion protein-metal hydroxide complex of claim 20, wherein

22. Xaa １ is F, and Xaa ２ The immunomodulatory fusion protein-metal hydroxide complex of claim 20 or 21, wherein

23. Xaa １ is M, and Xaa ２ The immunomodulatory fusion protein-metal hydroxide complex of claim 20 or 21, wherein is E.

24. Xaa １ is G; ２ The immunomodulatory fusion protein-metal hydroxide complex of claim 20 or 21, wherein

25. Xaa ４ is Q; ５ The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 20 to 24, wherein:

26. Xaa ４ is E, and Xaa ５ The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 20 to 24, wherein is S.

27. Xaa ４ is G; ５ The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 20 to 24, wherein is G.

28. 21. The immunomodulatory fusion protein-metal hydroxide complex of claim 20, wherein the metal hydroxide binding peptide comprises the amino acid sequence FQSEEQQ (SEQ ID NO: 129), MESEESN (SEQ ID NO: 130), or GGSEEGG (SEQ ID NO: 131).

29. The metal hydroxide binding peptide has the amino acid sequence Xaa １ -Xaa ２ -S-Xaa ３ -E-Xaa ４ -Xaa ５ -[L]-S-Xaa ３ -E-Xaa ６ -Xaa ７ (SEQ ID NO: 133), １ is F, M or G, and Xaa ２ is Q, E, or G; ３ is E, S, V, H, Q, and G; ４ is Q, S, or G; ５ is Q, N, or G; ５ is G; ６ The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein: is G; and L is a peptide linker, optionally a G / S linker, optionally GGGS.

30. The metal hydroxide binding peptide comprises a sequence of linked amino acids comprising the formula [A]x, where A is an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and x is a linked amino acid represented by A. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein x is an integer value representing the number of amino acid sequences, and x is 1 to 4.

31. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein the metal hydroxide-binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[B], where A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:

131.

32. The metal hydroxide-binding peptide has the formula ([A]-[B]): Ｘ wherein A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[B], and x is 1 to 4.

33. 15. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein the metal hydroxide binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[L]-[A], where A is an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

34. The metal hydroxide binding peptide has the formula ([A]-[L]-[A]) ｘ wherein A is an amino acid sequence selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131; and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[L]-[A], where x is 1 to 4, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

35. 15. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein the metal hydroxide binding peptide comprises a sequence of linked amino acids comprising the formula [A]-[L]-[B], where A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

36. The metal hydroxide binding peptide has the formula ([A]-[L]-[B]): ｘ wherein A and B are the same or different amino acid sequences selected from the group consisting of SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, and x is an integer with a value indicating the number of linked amino acid sequences represented by [A]-[L]-[B], where x is 1 to 4, and L is a peptide linker, optionally a G / S linker, optionally GGGS.

37. 15. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:

101.

38. The metal hydroxide-binding peptide has the formula [C] ｘ wherein C is an amino acid sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101; and x is an integer having a value indicating the number of linked amino acid sequences represented by C, and x is 1 to 4. Immunoregulatory fusion protein-metal hydroxide complexes.

39. The metal hydroxide-binding peptide has the formula [C] ｘ -[D] ｙ wherein C and D are the same or different amino acid sequences, C and D are selected from the group consisting of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101, x is an integer with a value representing the number of linked amino acid sequences represented by C, y is an integer with a value representing the number of linked amino acid sequences represented by D, x is 1 to 4, y is 1 to 4, and x and y are the same or different.

40. 15. The immunomodulatory fusion protein-metal hydroxide complex of claim 14, wherein the metal hydroxide binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:

115.

41. 41. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 40, wherein the immunomodulatory fusion protein comprises a metal hydroxide binding peptide comprising about 1-5, 1-10, 1-15, 1-20 phosphoserine residues, and the immunomodulatory fusion protein is adsorbed to the metal hydroxide by ligand exchange of the phosphoserine residues.

42. 1. An immunomodulatory fusion protein-metal hydroxide complex comprising: (a) an immunomodulatory fusion protein, (i) an immunomodulatory domain, optionally linked to a stabilization domain; (ii) an immunomodulatory fusion protein comprising a terminal metal hydroxide binding peptide comprising one or more hydroxyl substituents coupled by a protein reactive moiety, optionally via a linker; (b) a metal hydroxide; An immunomodulatory fusion protein-metal hydroxide complex is provided, wherein the immunomodulatory fusion protein is adsorbed to a metal hydroxide by ligand exchange via the at least one hydroxyl substituent of the metal hydroxide-binding peptide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

43. 43. The immunomodulatory fusion protein-metal hydroxide complex of claim 42, wherein said protein reactive moiety comprises a sulfhydryl reactive moiety, optionally, said sulfhydryl reactive moiety is a maleimide.

44. 43. The immunomodulatory fusion protein-metal hydroxide complex of claim 42, wherein the protein-reactive moiety comprises a sortase recognition motif.

45. 45. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 42-44, wherein the metal hydroxide binding peptide comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more hydroxyl substituents.

46. 46. ​​The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 42-45, wherein the hydroxyl substituent is selected from the group consisting of a fluoride group, a citrate group, a phosphate group, a carbonate group, and a sulfate group, optionally wherein the hydroxyl substituent is a phosphate group.

47. The hydroxyl substituent comprises at least one phosphorylated amino acid residue, and optionally the phosphorylated amino acid residue is selected from the group consisting of phosphoserine, phosphotyrosine, and phosphothreo.

47. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 42 to 46, wherein the phosphorylated amino acid residue is selected from the group consisting of phosphoserine, phosphoaminoserine, and phosphoaminosparin.

48. 46. ​​The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 45, wherein the metal hydroxide is selected from aluminum hydroxide, aluminum phosphate, calcium hydroxide, calcium phosphate, iron hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, zinc hydroxide, and zirconium hydroxide, optionally wherein the metal hydroxide is aluminum hydroxide (alum).

49. 1. An immunomodulatory fusion protein comprising: (a) an immunomodulatory domain; (b) a metal hydroxide binding peptide comprising at least one kinase target motif of the secretory pathway kinase Fam20C comprising the amino acid sequence S-X-E, where X is any amino acid, and optionally X is E, S, V, H, Q, or G; (c) optionally, a stabilization domain; The present invention provides an immunomodulatory fusion protein, wherein the at least one kinase targeting motif of the metal hydroxide binding peptide comprises a serine modified with a phosphate group, and the immunomodulatory fusion protein undergoes ligand exchange with alum via the at least one phosphoserine of the metal hydroxide binding peptide, thereby coupling the immunomodulatory fusion protein to alum and forming an immunomodulatory fusion protein-metal hydroxide complex.

50. 1. An immunomodulatory fusion protein comprising: (a) an immunomodulatory domain, optionally linked to a stabilization domain, and (b) a metal hydroxide-binding peptide comprising one or more phosphorylated amino acids coupled by a protein-reactive moiety, optionally via a linker; An immunomodulatory fusion protein, wherein the immunomodulatory fusion protein undergoes ligand exchange with alum via the at least one hydroxyl substituent of the metal hydroxide binding peptide, thereby coupling the immunomodulatory fusion protein to alum and forming an immunomodulatory fusion protein-metal hydroxide complex.

51. 51. The immunomodulatory fusion protein of claim 50, comprising an immunomodulatory domain and a metal hydroxide-binding peptide, wherein the metal hydroxide-binding peptide is coupled to the N-terminus or C-terminus of the immunomodulatory domain by a protein-reactive moiety.

52. 51. The immunomodulatory fusion protein of claim 50, comprising an immunomodulatory domain, a stabilizing domain, and a metal hydroxide-binding peptide, wherein the stabilizing domain is operably linked to either the N-terminus or C-terminus of the immunomodulatory domain, optionally via an amino acid linker, and the metal hydroxide-binding peptide is coupled to a terminus of the immunomodulatory domain or the stabilizing domain by a protein-reactive moiety.

53. 1. A method for increasing phosphorylation of an immunomodulatory fusion protein, comprising: (a) Immunomodulatory domain, Metal hydroxide binding peptides comprising one or more kinase targeting motifs; a nucleotide sequence encoding an immunomodulatory fusion protein, optionally including a stabilization domain; and (b) ER targeting leader sequence, operably linked thereto a kinase domain, operably linked thereto contacting the cell with a nucleotide sequence encoding a kinase comprising an anchor peptide; The method, wherein the kinase is localized in the secretory pathway and the one or more kinase target motifs of the metal hydroxide binding peptide are phosphorylated by the kinase in the secretory pathway, thereby increasing phosphorylation of the immunomodulatory fusion protein.

54. 54. The method of claim 53, wherein the kinase comprises an ER targeting leader sequence that directs the kinase to the secretory pathway, and optionally, the kinase comprises a kinase domain selected from the group consisting of protein kinase A, cAMP-dependent protein kinase, cyclin-dependent kinase, extracellular regulated kinase-2, casein kinase 1, casein kinase 2, glycogen synthase kinase-3, calmodulin-dependent protein kinase-2, Abelson murine leukemia virus tyrosine kinase, Rous sarcoma virus tyrosine kinase, insulin receptor tyrosine kinase, protein kinase B, protein kinase D, proviral integration site kinases 1-3, AMP-activated protein kinase, mitogen-activated protein kinase, or NimA-related kinase.

55. 55. The method of claim 53 or 54, wherein the kinase comprises Fam20C, wherein Fam20C comprises the amino acid sequence set forth in SEQ ID NO:

135.

56. 56. The method of any one of claims 53 to 55, wherein the kinase comprises an anchor peptide that inhibits secretion of the kinase, optionally wherein the anchor peptide comprises the amino acid sequence KDEL or HDEL.

57. 57. The method of any one of claims 53 to 56, wherein the cell is contacted with an expression vector comprising a nucleic acid encoding the immunomodulatory fusion protein.

58. 58. The method of any one of claims 53 to 57, wherein the cell is contacted with an expression vector comprising a nucleic acid encoding the kinase.

59. 59. The method of any one of claims 53 to 58, wherein the cell is contacted with an expression vector comprising a nucleic acid encoding the kinase and a nucleic acid encoding the immunoregulatory fusion protein.

60. 1. A method for increasing phosphorylation of an immunomodulatory fusion protein, comprising: (i) an immunomodulatory domain, (ii) a metal hydroxide binding peptide comprising one or more kinase target motifs of the secretory pathway kinase Fam20C; (iii) optionally, a stabilization domain; The method comprises contacting a cell with an expression vector comprising a nucleic acid encoding the immunomodulatory fusion protein and an expression vector comprising a nucleic acid encoding the secretory pathway kinase Fam20C operably linked to an anchor peptide, wherein the secretory pathway kinase Fam20C is localized to the secretory pathway by the anchor peptide and the one or more kinase target motifs are phosphorylated by Fam20C within the secretory pathway, thereby increasing phosphorylation of the immunomodulatory fusion protein.

61. 61. The method of any one of claims 53 to 60, comprising maintaining said cells under conditions that allow expression of said immunomodulatory fusion protein.

62. 62. The method of claim 61, further comprising isolating the immunomodulatory fusion protein.

63. 61. An immunomodulatory fusion protein produced by the method of any one of claims 51-60, comprising at least one phosphorylated amino acid, wherein the immunomodulatory fusion protein is adsorbed by ligand exchange with alum via the at least one phosphorylated amino acid, thereby coupling the immunomodulatory fusion protein to alum and forming an immunomodulatory fusion protein-metal hydroxide complex.

64. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 48, or the immunomodulatory fusion protein of any one of claims 49 to 52 and 63, wherein the immunomodulatory domain comprises a polypeptide that activates, enhances or promotes a response by an immune cell.

65. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 48, or the immunomodulatory fusion protein of any one of claims 49 to 52 and 63, wherein the immunomodulatory domain comprises a polypeptide that inhibits, reduces or suppresses a response by an immune cell.

66. 66. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 64 or 65, wherein the immune cell is a lymphoid cell selected from an innate lymphoid cell, a T cell, a B cell, an NK cell, and combinations thereof.

67. 67. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of any one of claims 64 to 66, wherein the immune cell is a myeloid cell selected from a monocyte, a neutrophil, a granulocyte, a mast cell, a macrophage, a dendritic cell, and combinations thereof.

68. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of any one of claims 64 to 67, wherein the response by the immune cells comprises cytokine production, antibody production, production of antigen-specific immune cells, increased effector function and / or cytotoxicity, and combinations thereof.

69. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-68, or the immunomodulatory fusion protein of any one of claims 49-52 and 63-68, wherein the immunomodulatory domain comprises one or more selected from a cytokine, a chemokine, an activating ligand / receptor, an inhibitory ligand / receptor, or a combination thereof.

70. 70. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 69, wherein the immunomodulatory domain comprises one or more cytokines.

71. 71. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 70, wherein the cytokine is 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, and combinations thereof.

72. 72. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 71, wherein the cytokine is IL-2.

73. 72. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 71, wherein the cytokine is IL-15 / IL15RA.

74. 71. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 70, wherein the cytokine is 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, and combinations thereof.

75. 75. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 74, wherein the cytokine is a single chain fusion of IL-12(p35) / IL-12(p40).

76. 71. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 70, wherein the cytokine is a human IL-1 family member selected from IL-1, IL-18, IL-33, and combinations thereof.

77. 77. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 76, wherein the cytokine is IL-18.

78. 71. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 70, wherein the cytokine is selected from TNFα, INFα, IFN-γ, GM-CSF, FLT3L, G-CSF, M-CSF, and combinations thereof.

79. 70. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 69, wherein the immunomodulatory domain comprises one or more chemokines.

80. 80. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 79, wherein the chemokine is selected from LIF, MIP-2, MIP-1α, MIP-1β, CXCL1, CXCL9, CXCL10, MCP-1, eotaxin, RANTES, LIX, and combinations thereof.

81. 80. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 79, wherein the chemokine is selected from CCL3, CCL4, CCL5, eotaxin, and combinations thereof.

82. 70. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 69, wherein the immunomodulatory domain comprises one or more activating ligands / receptors.

83. 83. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 82, wherein the activating ligand / receptor is selected from the TNF superfamily, the CD28 receptor superfamily, the B7 ligand family, and a T cell receptor.

84. 83. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 82, wherein the activating ligand / receptor is a TNF superfamily ligand selected from TNF-alpha, CD40L, 4-1BBL, OX40, and combinations thereof.

85. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 83, wherein the activating ligand / receptor is a TNF superfamily receptor and the immunomodulatory domain comprises 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.

86. 84. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 83, wherein the activating ligand / receptor is a CD28 superfamily member or a B7 family member selected from ICOS ligand, CD80, and CD86, and combinations thereof.

87. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 83, wherein said activating ligand / receptor is a CD28 superfamily member and said immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from an anti-ICOS antibody and an anti-CD28 antibody.

88. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 83, wherein the activating ligand / receptor is a T cell receptor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-CD3 gamma antibody, an anti-CD3 delta antibody, an anti-CD3 zeta antibody, and an anti-CD3 epsilon antibody.

89. 70. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 69, wherein the immunomodulatory domain comprises one or more inhibitory ligands / receptors.

90. 90. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 89, wherein the inhibitory ligand / receptor is selected from the CD28 receptor superfamily, the TNF superfamily, and checkpoint inhibitors.

91. 91. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 90, wherein the inhibitory ligand / receptor is a CD28 superfamily member and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, and an anti-CTLA4 antibody.

92. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 90, wherein the inhibitory ligand / receptor is a TNF superfamily member and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-TIGIT antibody and an anti-BTLA antibody.

93. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 92, wherein the inhibitory ligand / receptor is a TNF superfamily member and the immunomodulatory domain comprises an antibody or antigen-binding fragment that is an anti-TIGIT antibody.

94. The immunomodulatory fusion protein-metal hydroxide complex or immunomodulatory fusion protein of claim 90, wherein the inhibitory ligand / receptor is a checkpoint inhibitor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof 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.

95. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 48 and 64 to 94, or the immunomodulatory fusion protein of any one of claims 49 to 52 and 63 to 94, comprising a stabilization domain, said stabilization domain comprising human serum albumin or a fragment thereof.

96. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1 to 48 and 64 to 92, or the immunomodulatory fusion protein of any one of claims 49 to 52 and 63 to 94, comprising a stabilization domain, said stabilization domain comprising an Fc domain or a mutant Fc domain with reduced FcR interaction.

97. The immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-96, or the immunomodulatory fusion protein of any one of claims 49-52 and 63-96, wherein the immunomodulatory fusion protein-metal hydroxide complex is of sufficient mass to reduce size-dependent diffusion away from an injection site upon administration in vivo.

98. A pharmaceutical composition comprising an immunomodulatory fusion protein-metal hydroxide complex according to any one of claims 1 to 48 and 64 to 96, or an immunomodulatory fusion protein according to any one of claims 49 to 52 and 63 to 97, and a pharma- ceutically acceptable carrier.

99. A nucleic acid comprising a nucleotide sequence encoding an immunomodulatory fusion protein according to any one of claims 49 and 62 to 97.

100. 100. An expression vector comprising the nucleic acid of claim 99.

101. A cell transformed with the expression vector described in claim 100.

102. 102. A method for producing an immunomodulatory fusion protein, comprising maintaining a cell of claim 101 under conditions that allow expression of said immunomodulatory fusion protein.

103. 103. The method of claim 102, further comprising obtaining the immunomodulatory fusion protein and adsorbing the immunomodulatory fusion protein to a metal hydroxide, thereby forming an immunomodulatory fusion protein-metal hydroxide complex.

104. 10. A method for activating, enhancing or promoting an immune cell mediated response in a subject, comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98.

105. 10. A method for inhibiting, reducing or suppressing an immune cell mediated response in a subject, comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-95, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98.

106. 106. The method of claim 104 or 105, wherein the immune cell is a lymphoid cell selected from an innate lymphoid cell, a T cell, a B cell, a NK cell, and combinations thereof.

107. 106. The method of claim 104 or 105, wherein the immune cells are myeloid cells selected from monocytes, neutrophils, granulocytes, mast cells, macrophages, dendritic cells, and combinations thereof.

108. 107. The method of any one of claims 104 to 106, wherein the response by the immune cells comprises cytokine production, antibody production, production of antigen-specific immune cells, increased effector function and / or cytotoxicity, and combinations thereof.

109. The method of any one of claims 104 to 107, wherein the response by the immune cells occurs in a tumor microenvironment.

110. 10. A method for reducing or inhibiting tumor growth comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98.

111. 10. A method for treating cancer in a subject, comprising administering to a subject in need of cancer treatment an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98.

112. 112. The method of claim 110 or 111, wherein an anti-tumor immune response is induced in the subject following administration of the immunomodulatory fusion protein-metal hydroxide complex, the immunomodulatory fusion protein, or the pharmaceutical composition.

113. 113. The method of any one of claims 108-112, wherein the immunomodulatory fusion protein-metal hydroxide complex, the immunomodulatory fusion protein, or the pharmaceutical composition is administered intratumorally.

114. 100. A kit comprising a container containing an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, or an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition of claim 98, for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, and a package insert containing instructions for administering said fusion protein or pharmaceutical composition.

115. 100. A kit comprising a container containing an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, or an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition of claim 98, for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, and a package insert containing instructions for administering the antibody or pharmaceutical composition alone or in combination with another agent.

116. 13. Use of an immunomodulatory fusion protein-metal hydroxide complex according to any one of claims 1 to 48 and 64 to 97, or an immunomodulatory fusion protein according to any one of claims 49 to 52 and 63 to 97, and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition according to claim 98, for the manufacture of a medicament for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

117. An immunomodulatory fusion protein-metal hydroxide complex according to any one of claims 1 to 48 and 64 to 97, or an immunomodulatory fusion protein according to any one of claims 49 to 52 and 63 to 97, and optionally a pharma- ceutically acceptable carrier, or a pharmaceutical composition according to claim 98, in the manufacture of a medicament for treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof.

118. An immunomodulatory fusion protein-metal hydroxide complex according to any one of claims 1 to 48 and 64 to 97, or an immunomodulatory fusion protein according to any one of claims 49 to 52 and 63 to 97, and optionally a pharma- ceutically acceptable salt thereof, for use as a medicament. or the pharmaceutical composition of claim 98.

119. 106. A method for reducing or inhibiting tumor growth or treating cancer in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98, and an effective amount of a second composition comprising a tumor antigen-targeting antibody or antigen-binding fragment thereof, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

120. 120. The method of claim 119, wherein the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen.

121. The method of claim 119 or 120, wherein the tumor antigen-targeting antibody specifically binds to human HER-2 / neu, EGFR, VEGFR, CD20, CD33, or CD38.

122. 106. A method for reducing or inhibiting tumor growth or treating cancer in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98, and an effective amount of a second composition comprising a cancer vaccine, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

123. 123. The method of claim 122, wherein the cancer vaccine is a population of cells immunized in vitro with a tumor antigen and administered to the subject.

124. 124. The method of claim 123, wherein the cancer vaccine is a peptide comprising one or more tumor-associated antigens.

125. The method of claim 123, wherein the cancer vaccine is an amphipathic peptide conjugate comprising a tumor-associated antigen, a lipid, and optionally a linker, and the amphipathic peptide conjugate binds to albumin under physiological conditions.

126. 126. The method of any one of claims 122-125, wherein the cancer vaccine further comprises an adjuvant.

127. 106. A method for reducing or inhibiting tumor growth or treating cancer in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98, and an effective amount of a second composition comprising an immune checkpoint inhibitor, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

128. 128. The method of claim 127, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof that binds to PD-1, PD-L1, CTLA-4, LAG3, or TIM3.

129. 106. A method for reducing or inhibiting tumor growth or treating cancer in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein-metal hydroxide complex of any one of claims 1-48 and 64-97, an immunomodulatory fusion protein of any one of claims 49-52 and 63-97, or a pharmaceutical composition of claim 98, and an effective amount of a second composition comprising adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

130. 130. The method of claim 129, wherein the adoptive cell therapy comprises immune effector cells comprising a chimeric antigen receptor (CAR) molecule that binds to a tumor antigen.

131. The method of claim 129 or 130, 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.

132. 132. The method of claim 131 , wherein the antigen binding domain binds to the tumor antigen associated with the disease.

133. 132. The method of claim 131, wherein the tumor antigen is selected from CD19, EGFR, Her2 / neu, CD30, and BCMA.

134. 134. The method of any one of claims 130 to 133, wherein the immune effector cell is a T cell, such as a CD8+ T cell.

135. The method of any one of claims 130 to 133, wherein the immune effector cells are natural killer (NK) cells.

136. 136. The method of any one of claims 104-113 and 118-135, wherein the immunomodulatory fusion protein-metal hydroxide complex, the immunomodulatory fusion protein, or the pharmaceutical composition is administered intratumorally.

137. 137. The method of any one of claims 119 to 136, wherein the immunomodulatory fusion protein-metal hydroxide complex, the immunomodulatory fusion protein or the pharmaceutical composition, and the second composition are administered simultaneously or sequentially.

138. 137. The method of any one of claims 104-113 and 118-136, comprising administering a plurality of immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins or pharmaceutical compositions, wherein the immunomodulatory domains are different.

139. The method of claim 138, wherein the immunomodulatory domains are different cytokines.

140. 140. The method of claim 138 or 139, wherein the multiple immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins or pharmaceutical compositions are formulated together.

141. 140. The method of claim 138 or 139, wherein the multiple immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins or pharmaceutical compositions are formulated separately.

142. 142. The method of claim 141, wherein the multiple immunomodulatory fusion protein-metal hydroxide complexes, immunomodulatory fusion proteins or pharmaceutical compositions are administered simultaneously or sequentially.

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