Methods and compositions comprising engineered il-7 and il-12 polypeptides for treating cancer

Abstract

The dual therapy involving tumor stroma-binding IL-7 and IL-12 variants led to synergistic antitumor efficacy through suppression of immune exhaustion and promotion of immune memory without compromised tolerability. The disclosure describes a polypeptide comprising an IL-7 polypeptide, or a functional fragment thereof, linked to a extracellular matrix (ECM)-affinity domain. Also described is a nucleic acid encoding a polypeptide of the disclosure and cells comprising the polypeptide(s) or nucleic acid(s) of the disclosure.

Description

METHODS AND COMPOSITIONS COMPRISING ENGINEERED IL-7 AND IL-12 POLYPEPTIDES FOR TREATING CANCER

[0001] This application claims priority of U.S. Provisional Application No. 63 / 507,780 filed June 13, 2023, which is hereby incorporated by reference in its entirety.BACKGROUNDII. Field of the Invention

[0002] The invention generally relates to the field of medicine. More particularly, it concerns compositions and methods involving nucleotide constructs and proteins for the treatment of cancer.III. Background

[0003] Immunotherapy has become one of the dominant pillars of modem cancer care, and combination immunotherapy is a promising approach to promote antitumor immunity by activating complementary pathways (1-3). For example, combining checkpoint inhibitor (CPI) antibodies blocking the PD-1 (e.g., with nivolumab) and CTLA-4 (e.g, ipilimumab) axes increases the overall survival of melanoma patients when compared to blockade with either agent alone (4-6). However, a major limitation of combination immunotherapies is that the rate of immune-related adverse events (irAEs) also increases when compared to the monotherapy regimen (7-9). In the same trial examining the combination of nivolumab and ipilimumab, 55%-60% of patients experienced Grade 3 or 4 adverse events, while monotherapy led to 10%- 20% experiencing Grade 3 or 4 adverse events. A similar observation was made in another trial examining IL- 12 in combination with IL-2 (10). Thus, the selection of agents that enhance clinical outcome without exacerbating toxicity is highly desired.INCORPORATION OF SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 13, 2024, is named ARCDP0803WO.xml and is 50,520 bytes in size.SUMMARY OF INVENTION

[0005] The dual therapy involving tumor stroma-binding IL-7 and IL- 12 variants led to synergistic antitumor efficacy through suppression of immune exhaustion and promotion of immune memory without compromised tolerability. The disclosure describes a polypeptidecomprising an IL-7 polypeptide, or a functional fragment thereof, linked to a extracellular matrix (ECM)-affinity domain. Also described is a nucleic acid encoding a polypeptide of the disclosure and cells comprising the polypeptide(s) or nucleic acid(s) of the disclosure. Methods relate to a method of making a polypeptide comprising expressing a nucleic acid of the disclosure in a cell. Also described is a method of making a cell comprising transferring a nucleic acid of the disclosure into a cell. Also provided are compositions comprising nucleic acids, cells, or polypeptides of the disclosure. Also described is a composition comprising i) IL- 12 polypeptide(s) conjugated to the collagen binding domain of SEQ ID NO:6 and ii) an IL-7 polypeptide conjugated to the collagen binding domain of SEQ ID NO:6. Methods also include treating cancer in a subject comprising administering a polypeptide or composition described herein.

[0006] The ECM-affinity domain may be further described as a collagen binding domain (CBD). The polypeptide may comprise or exclude a CBD from decorin or von Willebrand factor (VWF). The ECM-affinity domain may comprise or exclude a peptide from placenta growth factor-2 (P1GF-2) or CXCL-12y. The ECM-affinity domain may comprise a peptide that has the amino acid sequence of one of SEQ ID NOs:l-19. The ECM-affinity domain may comprise a peptide that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to the amino acid sequence of one of SEQ ID NOs: 1- 19. The ECM-affinity domain may comprise or exclude a peptide having the amino acid sequence of SEQ ID NO:6.

[0007] The term “functional fragment” as used herein refers to a fragment of the protein or polypeptide that can bind to and / or activate it’s endogenous binding proteins, such as a receptor or effector proteins. The functional fragments of the disclosure include or exclude those that bind to the receptor, such as the IL-7 or IL- 12 receptor. The IL- 12 functional fragment may be or may exclude one that can stimulate the growth and function of T cells. The IL- 12 functional fragment may be or may exclude one that stimulates the production of interferon-gamma and / or tumor necrosis factor alpha. The IL- 12 functional fragment may be or may exclude one that reduces IL-4 mediated suppression of IFN-gamma. The IL- 12 functional fragment may be or may exclude one that binds to the IL- 12 receptor and activates signal transduction upon binding to the IL- 12 receptor. The IL-7 functional fragment may be or may exclude one that binds to the IL-7 receptor and activates signal transduction upon binding to the IL-7 receptor.

[0008] The ECM-affinity domain may be carboxy-proximal to the IL-7 polypeptide or functional fragment thereof. The ECM-affinity domain may be linked, through a peptide bond,to the carboxy terminus of the IL-7 polypeptide. The ECM-affinity domain may be amino- proximal to the IL-7 polypeptide or functional fragment thereof. The ECM-affinity domain may be linked, through a peptide bond, to the amino terminus of the IL-7 polypeptide. The ECM-affinity domain may be amino-proximal to the p40 polypeptide or functional fragment thereof. The ECM-affinity domain may be amino-proximal to the p35 polypeptide or functional fragment thereof. The ECM-affinity domain may be carboxy-proximal to the p35 polypeptide or functional fragment thereof. The ECM-affinity domain may be carboxy-proximal to the p40 polypeptide or functional fragment thereof. The composition may comprise a p35 polypeptide and an ECM-affinity domain amino-proximal to the p35 polypeptide. The composition may comprise a p40 polypeptide and an ECM-affinity domain carboxy-proximal to the p40 polypeptide. The ECM-affinity domain may be linked, through a peptide bond to the carboxy terminus of the p40 and / or p35 polypeptide. A first region is carboxy-proximal to a second region when the first region is attached to the carboxy terminus of the second region. There may be further intervening amino acid residues between the first and second regions. Thus, the regions need not be immediately adjacent, unless specifically specified as not having intervening amino acid residues. The term “amino-proximal” is similarly defined in that a first region is amino-proximal to a second region when the first region is attached to the amino terminus of the second region. Similarly, there may be further intervening amino acid residues between the first and second regions unless stated otherwise.

[0009] The polypeptide may further comprise or may exclude a serum protein operatively linked to the IL-7 polypeptide or ECM-affinity domain. The polypeptide comprising the p35 polypeptide, a functional fragment of p35, p40 polypeptide, and / or a functional fragment of p40, linked to a ECM-affinity domain may further comprise or may exclude a serum protein operatively linked to the p40 and / or p35 polypeptide or ECM-affinity domain. The serum protein may be operatively linked, through a peptide bond, to the amino or carboxy terminus of the polypeptide comprising the p40 and / or p35 polypeptide, or a functional fragment of p40 and / or p35, linked to a ECM-affinity domain. The serum protein may be operatively linked, through a peptide bond, to the amino or carboxy terminus of the IL-7 polypeptide. The serum protein may comprise albumin. The albumin may comprise or exclude the amino acid sequence one of SEQ ID NOs:20-22. The albumin protein may comprise an amino acid sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to one of SEQ ID NOs:20-22. The IL-7 polypeptide may comprise or exclude the amino acid sequence of SEQ ID NO:23 or 24, a polypeptidehaving an amino acid sequence with at least 80% sequence identity to SEQ ID NO:23 or 24, or a polypeptide comprising a fragment of SEQ ID NO:23 or 24. The polypeptide may comprise or exclude the amino acid sequence of SEQ ID NO:27 or 28 or a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:27 or 28.

[0010] The ECM-affinity domain and / or serum protein may be linked to the IL-7 polypeptide, IL- 12 polypeptide, p35 polypeptide, p40 polypeptide, or fragments thereof through a linker. The linker may comprise or exclude a glycine serine linker. The linker may comprise (GGGS)n, wherein n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The linker may comprise or consist of or exclude SEQ ID NO:32. The linker may be composed of only glycine and serine residues (a glycine-serine linker). The linker may be a flexible linker or may exclude a flexible linker. Exemplary flexible linkers include or exclude glycine polymers (G)n, glycineserine polymers (including, for example, (GS)n, (GSGGS)n - SEQ ID NO:33, (GGGGS)n - SEQ ID NO:34 and (GGGS)n - SEQ ID NO:35, where n is an integer of at least one. n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art and may be used as a linker in the polypeptides of the disclosure. Exemplary linkers can comprise or consist of GGSG (SEQ ID NO:36), GGSGG (SEQ ID NO:37), GSGSG (SEQ ID NO:38), GSGGG (SEQ ID NO:39), GGGSG (SEQ ID NO:40), GSSSG (SEQ ID NO:41), and the like. Also included are linkers comprising or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeated units of any one of SEQ ID NOS:36-41. Further linkers useful in the polypeptides of the disclosure are described herein.

[0011] The ECM-affinity domain and / or serum may be are linked to the IL-7 polypeptide through a peptide bond. The ECM-affinity domain and / or serum protein may be linked to the p35 and / or p40 polypeptide through a peptide bond. The polypeptide may comprise or further comprise or may exclude a molecular tag. The molecular tag may comprise or exclude SEQ ID NOG 1 or 42. The ratio of ECM-affinity domain to IL-7 is about 1 : 1 to 5: 1. The ratio of ECM- affinity domain to IL-7, IL-12, p35, or p40 may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 to about 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10, or any derivable range therein. The molar ratio or ratio of mg, ug, ng, or pg of IL-7 to mg, ug, ng, or pg IL- 12, p35, and / or p40 may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 to about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10, or any derivable range therein.

[0012] Methods of the disclosure may further comprise or exclude isolating polypeptide from the cell. Compositions of the disclosure may comprise or further comprise a polypeptide comprising an IL- 12 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain. The ECM-affinity domain linked to the IL- 12 polypeptide or functional fragment thereof may be further defined as a CBD (collagen binding domain). The IL- 12 polypeptide may comprise or exclude a p35 polypeptide, or a functional fragment thereof, and / or a p40 polypeptide, or a functional fragment thereof. The p35 polypeptide may be linked to an ECM- affinity domain. The p40 polypeptide may be linked to an ECM-affinity domain.

[0013] The composition may be formulated for intravenous administration. The composition may be formulated for intratumoral or peritumoral administration.

[0014] The p35 polypeptide may comprise or exclude the amino acid sequence of SEQ ID NO:25 or 26, a polypeptide with an amino acid sequence that has or has at least 80% sequence identity to SEQ ID NO:25 or 26, or a polypeptide with an amino acid sequence that comprises a fragment of SEQ ID NO:25 or 26. The p40 polypeptide may comprise or exclude the amino acid sequence of SEQ ID NO:43 or 44, a polypeptide with an amino acid sequence that has or has at least 80% sequence identity to SEQ ID NO:43 or 44, or a polypeptide with an amino acid sequence that comprises a fragment of SEQ ID NO:43 or 44. The p35 polypeptide may comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO:25 or 26. The p40 polypeptide may comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO:43 or 44. The ECM-affinity domain linked to the p35 polypeptide may comprise or exclude the amino acid sequence of SEQ ID NO:29 or 30 or a polypeptide with an amino acid sequence that has or has at least 80% sequence identity to SEQ ID NO:29 or 30. The ECM-affinity domain linked to the p40 polypeptide may comprise or exclude the amino acid sequence of SEQ ID NO:45 or 46 or a polypeptide with an amino acid sequence that has or has at least 80% sequence identity to SEQ ID NO:45 or 46. The ECM-affinity domain linked to the p35 polypeptide may comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO:29 or 30, or a fragment thereof. The ECM-affinity domain linked to the p40 polypeptide may comprise an amino acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity (or any derivable range therein) to SEQ ID NO:45 or 46, or a fragment thereof. The ECM-affinity domain and / or serum protein may be linked to the p35 and / or p40 polypeptide through a peptide bond.

[0015] The p35 polypeptide may be a human p35 polypeptide or derived from the human p35 gene. The p40 polypeptide may be a human p40 polypeptide or derived from the human p40 gene. The p35 polypeptide may be a mouse p35 polypeptide or derived from the mouse p35 gene. The p40 polypeptide may be a mouse p40 polypeptide or derived from the mouse p40 gene. The IL-7 polypeptide may be a human IL-7 polypeptide or derived from the human IL-7 gene. The IL-7 polypeptide may be a mouse IL-7 polypeptide or derived from the mouse IL-7 gene.

[0016] The methods may exclude administration of IL- 12. The subject may be administered a dose of 60 pg / kg or less of IL-7. The subject may be administered a dose of, a dose of at most, or a dose of at least 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100 pg / kg, mg / kg, ng / kg, pg, mg, g, or ng (or any derivable range therein) of IL-7, IL- 12, p35, and / or p40 polypeptide. The methods may comprise or further comprise or may exclude administration a polypeptide comprising an IL- 12 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain. The methods may comprise or exclude administration of a p35 polypeptide linked to an ECM-affinity domain. The methods may comprise or exclude administration of a p40 polypeptide linked to an ECM-affinity domain.

[0017] The cancer may comprise or exclude melanoma, colon cancer, or breast cancer. The cancer may comprise or exclude metastatic cancer and / or the subject may be one that has been diagnosed with metastatic cancer. The subject may be one that has not been diagnosed with metastatic cancer or one that is has not been . The breast cancer may be further defined as or exclude triple negative breast cancer (TNBC). The subject may be one that has been determined to be resistant and / or non-responsive to a prior therapy. The subject may exclude one that has been determined to be resistant and / or non-responsive to a prior therapy. The cancer may be one that is determined to be or is known in the art to be resistant to a prior therapy. The cancer may exclude one that is determined to be or is known in the art to be resistant to a prior therapy. The prior therapy may comprise or exclude a nucleotide analog. The nucleotide analog may comprise or exclude 6-thioguanine. The prior therapy may comprise or exclude immune checkpoint inhibitor (ICI) therapy. The method may comprise or exclude administration of an additional agent. The subject may be one that has been administered or has been prescribed administration of an additional agent or exclude one that has been administered or has been prescribed administration of an additional agent. The additional agent may comprise or exclude an immunotherapy. The immunotherapy may comprise or exclude ICI therapy. The ICI therapy may comprise or exclude a monotherapy or a combination ICI therapy. The subject may be one that is receiving ICI monotherapy or ICI combination therapy. The term ICI monotherapy refers to a subject receiving only one of: an inhibitor of PD-1, PDL1, PDL2, CTLA-4, B7-1, and B7-2 and excludes subjects receiving more than one of: an inhibitor of PD-1, PDL1, PDL2, CTLA-4, B7-1, and B7-2. The ICI therapy may comprise or exclude an anti-PD-1 monoclonal antibody and / or an anti-CTLA-4 monoclonal antibody. The ICI therapy may comprise or exclude one or more of nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, pembrolizumab, pidilizumab, ipilimumab or tremelimumab.

[0018] The composition or polypeptide may be administered systemically. The composition or polypeptide may be administered intratumorally or peritumorally. Systemic, intraumoral, and / or peritumoral may be excluded as routes of administration in the methods described herein. The composition or polypeptide may be administered intravenously. The methods may exclude intravenous administration. The cancer may comprise or exclude stage I, II, III, and / orIV cancer or wherein the subject has or has been diagnosed as having stage I, II, III, or IV cancer. The subject may exclude one that has or has been diagnosed as having stage I, II, III, and / or IV cancer. The cancer may comprise or exclude metastatic cancer. The cancer may comprise or exclude recurrent cancer. The subject may be one that has or has been diagnosed as having metastatic and / or recurrent cancer. The subject may exclude one that has or has been diagnosed as having metastatic and / or recurrent cancer. The method may comprise or exclude reducing T cell exhaustion, inhibiting metastasis, reducing relapse, increasing survival, reducing tumor size, reducing toxicity, and / or reducing or inhibiting immune related adverse events (irAEs). The method may comprise or exclude administration of a polypeptide comprising an IL-7 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain and a polypeptide comprising an IL- 12 polypeptide, or a functional fragment thereof, linked to an ECM-affinity domain, and wherein the administered dose of one or both polypeptides is less than one half of the effective dose of a monotherapy, wherein the monotherapy comprises the administration of only one of the polypeptides.

[0019] The term “cytokine polypeptide” as used herein refers to a polypeptide, which is cytokine or a receptor binding domain thereof and retains at a portion of cytokine activity.

[0020] The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product comprising a polymer of amino acids.

[0021] The terms “subject,” “mammal,” and “patient” are used interchangeably. The subject may be a mammal. The subject may comprise or exclude a human. The subject may comprise or exclude a mouse, rat, rabbit, dog, donkey, or a laboratory test animal such as fruit fly, zebrafish, etc. The subject may comprise or exclude a non-human primate.

[0022] It is contemplated that the methods and compositions include exclusion of any of the embodiments described herein.

[0023] The references to the methods of treatment by therapy or surgery or in vivo diagnosis methods in example 1 of this description and in the claims and disclosure of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods.

[0024] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0025] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0026] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0027] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0028] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0029] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0030] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0032] FIG. 1A-1E. In vitro characterization of IL-7-CBD, and intravenously administrated CBD-IL- 12 and IL-7-CBD combination therapy exhibit synergistic antitumor effect and reduce toxicity compared to high dose of CBD-IL-12 (10 pg). A. Molecular schematic illustration indicating the fusion of CBD to the C-terminal of recombinant IL-7. B. Dose-response of phosphorylated STAT5 with mouse IL-7 and IL-7-CBD fusion protein in CD3+ T cells. C. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and treated with either PBS (i.v., n=10), 33.3 pmol IL- 12 + 666 pmol IL-7 (i.v., n=10), 33.3 pmol CBD-IL-12 (i.v., n=10) or with 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD (i.v., n=10) on day 7, 13, 19, and 25. Survival curves (C) are shown. D. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and treated with either PBS (i.v., n=8), 33.3 pmol CBD-IL-12 (i.v., n=8), 166.5 pmol CBD-IL-12 (i.v., n=8), or with 33.3 pmol CBD-IL-12 + 1332 pmol IL-7-CBD (i.v., n=8) on day 9. Survival curves (D) are shown. E. C57BL / 6 mice were administrated with PBS (i.v., n=8), 33.3 pmol CBD-IL-12 (i.v., n=8), 166.5 pmol CBD-IL-12 (i.v., n=8), or 33.3 pmol CBD-IL-12 + 1332 pmol IL-7-CBD (i.v., n=8), and blood was collected on day 2 for cytokine expression level analysis (IFNy, IL-6, and TNFa), on day 3 for blood chemistry analysis (AST, ALT, Amylase, and total bilirubin), and on day 4 for circulating blood cell count (white blood cells, red blood cells, and hemoglobin). Overall survival rates complied for two independent experiments, and statistical analyses were performed using log-rank (Mantel-Cox) tests. Blood toxicity analysis complied for multiple comparisons, and statistical analyses were performed using one-way ANOVA tests. (*: 0.05>P, **: 0.01>P, and ***: 0.001>P)

[0033] FIG. 2A-2I. IL-7-CBD synergizes with CBD-IL-12 to antagonize CD8+ T cell exhaustion and promote CD8+ effector T cells, but does not alter CD8+ T cell infiltration, and CBD-IL-12 and IL- 7-CBD combination therapy induces intratumoral inflammation. B16F10 bearing mice were treated with either PBS (i.t., n=7), 33.3 pmol CBD-IL-12, 333 pmol IL-7- CBD (i.t., n=7), or 33.3 pmol CBD-IL-12 + 333 pmol IL-7-CBD (i.t., n=7) on day 7 and tumors were harvested day 13. Cells were digested into a single cell suspension, stained, and run by flow cytometry. A. Overall counts of CD45+ and CD8+ T Cells. B. UMAP of concatenatedCD8+ T cells with FlowSom clustering displayed as an overlay. C. UMAP displaying individual groups as labelled with clusters overlaid. D. Stacked bar graph displaying the percentage of each group per cluster. E. Heatmap of FlowSom clusters. F. Expression of KERG1 and Tox for each FlowSom cluster. G. Percent population of each cluster for each sample. The tumors were homogenized for protein extraction, and H. cytokine or chemokine expression levels were quantified using Eegendplex and normalized by total protein content. I. The tumors were harvested after day 6 of the injection and performed with a paraffin embedding process for CD8, PD-1, and EAG-3 fluorescence staining. The fluorescence imaging was conducted using a STEEEARIS 8 confocal microscope (Eecia Microsystems). The tumors were homogenized for protein extraction, and cytokines or chemokines expression levels were quantified using Eegendplex and normalized by total protein content. Statistical analyses were performed using one-way ANOVA tests. (*: 0.05>P, **: 0.01>P, and ***: 0.001>P)

[0034] FIG. 3A-3G. Intratumorally administered CBD-IE-12 and IE-7-CBD combination therapy inhibits lung metastasis of B16F10 and B16F10 rechallenge via an induced systemic tumor- specific immune response. A, B. C57BE / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0. The B16F10 bearing mice were administrated with 3 x 105B16F10 melanoma intravenously on day 7 and treated with either PBS (i.t., n=10), 33.3 pmol CBD-IE-12 (i.t., n=10) or with 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD (i.t., n=10) on day 7, 13, and 19. The mice lung were harvested for measurement of metastasis nodules on day 25. Designed experiments schedules (A), lung metastasis nodules, weight data, and histological images (B) are shown. C, D. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and treated with either 166.5 pmol CBD-IL-12 (i.t.), or with 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD (i.t.) on day 7, 13, 19, and 25. After 60 days of the melanoma inoculation, tumor- free mice were rechallenged with 2 x 105B16F10 melanoma intradermally. Designed experimental schedule (C) and overall survival rate (D) are shown. E. The tumor-free mice due to 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD combination therapeutic schedule, were rechallenged with 2 x 105B16F10 melanoma and injected with either 300 pg IgG isotype (i.p., n=7), 300 pg mouse CD8 antibody (i.p., n=7), 300 pg mouse CD4 antibody (i.p., n=7), or 300 pg mouse IFNy antibody (i.p., n=7) on day -1, 2, 5, and 8. The overall survival rate (E) is shown. F, G. The tumor-free mice due to 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD combination therapeutic schedule, were rechallenged with 2 x 105B16F10 and the mice blood was collected on the scheduled time point on day 0, 3, and 7 for quantification of the melanoma antigen (TRP2) specific CD8+ T cells in the circulatinglymphocyte. The primary tumor-draining lymph nodes were harvested on day 11 for quantification of the melanoma antigen (TRP2) specific central memory (CD62L+ and CD44+) CD8+ T cells. The population of TRP2-specific CD8+ T cells in the blood (F) is shown and the population of TRP2-specific central memory CD8+ T cells in the tumor draining lymph nodes (G) is shown. Overall survival rates were compiled for two independent experiments, and statistical analyses were performed using log-rank (Mantel-Cox) tests. Melanoma lung metastasis nodules and TRP2-specific CD8+ T cells analysis was compiled for multiple comparisons, and statistical analyses were performed using one- way ANOVA tests or t-test. (*P < 0.05, **P < 0.01, and ***P < 0.001)

[0035] FIG. 4A-4D. IL-7-CBD + CBD-IL-12 combination therapy synergizes with CPI and effectively suppresses tumor growth even for the tumors with low objective responsive rate to aPD-1 immunotherapy such as 4T1 triple-negative breast cancer and inducible Bral'V600l7Ptcn-Z7PCATSTAmelanoma models. A. Balb / C mice were inoculated with 5 x 1054T1 breast cancer cells on mammary pad and treated with either PBS (i.t., n=10), 100 pg CPI (i.p., n=10), 33.3 CBD-IL-12 (i.t., n=10), 666 pmol IL-7-CBD (i.t., n=10), 33.3 CBD-IL-12 + 666 pmol IL-7- CBD (i.t., n=10), or 100 pg CPI + 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD (i.t., n=10) on day 7, 13, 19, and 25. The tumor growth curve and overall survival rate are shown (A). B, C, D. To demonstrate the superiority of intravenous IL7- CBD + CBD-IL-12 combination therapy, Braf^^ / Pten^ / pCAT8mice were applied 50 pg of 4-OH- tamoxifen on the back skin and treated with either 100 pg CPI (i.p., n=6), 100 pg CPI (i.p.) + 33.3 pmol CBD-IL-12 (i.v., n=8), or 100 pg CPI (i.p.) + 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD (i.v., n=8). The tumor size growth curve overall survival rate (B), the photograph of melanoma induced mice (C), and body weight exchange graph of therapeutic approach mice (D) are shown. Mice body weight analysis on day 53 was compiled for multiple comparisons, and statistical analyses were performed using one-way ANOVA tests. (*P < 0.05, **P < 0.01, and ***P < 0.001)

[0036] FIG. 5A-5B. Biophysical characterization of IL-7 and IL-7-CBD. A. SDS-PAGE for IL- 7 and IL-7-CBD under a non-reducing condition. As expected, IL-7 shows -16.0 kDa and IL-7-CBD shows 37.1 kDa size. B. Collagen-binding affinity of IL-7-CBD to collagen I.

[0037] FIG. 6. Therapeutic evaluation of intravenously administered IL-7-CBD monotherapy in MC38 model. C57BL / 6 mice were inoculated with 5 x 105MC38 cancer cells subcutaneously and injected with 100 pL PBS (n=10), 1.3 nmol IL-7 (i.v., n=10), or 1.3 nmol IL-7- CBD (i.v., n=10) on day 7 and 13.

[0038] FIG. 7. Intravenously administered CBD-fused cytokines show a stronger overall antitumor effect than unmodified cytokines in B16F10 model. C57BL / 6 mice were inoculatedwith 5 x 105B16F10 melanoma intradermally and injected with 30 pL PBS (n=10), 33.3 pmol CBD-IL-12 (i.v., n=10), 33.3 pmol IL- 12 (i.v.) + 666 pmol IL-7 (i.v., n=10), or 33.3 pmol CBD-IL-12 (i.v.) + 666 pmol IL-7-CBD (i.v., n=10) on day 7, 13, 19, and 25.

[0039] FIG. 8. Addition of IL-7-CBD to CBD-IL-12 significantly increases therapeutic scores in B16F10 model. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 100 pL PBS (n=8), 33.3 pmol CBD-IL-12 (i.v., n=8), 166.5 pmol CBD-IL-12 (i.v., n=8), or 1.3 nmol IL-7-CBD (i.v.) + 33.3 pmol CBD-IL-12 (i.v., n=8) on day 9.

[0040] FIG. 9. Escalating the dose of IL-7-CBD combined with CBD-IL-12 increased the overall antitumor effect depending on IL-7-CBD dose. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 100 pL PBS (n=8), 33.3 pmol CBD- IL-12 (i.v., n=8), 333 pmol IL-7-CBD (i.v.) + CBD-IL-12 (i.v., n=8), 666 pmol IL-7-CBD (i.v.) + 33.3 pmol CBD-IL-12 (i.v., n=8), or 1.3 nmol IL-7-CBD (i.v.) + 33.3 pmol CBD-IL-12 (i.v., n=8) on day 7.

[0041] FIG. 10A-10B. Blood toxicology study of IL- 12, IL- 15, and IL-7. C57BL / 6 mice were injected with 83.3 pmol IL-12 (i.v., n=5), 83.3 pmol IL-12 (i.v.) + 375 pmol IL-15 superagonist (i.v., n=5), or 83.3 pmol IL-12 (i.v.) +333 pmol IL-7 (i.v., n=5) and the mice blood was collected on the scheduled timepoints for toxicity tests. A. Systemic inflammation cytokines or chemokines (IFNy, CXCL9, and CXCL10) expression level and B. Blood chemistry assay (ALT, AST, Total bilirubin, BUN, and Creatine kinase) on day 3. Statistical analyses were performed using one-way ANOVA tests or t- test.

[0042] FIG. 11. Blood inflammatory cytokine and chemokine expression levels simulated by CBD-fused cytokines or unmodified cytokines. C57BL / 6 mice were injected with 100 pL PBS (i.v., n=5), 33.3 pmol IL-12 (i.v.) + 666 pmol IL-7 (i.v., n=5), or 33.3 pmol CBD-IL-12 (i.v.) + 666 pmol IL-7-CBD (i.v., n=5) and the mice blood was collected on the scheduled timepoints for the analysis of inflammatory molecules such as IFNy, IL-6, TNF-a, CXCL9, and CXCL10.

[0043] FIG. 12. Systemic toxicity induced by CBD-fused cytokines or unmodified cytokines. C57BL / 6 mice were injected with 100 pL PBS (i.v., n=5), 33.3 pmol IL-12 (i.v.) + 666 pmol IL-7 (i.v., n=5), or 33.3 pmol CBD-IL-12 (i.v.) + 666 pmol IL-7-CBD (i.v., n=5) and the mice blood was collected on the scheduled timepoints for the blood chemistry analysis such as AST, ALT, Amylase, Total bilirubin, albumin, BUN, Creatinine, and Total protein.

[0044] FIG. 13. The organ histology in CBD-fused cytokines or unmodified cytokines administered mice. C57BL / 6 mice were injected with 100 pL PBS (i.v., n=5), 33.3 pmol IL-12 (i.v.) + 666 pmol IL-7 (i.v., n=5), or 33.3 pmol CBD-IL-12 (i.v.) + 666 pmol IL-7-CBD (i.v., n=5) and the major organs (heart, liver, lung, spleen, and kidney) in the mice were harvested on day 4 for immunohistochemistry of mouse CD8. The organs were incubated in 2% PFA for 2 days at 4 oC, and the paraffin embedding, section, and H&E staining process were performed at the Human Tissue Resource Center, The University of Chicago. The scale bar is 400 pm.

[0045] FIG. 14A-14C. CBD-fused cytokines recruit fewer CD8+ T cells into the hepatic region compared to unmodified cytokines. C57BL / 6 mice were injected with 100 pL PBS (i.v., n=5), 33.3 pmol IL- 12 (i.v.) + 666 pmol IL-7 (i.v., n=5), or 33.3 pmol CBD-IL-12 (i.v.) + 666 pmol IL-7-CBD (i.v., n=5) and the major organs (heart, liver, lung, spleen, and kidney) in the mice were harvested on day 4 for immunohistochemistry of mouse CD8. The organs were incubated in 2% PFA for 2 days at 4 oC, and the paraffin embedding, section, and CD8 staining process were performed at the Human Tissue Resource Center, The University of Chicago. A. Representative images of hepatic recruited CD8+ T cells and B. Quantification data. C. CD8 staining images of heart, lung, kidney, and spleen. The scale bar is 400 pm. Statistical analyses were performed using one-way ANOVA tests. (*: 0.05>P, **: 0.01>P, and ***: 0.001>P)

[0046] FIG. 15. IL-7-CBD + CBD-IL-12 combination therapy significantly stimulated intratumoral IFN-y compared CBD-IL-12 monotherapy. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 30 pL PBS (i.t., n=7), 333 pmol IL- 7-CBD (i.t., n=7), 33.3 pmol CBD-IL-12 (i.t., n=7), or 333 pmol IL-7-CBD (i.t.) + 33.3 pmol CBD-IL-12 (i.t., n=10) on day 7. The tumors were harvested on day 13 for analysis of cytokine expression level.

[0047] FIG. 16. Intratumorally administered combination therapy show sufficient antitumor effect even with only immune cells in the circulating system. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 30 pL PBS (i.t., n=10) or 333 pmol IL-7-CBD (i.t.) + 33.3 pmol CBD-IL-12 (i.t., n=10) on day 7, 13, 19, and 25. we injected 25 pg FTY720 intraperitoneally daily from days 6 to 25 after B16F10 inoculation.

[0048] FIG. 17. Therapeutic evaluation of intratumorally administered CBD-fused cytokines in B16F10 model. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 30 pL PBS (i.t., n=10), 33.3 pmol IL-12 (i.t.) + 333 pmol IL-7 (i.t., n=10), or 33.3 pmol CBD-IL-12 (i.t.) + 333 pmol IL-7-CBD (i.t., n=10) on day 7, 13, 19, and 25.

[0049] FIG. 18. Synergistic effect of intratumorally administered CBD-fused cytokines in B16F10 model. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 30 pL PBS (i.t., n=10), 33.3 pmol CBD-IL-12 (i.t., n=10), 333 pmol IL-7- CBD (i.t., n=10), or 333 pmol IL-7-CBD (i.t.) + 33.3 pmol CBD-IL-12 (i.t., n=10) on day 7, 13, 19, and 25.

[0050] FIG. 19. Percent survival of intratumorally administered high-dose CBD-IL-12 (10 pg) and low-dose CBD-IL-12 + IL-7-CBD in B16F10 model. C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma intradermally and injected with 30 pL PBS (i.t., n=10), 166.5 pmol CBD-IL-12 (i.t., n=10), or 33.3 pmol CBD-IL-12 (i.t.) + 333 pmol IL-7-CBD (i.t, n=10) on day 7, 13, 19, and 25.

[0051] FIG. 20. Combination therapy has a synergistic effect with aPD-1, and IL-7-CBD + CBD-IL-12 + aPD-1 significantly increases therapeutic scores in poorly immunogenic and CPI- unresponsive 4T1 breast cancer model. Balb / C mice were inoculated with 5 x 1054T1 breast cancer cells into the mammary fat pad on day 0 and injected with 100 pg aPD-1 (i.p., n=10), 83.3 pmol CBD- IL-12 (i.t., n=10), 666 pmol IL-7-CBD (i.t., n=8), or 83.3 pmol CBD- IL-12 (i.t.) + 666 pmol IL-7-CBD (i.t., n=10) on days 7, 13, 19, and 25.

[0052] FIG. 21. Combination therapy potently synergizes aPD-1, and IL-7-CBD + CBD- IL-12 + aPD-1 significantly increases therapeutic scores in poorly immunogenic and genetically engineered Bral'V600l7Ptcn- / 7pCATS I Amelanoma model. Brafv600E / Pten‘ / 7pCATSTAmice were applied 50 pg of 4-OH-tamoxifen on the back skin and treated with either 100 pg aPD-1 (i.p., n=6), 100 pg aPD-1 (i.p.) + 33.3 pmol CBD-IL-12 (i.v., n=8), or 100 pg aPD-1 (i.p.) + 33.3 pmol CBD-IL-12 + 666 pmol IL-7-CBD (i.v., n=8).

[0053] FIG. 22. Representative gating strategy for gating CD8 T cells in the tumor.

[0054] FIG. 23A-23F. Blood toxicology study of single-dose injection in B16F10-bearing mice. B16F10-bearing C57BL / 6 mice were injected with PBS (i.v., n=4), 33.3 pmol CBD-IL- 12 (i.v., n=4), 166.5 pmol CBD-IL-12 (i.v., n=4), or 1.3 nmol IL-7-CBD + 33.3 pmol CBD- IL-12 (i.v., n=4), and the blood was collected for the blood cell counts or blood chemistry analysis. A. Designed experiment schedule. B. Body weight was measured during the experiment schedule. C. Blood cell counts and D. Statistical analysis on day 3. E. Blood chemistry analysis and F. Statistical analysis on day 3. Statistical analyses were performed using one-way ANOVA tests or t-tests. (*: 0.05>P, **: 0.01>P, and ***:0.001>P).

[0055] FIG. 24A-24F. Blood toxicology study of multiple(dual)-dose injection in B 16F10- bearing mice. B16F10-bearing C57BL / 6 mice were injected with PBS (i.v., n=4), 33.3 pmol CBD-IL-12 (i.v., n=4), 666 pmol IL-7 -CBD (i.v., n=4), 666 pmol IL-7 -CBD + 33.3 pmol CBD-IL-12 (i.v., n=4) on day 0 and 6, and the blood was collected for the blood cell counts or blood chemistry analysis. A. Designed experiment schedule. B. Body weight was measured during the experiment schedule. C. Blood cell counts and D. Statistical analysis on day 3. E. Blood chemistry analysis and F. Statistical analysis on day 3. Statistical analyses were performed using one-way ANOVA tests or t-tests. (*: 0.05>P, **: 0.01>P, and ***:0.001>P).

[0056] FIG. 25A-25B. Pharmacokinetic study and tumor accumulation efficacy of IL-7- CBD and CBD-IL-12. B16F10-bearing C57BL / 6 mice were injected with 666 nmol IL-7 (i.v., n=3), 666 nmol IL-7-CBD (i.v., n=3), 166.5 pmol IL- 12 (i.v., n=3), or 166.5 pmol CBD-IL-12 (i.v., n=3). A. The blood was collected at the scheduled time points for the pharmacokinetics study and B. The tumors were harvested 24 hr after the injection for analysis of the cytokine concentration. Statistical analyses were performed using one-way ANOVA tests or t-tests. (*: 0.05>P, **: 0.01>P, and ***:0.001>P).

[0057] FIG. 26. Combination therapy reduces NK and NKT cell numbers compared to CBD-IL-12 monotherapy, but does not alter CD4+ T cell populations compared to CBD-IL-12 alone B16F10 bearing mice were treated i.t. with either PBS, 33.3 pmol CBD-IL-12, 333 pmol IL-7-CBD, or 33.3 pmol CBD-IL-12 + 333 pmol IL-7-CBD, on day 7 and tumors were harvested day 13. Cells were digested into a single cell suspension, stained, and run by flow cytometry. Overall counts of NK cells, NKT cells, Tregs, and conventional CD4+ T cells. Statistical analyses were performed using one-way ANOVA tests. (*: 0.05>P, **: 0.01>P, and ***: 0.001>P).

[0058] FIG. 27. The CBD fusion to IL-7 or IL- 12 prolongates the intratumoral retention of the cytokines. B16F10-bearing C57BL / 6 mice were injected with 666 nmol IL-7, 666 nmol IL- 7-CBD, 166.5 pmol IL- 12, or 166.5 pmol CBD-IL-12 intratumorally. The tumors were harvested 72 hr post intratumoral injection of the cytokines for analysis of the cytokine concentration. Statistical analyses were performed using one-way ANOVA tests or t-tests. (*: 0.05>P, **: 0.01>P, and ***:0.001>P).

[0059] FIG. 28A-28B. Dose-dependent phosphorylated STAT5 with mouse IL-7 and IL-7 receptor (CD 127) expression level in mouse CD8+ T cells or NK cells. Mouse CD8+ T cells or NK cells were isolated from the mouse spleen. A. The cells were treated with various concentrations of mouse IL-7 for evaluating STAT5 phosphorylation. B. The cell surface markers were stained for evaluating IL-7 receptor (CD 127) expression level. Statistical analyses were performed using one-way ANOVA tests or t-tests. (*: 0.05>P, **: 0.01>P, and ***:0.001>P).DETAILED DESCRIPTION

[0060] Identifying immunotherapy agents with complementary mechanisms of action that do not elicit an additive toxicity profile is challenging. In Example 1, a dual therapy involving tumor stroma-binding IL-7 and IL- 12 variants was used to guide the design of next-generation cytokine combinations, which led to synergistic antitumor efficacy without compromised tolerability. Systemic treatment with IL-7-CBD and CBD-IL-12 significantly improved the survival of poorly immunogenic B16E10 melanoma-bearing mice when compared to either agent in monotherapy. Additionally, treatment of the primary tumor with IL-7-CBD + CBD- IL-12 exhibited striking benefits when compared to CBD-IL-12 monotherapy in long-term anticancer immune memory, as evidenced by the rejection in rechallenge experiments. Supplementing CBD-IL-12 therapy with IL-7-CBD also demonstrated an advantage in a CBD- IL-12-resistant, immunosuppressive 4T1 breast cancer model, highlighting how the spectrum of treatable cancer types expands upon such dual therapy.I. ECM-affinity domains

[0061] Collagen is an extracellular matrix (ECM)-protein that regulates a variety of cellular biological functions, such as proliferation, differentiation, and adhesion in both normal and tumor tissue (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). Collagen is the most abundant protein in the mammalian body and exists in almost all tissues in one or more of 28 isoforms (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). The blood vessel sub-endothelial space is rich in collagen. Because of its insolubility under physiological conditions, collagen barely exists within the blood (Dubois et al., Blood 107:3902-06, 2006; Bergmeier and Hynes, Cold Spring Harb Perspect Biol 4:a005132, 2012). Tumor vasculature is reported to be permeable due to an abnormal structure (Nagy et al., British journal of cancer 100:865, 2009). Thus, with its leaky vasculature, collagen is exposed in the tumor (Liang et al., Journal of controlled release 209:101-109, 2015; Liang et al., Sci Rep 6:18205, 2016; Yasunaga et al., Bioconjugate Chemistry 22:1776-83, 2011; Xu et al. The Journal of cell biology 154:1069-80, 2001; Swartz and Lund, Nat Rev Cancer 12:210-19). Also, tumor tissue contains increased amounts of collagen compared to normal tissues (Zhou et al. J Cancer 8:1466-76, 2017; Provenzano et al. BMC Med 6:11, 2008).

[0062] von Willebrand factor (vWF) is a blood coagulation factor and binds to both type I and type III collagen, and the adhesion receptor GPIb on blood platelets (Lenting et al., Journal of thrombosis and haemostasis :JTH 10:2428-37, 2012; Shahidi Advances in experimental medicine and biology 906:285-306, 2017). When injured, collagen beneath endothelial cells isexposed to blood plasma, and vWF-collagen binding initiates the thrombosis cascade (Shahidi Advances in experimental medicine and biology 906:285-306, 2017; Wu et al. Blood 99:3623- 28, 2002). The vWF A domain has the highest affinity against collagen among reported non- bacterial origin proteins / peptides (Addi et al., Tissue Engineering Part B: Reviews, 2016). Particularly within the A domain, the A3 domain of vWF has been reported as a collagen binding domain (CBD) (Ribba et al. Thrombosis and Haemostasis 86:848-54, 2001). As described above, the inventors contemplated that a fusion protein with the vWF A3 CBD may achieve targeted cytokine immunotherapy even when injected systemically due to exposure of collagen via the leaky tumor vasculature.

[0063] The ECM-affinity domain may comprise or exclude a collagen binding domain from decorin. The ECM-affinity domain may comprise or exclude a decorin peptide such as LRELHLNNNC (SEQ ID NO:1), which is derived from bovine or LRELHLDNNC (SEQ ID NOG), which is derived from human.

[0064] The ECM -peptide may comprise or exclude a peptide fragment from human decorin, which is represented by the following amino acid sequence:CGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGL DKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVK LERLYLSKNQLKELPEKMPKTLQELRAHENEITKVRKVTFNGLNQMIVIELGTNPLKS SGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASLKGLNNL AKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNI SVVGSSDFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYK (SEQ ID NOG).

[0065] The ECM-peptide may comprise or exclude a peptide fragment from vWF. The ECM-peptide may comprise or exclude vWF Al derived from human sequence, residues 1237- 1458 (474-695 of mature VWF) or a fragment thereof, which is represented by the amino acid sequenceCQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEV LKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYA GSQVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKK VIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLC (SEQ ID NO:4).

[0066] The ECM-peptide may comprise or exclude all or a fragment of vWF A3, which is represented by the following amino acid sequences: CSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVIL VTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIED LPTMVTLGNSFLHKLCSGFVRICTG (SEQ ID N0:5) and CSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVP WNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVIL VTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIED LPTMVTLGNSFLHKLCSGFVRI (SEQ ID N0:6).

[0067] The ECM-peptide may comprise or exclude all or a fragment of vWF A3, which is represented by the following amino acid sequences: CSQPLDVVLLLDGSSSLPESSFDKMKSFAKAFISKANIGPHLTQVSVIQYGSINTIDVP WNVVQEKAHLQSLVDLMQQEGGPSQIGDALAFAVRYVTSQIHGARPGASKAVVIII MDTSLDPVDTAADAARSNRVAVFPVGVGDRYDEAQLRILAGPGASSNVVKLQQVE DLSTMATLGNSFFHKLCSGFSGV (SEQ ID NO:7).

[0068] The ECM-affinity domain may comprise or exclude a peptide from von Willebrand factor (vWF). The sequence of human vWF comprises the following: MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCS YLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYAS KGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDD FMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLL KSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYG WTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEG LCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKS FDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLV KLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLV KLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQ HSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECL CGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDE ECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDG FMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGC NTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSN PGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVES GRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVE EDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEP GGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERERISQKWVRVAVVEYHDGSHAYIGEKDRKRPSEERRIASQVKYAGSQV ASTSEVEKYTEFQIFSKIDRPEASRITEEEMASQEPQRMSRNFVRYVQGEKKKKVIVIP VGIGPHANEKQIREIEKQAPENKAFVESSVDEEEQQRDEIVSYECDEAPEAPPPTEPPDMAQVTVGPGEEGVSTEGPKRNSMVEDVAFVEEGSDKIGEADFNRSKEFMEEVIQRM DVGQDSIHVTVEQYSYMVTVEYPFSEAQSKGDIEQRVREIRYQGGNRTNTGEAERYE SDHSFEVSQGDREQAPNEVYMVTGNPASDEIKREPGDIQVVPIGVGPNANVQEEERIGWPNAPIEIQDFETEPREAPDEVEQRCCSGEGEQIPTESPAPDCSQPEDVIEEEDGSSSF PASYFDEMKSFAKAFISKANIGPRETQVSVEQYGSITTIDVPWNVVPEKAHEESEVDV MQREGGPSQIGDAEGFAVRYETSEMHGARPGASKAVVIEVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQERIEAGPAGDSNVVKEQRIEDEPTMVTEGNSFEHKECS GFVRICMDEDGNEKRPGDVWTEPDQCHTVTCQPDGQTEEKSHRVNCDRGERPSCPN SQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKETGSCSYVEFQNKEQDEEVIEHNGACSPGARQGCMKSIEVKHSAESVEEHSDMEVTVNGREVSVPYVGGNMEVNVYGAIMHEVRFNHEGHIFTFTPQNNEFQEQESPKTFASKTYGECGICDENGANDFMERDGTVTTDWKTEVQEWTVQRPGQTCQPIEEEQCEVPDSSHCQVEEEPEFAECHKVEAPATFYAICQQDSCHQEQVCEVIASYAHECRTNGVCVDWRTPDFCAMSCPPSEVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMEEGSCVPEEACTQCIGEDGVQHQFEEAWVPDHQPCQICTCESGRKVNCTTQPCPTAKAPTCGECEVARERQNADQCCPE YECVCDPVSCDEPPVPHCERGEQPTETNPGECRPNFTCACRKEECKRVSPPSCPPHRE PTERKTQCCDEYECACNCVNSTVSCPEGYEASTATNDCGCTTTTCEPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGERVAQCSQKPCEDSCRSGFTYVEHEGECCGRCEPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCEINECVRVKEEVFIQQRNVSC PQEEVPVCPSGFQESCKTSACCPSCRCERMEACMENGTVIGPGKTVMIDVCTTCRCM VQVGVISGFKEECRKTTCNPCPEGYKEENNTGECCGRCEPTACTIQERGGQIMTEKRDETEQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCEAEGGKIMKIPGTCCDTC EEPECNDITAREQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSP TRTEPMQVAEHCTNGSVVYHEVENAMECKCSPRKCSK (SEQ ID NO:8).

[0069] The peptide may comprise or exclude a peptide from the vWF A3 domain. The vWF A3 domain is derived from the human sequence, residues 1670-1874 (907-1111 of mature vWF) and has the following sequence:CSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQ VSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEM HGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGP AGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSG (SEQ ID N0:9).

[0070] The ECM-affinity domain may comprise or exclude a peptide from P1GF-2. P1GF- 2 has the following sequence:MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALE RLVDVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGD RPSYVELTFSQHVRCECRPLREKMKPERRRPKGRGKRRREKQRPTDCHLCGDAVPR R (SEQ ID NO: 10).

[0071] Exemplary P1GF-2 ECM affinity peptides include: RRRPKGRGKRRREKQRPTDCHLCGDAVPRR (SEQ ID NO: 11); RRRPKGRGKRRREKQRPTDCHL (SEQ ID NO: 12); RRPKGRGKRRREKQRPTD (SEQ ID NO: 13); RRRPKGRGKRRREKQ (SEQ ID NO: 14); GKRRREKQ (SEQ ID NO: 15); RRRPKGRG (SEQ ID NO: 16); and RRKTKGKRKRSRNSQTEEPHP (SEQ ID NO: 17).

[0072] The ECM-affinity domain may comprise or exclude a peptide from CXCL- 12y. The sequence of CXCL-12y is the following: CXCL-12y:KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKW IQEYLEKALNKGRREEKVGKKEKIGKKKRQKKRKAAQKRKN (SEQ ID NO: 18). An exemplary peptide includes all or part of SEQ ID NO: 18 and the following peptide: GRREEKVGKKEKIGKKKRQKKRKAAQKRKN (SEQ ID NO: 19).

[0073] The ECM-affinity domain may be a peptide with 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity (or any derivable range therein) to an ECM or CBD peptide or fragment of the peptides described above.

[0074] A linker sequence may be included in the polypeptides of the disclosure or may be exclude in polypeptides of the disclosure or may be excluded between two domains or regions in the polypeptides of the disclosure. For example, a linker having at least, at most, or exactly 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (or any derivable range therein) may separate the cytokine and the ECM-affinity peptide.

[0075] The ECM-affinity domains of the disclosure may comprise or exclude peptides that have affinity to one or more components of the extracellular matrix such as fibronectin,collagen, (collagen type I, collagen type III, and / or collagen type IV) tenascin C, fibrinogen, and fibrin. In certain aspects the ECM-affinity domain has an affinity for collagen. And in other aspects the ECM-affinity domain does not bind fibronectin.

[0076] In some embodiments, the ECM-affinity domains, IL-7, IL-12, and / or functional fragment thereof, of the disclosure is further linked to a serum protein. Serum proteins useful in the methods, compositions, and polypeptides of the disclosure include or exclude albumin, globulin, and fibrinogen. Globulins may include or exclude alpha 1 globulins, alpha 2 globulins, beta globulins, and gamma globulins. The albumin may comprise or exclude mouse, human, bovine, or any other homologous albumin protein. The albumin may comprise or exclude human serum albumin, which is encoded by the ALB gene, and exemplified by the following amino acid sequence:KWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC PFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCAS LQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRA DLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYA KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNR RPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:20). Serum albumin may comprise or exclude a polypeptide having the following sequence: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECC QAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEK PLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARR HPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL FEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETF TFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADD KETCFAEEGKKLVAASQAALGL (SEQ ID NO:21)

[0077] The albumin may comprise or exclude mouse albumin having the following sequence:EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVAD ESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLP PFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCA EADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPN ADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDK PLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRR HPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDL YEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVE DYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFT FHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADK DTCFSTEGPNLVTRCKDALA (SEQ ID NO:22).II. Proteinaceous Compositions

[0078] The polypeptides or polynucleotides of the disclosure, such as the ECM-affinity domain, serum protein, or cytokine polypeptide, may include or exclude 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, or 50 or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with at least, or at most 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, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,I I I, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs:l-46.

[0079] The polypeptides or polynucleotides of the disclosure, such as the ECM-affinity domain, serum protein, or cytokine polypeptide, may include or exclude 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, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or more contiguous amino acids, or any range derivable therein, of SEQ ID NO: 1-46.

[0080] In some embodiments, a polypeptide of the disclosure may comprise or exclude amino acids 1 to 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517,518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536,537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555,556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574,575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593,594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 (or any derivable range therein) of SEQ ID NOs:l-46.

[0081] In some embodiments, a polypeptide of the disclosure, such as the ECM-affinity domain, serum protein, or cytokine polypeptide, may comprise or exclude at least, at most, or exactly 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 650, 700, 750, 800,850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 (or any derivable range therein) contiguous amino acids of SEQ ID NOs:l-46.

[0082] In some embodiments, the polypeptide, such as the ECM-affinity domain, serum protein, or cytokine polypeptide, may comprise or exclude at least, at most, or exactly 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 (or any derivable range therein) contiguous amino acids of SEQ ID NOs:l-46 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with any one of SEQ ID NOS: 1-46.

[0083] A polypeptide of the disclosure, such as an ECM-affinity domain, serum protein, or cytokine polypeptide, may be at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 198%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous with one of SEQ ID NOS: 1-46.

[0084] The disclosure includes or excludes a nucleic acid molecule or polypeptide starting at position 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575,576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 650, 700, 750,800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 of any of SEQ ID NOs:l-46 and comprising 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 650, 700, 750, 800,850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 contiguous nucleotides or amino acids of any of SEQ ID NOs:l-46.

[0085] The polypeptides and nucleic acids of the disclosure may include or exclude at least, at most, or exactly 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 substitutions (or any range derivable therein).

[0086] The substitution may be or may exclude an amino acid position or nucleic acid position 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,or 615 (or combinations thereof) of one of SEQ ID NO: 1-46. One or more of these substitutions may be specifically excluded from an embodiment.

[0087] The amino acid at position 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 , 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 , 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 , 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 , 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 , 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 , 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, or 650 of one of SEQ ID NOs:l-46 may be substituted with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0088] Peptides, polypeptides, and proteins of the disclosure, such as the ECM-affinity domain, serum protein, or cytokine polypeptide, having at least, having at least, or having 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% identity to any one of SEQ ID NO: 1-46 includes a fragment or segment starting at amino acid 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, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120 , 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547,548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642,643, 644, 645, 646, 647, 648, 649, or 650 (or any range derivable therein) and ending at amino acid 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, or 650 (or any range derivable therein) of one of SEQ ID NOs:l-46.

[0089] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Nonconservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. One or more of these substitutions may be specifically excluded from an embodiment.

[0090] Proteins may be recombinant, or synthesized in vitro. Alternatively, a nonrecombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.

[0091] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0092] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either of the 5' or 3' portions of the coding region.

[0093] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.

[0094] In other embodiments, alteration of the function of a polypeptide is intended by introducing one or more substitutions. For example, certain amino acids may be substituted for other amino acids in a protein structure with the intent to modify the interactive binding capacity of interaction components. Structures such as, for example, protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to alter such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with different properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes with appreciable alteration of their biological utility or activity.

[0095] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0096] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0097] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0098] In specific embodiments, all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide or polypeptide is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

[0099] One embodiment includes the use of gene transfer to cells, including microorganisms, for the production and / or presentation of proteins. The gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions. A nucleic acid encoding virtually any polypeptide may be employed. The generation of recombinant expression vectors, and the elements included therein, are discussed herein. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell used for protein production.III. Nucleic Acids

[0100] In certain embodiments, the current disclosure concerns recombinant polynucleotides encoding the proteins, polypeptides, and peptides of the invention, such as ECM-affinity domain operatively linked to IL-7, IL- 12, and / or other molecules. Therefore, certain embodiments relate to nucleotides encoding for an ECM-affinity polypeptide and / or an ECM-affinity polypeptide or fragment thereof fused to IL-7, IL- 12, or a functional fragment thereof.

[0101] As used in this application, the term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid.Included within the term “polynucleotide” are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0102] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence of: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more nucleotides, nucleosides, or base pairs (or any range derivable therein), including all values and ranges there between, of a polynucleotide encoding one or more amino acid sequence described or referenced herein. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0103] In particular embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide or peptide of the disclosure. The term “recombinant” may be used in conjunction with a polynucleotide or polypeptide and generally refers to a polypeptide or polynucleotide produced and / or manipulated in vitro or that is a replication product of such a molecule.

[0104] In other embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide or peptide of the disclosure.

[0105] The nucleic acid segments used in the current disclosure can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post- translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0106] In certain embodiments, the current disclosure provides polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence of this disclosure using the methods described herein (e.g., BLAST analysis using standard parameters).

[0107] The disclosure also contemplates the use of polynucleotides which are complementary to all the above described polynucleotides.A. Vectors

[0108] Polypeptides of the disclosure may be encoded by a nucleic acid molecule comprised in a vector. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for exampleSambrook et al., 2001 ; Ausubel et al., 1996, both incorporated herein by reference). In addition to encoding a polypeptide of the disclosure, the vector can encode other polypeptide sequences such as a one or more other bacterial peptide, a tag, or an immunogenicity enhancing peptide. Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al., 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S- transferase (GST) soluble fusion proteins for later purification and separation or cleavage.

[0109] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.B. Promoters and Enhancers

[0110] A “promoter” is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0111] Naturally, it may be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al., 2001, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, or inducible and in certain embodiments may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.

[0112] The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter.C. Initiation Signals and Internal Ribosome Binding Sites (IRES)

[0113] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.

[0114] In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5’ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference).D. Selectable and Screenable Markers

[0115] In certain embodiments of the invention, cells containing a nucleic acid construct of the current disclosure may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.E. Host Cells

[0116] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequentgenerations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

[0117] Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).F. Expression Systems

[0118] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

[0119] The insect cell / baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULO VIRUS EXPRESSION SYSTEM FROM CLONTECH®.

[0120] In addition to the disclosed expression systems of the invention, other examples of expression systems include STRATAGENE®’s COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level productionof recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.IV. Administration of Therapeutic Compositions

[0121] The therapy provided herein may comprise or exclude administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, the first and second cancer treatments are administered in a separate composition. In some aspects, the first and second cancer treatments are in the same composition.

[0122] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0123] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some aspects, the additional therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The cancer therapy or additional therapy may be administered by a route of administration that excludes administration intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0124] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuousinfusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0125] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.V. Therapeutic Methods

[0126] The compositions of the disclosure may be used for in vivo, in vitro, or ex vivo administration. The compositions and methods relate to the treatment of cancer.

[0127] The cancer may include, but are not limited to, cancers and tumors of all types, locations, sizes, and characteristics. The methods and compositions of the disclosure include or exclude treating, for example, pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, colorectal cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, glioma, glioblastoma multiforme, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumors brain tumor, visual pathway and hypothalamic glioma, breast cancer, lymphoid cancer, bronchial adenomas / carcinoids, tracheal cancer, Burkitt lymphoma, carcinoid tumor, childhood carcinoid tumor, glioblastoma, neuroblastoma, gastrointestinal carcinoma of unknown primary, central nervous system lymphoma, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's, childhood extragonadal Germ cell tumor, extrahepatic bile duct cancer, eye Cancer, intraocular melanoma eye Cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, glioma of the brain stem, glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkinlymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer , leukemia, acute lymphoblastic (also called acute lymphocytic leukemia) leukemia, acute myeloid (also called acute myelogenous leukemia) leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia) leukemia, chronic myelogenous (also called chronic myeloid leukemia) leukemia, hairy cell lip and oral cavity cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphomas, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Non-Hodgkin (an old classification of all lymphomas except Hodgkin's) lymphoma, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, childhood medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant, fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, childhood Salivary gland cancer Sarcoma, Ewing family of tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sezary syndrome sarcoma, skin cancer (nonmelanoma), skin cancer (melanoma), skin carcinoma, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, metastatic stomach cancer, supratentorial primitive neuroectodermal tumor, childhood T-cell lymphoma, testicular cancer, throat cancer, thymoma, childhood thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrialuterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, childhood vulvar cancer, and wilms tumor (kidney cancer).

[0128] In some aspects, the cancer comprises or excludes aggressive cancer. In some aspects, the cancer comprises or excludes Stage I cancer. In some aspects, the cancer comprises or excludes Stage II cancer (e.g., IIA, IIB, IIC). In some aspects, the cancer comprises or excludes Stage III cancer (e.g., IIIA, IIIB, IIIC). In some aspects, the cancer comprises or excludes Stage IV cancer (e.g., IVA, IVB).

[0129] Methods may comprise or exclude the determination, administration, or selection of an appropriate cancer “management regimen” and predicting the outcome of the same. As used herein the phrase “management regimen” refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and / or duration of a treatment) provided to a subject in need thereof (e.g., a subject diagnosed with cancer).VI. Pharmaceutical Compositions and Methods

[0130] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects involve administering an effective amount of a composition to a subject. In some embodiments, a composition comprising the polypeptides of the disclosure agent may be administered to the subject or patient to treat cancer. Additionally, such compounds can be administered in combination with an additional treatment.

[0131] Compositions can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, transcatheter injection, intraarterial injection, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified. The preparation of such formulations will be known to those of skill in the art in light of the present disclosure. Other routes of administration include intratumoral, peri-tumoral, intralymphatic, injection into inflamed tissue, or into lymph nodes. In some embodiments, the administration is systemic.

[0132] Other routes of administration are also contemplated. For example, the constructs and agents may be administered in association with a carrier. In some embodiments, the carrier is a nanoparticle or microparticle.

[0133] Particles can have a structure of variable dimension and known variously as a microsphere, microparticle, nanoparticle, nanosphere, or liposome. Such particulateformulations can be formed by covalent or non-covalent coupling of the construct to the particle. By “particle,” “microparticle,” “bead,” “microsphere,” and grammatical equivalents herein is meant small discrete particles that are administrable to a subject. In certain embodiments, the particles are substantially spherical in shape. The term “substantially spherical,” as used herein, means that the shape of the particles does not deviate from a sphere by more than about 10%. The particles typically consist of a substantially spherical core and optionally one or more layers. The core may vary in size and composition. In addition to the core, the particle may have one or more layers to provide functionalities appropriate for the applications of interest. The thicknesses of layers, if present, may vary depending on the needs of the specific applications. For example, layers may impart useful optical properties.

[0134] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0135] The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0136] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0137] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit / risk ratio. The term “pharmaceutically acceptable carrier,” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[0138] As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.

[0139] Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effects desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.

[0140] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactic ally effective. Theformulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0141] Typically, for a human adult (weighing approximately 70 kilograms), from about 0.1 mg to about 3000 mg (including all values and ranges there between), or from about 5 mg to about 1000 mg (including all values and ranges there between), or from about 10 mg to about 100 mg (including all values and ranges there between), of a compound are administered. It is understood that these dosage ranges are by way of example only, and that administration can be adjusted depending on the factors known to the skilled artisan.

[0142] In certain embodiments, a subject is administered about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 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, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450,460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610,620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770,775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925,930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (mcg) or pg / kg or micrograms / kg / minute or mg / kg / min or micrograms / kg / hour or mg / kg / hour, or pM or mM of an agent discussed herein. Any range derivable therein is contemplated.

[0143] A dose may be administered on an as needed basis or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9, or timesper day (or any range derivable therein). A dose may be first administered before or after signs of a condition. In some embodiments, the patient is administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 3, 4, or 5 days after the patient experiences or exhibits signs or symptoms of the condition (or any range derivable therein). The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of the condition have disappeared or been reduced or after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days after symptoms of an infection have disappeared or been reduced.VII. Sequences

[0144] The table below provides exemplary polypeptides useful in the compositions and methods of the disclosure.VIII. Examples

[0145] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.EXAMPLE 1: Collagen-binding IL-7 reduces exhaustion and enhances memory in IL- 12 cancer immunotherapy

[0146] Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, since this is often associated with increased overall toxicity, identifying regimens with high tolerability is a main objective in combination therapy. Here, the inventors show that combination of IL-7-CBD, an IL-7 domain fused to a collagen -binding domain (CBD) to enhance retention in the tumor stroma, with CBD-IL-12 prevents immune cell exhaustion and leads to a significant benefit in therapeutic response in aggressive and immunosuppressive B16F10 melanoma and 4T1 breast cancer models. IL-7-CBD when combined with CBD-IL- 12 did not induce any additional immune-related adverse effects. Furthermore, IL-7-CBD played a key role in the development of immunological memory, as evidenced by the eradication of rechallenged melanoma in mice cured by IL-7-CBD + CBD-IL-12 combination therapy. IL-7-CBD combined with CBD-IL-12 (both i.v.) potently synergized with checkpoint inhibitor (CPI) therapy and suppressed tumor growth in the poorly immunogenic, CPI- unresponsive autochthonous Brafv600E / Pten' / 7pCATSTAmelanoma model. The data providessupport for IL-7-CBD as an immune potentiator for combination with an immune agonist such as CBD-IL-12 to achieve strong and durable immunotherapy, which has clinical applicability.A. Introduction

[0147] Immunotherapy has become one of the dominant pillars of modem cancer care, and combination immunotherapy is a promising approach to promote antitumor immunity by activating complementary pathways (1-3). For example, combining checkpoint inhibitor (CPI) antibodies blocking the PD-1 (e.g., with nivolumab) and CTLA-4 (e.g, ipilimumab) axes increases the overall survival of melanoma patients when compared to blockade with either agent alone (4-6). However, a major limitation of combination immunotherapies is that the rate of immune-related adverse events (irAEs) also increases when compared to the monotherapy regimen (7-9). In the same trial examining the combination of nivolumab and ipilimumab, 55%-60% of patients experienced Grade 3 or 4 adverse events, while monotherapy led to 10%- 20% experiencing Grade 3 or 4 adverse events. A similar observation was made in another trial examining IL- 12 in combination with IL-2 (10). Thus, the selection of agents that enhance clinical outcome without exacerbating toxicity is highly desired.

[0148] Interleukin-7 (IL-7) is a common gamma chain (yc) binding cytokine that plays a crucial role in T cell development, expansion, and memory (11-14). IL-7 signals through IL-7 receptor a (IL-7R a) and yc, the former being predominantly expressed on mature T cells and dictating the cell-type specificity. Due to the immunomodulatory properties of recombinant IL- 7, it has been tested in a clinical trial in patients with advanced cancers as monotherapy (15). IL-7 was well-tolerated up to a dose of 60 pg / kg and induced robust expansion of peripheral CD8+ T cells, but not regulatory T cells (Tregs) or natural killer (NK) cells (16-18). Despite demonstrating obvious biological activity at well-tolerated doses, IL-7 did not show clinical benefit in a monotherapy setting. A possible explanation for the lack of tumor regression is that IL-7 by itself does not induce potent effector functions in CD8+ T cells (19) and would likely require a combination approach involving an immune- stimulatory factor to fully unlock its therapeutic potential.

[0149] Here, the inventors designed a combination therapy approach consisting of IL-7 and an immune-stimulatory cytokine, IL- 12, which exerts potent antitumor effects but also induces severe irAEs. The inventors used an engineered form for both cytokines to further improve their biodistribution, leveraging the reported tumor stroma-binding technology, which uses a collagen-binding domain (CBD) derived from the A3 domain of von Willebrand factor as a tumor-retention domain (20-23). The inventors show that the combination of IL-7-CBD withCBD-IL-12 addresses a key challenge in combination therapy: increased therapeutic efficacy without compromised safety. The inventors demonstrate this result by conducting extensive toxicology studies and paralleling those with the antitumor efficacy studies of the IL-7-CBD + CBD-IL-12 combination therapy. The inventors also show that IL-7-CBD and CBD-IL-12 activate nonredundant immunological pathways, with IL-7-CBD providing the memory and survival signal for intratumoral T cells, while CBD-IL-12 supports the activation and effector programs in the T cells.B. Results1. IL-7-CBD synergizes with CBD-IL-12 without increasing systemic toxicity

[0150] Motivated by the improved therapeutic properties of intravenously or intratumorally administered immunotherapy agents when fused to the collagen-binding domain derived from the A3 domain of von Willebrand factor (CBD) (20), which leads to active retention in the exposed tumor stroma, the inventors fused the CBD domain to the C-terminus (IL-7-CBD), N- terminus (CBD-IL-7), or C and N-termini (CBD- IL-7-CBD) of murine IL-7 and chose to proceed with IL-7-CBD, which had a higher yield (5.8 mg / L) than other candidates (CBD-IL- 7: 4.8 mg / L and CBD-IL-7-CBD: 3 mg / L) (FIG. 1A, 5A). C-terminal fusion of CBD did not abrogate the activity of IL-7 as assessed by STAT5 phosphorylation on mouse T cells (FIG. IB). The inventors then performed a collagen-coated enzyme-linked immunosorbent assay to confirm that IL-7 -CBD has adequate binding to collagen I, as they have observed with other cytokine fusions to CBD (21) (FIG. 5B). Finally, the inventors performed an in vivo tumor growth inhibition study using the MC38 colorectal adenocarcinoma model, which is known to respond to IL-7 treatment (24), to compare the antitumor activity of IL-7 versus IL-7-CBD FIG. 6). In line with previous observations, intravenous administration of an equimolar dose of IL- 7-CBD significantly extended the survival of MC38-bearing mice when compared to the unmodified IL-7. Collectively, these data indicate that the fusion of CBD to IL-7 retains both IL-7 receptor-stimulating activity and collagen-binding properties, as well as improves the monotherapy efficacy in vivo.

[0151] After demonstrating the superior antitumor efficacy of IL-7-CBD over unmodified IL-7 in the MC38 model, which is known to be CPI-responsive (25, 26), the inventors sought to combine it with CBD-IL-12 (21) and assess the efficacy of the combination therapy in the CPI-unresponsive B16F10 melanoma model (FIG. 1C, 7). In comparison with the unmodified IL-7 + IL- 12, CBD-fused cytokine combination therapy significantly increased the survivalwhen administered intravenously. To understand the therapeutic benefit of the addition of IL- 7-CBD to CBD-IL-12, the inventors designed an experiment in which mice either received a 10 pg CBD-IL-12 monotherapy (166.5 pmol, or 10 pg on an IL-12 basis) or 2 pg CBD-IL-12 (33.3 pmol, or 2 pg on an IL-2 basis) supplemented with IL-7-CBD (FIG. ID, 8). A single i.v. administration of 10 pg CBD-IL-12 monotherapy and 2 pg CBD-IL-12 + IL-7-CBD combination therapy yielded equally significant extension in the survival of B16F10 melanoma-bearing mice when compared to phosphate-buffered saline (PBS). This result indicates that the inventors are able to reduce the dose of CBD-IL-12 fivefold by supplementing the CBD-IL-12 regimen with IL-7-CBD at no detriment to antitumor efficacy. The inventors also observed that escalating the dose of IL-7-CBD proportionally increases the antitumor efficacy when combined with CBD-IL-12, indicating that synergistic therapeutic efficacy is dependent on the dose of IL-7-CBD (FIG. 9).

[0152] A key hurdle in the majority of combination therapy trials is that, despite the overall increased survival, such combination approaches often lead to higher rates of irAEs. To investigate whether addition of IL- 7-CBD to CBD-IL-12 worsens the tolerability of the therapy, the inventors performed extensive toxicology studies in mice receiving either 10 pg CBD-IL-12 monotherapy, 2 pg CBD-IL-12 monotherapy or combination of 2 pg CBD-IL-12 with 20 pg IL-7-CBD systemically (FIG. IE). The inventors collected the blood of treated mice at various timepoints to quantify the levels of circulating leukocytes, erythrocytes, organ damage markers (alanine aminotransferase, aspartate aminotransferase, amylase, and total bilirubin) and proinflammatory cytokines (IFNy, IL-6 and TNFa). The data demonstrate that IL-7-CBD + CBD-IL-12-treated animals experienced these irAEs similar to 2 pg (based on IL- 12 molar equivalents) CBD-IL-12 monotherapy -treated animals, which was significantly lower than in the 20 pg CBD-IL-12-treated cohort. This lack of stimulation of systemic inflammation upon IL-7 combination with IL- 12 was likely an IL-7-specific phenomenon, since combination of IL- 15, another actively studied yccytokine, with IL- 12 led to significantly higher systemic inflammation and toxicity (FIG. 10). ). To further evaluate toxicity, we performed a singledose or multiple-dose toxicity test in tumor-bearing mice (FIGS. 23-24), where the dose and injection interval were based on the single-dose or multiple-dose therapeutic evaluation (FIG. 1, C and D). The CBD-IL-12 monotherapy and the IL-7-CBD + CBD-IL-12 combination therapy induced liver toxicity in both single-dose and multiple-dose injections, but the combination therapy showed toxicity similar to the 2-pg CBD-IL-12 monotherapy, and IL-7- CBD in the combination therapy did not lead to the additional irAEs. To demonstrate that the CBD-fused cytokine combination was superior and safer than the unmodified cytokinecombination, the inventors designed a similar pharmacokinetic and toxicity study (FIGs. 11- 13). In line with previous results with IL-12 forms in monotherapy (21, 27), IL-7-CBD + CBD- IL-12 combination therapy exhibited short systemic circulation and promoted tumor accumulation via tumor stroma targeting of the CBD (FIG. 25) and induced fewer systemic side effects, including less hepatic CD8+ T cell infiltration, versus unmodified IL-7 + IL- 12 therapy (FIG. 14). It is crucial to note that the lack of systemic toxicity is a key feature of IL- 7-CBD, unlike another reported engineered IL-7-Fc fusion protein (46). This long-acting engineered IL-7-Fc protein induced splenomegaly and caused plasma IL-6 elevation, a sign of systemic toxicity (Kim et al., 2022). The inventors of IL-7-CBD did not observe any organ weight change or serum cytokine elevation, likely due to the lack of half-life extension domains such as Fc. Combined, these results indicate that IL-7-CBD and CBD-IL-12 display a significant synergy in promoting antitumor immunity without increasing irAEs in healthy organs.2. Addition of IL-7-CBD to CBD-IL-12 therapy reduces the frequency of terminally exhausted CD8+ T cells

[0153] To understand the mechanism of action and the therapeutic benefit of the addition of IL-7-CBD to CBD- IL- 12, the inventors performed spectral flow cytometry to provide an in-depth look at the tumor-infiltrating lymphocytes. While CBD-IL-12 induced an increase in total CD45+ immune cell infiltration, but the addition of IL-7-CBD did not further increase this. Furthermore, CBD-IL-12 increases conventional (Foxp3-) CD4+ T cell counts, although not significantly, and decreases Treg infiltration, but IL-7-CBD did not further alter either of these populations in the combination therapy (FIG 26). Neither IL-7-CBD nor CBD-IL-12 monotherapies altered NK or NK T cell infiltration, but combination therapy decreased both NK and NKT cell infiltration compared to the CBD-IL-12 monotherapy (FIG. 26). In terms of CD8+ T cell infiltration, addition of IL-7-CBD did not alter CD8+ T cell infiltration either when added as a monotherapy, compared to untreated mice, or as a combination therapy when compared to the CBD- IL- 12 monotherapy (FIG. 2A). However, because CD8+ T cells are considered so important for immunotherapy responses and can be so heterogenous, the inventors next assessed whether the specific phenotype of the CD8+ T cell infiltrates was altered. The inventors performed unsupervised clustering using FlowSom, an algorithm specifically developed to analyze flow cytometry data, on concatenated CD8+ T cells. The inventors observed 7 distinct clusters as determined by the algorithm, and they segregated distinctly when displayed on the Uniform Manifold Approximation and Projection (UMAP) of the same CD8+ T cells (FIG. 2B). The dimensionality -reduced data demonstrate that, whileIL-7-CBD monotherapy does not seem to alter the CD8+ T cell phenotype significantly compared to saline treatment, combining it with CBD- IL- 12 alters the T cell landscape due to distinct distribution of the cells in each cluster (FIG. 2C). Specifically, Clusters 2, 4, and 6 were significantly influenced by the IL-7 -CBD combination when compared against CBD-IL-12 monotherapy (FIG. 2D). Cluster 6 expressed high levels of canonical exhaustion markers such as T cell immunoglobulin and mucin domain 3 (Tim3) and PD-1 (28, 29), and it also uniquely expressed high levels of Tox, a marker known to be associated with terminal exhaustion (30) (FIG. 2E). This indicates that supplementing CBD-IL-12 therapy with IL-7-CBD significantly reduces the frequency of exhausted CD8+ T cells. Contrastingly, Cluster 2 and 4 both express high levels of killer cell lectin like receptor G1 (KLRG1), a marker of effector function (31) (FIG. 2E, F). In addition, cluster 4 has higher expression of IL-7Ra (CD127) as well as activation markers such as CD44, CD25, and inducible costimulator (ICOS) and higher expression of PD-1, lymphocyte-activation gene 3 (Lag3), and Tim3 compared to cluster 2. Both clusters 2 and 4 are distinctly enriched upon CBD-IL-12 treatment, but upon the addition of IL-7 -CBD, these clusters are enriched even further and constitute about 70% of total CD8+ T cells. (FIG. 2G) Thus, the IL-7 -CBD + CBD-IL-12 combination therapy alters the CD8+ T cell landscape as evidenced by reduced abundance of the terminally exhausted Cluster 6 and increased abundance of the “effector-like” clusters 2 and 4.

[0154] The inventors also visualized the presence of CD8+ T cells throughout the tumor stroma (FIG. 2H) after IL-7-CBD + CBD-IL-12 therapy, further corroborating the abovementioned flow cytometric findings. The inventors then sought to characterize the landscape of intratumoral inflammatory cytokines and chemokines upon treatment with the combination of IL-7-CBD + CBD-IL-12 (FIG. 21). Consistent with flow cytometry data, IL-7- CBD monotherapy did not result in substantial inflammation when compared to saline treatment. Although CBD-IL-12 monotherapy-treated mice had significant elevation of certain markers, combination with IL-7-CBD increased the breadth and the magnitude of the cytokine secretion within the tumor. Notably, compared with CBD-IL-12 monotherapy, the normalized amount of intratumoral interferon-y (IFNy) was higher in the dual therapy-treated animals (FIG. 15). Contrasting this result with the level of circulating IFNy (FIG. IE) indicates that addition of IL-7-CBD boosts only tumor- specific inflammation but not systemic inflammation. To verify whether the combination therapy required immune cell migration from tumor-draining lymph nodes or whether immune cells in the circulation were sufficient to elicit an anti-tumoral immune response, the inventors evaluated the anti-tumoral effect of combination therapy in mice injected with FTY720, a sphingosine- 1 -phosphate receptor modulator that sequesterslymphocytes in lymph nodes (FIG. 16). Intratumoral treatment with IL-7- CBD + CBD-IL-12 led to a similar extension in survival in mice receiving FTY720 or vehicle control, pointing out that tumor-resident T cells are sufficient to drive the antitumor response.3. Locally administered IL-7-CBD + CBD-IL-12 generates potent systemic immunity and effective long-term immune response

[0155] Intratumoral administration of immunotherapeutic s has emerged as an alternative to systemic administration, especially for intratumorally-accessible cancers, so as to limit the systemic exposure and reduce side effects (32-34). Given the fusion of the CBD to IL-7 and IL- 12, local administration may provide further advantages in certain indications in a clinical setting due to the retention of the cytokines in the tumor matrix. The inventors first compared the therapeutic efficacy of the intratumoral injection of CBD-fused cytokine combination therapy to the unmodified IL-7 + IL- 12 in the B16F10 melanoma model (FIG. 17). CBD-fused cytokine combination therapy yielded a significantly extended survival of the melanomabearing mice compared to the unmodified combination, with an 80% complete response (CR) rate. The extension in survival can be attributed to the prolonged intratumoral residence of the cytokines (FIG. 27). The inventors then tested local administration of monotherapy of CBD- IL-12 or IL-7- CBD, compared to the combination, which was superior, at around 70% CR in this instance (FIG. 18). It is crucial for locally administered immunotherapies to not only work against the primary tumors, but to also produce systemic antitumor effects. To investigate this, the inventors designed an experiment in which the primary B16F10 tumor was treated at the same time as an intravenous challenge of Bl 6F 10 cells, which are reported to colonize the lungs (35) (FIG. 3A). Intratumoral treatment with CBD-IL-12 alone did not completely abrogate the formation of such metastases, but therapy with IL-7-CBD + CBD-IL-12 combination completely prevented colonization of the lungs as evidenced by the lung weight and hematoxylin and eosin (H&E) staining, which were not different from the healthy lungs (FIG. 3B).

[0156] Since IL-7 plays a major role in generation of immunological memory, the inventors next sought to dissect the contribution of the IL-7-CBD portion in IL-7-CBD + CBD-IL-12 combination therapy and compare it with the immune memory established by CBD-IL-12 monotherapy. To match the CR rate of CBD-IL- 12 monotherapy to the IL-7-CBD + CBD-IL- 12 combination therapy, the inventors used a 5-fold higher dose (10 pg on an IL- 12 basis) of CBD-IL-12 than the dose used in the combination regimen. Both 10 pg CBD- IL- 12 monotherapy and IL-7-CBD + CBD-IL-12 combination therapy led to about 80% CR rate against the primary B16F10 tumor (FIG. 19). The inventors then rechallenged these mice onthe contralateral side of the back 60 days after the primary tumor inoculation (FIG. 3C). All mice whose primary tumors were cured by 10 pg CBD-IL-12 monotherapy succumbed to this rechallenge with B16F10 melanoma cells (FIG. 3D). In striking contrast, all of the mice that were initially cured by the combination regimen rejected the rechallenge, indicating that longterm immune memory was established against poorly immunogenic B16F10 melanoma. Next, the inventors performed a depletion study using aCD8, aCD4, and alFNy to identify the immune players that mediate rejection of the rechallenge in combination-treated animals (FIG. 3E). The results indicate that CD8+ T cells, but not CD4+ T cells or IFNy, have the most profound effect on the prevention of regrowth of the B16F10 tumor.

[0157] The inventors hypothesized that, upon a rechallenge with B16F10 cells, there is an expansion of tumor- specific memory CD8+ T cells, which mediates the rejection of the delayed contralateral challenge. To study this, the inventors evaluated the changes in the population of CD8+ T cells specific to tyrosinase-related protein 2 (TRP2), a well-known Bl 6F 10 melanoma antigen, in the blood and primary tumor-draining lymph nodes in the cured mice. Mice that were cured by the combination therapy showed significantly greater expansion of TRP2- specific CD8+ T cells in the blood compared to CBD-IL-12 monotherapy-cured mice 7 days after re-inoculation (FIG. 3F). Additionally, in the tumor-draining lymph node, there were more TRP2-specific central memory CD8+ T cells in mice cured by the combination therapy versus 10 pg CBD-IL-12 monotherapy (FIG. 3G). These data demonstrate that intratumorally administered IL-7- CBD combined with CBD-IL-12 could boost systemic anti-tumoral immune response and inhibit pulmonary metastasis and could promote tumor- specific anamnestic response, especially tumor- specific CD8+ T cells, and prevent tumor re-growth.4. Combining IL-7-CBD + CBD-IL-12 regimen with aPD-1 elicits longterm tumor control of immunosuppressive breast tumors and genetically engineered melanoma

[0158] CPI therapy targeting programmed cell death protein 1 (PD-1) is a representative immuno-oncology approach and has led to favorable results in the treatment of certain carcinomas. However, the number of reports that show insufficient therapeutic index in poorly inflamed (or cold) tumors is continuously increasing (36-38). To investigate whether IL-7- CBD + CBD-IL-12 dual therapy can synergize with aPD-1 immunotherapy, the inventors designed an experiment in which mice received i.v. cytokines of either of the single agents, or the dual therapy, or the triple combination with aPD-1 in the orthotopic 4T1 highly immunosuppressive breast cancer model (FIG. 4A, 20). Neither of the tested monotherapies nor aPD- 1 monotherapy resulted in any therapeutic benefit when compared to the salinetreatment. The dual engineered cytokine therapy significantly extended the survival of 4T1- bearing animals, corroborating the synergistic effect between IL-7-CBD and CBD-IL-12. Upon further addition of aPD- 1 antibody to the dual therapy regimen, mice experienced even greater extension in the overall survival, highlighting that all three agents act on nonredundant immune pathways. Then, the inventors further investigated the CPI potentiating effect of the dual therapy in the genetically engineered Bral'V600l7Ptcn- / 7pCATS I Amelanoma, an immune-desert and CPI-resistant model (39) (FIG. 4B-D, 21). Systemic administration of the triple therapy led to striking, long-term tumor control, without any loss in body weight during the course of the treatment. Together, the data indicate that combining IL-7-CBD + CBD-IL-12 dual therapy with aPD-1 can overcome CPI resistance and deliver remarkable therapeutic outcomes in very challenging and clinically relevant cancer models.C. Discussion

[0159] Combinatorial immunotherapy is a promising approach that can overcome the limitations of monotherapy by enhancing overall survival rate. However, a major hurdle of the combination therapies is the increased cumulative toxicity. Hence, current immunotherapy combinations benefit only those patients who can tolerate the additional side effects. To achieve a net gain in the therapeutic index, it is desired to identify immunotherapy agents with complementary mechanisms of action that do not elicit an additive toxicity profile. In this study, a dual therapy involving tumor stroma-binding IL- 7 and IL- 12 variants was used to guide the design of next-generation immunotherapy combinations, which led to synergistic antitumor efficacy without compromised tolerability. Systemic treatment with IL-7-CBD and CBD-IL- 12 significantly improved the survival of poorly immunogenic B16F10 melanoma-bearing mice when compared to either agent in monotherapy. IL-7-CBD did not induce additional irAEs in the combination therapy. Local treatment with IL-7-CBD and CBD-IL-12 induced an antitumor immunity not only at the injection site but also systemically, leading to the eradication of pulmonary B16F10 metastases. During combination therapy, IL-7-CBD played a key role in the development of immunological memory and prevented B16F10 melanoma regrowth upon rechallenge. Combination of IL-7-CBD and CBD-IL-12 significantly extended the survival in the 4T1 triple-negative breast cancer model, which was completely resistant to either agent alone. Addition of aPD-1 to the dual therapy regimen further increased the response rates, demonstrating the importance of activating multiple immunological pathways to boost antitumor immunity.

[0160] IL-7-CBD provided prosurvival signals mediated by STAT5 activity to tumorresident T cells, while CBD-IL-12 provided immune-activating signals mediated by STAT4 activation. Potent immune stimulators such as IL- 12 may lead to the induction of exhausted T cells, which are no longer able to kill the cancer cells. The inventors hypothesize that the IL- 7-CBD as a combination agent might limit T cell exhaustion. In-depth characterization of the phenotype of tumor-resident T cells revealed that, when compared with the CBD-IL-12 monotherapy, the dual therapy can regulate the immune cell fitness and lead to the decrease of terminally exhausted ToxhlghLag3hlghTim3hlghCD8+ T cell (30, 40, 41) population. Instead, the dual therapy expanded the fraction of KLRGlhlghPD-lhlghCD8+ T cells, which maintain a high tumor-killing potential. Another noteworthy feature of the dual therapy is that the amount of certain inflammatory markers produced by T cells, such as IFN-y, was elevated in the tumor but not in the blood when compared to the CBD-IL-12 monotherapy. Given that the dual combination skews the tumor-resident T cells from an exhausted state to a more functional state, we hypothesize that these T cells are able to sustain a high secretion level of IFN-y for extended period of time.

[0161] In addition, treatment of the primary tumor with IL-7-CBD and CBD-IL-12 exhibited notable benefits when compared to the CBD-IL-12 monotherapy in long-term anticancer immune response. The mice cured by the combination therapy showed that circulating CD8+ T cells specific for the B16F10 antigen TRP2 were significantly expanded after B16F10 rechallenge, consistent with the known role of IL-7 as a key player in the development of long-term immunity. These mice also had more TRP2-specific CD62L+CD44+CD8+ T cells in the primary tumor-draining lymph node compared to the CBD-IL-12 monotherapy. Thus, all the mice cured by the combination survived in rechallenge experiments, whereas none of the mice cured by CBD-IL-12 survived the rechallenge.

[0162] A lack of additive toxicity is certainly a critical feature of the design of the combination approach. The inventors believe that IL-7 receptor expression by CD8+ T cells is critical for antitumor efficacy, whereas minimal expression of it on NK cells prevents additional systemic inflammation (17). The inventors have previously shown that NK cells are the main contributors to the toxicity in IL-12 monotherapy (27). However, because murine NK cells have a minimal expression level of the IL-7 receptor and low responsiveness to IL-7 (FIG. 28), IL-7-CBD does not significantly contribute to NK cell activation and thus systemic inflammation. Therefore, the wide dose range of IL-7-CBD (5, 10, and 20 pg) combined with CBD-IL-12 (2 pg) did not induce dose-dependent systemic toxicity and increased therapeutic efficacy similarly as the 10-pg CBD-IL-12 monotherapy. This further corroborates the choiceof IL-7 as the partner cytokine to IL-12. Conversely, IL-15, another common yc family member and representative candidate for combination therapy with IL- 12, significantly induced additive toxicity upon the combination with IL- 12, likely due to the constitutive expression of its receptor complex on NK cells.

[0163] For clinical translation, an important feature of the IL-7-CBD + CBD-IL-12 combination therapy is that synergistic antitumor efficacy is observed upon simultaneous administration of the two agents. In the clinical use, different agents of combination therapies may have different administration schedules due to their respective mechanisms of action and pharmacokinetics, which can increase the burden on medical staff and patients. The simultaneous administration observed to be effective here may be more straightforward and clinically translatable than other described combinations that may require the administration of dual drugs on different schedules (42-44).

[0164] Systemic administration of IL-7-CBD and CBD-IL-12 in conjunction with suboptimal aPD-1 dose showed unprecedented tumor control without any overt toxicity in the autochthonous Braf^^ / Pten^’ / pCAT8melanoma model. This genetically engineered mouse model recapitulates many aspects of the CPI-resistant melanoma seen in patients, highlighting the translational potential of our combination therapy. In summary, the inevntors demonstrated that the combination of IL-7-CBD, across a wide dose range, with CBD- IL- 12 exhibited superior antitumor efficacy by minimizing T cell exhaustion without eliciting additional irAEs and allowed reduction of CBD-IL-12 dose without loss in efficacy. Such therapeutic index enhancement may serve as the basis for designing future combination therapy regimens.D. Experimental1. Mice and cancer cell line

[0165] Female C57BL / 6 mice (age 8-12 weeks) were purchased from Charles River Laboratory. Female Balb / C mice (aged 8-12 weeks) were purchased from Jackson Laboratory. B16F10 melanoma and 4T1 breast cancer cell lines were obtained from ATCC and were cultured according to instructions. Cell lines were routinely checked for my mycoplasma contamination. Brafv600E / Pten' / 7pCATSTAmice (aged 8-12 weeks) were bred at the animal facility of the University of Chicago. All the animal experiments performed in this research were approved by the Institutional Animal Care and Use Committee of the University of Chicago.2. Production and purification of recombinant cytokines

[0166] To produce naive IL-7, sequence optimized mouse IL-7 was cloned into mammalian expression vector pcDN A3.1(+) by GenScript. A His-tag sequence (His)6 was added to the C- terminal of IL-7 to enable affinity chromatography. To produce IL-7-CBD (SEQ ID NO:28), a CBD (A3 domain of VWF (20)) encoding sequence was inserted into C-terminal of IL-7 with a linker (GGGS)2, and the His-tag sequence was inserted into C-terminal of CBD. Sequence optimized IL-7-CBD was cloned into mammalian expression vector pcDNA3.1(+) by GenScript. The proteins were expressed in HEK293F system (Invitrogen). 1 mg / L pDNA with 2 mg / L 25kDa polyethyleneimine (Polysciences) were co-incubated in OptiPro SFM medium (4% final volume) for 10 min. The mixture was then transfected into HEK293F cells (IxlO6cells / mL). After 5 days of the transfection, the supernatant was collected and filtered using 0.2 pm filter unit. The protein purification was performed as described previously (20, 22). Purified proteins were tested for endotoxin using HEK-Blue TLR4 reporter cell line and endotoxin levels were below 0.01 endotoxin units / mL. Protein purity was confirmed using SDS-PAGE as described previously (20, 45). Protein concentration was determined by absorbance method using NanoDrop (Thermofisher Scientific). CBD-IL-12 (SEQ ID NO:30) was produced as described previously (21). Recombinant mouse IL- 15 was purchased from PeproTech (NJ, US), Inc. and recombinant mouse IL-15R alpha Fc chimera protein was purchased from R&D systems (MN, US).3. Analysis of pSTAT5 activity of IL-7 and IL-7-CBD using flow cytometry

[0167] Mouse CD3+ T cells were purified from the blood of female C57BL / 6 mice using EasySep mouse total (CD3+) T-cell isolation kit (Stem Cell). Purified CD3+ T cells (1 x 106cells / mL were rested in cell culture media for 2 hr and transferred into 96-well plates (50,000 cells / well). The cell culture media was IMDM (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin- streptomycin. Mouse CD3+ T cells were stimulated for 20 min at 37 °C with various concentrations of IL-7 or IL-7-CBD. Then, cells were permeabilized using BD Phosflow Perm Buffer III for 30 min in 4°C. The cells were stained with Alexa Fluor 647-conjugated pSTAT5 antibody (clone: 47, BD) for overnight at 4°C (Dilution: 1 :50). The cells were acquired using BD LSR flow cytometer and data were analyzed using FlowJo (TreeStar).4. In vitro collagen I binding affinity of IL-7-CBD

[0168] 100 pL Human collagen I (10 pg / mL in PBS, EMD Millipore) was coated in medium-binding 96-well plate (Greiner Bio) overnight at 37 oC. The collagen-coated 96-well was washed 3 times with PBS-T (IxPBS and 0.05% tween 20) and blocked with blocking buffer (2% BSA in PBS-T) at room temperature for 2 hrs. Then, the plate was washed 3 times with PBS-T and added IL-7-CBD or IL-7 at serial dilution in dilution buffer (0.1% BSA in PBS-T) at room temperature for 3 hrs. After the incubation, the plate was washed 3 times with PBS-T and added IL-7 detection antibody (biotinylated) diluted in dilution buffer (dilution factor is 1 : 100) at room temperature for 1 hr. After the incubation, the plate was washed 3 times with PBS-T and added avidin-HRP diluted in dilution buffer at room temperature for 30 min. Then, the plate was washed 3 times with PBS-T and added 100 pL TMB substrate (EMD Millipore) for colorimetry analysis.5. In vivo evaluation of IL-12 + IL-7 and CBD-IL-12 + IL-7-CBD combination therapies administered intravenously or intratumorally

[0169] C57BL / 6 mice were inoculated with 5 x 105B16E10 melanoma cells intradermally on day 0. Tumor- bearing animals were treated with 100 pL PBS (n=10), 33.3 pmol CBD-IL- 12 (n=10), 33.3 pmol IL- 12 + 666 pmol IL-7 (n=10), or 33.3 pmol CBD-IL-12 + 666 pmol IL- 7-CBD (n=10) on days 7, 13, 19, and 25 intravenously. Alternatively, tumor-bearing mice were treated with 30 pL PBS (n=10), 33.3 pmol IL-12 + 333 pmol IL-7 (n=10), or 33.3 pmol CBD- IL-12 + 333 pmol IL-7-CBD (n=10) on day 7, 13, 19, and 25 intratumorally. The tumor size was calculated using the following formula: height x width x thickness x (K / 6). The mice were euthanized when the tumor size reached 1,000 m3and / or based on humane end-point criteria.6. Therapeutic evaluation of IL-7-CBD monotherapy administered intravenously

[0170] C57BL / 6 mice were inoculated with 5 x 105MC38 colon cancer cells subcutaneously on day 0 and injected with 100 pL PBS (n=10), 1.3 nmol IL-7 (n=10), or 1.3 nmol IL-7-CBD (n=10) on day 7 and 13. The tumor size was calculated using the following formula: height x width x thickness x (K / 6). The mice were euthanized when the tumor size reached 1,000 m3and / or based on humane end-point criteria.7. Therapeutic evaluation of high dose CBD-IL-12 vs CBD-IL-12 + IL-7- CBD administered intravenously or intratumorally

[0171] C57BL / 6 mice were inoculated with 5 x 105B16E10 melanoma cells intradermally on day 0 and injected with 100 pL PBS (n=8), 33.3 pmol CBD-IL-12 (n=8), 166.5 pmol CBD- IL-12 (n=8), or 33.3 pmol CBD-IL-12 + 1.3 nmol IL-7-CBD (n=8) on day 8. The tumor sizewas calculated using the following formula: height x width x thickness x (K / 6). The mice were euthanized when the tumor size reached 1,000 m3 and / or based on humane end-point criteria.

[0172] To evaluate the effect of lymphocyte egress from the lymph nodes, the inventors injected 25 pg FTY720 (Selleck Chemical) intraperitoneally daily from days 6 to 25 after B16F10 inoculation.

[0173] To study the effects of immunological memory formation, mice that were cured from either 10 pg CBD- IL-12 monotherapy or 2 pg CBD-IL-12 + IL-7-CBD combination therapy were rechallenged with 2 x 105B16F10 tumor cells on day 60 (60 days after the primary tumor inoculation) on the contralateral side of the back.

[0174] To understand which immune cell subsets were responsible for the rejection of B16F10 rechallenge, mice that were cured with 2 pg CBD-IL-12 + IL-7-CBD combination therapy were rechallenged with 2 x 105B16F10 melanoma cells intradermally on day 60 (60 days after primary tumor inoculation) and injected intraperitoneally with IgG isotype antibody, 300 pg CD8 antibody, 300 pg CD4 antibody, and 300 pg IFNy antibody on days 59, 62, 65, and 68 (post primary tumor inoculation). The following anti- mouse antibodies were used for inhibition: IgG isotype antibody (clone: MOPC-21, BioXCell), aCD8 (clone: 2.43, BioXCell), aCD4 (clone: GK1.5, BioXCell), a.IFNy (clone: XMG1.2, BioXCell).8. Synergistic effect of CBD-IL-12 combined with IL-7-CBD administered intratumorally

[0175] For making B16F10 primary tumor model, C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and injected with 30 pL PBS (n=10), 33.3 pmol CBD-IL-12 (n=10), 333 pmol IL-7-CBD (n=10), or 33.3 pmol CBD-IL-12 + 333 pmol IL-7-CBD (n=10) on day 7. The tumor size was calculated using the following formula: height x width x thickness x (K / 6). The mice were euthanized when the tumor size reached 1,000 m3 and / or based on humane end-point criteria.9. In vivo pharmacokinetic studies of IL-7-CBD + CBD-IL-12 administered intravenously or intratumorally

[0176] C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and injected with 166.5 pmol of IL- 12, 166.5 pmol of CBD-IL-12, 666 pmol of IL-7, or 666 pmol of IL-7-CBD intravenously or intratumorally on day 9. The blood was collected at the scheduled time points for the pharmacokinetic studies. The tumors were harvested 24 hours after intravenous injection, and the tumors were harvested 72 hours after intratumoral injection for analysis of the cytokine concentration.10. Blood toxicity testing of high dose CBD-IL-12 vs CBD-IL-12 + IL-7- CBD administered intravenously

[0177] C57BL / 6 mice were treated with 100 pL PBS (n=8), 33.3 pmol CBD-IL-12 (n=8),166.5 pmol CBD- IL-12 (n=8), or 33.3 pmol CBD-IL-12 + 1.3 nmol IL-7-CBD (n=8) on day 0. Mice were bled on day 2 for the quantification of cytokine or chemokine levels (IFNy, IL-6, TNFa, CXCL9, and CXCL10) using Legendplex (Biolegend), on day 3 for blood chemistry assay (AST, ALT, amylase, and total bilirubin) using Vet Axcel Blood Chemistry Analyzer (Alfa Wasserman), and on day 4 for circulating blood cell count (white blood cells, red blood cells, and hemoglobin) using COULTER Ac*T 5diff CP hematology analyzer (Beckman). For IL- 15 superagonist experiment, the inventors used the same Legendplex kit and blood chemistry analyzer as for other toxicity evaluation experiments (83.3 pmol IL- 12, 376 pmol IL-15, and 333 pmol IL-7).11. Evaluation of cytokine and chemokine expression level in tumor

[0178] C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and injected with 30 pL PBS (n=8), 33.3 pmol CBD-IL-12 (n=8), 333 pmol IL-7- CBD, or 33.3 pmol CBD- IL- 12 + 333 pmol IL-7-CBD (n=8) on day 7. The tumors were harvested and homogenized using T- PER (ThermoFisher Scientific) with protease inhibitor tablet (Thermofisher Scientific) on day 13. The cytokine and chemokine expression levels in the tumor were analyzed using Legendplex (Biolegend).12. High-resolution microscopy of tumor infiltrating lymphocytes in B16F10 melanoma

[0179] C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and injected with 30 pL PBS (n=8), 33.3 pmol CBD-IL-12 (n=8), 333 pmol IL-7- CBD, or 33.3 pmol CBD- IL- 12 + 333 pmol IL-7-CBD (n=8) on day 7. The tumors were harvested on 13 day and incubated in 2% PFA for 2 days at 4°C. The fixed tumors were embedded in paraffin and sectioned to a thickness of 5 pm. The slide staining was performed according to the manufacturer’s protocols. Primary antibody dilution was 1:100 and secondary was 1:200. The following anti-mouse antibodies were used for imaging: CD8 Alexa Fluor 647 (clone: 53-6.7, Thermofisher Scientific), PD-1 Alexa Fluor 555 (clone: J43, Novus Biologicals), and LAG-3 Alexa Fluor 594 (clone: EPR20294-77, Abeam).13. Analysis of tumor infiltrating lymphocytes in the B16F10 melanoma

[0180] C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0 and injected with 30 pL PBS (n=8), 33.3 pmol CBD-IL-12 (n=8), 333 pmol IL-7-CBD, or 33.3 pmol CBD- IL- 12 + 333 pmol IL-7-CBD (n=8) on day 7. The tumors were harvested and digested on day 13. In brief, the tumors were cut into small pieces and then digested in 1 mL digest solution (DMEM with 2% FBS, 1 mg / mL collagenase IV (Worthington Biochemical), 3.3 mg / mL collagenase D (Sigma- Aldrich), 20 pg per mL DNase I (Worthington Biochemical), and 1.2 mM CaCh for 60 min at 37°C on a shaker. Then, the tumor mixture was quenched by 5 mM EDTA, and single cell suspension was prepared using a cell strainer (70 pm). The single cells were resuspended in DMEM with 2% FBS, and cell staining was performed according to the manufacturer’s protocols (00-5523-00, Thermo Fisher Scientific). The following anti-mouse antibodies were used for flow cytometry: CD45 Alexa Fluor (AF)532 (clone 30- Fl l, Thermo Fisher Scientific), CD3e Brilliant Ultra Violet (BUV)395 (clone 145-2C11, BD), CD4 BUV496 (clone GK1.5, BD), CD8a BUV805 (clone 53-6.7, BD), CD62E BUV737 (clone MEE- 14,

[0181] BD), NK-1.1 APC / Fire810 (clone S17061D, BioLegend), CD44 PerCp-Cy5.5 (clone IM7, BioLegend), PD-1 Brilliant Violet (BV)605 (clone 29F.1A12,, BioLegend), Lag3 BV421 (clone C9B7W, BioLegend), Tim3 BV480 (clone 5D12, BD), ICOS BV650 (clone C398.4A, BioLegend), CD25 BV785 (clone PC61, BioLegend), CD127 AF647 (clone A7R34, BioLegend), KLRG1 APC-Cy7 (clone 2F1 / KLRG1, BioLegend), CD69 PE-Dazzle (clone H1.2F3, BioLegend), Foxp3 AF488 (clone MF23, BD), Tox PE (clone TXRX10, Thermo Fisher Scientific), TCF1 Pacific Blue (clone C63D9, Cell Signaling Technologies), Ki67 PE- Cy7 (clone B56 ,BD). Viability was determined using a fixable, amine reactive dye: Live / Dead Blue Fixable Cell Stain Kit (Thermo Fisher Scientific). Cells were acquired using the Cytek Aurora spectral flow cytometer and data analysis was performed using Flow Jo (TreeStar Inc).14. Flow cytometry clustering

[0182] After spectral unmixing and conventional compensation was performed using a combination of single stained cells and compensation beads (Cat # 01-3333-42, Thermo Fisher Scientific), CD8+ T cells were manually gated as shown (FIG. 22). Equal sampling of 1800 CD8+ T cells per sample were then concatenated together into one file, and all subsequent analysis was performed. Individual samples could be identified from the concatenated file due to unique keyword identifiers added to the individual samples before concatenation. Dimensionality reduction was performed via Uniform Manifold Approximation and Projection (UMAP) while FlowSom was used for unsupervised clustering. Both UMAP and FlowSom are available as FlowJo plugins and can be freely downloaded from the FlowJo Exchange. Both dimensionality reduction and clustering were performed using the following markers: CD44,CD62L, Lag3, PD-1, Tim3, TCF1, Tox, CD69, CD127, CD25, ICOS, Ki67, KLRG1. UMAP was used to visualize the high-dimensional data into one 2-dimensional space. Unsupervised clustering was performed using FlowSom. The number of clusters was determined automatically by the algorithm, but the quality of the clustering was also visually confirmed by overlaying the FlowSom clusters on the UMAP. Thus, no modifications to the FlowSom default program were necessary for this data.15. In vitro pSTAT5 activity and IL-7 receptor (CD127) expression in mouse NK cells or CD8+ T cells

[0183] Mouse NK cells or CD8+ T cells were purified from the spleen of female C57BL / 6 mice using an EasySep mouse CD8+ T cell or NK cell isolation kit (STEMCELL Technologies). Purified mouse cells (1 x 106cells / ml) were rested in cell culture medium for 2 hours and transferred into 96-well plates (50,000 cells per well). The cell culture medium was RPMI 1640 (Gibco) containing 10% heat-inactivated FBS and 1% penicillin- streptomycin. Mouse cells were stimulated for 20 min at 37 °C with various concentrations of IL-7. Then, cells were permeabilized using BD Phosflow Perm Buffer III for 30 min in 4°C. The cells were conducted intracellular staining with pSTAT5 AF647 (clone 47, BD Biosciences) (dilution, 1:50) or cell membrane staining with CD8a BUV805 (clone 53-6.7, BD Biosciences), NK-1.1 APC / Fire810 (clone S 1706 ID, BioLegend), and CD 127 AF647 (clone A7R34, BioLegend) (dilution, 1:200) for overnight at 4°C. The cells were acquired using BD LSR flow cytometer, and data were analyzed using Flow Jo (TreeStar).16. Inhibitory effect of pulmonary metastatic melanoma

[0184] C57BL / 6 mice were inoculated with 5 x 105B16F10 melanoma cells intradermally on day 0. The tumor bearing mice were administrated 2 x 105B16F10 melanoma cells intravenously on day 7 and injected with 33.3 pmol CBD-IL-12 (n=10) or 33.3 pmol CBD-IL- 12 + 333 pmol IL-7-CBD (n=10) on day 7, 13, and 19. The lungs were harvested on day 25 for measuring the lung weight.17. Histological analysis of IL-12 + IL-7 versus CBD-IL-12 + IL-7-CBD combination therapies

[0185] C57BL / 6 mice were injected with 100 pL PBS, 33.3 pmol IL- 12 + 333 pmol IL-7, or 33.3 pmol CBD- IL-12 + 333 pmol IL-7-CBD on day 0 intravenously. Mice were bled days 0, 2, and 4 for blood toxicity assay. The major organs (heart, liver, lung, spleen, and kidney) and tumor were harvested on day 4 for histological analysis. The harvested tissues were incubated in 2% PFA for 2 days at 4°C. The fixed tumors were embedded in paraffin andsectioned to a thickness of 5 p m. H&E and CD8+ T cell staining were performed Human Tissue Resource Center at The University of Chicago. CD8+ T cell-stained area in the liver was analyzed using ImageJ.18. Melanoma antigen (TRP2)-specific immune cell population

[0186] Mice whose B16F10 melanoma tumors were cured either by 166.5 pmol CBD-IL- 12 monotherapy (10 pg CBD-IL-12 monotherapy) or 33.3 pmol CBD-IL-12 + 333 pmol IL-7- CBD combination therapy were rechallenged with 2 x 105B16F10 melanoma cells intradermally on day 0. Mice were bled on days 0, 3, and 7 after the rechallenge. Red blood cells were lysed by ACK buffer (Gibco). The single cells were resuspended in DMEM with 2% FBS, and cell staining was performed according to the manufacturer’s protocols (00-5523-00, Thermo Fisher Scientific). BD HorizonTM Brilliant Stain Buffer was used as a staining buffer. The following anti-mouse antibodies and other reagents were used for flow cytometry: CD45 APC-Cy7 (clone: 30-F11, Biolegend), CD3s FITC (clone: 500A2, Biolegend), CD4 BV605 (clone: GK1.5, Biolegend), CD8 BV421 (clone: 53-6.7, Biolegend), CD44 PerCp-Cy5.5 (clone: 1M7, Thermofisher Scientific), CD62L PE-Cy7 (clone: MEL- 14, Biolegend), PD-1 BV711 (clone: 29F.1A12, Biolegend), TRP2 pentamer APC (Proimmune), TRP2 pentamer PE (Proimmune), Live / dead aqua blue (Thermofisher Scientific). Cells were acquired using the BD LSR flow cytometer and data analysis was performed using FlowJo (TreeStar Inc).19. Therapeutic evaluation of CBD-IL-12 + IL-7-CBD combined with CPI in 4T1 breast cancer model

[0187] Balb / C mice were inoculated with 5 x 1054T1 breast cancer cells into the mammary fat pad on day 0 and injected with 100 pg aPD-1, 83.3 pmol CBD-IL-12, 666 pmol IL-7-CBD, or 83.3 pmol CBD-IL- 12 + 666 pmol IL-7-CBD on days 7, 13, 19, and 25.20. Therapeutic evaluation of CBD-IL-12 + IL-7-CBD combined with CPI in BrafV600E / Pten' / 7pCATSTAmelanoma model

[0188] Braf^^ / Pten^' / pCAT8mice were intratumorally treated with 25 pg tamoxifen on day 0 and injected intravenously with 100 pg aPD-1, 83.3 pmol CBD-IL-12, or 83.3 pmol CBD-IL-12 + 666 pmol IL-7- CBD on days 25, 32, 39, 46, and 53. Tumor size and body weight measurement were conducted twice a week, and photograph of the tumor was taken on day 53.

[0189] Although certain embodiments have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention. Further, where appropriate, aspects of any of the examples describedabove may be combined with aspects of any of the other examples described to form further examples having comparable or different properties and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. Any reference to a patent publication or other publication is a herein a specific incorporation by reference of the disclosure of that publication. The claims are not to be interpreted as including means-plus- or step-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase(s) “means for” or “step for,” respectively.References

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Claims

CLAIMS1. A polypeptide comprising an IL-7 polypeptide, or a functional fragment thereof, linked to a extracellular matrix (ECM)-affinity domain.

2. The polypeptide of claim 1, wherein the ECM-affinity domain comprises a collagen binding domain (CBD).

3. The polypeptide of claim 1 or 2, wherein the ECM-affinity domain is carboxy-proximal to the IL-7 polypeptide or functional fragment thereof.

4. The polypeptide of claim 3, wherein the ECM-affinity domain is linked, through a peptide bond, to the carboxy terminus of the IL-7 polypeptide.

5. The polypeptide of claim 1, wherein the ECM-affinity domain is amino-proximal to the IL-7 polypeptide or functional fragment thereof.

6. The polypeptide of claim 5, wherein the ECM-affinity domain is linked, through a peptide bond, to the amino terminus of the IL-7 polypeptide.

7. The polypeptide of any one of claims 1-6, wherein the polypeptide further comprises a serum protein operatively linked to the IL-7 polypeptide or ECM-affinity domain.

8. The polypeptide of claim 7, wherein the serum protein is operatively linked, through a peptide bond, to the amino or carboxy terminus of the polypeptide.

9. The polypeptide of claim 7 or 8, wherein the serum protein comprises albumin.

10. The polypeptide of claim 9, wherein the albumin comprises the amino acid sequence of one of SEQ ID NOs:20-22 or an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOs:20-22.

11. The polypeptide of any one of claims 2-10, wherein the polypeptide comprises a CBD from decorin or von Willebrand factor (VWF).

12. The polypeptide of any one of claims 1-10, wherein the ECM-affinity domain comprises a peptide from placenta growth factor-2 (P1GF-2) or CXCL-12y.

13. The polypeptide of any one of claims 1-12, wherein the ECM-affinity domain comprises a peptide having the amino acid sequence of one of SEQ ID NOS: 1-19 or a peptide with an amino acid sequence that is at least 85% identical to a fragment of one of SEQ ID NOS:1-19.

14. The polypeptide of claim 13, wherein the ECM-affinity domain comprises a peptide with the amino acid sequence of SEQ ID NO:6.

15. The polypeptide of any one of claims 1-14, wherein the IL-7 polypeptide is a human IL-7 polypeptide or derived from the human IL-7 gene or wherein the IL-7 polypeptide is a mouse IL-7 polypeptide or derived from the mouse IL-7 gene.

16. The polypeptide of any one of claims 1-15, wherein the IL-7 polypeptide comprises the amino acid sequence of SEQ ID NO:23 or 24, a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:23 or 24, or a polypeptide comprising an amino acid sequence of a fragment of SEQ ID NO:23 or 24.

17. The polypeptide of any one of claims 1-16, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:27 or 28 or a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:27 or 28.

18. The polypeptide of any one of claims 1-17, wherein the ECM-affinity domain and / or serum protein are linked to the IL-7 polypeptide through a linker.

19. The polypeptide of claim 18, wherein the linker comprises a glycine serine linker.

20. The polypeptide of claim 19, wherein the linker comprises (GGGS)n, wherein n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

21. The polypeptide of claim 20, wherein n = 2.

22. The polypeptide of any one of claims 1-21, wherein the ECM-affinity domain and / or serum protein are linked to the IL-7 polypeptide through a peptide bond.

23. The polypeptide of any one of claims 1-22, wherein the polypeptide comprises a molecular tag.

24. The polypeptide of claim 23, wherein the molecular tag comprises the amino acid sequence of SEQ ID NO:31 or 42.

25. The polypeptide of any one of claims 1-24, wherein the ratio of ECM-affinity domain to IL-7 is about 1:1 to 5:1.

26. A nucleic acid encoding the polypeptide of any one of claims 1-25.

27. A cell comprising the polypeptide of any one of claims 1-25 or the nucleic acid of claim 26.

28. A method of making a polypeptide comprising expressing the nucleic acid of claim 26 in a cell.

29. The method of claim 28, wherein the method further comprises isolating the polypeptide from the cell.

30. A method of making a cell comprising transferring the nucleic acid of claim 26 into a cell.

31. A composition comprising the polypeptide of any one of claims 1-25, the nucleic acid of claim 26, or the cell of claim 27.

32. The composition of claim 31, wherein the composition further comprises one or more polypeptide(s) comprising an IL- 12 polypeptide, or a functional fragment thereof, linked to a ECM- affinity domain.

33. The composition of claim 32, wherein the ECM-affinity domain linked to the IL- 12 polypeptide or functional fragment thereof comprises a CBD.

34. The composition of claim 32 or 33, wherein the IL- 12 polypeptide(s) comprises a p35 polypeptide, or a functional fragment thereof and a p40 polypeptide, or a functional fragment thereof.

35. The composition of any one of claims 31-34, wherein the p35 polypeptide is a human p35 polypeptide or derived from the human p35 gene; and / or wherein the p40 polypeptide is a human p40 polypeptide or derived from the human p40 gene; and / or wherein the p35 polypeptide is a mouse p35 polypeptide or derived from the mouse p35 gene; and / or wherein the p40 polypeptide is a mouse p40 polypeptide or derived from the mouse p40 gene.

36. The composition of claim 34 or 35, wherein the composition comprises a p35 polypeptide linked to an ECM-affinity domain.

37. The composition of any one of claims 34-36, wherein the composition comprises a p40 polypeptide linked to an ECM-affinity domain.

38. The composition of any one of claims 34-37, wherein the ECM-affinity domain is amino-proximal to the p40 and / or p35 polypeptide or functional fragment thereof.

39. The composition of any one of claims 34-38, wherein the ECM-affinity domain is carboxy-proximal to the p35 and / or p40 polypeptide or functional fragment thereof.

40. The composition of any one of claims 34-38, wherein the composition comprises a p35 polypeptide and a ECM-affinity domain amino-proximal to the p35 polypeptide.

41. The composition of any one of claims 34-40, wherein the composition comprises a p40 polypeptide and a ECM-affinity domain carboxy-proximal to the p40 polypeptide.

42. The composition of any one of claims 34-41, wherein the ECM-affinity domain is linked, through a peptide bond to the carboxy terminus of the p40 and / or p35 polypeptide.

43. The composition of any one of claims 34-42, wherein the polypeptide comprising the p35 polypeptide, a functional fragment of p35, p40 polypeptide, or a functional fragment of p40, linked to a ECM-affinity domain further comprises a serum protein operatively linked to the p40 and / or p35 polypeptide or ECM-affinity domain.

44. The composition of claim 43, wherein the serum protein is operatively linked, through a peptide bond, to the amino or carboxy terminus of the polypeptide comprising the p40 and / or p35 polypeptide, or a functional fragment of p40 and / or p35, linked to a ECM-affinity domain.

45. The composition of claim 43 or 44, wherein the serum protein comprises albumin.

46. The composition of claim 45, wherein the albumin comprises the amino acid sequence of one of SEQ ID NOs:20-22 or an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOs:20-22.

47. The composition of any one of claims 33-46, wherein the CBD comprises a CBD from decorin or von Willebrand factor (VWF).

48. The composition of any one of claims 32-46, wherein the ECM-affinity domain linked to the IL- 12 polypeptide, or functional fragment thereof, comprises a peptide from placenta growth factor-2 (P1GF-2) or CXCL-12y.

49. The composition of any one of claims 32-48, wherein the ECM-affinity domain linked to the IL- 12 polypeptide, or functional fragment thereof, comprises a peptide having the amino acid sequence of one of SEQ ID NOS: 1-19 or a peptide with an amino acid sequence that is at least 85% identical to a fragment of one of SEQ ID NOS: 1-19.

50. The composition of claim 49, wherein the ECM-affinity domain comprises a peptide with the amino acid sequence of SEQ ID NO:6.

51. The composition of any one of claims 31-50, wherein the composition is formulated for intravenous administration.

52. The composition of any one of claims 31-49, wherein the composition is formulated for intratumoral or peritumoral administration.

53. The composition of any one of claims 34-52, wherein the serum protein and / or ECM- affinity domain are linked to the p35 and / or p40 polypeptide through a linker.

54. The composition of claim 53, wherein the linker comprises a glycine serine linker.

55. The composition of claim 54, wherein the linker comprises (GGGS)n, wherein n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

56. The composition of claim 55, wherein n = 2.

57. The composition of any one of claims 34-56, wherein the ECM-affinity domain and / or serum protein are linked to the p35 and / or p40 polypeptide through a peptide bond.

58. The composition of any one of claims 34-57, wherein the p35 polypeptide comprises the amino acid sequence SEQ ID NO:25 or 26, a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:25 or 26, or a polypeptide with an amino acid sequence of a fragment of SEQ ID NO:25 or 26.

59. The composition of any one of claims 34-58, wherein the p40 polypeptide comprises the amino acid sequence of SEQ ID NO:43 or 44, a polypeptide with an amino acid sequence having at least 80% sequence identity to SEQ ID NO:43 or 44, or a polypeptide with an amino acid sequence that comprises a fragment of SEQ ID NO:43 or 44.

60. The composition of any one of claims 34-58, wherein the ECM-affinity domain linked to a p35 polypeptide comprises the amino acid sequence of SEQ ID NO:29 or 30 or a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:29 or 30.

61. The composition of any one of claims 34-58, wherein the ECM-affinity domain linked to a p40 polypeptide comprises the amino acid sequence of SEQ ID NO:45 or 46 or a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:45 or 46.

62. The composition of any one of claims 32-61, wherein the IL- 12 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain comprises a molecular tag.

63. The composition of claim 62, wherein the molecular tag comprises the amino acid sequence of SEQ ID NO:31 or 42.

64. The composition of any one of claims 31-62, wherein the ratio of ECM-affinity domain to IL- 12 and / or IL-7 is about 1:1 to 5:1.

65. A method for treating cancer in a subject comprising administering the polypeptide of any one of claims 1-25 or the composition of any one of claims 31-64.

66. The method of claim 65, wherein the method comprises administration of a polypeptide of any one of claims 1-25 and wherein the method excludes administration of IL- 12.

67. The method of claim 66, wherein the subject is administered a dose of 60 pg / kg or less.

68. The method of any one of claims 65 or 67, wherein the method further comprises administration a polypeptide comprising an IL- 12 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain.

69. The method of claim 68, wherein the ECM-affinity domain linked to the IL- 12 polypeptide or functional fragment thereof comprises a CBD.

70. The method of claim 68 or 69, wherein the IL- 12 polypeptide comprises a p35 polypeptide, or a functional fragment thereof and a p40 polypeptide, or a functional fragment thereof.

71. The method of claim 70, wherein the method comprises administration of a p35 polypeptide linked to an ECM-affinity domain.

72. The method of claim 70 or 71, wherein the method comprises administration of a p40 polypeptide linked to an ECM- affinity domain.

73. The method of any one of claims 70-72, wherein the ECM-affinity domain is amino- proximal to the p40 and / or p35 polypeptide or functional fragment thereof.

74. The method of any one of claims 70-73, wherein the ECM-affinity domain is carboxy- proximal to the p35 and / or p40 polypeptide or functional fragment thereof.

75. The method of any one of claims 70-74, wherein the composition comprises a p35 polypeptide and a ECM-affinity domain amino-proximal to the p35 polypeptide.

76. The method of any one of claims 70-75, wherein the composition comprises a p40 polypeptide and a ECM-affinity domain carboxy-proximal to the p40 polypeptide.

77. The method of any one of claims 70-76, wherein the ECM-affinity domain is linked, through a peptide bond to the carboxy terminus of the p40 and / or p35 polypeptide.

78. The method of any one of claims 70-77, wherein the polypeptide comprising the p35 polypeptide, a functional fragment of p35, p40 polypeptide, or a functional fragment of p40, linked to a ECM-affinity domain further comprises a serum protein operatively linked to the p40 and / or p35 polypeptide or ECM-affinity domain.

79. The method of claim 78, wherein the serum protein is operatively linked, through a peptide bond, to the amino or carboxy terminus of the polypeptide comprising the p40 and / or p35 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain.

80. The method of claim 78 or 79, wherein the serum protein comprises albumin.

81. The method of claim 80, wherein the albumin comprises the amino acid sequence of one of SEQ ID NOs:20-22 or an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOs:20-22.

82. The method of any one of claims 69-81, wherein the CBD comprises a CBD from decorin or von Willebrand factor (VWF).

83. The method of any one of claims 68-82, wherein the ECM-affinity domain linked to the IL- 12 polypeptide, or functional fragment thereof, comprises a peptide from placenta growth factor-2 (P1GF-2) or CXCL-12y.

84. The method of any one of claims 68-83, wherein the ECM-affinity domain linked to the IL- 12 polypeptide, or functional fragment thereof, comprises a peptide having the amino acid sequence of one of SEQ ID NOs:l-19 or a peptide with an amino acid sequence that is at least 85% identical to a fragment of one of SEQ ID NOs:l-19.

85. The method of claim 84, wherein the ECM-affinity domain comprises a peptide having the amino acid sequence of SEQ ID NO:6.

86. The method of any one of claims 68-85, wherein the serum protein and / or ECM-affinity domain are linked to the IL- 12 polypeptide through a linker.

87. The method of claim 86, wherein the linker comprises a glycine serine linker.

88. The method of claim 87, wherein the linker comprises (GGGS)n, wherein n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

89. The method of claim 88, wherein n = 2.

90. The method of any one of claims 68-89, wherein the ECM-affinity domain and / or serum protein are linked to the IL- 12 polypeptide through a peptide bond.

91. The method of any one of claims 70-90, wherein the p35 polypeptide comprises the amino acid sequence SEQ ID NO:25 or 26, a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:25 or 26, or a polypeptide with an amino acid sequence of a fragment of SEQ ID NO:25 or 26.

92. The method of any one of claims 70-91, wherein the p40 polypeptide comprises the amino acid sequence of SEQ ID NO:43 or 44, a polypeptide with an amino acid sequence having at least 80% sequence identity to SEQ ID NO:43 or 44, or a polypeptide with an amino acid sequence that comprises a fragment of SEQ ID NO:43 or 44.

93. The method of any one of claims 70-92, wherein the ECM-affinity domain linked to a p35 polypeptide comprises the amino acid sequence of SEQ ID NO:29 or 30 or a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:29 or 30.

94. The method of any one of claims 70-93, wherein the ECM-affinity domain linked to a p40 polypeptide comprises the amino acid sequence of SEQ ID NO:45 or 46 or a polypeptide with an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:45 or 46.

95. The method of any one of claims 68-94, wherein the IL- 12 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain comprises a molecular tag.

96. The method of claim 95, wherein the molecular tag comprises the amino acid sequence of SEQ ID NO:31 or 42.

97. The method of any one of claims 68-96, wherein the ratio of ECM-affinity domain to IL- 12 and / or IL-7 is about 1:1 to 5:1.

98. The method of any one of claims 65-97, wherein the cancer comprises melanoma, colon cancer, or breast cancer.

99. The method of any one of claims 65-98, wherein the cancer comprises metastatic cancer and / or the subject has been diagnosed with metastatic cancer.

100. The method of claim 98 or 99, wherein the breast cancer comprises triple negative breast cancer (TNBC).

101. The method of any one of claims 65-100, wherein the subject has been determined to be resistant and / or non-responsive to a prior therapy.

102. The method of any one of claims 65-101, wherein the cancer is one that is resistant to a prior therapy.

103. The method of claim 101 or 102, wherein the prior therapy comprises a nucleotide analog.

104. The method of claim 103, wherein the nucleotide analog comprises 6-thioguanine.

105. The method of any one of claims 101-104, wherein the prior therapy comprises immune checkpoint inhibitor (ICI) therapy.

106. The method of any one of claims 65-105, wherein the method comprises administration of an additional agent.

107. The method of any one of claims 65-106, wherein the subject is one that has been administered or has been prescribed administration of an additional agent.

108. The method of claim 106 or 107, wherein the additional agent comprises an immunotherapy .

109. The method of claim 108, wherein the immunotherapy comprises ICI therapy.

110. The method of any one of claims 105-109, wherein the ICI therapy comprises a monotherapy or a combination ICI therapy.

111. The method of any one of claims 105-110, wherein the ICI therapy comprises an inhibitor of PD-1, PDL1, PDL2, CTLA-4, B7-1, and / or B7-2.

112. The method of any one of claims 105-111, wherein the ICI therapy comprises an anti- PD-1 monoclonal antibody and / or an anti-CTLA-4 monoclonal antibody.

113. The method of any one of claims 105-112, wherein the ICI therapy comprises one or more of nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, pembrolizumab, pidilizumab, ipilimumab or tremelimumab.

114. The method of any one of claims 65-113, wherein the composition or polypeptide is administered systemically.

115. The method of any one of claims 65-113, wherein the composition or polypeptide is administered intratumorally or peritumorally.

116. The method of any one of claims 65-114, wherein the composition or polypeptide is administered intravenously.

117. The method of any one of claims 65-116, wherein the cancer comprises stage I, II, III, or IV cancer or wherein the subject has or has been diagnosed as having stage I, II, III, or IV cancer.

118. The method of any one of claims 65-116, wherein the cancer comprises metastatic and / or recurrent cancer or wherein the subject has or has been diagnosed as having metastatic and / or recurrent cancer.

119. The method of any one of claims 65-118, wherein the method comprises reducing T cell exhaustion, inhibiting metastasis, reducing relapse, increasing survival, reducing tumor size, reducing toxicity, and / or reducing or inhibiting immune related adverse events (irAEs).

120. The method of any one of claims 65-119, wherein the method comprises administration of a polypeptide comprising an IL-7 polypeptide, or a functional fragment thereof, linked to a ECM-affinity domain and a polypeptide comprising an IL- 12 polypeptide, or a functional fragment thereof, linked to an ECM-affinity domain, and wherein the administered dose of one or both polypeptides is less than one half of the effective dose of a monotherapy, wherein the monotherapy comprises the administration of only one of the polypeptides.

121. The method of any one of claims 65-120, wherein the subject is a human subject.

122. A composition comprising i) IL- 12 polypeptide(s) conjugated to the collagen binding domain of SEQ ID NO:6 and ii) an IL-7 polypeptide conjugated to the collagen binding domain of SEQ ID NO:6.

123. The composition of claim 122, wherein a) comprises a polypeptide comprising a collagen binding domain having the amino acid sequence of SEQ ID NO:6 conjugated to the N-terminus of the p35 subunit and of IL- 12 and a polypeptide comprising a collagen binding domain having the amino acid sequence of SEQ ID NO:6 conjugated to the C-terminus of the p40 subunit and of IL- 12; and wherein b) comprises a collagen binding domain having the amino acid sequence of SEQ ID NO:6 conjugated to the C-terminus of IL-7.

124. The composition of claim 123, wherein a) comprises i) a polypeptide having the amino acid sequence of SEQ ID NO:29 or 30 and ii) a polypeptide having the amino acid sequence of SEQ ID NO:45 or 46 and wherein b) comprises a polypeptide having the amino acid sequence of SEQ ID NO:27 or 28.

125. A method for treating a subject for cancer comprising administering to the subject the composition of any one of claims 121-124 to the subject, wherein the cancer comprises breast cancer or melanoma.

126. The method of claim 125, wherein the subject is being treated with an anti-PDl immunotherapeutic antibody.

127. The method of claim 125, wherein the method further comprises administration of an anti-PDl immunotherapeutic antibody to the subject.

128. The method of claim 126, wherein the subject is not being treated with an anti-CTLA4 antibody and wherein the cancer comprises triple negative breast cancer.

129. The method of claim 127, wherein the method excludes administration of an anti- CTLA4 antibody and wherein the cancer comprises triple negative breast cancer.

130. The method of claims 129, wherein the administered dose of at least one of the IL-7 or IL- 12 polypeptides is less than one half of the effective dose of a monotherapy, wherein the monotherapy comprises the administration of only one of an IL-7 or an IL- 12 polypeptide unconjugated to a collagen binding domain.

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