Bifunctional linear fusion collagen localization immunomodulatory molecule and method thereof

Abstract

Disclosed herein is an immunomodulatory fusion protein comprising IL-2;IL-12, a collagen binding domain, and a linear polypeptide spacer, and methods of making and using the same. Disclosed herein is an immunomodulatory fusion protein useful for treating cancer. Described herein are compounds, compositions, and methods for treating cancer. The compounds include fusion proteins comprising each of IL-2, IL-12, a collagen binding domain, and a linear polypeptide spacer.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit of and priority thereto of U.S. Provisional Patent Application No. 63 / 127,995, filed on 18 December 2020, whose disclosure is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] background Immunotherapy has revolutionized oncology by demonstrating sustained healing responses in a small number of patients, but its widespread application is limited by immune-related adverse events (irAEs) (Michot et al. 2016, Eur J Cancer, 54: 139-148). Ideally, the most potent immune activation events should be limited to tumor tissue, while healthy, non-tumor tissue should be preserved. Various tumor localization approaches have been proposed: linking immunomodulators to tumor targeting modules of immune cytokines (Hutmacher and Neri 2018, Adv Drug Deliv Rev); systemic activity of masking agents and activation of tumor-localized proteolytic degradation (Thomas and Daugherty 2009, Protein Sci 18:2053-2059); intratumoral injection of drugs (Singh and Overwijk 2015, Nat Commun 8:1447; Ager et al. 2017, Cancer Immunol Res 5:676-684; Bommareddy et al. 2017, Cancer J 23:40-47; Milling et al. 2017, Adv Drug Deliv Rev 114:79-101; Singh et al. 2017, Nat Commun 8:1447; Sagiv-Barfi et al. 2018, Sci Transl Med 10:eaan4488); peritumoral injection of solid biomaterials to capture drugs (Park et al. 2018, Sci Transl Med, 10:eaar1916); conjugation to solid particles (Kwong et al. 2013, Cancer Res 73:1547-1558) or conjugation of basic charged peptides to promote partially nonspecific adhesion of drugs to the extracellular matrix of tumor cells (Ishihara et al. 2017, Sci Transl Med 9:eaan0401; Ishihara et al. 2018, Mal Cancer Ther 17:2399-2411).A related but different approach is to localize growth factors within the tissue to regenerate it (Nishi et al. 1998, Proc Natl Acad Sci 95:7018-7023; Martino et al. 2014, Science 343:885-888; Mitchell et al. 2016, Acta Biomater 30:1-12).

[0003] Each of the current approaches described above has significant problems. Immune cytokines systemically expose immune cells to immunomodulators (Tzeng et al. 2015, Proc Natl Acad Sci 112:3320-3325). Masking agents may not mask outside the target tissue, and masking agents can complicate manufacturing and immunogenicity. Intratumoral injections often result in rapid diffusion outside the tumor compartment. Peptide conjugation to random sites is difficult to reproduce, can negatively impact specific activity, does not completely prevent tumor exit, and causes significant CMC problems due to the heterogeneity of the products of random conjugation methods. Therefore, there remains a demand for novel immunotherapeutic approaches that promote tumor localization and increase efficacy while preventing systemic toxicity. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Michot et al. 2016, Eur J Cancer, 54: 139-148 [Non-Patent Document 2] Hutmacher and Neri 2018, Adv Drug Deliv Rev. [Non-Patent Document 3] Thomas and Daugherty 2009, Protein Sci 18:2053-2059 [Non-Patent Document 4] Singh and Overwijk 2015, Nat Commun 8:1447

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Summary of the Invention

Means for Solving the Problems

[0005] Summary Described herein are compounds, compositions, and methods for treating cancer. The compounds include fusion proteins comprising each of IL-2, IL-12, a collagen-binding domain, and a linear polypeptide spacer. When administered to a subject, the compounds can have a preferred residence time within the tumor and, in some embodiments, can provide a treatment having an acceptable toxicity or an enhanced therapeutic index. In some embodiments, the collagen-binding domain binds to collagen within the tumor and maintains localization of the compound within the tumor for an extended period of time.

[0006] (i) IL-2; (ii) IL-12; (iii) a collagen-binding domain, and (iv) a linear polypeptide spacer are disclosed herein.

[0007] In various embodiments, the immunomodulatory fusion protein is linear. In various embodiments, the immunomodulatory fusion protein is a continuous chain. In various embodiments, the immunomodulatory fusion protein is a continuous polypeptide chain.

[0008] In various embodiments, IL-2 is present at the N-terminus of the immunomodulatory fusion protein. In various embodiments, IL-12 is present at the C-terminus of the immunomodulatory fusion protein. In various embodiments, IL-2 is present at the N-terminus of the immunomodulatory fusion protein and IL-12 is present at the C-terminus of the immunomodulatory fusion protein.

[0009] In various embodiments, the linear polypeptide spacer is disposed between IL-2 and the collagen-binding domain. In various embodiments, the collagen-binding domain is disposed between IL-12 and the linear polypeptide spacer.

[0010] In various embodiments, the C-terminus of IL-2 is operably connected to the N-terminus of a linear polypeptide spacer. In various embodiments, the C-terminus of IL-2 is operably connected to the N-terminus of a linear polypeptide spacer by a linker.

[0011] In various embodiments, the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of the collagen-binding domain. In various embodiments, the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of the collagen-binding domain by a linker.

[0012] In various embodiments, the C-terminus of the collagen-binding domain is operably linked to the N-terminus of IL-12. In various embodiments, the C-terminus of the collagen-binding domain is operably linked to the N-terminus of IL-12 by a linker.

[0013] In various embodiments, the collagen-binding domain is located between IL-2 and a linear polypeptide spacer. In various embodiments, the linear polypeptide spacer is located between IL-12 and the collagen-binding domain. In various embodiments, the C-terminus of IL-2 is operably ligated to the N-terminus of the collagen-binding domain.

[0014] In various embodiments, the C-terminus of IL-2 is operably linked to the N-terminus of a collagen-binding domain by a linker. In various embodiments, the C-terminus of a collagen-binding domain is operably linked to the N-terminus of a linear polypeptide spacer.

[0015] In various embodiments, the C-terminus of the collagen-binding domain is operably linked to the N-terminus of the linear polypeptide spacer by a linker. In various embodiments, the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of IL-12. In various embodiments, the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of IL-12 by a linker.

[0016] In various embodiments, IL-2 is located at the C-terminus of the immunomodulatory fusion protein. In various embodiments, IL-12 is located at the N-terminus of the immunomodulatory fusion protein. In various embodiments, IL-2 is located at the C-terminus of the immunomodulatory fusion protein, and IL-12 is located at the N-terminus of the immunomodulatory fusion protein.

[0017] In various embodiments, the N-terminus of IL-2 is operably connected to the C-terminus of a linear polypeptide spacer. In various embodiments, the N-terminus of IL-2 is operably connected to the C-terminus of a linear polypeptide spacer by a linker.

[0018] In various embodiments, the N-terminus of the linear polypeptide spacer is operably linked to the C-terminus of the collagen-binding domain. In various embodiments, the N-terminus of the linear polypeptide spacer is operably linked to the C-terminus of the collagen-binding domain by a linker.

[0019] In various embodiments, the N-terminus of the collagen-binding domain is operably linked to the C-terminus of IL-12. In various embodiments, the N-terminus of the collagen-binding domain is operably linked to the C-terminus of IL-12 by a linker.

[0020] In various embodiments, the collagen-binding domain is located between IL-2 and the linear polypeptide spacer. In various embodiments, the linear polypeptide spacer is located between IL-12 and the collagen-binding domain.

[0021] In various embodiments, the N-terminus of IL-2 is operably linked to the C-terminus of the collagen-binding domain. In various embodiments, the N-terminus of IL-2 is operably linked to the C-terminus of the collagen-binding domain by a linker.

[0022] In various embodiments, the N-terminus of the collagen-binding domain is operably linked to the C-terminus of the linear polypeptide spacer. In various embodiments, the N-terminus of the collagen-binding domain is operably linked to the C-terminus of the linear polypeptide spacer by a linker.

[0023] In various embodiments, the N-terminus of the linear polypeptide spacer is operably connected to the C-terminus of IL-12. In various embodiments, the N-terminus of the linear polypeptide spacer is operably connected to the C-terminus of IL-12 by a linker.

[0024] In various embodiments, one or more of the linkers are identical. In various embodiments, one or more of the linkers are different.

[0025] In various embodiments, IL-12 is located at the C-terminus of an immunomodulatory fusion protein and is operably linked to a collagen-binding domain, the collagen-binding domain is operably linked to a linear polypeptide spacer, the linear polypeptide spacer is operably linked to IL-2 at the N-terminus of the protein, and the protein is linear.

[0026] In various embodiments, IL-12 is located at the N-terminus of an immunomodulatory fusion protein and is operably linked to a collagen-binding domain, the collagen-binding domain is operably linked to a linear polypeptide spacer, the linear polypeptide spacer is operably linked to IL-2 at the C-terminus of the protein, and the protein is linear.

[0027] In various embodiments, IL-12 is located at the C-terminus of the immunomodulatory fusion protein and is operably linked to a linear polypeptide spacer, the linear polypeptide spacer is operably linked to a collagen-binding domain, the collagen-binding domain is operably linked to IL-2 at the N-terminus of the protein, and the protein is linear.

[0028] In various embodiments, IL-12 is located at the N-terminus of an immunomodulatory fusion protein and is operably linked to a linear polypeptide spacer, the linear polypeptide spacer is operably linked to a collagen-binding domain, the collagen-binding domain is operably linked to IL-2 at the C-terminus of the protein, and the protein is linear.

[0029] In various embodiments, the immunomodulatory fusion protein further comprises a second linear polypeptide spacer.

[0030] In various embodiments, IL-12 is located at the N-terminus of the immunomodulatory fusion protein and is operably linked to a first linear polypeptide spacer, the first linear polypeptide spacer is operably linked to a collagen-binding domain, the collagen-binding domain is operably linked to a second linear polypeptide spacer, the second linear polypeptide spacer is operably linked to IL-2 at the C-terminus of the protein, and the protein is linear.

[0031] In various embodiments, IL-12 is located at the C-terminus of the immunomodulatory fusion protein and is operably linked to a first linear polypeptide spacer, the first linear polypeptide spacer is operably linked to a collagen-binding domain, the collagen-binding domain is operably linked to a second linear polypeptide spacer, the second linear polypeptide spacer is operably linked to IL-2 at the N-terminus of the protein, and the protein is linear.

[0032] In various embodiments, the immunomodulatory fusion protein is a continuous chain. In various embodiments, the immunomodulatory fusion protein is a continuous polypeptide chain.

[0033] In various embodiments, the collagen-binding domain comprises (i) a leucine-rich repeat derived from a member of human proteoglycan class II of the low molecular weight leucine-rich proteoglycan (SLRP) family, including Lumican; or (ii) a human type I glycoprotein having an Ig-like domain selected from LAIR1 and LAIR2.

[0034] In various embodiments, the collagen-binding domain includes lumican. In various embodiments, lumican has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 11.

[0035] In various embodiments, the collagen-binding domain includes LAIR 1. In various embodiments, LAIR1 has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 13. In various embodiments, LAIR1 has at least 80% identity to the amino acids shown in SEQ ID NO: 14.

[0036] In various embodiments, the collagen-binding domain includes LAIR 2. In various embodiments, LAIR2 has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 15.

[0037] In various embodiments, IL-2 includes human IL-2. In various embodiments, IL-2 includes human wild-type IL-2. In various embodiments, IL-2 has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. In various embodiments, IL-2 has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0038] In various embodiments, IL-12 includes human IL-12. In various embodiments, IL-12 includes human wild-type IL-12. In various embodiments, IL-12 has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 5. In various embodiments, IL-12 has at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 6.

[0039] In various embodiments, the linear polypeptide spacer is albumin. In various embodiments, the linear polypeptide spacer is an albumin-binding domain. In various embodiments, the albumin includes human albumin.

[0040] In various embodiments, the albumin contains at least about 80% sequence identity to the amino acid sequences shown in SEQ ID NOs: 16-18. In various embodiments, the albumin-binding domain contains at least about 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 19.

[0041] In various embodiments, the molecular weight of the immunomodulatory fusion protein is at least about 100 to 1000 kDa.

[0042] Further disclosed herein are pharmaceutical compositions comprising one of the immunomodulatory fusion proteins disclosed herein and a pharmaceutically acceptable carrier.

[0043] A method for activating, enhancing, or promoting an immune cell response in a subject, or inhibiting, reducing, or suppressing an immune cell response in a subject, further comprising the step of administering an effective amount of any one of the pharmaceutical compositions disclosed herein to a subject in need thereof.

[0044] A method for treating cancer or reducing or inhibiting tumor growth is further disclosed herein, comprising the step of administering an effective amount of any one of the pharmaceutical compositions disclosed herein to a subject in need thereof.

[0045] In various embodiments, the subject has at least one tumor. In various embodiments, the composition is administered to at least one tumor either intratumorally (i.tu) or peritumorally (peri.tu). In various embodiments, the size of at least one tumor is reduced to or substantially the same as a reference standard. In various embodiments, the reference standard is the size of the tumor before administration.

[0046] In various embodiments, the composition is administered by injection.

[0047] In various embodiments, the composition is retained in the tumor for more than 24 hours. 1 / 2 It has.

[0048] In various embodiments, less than 25% of the injected dose is detected in the serum 12 hours after intratumor injection.

[0049] In various embodiments, at least one tumor is 1 mm 2 Each tumor has 50 or fewer stromal CD8+ cytotoxic T cells (CTLs). In various embodiments, at least one tumor is 1 mm 2 More than 50 stromal CD8+ cytotoxic T cells (CTLs) and 1 mm 2 Each cell has 500 or fewer intraepithelial compartments containing CD8+ cytotoxic T cells (CTLs). In various embodiments, at least one tumor is 1 mm 2 Each cell has more than 500 intraepithelial compartments containing CD8+ cytotoxic T cells (CTLs).

[0050] In various embodiments, the method does not result in cytokine release syndrome in the subjects. In various embodiments, the subjects do not experience grade 4 cytokine release syndrome.

[0051] Further disclosed herein are methods for reducing or inhibiting tumor growth or treating cancer in a subject, comprising the step of administering to a subject in need of such treatment an effective amount of any one of the pharmaceutical compositions disclosed herein and an effective amount of a second composition comprising (i) an antibody targeting a tumor antigen, (ii) a cancer vaccine, (iii) an immune checkpoint inhibitor, or (iv) adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

[0052] In various embodiments, the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen, and / or an antibody targeting the tumor antigen specifically binds to human HER-2 / neu, EGFR, VEGFR, CD20, CD33, CD38, or its antigen-binding fragment. In various embodiments, the cancer vaccine is a peptide containing one or more tumor-associated antigens, or a population of cells immunized with the tumor antigen in vitro and administered to a target. In various embodiments, the immune checkpoint inhibitor is an antibody or its antigen-binding fragment that binds to PD-1, PD-L1, CTLA-4, LAG3, or TIM3. In various embodiments, the immune effector cells contain a chimeric antigen receptor (CAR) molecule that binds to the tumor antigen. In certain embodiments, the immunomodulatory fusion protein described herein comprises an IL-2;IL-12;LAIR2 collagen-binding domain (LAIR2 having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 15); and albumin (albumin having at least about 80% sequence identity to the amino acid sequences shown in SEQ ID NOs: 16-18). [Brief explanation of the drawing]

[0053] [Figure 1] Figure 1 illustrates an exemplary bifunctional linear fusion collagen localization immunomodulatory construct containing IL-12, a collagen-binding domain, albumin, and IL-2.

[0054] [Figure 2-1] Figures 2A-2F are graphs showing recombinant proteins purified with NiNTA resin and evaluated for product quality using analytical size exclusion chromatography (SEC). Figure 2A shows the SEC profile of the 12-MSA-Lum-MSA-2 construct (HMW=19%; main=79%; LMW=2%). Figure 2B shows the SEC profile of the 12-Lum-MSA-2 construct (HMW=9%; main=90%; LMW=1%). Figure 2C shows the SEC profile of the 12-MSA-Lum-2 construct (HMW=34%; main=64%; LMW=2%). Figure 2D shows the SEC profile of the 12-MSA-LAIR-MSA-2 construct (HMW=11%; main=89%; LMW=0%). Figure 2E shows the SEC profile of the 12-LAIR-MSA-2 construct (HMW=11%; main=89%; LMW=1%). Figure 2F shows the SEC profile of the 12-MSA-LAIR-2 structure (HMW=16%; Main=84%; LMW=0%). [Figure 2-2] Same as above.

[0055] [Figure 3] Figure 3 is a bar graph showing the product yield and product quality of various constructs. Product yield and product quality (percentage of main peaks) are highest when only a single MSA is present in the construct, and such an MSA is located between the collagen-binding domain (lumican or LAIR) and IL-2.

[0056] [Figure 4]Figures 4A–4B are graphs showing the binding of bifunctional linear fusion immunomodulatory constructs containing collagen-binding domains to collagen as a function of concentration. Binding was determined by ELISA. Figure 4A shows a construct containing LAIR (e.g., 12-MSA-LAIR-MSA-2 construct) that yielded higher affinity binding to collagen compared to a construct containing lumican (e.g., 12-MSA-Lum-MSA-2 construct). Furthermore, placing lumican between MSA and IL-2 (e.g., 12-MSA-Lum-2 construct) enabled higher affinity binding to collagen than placing lumican between MSA and IL-2. Figure 4B shows three constructs containing LAIR, each containing a different spacer between LAIR and IL-2, namely MSA, ABD, and MSA_Mut1-2, resulting in equivalent levels of collagen binding. MSA_Mut1-2 contains the H464Q mutation, which inhibits FcRn binding.

[0057] [Figure 5A-B] Figures 5A-5B are graphs showing that the IL-2 cytokine activity of various constructs is maintained in the presence of collagen. The bioactivity of IL-2 was measured for the following: (1) IL-2 alone, (2) IL-12 alone, (3) a combination of an IL-2 monofunctional linear construct containing a collagen-binding domain and an IL-12 monofunctional linear construct containing a collagen-binding domain, and (4) two bifunctional linear constructs, each containing a collagen-binding domain: 12-Lum-MSA-2 and 12-LAIR-MSA-2. Figure 5A shows the absorbance readings of the constructs in a normal tissue culture plate. Figure 5B shows the absorbance readings of the constructs in a plate coated with collagen I (Corning).

[0058] [Figure 5C-D]Figures 5C-5D are graphs showing that the IL-2 cytokine activity of various constructs is maintained in the presence of collagen. IL-2 bioactivity was measured for three bifunctional linear constructs containing the following collagen-binding domains: (1) 12-LAIR-MSA-2, (2) 12-LAIR-MSA-2, and (3) 12-LAIR-MSA_H464Q-2, which contains the H464Q mutation that inhibits FcRn binding. Figure 5C shows the absorbance readings of the constructs in a normal tissue culture plate. Figure 5D shows the absorbance readings of the constructs in a plate coated with collagen I (Corning).

[0059] [Figure 6] Figures 6A-6B are graphs showing that the IL-12 activity of various constructs is maintained in the presence of collagen. The bioactivity of IL-12 was measured for the following: (1) IL-2 alone, (2) IL-12 alone, (3) a combination of an IL-2 monofunctional linear construct containing a collagen-binding domain and an IL-12 monofunctional linear construct containing a collagen-binding domain, and (4) two bifunctional linear constructs, each containing a collagen-binding domain: 12-Lum-MSA-2 and 12-LAIR-MSA-2. Figure 6A shows the absorbance readings of the constructs in a normal tissue culture plate. Figure 6B shows the absorbance readings of the constructs in a plate coated with collagen I (Corning).

[0060] [Figure 7A]Figure 7A is a graph of tumor growth curves (average tumor volume over time) showing tumor volume growth in C57BL / 6 mice that were inoculated with B16F10 cells and then treated on days 0 and 6 by intratumoral injection of 100 pmol of: (1) PBS, (2) a combination of IL-2 monofunctional linear construct containing MSA (MSA-2) and IL-12 monofunctional linear construct containing MSA (12-MSA), (3) a combination of IL-2 monofunctional linear construct containing MSA and collagen-binding domains (LAIR-MSA-2) and IL-12 monofunctional linear construct containing MSA and collagen-binding domains (12-MSA-LAIR), (4) a bifunctional linear construct 12-Lum-MSA-2 containing MSA and collagen-binding domains, and (5) a bifunctional linear construct 12-LAIR-MSA-2 containing MSA and collagen-binding domains.

[0061] [Figure 7B] Figure 7B is a graph showing the percentage change in body weight of C57BL / 6 mice that were inoculated with B16F10 cells and then treated on days 0 and 6 by intratumoral injection of 100 pmol of: (1) PBS, (2) a combination of IL-2 monofunctional linear construct containing MSA (MSA-2) and IL-12 monofunctional linear construct containing MSA (12-MSA), (3) a combination of IL-2 monofunctional linear construct containing MSA and a collagen-binding domain (LAIR-MSA-2) and IL-12 monofunctional linear construct containing MSA and a collagen-binding domain (12-MSA-LAIR), (4) a bifunctional linear construct 12-Lum-MSA-2 containing MSA and a collagen-binding domain, and (5) a bifunctional linear construct 12-LAIR-MSA-2 containing MSA and a collagen-binding domain.

[0062] [Figure 8]Figures 8A-8B show tumor growth curves (mean tumor volume over time) illustrating the dose-response therapeutic efficacy of 12-LAIR-MSA-2, a bifunctional linear construct containing MSA and collagen-binding domains, in a subcutaneous B16F10 melanoma syngeneic model inoculated into both flanks of C57BL / 6 mice. At all dose levels tested, the bifunctional linear construct 12-LAIR-MSA-2 demonstrated an abscopal effect, resulting in significant inhibition of tumor growth in both treated (Figure 8A) and untreated (Figure 8B) tumors.

[0063] [Figure 9] Figures 9A–9C show the efficacy and toxicity of various bifunctional constructs in the B16F10 mouse model. C57BL / 6 mice were inoculated with B16F10 cells and treated with 400 pmol of (1) PBS control, (2) 12-LAIR-MSA-2, (3) 12-LAIR-MSA_H464Q-2, (4) 12-LAIR-ABD-2, and (5) 12-Lum-MSA-2 via intratumoral injection. Figure 9A is a graph of tumor growth curves (mean tumor volume over time) showing that all bifunctional constructs tested resulted in significant inhibition of tumor growth compared to the PBS control group. Figure 9B is a graph of survival rates showing that animals treated with bifunctional constructs via intratumoral injection had extended survival rates compared to the PBS control group. Figure 9C is a graph of weight change rates showing that all bifunctional constructs tested demonstrated a good safety profile, reflecting the absence of weight loss.

[0064] [Figure 10-1]Figures 10A–10C show the efficacy and toxicity of 12-LAIR-MSA-2 in combination with the checkpoint inhibitors anti-PD1 or anti-CTLA. C57BL / 6 mice were inoculated with B16F10 cells and treated as instructed by intratumor (IT) injection of PBS or 400 pmol of 12-LAIR-MSA-2 and intraperitoneal (IP) injection of isotype control (rat IgG2a), anti-PD1 (clonal RMP1-14), or anti-CTLA4 (9D9). Figures 10A–10B show that treatment with either anti-PD1 or anti-CTLA4 alone did not affect tumor growth inhibition, treatment with the bifunctional construct 12-LAIR-MSA-2 alone resulted in significant tumor growth inhibition, and the antitumor activity of 12-LAIR-MSA-2 was further enhanced by combination with either anti-PD1 or anti-CTLA4. Figure 10C shows that adding either anti-PD1 or anti-CTLA4 to the bifunctional construct 12-LAIR-MSA-2 did not result in further weight loss compared to treatment with 12-LAIR-MSA-2 alone. [Figure 10-2] Same as above.

[0065] [Figure 11A] Figure 11A is a graph of tumor growth curves (mean tumor volume over time) showing tumor volume growth in C57BL / 6 mice inoculated with MC38 cells and then treated with PBS or 12-LAIR-MSA-2 by intratumoral injection on days 0 and 6.

[0066] [Figure 11B] Figure 11B is a graph showing the percentage change in body weight of C57BL / 6 mice inoculated with MC38 cells and then treated with PBS or 12-LAIR-MSA-2 by intratumoral injection on days 0 and 6.

[0067] [Figure 12A]Figure 12A is a graph of tumor growth curves (mean tumor volume over time) showing tumor volume growth in C57BL / 6 mice inoculated with MC38 cells and then treated with specified doses of test products (PBS, 12-LAIR-MSA-2, 12-LAIR-ABD-2, and 12-Lum-MSA-2) by intratumoral injection on days 0 and 6. Where specified, mice were treated with an isotype control (rat IgG2a) or anti-PD1 (clone RMP1-14) by intraperitoneal injection for 3 weeks in BIW.

[0068] [Figure 12B] Figure 12B is a graph showing the percentage change in body weight of C57BL / 6 mice inoculated with MC38 cells and then treated with specified doses of test products (PBS, 12-LAIR-MSA-2, 12-LAIR-ABD-2, and 12-Lum-MSA-2) by intratumoral injection on days 0 and 6. Where specified, mice were treated with an isotype control (rat IgG2a) or anti-PD1 (clone RMP1-14) by intraperitoneal injection for 3 weeks in BIW.

[0069] [Figure 13A] Figure 13A is a graph of tumor growth curves (mean tumor volume over time) showing tumor volume growth in BALB / c mice inoculated with CT6 cells and then treated with PBS or 12-LAIR-MSA-2 by intratumoral injection on days 0 and 6.

[0070] [Figure 13B] Figure 13B is a graph showing the percentage change in body weight of BALB / c mice inoculated with CT26 cells and then treated with PBS or 12-LAIR-MSA-2 by intratumoral injection on days 0 and 6.

[0071] [Figure 14A]Figure 14A is a graph of tumor growth curves (mean tumor volume over time) showing tumor volume growth in BALB / c mice inoculated with CT26 cells and then treated with specified doses of test products (PBS, 12-LAIR-MSA-2, 12-LAIR-ABD-2, and 12-Lum-MSA-2) by intratumoral injection on days 0 and 6. Where specified, mice were treated with an isotype control (rat IgG2a) or anti-PD1 (clone RMP1-14) by intraperitoneal injection for 3 weeks in BIW.

[0072] [Figure 14B] Figure 14B is a graph showing the percentage change in body weight of BALB / c mice inoculated with CT26 cells and then treated with specified doses of test products (PBS, 12-LAIR-MSA-2, 12-LAIR-ABD-2, and 12-Lum-MSA-2) by intratumoral injection on days 0 and 6. Where specified, mice were treated with an isotype control (rat IgG2a) or anti-PD1 (clone RMP1-14) by intraperitoneal injection for 3 weeks in BIW.

[0073] [Figure 15A] Figure 15A is a graph showing 12-LAIR-MSA-2 levels in the serum of C57BL / 6 mice inoculated with B16F10 cells in the right posterior flank. Seven days after inoculation (day 0), mice were randomized to a treatment group (n=10) and treated with either 400 pmol of PBS control or 12-LAIR-MSA-2 by intravenous or intratumoral injection. Serum levels of 12-LAIR-MSA-2 were measured 2 or 24 hours after administration.

[0074] [Figure 15B] Figure 15B is a graph showing interferon-gamma (INF-γ) levels 2 hours or 24 hours after administration of the 12-LAIR-MSA-2 fusion protein via intravenous (IT) or intravenous (IV) administration.

[0075] [Figure 15C]Figure 15C is a graph showing IP-10 levels 2 or 24 hours after administration of the 12-LAIR-MSA-2 fusion protein via intravenous (IT) or intravenous (IV) administration.

[0076] [Figure 15D] Figure 15D is a graph showing MCP-1 levels 2 or 24 hours after administration of the 12-LAIR-MSA-2 fusion protein via intravenous (IT) or intravenous (IV) administration.

[0077] [Figure 15E] Figure 15E is a graph showing the effectiveness of the treatment, as measured by survival rate, in mice administered 12-LAIR-MSA-2 fusion protein via initiation (IT) administration compared to intravenous administration (IV). [Modes for carrying out the invention]

[0078] Detailed explanation Cytokines that amplify and modulate immune cell responses for tumor control can exert robust synergistic effects with other immunotherapies. Two such cytokines are interleukin-2 (IL-2) and IL-12, which enlarge and stimulate T cells and natural killer (NK) cells, mediating anti-tumor immunity. Despite their promising therapeutic effects, dose-limiting toxicity in some embodiments limits the efficacy and clinical application of these cytokine therapies.

[0079] Ultimately, the therapeutic efficacy of cytokines can be improved by localizing their action to the tumor and detaching them from healthy tissue. However, even when administered directly to the tumor, cytokines rapidly leak into the systemic circulation, making it difficult to adequately address toxicity and limited efficacy issues. The compounds described herein, in some embodiments, when injected into a tumor, extend and localize their therapeutic antitumor activity while limiting their systemic spread (imiting), thereby improving efficacy while improving the safety profile. In some embodiments, the compounds bind to collagen present in many tumor types that are abundantly expressed therein. Bifunctional linear fusion construct

[0080] To devise collagen-binding cytokines, IL-2 and IL-12 were combined with collagen-binding proteins in a single fusion protein.

[0081] When administered intratumorally, the bifunctional linear immunomodulatory fusion protein containing a collagen-binding domain, IL-2, and IL-12 demonstrated reduced systemic exposure and improved therapeutic index compared to administration of either the linear immunomodulatory fusion protein containing a collagen-binding domain and IL-2, or the linear immunomodulatory fusion protein containing a collagen-binding domain and IL-12. In some embodiments, reduced systemic exposure resulted in reduced toxicity or improved therapeutic index. When administered intratumorally, the bifunctional linear immunomodulatory fusion protein containing a collagen-binding domain, IL-2, and IL-12 demonstrated reduced systemic exposure compared to administration of either the immunomodulatory fusion protein containing a collagen-binding domain and IL-2, or the immunomodulatory fusion protein containing a collagen-binding domain and IL-12. In some embodiments, reduced systemic exposure resulted in reduced toxicity or improved therapeutic index. Intratumor retention

[0082] Several factors necessitate the intratumoral retention of cytokine fusion proteins: collagen binding affinity, collagen concentration, size-dependent leakage by diffusion or convection, and consumption mediated by cytokine receptors. Increased affinity for collagen and molecular weight contribute to intratumoral retention and systemic distribution of collagen-binding fusion proteins. In some embodiments, increased affinity for collagen or increased molecular weight of the collagen-binding immunomodulatory molecule results in the therapeutic effect of a composition containing an immunomodulatory fusion protein administered to a subject by increasing intratumoral retention and decreasing systemic distribution.

[0083] Accordingly, immunomodulatory fusions to domains having a specific affinity for collagen are provided herein, which, in some embodiments, result in greater retention within specific collagen-rich tumors. In some embodiments described herein, the immunomodulatory fusion protein comprises IL-2, IL-12, and collagen-binding domains, the collagen-binding domains increasing tumor retention and reducing systemic exposure to IL-2 and IL-12 after intratumoral administration in the subject, thereby reducing treatment-related toxicity.

[0084] Unless otherwise specified in the context, it is specifically intended that the various features described herein may be used in any combination. Furthermore, in some embodiments, any feature or combination of features shown herein may be excluded or omitted. For example, where it is stated herein that a complex consists of components A, B, and C, it is specifically intended that A, B, C, or any combination thereof may be omitted and discarded individually or in any combination. definition

[0085] As used herein, the following terms are intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0086] As used herein and in the claims, the singular forms “a,” “an,” and “the” should be noted as including multiple references unless the context explicitly indicates otherwise.

[0087] As used herein, “and / or” encompasses any possible combination of one or more of the enumerated items relating to each other, and “reder to” which refers to the absence of a combination when interpreted as an alternative ("or"). Furthermore, any feature or combination of features shown herein may be excluded or omitted.

[0088] When used herein, the term "about" means, for example, when referring to measurable values ​​such as the amount, dose, time, or temperature of a compound or drug, it encompasses a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of a particular quantity.

[0089] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0090] As used herein, the term “fusion protein” refers to a protein produced by conjugating two or more elements, components, or domains and / or polypeptides to create a larger polypeptide. As used herein, the terms “linked,” “operably linked,” “fused,” and “fusion” are interchangeable and refer to the conjugation of two or more elements, components, domains, and / or polypeptides within a fusion protein that, when expressed in the fusion protein, enables at least one of the elements, components, domains, and / or polypeptides to have at least some of the biological function or cellular activity as it does in its native state and / or when expressed without conjugation. The conjugation of two or more elements, components, or domains can be carried out by any means known in the art, including chemical conjugation, non-covalent complex formation, or recombination means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art. Therefore, elements, components, domains, and / or polypeptides can be joined by covalent bonds (e.g., peptide bonds) or non-covalent bonds. Elements, components, domains, and / or polypeptides can be joined by peptide bond formation within ribosomes during or after translation.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terms used herein are intended to describe only specific embodiments and are not intended to be limiting. immunomodulatory fusion protein

[0092] As used herein, the term “immunomodulatory fusion protein” refers to a polypeptide comprising collagen-binding domains operably linked to IL-2 and IL-12. In some embodiments, the collagen-binding domains are operably linked to IL-2 and IL-12 by linear polypeptide spacers. In some embodiments, the collagen-binding domains are operably linked to IL-2 and IL-12 by linear polypeptide spacers. In some embodiments, the collagen-binding domains are operably linked to IL-2 and IL-12 by linkers.

[0093] In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a collagen-binding domain that is operably linked to an IL-2 and IL-12. In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a collagen-binding domain that is operably linked to IL-2 and IL-12 by a linear polypeptide spacer. In some embodiments, the immunomodulatory fusion protein further comprises a linker. In some embodiments, the immunomodulatory fusion protein further comprises a plurality of linkers. I. Collagen-binding domain

[0094] In some embodiments, the disclosure provides an immunomodulatory fusion protein comprising a collagen-binding domain. In some embodiments, the collagen-binding domain has a MW of approximately 5–1,000 kDa, approximately 5–100 kDa, approximately 10–80 kDa, approximately 20–60 kDa, approximately 30–50 kDa, or approximately 10 kDa, approximately 20 kDa, approximately 30 kDa, approximately 40 kDa, approximately 50 kDa, approximately 60 kDa, approximately 70 kDa, approximately 80 kDa, approximately 90 kDa, or approximately 100 kDa. In some embodiments, the collagen-binding domain is approximately 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, or 1,000 kDa. In some embodiments, the collagen-binding domain is approximately 30 kDa. In some embodiments, the collagen-binding domain is approximately 40 kDa.

[0095] In some embodiments, the collagen-binding domain is approximately 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10-50, or 10-20 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 10 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 15 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 20 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 30 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 40 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 50 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 60 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 70 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 80 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 90 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 100 amino acid lengths. In some embodiments, the collagen-binding domain is approximately 120 amino acids long. In some embodiments, the collagen-binding domain is approximately 150 amino acids long. In some embodiments, the collagen-binding domain is approximately 200 amino acids long. In some embodiments, the collagen-binding domain is approximately 250 amino acids long. In some embodiments, the collagen-binding domain is approximately 300 amino acids long. In some embodiments, the collagen-binding domain is approximately 350 amino acids long.

[0096] In some embodiments, the collagen-binding domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) leucine-rich repeats that bind to collagen. In some embodiments, the collagen-binding domain comprises a proteoglycan. In some embodiments, the collagen-binding domain comprises a proteoglycan, which is selected from the group consisting of decorin, biglycan, testican, bikunin, fibromodulin, lumican, chondroadherin, keratin, ECM2, epilytican, asporin, PRELP, keratocan, osteoadherin, optisin, osteoglycan, nictaropine, tsukushi, podocan, podocan-like protein 1 versican, perlecan, nidogen, neurocan, agrecan, and brevican.

[0097] In some embodiments, the collagen-binding domain comprises a class I low molecular weight leucine-rich proteoglycan (SLRP). In some embodiments, the collagen-binding domain comprises a class II SLRP. In some embodiments, the collagen-binding domain comprises a class III SLRP. In some embodiments, the collagen-binding domain comprises a class IV SLRP. In some embodiments, the collagen-binding domain comprises a class V SLRP. A further description of the SLRP classes is disclosed in Schaefer & Iozzo (2008) J Biol Chem 283(31):21305-21309, which is incorporated herein by reference in its entirety.

[0098] In some embodiments, the collagen-binding domain comprises one or more leucine-rich repeats derived from members of the human proteoglycan class II of the low molecular weight leucine-rich proteoglycan (SLRP) family. In some embodiments, the SLRP is selected from lumican, decorin, biglycan, fibromodulin, keratin, epigenican, asporin, and osteoglycin.

[0099] "k d ”(seconds -1The term "k off " refers to the dissociation rate constant of a specific protein-protein interaction. This value is also referred to as the k off value.

[0100] "k a " (M -1 × sec -1 ) refers to the association rate constant of a specific protein-protein interaction. This value is also referred to as the k a value. -1 × sec -1 "K D " (M) refers to the dissociation equilibrium constant of a specific protein-protein interaction. K D = k d / k a . In some embodiments, the affinity of a protein (e.g., binding domain) is described in terms of K D for the interaction between two proteins. For clarity, as is known in the art, a lower K D value indicates a higher affinity interaction, and a higher K D value indicates a lower affinity interaction. on value.

[0101] "K D " (M) refers to the dissociation equilibrium constant of a specific protein-protein interaction. K D = k d / k a . In some embodiments, the affinity of a protein (e.g., binding domain) is described in terms of K D for the interaction between two proteins. For clarity, as is known in the art, a lower K D value indicates a higher affinity interaction, and a higher K D value indicates a lower affinity interaction. D "K D " (M) refers to the dissociation equilibrium constant of a specific protein-protein interaction. K D = k d / k a . In some embodiments, the affinity of a protein (e.g., binding domain) is described in terms of K D for the interaction between two proteins. For clarity, as is known in the art, a lower K D value indicates a higher affinity interaction, and a higher K D value indicates a lower affinity interaction. D = k d / k a In some embodiments, the affinity of a protein (e.g., binding domain) is described in terms of K D for the interaction between two proteins. For clarity, as is known in the art, a lower K D value indicates a higher affinity interaction, and a higher K D value indicates a lower affinity interaction. D For clarity, as is known in the art, a lower K D value indicates a higher affinity interaction, and a higher K D value indicates a lower affinity interaction. D value indicates a higher affinity interaction, and a higher K D D value indicates a lower affinity interaction.

[0102] In some embodiments, when measured by suitable methods known in the art for determining protein binding affinity, such as ELISA, surface plasmon resonance (BIAcore), FACS analysis, etc., the collagen binding domain binds to collagen (e.g., type 1 or type 3 collagen) with a binding affinity K D value of 0.1 - 1,000 nM. In some embodiments, when determined by suitable methods known in the art, the collagen binding domain has a binding affinity K D value of 0.1 - 1.0 nM, 1.0 - 10 nM, 10 - 20 nM, 20 - 30 nM, 30 - 40 mM, 40 - 50 nM, 50 - 60 nM, 70 - 80 nM, 90 - 100 nM, 10 - 50 nM, 50 - 100 nM, 100 - 1,000, or 1,000 - 10,000 nM. D value for binding to collagen (e.g., type 1 or type 3 collagen). In some embodiments, when determined by suitable methods known in the art, the collagen binding domain has a binding affinity K D value of 0.1 - 1.0 nM, 1.0 - 10 nM, 10 - 20 nM, 20 - 30 nM, 30 - 40 mM, 40 - 50 nM, 50 - 60 nM, 70 - 80 nM, 90 - 100 nM, 10 - 50 nM, 50 - 100 nM, 100 - 1,000, or 1,000 - 10,000 nM. DIt binds to collagen in a specific value. In some embodiments, the immunomodulatory fusion protein has a binding affinity K of 0.1-1.0 nM, 1.0-10 nM, 10-20 nM, 20-30 nM, 30-40 mM, 40-50 nM, 50-60 nM, 70-80 nM, 90-100 nM, 10-50 nM, 50-100 nM, 100-1,000, or 1,000-10,000 nM, as determined by preferred methods known in the art. D It binds to collagen by value. In some embodiments, the collagen-binding domain binds to a trimer peptide containing a repeating GPO triplet. In some embodiments, the collagen-binding domain binds to a common collagen motif in a hydroxyproline-dependent manner. A. Rumikan

[0103] Lumican, also known as LUM, is an extracellular matrix protein encoded by the LUM gene on chromosome 12 in humans (Chakravarti et al., (1995) Genomics 27(3):481-488). Lumican is a member of proteoglycan class II of the small leucine-rich proteoglycan (SLRP) family, which also includes decorin, biglycan, fibromodulin, keratocan, epigenican, and osteoglycin (Iozzo & Schaefer (2015) Matrix Biology 42: 11-55). Lumican is a stable protein that specifically binds to collagen type I and type IV.

[0104] Lumican has a molecular weight of approximately 40 kDa and possesses four major intramolecular domains: 1) a 16-amino acid signal peptide, 2) a negatively charged N-terminal domain containing sulfated tyrosine and disulfide bonds, 3) a 10-tandem leucine-rich repeat that binds lumican to collagen, and 4) a 50-amino acid carboxyl-terminal domain containing two conserved cysteines 32 residues apart. (Kao et al., (2006) Experimental Eye Research 82(1):3-4). Four N-linking sites are located within the leucine-rich repeat domain of the protein core, which can be substituted with keratan sulfate. The core protein of lumican (like decorin and fibromodulin) is horseshoe-shaped. This allows lumican to bind to collagen molecules within collagen fibrils, thus helping to keep adjacent fibrils detached, Scott (1996) Biochemistry 35(27): 8795-8799.

[0105] In some embodiments, the collagen-binding domain comprises a class II low molecular weight leucine-rich proteoglycan (SLRP). A further description of the SLRP class is disclosed in Schaefer & Iozzo (2008) J Biol Chem 283(31):21305-21309, which is incorporated herein by reference in its entirety. In some embodiments, the collagen-binding domain comprises one or more leucine-rich repeats derived from members of the human proteoglycan class II of the low molecular weight leucine-rich proteoglycan (SLRP) family. In some embodiments, the SLRP is lumican. In some embodiments, the lumican is human lumican. In some embodiments, lumican includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 11.

[0106] In some embodiments, Lumican is a variant that includes one or more amino acid substitutions, additions, or deletions, optionally two, three, four, five, six, seven, eight, nine, ten, or more, compared to the Lumican protein containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the Lumican variant has increased collagen binding affinity compared to the collagen binding affinity of the Lumican protein containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the Lumican variant has decreased collagen binding affinity compared to the collagen binding affinity of the Lumican protein containing the amino acid sequence of SEQ ID NO: 11. B. LAIR1 and LAIR2

[0107] Leukocyte-associated immunoglobulin-like receptors (LAIR- and LAIR-2): Leukocyte-associated IgG-like receptor (LAIR)-1 is a collagen receptor that inhibits immune cell function upon collagen binding. Following LAIR-I, the human genome encodes LAIR-2, a soluble homolog. Human (h)LAIR-I is expressed in the majority of PBMCs and thymocytes (Maasho et al., (2005) Mal Immunol 42: 1521-1530). In vitro crosslinking of LAIR-1 with mAbs delivers a potent inhibitory signal capable of inhibiting immune cell function. Collagen is known to be a native, high-affinity ligand for the LAIR molecule. The interaction of hLAIR-1 with collagen directly inhibits immune cell activation in vitro (Meyaard et al., (1997) Immunity 7:283-290; Poggi (1998) Eur J Immunol 28:2086-2091; Van der Vuurst de Vries et al., (1999) Eur J Immunol 29:3160-3167; Lebbink et al., (2006) J Exp Med 203:1419-1425).

[0108] In some embodiments, the collagen-binding domain includes a human type I glycoprotein having an Ig-like domain, or its extracellular portion that binds to collagen. In some embodiments, the type I glycoprotein competes with lumican for binding to type I collagen. In some embodiments, the human type I glycoprotein is selected from LAIR, LAIR1, and LAIR2.

[0109] In some embodiments, the human type I glycoprotein is LAIR1. In some embodiments, LAIR1 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the human type I glycoprotein is LAIR1, and the collagen-binding domain includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with amino acid residues 22-122 of the amino acid sequence shown in SEQ ID NO: 13.

[0110] In some embodiments, the human type I glycoprotein is LAIR1. In some embodiments, LAIR1 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 14.

[0111] In some embodiments, LAIR includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 12.

[0112] In some embodiments, LAIR1 is a variant that includes one or more amino acid substitutions, additions, or deletions, optionally two, three, four, five, six, seven, eight, nine, ten, or more, compared to the LAIR1 protein containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, the LAIR1 variant has increased collagen binding affinity compared to the collagen binding affinity of the LAIR1 protein containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, the LAIR1 variant has decreased collagen binding affinity compared to the collagen binding affinity of the LAIR1 protein containing the amino acid sequence of SEQ ID NO: 13.

[0113] In some embodiments, LAIR1 is a variant that includes one or more amino acid substitutions, additions, or deletions, optionally two, three, four, five, six, seven, eight, nine, ten, or more, compared to the LAIR1 protein containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the LAIR1 variant has increased collagen binding affinity compared to the collagen binding affinity of the LAIR1 protein containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the LAIR1 variant has decreased collagen binding affinity compared to the collagen binding affinity of the LAIR1 protein containing the amino acid sequence of SEQ ID NO: 14.

[0114] In some embodiments, LAIR is a variant that includes one or more amino acid substitutions, additions, or deletions, optionally two, three, four, five, six, seven, eight, nine, ten, or more, compared to the LAIR protein containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the LAIR1 variant has increased collagen binding affinity compared to the collagen binding affinity of the LAIR protein containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the LAIR variant has decreased collagen binding affinity compared to the collagen binding affinity of the LAIR protein containing the amino acid sequence of SEQ ID NO: 12.

[0115] In some embodiments, the human type I glycoprotein is LAIR2. In some embodiments, LAIR2 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 15.

[0116] In some embodiments, LAIR2 is a variant that includes one or more amino acid substitutions, additions, or deletions, optionally two, three, four, five, six, seven, eight, nine, ten, or more, compared to the LAIR2 protein containing the amino acid sequence of SEQ ID NO: 15. In some embodiments, the LAIR2 variant has increased collagen binding affinity compared to the collagen binding affinity of the LAIR2 protein containing the amino acid sequence of SEQ ID NO: 15. In some embodiments, the LAIR2 variant has decreased collagen binding affinity compared to the collagen binding affinity of the LAIR2 protein containing the amino acid sequence of SEQ ID NO: 15. [Table 1-1] [Table 1-2] II. Immunomodulatory Domains

[0117] The immunomodulatory fusion proteins disclosed herein comprise at least one IL-2 and at least one IL-12. In certain embodiments, the immunomodulatory fusion proteins disclosed herein comprise IL-2, IL-12, and a collagen-binding domain. In certain embodiments, the immunomodulatory fusion proteins disclosed herein comprise IL-2, IL-12, a collagen-binding domain, and at least one linear polypeptide spacer. In some embodiments, IL-2 is operably linked to the collagen-binding domain. In some embodiments, IL-2 is operably linked to the linear polypeptide spacer. In some embodiments, IL-12 is operably linked to the collagen-binding domain. In some embodiments, IL-12 is operably linked to the linear polypeptide spacer. A.IL-2

[0118] As used herein, “interleukin (IL)-2” (IL-2) refers to a pleomorphic cytokine that activates T cells and natural killer (NK) cells and induces their proliferation. The biological activity of IL-2 is mediated through the multi-subunit IL-2 receptor complex (IL-2R), which consists of three polypeptide subunits across the cell membrane: p55 (IL-2Rα, alpha subunit, also known as CD25 in humans), p75 (IL-2Rβ, beta subunit, also known as CD122 in humans), and p64 ​​(IL-2Rγ, gamma subunit, also known as CD132 in humans).

[0119] In some embodiments, the immunomodulatory fusion protein includes IL-2. In some embodiments, IL-2 is operably linked to a collagen-binding domain. In some embodiments, the immunomodulatory fusion protein includes a member of the IL-2 family operably linked to a collagen-binding domain.

[0120] The T cell response to IL-2 depends on various factors, including: (1) the concentration of IL-2; (2) the number of IL-2R molecules on the cell surface; and (3) the number of IL-2R molecules dedicated to IL-2 (i.e., the affinity of the binding interaction between IL-2 and IL-2R (Smith, "Cell Growth Signal Transduction is Quanta!" In Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)).

[0121] In some embodiments, IL-2 is wild-type IL-2 (e.g., its precursor form, human IL-2, or mature IL-2). In some embodiments, IL-2 is human IL-2. In some embodiments, IL-2 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 1 or 2. In some embodiments, IL-2 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4.

[0122] In other embodiments, IL-2 is a mutant human IL-2. The terms “IL-2 mutant” or “mutant IL-2 polypeptide,” as used herein, are intended to encompass any mutant form of the various forms of the IL-2 molecule, including full-length IL-2, the truncated form of IL-2, and forms in which IL-2 is linked to another molecule by fusion or chemical conjugation, etc. Various forms of IL-2 mutants are characterized by having at least one amino acid mutation that affects the interaction of IL-2 with CD25. This mutation may involve substitution, deletion, cleavage, or modification of a wild-type amino acid residue that is normally located in that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise specified, IL-2 mutants may be referred to herein as IL-2 mutant peptide sequences, IL-2 mutant polypeptides, IL-2 mutant proteins, or IL-2 mutant analogs.

[0123] In some embodiments, the IL-2 mutant contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 or 2 that binds to CD25. For example, in some embodiments, the IL-2 mutant has at least one mutation (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) that increases affinity for the alpha subunit of the IL-2 receptor compared to wild-type IL-2. It should be understood that the mutations identified in mouse IL-2 may be made at the corresponding residues of full-length human IL-2 (nucleic acid sequence (accession: NM000586); amino acid sequence (accession: P60568)) or human IL-2 that does not contain the signal peptide. Therefore, in some embodiments, IL-2 is human IL-2. In other embodiments, IL-2 is mutant human IL-2. The amino acid sequence of human IL-2 (SEQ ID NO: 1; full length) can be found in the Genbank accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is shown in SEQ ID NO: 2 (human wild-type mature). The amino acid sequence of mouse (Mus musculus) IL-2 can be found in the Genbank accession locator (SEQ ID NO: 3). The amino acid sequence of mature mouse IL-2 is shown in SEQ ID NO: 4.

[0124] In certain embodiments, IL-2 is mutated to alter its affinity for the IL-2R alpha receptor compared to unmodified IL-2 (e.g., reduced affinity). Site-directed mutagenesis can be used to isolate an IL-2 mutant, i.e., IL-2Ra, that exhibits reduced binding affinity to CD25 compared to wild-type IL-2. Increasing the affinity of IL-2 for IL-2Ra on the cell surface increases receptor occupancy within a limited range of IL-2 concentrations, and similarly increases the local concentration of IL-2 on the cell surface.

[0125] In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include: L80F, R81D, L85V, I86V, and I92F. [Table 2-1] [Table 2-2] B.IL-12

[0126] Interleukin-12 (IL-2) plays a crucial role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16: 495-521 (1998). IL-12 primarily functions as a 70 kDa heterodimeric protein composed of two disulfide-linked p35 and p40 subunits. The precursor form of the IL-12 p40 subunit (NM_002187; P29460; also known as IL-12B, natural killer cell-stimulating factor 2, or cytotoxic lymphocyte maturation factor 2) is 328 amino acids long, while its mature form is 306 amino acids long. The precursor form of the IL-12 p35 subunit (NM_000882; P29459; also known as IL-12A, natural killer cell-stimulating factor 1, or cytotoxic lymphocyte maturation factor 1) is 219 amino acids long, while the mature form is 197 amino acids long.

[0127] In some embodiments, the immunomodulatory fusion protein includes IL-12. In some embodiments, the immunomodulatory fusion protein includes IL-12 operably linked to a collagen-binding domain.

[0128] In some embodiments, IL-12 includes IL-12A (e.g., SEQ ID NO: 6). In some embodiments, IL-12 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of IL-12A shown in SEQ ID NO: 6.

[0129] In some embodiments, IL-12 includes IL-12A (e.g., SEQ ID NO: 8). In some embodiments, IL-12 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of IL-12A shown in SEQ ID NO: 8.

[0130] In some embodiments, IL-12 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of IL-12A shown in SEQ ID NO: 10.

[0131] In some embodiments, IL-12 includes IL-12B (e.g., SEQ ID NO: 5). In some embodiments, IL-12 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of IL-12B shown in SEQ ID NO: 5.

[0132] In some embodiments, IL-12 includes IL-12B (e.g., SEQ ID NO: 7). In some embodiments, IL-12 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of IL-12B shown in SEQ ID NO: 7. In some embodiments, IL-12 includes IL-12B (e.g., SEQ ID NO: 7).

[0133] In some embodiments, IL-12 includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of IL-12B shown in SEQ ID NO: 9.

[0134] In some embodiments, IL-12 comprises both IL-12A and IL-12B. In some embodiments, IL-12 comprises both IL-12A and IL-12B and a linker. In some embodiments, the immunomodulatory fusion protein comprises IL-12 comprising the amino acid sequences shown in SEQ ID NOs. 5-10. In some embodiments, IL-12 comprises an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequences of IL-12A and IL-12B shown in SEQ ID NOs. 5-10.

[0135] The terms “IL-12 variant” or “variant IL-12 polypeptide,” as used herein, are intended to encompass any variant forms of the various forms of the IL-12 molecule, including full-length IL-12, cleaved forms of IL-12, and forms in which IL-12 is linked to another molecule by fusion or chemical conjugation, etc. Various forms of IL-12 variants are characterized by having at least one amino acid mutation. This mutation may involve substitution, deletion, cleavage, or modification of a wild-type amino acid residue that is normally located in that position. Variants obtained by amino acid substitution are preferred. Unless otherwise specified, IL-12 variants may be referred to herein as IL-12 variant peptide sequences, IL-12 variant polypeptides, IL-12 variant proteins, or IL-12 variant analogs. [Table 3-1] [Table 3-2] III. Linear Polypeptide Spacer

[0136] In some embodiments, the linear polypeptide spacer is a polypeptide containing an "N" amino acid length, where N = 1 to 1000, 50 to 800, 100 to 600, or 200 to 500. In some embodiments, the linear polypeptide spacer contains about 1 to about 100 amino acid residues. In some embodiments, the linear polypeptide spacer contains more than 100 amino acid residues. In certain embodiments, the linear polypeptide spacer contains about 1 to about 100 amino acid residues.

[0137] In some embodiments, the linear polypeptide spacer is a soluble polypeptide. In some embodiments, the linear polypeptide spacer has a molecular weight between 1 and 200 kDa. In some embodiments, the linear polypeptide spacer has molecular weights of 1-10 kDa, 10-20 kDa, 20-30 kDa, 30-40 kDa, 40-50 kDa, 50-60 kDa, 60-70 kDa, 70-80 kDa, 80-90 kDa, 90-100 kDa, 100-110 kDa, 110-120 kDa, 120-130 kDa, and 130-140 kDa. It has a molecular weight of Da, 140-150 kDa, 150-160 kDa, 160-170 kDa, 170-180 kDa, 180-190 kDa, 190-200 kDa, 10-100, 100-200 kDa, 200-300 kDa, 300-400 kDa, 400-500 kDa, 500-1,000 kDa, or 100-1,000 kDa.

[0138] In certain embodiments, a linear polypeptide spacer results in steric separation between one element of a fusion protein and another. In certain embodiments, a linear polypeptide spacer results in steric separation between one domain of a fusion protein and another. In some embodiments, a linear polypeptide spacer between IL-2 and a collagen-binding protein results in steric separation so that IL-2 retains its activity (e.g., promotes receptor / ligand engagement). In some embodiments, a linear polypeptide spacer between IL-12 and a collagen-binding protein results in steric separation so that IL-12 retains its activity (e.g., promotes receptor / ligand engagement). In certain embodiments, a linear polypeptide spacer between IL-2 and a collagen-binding protein and / or IL-12 and a collagen-binding protein results in steric separation so that IL-2 and / or IL-12 bind to the same receptor on the same cell (wuch that). In certain embodiments, a linear polypeptide spacer between IL-2 and collagen-binding protein and / or IL-12 and collagen-binding protein results in steric separation so that IL-2 and / or IL-12 bind to receptors on different cells.

[0139] In some embodiments, the linear polypeptide spacer between IL-2 and the collagen-binding protein is of sufficient length or mass to reduce the adsorption of the immunomodulatory domain to the collagen fibrils. In some embodiments, the linear polypeptide spacer between IL-12 and the collagen-binding protein is of sufficient length or mass to reduce the adsorption of the immunomodulatory domain to the collagen fibrils. Methods for measuring adsorption are known to those skilled in the art. For example, adsorption can be measured by ellipsometry (ELM), surface plasmon resonance (SPR), optical waveguide light-mode spectroscopy (OWLS), attenuated total internal reflection infrared spectroscopy (ATR-IR), circular dichroism (CD), total internal reflection infrared spectroscopy (TIRF), and other high-resolution microscopy techniques. In some embodiments, these methods indicate the spatial arrangement between the domains of the immunomodulatory fusion protein.

[0140] In certain embodiments, the linear polypeptide spacer provides one of several functional benefits, including but not limited to: i) separating IL2 and IL12, allowing both cytokines to access either receptor on the same cell or on separate cells; ii) separating collagen from IL2, improving the geometric shape of their interaction in vivo; iii) increasing the hydrodynamic radius of the fusion construct, thereby slowing the burst release rate upon administration by utilizing size exclusion; and / or iv) stabilizing and / or improving the solubility of domains that are relatively insoluble. In certain embodiments, the linear polypeptide spacer improves the retention of the fusion product in target tissue upon administration to a subject.

[0141] In some embodiments, a linear polypeptide spacer between IL-2 and collagen-binding protein provides a molecular weight sufficient to slow or reduce diffusion from tissue. In some embodiments, a linear polypeptide spacer between IL-12 and collagen-binding protein provides a molecular weight sufficient to slow or reduce diffusion from tissue. Methods for measuring diffusion from tissue are known to those skilled in the art. For example, diffusion can be measured by in vivo imaging or by microscopic examination of tissue sections over time. Exemplary methods are described at least in Schmidt & Wittrup, Mol. Canc. Ther. 2009' and Wittrup et al., Methods in Enzymol 2012, which are incorporated herein by reference in their entirety. albumin

[0142] The term "albumin" refers to a protein that has a three-dimensional structure identical to or very similar to human albumin (SEQ ID NO: 16) and has a long serum half-life. Exemplary albumin proteins include human serum albumin (HSA; SEQ ID NOs: 17 and 18), primate serum albumin (such as chimpanzee serum albumin), gorilla serum albumin or macaque serum albumin, rodent serum albumin (such as hamster serum albumin), guinea pig serum albumin, mouse serum albumin and rat serum albumin, bovine serum albumin (such as cow serum albumin), equine serum albumin (such as horse serum albumin or donkey serum albumin), rabbit serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, chicken serum albumin and pig serum albumin.

[0143] In some embodiments, the linear polypeptide spacer is albumin, an albumin binder, an albumin-binding domain, or an albumin mutation. In some embodiments, the linear polypeptide spacer comprises albumin or a fragment thereof. In some embodiments, the linear polypeptide spacer is human albumin. In some embodiments, the albumin is serum albumin, e.g., human serum albumin (SEQ ID NO: 17). In some embodiments, the linear polypeptide spacer is an albumin-binding domain.

[0144] In some embodiments, the linear polypeptide spacer includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of human albumin shown in SEQ ID NO: 16.

[0145] In some embodiments, the linear polypeptide spacer includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of human serum albumin shown in SEQ ID NO: 17.

[0146] In some embodiments, the linear polypeptide spacer includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of human serum albumin shown in SEQ ID NO: 18.

[0147] In some embodiments, albumin is a variant that, compared to the albumin protein containing the amino acid sequences of SEQ ID NOs. 16-18, includes one or more amino acid substitutions, additions, or deletions, optionally two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions, additions, or deletions. In some embodiments, the albumin mutation includes at least one amino acid mutation compared to wild-type albumin. This mutation may involve substitution, deletion, cleavage, or modification of a wild-type amino acid residue that is normally located in that position.

[0148] In certain embodiments, the linear polypeptide is a serum protein-binding domain. In some embodiments, the linear polypeptide spacer is an albumin-binding domain. In some embodiments, the linear polypeptide spacer contains an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of the albumin-binding domain shown in SEQ ID NO: 19. In some embodiments, the albumin-binding domain, upon administration to a subject, binds non-covalently to serum albumin. In some embodiments, the albumin-binding domain demonstrates a non-covalent means of enhancing the hydrodynamic radius of the fusion construct in situ. In certain embodiments, the albumin-binding domain, when administered to a subject, improves the retention of the fusion construct in the target tissue. [Table 4-1] [Table 4-2] IV. Linker

[0149] In certain embodiments, the fusion protein described herein includes one or more linkers. In certain embodiments, a linker connects one element of the fusion protein to another element. In certain embodiments, a linker connects one domain of the fusion protein to another domain. In certain embodiments, the fusion protein described herein includes one, two, three, four, five or more linkers. In some embodiments, the linker consists of "short" amino acid residues, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Thus, in certain cases, the linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the linker is a peptide bond. In some embodiments, the linker consists of about 3 to about 50 consecutive amino acid residues, e.g., 8, 9, or 10. In some embodiments, the linker contains 0 to about 100 amino acid residues. In some embodiments, the linker contains about 5 to about 50 amino acid residues. In some embodiments, the linker contains about 5 to about 15 amino acid residues. In certain embodiments, the linker is a non-peptide linker. In certain embodiments, the linker covalently connects one element of the fusion protein to another element. In certain embodiments, the linker noncovalently connects one element of the fusion protein to another element. In certain embodiments, the fusion protein described herein contains two or more types of linkers and / or two or more linkers of the same or different lengths (e.g., number of amino acid residues). Peptide linker

[0150] Exemplary linkers include gly-ser polypeptide linkers, glycine-praline polypeptide linkers, and praline-alanine polypeptide linkers. In certain embodiments, the linear polypeptide spacer is a gly-ser polypeptide linker, i.e., a peptide consisting of a glycine residue and a serine residue.

[0151] In some embodiments, the linker is a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids, which are described herein or known in the art. A preferred non-immunogenic linker peptide is, for example, (G4S) n (SG4) n Or G4 (SG4) n It contains a linker peptide, where n is generally a number between 1 and 10, typically between 2 and 4.

[0152] An example of a gly-ser polypeptide linker is the amino acid sequence Ser(Gly4Ser). n Includes. In certain embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3, i.e., Ser(Gly4Ser)3. In certain embodiments, n=4, i.e., Ser(Gly4Ser)4. In certain embodiments, n=5. In certain embodiments, n=6. In certain embodiments, n=7. In certain embodiments, n=8. In certain embodiments, n=9. In certain embodiments, n=10. Another exemplary gly-ser polypeptide linker is the amino acid sequence Ser(Gly4Ser) n Includes. In a particular embodiment, n=1. In a particular embodiment, n=2. In a particular embodiment, n=3. In a particular embodiment, n=4. In a particular embodiment, n=5. In a particular embodiment, n=6. Another exemplary gly-ser polypeptide linker is (Gly4Ser) n Includes. In a particular embodiment, n=1. In a particular embodiment, n=2. In a particular embodiment, n=3. In a particular embodiment, n=4. In a particular embodiment, n=5. In a particular embodiment, n=6. Another exemplary gly-ser polypeptide linker is (Gly3Ser) nThis includes n=1 in a particular embodiment. In a particular embodiment, n=2. In a particular embodiment, n=3. In a particular embodiment, n=4. In a particular embodiment, n=5. In a particular embodiment, n=6.

[0153] In some embodiments, IL-2 is operably linked to a collagen-binding domain by a linker, such as a gly-ser linker. In some embodiments, IL-2 is operably linked to a linear peptide spacer by a linker, such as a gly-ser linker. In some embodiments, IL-12 is operably linked to a collagen-binding domain by a linker, such as a gly-ser linker. In some embodiments, IL-12 is operably linked to a linear polypeptide spacer by a linker, such as a gly-ser linker. In some embodiments, the collagen-binding domain is operably linked to a linear peptide spacer by a linker, such as a gly-ser linker. V. Exemplary immunomodulatory fusion proteins

[0154] This disclosure provides immunomodulatory fusion proteins comprising an immunomodulatory domain and a collagen-binding domain. The immunomodulatory fusion proteins of this disclosure are modular and can be configured to incorporate various individual domains. A. IL-2 and IL-12 fusion protein

[0155] In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, lumican, and a linear polypeptide spacer, where IL-2 is operably linked to the lumican. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, lumican, and a linear polypeptide spacer, where IL-2 is operably linked to the linear polypeptide spacer. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, lumican, and a linear polypeptide spacer, where IL-12 is operably linked to the lumican. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, lumican, and a linear polypeptide spacer, where IL-12 is operably linked to the linear polypeptide spacer.

[0156] In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR1, and a linear polypeptide space, where IL-2 is operably linked to LAIR1. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR1, and a linear polypeptide space, where IL-2 is operably linked to the linear polypeptide space. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR1, and a linear polypeptide space, where IL-12 is operably linked to LAIR1. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR1, and a linear polypeptide space, where IL-12 is operably linked to the linear polypeptide space.

[0157] In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR2, and a linear polypeptide space, where IL-2 is operably linked to LAIR2. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR2, and a linear polypeptide space, where IL-2 is operably linked to the linear polypeptide space. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR2, and a linear polypeptide space, where IL-12 is operably linked to LAIR2. In some embodiments, the immunomodulatory fusion protein comprises IL-2, IL-12, LAIR2, and a linear polypeptide space, where IL-12 is operably linked to the linear polypeptide space.

[0158] In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence, or a portion thereof, that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs. 23-70.

[0159] In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence having the leader sequence: MRVPAQLLGLLLLWLPGARCA shown in SEQ ID NO: 71.

[0160] In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence having the His tag sequence:HHHHHHHHHH, as shown in SEQ ID NO: 72.

[0161] In some embodiments, the immunomodulatory fusion protein comprises an amino acid sequence or a portion thereof having at least 80% identity with the amino acid sequences shown in SEQ ID NOs. 23-70, wherein the immunomodulatory fusion protein excludes the leader sequence of SEQ ID NO: 71: MRVPAQLLGLLLLWLPGARCA.

[0162] In some embodiments, the immunomodulatory fusion protein comprises an amino acid sequence or a portion thereof that has at least 80% identity with the amino acid sequences shown in SEQ ID NOs. 23-70, wherein the immunomodulatory fusion protein excludes the His tag sequence:HHHHHHHHHH of SEQ ID NO. 72.

[0163] In some embodiments, the immunomodulatory fusion protein comprises an amino acid sequence or a portion thereof having at least 80% identity with the amino acid sequences shown in SEQ ID NOs. 23-70, wherein the immunomodulatory fusion protein excludes the leader sequence of SEQ ID NO: 71: MRVPAQLLGLLLLWLPGARCA and the His tag sequence of SEQ ID NO: 72: HHHHHHHHHH.

[0164] In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence having at least 80% identity with a portion of the amino acid sequences shown in SEQ ID NOs. 23-70, where a portion excludes a leader sequence having the amino acid sequence shown in SEQ ID NO. 71.

[0165] In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence having at least 80% identity with a portion of the amino acid sequences shown in SEQ ID NOs. 23-70, where a portion excludes the His-tagged sequence having the amino acid sequence shown in SEQ ID NO. 72.

[0166] In some embodiments, the immunomodulatory fusion protein comprises an amino acid sequence having at least 80% identity with a portion of the amino acid sequences shown in SEQ ID NOs: 23-70, wherein a portion excludes a leader sequence having the amino acid sequence shown in SEQ ID NO: 71, and a portion further excludes a His-tag sequence having the amino acid sequence shown in SEQ ID NO: 72.

[0167] In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence having at least 80% identity with a portion of the amino acid sequence shown in SEQ ID NO: 73. In some embodiments, the immunomodulatory fusion protein includes an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 73. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 [Table 6-18] [Table 6-19] [Table 6-20] [Table 6-21] VI. Methods for producing immunomodulatory fusion proteins

[0168] The immunomodulatory fusion proteins of the present invention are prepared using recombinant DNA technology. In some embodiments, the domains of the immunomodulatory fusion proteins described herein (e.g., collagen-binding domains, cytokines) are prepared in transformed host cells using recombinant DNA techniques. Methods for preparing such DNA molecules are well known in the art. For example, the peptide-encoding sequence can be excised from DNA using a suitable restriction enzyme. Alternatively, the DNA molecule can be synthesized using chemical synthesis techniques such as the phosphoramidate method. Combinations of these techniques can also be used.

[0169] The immunomodulatory fusion proteins of the present invention are isolated and purified using one or more methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, size-exchange chromatography, ion-exchange chromatography, hydrophobic interaction-exchange chromatography, and mixed-mode chromatography. In certain embodiments, the fusion proteins described herein are purified by size-exchange chromatography using Protein A resin. In certain embodiments, the fusion proteins described herein are purified by size-exchange chromatography using Capto® Blue resin. In certain embodiments, the fusion proteins described herein are purified by size-exchange chromatography using CaptureSelect® HSA resin. In certain embodiments, the purified fusion proteins described herein are concentrated by any preferred method known in the art. In certain embodiments, the purified fusion proteins are concentrated to concentrations of 0.1–100 mg / ml, 1–50 mg / ml, or 10–30 mg / ml. In certain embodiments, the purified fusion protein is concentrated to concentrations of 0.1–100 mg / ml, 1–50 mg / ml, or 10–30 mg / ml without any detectable aggregation of the fusion protein. In certain embodiments, the purified fusion protein is concentrated to a concentration of approximately 20 mg / ml without any detectable aggregation of the fusion protein.

[0170] In one exemplary embodiment, codon-optimized DNA sequences encoding IL-12, IL-2, collagen-binding protein, and albumin were synthesized and cloned into the pD2610-v1 vector. For expansion, the plasmid was transformed into DH10B competent cells. The purified expression vector was transiently transfected into HEK293 cells. Recombinant proteins were purified by anion exchange and preparative size exclusion chromatography (SEC) using Q Sepharose resin. VII. Pharmaceutical Compositions and Dosage Methods

[0171] As used herein, the term “pharmaceutical composition” refers to a combination of an activator and an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0172] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0173] As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt (e.g., an acid or a base) of the compounds of the present invention that, when administered to a subject, can provide the compounds of the present invention or their active metabolites or residues. As is known to those skilled in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid (toluene-psulfonic acid), tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Other acids, such as oxalic acid, are not pharmaceutically acceptable in themselves, but may be used in the preparation of salts that are useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts. In certain embodiments, the disclosure provides a pharmaceutical composition comprising an immunomodulatory fusion protein together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0174] In certain embodiments, the disclosure provides a pharmaceutical composition comprising an immunomodulatory fusion protein together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0175] In certain embodiments, the effective amount of a pharmaceutical composition containing a therapeutically used immunomodulatory fusion protein depends, for example, on the therapeutic situation and purpose. Those skilled in the art will therefore recognize that the appropriate dose level for treatment according to a particular embodiment varies in part depending on the molecule being delivered, the indication for which the immunomodulatory fusion protein is used, the route of administration, and the patient's size (body weight, body surface or organ size) and / or condition (age and overall health). In certain embodiments, the clinician may set the dose and modify the route of administration to obtain the optimal therapeutic effect. VIII. Treatment Methods

[0176] The immunomodulatory fusion proteins and / or nucleic acids expressing them described herein are useful for treating disorders associated with abnormal apoptosis or differentiation processes (e.g., cell proliferation disorders (e.g., hyperproliferative disorders) or cell differentiation disorders, e.g., cancer). Non-limiting examples of cancers that can be treated by the methods of this disclosure are listed below.

[0177] Examples of cell proliferation and / or differentiation disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorder, or hematopoietic neoplasm disorder, e.g., leukemia). Metastatic tumors can arise from a number of primary tumor types, including but not limited to those of the prostate, colon, lung, breast, and liver. Therefore, compositions used herein, for example, containing immunomodulatory fusion proteins, may be administered to patients with cancer.

[0178] As used herein, the terms “cancer” (or “cancerous”), “hyperproliferative,” and “neoplasm” refer to cells that possess the capacity for autonomous proliferation (i.e., an abnormal state or condition characterized by rapidly growing cell growth). Disease states of hyperproliferative and neoplasms may be classified as pathological (i.e., characterizing or constituting a disease state), or they may be classified as nonpathological (i.e., deviating from normal but not associated with a disease state). These terms mean that all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. “Pathological hyperproliferative” cells arise in disease states characterized by the growth of malignant tumors. An example of nonpathological hyperproliferative cells is the proliferation of cells associated with wound repair.

[0179] The terms “cancer” or “neoplasm” are used to refer to malignant tumors of various organ systems, including those affecting the lungs, breasts, thyroid gland, lymph nodes and lymphoid tissue, digestive organs, and genitourinary tract, as well as adenocarcinomas, which are generally considered to include most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestine cancer and esophageal cancer.

[0180] The term “cancer” is as recognized in the art and refers to malignant tumors of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. Immunomodulatory fusion proteins may be used to treat patients who have, are suspected of having, or are at high risk of developing any type of cancer, including renal cancer or melanoma, or any viral disease. Exemplary cancers include those formed from the tissues of the cervix, lungs, prostate, breast, head and neck, colon, and ovaries. The term also includes carcinosarcoma, which is a malignant tumor composed of cancerous and sarcoma tissues. “Adenocarcinoma” refers to cancer that originates from glandular tissue or in which tumor cells form recognizable glandular structures.

[0181] In certain embodiments, the immunomodulatory fusion proteins disclosed herein are used to treat cancer. In certain embodiments, the immunomodulatory fusion proteins disclosed herein are used to treat melanoma, leukemia, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, and brain cancer.

[0182] In certain embodiments, the immunomodulatory fusion proteins disclosed herein inhibit the growth and / or proliferation of tumor cells. In certain embodiments, the immunomodulatory fusion proteins disclosed herein reduce tumor size. In certain embodiments, the immunomodulatory fusion proteins disclosed herein inhibit metastasis of primary tumors.

[0183] In certain embodiments, administration of the immunomodulatory fusion protein disclosed herein to a subject does not result in cytokine release syndrome after administration to the subject. In certain embodiments, the subject does not experience Grade 4 cytokine release syndrome. In certain embodiments, the subject does not experience one or more symptoms associated with Grade 4 cytokine release syndrome, selected from the group consisting of hypotension, organ toxicity, fever, and / or dyspnea resulting in the need for oxygen supplementation.

[0184] In certain embodiments, when the fusion protein disclosed herein is administered intravenously or intratumorally to a subject with cancer, cytokine levels increase in the subject's serum after administration compared to IV or IT administration of recombinant IL-2 and / or IL-12. In certain embodiments, the cytokines that increase in the subject's serum are selected from INFγ, IP-10, and MCP-1. Combination therapy

[0185] In some embodiments, immunomodulatory fusion proteins are used in combination with other therapies. In some embodiments, immunomodulatory fusion proteins are used in combination with further therapeutic agents to treat cancer. For example, in some embodiments, immunomodulatory fusion proteins are used in combination with another immunotherapy. Exemplary immunotherapies include, but are not limited to, chimeric antigen receptor (CAR) T-cell therapy, tumor-associated antigen-targeting antibodies, immune checkpoint inhibitors, and cancer vaccines. I. Tumor-associated antigen-targeted antibodies

[0186] In some embodiments, the disclosure provides immunomodulatory fusion proteins used or implemented in conjunction with antibodies targeting tumor antigens.

[0187] Therapeutic monoclonal antibodies have been considered a class of pharmaceutically active drugs that enable tumor-selective treatment by targeting tumor-selective antigens or epitopes.

[0188] Methods for generating antibodies and their antigen-binding fragments are well known in the art, and all of them are disclosed, for example, in U.S. Patent No. 7,247,301, U.S. Patent No. 7,923,221, and U.S. Patent Application No. 2008 / 0138336, which are incorporated herein by reference in their entirety.

[0189] The therapeutic antibodies that can be used in the methods of this disclosure include, but are not limited to, any anti-cancer antibodies recognized in the art that are approved for use, in clinical trials, or under development for clinical use. In certain embodiments, two or more anti-cancer antibodies may be included in the combination therapy of this disclosure.

[0190] Non-exclusive examples of anti-cancer antibodies include, but are not limited to, trastuzumab (HERCEPTIWM, Genentech, South San Francisco, Calif.) used to treat HER-2 / neu-positive breast cancer or metastatic breast cancer; bevacizumab (AVASTIWM, Genentech) used to treat colorectal cancer, metastatic colorectal cancer, breast cancer, metastatic breast cancer, non-small cell lung cancer, or renal cell carcinoma; rituximab (RITUXAWM, Genentech) used to treat non-Hodgkin lymphoma or chronic lymphocytic leukemia; and used to treat breast cancer, prostate cancer, non-small cell lung cancer, or ovarian cancer.

[0191] Pertuzumab (OMNITARG®, manufactured by Genentech); Cetuximab (ERBITUX®, manufactured by ImClone Systems Incorporated, New York, NY), which can be used to treat colorectal cancer, metastatic colorectal cancer, lung cancer, head and neck cancer, colon cancer, breast cancer, prostate cancer, stomach cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, renal cell carcinoma, prostate cancer, cervical cancer, or bladder cancer; IMC-1 Cl 1 (ImClone Systems Incorporated), used to treat colorectal cancer, head and neck cancer, and other potential cancer targets; Tositumomab and Tositumomab and Iodine I 131 (BEXXAR, manufactured by Corixa Corporation), used to treat non-Hodgkin lymphoma that may be refractory to rituximab, relapsed after chemotherapy, transformed or untransformed, CD20-positive, follicular non-Hodgkin lymphoma; XM, Seattle, Wash.); In111 ibritumomab tiuxetan; Y90 ibritumomab tiuxetan; In111 ibritumomab tiuxetan and Y90 ibritumomab tiuxetan (ZEVALIN®, manufactured by Biogen Idee, Cambridge, Mass.) used to treat non-Hodgkin lymphoma, which may include lymphoma or relapsed follicular lymphoma; relapsed or refractory, low-grade or follicular non-Hodgkin lymphoma; or transformed B-cell non-Hodgkin lymphoma; EMD 7200 (EMD Pharmaceuticals, Durham, NC) used to treat non-small cell lung cancer or cervical cancer; SGN-30 (genetically engineered monoclonal antibody targeting the CD30 antigen, manufactured by Seattle Genetics, Bothell, Wash.) used to treat Hodgkin lymphoma or non-Hodgkin lymphoma.); SGN-15 (a genetically engineered monoclonal antibody targeting Lewisy-related antigen conjugated to doxorubicin, manufactured by Seattle Genetics), used for treating non-small cell lung cancer; SGN-33 (a humanized antibody targeting CD33 antigen, manufactured by Seattle Genetics), used for treating acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS); SGN-40 (a humanized monoclonal antibody targeting CD40 antigen, manufactured by Seattle Genetics), used for treating multiple myeloma or non-Hodgkin lymphoma; SGN-35 (a genetically engineered monoclonal antibody targeting CD30 antigen conjugated to auristatin E, manufactured by Seattle Genetics), used for treating non-Hodgkin lymphoma; SGN-70 (a humanized antibody targeting CD70 antigen, manufactured by Seattle Genetics), used for treating renal cancer and nasopharyngeal cancer; SGN-75 (a conjugate composed of SGN70 antibody and auristatin derivative, manufactured by Seattle Genetics); and SGN-17 / 19 (a fusion protein containing an antibody and an enzyme conjugated to melphalan prodrug, manufactured by Seattle Genetics), used for treating melanoma or metastatic melanoma). II. Immune checkpoint blockade

[0192] In some embodiments, the present disclosure provides an immunomodulatory fusion protein that is used or implemented in combination with an immune checkpoint inhibitor or an immune checkpoint blocking agent.<00009,04>

[0193] The activation and effector functions of T cells are balanced by co-stimulatory and inhibitory signals called "immune checkpoints". Inhibitory ligands and receptors that regulate the effector functions of T cells are overexpressed in tumor cells. Subsequently, amplification of antigen-specific T cell responses is brought about by agonists of co-stimulatory receptors or antagonists of inhibitory signals. In contrast to therapeutic antibodies that directly target tumor cells, immune checkpoint blocking agents enhance endogenous anti-tumor activity.

[0194] In certain embodiments, immune checkpoint blockade agents suitable for use in the methods disclosed herein are antibodies that target antagonists of inhibitory signals, such as PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM3. These ligands and receptors are reviewed in Pardall, D., Nature. 12: 252-264, 2012.

[0195] In certain embodiments, the immune checkpoint blockade agent is an antibody or an antigen-binding portion thereof that disrupts or inhibits signal transduction from an inhibitory immunomodulatory agent. In certain embodiments, the immune checkpoint blockade agent is a small molecule that disrupts or inhibits signal transduction from an inhibitory immunomodulatory agent.

Example

[0196] The invention generally described herein is more readily understood by reference to the following examples, which are included to merely illustrate certain aspects and embodiments of the invention and are not intended to limit the invention. (Example 1) Method for Preparing a Linear Construct

[0197] The proteins of the present invention are typically produced using recombinant DNA technology. In one exemplary embodiment, codon-optimized DNA sequences encoding IL-12, IL-2, collagen-binding protein, and albumin were synthesized and cloned into the pD2610-v1 vector. For expansion, the plasmid was transformed into DH10B competent cells. The purified expression vector was transiently transfected into HEK293 cells. The recombinant proteins were purified by anion exchange and preparative size exclusion chromatography (SEC) using Q Sepharose resin. The enriched proteins were evaluated for product quality using analytical SEC. The proteins were then finalized by another preparative SEC before in vitro and in vivo evaluation.

[0198] Proteins are isolated and purified using methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. (Example 2) Recombinant collagen-binding fusion proteins bind to collagen in vitro.

[0199] To evaluate the ability of collagen-binding immunomodulatory molecules to bind to collagen, the expressed and purified collagen-binding fusion proteins described in Example 1 were tested for their ability to bind to a collagen I-coated plate using linear fusion constructs and ELISA with anti-His detection. Briefly, a 96-well plate coated with collagen I (Corning) was blocked with 1% wt / vol bovine serum albumin (BSA) at room temperature for 1 hour. Proteins containing Hisx10 were incubated in the plate at increasing concentrations for 1.5 hours. The wells were then washed and incubated with anti-His tag detection antibody (Abcam) for 1.5 hours. The bound Hisx10-tagged collagen-binding fusion proteins were visualized by TMB color development, followed by subtraction of the absorbance reading at 650 nm from the absorbance reading at 450 nm. As shown in Figure 4A, constructs containing LAIR bound more strongly to collagen than constructs containing Lum. Furthermore, positioning lumican between MSA and IL-2 allowed for tighter binding to collagen than positioning lumican between MSA and IL-2. As shown in Figure 4B, three LAIR-containing constructs using different spacers between LAIR and IL-2 resulted in comparable levels of collagen binding.

[0200] The LAIR fusion strongly binds to collagen. The LAIR fusion binds with a tighter affinity than the lumican fusion. In vivo data and bioactivity are left undetermined to allow for the selection of weak or strong binding as needed. (Example 3) Recombinant collagen-binding fusion proteins maintain IL-2 cytokine activity.

[0201] To evaluate the ability of collagen-binding immunomodulatory molecules to maintain IL-2 cytokine activity in the presence of collagen, samples were serially diluted in assay medium, and 50 μl of diluted sample and 50 μl of assay medium were added to either a normal tissue culture plate or a plate coated with Collagen I (Corning) and incubated for 1 hour. Next, approximately 25,000 CTLL-2 cells were transferred to each well in 100 μl of assay medium and incubated for 3 days. After incubation, 20 μl of Promega Substrate Cell Titer 96 Aqueous One Solution Reagent was added to each well, incubated at 37°C, and absorbance was read at 490 nm.

[0202] As shown in Figures 5A–5D, bifunctional constructs containing both IL-2 and IL-12 yielded IL-2 activity at levels equivalent to IL-2 alone. Furthermore, IL-2 activity was not affected by collagen binding and was independent of the choice of spacer or collagen-binding domain. (Example 4) Recombinant collagen-binding fusion proteins maintain IL-12 cytokine activity.

[0203] To evaluate the ability of collagen-binding immunomodulatory molecules to maintain IL-2 cytokine activity in the presence of collagen, samples were serially diluted in assay medium, and 50 μl of diluted sample and 50 μl of assay medium were added to either a standard tissue culture plate or a plate coated with Corning's Collagen I, and incubated for 1 hour. Next, approximately 15,000 2D6 cells were transferred to each well containing 100 μl of assay medium and incubated for 4 days. After incubation, 20 μl of Promega Substrate Cell Titer 96 Aqueous One Solution Reagent was added to each well, incubated at 37°C, and absorbance was read at 490 nm.

[0204] As shown in Figures 6A-6B, bifunctional constructs containing both IL-2 and IL-12 yielded IL-12 activity at levels equivalent to IL-12 alone. Furthermore, IL-12 activity was not affected by collagen binding and was independent of the selection of the collagen-binding domain. (Example 5) Synergistic effects of immunomodulatory collagen-binding molecules and antitumor antigen antibodies in a mouse melanoma tumor model

[0205] To evaluate the efficacy and toxicity of combinations of bifunctional and monofunctional constructs, 200,000 B16F10 cells were inoculated into the right posterior flank of C57BL / 6 mice in 0.1 ml of PBS. Nine days after inoculation (day 0), the mice were randomized to the treatment group (n=10). On days 0 and 6, mice were treated with 100 pmol intratumoral injection of 100 pmol each of the following: (1) PBS, (2) a combination of an IL-2 monofunctional linear construct containing MSA (MSA-2) and an IL-12 monofunctional linear construct containing MSA (12-MSA), (3) a combination of an IL-2 monofunctional linear construct containing MSA and a collagen-binding domain (LAIR-MSA-2) and an IL-12 monofunctional linear construct containing MSA and a collagen-binding domain (12-MSA-LAIR), (4) a bifunctional linear construct 12-Lum-MSA-2 containing MSA and a collagen-binding domain, and (5) a bifunctional linear construct 12-LAIR-MSA-2 containing MSA and a collagen-binding domain. Mice were monitored at least twice a week for tumor growth and weight loss, and were treated if they were mortal, had lost more than 20% of their body weight, or had a tumor volume of 3,000 mm³. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized.

[0206] As shown in Figures 7A–7B, tumor growth and body weight during treatment with a combination of bifunctional or monofunctional constructs demonstrate that both the bifunctional linear constructs 12-Lum-MSA-2 and 12-LAIR-MSA-2 exhibited a superior safety profile compared to combinations of monofunctional constructs, regardless of whether the monofunctional constructs contained collagen-binding domains. This is indicated by the absence of body weight loss, which is associated with systemic exposure to cytokines. Both 12-Lum-MSA-2 and 12-LAIR-MSA-2 resulted in significant inhibition of tumor growth. (Example 6) Linear construction monotherapy in the B16F10 model - Abscopal effect and bilateral flank model

[0207] To further evaluate the dose-response therapeutic efficacy of a bifunctional linear construct containing MSA and collagen-binding domains, 12-LAIR-MSA-2 was evaluated in a subcutaneous B16F10 melanoma syngeneic model in which C57BL / 6 mice were inoculated bilaterally into the flanks. Control C57BL / 6 mice were inoculated with 200,000 B16F10 cells in 0.1 mL of PBS into either the right posterior flank (treated tumor plot) or, after 10 days, the left posterior flank (untreated tumor plot). Other mice in the study were also inoculated with 200,000 B16F10 cells in 0.1 mL of PBS into the right posterior flank and, after 10 days, the left posterior flank. Eight days after tumor inoculation in the right posterior flank (day 0), mice were randomized to the treatment group (n=15). On days 0, 6, and 12, the right posterior flank tumors of the mice were treated by intratumoral injection of a specified dose of 12-LAIR-MSA-2. Mice are monitored at least twice a week for tumor growth and weight loss in both flanks. If the mouse is in a near-fatal condition, has lost more than 20% of its body weight, or the total tumor volume exceeds 3,000 mm³, then the mouse is monitored. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized.

[0208] As shown in FIGS. 8A-8B, at all tested dose levels, the bifunctional linear construct 12-LAIR-MSA-2 produced significant tumor growth inhibition and demonstrated an abscopal effect in both treated tumors (FIG. 8A) and untreated tumors (FIG. 8B). (Example 7) Comparison of linear constructs in the B16F10 model

[0209] The efficacy and toxicity of various bifunctional constructs were evaluated in a B16F10 mouse model. 200,000 B16F10 cells in 0.1 ml of PBS were inoculated into the right hind flank of C57BL / 6 mice. Seven days after inoculation (day 0), the mice were randomized into treatment groups (n = 10). On days 0 and 6, the mice were treated by intratumoral injection of 400 pmol of (1) PBS control, (2) 12-LAIR-MSA-2, (3) 12-LAIR-MSA_H464Q-2, (4) 12-LAIR-ABD-2, and (5) 12-Lum-MSA-2. The mice were monitored at least twice a week for tumor growth and weight loss, and euthanized when they were in a moribund state, had a weight loss exceeding 20%, or a tumor volume exceeding 3,000 mm 3 was found to exceed.

[0210] As shown in FIGS. 9A-9C, all tested bifunctional constructs produced significant tumor growth inhibition, demonstrated a good safety profile reflected by the lack of weight loss, and an extended survival of the animals compared to the PBS control group. (Example 8) Combination of linear constructs - checkpoint in the B16F10 model

[0211] To evaluate 12-LAIR-MSA-2 in combination with the checkpoint inhibitors anti-PD1 or anti-CTLA, 200,000 B16F10 cells in 0.1 ml of PBS were inoculated into the right posterior flank of C57BL / 6 mice. Seven days after inoculation (day 0), mice were randomized to treatment groups (n=10). Mice were treated as instructed by intratumoral (IT) injection of PBS or 400 pmol of 12-LAIR-MSA-2 and intraperitoneal (IP) injection of an isotype control (rat IgG2a), anti-PD1 (clonal RMP1-14), or anti-CTLA4 (9D9). IT injections were administered on days 0, 6, and 12, while IP injections were administered via BIW until the end of the study. Mice were monitored at least twice a week for tumor growth and weight loss, and were not mortal, had a weight loss exceeding 20%, or had a tumor volume of 3,000 mm³. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized.

[0212] As shown in Figures 10A-10B, treatment with either anti-PD1 or anti-CTLA4 alone did not affect tumor growth inhibition. Treatment with the bifunctional construct 12-LAIR-MSA-2 alone resulted in significant tumor growth inhibition. The antitumor activity of 12-LAIR-MSA-2 was further enhanced by its combination with either anti-PD1 or anti-CTLA4. As shown in Figure 10C, adding either anti-PD1 or anti-CTLA4 to the bifunctional construct 12-LAIR-MSA-2 did not result in further weight loss compared to treatment with 12-LAIR-MSA-2 alone. (Example 9) Linear construction monotherapy in the MC38 model - Safety and efficacy

[0213] The dose-response therapeutic efficacy of 12-LAIR-MSA-2, a bifunctional linear construct containing MSA and collagen-binding domains, was evaluated in an MC38 model using C57BL / 6 mice. 1,000,000 MC38 cells were inoculated into the right posterior flank of C57BL / 6 mice in 0.1 ml of PBS. Six days after inoculation (day 0), mice were randomized to the treatment group (n=10). On days 0 and 6, mice were treated with intratumoral injection of a specified dose of 12-LAIR-MSA-2. Tumor growth and weight loss were monitored at least twice a week. If a mouse was mortal, had a weight loss exceeding 20%, or had a tumor volume of 3,000 mm³, treatment was initiated. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized.

[0214] As shown in Figure 11A, treatment with 12-LAIR-MSA-2 at all dose levels resulted in significant inhibition of tumor growth. Furthermore, dose-response was observed for treatment at the highest dose level that yielded the highest complete response (CR) rate. As shown in Figure 11B, none of the treatment groups showed significant weight loss. (Example 10) Comparison of linear structures in the MC38 model

[0215] The efficacy and toxicity of various bifunctional constructs were evaluated in the B16F10 mouse model. Mice were treated on days 0 and 6 with specified doses of PBS, 12-LAIR-MSA-2, 12-LAIR-ABD-2, and 12-Lum-MSA-2 via intratumoral injection. Where specified, mice were treated with an isotype control (rat IgG2a) or anti-PD1 (clone RMP1-14) via intraperitoneal injection in BIW for 3 weeks. Mice were monitored at least twice a week for tumor growth and weight loss, and were deemed mortal, had a weight loss exceeding 20%, or had a tumor volume of 3,000 mm³. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized.

[0216] As shown in Figure 12A, all bifunctional constructs containing different collagen-binding domains or spacers between IL-2 and the collagen-binding domain resulted in significant tumor growth inhibition and complete response (CR) rates. In comparison, anti-PD1 treatment in the same model did not achieve a similar level of tumor growth control and did not result in any cure. As shown in Figure 12B, none of the treatment groups showed significant weight loss. (Example 11) Linear construction monotherapy in the CT26 model - Safety and efficacy

[0217] The dose-response therapeutic efficacy of 12-LAIR-MSA-2, a bifunctional linear construct containing MSA and collagen-binding domains, was evaluated in a CT26 model of BALB / c mice. 500,000 CT26 cells were inoculated into the right posterior flank of BALB / c mice in 0.1 ml of PBS. Six days after inoculation (day 0), mice were randomized to the treatment group (n=10). Treatment was performed on days 0, 6, and 12 by intratumoral injection of specified doses of PBS or 12-LAIR-MSA-2. Where specified, mice were treated with isotype control (rat IgG2a) or anti-PD1 (clone RMP1-14) by intraperitoneal injection via BIW for 3 weeks. Mice were monitored at least twice a week for tumor growth and weight loss. If mortal, weight loss exceeding 20%, or tumor volume exceeding 3,000 mm³ occurred, treatment was discontinued. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized. As shown in Figures 13A-13B, tumor growth inhibition and weight changes were observed with 12-LAIR-MSA-2 treatment at various dose levels or administration frequencies. None of the treatment groups showed weight loss, and dose-dependent antitumor activity was observed. (Example 12) Comparison of linear structures in the MC38 model

[0218] The efficacy and toxicity of various bifunctional constructs were evaluated in the B16F10 mouse model. 500,000 CT26 cells in 0.1 ml of PBS were inoculated into the right posterior flank of BALB / c mice. Six days after inoculation (day 0), mice were randomized to the treatment group (n=10). Treatment was performed on days 0, 6, and 12 by intratumoral injection of specified doses of PBS, 12-LAIR-MSA-2, 12-LAIR-ABD-2, and 12-Lum-MSA-2 at specified rates. Where specified, mice were treated with isotype control (rat IgG2a) or anti-PD-1 (clone RMP1-14) by intraperitoneal injection in BIW for 3 weeks. Mice were monitored at least twice a week for tumor growth and weight loss, and were treated if they were mortal, had a weight loss of more than 20%, or had a tumor volume of 3,000 mm³. 3 If it was determined that the condition exceeded a certain limit, the animal was euthanized.

[0219] As shown in Figures 14A–14B, all bifunctional constructs containing different collagen-binding domains or spacers between IL-2 and collagen-binding domains resulted in significant inhibition of tumor growth. In comparison, treatment with anti-PD1 in the same model did not result in inhibition of tumor growth. None of the treatment groups showed weight loss. (Example 13) Linear structures in the B16F10 model - IT and IV administration

[0220] The efficacy of intratumor (IT) administration of the 12-LAIR-MSA-2 construct compared to intravenous (IV) administration was evaluated in a B16F10 mouse model. 200,000 B16F10 cells were inoculated into the right posterior flank of C57BL / 6 mice in 0.1 ml of PBS. Seven days after inoculation (day 0), mice were randomized to treatment groups (n=10). Mice were treated with either 400 pmol of PBS control or 12-LAIR-MSA-2 via intravenous or intratumor injection. Serum levels of 12-LAIR-MSA-2 were measured 2 or 24 hours after administration (Figure 15A). At 2 hours, a significant decrease in serum levels of the fusion protein was observed when delivered via IT compared to IV. At 24 hours, very low levels of the fusion protein were detected in mice administered via either IT or IV. Cytokines, including interferon-gamma (INF-γ), interferon-gamma-inducible protein (IP-10), and monocyte chemotactic protein-1 (MCP-1), were also measured 2 or 24 hours after administration of the fusion protein via intratumoral (IT) or intravenous (IV) administration (Figures 15B-15D). Cytokine levels at 24 hours were not significantly different compared to mice administered the fusion protein via IT or IV. However, the effectiveness of the treatment, as measured by survival rate, was significantly improved in mice administered the fusion protein via IT (aministration) compared to those administered via IV (Figure 15E). These results confirm that the fusion protein described herein is effective in reducing serum concentrations of the fusion protein and improving the survival rate of subjects when administered intratumorally. Embedding by reference

[0221] The full disclosures of each patent document and scientific literature cited herein are incorporated by reference for all purposes. Equal portions

[0222] This disclosure can be embodied in other specific forms, deviating from its essential characteristics. Therefore, the embodiments described herein are not limiting to the disclosures set forth herein, but are illustrative. The scope of this disclosure is indicated not by the foregoing description but by the appended claims, and all changes within the meaning of the claims and their equivalents are intended to be encompassed therein. In certain embodiments, for example, the following items are provided: (Item 1) (i) IL-2; (ii) IL-12; (iii) Collagen-binding domain, and (iv) Linear polypeptide spacer An immunomodulatory fusion protein containing [this protein]. (Item 2) A linear immunomodulatory fusion protein, as described in item 1. (Item 3) An immunomodulatory fusion protein, which is a continuous chain, as described in any one of items 1 or 2. (Item 4) An immunomodulatory fusion protein, which is a continuous polypeptide chain, as described in any one of items 1 to 3. (Item 5) An immunomodulatory fusion protein according to any one of items 1 to 4, wherein the aforementioned IL-2 is located at the N-terminus. (Item 6) An immunomodulatory fusion protein according to any one of items 1 to 5, wherein the aforementioned IL-12 is located at the C-terminus. (Item 7) An immunomodulatory fusion protein according to any one of items 1 to 6, wherein IL-2 is located at the N-terminus and IL-12 is located at the C-terminus. (Item 8) The immunomodulatory fusion protein according to any one of items 1 to 7, wherein the linear polypeptide spacer is positioned between the IL-2 and the collagen-binding domain. (Item 9) The immunomodulatory fusion protein according to any one of items 1 to 8, wherein the collagen-binding domain is positioned between IL-12 and the linear polypeptide spacer. (Item 10) The immunomodulatory fusion protein according to any one of items 1 to 9, wherein the C-terminus of IL-2 is operably ligated to the N-terminus of the linear polypeptide spacer. (Item 11) The immunomodulatory fusion protein according to item 10, wherein the C-terminus of IL-2 is operably linked to the N-terminus of the linear polypeptide spacer by a linker. (Item 12) The immunomodulatory fusion protein according to any one of items 1 to 11, wherein the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of the collagen-binding domain. (Item 13) The immunomodulatory fusion protein according to item 12, wherein the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of the collagen-binding domain by a linker. (Item 14) The immunomodulatory fusion protein according to any one of items 1 to 13, wherein the C-terminus of the collagen-binding domain is operably linked to the N-terminus of IL-12. (Item 15) The immunomodulatory fusion protein according to item 14, wherein the C-terminus of the collagen-binding domain is operably linked to the N-terminus of IL-12 by a linker. (Item 16) The immunomodulatory fusion protein according to any one of items 1 to 6, wherein the collagen-binding domain is located between the IL-2 and the linear polypeptide spacer. (Item 17) The immunomodulatory fusion protein according to item 16, wherein the linear polypeptide spacer is positioned between the IL-12 and the collagen-binding domain. (Item 18) The immunomodulatory fusion protein according to any one of items 16 to 17, wherein the C-terminus of IL-2 is operably linked to the N-terminus of the collagen-binding domain. (Item 19) The immunomodulatory fusion protein described in item 18, wherein the C-terminus of IL-2 is operably linked to the N-terminus of the collagen-binding domain by a linker. (Item 20) The immunomodulatory fusion protein according to any one of items 16 to 19, wherein the C-terminus of the collagen-binding domain is operably linked to the N-terminus of the linear polypeptide spacer. (Item 21) The immunomodulatory fusion protein according to item 20, wherein the C-terminus of the collagen-binding domain is operably linked to the N-terminus of the linear polypeptide spacer by a linker. (Item 22) The immunomodulatory fusion protein according to any one of items 16 to 21, wherein the C-terminus of the linear polypeptide spacer is operably ligated to the N-terminus of IL-12. (Item 23) The immunomodulatory fusion protein according to item 22, wherein the C-terminus of the linear polypeptide spacer is operably linked to the N-terminus of IL-12 by a linker. (Item 24) An immunomodulatory fusion protein according to any one of items 1 to 3, wherein the aforementioned IL-2 is located at the C-terminus. (Item 25) The immunomodulatory fusion protein described in item 24, wherein IL-12 is located at the N-terminus. (Item 26) The immunomodulatory fusion protein according to any one of items 24 to 25, wherein IL-2 is located at the C-terminus and IL-12 is located at the N-terminus. (Item 27) The immunomodulatory fusion protein according to any one of items 24 to 27, wherein the N-terminus of IL-2 is operably ligated to the C-terminus of the linear polypeptide spacer. (Item 28) The immunomodulatory fusion protein according to item 27, wherein the N-terminus of IL-2 is operably linked to the C-terminus of the linear polypeptide spacer by a linker. (Item 29) The immunomodulatory fusion protein according to any one of items 24 to 28, wherein the N-terminus of the linear polypeptide spacer is operably linked to the C-terminus of the collagen-binding domain. (Item 30) The immunomodulatory fusion protein according to item 27, wherein the N-terminus of the linear polypeptide spacer is operably linked to the C-terminus of the collagen-binding domain by a linker. (Item 31) The immunomodulatory fusion protein according to any one of items 24 to 30, wherein the N-terminus of the collagen-binding domain is operably linked to the C-terminus of IL-12. (Item 32) The immunomodulatory fusion protein according to item 31, wherein the N-terminus of the collagen-binding domain is operably linked to the C-terminus of IL-12 by a linker. (Item 33) The immunomodulatory fusion protein according to item 26, wherein the collagen-binding domain is positioned between IL-2 and the linear polypeptide spacer. (Item 34) The immunomodulatory fusion protein according to item 27, wherein the linear polypeptide spacer is positioned between the IL-12 and the collagen-binding domain. (Item 35) The immunomodulatory fusion protein according to any one of items 33 to 34, wherein the N-terminus of IL-2 is operably linked to the C-terminus of the collagen-binding domain. (Item 36) The immunomodulatory fusion protein according to item 35, wherein the N-terminus of IL-2 is operably linked to the C-terminus of the collagen-binding domain by a linker. (Item 37) The immunomodulatory fusion protein according to any one of items 33 to 36, wherein the N-terminus of the collagen-binding domain is operably linked to the C-terminus of the linear polypeptide spacer. (Item 38) The immunomodulatory fusion protein according to item 37, wherein the N-terminus of the collagen-binding domain is operably linked to the C-terminus of the linear polypeptide spacer by a linker. (Item 39) The immunomodulatory fusion protein according to any one of items 33 to 38, wherein the N-terminus of the linear polypeptide spacer is operably ligated to the C-terminus of IL-12. (Item 40) The immunomodulatory fusion protein according to item 39, wherein the N-terminus of the linear polypeptide spacer is operably linked to the C-terminus of IL-12 by a linker. (Item 41) An immunomodulatory fusion protein according to any one of items 11, 13, 15, 19, 21, 23, 28, 30, 32, 36, 38, or 40, wherein one or more of the linkers are identical. (Item 42) An immunomodulatory fusion protein as described in any one of items 11, 13, 15, 19, 21, 23, 28, 30, 32, 36, 38, or 40, wherein one or more of the linkers are different. (Item 43) The immunomodulatory fusion protein according to item 1, wherein IL-12 is located at the C-terminus and is operably linked to the collagen-binding domain, the collagen-binding domain is operably linked to a linear polypeptide spacer, the linear polypeptide spacer is operably linked to IL-2 at the N-terminus of the protein, and the protein is linear. (Item 44) The immunomodulatory fusion protein according to item 1, wherein IL-12 is located at the N-terminus and is operably linked to the collagen-binding domain, the collagen-binding domain is operably linked to a linear polypeptide spacer, the linear polypeptide spacer is operably linked to IL-2 at the C-terminus of the protein, and the protein is linear. (Item 45) The immunomodulatory fusion protein according to item 1, wherein IL-12 is located at the C-terminus and is operably linked to the linear polypeptide spacer, the linear polypeptide spacer is operably linked to the collagen-binding domain, the collagen-binding domain is operably linked to IL-2 at the N-terminus of the protein, and the protein is linear. (Item 46) The immunomodulatory fusion protein according to item 1, wherein IL-12 is located at the N-terminus and is operably linked to the linear polypeptide spacer, the linear polypeptide spacer is operably linked to the collagen-binding domain, the collagen-binding domain is operably linked to IL-2 at the C-terminus of the protein, and the protein is linear. (Item 47) An immunomodulatory fusion protein according to any one of items 1 to 46, further comprising a second linear polypeptide spacer. (Item 48) The immunomodulatory fusion protein according to item 47, wherein IL-12 is located at the N-terminus and is operably linked to a first linear polypeptide spacer, the first linear polypeptide spacer is operably linked to the collagen-binding domain, the collagen-binding domain is operably linked to a second linear polypeptide spacer, the second linear polypeptide spacer is operably linked to IL-2 at the C-terminus of the protein, and the protein is linear. (Item 49) The immunomodulatory fusion protein according to item 47, wherein IL-12 is located at the C-terminus and operably linked to a first linear polypeptide spacer, the first linear polypeptide spacer operably linked to the collagen-binding domain, the collagen-binding domain operably linked to a second linear polypeptide spacer, the second linear polypeptide spacer operably linked to IL-2 at the N-terminus of the protein, and the protein is linear. (Item 50) An immunomodulatory fusion protein, which is a continuous chain, as described in any one of items 43 to 49. (Item 51) An immunomodulatory fusion protein, as described in any one of items 43 to 50, which is a continuous polypeptide chain. (Item 52) The collagen-binding domain, (i) Leucine-rich repeats derived from members of human proteoglycan class II of the low molecular weight leucine-rich proteoglycan (SLRP) family, including Lumican; or (ii) Human type I glycoprotein having an Ig-like domain selected from LAIR1 and LAIR2 An immunomodulatory fusion protein, including any one of items 1 through 51. (Item 53) The immunomodulatory fusion protein described in item 52, wherein the collagen-binding domain contains lumican. (Item 54) The immunomodulatory fusion protein described in item 53, wherein the lumican has at least about 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 11. (Item 55) The immunomodulatory fusion protein described in item 52, wherein the collagen-binding domain contains LAIR 1. (Item 56) LAIR1 is an immunomodulatory fusion protein as described in item 55, which has at least approximately 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 13. (Item 57) LAIR1 is an immunomodulatory fusion protein as described in item 55, having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 14. (Item 58) The immunomodulatory fusion protein described in item 52, wherein the collagen-binding domain includes LAIR 2. (Item 59) LAIR2 is an immunomodulatory fusion protein as described in item 58, having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 15. (Item 60) The immunomodulatory fusion protein according to any one of items 1 to 59, wherein the IL-2 comprises human IL-2. (Item 61) The immunomodulatory fusion protein according to any one of items 1 to 60, wherein the IL-2 includes human wild-type IL-2. (Item 62) The immunomodulatory fusion protein according to any one of items 1 to 61, wherein the IL-2 has at least about 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1. (Item 63) The immunomodulatory fusion protein according to any one of items 1 to 62, wherein the IL-2 has at least about 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2. (Item 64) The immunomodulatory fusion protein according to any one of items 1 to 63, wherein the IL-12 comprises human IL-12. (Item 65) An immunomodulatory fusion protein according to any one of items 1 to 64, wherein the IL-12 comprises human wild-type IL-12. (Item 66) The immunomodulatory fusion protein according to any one of items 1 to 65, wherein the IL-12 has at least about 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. (Item 67) The immunomodulatory fusion protein according to any one of items 1 to 66, wherein the linear polypeptide spacer is albumin. (Item 68) The immunomodulatory fusion protein according to any one of items 1 to 66, wherein the linear polypeptide spacer is an albumin-binding domain. (Item 69) The immunomodulatory fusion protein described in item 67, wherein the albumin includes human albumin. (Item 70) The immunomodulatory fusion protein described in item 67, wherein the albumin includes human serum albumin. (Item 71) The immunomodulatory fusion protein described in item 67, wherein the albumin contains at least about 80% sequence identity with respect to the amino acid sequences shown in SEQ ID NOs. 16-18. (Item 72) The immunomodulatory fusion protein according to item 68, wherein the albumin-binding domain has at least about 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 19. (Item 73) An immunomodulatory fusion protein as described in any one of items 1 to 72, having a molecular weight of at least 100 to 1000 kDa. (Item 74) A pharmaceutical composition comprising an immunomodulatory fusion protein as described in any one of items 1 to 73, and a pharmaceutically acceptable carrier. (Item 75) A method for activating, enhancing, or promoting an immune cell response in a subject, or for inhibiting, reducing, or suppressing an immune cell response in a subject, comprising the step of administering an effective amount of the pharmaceutical composition described in item 74 to a subject in need thereof. (Item 76) A method for treating cancer or reducing or inhibiting tumor growth, comprising the step of administering an effective amount of the pharmaceutical composition described in item 74 to a subject in need thereof. (Item 77) The method according to item 76, wherein the subject has at least one tumor. (Item 78) The method according to item 77, wherein the composition is administered to the at least one tumor either intratumorally (i.tu) or peritumorally (peri.tu). (Item 79) The method according to item 78, wherein the composition is administered by injection. (Item 80) The method according to any one of items 77 to 79, wherein at least one tumor size is reduced to or substantially the same as a reference standard. (Item 81) The method according to item 80, wherein the reference standard is the size of the tumor before administration. (Item 82) If the composition is retained in the tumor for more than 24 hours 1 / 2 The method according to any one of items 75 to 81, having the characteristics of: (Item 83) The method described in item 78, wherein less than 25% of the injected dose is detected in the serum 12 hours after intratumor injection. (Item 84) The aforementioned at least one tumor is 1 mm 2 The method according to any one of items 77 to 83, having 50 or fewer stromal CD8+ cytotoxic T cells (CTLs) per cell. (Item 85) The aforementioned at least one tumor is 1 mm 2 More than 50 stromal CD8+ cytotoxic T cells (CTLs) and 1 mm 2 The method according to any one of items 77 to 83, wherein each cell has 500 or fewer intraepithelial compartment CD8+ cytotoxic T cells (CTLs). (Item 86) The aforementioned at least one tumor is 1 mm 2 The method according to any one of items 77 to 83, wherein each cell has 500 or more intraepithelial compartment CD8+ cytotoxic T cells (CTLs). (Item 87) A method according to any one of items 75 to 86, which does not cause cytokine release syndrome in the subject. (Item 88) The method described in any one of items 75 to 87, wherein the subject does not experience Grade 4 cytokine release syndrome. (Item 89) A method for reducing or inhibiting tumor growth or treating cancer in a subject, comprising the step of administering to a subject in need an effective amount of a pharmaceutical composition described in item 74 and an effective amount of a second composition comprising (i) an antibody targeting a tumor antigen, (ii) a cancer vaccine, (iii) an immune checkpoint inhibitor, or (iv) adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject. (Item 90) The method according to item 89, wherein the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen, and / or an antibody targeting the tumor antigen specifically binds to human HER-2 / neu, EGFR, VEGFR, CD20, CD33, CD38, or an antigen-binding fragment thereof. (Item 91) The method according to item 89, wherein the cancer vaccine is a peptide containing one or more tumor-associated antigens, or a population of cells immunized with tumor antigens in vitro and administered to the subject. (Item 92) The method according to item 89, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment that binds to PD-1, PD-L1, CTLA-4, LAG3, or TIM3. (Item 93) The method according to item 89, wherein the immune effector cells contain a chimeric antigen receptor (CAR) molecule that binds to a tumor antigen. (Item 94) (i) IL-2; (ii) IL-12; (iii) LAIR2 collagen-binding domain, LAIR2 has a collagen-binding domain that contains at least 80% identity with the amino acid sequence shown in SEQ ID NO: 15; and (iv) Albumin, Albumin containing at least approximately 80% sequence identity with the amino acid sequences shown in SEQ ID NOs. 16-18. An immunomodulatory fusion protein containing [this protein].

Claims

1. It is an immunomodulatory fusion protein, (i) IL-2; (ii) IL-12; (iii) LAIR2 collagen-binding domain, LAIR2 is a collagen-binding domain consisting of the amino acid sequence shown in SEQ ID NO: 15; and (iv) Albumin, Albumin, consisting of the amino acid sequences shown in SEQ ID NOs. 16-18 An immunomodulatory fusion protein comprising, wherein IL-2 is located at the N-terminus, IL-12 is located at the C-terminus, the C-terminus of LAIR2 is operably linked to the N-terminus of IL-12, the albumin is positioned between IL-2 and LAIR2, the C-terminus of IL-2 is operably linked to the N-terminus of albumin, and the protein is linear.

2. A pharmaceutical composition comprising the immunomodulatory fusion protein described in claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, for activating, enhancing, or promoting the immune cell response in a target, or for inhibiting, reducing, or suppressing the immune cell response in a target.

4. The pharmaceutical composition according to claim 2 for treating cancer or for reducing or inhibiting tumor growth.

5. The pharmaceutical composition according to claim 4, wherein the subject has at least one tumor.

6. The pharmaceutical composition according to claim 5, characterized in that the composition is administered to at least one tumor either intratumor (i.tu) or peritumor (peri.tu).

7. The pharmaceutical composition according to claim 6, characterized in that the composition is administered by injection.

8. If the above composition is retained in the tumor for more than 24 hours １／２ A pharmaceutical composition according to claims 6 to 7, having the following characteristics.

9. The pharmaceutical composition according to claim 9, wherein less than 25% of the injected dose is detectable in the serum 12 hours after intratumor injection.

10. The aforementioned at least one tumor is 1 mm ２ A pharmaceutical composition according to any one of claims 5 to 9, comprising fewer than 50 interstitial CD8+ cytotoxic T cells (CTLs) per cell.

11. The aforementioned at least one tumor is 1 mm ２ More than 50 stromal CD8+ cytotoxic T cells (CTLs) per cell and 1 mm ２ A pharmaceutical composition according to any one of claims 5 to 9, having fewer than 500 intraepithelial compartment CD8+ cytotoxic T cells (CTLs) per cell.

12. The aforementioned at least one tumor is 1 mm ２ The pharmaceutical composition according to any one of claims 5 to 9, having more than 500 intraepithelial compartment CD8+ cytotoxic T cells (CTLs) per cell.

13. A pharmaceutical composition according to any one of claims 5 to 12, which does not cause cytokine release syndrome in the subject.

14. The pharmaceutical composition according to any one of claims 5 to 12, wherein the subject does not experience Grade 4 cytokine release syndrome.

15. A combination for reducing or inhibiting tumor growth or treating cancer, wherein the combination comprises an effective amount of the pharmaceutical composition described in claim 5 and a second composition comprising (i) an antibody targeting a tumor antigen, (ii) a cancer vaccine, or (iii) an immune checkpoint inhibitor, wherein the cancer vaccine is for treating cancer, and the immune checkpoint inhibitor is an antibody or antigen-binding fragment that binds to PD-1, PD-L1, CTLA-4, LAG3, or TIM3.

16. The combination according to claim 15, wherein the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen, and / or an antibody targeting the tumor antigen specifically binds to human HER-2 / neu, EGFR, VEGFR, CD20, CD33, CD38, or an antigen-binding fragment thereof.

17. The combination according to claim 15, wherein the cancer vaccine is a peptide containing one or more tumor-associated antigens, or a population of cells immunized with tumor antigens in vitro and administered to the target.

Images