Combination therapies for cancer

Abstract

Provided herein are combination therapies including a checkpoint inhibitor and an immunomodulatory fusion protein, as well as corresponding compositions. Methods of administering the combination therapies and kits also are described.

Description

COMBINATION THERAPIES FOR CANCERREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (67280W001_Seq.xml; Size: 100,468 bytes; and Date of Creation: August 15, 2024) is herein incorporated by reference in its entirety.RELATED APPLICATIONS

[0002] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 579,429, filed August 29, 2023 and U.S. Provisional Patent Application Ser. No. 63 / 593,615, filed October 27, 2023, the content of each is hereby incorporated by reference in its entirety into this disclosure.BACKGROUND

[0003] Despite clinical evidence of anti-tumor activity, cytokine therapies have been hampered by a narrow therapeutic window and limited response rate. Two such cytokines are interleukin 2 (IL- 2) and interleukin 12 (IL- 12), which potently synergize to activate and proliferate T cells and natural killer (NK) cells.

[0004] IL-2 was initially associated with treatment-related mortality, and IL-2 toxicity can manifest in multiple organ systems, most significantly the heart, lungs, kidneys, and central nervous system. The most common manifestation of IL-2 toxicity is capillary leak syndrome, resulting in a hypovolemic state and fluid accumulation in the extravascular space.

[0005] As for IL- 12, the most common adverse events reported in clinical trials were fever and flu-like symptoms. Other common toxicities included fatigue, nausea, vomiting, diarrhea, and headache. Hepatic toxicities were also frequently observed, including elevated alanine transaminase (ALT) and aspartate transaminase (AST). However, the only approved human IL-2 product (Proleukin) is rarely used in the clinic due to systemic toxicities, and no IL- 12 product has been approved to date due to severe dose-limiting toxicities.

[0006] A popular strategy used to trigger a systemic immune response against tumor cells that have recently produced excellent results in the clinical setting is the use of checkpoint inhibitors either alone or in combination with other anti-cancer modalities. Checkpoint inhibitors are a class of immunotherapy drugs that work by targeting specific molecules known as checkpoint proteins.These checkpoint proteins regulate the immune system's response to prevent excessive activation and damage to normal cells. At the same time, these checkpoint proteins often play a role in suppressing the immune system's ability to recognize and attack cancer cells.

[0007] Probably the most well-known of the checkpoint proteins is programmed cell death protein 1 (PD-1) and its ligand, programmed cell death ligand 1 (PD-L1). Checkpoint inhibitors are designed to block the interaction between PD-1 and PD-L1, which stops the inhibition of the immune system. This allows T cells to recognize and attack cancer cells more effectively. PD-1 is found on the surface of T cells, while PD-L1 is often expressed on the surface of cancer cells and other cells within the tumor microenvironment. When PD-1 on T cells binds to PD-L1 on cancer cells, it can inhibit the immune response and allow the cancer cells to evade destruction by the immune system.

[0008] To reduce the chances of triggering systemic toxicities, strategies are being developed to localize the action of IL-2 and IL- 12, and trigger an abscopal effect. An abscopal effect is a phenomenon in cancer treatment where localized treatment, applied to one tumor site, not only affects the targeted tumor but also leads to a systemic immune response. While intratumoral injection reduces the chances of systemic toxicities, IL-2 and IL- 12 may still leak from the tumor at the injection site. There remains a need for the improvement of the efficacy of cancer immunotherapy, including immunotherapy approaches that restrict the most potent immune activation events to tumor tissue while preventing systematic toxicity. Therefore, it is desirable to explore combination therapies that combine checkpoint inhibitors and cytokines.BRIEF SUMMARY

[0009] Described herein is a method of treating cancer in a subject in need thereof, the method including administering a combination therapy which comprises a therapeutically effective amount of a checkpoint inhibitor and a therapeutically effective amount of an immunomodulatory fusion protein, the immunomodulatory fusion protein comprising: an interleukin 2 (IL-2); an interleukin (IL-12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain.

[0010] In an aspect, the checkpoint inhibitor comprises an antagonist of PD- 1 , an antibody which binds PD-1, PD-L1, CTLA-4, LAG3, or TIM3 or an antigen binding fragment thereof. In an aspect,the antagonist of PD-1 is a humanized IgG4 anti-PD-1 monoclonal antibody. In an aspect, the antagonist of PD-1 is pcmbrolizumab.

[0011] In an aspect, the combination therapy comprises an additional checkpoint inhibitor.

[0012] In an aspect, the subject is receiving or has received standard of care (SoC) chemotherapy, radiation, targeted cancer therapy, a cancer vaccine, and / or an immunomodulatory therapy

[0013] In an aspect, the cancer is a solid tumor. In an aspect, the solid tumor is an advanced or metastatic solid tumor that tests positive for PD-L1 expression and is selected from the group consisting of bladder cancer, breast cancer, including triple negative breast cancer (TNBC), renal cell cancer, including clear cell renal cell carcinoma (ccRCC), kidney cancer, head / neck squamous cell carcinoma, malignant melanoma, non- small-cell lung cancer (NSCLC), including squamous cell carcinoma (SCC), ovarian cancer, pancreatic cancer, prostate cancer, small-cell lung cancer (SCLC), colorectal cancer, endometrial cancer, cervical cancer, sarcoma, and hepatocellular carcinoma (HCC).

[0014] In an aspect, the IL-2 comprises human IL-2 or human wild-type IL-2. In an aspect, the IL- 2 comprises at least about 80% sequence identity to the amino acid sequence as set forth in SEQ ID NOri or SEQ ID NO:2.

[0015] In an aspect, the IL-12 comprises human IL-12 or human wild-type IL-12. In an aspect, the IL- 12 comprises at least about 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6

[0016] In an aspect, the albumin comprises a human albumin or human serum albumin. In an aspect, the albumin comprises at least about 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 16-18. In an aspect, the albumin binding domain comprises at least about 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 19-22.

[0017] In an aspect, the immunomodulatory fusion protein further comprises a glycine- serine (GS) repeat.

[0018] In an aspect, the LAIR1 has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14. In an aspect, the LAIR2 has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO: 15. In an aspect, the immunomodulatory fusion protein comprises at least about 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs:39, 45, 50, 51, 57, 63, 66, 69, or 73.

[0019] In an aspect, the combination therapy is administered via intratumoral, intraperitoneal, intranodal, pcritumoral, subcutaneous, or intravenous delivery. In an aspect, the immunomodulatory fusion protein is administered simultaneously, concurrently, sequentially, successively, alternatively with or separately from the checkpoint inhibitor.

[0020] In an aspect, the combination therapy results in a synergistic reduction in tumor burden, tumor regression, and / or tumor development of said cancer.

[0021] In an aspect, the method does not result in cytokine release syndrome in the subject.

[0022] In an aspect, the subject does not experience grade 4 cytokine release syndrome.

[0023] One aspect of the disclosure is a medicament comprising an immunomodulatory fusion protein for use in combination with a PD-1 antagonist for treating cancer in an individual, said immunomodulatory fusion protein comprising an interleukin 2 (IL-2); an interleukin (IL- 12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain.

[0024] In an aspect, the medicament further comprises a pharmaceutically acceptable excipient.

[0025] One aspect of the disclosure is the use of an immunomodulatory fusion protein comprising an interleukin 2 (IL-2); an interleukin (IL- 12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain in the manufacture of a medicament for treating a cancer in an individual when administered in combination with a PD-1 antagonist.

[0026] One aspect of the disclosure is a kit which comprises a first container, a second container and a package insert, wherein the first container comprises at least one dose of a medicament comprising an anti-PD-1 antagonist, the second container comprises at least one dose of a medicament comprising an immunomodulatory fusion protein comprising an interleukin 2 (IL-2); an interleukin (IL-12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain, and the package insert comprises instructions for treating an individual for cancer using the medicaments.

[0027] In an aspect, the instructions state that the medicaments are intended for use in treating an individual having a cancer that tests positive for PD-L1 expression by an immunohistochemical (IHC) assay.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0029] FIGs 1A-1F depict the abscopal effect mediated by mCLN-617. FIG. 1A is a schematic of the dual flank MC38 tumor model used in FIGs. IB- ID. FIGs. IB and ID show mice that were IT-treated in their right MC38 flank tumor with 400 pmol mCLN-617 or PBS on days 0 and 5 with or without IP-treatment with 10 mg / kg anti-PDl antibody BIW. FIG. 1C depicts mice were IT- treated in their right MC38 flank tumor with 50 (low), 100 (mid), or 200 pmol (high) mCLN-617 on days 0 and 5, and peripheral blood evaluated by flow cytometry on day 6. FIG. IE depicts a schematic of MC38 liver metastasis model used in FIG. IF. FIG. IF depicts mice that were implanted with MC38 flank tumors and intrasplenically injected with MC38-luc cells, which seeded to the liver after resection of the spleen. The flank tumor was IT-treated with 200 pmol mCLN-617 or PBS on days 0 and 5 with or without IP-treatment with 10 mg / kg anti-PDl antibody BIW. Shown are curves tracking flank tumor volume and luciferase signal.

[0030] FIG. 2 depicts a dose response in the B16F10 model Mice were inoculated with two B16F10 flank tumors ten days apart. The first inoculated tumor was IT-treated with the indicated dose of mCLN-617 or PBS on days 0, 6, and 12. Shown are tumor growth curves of both the treated and untreated flank tumors.

[0031] FIG. 3A shows mice bearing two MC38 tumors were IT-treated in the right flank with the indicated dose of PBS or mCLN-617 using the indicated volume on days 0 and 5. Shown are tumor growth curves of both the treated and untreated MC38 flank tumors. FIG. 3B shows 30 minutes after the first mCLN-617 treatment, serum samples were evaluated for IL-12p70 concentration by MSD analysis. The untreated tumor volume on day 21 is plotted against the measured IL-12p70 concentration on a per mouse basis. Data is pooled across dosing volumes.

[0032] FIG. 4A-4E shows an immunomodulatory fusion protein mediated clonal expansion of tumor-associated T cell clonotypes. Mice were implanted with MC38 tumors in both flanks and treated in the right flank IT with an immunomodulatory fusion protein and IP with isotype control (Group 1), IT with an immunomodulatory fusion protein and IP with anti-PDl (Group 2), IT with vehicle and IP with anti-PDl (Group 3), or IT with vehicle and IP with isotype control (Group 4). IT treatments were performed on days 0 and 5 and IP treatments on days 0 and 3. The immunomodulatory fusion protein was administered at 200 pmol and antibodies at 10 mg / kg.Peripheral was collected on days 0 and 6, and tumors collected on day 6 for TCRB sequencing (n=4-5 per group) FIG. 4A depicts Simpson clonality was calculated for each group on day 0 (predose) and day 6 (postdose) FIG. 4B depicts individual clonotypes were correlated between predose and postdose peripheral blood samples. Shown are scatterplots for individual representative mice, and each point represents the frequencies of a unique clonotype. Clonotypes are annotated based on association with the treated tumor. FIG. 4C shows the Morisita index calculated as a measure of correlation between predose and postdose peripheral blood samples. FIG. 4D depicts newly expanded clones in each group were characterized as previously detected or newly detected postdose. FIG. 4E depicts the number of newly expanded clones in each mouse per group that is right (treated) or left (untreated) tumor associated.

[0033] FIGs.5A-5E shows Tumor clonotype correlations. Mice were treated and samples collected as in FIG. 4. FIG. 5A depicts individual clonotypes correlated between the treated and untreated tumor samples. Shown are scatterplots for individual representative mice, and each point represents the frequencies of a unique clonotype. FIG. 5B depicts a Morisita index calculated as a measure of correlation between treated and untreated tumor samples. FIG. 5C depicts clonotype correlation but clonotypes are annotated based on association with the untreated tumor. FIGs. 5D- E shown the fraction of newly expanded clones that are associated with the treated (FIG. 5D) and untreated (FIG. 5E) tumor.

[0034] FIGs. 6A-6J depicts a characterization of CLN-617 according to an aspect of this disclosure. FIG. 6A depicts a schematic of mCLN-617 design. As shown in FIG. 6B, CLN-617 was incubated in the indicated matrix for the indicated duration at 37C, and a Western Blot performed detecting IL-2 or IL- 12. FIGs. 6C - 6J shows Human PBMCs that were CD3- stimulated overnight, then treated with the indicated test articles for 10 min (FIGs. 6D-6E), 2h (FIG. 6C), 48h (FIG. 6F-6H,6J), or 96h (FIG. 61). PBMC lysate was analyzed by signaling microarray (FIG. 6C) or CD8+assessed by flow cytometry (FIG. 6D-FIG. 6J). Data from one representative donor is shown.

[0035] FIGs.7A-7D shows the bioactivity of immunomodulatory fusion proteins. FIG. 7A-7B ELISA based binding assay on rat collagen I coated plates with the indicated test articles. FIG. 7C CTLL-2 proliferation assay with normal or collagen-coated tissue culture plates. FIG. 7D HEK- Blue IL- 12 alkaline phosphatase secretion assay with normal or collagen-coated tissue culture plates.

[0036] FIGs.8A-8C show activity of a human immunomodulatory fusion protein (h-IMFP) on PBMCs. In FIGs. 8A-8C studies were performed as described in FIG. 16D-I Shown here arc data with CD4+ T cells, CD8+ T cells, and CD56+ NK cells. ”Amber-IL-2” refers to LAIR2-HSA-IL2 and “Amber-IL-12” refers to IL12-HSA-LAIR2. Data from one representative data is shown.

[0037] FIG. 9 is a schematic showing a multi-phase, open-label, dose-escalation, optimization, and expansion treatment period of a study that will assess the safety, tolerability, and efficacy of IT CLN-617 in combination with pembrolizumab.

[0038] FIG. 10A is a schematic showing the dosing schema for Part 1 of the treatment period depicted in FIG. 9.

[0039] FIG. 10B is a schematic showing the dosing schema for Part 2 and Part 3 of the treatment period depicted in FIG. 9.

[0040] FIG. 11 A is a table showing Part 1 dose escalation treatment groups as depicted in FIG. 9.

[0041] FIG. 1 IB is a table showing Part 2 optimization treatment groups as depicted in FIG. 9.

[0042] FIG. 11C is a table showing Part 3 dose expansion treatment groups as depicted in FIG. 9.

[0043] FIGs. 12A and 12B show representative images of Screening and C2D1 Pre-dose Injected tumor biopsy samples stained with T / dendritic cell (FIG. 12A) and NK / macrophage immunofluorescence panels (FIG. 12B).

[0044] FIG. 13 summarizes the data from patient #1 (top) and patient #2 (bottom) in C2D1 and C4D1 injected and uninjected tumor biopsies compared to the screening biopsy in the tumor compartment.DETAILED DESCRIPTION

[0045] I. Introduction

[0046] To avoid systemic toxicities and promote anti-tumor activity, it is attractive to inject cytokines directly into the tumor, with the aim of maximizing bioavailability in the lesion and reducing systemic exposures. Recent advances in interventional radiology now allow for intratumoral (IT) injection across a range of solid tumors and anatomical locations. Furthermore, local treatment with immunomodulatory agents can stimulate a therapeutic systemic response, or abscopal effect, in both mice and humans, mediating the control of distal metastases. However, locally administered agents rapidly leak into circulation upon injection unless specifically retained,as noted by high measurable systemic drug concentrations and poor tolerability across multiple studies. For IL-2 and IL- 12 in particular, the clinical maximum tolerated dose (MTD) has not been improved by IT administration. In a previous study, murine IL-2 and IL- 12 were separately fused to collagen binding domains to promote retention in the tumor by anchoring the cytokines to collagen, which is in high abundance in solid tumors. In the present application, the inventors show that an immunomodulatory fusion protein comprised of human IL-2, human LAIR2, human serum albumin, and human IL- 12 (also referred to as CLN-617) can be used in synergy with checkpoint inhibition in a clinical setting.

[0047] The present invention is based, in part, on the surprising finding that concurrent administration of an immunomodulatory fusion protein of the present invention and a PD-1 antibody to tumor-bearing mice resulted in significantly higher anti-tumor efficacy compared to either agent alone. Thus, in one example, the invention provides a method for treating a cancer in an individual by administering to the individual a combination therapy that comprises a PD-1 antagonist and an immunomodulatory fusion protein of the present invention. In another example, the invention provides a medicament comprising an immunomodulatory fusion protein of the present invention for use in combination with a PD- 1 antagonist for treating a cancer.

[0048] Other embodiments provide the use of an immunomodulatory fusion protein of the present invention in the manufacture of a medicament for treating a cancer in an individual when administered in combination with a PD-1 antagonist. In some preferred embodiments, the medicaments comprise a kit, and the kit also comprises a package insert comprising instructions for using the combination to treat a cancer in an individual. In the aforementioned treatment methods, medicaments and uses, the PD-1 antagonist inhibits the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD- 1. In some preferred embodiments of the above treatment method, medicaments and uses, the PD-1 antagonist is a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to PD-1 or to PD-L1 and blocks the binding of PD-L1 to PD- 1.

[0049] II. Definitions

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0051] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0052] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0053] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0054] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0055] By " consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0056] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three -element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0057] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0058] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0059] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0060] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples arereported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0061] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0062] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0063] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0064] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0065] The terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and / or decrease the number, frequency, or severity of clinical symptoms and / or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms “treatment of a disease” and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.

[0066] Thus, to “treat” or “treating” a cancer as used herein means to administer a combination therapy of checkpoint inhibitor, such as a PD-1 antagonist, and an immunomodulatory fusion protein of the present invention to a subject having a cancer, or diagnosed with a cancer, to achieve at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth. Positive therapeutic effects in cancer can be measured in a number of ways (See, W. A. Weber, J. Nucl. Med. 5O:1S-1OS (2009)). For example, with respect to tumor growth inhibition, according to NCI standards, a T / CI=42% is the minimum level of antitumor activity. A T / C<10% is considered a high anti-tumor activity level, with T / C (%)=Median tumor volume of the treated / Median tumor volume of the control 100. In some embodiments, the treatment achieved by a therapeutically effective amount is any of progression free survival (PFS), disease free survival (DFS) or overall survival (OS). PFS, also referred to as “Time to Tumor Progression” indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. DFS refers to the length of time during and after treatment that the patient remains free of disease. OS refers to a prolongation in life expectancy as compared to naive or untreated individuals or patients. The dosage regimen of a combination therapy described herein that is effective to treat a cancer patient may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. While an embodiment of the treatment method, medicaments and uses of the present invention may not be effective in achievinga positive therapeutic effect in every subject, it should do so in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the chi2-test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere- Terpstra-test and the Wilcoxon-test.

[0067] The terms “prophylactic”, “preventive”, “preventing”, and “prevention” refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.

[0068] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0069] III. Combination Therapy and Methods of Use

[0070] One aspect of the invention provides for methods of treating cancer in a subject by administering a combination therapy, which includes a therapeutically effective amount of a checkpoint inhibitor and a therapeutically effective amount of an immunomodulatory fusion protein. As used herein, the term “therapeutically effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “therapeutically effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats a tumor, an effective amount of an agent is, for example, an amount sufficient to reduce or decrease a size of a tumor or to inhibit a tumor growth, as compared to the response obtained without administration of the agent. The term “therapeutically effective amount” can be used interchangeably with “effective dose,” “effective amount,” or “effective dose.”

[0071] The combination therapy may be administered in a variety of modes. The combination therapy may be administered via intratumoral delivery (IT). An IT delivery involves the direct injection of the combination therapy into the tumor. This primes tumor infiltrating T cells, whichthen circulate systemically to generate a global antitumor response. The combination therapy may be administered via intraperitoneal delivery (IP), which involves the injection of the combination therapy into the peritoneal cavity. The combination therapy may be administered via intravenous delivery (IV), which involves the combination therapy being injected into a vein and directly into the bloodstream. The combination therapy may be administered via intranodal (IN) delivery. IN delivery involves direct administration of the combination therapy to immune cells in lymph nodes. The combination therapy may be administered via peritumoral delivery which allows for delivery of the combination therapy directly into the tissue surrounding a tumor. The combination therapy may be administered via subcutaneous (SQ or Sub-Q) delivery. SQ delivery involves injecting or infusing the combination therapy between the skin and the muscle. SQ delivery of the combination therapy may provide several advantages including, but not limited to, fast administration times and increased patient access and compliance.

[0072] The checkpoint inhibitor and the immunomodulatory fusion protein may be administered in a variety of ways. For example, the immunomodulatory fusion protein may be administered simultaneously, concurrently, sequentially, successively, alternatively with or separately from the checkpoint inhibitor.

[0073] Cancer tumors are generally classified based on clinical and pathological features, such as stage and grade. The disclosed invention could be administered to a subject with an advanced- stage cancer.

[0074] In some aspects, the cancer being treated is a solid tumor. Advanced stage solid tumor cancers are particularly difficult to treat. Current treatments include surgery, radiotherapy, immunotherapy, and chemotherapy. Surgery alone may be an appropriate treatment for small localized tumors, but large invasive tumors may be unresectable by surgery. Other common treatments, such as radiotherapy and chemotherapy, are associated with undesirable side effects and damage to healthy cells. However, in some non-limiting aspects, the subject receiving the combination therapy is also receiving or has received another cancer treatment such as chemotherapy, including standard of care (SoC) chemotherapy, radiation, targeted cancer therapy, a cancer vaccine, and / or an immunomodulatory therapy.

[0075] “Chemotherapy” refers to the administration of one or more chemotherapeutic drugs and / or other agents to a subject with or suspected to have cancer by various methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal,or inhalation or in the form of a suppository. The term “Standard of Care (SoC) Chemotherapy” refers to the conventional or widely accepted chemotherapy treatment regimen that is typically used for a particular type of cancer. “Radiation”, “radiation therapy”, or “radiotherapy” refers the use of directed X-rays, subatomic particles, or ionizing radiation cancer management in both curative and palliative settings. “Targeted cancer therapy” refers is a type of cancer treatment that specifically targets the molecular and genetic abnormalities driving the growth and survival of cancer cells and are designed to interfere with specific molecules or pathways that are critical for cancer cell proliferation and survival. Non-limiting examples include monoclonal antibodies and small molecule inhibitors. A “cancer vaccine” refers to an immunotherapy designed to either prevent (prophylactic) or treat (therapeutic) cancer by stimulating the body's immune system to recognize and attack cancer cells. Therapeutic vaccines may contain cancer-specific antigens, pieces of cancer cells, or whole cancer cells that have been killed or weakened. “Immunomodulatory therapy” refers to a type of treatment that modifies or regulates the immune system's response to diseases, including, but not limited to, cancer and autoimmune conditions. In the context of cancer, immunomodulatory therapy typically aims to boost the immune system's ability to recognize and attack cancer cells. This can involve the administration of checkpoint inhibitors, cytokines, and / or CAR-T cell therapy. In some aspects, the combination therapy is given to a subject who failed to respond to the previous cancer treatment or had disease progression after administration of previous cancer treatment.

[0076] “Solid tumors” are tumors that do not contain any liquid or cysts. Non-limiting examples of solid tumors include sacromas and carcinomas. Sarcomas are tumors in a blood vessel, bone, fat tissue, ligament, lymph vessel, muscle, or tendon. Carcinoma refers to a malignant neoplasm of epithelial origin or cancer of the internal or external lining of the body. Carcinomas are divided into two major subtypes: adenocarcinoma, which develops in an organ or gland, and squamous cell carcinoma, which originates in the squamous epithelium. Adenocarcinomas generally occur in mucus membranes and are first seen as a thickened plaque-like white mucosa. They often spread easily through the soft tissue where they occur. Squamous cell carcinomas occur in many areas of the body. Most carcinomas affect organs or glands capable of secretion, such as the breasts, which produce milk, or the lungs, which secrete mucus, or colon or prostate or bladder.

[0077] In an aspect, the solid tumor is an advanced or metastatic solid tumor that tests positive for PD-L1 expression and is selected from the group including bladder cancer, breast cancer, includingtriple negative breast cancer (TNBC), renal cell cancer, including clear cell renal cell carcinoma (ccRCC), kidney cancer, hcad / ncck squamous cell carcinoma, malignant melanoma, non-small- cell lung cancer (NSCLC), including squamous cell carcinoma (SCC), ovarian cancer, pancreatic cancer, prostate cancer, small-cell lung cancer (SCLC), colorectal cancer, endometrial cancer, cervical cancer, sarcoma, and hepatocellular carcinoma (HCC).The combination therapies of the present invention are designed to treat such tumors.

[0078] “Tumor” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0079] “Tumor burden” also referred to as “tumor load”, refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumor(s), throughout the body, including lymph nodes and bone marrow. Tumor burden can be determined by a variety of methods known in the art, such as, e.g., by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scans.

[0080] The term “tumor size” refers to the total size of the tumor, which can be measured as the length and width of a tumor. Tumor size may be determined by a variety of methods known in the art, such as, e.g., by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., bone scan, ultrasound, CT or MRI scans.

[0081] In some embodiments, the combinations also may be useful in the treatment of heme malignancies, including, but not limited to a heme malignancy is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM),myeloid cell leukemia-1 protein (Mcl -1 ), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL).

[0082] “Checkpoint inhibitors”, also referred to as “immune checkpoint inhibitors” are a class of immunotherapy compounds that are configured to block checkpoint proteins from binding from their partner proteins. Checkpoint inhibitors may be antibodies or antigen binding fragments configured to, for example, inhibit PD-1, PD-L1, CTLA-4, LAG-3, and TIM3. In a non-limiting example, the checkpoint inhibitor may be an antagonist of PD- 1.

[0083] The PD-1 pathway, comprising the immune cell co-receptor Programmed Death 1 (PD-1) and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), mediates local immunosuppression in the tumor microenvironment. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2.

[0084] “PD-1 antagonist” means any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDLL, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the treatment method, medicaments and uses of the present invention in which a human individual is being treated, the PD-1 antagonist blocks binding of human PD-L1 to human PD-1, and preferably blocks binding of both human PD-L1 and PD-L2 to human PD-1. Human PD-1 amino acid sequences can be found in NCBI Locus No.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.: NP_054862 and NP_079515, respectively.

[0085] A PD-1 antagonist thus generally refers to a drug, antibody (including monoclonal antibodies), or antigen binding fragments thereof, or other inhibitor that can or is configured to block binding of PD-L1 to PD-1. An antagonist of PD-1 may be a monoclonal antibody (mAb), including, but not limited to, a human antibody, a humanized antibody, or a chimeric antibody. The mAb may also include a human constant region. The mAb may be a humanized IgG4. Nonlimiting examples of mAbs include MEDI0680 (Medlmmune / AstraZeneca), Nivolumab (Bristol- Myers Squibb / Ono Pharmaceuticals), Pembrolizumab (Merck), and Pidilizumab (CureTech). Pembrolizumab (Keytruda, MK-3475, formerly known as lambrolizumab; Merck, WhitehouseStation, NJ) is a distinct anti-PD-1 antibody FDA-approved in the US for the treatment of Melanoma, Non-Small Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Cancer (HNSCC), Classical Hodgkin Lymphoma (cHL), Primary Mediastinal Large B-Cell Lymphoma, Urothelial Carcinoma, Micro satellite Instability-High or Mismatch Repair Deficient Cancer, Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer (CRC), Gastric Cancer, Esophageal Cancer, Cervical Cancer, Hepatocellular Carcinoma (HCC), Merkel Cell Carcinoma (MCC), Renal Cell Carcinoma (RCC), Endometrial Carcinoma, Tumor Mutational Burden-High (TMB-H) Cancer, Cutaneous Squamous Cell Carcinoma (cSCC), Triple-Negative Breast Cancer (TNBC), and Adult Classical Hodgkin Lymphoma and Adult Primary Mediastinal Large B-Cell Lymphoma.

[0086] “PD-L1” or “PD-L2” expression, as used herein, means any detectable level of expression of the designated PD-L protein on the cell surface or of the designated PD-L mRNA within a cell or tissue. PD-L protein expression may be detected with a diagnostic PD-L antibody in an IHC assay of a tumor tissue section or by flow cytometry. Alternatively, PD-L protein expression by tumor cells may be detected by PET imaging, using a binding agent (e.g., antibody fragment, affibody and the like) that specifically binds to the desired PD-L target, e.g., PD-L1 or PD-L2. Techniques for detecting and measuring PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0087] Several approaches have been described for quantifying PD-L1 protein expression in IHC assays of tumor tissue sections. See, e.g., Thompson, R. H., et al., PNAS 101 (49); 17174-17179 (2004); Thompson, R. H. et al., Cancer Res. 66:3381-3385 (2006); Gadiot, J., et al., Cancer 117:2192-2201 (2011); Taube, J. M. et al., Sci Transl Med 4, 127ra37 (2012); and Toplian, S. L. et al., New Eng. J Med. 366 (26): 2443-2454 (2012).

[0088] One approach employs a simple binary end-point of positive or negative for PD-L1 expression, with a positive result defined in terms of the percentage of tumor cells that exhibit histologic evidence of cell- surface membrane staining. A tumor tissue section is counted as positive for PD-L1 expression is at least 1%, and preferably 5% of total tumor cells.

[0089] In another approach, PD-L1 expression in the tumor tissue section is quantified in the tumor cells as well as in infiltrating immune cells. The CPS (combined positive score), which includes PD-L1 expression on immune cells and percentage of tumor cells and infiltrating immune cells that exhibit membrane staining are separately quantified as <5%, 5 to 9%, and then in 10%increments up to 100%. For tumor cells, PD-L1 expression is counted as negative if the score is <5% score and positive if the score is ^5%. PD-L1 expression in the immune infiltrate is reported as a semi-quantitative measurement called the adjusted inflammation score (AIS), which is determined by multiplying the percent of membrane staining cells by the intensity of the infiltrate, which is graded as none (0), mild (score of 1, rare lymphocytes, ), moderate (score of 2, focal infiltration of tumor by lymphohistiocytic aggregates), or severe (score of 3, diffuse infiltration). A tumor tissue section is counted as positive for PD-L1 expression by immune infiltrates if the AIS is ^5.

[0090] A tissue section from a tumor that has been stained by IHC with a diagnostic PD-L1 antibody may also be scored for PD-L1 protein expression by assessing PD-L1 expression in both the tumor cells and infiltrating immune cells in the tissue section, using a novel scoring process described in application 61 / 807,581, filed 2 Apr. 2013. This PD-L1 scoring process comprises examining each tumor nest in the tissue section for staining and assigning to the tissue section one or both of a modified H score (MHS) and a modified proportion score (MPS). To assign the MHS, four separate percentages are estimated across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests: (a) cells that have no staining (intensity=O), (b) weak staining (intensity=l+), (c) moderate staining (intensity=2+) and (d) strong staining (intensity=3+). A cell must have at least partial membrane staining to be included in the weak, moderate, or strong staining percentages. The estimated percentages, the sum of which is 100%, are then input into the formula of lx(percent of weak staining cells)+2x(percent of moderate staining cells)+3x(percent of strong staining cells), and the result is assigned to the tissue section as the MHS. The MPS is assigned by estimating, across all of the viable tumor cells and stained mononuclear inflammatory cells in all of the examined tumor nests, the percentage of cells that have at least partial membrane staining of any intensity, and the resulting percentage is assigned to the tissue section as the MPS. In some embodiments, the tumor is designated as positive for PD-L1 expression if the MHS or the MPS is positive.

[0091] The level of PD-L mRNA expression may be compared to the mRNA expression levels of one or more reference genes that are frequently used in quantitative RT-PCR, such as ubiquitin C, or RNAscq.

[0092] In some embodiments, a level of PD-L1 expression (protein and / or mRNA) by malignant cells and / or by infiltrating immune cells within a tumor is determined to be “overexpressed” or“elevated” based on comparison with the level of PD-L1 expression (protein and / or mRNA) by an appropriate control. For example, a control PD-L1 protein or mRNA expression level may be the level quantified in nonmalignant cells of the same type or in a section from a matched normal tissue. In some preferred embodiments, PD-L1 expression in a tumor sample is determined to be elevated if PD-L1 protein (and / or PD-L1 mRNA) in the sample is at least 10%, 20%, or 30% greater than in the control.

[0093] “Sustained response” means a sustained therapeutic effect after cessation of treatment with a therapeutic agent or a combination therapy described herein. In some embodiments, the sustained response has a duration that is at least the same as the treatment duration, or at least 1.5, 2.0, 2.5, or 3 times longer than the treatment duration.

[0094] “Tissue Section” refers to a single pail or piece of a tissue sample, e.g., a thin slice of tissue cut from a sample of a normal tissue or of a tumor.

[0095] Other checkpoint proteins exist with different inhibitory pathways. Cytotoxic T- Lymphocyte-Associated Protein 4 (CTLA-4) is a protein that competes with another receptor (CD28) for binding to the antigen-presenting cells. CD28 promotes T cell activation, while CTLA- 4 inhibits it. Lymphocyte- Activation Gene 3 (LAG3) LAG3 is a receptor found on T cells that can negatively regulate T cell function by inhibiting T cell activation and proliferation. In addition, T- cell Immunoglobulin and Mucin Domain 3 (TIM3) is another immune checkpoint protein found on T cells and other immune cells. TIM3 is often expressed on exhausted T cells in chronic infections and in the tumor microenvironment. Like antagonists of PD-1, therapies that block these checkpoints aim to release the brakes on the immune response, enabling immune cells to more effectively target and destroy cancer cells.

[0096] Cytokines are produced in response to stimuli such as infection, whereby they typically act in an autocrine (cell signaling itself) or paracrine (cell signaling neighboring cells within close proximity) but not endocrine fashion (specialized cells releasing signaling molecules into the bloodstream targeting cells or tissues throughout the body). Due to their local expression and consumption, cytokines rarely achieve high levels in circulation; however, when they do, cytokines can trigger severe and detrimental inflammatory conditions such as cytokine release syndrome.

[0097] Cytokine release syndrome (CRS) is a potentially severe immune reaction when rapid activation and proliferation of immune cells occur triggering the release of a large amount of cytokines systematically. These cytokines can lead to systemic inflammation and a range ofsymptoms that can vary in severity. CRS is categorized into different grades based on the severity of symptoms. The grades range from 1 to 4, with grade 4 being the most severe. Grade 4 CRS is characterized by extremely high fever, hypotension (low blood pressure), hypoxia (low oxygen levels), and organ dysfunction. Grade 4 CRS is rare but can be life-threatening if not promptly recognized and managed. To avoid CRS, treatments limit dosage to the clinical maximum tolerated dose (MTD). In addition, premedication with acetaminophen (paracetamol) or non-steroidal antiinflammatory drugs (NSAIDs) is a common strategy to mitigate cytokine induction and the risk of developing CRS. Alternatively, local expression and consumption are encouraged and helps prevent elevated levels of cytokines in the bloodstream. In some embodiments, subjects receiving the combination therapy do not experience cytokine release syndrome, including, but not limited to, a grade 4 CRS.

[0098] IV. Immunomodulatory Fusion Proteins and Components Thereof

[0099] One of the components of the combination therapies of the present invention is an immunomodulatory fusion protein. The immunomodulatory fusion proteins and components thereof are now described below. As used herein, "interleukin (IL)-2," (IL-2) refers to a pleiotropic cytokine that activates and induces proliferation of T cells and natural killer (NK) cells. The biological activity of IL-2 is mediated through a multi-subunit IL-2 receptor complex (IL-2R) of three polypeptide subunits that span the cell membrane: p55 (IL-2Ra, the alpha subunit, also known as CD25 in humans), p75 (IL-2R[3, the beta subunit, also known as CD 122 in humans) and p64 (IL-2Ry, the gamma subunit, also known as CD 132 in humans).

[0100] In some embodiments, the immunomodulatory fusion protein comprises an IL-2. In some embodiments, the IL-2 is operably linked to a collagen binding domain. In some embodiments, the immunomodulatory fusion protein comprises a member of the IL-2 family operably linked to a collagen binding domain.

[0101] T cell response to IL-2 depends on a variety of factors, including: (1) the concentration of IL-2; (2) the number of IL-2R molecules on the cell surface; and (3) the number of IL-2R occupied by 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)).

[0102] In some embodiments, the IL-2 is wild-type IL-2 (e.g., human IL-2 in its precursor form or mature IL-2. In some embodiments, the IL-2 is human IL-2. In some embodiments, the IL-2comprises an amino acid sequence having 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 to the amino acid sequence as set forth in SEQ ID NOs: 1 or 2, or a portion thereof. In some embodiments, the IL-2 comprises an amino acid sequence having 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 to the amino acid sequence as set forth in SEQ ID NOs: 3 or 4, or a portion thereof.

[0103] In other embodiments, the IL-2 is a mutant human IL-2. The term “IL-2 mutant” or “mutant IL-2 polypeptide” as used herein is intended to encompass any mutant forms of various forms of the IL-2 molecule including full-length IL-2, truncated forms of IL-2 and forms where IL-2 is linked to another molecule such as by fusion or chemical conjugation. The various forms of IL-2 mutants are characterized in having a at least one amino acid mutation affecting the interaction of IL-2 with CD25. This mutation may involve substitution, deletion, truncation, or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, an IL-2 mutant may be referred to herein as an IL-2 mutant peptide sequence, an IL-2 mutant polypeptide, IL-2 mutant protein or IL- 2 mutant analog.

[0104] In some embodiments, IL-2 mutants comprise an amino acid sequence that is at least 80% identical to SEQ ID NOs: 1 or 2 that bind CD25. For example, some embodiments an IL-2 mutant has at least one mutation (e.g., a 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 the affinity for the alpha subunit of the IL-2 receptor relative to wild-type IL-2. It should be understood that mutations identified in mouse IL-2 may be made at corresponding residues in full length human IL-2 (nucleic acid sequence (accession: NM000586); amino acid sequence (accession: P60568) or human IL-2 without the signal peptide. Accordingly, in some embodiments, the IL-2 is human IL-2. In other embodiments, the IL-2 is a mutant human IL-2. The amino acid sequence of human IL-2 (SEQ ID NO: 1; full length) is found in Genbank under accession locator NP 000577.2. The amino acid sequence of mature human IL-2 is depicted in SEQ ID NO:2 (human wild-type mature). Themurine (Mus musculus) IL-2 amino acid sequence is found in Genbank under accession locator (SEQ ID NOG). The amino acid sequence of mature murine IL-2 is depicted in SEQ ID NO:4.

[0105] In certain embodiments, IL-2 is mutated such that it has an altered affinity (e.g., a lower affinity) for the IL-2R alpha receptor compared with unmodified IL-2. Site-directed mutagenesis can be used to isolate IL-2 mutants that exhibit decreased affinity binding to CD25, i.e., IL-2Ra, as compared to wild-type IL-2. Increasing the affinity of IL-2 for IL-2Ra at the cell surface will increase receptor occupancy within a limited range of IL-2 concentration, as well as raise the local concentration of IL-2 at the cell surface.

[0106] In some embodiments, the amino acid substitutions increasing IL-2RP binding affinity include: L80F, R81D, L85V, I86V, and I92F. In some embodiments, the amino acid substitutions that increase IL-2Ry binding affinity include: L80F, R81D, L85V, I86V, and I92F.

[0107] Table 1. Exemplary sequences for IL-2

[0108] Interleukin- 12 (IL-2) plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16: 495-521 (1998). IL-12 functions primarily as a 70 kDa heterodimeric protein consisting of two disulfide-linked p35 and p40 subunits. The precursor formof the IL-12 p40 subunit (NM 002187; P29460; also referred to as IL-12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2) is 328 amino acids in length, while its mature form is 306 amino acids long. The precursor form of the IL- 12 p35 subunit (NM_000882; P29459; also referred to as IL-12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids in length and the mature form is 197 amino acids long.

[0109] In some embodiments, the immunomodulatory fusion protein comprises an IL- 12. In some embodiments, the immunomodulatory fusion protein comprises an IL- 12 operably linked to a collagen binding domain.

[0110] In some embodiments, the IL-12 comprises IL-12A (e.g., SEQ ID NO: 6). In some embodiments, the IL- 12 comprises an amino acid sequence having 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 to the amino acid sequence of IL- 12A as set forth in SEQ ID NO: 6, or a portion thereof.

[0111] In some embodiments, the IL-12 comprises IL-12A (e.g., SEQ ID NO: 8). In some embodiments, the IL- 12 comprises an amino acid sequence having 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 to the amino acid sequence of IL- 12A as set forth in SEQ ID NO: 8, or a portion thereof.

[0112] In some embodiments, the IL- 12 comprises an amino acid sequence having 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 to the amino acid sequence of IL-12A as set forth in SEQ ID NO: 10, or a portion thereof.

[0131] In some embodiments, the IL-12 comprises IL-12B (e.g., SEQ ID NOs: 5). In some embodiments, the IL-12 comprises an amino acid sequence having 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 to the amino acid sequence of IL- 126 as set forth in SEQ ID NO: 5, or a portion thereof.

[0113] In some embodiments, the IL- 12 comprises IL-12B (e.g., SEQ ID NO: 7). In some embodiments, the IL- 12 comprises an amino acid sequence having 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 to the amino acid sequence of IL- 126 as set forth in SEQ ID NOs: 7, or a portion thereof. In some embodiments, the IL-12 comprises IL-12B (e.g., SEQ ID NO: 7).

[0114] In some embodiments, the IL- 12 comprises an amino acid sequence having 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 to the amino acid sequence of IL-12B as set forth in SEQ ID NO: 9, or a portion thereof.

[0115] In some embodiments, the IL- 12 comprises both IL-12A and IL-12B. In some embodiments, the IL- 12 comprises both IL-12A and IL-12B and a linker. In some embodiments, the immunomodulatory fusion protein comprises an IL- 12 comprising the amino acid sequences set forth in SEQ ID NOs: 5-10. In some embodiments, the IL-12 comprises an amino acid sequence having 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 to the amino acid sequence of IL-12A and IL-12B as set forth in SEQ ID NOs: 5- 10, or a portion thereof.

[0116] The term “IL-12 mutant” or “mutant IL- 12 polypeptide” as used herein is intended to encompass any mutant forms of various forms of the IL- 12 molecule including full-length IL- 12, truncated forms of IL- 12 and forms where IL- 12 is linked to another molecule such as by fusion or chemical conjugation. The various forms of IL- 12 mutants are characterized in having a at least one amino acid mutation. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, an IL- 12 mutantmay be referred to herein as an IL- 12 mutant peptide sequence, an IL- 12 mutant polypeptide, IL- 12 mutant protein or IL- 12 mutant analog.

[0117] Table 2: Exemplary sequences for IL- 12

[0118] As used herein, the term "immunomodulatory fusion protein" refers to a polypeptide comprising a collagen-binding domain operably linked to an IL-2 and IL- 12. In some embodiments, the collagen binding domain is operably linked to the IL-2 and IL- 12 by a linear polypeptide spacer, for example albumin. In some embodiments, the collagen binding domain is operably linked to the IL-2 and IL-12 by a linear polypeptide spacer. In some embodiments, the collagen binding domain is operably linked to the IL-2 and IL- 12 by a linker. In some embodiments, the collagen binding domain comprises a Leukocyte- Associated Immunoglobulin- Like Receptors (LAIR).

[0119] In some aspects, the disclosure provides an immunomodulatory fusion protein comprising a collagen-binding domain is operably linked to an IL-2 and IL- 12. As used herein, “operably linked” means that the components of the immunomodulatory fusion protein are placed into a functional relationship with each other. In some aspects, the disclosure provides an immunomodulatory fusion protein comprising a collagen-binding domain is operably linked to an IL-2 and IL- 12 by a linear polypeptide spacer, such as albumin or an albumin binding domain. In some embodiments, the immunomodulatory fusion protein further comprises a linker. In some embodiments, the immunomodulatory fusion protein further includes a plurality of linkers. For example, the immunomodulatory fusion protein may include a glycine-serine (GS) repeat.

[0120] In some embodiments, and as shown in the appendix, the disclosed immunomodulatory fusion protein was designed for IT administration, with two modes of local retention, and combines IL-2 and IL- 12 in a single polypeptide.

[0121] In some embodiments, the collagen-binding domain has a MW of about 5-l,000kD, about 5-100 kDa, about 10-80 kDa, about 20-60 kDa, about 30-50 kDa, or about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa or about 100 kDa. In some embodiments, the collagen- binding domain is about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 150kDa, about 200kDa, about 300kDA, about400kDa, about 500kDa, about 600kDa, about 700kDa, about 800kDa, about 900kDa or about lOOOkDa. In some embodiments, the collagen-binding domain is about 30 kDa. In some embodiments, the collagen-binding domain is about 40 kDa.

[0122] In some embodiments, the collagen-binding domain is about 10-350, about 10-300, about 10-250, about 10-200, about 10-150, about 10-100, about 10-50, or about 10-20 amino acids in length. In some embodiments, the collagen-binding domain is about 10 amino acids in length. In some embodiments, the collagen-binding domain is about 15 amino acids in length. In some embodiments, the collagen- binding domain is about 20 amino acids in length. In some embodiments, the collagen-binding domain is about 30 amino acids in length. In some embodiments, the collagen-binding domain is about 40 amino acids in length. In some embodiments, the collagen-binding domain is about 50 amino acids in length. In some embodiments, the collagen- binding domain is about 60 amino acids in length. In some embodiments, the collagen-binding domain is about 70 amino acids in length. In some embodiments, the collagen- binding domain is about 80 amino acids in length. In some embodiments, the collagen-binding domain is about 90 amino acids in length. In some embodiments, the collagen- binding domain is about 100 amino acids in length. In some embodiments, the collagen-binding domain is about 120 amino acids in length. In some embodiments, the collagen-binding domain is about 150 amino acids in length. In some embodiments, the collagen-binding domain is about 200 amino acids in length. In some embodiments, the collagen-binding domain is about 250 amino acids in length. In some embodiments, the collagen-binding domain is about 300 amino acids in length. In some embodiments, the collagen-binding domain is about 350 amino acids in length.

[0123] An immunomodulatory fusion protein can be abbreviated as IMFP.

[0124] A murine immunomodulatory fusion protein can be abbreviated as m-IMFP.

[0125] A human immunomodulatory fusion protein can be abbreviated as h-IMFP.

[0126] The term "albumin" refers to a protein having the same, or very similar three dimensional structure as human albumin (SEQ ID NO: 16) and having a long scrum 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. In some embodiments, the albumin-binding domain is a small, three-helical protein domain found in various surface proteins.

[0127] In some embodiments, the linear polypeptide spacer is an albumin, an albumin binder, an albumin binding domain, or an albumin mutation. In some embodiments, the linear polypeptide spacer comprises albumin, or fragments thereof. In some embodiments, the linear polypeptide spacer is human albumin. In some embodiments, the albumin is a serum albumin, for example, a human serum albumin (SEQ ID NO: 17). In some embodiments, the linear polypeptide spacer is an albumin binding domain.

[0128] In some embodiments, the linear polypeptide spacer comprises an amino acid sequence having 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 to the amino acid sequence of human albumin as set forth in SEQ ID NO: 16, or a portion thereof.

[0145] In some embodiments, the linear polypeptide spacer comprises an amino acid sequence having 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 to the amino acid sequence of human serum albumin as set forth in SEQ ID NO: 17, or a portion thereof.

[0129] In some embodiments, the linear polypeptide spacer comprises an amino acid sequence having 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% identityto the amino acid sequence of human serum albumin as set forth in SEQ ID NO: 18, or a portion thereof.

[0130] In some embodiments, the albumin is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions relative to an albumin protein comprising the amino acid sequence of SEQ ID NOs: 16-18. In some embodiments, the albumin mutation comprises at least one amino acid mutation compared to wild-type albumin. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position.

[0131] 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 comprises an amino acid sequence having 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 to the amino acid sequence of albumin binding domain as set forth in SEQ ID NOs: 19-22, or a portion thereof. In some embodiments, the albumin binding domain non-covalently binds to serum albumin once administered to a subject. 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 improves retention of the fusion construct at the target tissue when administered to a subject.

[0132] Table 3. Exemplary Sequences for Albumin

[0133] Leukocyte- Associated Immunoglobulin-Like Receptors (LAIR1 and LAIR2) Leukocyte- associated Ig-like receptor (LAIR)-l is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR1, the human genome encodes LAIR2, a soluble homolog. Human (h) LAIR1 is expressed on the majority of PBMC and thymocytes (Maasho et al., (2005) Mai Immunol 42: 1521-1530). Cross-linking of LAIR1 by mAbs in vitro delivers a potent inhibitory signal that is capable of inhibiting immune cell function. Collagens are known to be natural, high- affinity ligands for the LAIR molecules. Interaction of hLAIRl with collagens 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; Lcbbink et al., (2006) I Exp Med 203: 1 19-1425).

[0134] In some embodiments, the LAIR1 comprises an amino acid sequence having 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 to the amino acid sequence as set forth in SEQ ID NO: 13, or a portion thereof. In some embodiments, the human type I glycoprotein is LAIR1 and the collagen-binding domain comprises an amino acid sequence having 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 to amino acid residues 22- 122 of the amino acid sequence as set forth in SEQ ID NO: 13, or a portion thereof.

[0135] In some embodiments, the LAIR1 comprises an amino acid sequence having 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 to the amino acid sequence as set forth in SEQ ID NO: 14, or a portion thereof.

[0136] In some embodiments, the LAIR 1 comprises an amino acid sequence having 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 to the amino acid sequence as set forth in SEQ ID NO: 12, or a portion thereof.

[0137] In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the LAIR1 variant has increased binding affinity to collagen relative to a collagen binding affinity of a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the LAIR1 variant has decreased binding affinity to collagen relative to a collagen binding affinity of a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 13.

[0138] In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions relative to a LAIR1 protein comprising theamino acid sequence of SEQ ID NO: 14. In some embodiments, the LAIR1 variant has increased binding affinity to collagen relative to a collagen binding affinity of a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the LAIR1 variant has decreased binding affinity to collagen relative to a collagen binding affinity of a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 14.

[0139] In some embodiments, the LAIR2 comprises an amino acid sequence having 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 to the amino acid sequence as set forth in SEQ ID NO: 15, or a portion thereof.

[0140] In some embodiments, the LAIR2 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions relative to a LAIR2 protein comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the LAIR2 variant has increased binding affinity to collagen relative to a collagen binding affinity of a LAIR2 protein comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the LAIR2 variant has decreased binding affinity to collagen relative to a collagen binding affinity of a LAIR2 protein comprising the amino acid sequence of SEQ ID NO: 15.

[0141] Table 4. Exemplary sequences for Leukocyte-Associated Immunoglobulin-Like Receptors

[0142] The combination of these agents generated broad tumor antigen- agnostic immunity in several syngeneic models after IT delivery and synergized with anti-PDl therapy to maximize activity. In these embodiments, the combination therapy results in a synergistic reduction in tumor burden, tumor regression, and / or tumor development of cancer. Here, the inventors capitalized on the synergy of IL-2 and IL- 12 by generating a novel therapeutic molecule, that combines IL-2 and IL-12 in a single agent. Tables 5 and 6 list exemplary immunomodulatory fusion proteins.

[0143] Table 5. Exemplary murine bi-functional linear constructs

[0144] Table 6. Exemplary human bi-functional linear constructs

[0145] In a non-limiting aspect, the immunomodulatory fusion protein is comprised of human IL- 2, human LA1R2, human serum, and human IL- 12. In some embodiments, this immunomodulatory fusion protein is referred to as CLN-617. The murine surrogate of CLN-617 (mCLN-617) demonstrates a robust abscopal effect and synergizes with an anti-PDl blockade. In some aspects, mCLN-617 may be referred to as m-IMFP. In some aspects, the canine surrogate of CLN-617 (cCLN-617) may be used. In these aspects, cCLN-617 may be referred to as c-IMFP.

[0146] LAIR2 and HSA serve to retain CLN-617 in the TME by collagen binding and enhanced molecular weight, respectively. Mechanism of action studies were conducted for CLN-617 and are included in the appendix. CLN-617 was found to bind to collagen and stimulate IFNy expression. Collagen binding is an emerging substrate to anchor cytokines because, unlike cellular tumor- associated antigens, collagens are frequently present across solid tumors and lymph nodes and are not prone to internalization. Despite the high affinity of LAIR2 to collagen, collagen substrate binding did not encumber IL-2 or IL- 12 bioactivity in vitro. Beyond retention, LAIR2 in CLN-617 may also serve a checkpoint inhibition role by out-competing the binding of collagen fibers to the immune inhibitory receptor LAIR1 on lymphocytes. In mice, IT injection of mCLN-617 mediated > 10-fold greater exposure in tumor than in serum, reversing the PK challenge associated with systcmically-dclivcrcd cytokines, and allowing for activity at doses significantly below the MTD, as defined by body weight loss. Across multiple syngeneic mouse models, no body weight loss were observed at doses of 200 pmoles or below, while anti-tumor activity, including control ofdistal lesions, was observed even at 10 pmoles, the lowest dose evaluated, demonstrating a therapeutic index of at least 20. In one study, as shown in the appendix, C57BL / 6 WT mice were implanted with single flank B16F10 tumors and treated with IT with mCLN-617. In this study no mice treated with mCLN-617 succumbed to body weight loss. This study further supports that mCLN-617 exhibits de-tuned IL-12 activity in vivo resulting in further improved safety.

[0147] mCLN-617 has been found to be effective in checkpoint refractory models as shown in the appendix. mCLN-617 drove robust single-agent activity across IT-injected B16F10, CT26, and MC38 tumors, all of which were refractory to anti-PDl therapy. Despite local drug retention and clearance from the TME over the course of 72 hours, a durable therapeutic memory response was generated that was protective against re-challenge in surviving animals in an antigen-dependent manner. The activation of systemic immunity is critical for IT-delivered immunotherapeutic agents, particularly in the clinical setting of advanced disease, where patients may present with inaccessible metastatic lesions. In mice, distal tumors were potently controlled by mCLN-617 monotherapy. At the lowest dose level of 10 pmoles, and in individual mice with undetectable levels of IL-2 and IL- 12 in serum after injection, distal tumor growth was significantly inhibited by mCLN-617, suggesting a mechanism driven by systemic immunity rather than distal mCLN- 617 uptake. Even in mice with one flank MC38 tumor and distal liver MC38-luc metastases, mCLN-617 drove highly aggressive liver metastases into stasis as a monotherapy. The abscopal effect driven by mCLN-617 was highly synergistic with anti-PDl treatment, mediating complete responses in 90% of mice bearing two flank tumors and reducing luciferase signal from MC38-luc liver metastases to the limit of detection, despite no significant activity from anti-PDl therapy alone. This is further supported by data in the appendix showing that local administration of mCLN-617 triggers systemic immunity and shows synergy in combination with anti-PDl. As shown in some studies, the efficacy of mCLN-617 is dependent on IFNy, T cells, and Batf3+dendritic cells.

[0148] The mCLN-617-driven abscopal effect was partially dependent on CD4+ and CD8+ T cells, IFNy, and Batf3+ DCs. CD4+ and CD8+ T cells appear to have compensatory roles, since statistically significant inhibition of mCLN-617 activity in both treated and untreated tumors was only observed when both cell types were depleted. While warranting future investigation, a significant abscopal effect even in the absence of CD8+ T cells suggests the potential for non- canonical cytotoxic CD4+ T cells, which have been previously described56. Despite the ability ofNK cells to be potently activated by both IL-2 and IL- 12, they were expendable for mCLN-617 activity.

[0149] Intriguingly, in mice bearing two MC38 tumors, only one of which was treated with mCLN-617, very similar immunological remodeling of both tumors was observed, as measured by CD8+:Treg ratio and DC infiltration. CD8+ T cells in peripheral blood were enriched for pl5e / MHC tetramer staining, suggesting the trafficking of tumor- specific effector cells from treated to non-treated tumor. TCR sequencing findings were consistent with this hypothesis, showing similar’ clonotypes between the treated and untreated tumors, and highlighting the role of mCLN-617 in mediating the clonal expansion of newly detected tumor-associated T clonotypes in peripheral blood. Although CD4+ T cells and cDCl cells were not expanded in the TME following mCLN-617 treatment, activity was dependent on both cell types, demonstrating the functional role played by relatively small numbers of key cell types in the tumor. Future investigation into DC modulation in the draining lymph node is warranted to further elucidate the mechanisms of antigen priming and clonal expansion.

[0150] IFNy was significantly released in the injected TME following mCLN-617 treatment, and importantly, was observed even after repeat dosing with mCLN-617, showcasing the absence of tachyphylaxis. While tachyphylaxis has been described previously for IL- 12 monotherapy, combination with IL-2 has been shown to preserve functional IL- 12 signaling, which provides for another potential form of synergy between the two cytokines. Tachyphylaxia may furthermore be less pronounced by IT delivery than by systemic administration, since each IT treatment may draw new effector cell infiltrates previously unexposed to IL- 12.

[0151] Because CD25 expression on regulatory T cells57 and endothelial tissue may hamper the efficacy and exacerbate the toxicity of IL-2, respectively, there has been significant effort to bias IL-2 away from CD25 engagement. IL-2 agents with reduced CD25 binding have thus far generated discouraging activity in the clinic. Furthermore, CD25 is upregulated in response to IL- 12 signaling, thereby providing a basis for synergistic cross-talk between IL-2 and IL- 12. The use of wild-type domains in CLN-617 reduces the risk of anti-drug-antibodies during repeat dosing. From an immunological perspective, recent data suggests that engagement of CD25 on stem-like memory T cells may facilitate the selective expansion and differentiation of tumor- specific CD8+ T cells, particularly in combination with anti-PDl checkpoint blockade60. Consistent with this phenomenon, IT-treatment of mice with mCLN-617 mediated a dose-dependent increase in theintratumoral CD8+:Treg ratio and expansion of the stem-like memory TCF1+PD1+CD8+ T cells in peripheral blood, potentially explaining the robust synergy seen with anti-PDl therapy.

[0152] In mice bearing B16F10 tumors, varying the stoichiometries of IT-administered and retained IL-2 and IL- 12 fusions posed no advantage over stoichiometric equivalent dosing by mCLN-617. Surprisingly, IL-2 and IL- 12 on separate molecules were significantly more toxic than dosing of mCLN-617 at equimolar dose levels, despite no significant difference in efficacy. The mechanism behind this phenomenon warrants further study, but one potential explanation is the differential abundance of IL-2 receptors and IL- 12 receptors in the TME. Because both receptor complexes trigger cytokine internalization and TMDD, mCLN-617 may be consumed and signal in proportion with the abundance of each receptor. While IL- 12 is typically more potent and toxic than IL-2, a greater abundance of IL-2 receptors may ultimately compensate for this.

[0153] The preclinical data presented herein suggests that IT delivery of CLN-617 should be safe yet capable of eradicating established injected and uninjected tumors in syngeneic models, synergizing with anti-PDl therapy, and generating a robust abscopal response dependent on cellular immunity, antigen cross-presentation, and interferon-gamma (IFNy).

[0154] A phase I trial of CLN-617 in patients with advanced solid tumors commenced in 2023.

[0155] METHODS, MEDICAMENTS, KITS AND USES

[0156] In one aspect of the invention, the invention provides a method for treating a cancer in an individual comprising administering to the individual a combination therapy which comprises a PD-1 antagonist and an immunomodulatory fusion protein of the present invention.

[0157] While the present invention describes a combination therapy comprising IMFPs and a PD- 1 antagonist, it should be understood that the combination therapy may also comprise one or more additional therapeutic agents. The additional therapeutic agent may be, e.g., a chemotherapeutic other than an immunomodulatory fusion protein of the present invention, a biotherapeutic agent (including but not limited to antibodies to VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4- IBB, and ICOS), an immunogenic agent (for example, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor derived antigen or nucleic acids, immune stimulating cytokines (for example, IL-2, IFNa2, GM-CSF), and cells transfected with genes encoding immune stimulating cytokines such as but not limited to GM-CSF).

[0158] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclo sphosphamidc; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocannycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gammall and calicheamicin phill, see, e.g., Agnew, Chem. Inti. Ed. Engl., 33:183- 186 (1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzino statin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; clformithinc; clliptinium acetate; an cpothilonc; ctoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are anti- hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0159] Each therapeutic agent in a combination therapy of the invention may be administered either alone or in a medicament (also referred to herein as a pharmaceutical composition) which comprises the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients and diluents, according to standard pharmaceutical practice.

[0160] Each therapeutic agent in a combination therapy of the invention may be administered simultaneously (i.e., in the same medicament), concurrently (i.e., in separate medicaments administered one right after the other in any order) or sequentially in any order. Sequential administration is particularly useful when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and another agent is a sterile liquid) and / orare administered on different dosing schedules, e.g., a chemotherapeutic that is administered at least daily and a biothcrapcutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.

[0161] In some embodiments, the immunomodulatory fusion protein of the present invention is administered before administration of the PD-1 antagonist, while in other embodiments, the immunomodulatory fusion protein of the present invention is administered after administration of the PD- 1 antagonist.

[0162] In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosage regimen (dose, frequency and duration of treatment) that is typically employed when the agent is used as monotherapy for treating the same cancer. In other embodiments, the patient receives a lower total amount of at least one of the therapeutic agents in the combination therapy than when the agent is used as monotherapy, e.g., smaller doses, less frequent doses, and / or shorter treatment duration.

[0163] Each therapeutic agent in a combination therapy of the invention can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration.

[0164] A combination therapy of the invention may be used prior to or following surgery to remove a tumor and may be used prior to, during, or after radiation therapy.

[0165] In some embodiments, a combination therapy of the invention is administered to a patient who has not been previously treated with a biotherapeutic or chemotherapeutic agent, i.e., is treatment-naive. In other embodiments, the combination therapy is administered to a patient who failed to achieve a sustained response after prior therapy with a biotherapeutic or chemotherapeutic agent, i.e., is treatment-experienced.

[0166] A combination therapy of the invention is typically used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan. In some preferred embodiments, a combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm3, 300 mm3, 400 mm3, 500 mm3, 750 mm3, or up to 1000 mm3

[0167] A combination therapy of the invention is preferably administered to a human patient who has a cancer that tests positive for PD-L1 expression. In some preferred embodiments, PD-L1 expression is detected using a diagnostic anti-human PD-L1 antibody, or antigen binding fragmentthereof, in an THC assay on an FFPE or frozen tissue section of a tumor sample removed from the patient. Typically, the patient's physician would order a diagnostic test to determine PD-L1 expression in a tumor tissue sample removed from the patient prior to initiation of treatment with the PD-1 antagonist and an immunomodulatory fusion protein of the present invention, but it is envisioned that the physician could order the first or subsequent diagnostic tests at any time after initiation of treatment, such as for example after completion of a treatment cycle.

[0168] Selecting a dosage regimen (also referred to herein as an administration regimen) for a combination therapy of the invention depends on several factors and can be unpredictable; the various factors that can influence dosage include but are not limited the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated. Preferably, a dosage regimen maximizes the amount of each therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each biotherapeutic and chemotherapeutic agent in the combination depends in pail on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of the appropriate dosage regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, the patient's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0169] Biotherapeutic agents in a combination therapy of the invention may be administered by continuous infusion, or by doses at intervals of, e.g., daily, every other day, three times per week,or one time each week, two weeks, three weeks, monthly, bimonthly, etc. A total weekly dose is generally at least 0.5 pg / kg, 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 30 ug / kg, 100 pg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg body weight or more.

[0170] In some embodiments that employ an anti-human PD- 1 mAb as the PD- 1 antagonist in the combination therapy, the dosing regimen will comprise administering the anti-human PD-1 mAb at a dose of 1, 2, 3, 5 or 10 mg / kg at intervals of about 14 days (±2 days) or about 21 days (±2 days) or about 30 days (+2 days) throughout the course of treatment.

[0171] In other embodiments that employ an anti-human PD-1 mAb as the PD-1 antagonist in the combination therapy, the dosing regimen will comprise administering the anti-human PD-1 mAb at a dose of from about 0.005 mg / kg to about 10 mg / kg, with intra-patient dose escalation. In other escalating dose embodiments, the interval between doses will be progressively shortened, e.g., about 30 days (±2 days) between the first and second dose, about 14 days (±2 days) between the second and third doses. In certain embodiments, the dosing interval will be about 14 days (±2 days), for doses subsequent to the second dose.

[0172] In certain embodiments, that employ an anti-human PD-1 mAb as the PD-1 antagonist in the combination therapy, the dosing regimen will comprise administering the anti-human PD-1 mAb at a dose of about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, or about 225 mg every two weeks or every three weeks. In certain embodiments, that employ an antihuman PD-1 mAb as the PD-1 antagonist in the combination therapy, the dosing regimen will comprise administering the anti -human PD-1 mAb at a dose of about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, or about 425 mg every four weeks or every five weeks.

[0173] In certain embodiments, a subject will be administered an intravenous (IV) infusion of a medicament comprising any of the PD- 1 antagonists described herein.

[0174] The anti-PD-1 and an immunomodulatory fusion protein of the present invention medicaments described herein may be provided as a kit which comprises a first container and a second container and a package insert. The first container contains at least one dose of a medicament comprising an anti-PD-1 antagonist, the second container contains at least one dose of a medicament comprising an immunomodulatory fusion protein of the present invention, and the package insert, or label, which comprises instructions for treating a patient for cancer using the medicaments. The first and second containers may be comprised of the same or different shape(e.g., vials, syringes, and bottles) and / or material (e.g., plastic or glass). The kit may further comprise other materials that may be useful in administering the medicaments, such as diluents, filters, IV bags and lines, needles and syringes. In some preferred embodiments of the kit, the anti- PD-1 antagonist is an anti-PD-1 antibody and the instructions state that the medicaments are intended for use in treating a patient having a cancer that tests positive for PD-L1 expression by an IHC assay.

[0175] EXAMPLES

[0176] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention.

[0177] Methods for the preparation of linear constructs are disclosed in PCT Publication No. WO2022133326A1, the entirety of which is incorporated by reference.

[0178] Example 1 - Evaluation of mCI.N-617, alone and in combination with an anti-PDl antibody

[0179] For patients with advanced metastatic disease, it is critical that IT injection eradicates not only the injected lesion, but also distal non-injected lesions, by mounting a systemic anti-tumor immune response, or abscopal effect. To evaluate the ability of mCLN-617 to drive an abscopal effect, a study was performed in C57BL / 6 mice bearing two subcutaneous flank MC38 tumors on opposing flanks, only one of which was IT treated with mCLN-617 (FIG. 1A). As observed in mice bearing a single treated MC38 tumor all injected lesions were eradicated. In the untreated flank tumor, tumor growth was transiently reversed (FIG. IB) and 3 / 7 tumors completely responded to therapy by end of study. The remaining 4 / 7 tumors grew out 28 days delayed relative to tumors in PBS vehicle-treated animals. A similar abscopal effect was observed in mice bearing two B16F10 tumors (FIG. 2). In order to confirm that the abscopal effect was not related to systemic exposure of mCLN-617, FIG. 3 shows that control of untreated MC38 flank tumors was not correlated to IT injection volume (FIG. 3A) nor to IL-12p70 concentrations in serum 30 minutes after injection (FIG. 3B). Evidence of a robust abscopal effect even in mice with the lowest serum exposure of mCLN-617 suggests that activity is not driven by exposure of the distal lesion to mCLN-617, but rather by mobilization of a systemic immune response.

[0180] Sampling of peripheral blood following IT treatment with mCLN-617 at the highest dose evaluated demonstrated a 14-fold enrichment of circulating CD8+ T cells expressing both TCF-1and PD1 , which marks the stem-like memory T cell population that drives responses to anti-PDl trcatmcnt33 (FIG. 1C). It was hypothesized that mCLN-617 monotherapy may therefore be dampened by PD1 expression on mobilized T cells, and that refractory mice may be sensitive to systemically delivered anti-PDl antibody. While mice bearing two MC38 tumors were poorly responsive to anti-PDl treatment alone, the combination of IT-delivered mCLN-617 and an intraperitoneally (IP)-delivered anti-PDl antibody led to the eradication of all injected lesions and 9 / 10 distal untreated lesions (FIG. ID).

[0181] While T cells mobilized from one subcutaneous flank tumor may more readily survey a second flank tumor, the question remained whether mCLN-617 could also drive an abscopal effect in a more challenging setting of distal liver metastasis. In this model, mice were SC implanted with MC38 cells and simultaneously intra- splenically injected with luciferase-tagged MC38 (MC38-luc) cells to seed liver metastases (FIG. IE). Only the SC tumor was treated with mCLN- 617, with or without an IP-delivered anti-PDl antibody. mCLN-617 alone readily controlled the injected tumor and drove liver metastases to stasis, as measured by luminescence signal (FIG. IF). When IT-delivered mCLN-617 was combined with systemic anti-PDl antibody, luciferase signals declined to background levels, suggesting eradication of distal liver metastases.

[0182] It was also discovered that the combination therapy drives the clonal expansion of tumor- associated T cells. Studies in the appendix also support the finding that mCLN-617 mediates clonal expansion of tumor associated T cells. The association of mCLN-617 with enhanced antigen presentation and expansion of tumor- specific T cells in the periphery suggested a potential impact of mCLN-617 on the clonality of the T cell response. Highly clonal T cell expansion is associated with neoantigen-specific T cell responses37, which drives the activity of anti-PDl therapy38. To investigate this further, mice bearing two MC38 tumors were treated IT in one tumor with mCLN- 617, with or without IV -delivered anti-PDl antibody, and compared to mice treated with anti-PDl antibody alone or vehicle control. Tumors were collected at the end of study, and blood was sampled pre- and post-dosing. The TCRB locus was sequenced in all samples to investigate the clonality of response and association between peripherally expanded clonotypes and tumor infiltrating lymphocytes (TILs).

[0183] In post-dose peripheral blood samples, the TCRB repertoire clonality was significantly enhanced by mCLN-617, and further improved synergistically upon combination with anti-PDl therapy, while anti-PDl alone had no impact on clonality (FIG. 4A). In mice that were treated withmCLN-617 or the combination with anti-PDl , an abundance of newly expanded and / or detected clones were identified post-dose (FIG. 4B). This poor correlation between clonotypcs detected pre- and post-dose, as measured by Morisita Index, was observed for mCLN-617 and further enhanced by the combination with anti-PD 1 , while anti-PD 1 alone failed to significantly impact the Morisita Index relative to vehicle control (FIG. 4C). In both mCLN-617 treated groups, the majority of clonotypes that were significantly expanded in the post-dose sample were newly detected clonotypes not observed in the pre-dose sample, demonstrating the generation of a novel immune response rather than simply an expansion of a pre-existing response (FIG. 4D). FIG. 4E depicts the number of newly expanded clones in each mouse per group that is right (treated) or left (untreated) tumor associated.

[0184] An abscopal effect is premised on the mobilization of T cells from the injected tumor, so the hypothesis was that expanded clonotypes in peripheral blood would be tumor-associated. To study this further, post-dose blood and tumor samples were cross-referenced to define tumor- associated clonotypes in peripheral blood (FIG. 4B). Due to a high degree of similarity between clonotypes in the treated and untreated tumors (FIGs. 5A and 5B) equivalent trends were observed regardless of which tumor was used as reference (FIG. 5C) and >80% of newly expanded clonotypes were tumor- associated across groups (FIGs. 5D and 5E). Compared to vehicle treated mice, mCLN-617 treated mice showed >30x enhancement in tumor-associated clonotypes, which was further doubled upon combination with anti-PDl. In mCLN-617 treated mice, the similarity of TCRB repertoire between treated and untreated tumors, along with the expansion of newly detected tumor-associated clonotypes in peripheral blood, is consistent with a tumor-specific abscopal effect.

[0185] Example 2 - Development of CLN-617

[0186] The robust preclinical efficacy of mCLN-617 motivated the development of the fully human clinical candidate CLN-617. The structure of CLN-617 is identical to mCLN-617 except that human domains were utilized instead of murine domains (FIG. 6A). Humans have two isoforms of LAIR, LAIR1 and LAIR2. LAIR2 was incorporated into CLN-617 on the basis of a similar collagen-binding affinity as murine LAIR1, while human LAIR1 exhibited 15-fold lower affinity than LAIR2 (FIG. 7A).

[0187] CLN-617 was produced in CHO cells and purified by Capto Blue resin. To assess stability in physiologically relevant conditions, CLN-617 was incubated for 7 days at 37oC in conditionedmedia from SK-MEL-2 (melanoma cell line) and SCC-25 (head and neck carcinoma cell line) or 10% human scrum, as a mimic of interstitial fluid. CLN-617 remained intact with no evidence of degradation as assessed by Western Blot analysis with anti-IL-2 and anti-IL-12 antibodies (FIG. 6B)

[0188] CLN-617 demonstrated high affinity binding to collagen (EC50 = 4.5 nM) (FIG. 7B). As with mCLN-617, CLN-617 exhibited similar IL-2 and IL- 12 bioactivity as commercially available IL-2 and IL- 12 when evaluated for proliferation activity with CTLL-2 cells (FIG. 7C) and HEK- Blue IL- 12 cells (FIG. 7D). This activity was unaffected by the presence of collagen. To confirm that the totality of IL-2 and IL-12 signaling was recapitulated with CLN-617, CD3- stimulated human PBMCs were incubated with CLN-617 or a combination of recombinant IL-2 and IL- 12 and evaluated by signaling microarray analysis. Upregulated analytes were indistinguishable between CLN-617 and the two cytokines (FIG. 6C). CLN-617 specifically triggered phosphorylation of both signal transducer and activator of transcription 4 (pSTAT4) (FIG. 6D) and pSTATS (FIG. 6E) mediators of IL-12 and IL-2 signaling. pSTAT4 and pSTAT5 levels were assessed in CD8+T-cells by FACS and resulting data shows that the IL-2 and IL- 12 activity was maintained on CLN-617. This was further supported by a protein signaling microarray, where a high correlation was shown between signaling mediated by CLN-617 vs. a combination of recombinant IL-2 and IL- 12.

[0189] Human CD8+ T cells were activated by CLN-617 in a manner consistent with in vivo pharmacodynamic effects observed with mCLN-617 in mice. CLN-617 stimulated IF y production by human CD8+ T cells in a manner that was dependent on both IL-2 and IL- 12 (FIG. 6F). CLN-617 also enhanced cytotoxicity, as measured by GzmB expression (FIG. 6G) and proliferation, as measured both by Ki67 expression (FIG. 6H) and proliferation dye dilution (FIG. 61) of human CD8+ T cells. Upregulation of PD1 on CLN-617 treated CD8+ T cells provides a strong rationale for combination with anti-PDl blocking antibodies during clinical development (FIG. 6J). Similar results were observed with CD4+ T cells and CD56+ NK cells (FIGs. 8A-C). In totality, the expectation is for CLN-617 to stimulate the activation and proliferation of T and NK cells in situ following IT injection in the clinic.

[0190] Example 3 - Study Design

[0191] CLN-617 is being evaluated as a single agent and in combination with Pembrolizumab in a phase I open-label, dose-escalation, optimization, and expansion study. Cohort 1 (70 ug dose) ofPart 1 dose escalation was completed. Multiplex immunofluorescence analysis was conducted using tumor biopsies from both injected and uninjcctcd tumors collected at screening, C2D1 (monotherapy) and C4D1 (in combination with Pembrolizumab). In addition, serum samples were collected at various time points to monitor cytokine induction by CLN-617 monotherapy and in combination with Pembrolizumab.

[0192] Up to 20 patients with restricted tumor types will be enrolled in Part 2 Dose Optimization. Up to 30 patients with specific tumor types will be enrolled in Part 3 Dose Expansion. The number of subjects provided for each phase of the study below is a non-limiting example. The particular dosing regimen and / or quantities are also non-limiting. Those of ordinary skill in the art would understand that the number of subjects participating in the study can be increased or decreased. Those of ordinary skill in the art would understand how to modify the dosing regimen and / or quantities for a particular patient or group of subjects.

[0193] The study consists of three periods, a screening period (28 days), treatment period and follow up period. The treatment period consists of three parts as shown in FIG. 9. FIGs. 9, 11A, 11B, and 11C additionally includes a dosing regimen according to one aspect of the invention. CLN-617 will be investigated in patients with advanced solid tumors in three parts to examine dose escalation (Part 1), dose optimization (Part 2) and dose expansion (Part 3). Tumor assessments will take place at Screening and approximately every 6 weeks in the first 3 months, every 9 weeks in the following 3 months, and every 12 weeks thereafter until disease progression. Patients will be treated until progressive disease, intolerable toxicity, no injectable lesions (continue if patient benefits on pembrolizumab treatment), or up to 24 months of treatment, whichever occurs first.

[0194] Part 1 of this study will use a multiple ascending dose design during a six-week DLT period to assess multiple IT injections of CLN-617 with the introduction of pembrolizumab starting from the fourth week of the DLT period. In Part I, CLN-617 will be administered intratumorally (IT) to patients on Day 1, Day 8, and Day 15 of a 21-day cycle (Cycle 1), then on Day 22 (Cycle 2, Day 1) and beyond in every three-week cycle (Q3W). Pembrolizumab, an anti-PD-1 (antiprogrammed cell death 1) monoclonal antibody, will be administered approximately 24 hours post the CLN-617 IT injection on Day 23 (Cycle 2, Day 2) on a Q3W schedule. Starting on Cycle 4 Day 1 and beyond, CLN-617 and pembrolizumab will be dosed on the same day on a Q3W schedule. FIG. 10A depicts an exemplary dosing schema for Part 1. Part 2 of the study will furtherexamine two dose levels of CLN-617 chosen from Part 1 for combination therapy with pcmbrolizumab in select tumor types. The expansion of these two dose levels of the study drug in Part 2 will allow more safety, tolerability, PK, and efficacy assessments prior to choosing an optimal CLN-617 dose for Part 3. In Part 2, selected doses of CLN-617 will be administered IT to patients on Day 1, Day 8, and Day 15 of a 21-day cycle (Cycle 1) followed by 200 mg of pembrolizumab approximately 24 hours post initial dose of CLN-617 (Cycle 1, Day 2). On Day 22 (Cycle 2, Day 1), CLN-617 will be dosed, followed by 200 mg pembrolizumab, approximately 24 hours post CLN-617 IT injection on Day 23. On Cycle 3 and beyond, CLN-617 and pembrolizumab will be dosed on the same day, on a Q3W schedule.

[0195] Initiation of Part 3 will be contingent upon review of totality of all available data, including post-dose safety assessments and exposure data from the proceeding cohorts in Part 1 and 2.1n Part 3, the optimal dose of CLN-617 will be expanded into patients with advanced melanoma or head and neck squamous cell carcinoma (HNSCC). CLN-617 will be administered intratumorally to patients on Day 1, Day 8, and Day 15 of a 21-day cycle (Cycle 1) followed by 200 mg of pembrolizumab approximately 24 hours post initial dose of CLN-617 (Cycle 1, Day 2). On Day 22 (Cycle 2, Day 1) CLN-617 will be dosed, followed by 200 mg pembrolizumab, approximately 24 hours post CLN-617 IT injection on Day 23. On Cycle 3 and beyond, CLN-617 and pembrolizumab will be dosed on the same day, on a Q3W schedule. FIG. 10B depicts an exemplary dosing schema for Part 2 and Part 3.

[0196] The primary objectives includes characterizing the safety, tolerability, Dose Liming Toxicity (DLT), and Maximum Tolerated Dose (MTD) (or Maximum Administered Dose (MAD), or Maximum Biologically Effective Dose (MB ED) if no MTD defined of CLN-617 administered by IT injection (Cycle 1 and beyond) and in combination with pembrolizumab intravenously (Cycle 2 and beyond) in patients with advanced solid tumors(Part 1), further characterizing the safety, tolerability, and preliminary efficacy of two dose levels of CLN-617 in combination with pembrolizumab in patients with advanced solid tumors (Part 2). Based on the totality of data, one dose level will be selected for dose expansion cohorts (Part 3), and evaluating the preliminary antitumor activity of a selected dose level of CLN-617 in combination with pembrolizumab, as assessed by the Overall Response Rate (ORR), Duration of Response (DOR), Disease Control Rate (DCR), Progression Free Survival (PFS), and Overall Survival (OS) per Response Evaluation Criteria in Solid Tumors (RECISTvl.l) in patients with advanced melanoma and HNSCC..Secondary endpoints involve the assessment of pharmacokinetics and immunogenicity across the three parts of the trial, including, but not limited to, assessing the PK profile of CLN-617 administered alone or in combination with pembrolizumab in patients with advanced solid tumors, assessing the immunogenicity of CLN-617 administered alone or in combination with pembrolizumab in patients with advanced solid tumors. Biomarkers are exploratory endpoints exploring the effect of CLN-617 administered alone or in combination with pembrolizumab on pharmacodynamic and immune biomarkers in peripheral blood and tumor tissue (injected and uninjected), exploring relationships between various PK / PD parameters and measures of clinical anti-tumor activity including ORR, DOR, PFS, and OS in patients treated with CLN-617 alone or in combination with pembrolizumab, exploring the relationship between baseline tumor tissue biomarkers and measures of clinical anti-tumor activity, including ORR, DOR, PFS, and OS in patients treated with CLN-617 alone or in combination with pembrolizumab, exploring the relationship between genomic biomarkers and measures of clinical anti-tumor activity, including ORR, DOR, PFS, and OS in patients treated with CLN-617 alone or in combination with pembrolizumab.

[0197] A subject may be included in the study if they previously received or had a contraindication to standard therapy that confers an overall survival benefit. The Part 1 dose escalation cohorts are subjects with histologically or cytologically confirmed advanced incurable or metastatic non- neurological solid tumors with accessible injectable lesions. The Pail 2 dose optimization cohorts are subjects with histologically or cytologically confirmed select advanced incurable or metastatic cancer types with accessible injectable lesions. The Part 3 dose expansions will have two cohorts. The first cohort will include subjects with histologically or cytologically confirmed metastatic or locally advanced, unresectable melanoma with accessible injectable lesions. The second cohort will include subjects with histologically or cytologically confirmed metastatic or locally advanced, unresectable HNSCC with accessible injectable lesions.

[0198] The subjects must have two or more measurable lesions for Part 1, or one or more measurable lesions for Part 2 and Part 3 that meet RECIST vl.l. The subjects must also have tumors amenable to direct injection with or without imaging guidance. For more information on RECIST, refer to Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). European Journal of Cancer. 2009. 45: 228-247; Seymour et al.iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017. 18: cl43-cl52; Pcrsigchl ct al. iRECIST: how to do it. Cancer Imaging. 2020. 20: 2.

[0199] A subject may be excluded if (i) they have concomitant second malignancies (except adequately treated non-melanomatous skin cancers, ductal carcinoma in situ, superficial bladder cancer, prostate cancer, or in situ cervical cancer), unless in complete remission two years prior to study entry, and no additional therapy is required or anticipated to be required during study participation, (ii) they have an active autoimmune disease or a history of known autoimmune disease, or history of a syndrome that required systemic corticosteroids or immunosuppressive medications, (iii) have a serious uncontrolled medical disorder that would impair the ability of the subject to receive protocol therapy or whose control may be jeopardized by the complication of therapy, (iv) requires active systemic anticoagulation at the time of IT injection or biopsy, or with significant bleeding diathesis due to risk of hematoma at the injection site, (v) risk of vascular catastrophe, (vi) treatment with systemic antiviral, antibacterial, or antifungial agents for acute infection with < 7 days of dosing on C1D1, (vii) diagnosed with HIV 1 / 2 primary immunodeficiency disease, (viii) diagnosed with hepatitis B or hepatitis C virus, (ix) prior organ allograft or allogeneic hematopoietic transplantation, (x) active central nervous system metastases and / or carcinomatous meningitis, (xi) active SARS-CoV-2 infection, (xii) has received immunosuppressive medications, (xiii) has undergone anticancer treatment, immunotherapy, radiotherapy, or major surgery within 14-28 days prior to the first dose of the study drug, (xiv) female of child-bearing potential who is pregnant or breast-feeding or plans to become pregnant within 120 days for last study drug administration, (xv) male subject who plans to father a child or donate sperm within 120 days or 5 half-lives of CLN-617.

[0200] Screening Period

[0201] At the screening visit, following the administration of the informed consent forms, assessments will be taken to determine eligibility, including demography, medical and cancer history, vital signs, height and weight, a physical exam and ECOG, an ECG, a tumor assessment, an assessment of pregnancy, and blood and urine samples to assess the patient's general health and adherence to inclusion / exclusion criteria.

[0202] Patients who meet eligibility criteria at the screening visit will be scheduled for their Day 1 visit and collect a baseline tumor biopsy sample anytime between the screening visit and before their Day 1 IT injection of CLN-617.

[0203] Part i (Dose Escalation)

[0204] Patients with advanced solid tumors received treatment with an initial 3 IT injections of CLN-617 (1 dose weekly [Q1W] for the first 3 weeks). Subsequent treatment cycles will be 3 weeks in duration with IT CLN-617 on Day 1 and IV infusion of pembrolizumab 200 mg on Day 2 for Cycle 2 and Cycle 3 (approximately 24 hours post CLN-617) administered every 3 weeks (Q3W). Cycle 4 and beyond CLN-617 and pembrolizumab will be dosed on the same day administered every 3 weeks. Dose levels of CLN-617 to be tested include 70 (dose level 1), 200, 400, 800, 1300, and 2000 pg. Dose escalation will follow a standard two-parameter logistic Continual Reassessment Model (CRM) to discover the Maximum Tolerated Dose (MTD) defined as the dose at which the probability of Dose-Limiting Toxicity (DLT) is at most 30%.

[0205] Tumor biopsies for select patients were analyzed by quantitative multiplex immunofluorescence panels containing the following markers.

[0206] T cell / dendritic cell panel: CD4, CD8, Ki67, FoxP3, PD-L1, CDllc

[0207] NK / macrophage panel: CD3, Granzyme B, CD56, CD16, CD68, CD163

[0208] Patient #1 (67-year-old female) and Patient #2 (67-year-old male) were both previously heavily treated for Stage IV melanoma prior to CLN-617 therapy. For Patient #1CLN-617 was injected into the tumor bearing inguinal lymph node and patient remained on therapy for 4 cycles. For Patient #2 CLN-617 was injected into a liver lesion and patient remained on therapy for 6 cycles. FIGs. 12A, 12B, and 13 show the biomarker data for Patients #1 and #2 after CLN-617 therapy.

[0209] FIGs. 12A and 12B show representative images of Screening and C2D1 Pre-dose Injected tumor biopsy samples stained with T / dendritic cell (FIG. 12A) and NK / macrophage immunofluorescence panels (FIG. 12B). Inlets on the right depict examples of co-staining with pairs of markers, as indicated. Cell density (number of positive cells / mm2) and percentage of positive cells for single marker or co-stained markers were quantified using an automated image analysis algorithm. FIG. 13 summarizes the data from patient #1 (top) and patient #2 (bottom) in C2D1 and C4D1 injected and uninjected tumor biopsies compared to the screening biopsy in the tumor compartment. Multiplex immunofluorescence findings are also summarized below:

[0210] Increased CD3+ T cell and CD8+ T cell infiltration, proliferation (CD8+Ki67+) and CD8+ / Treg (CD4+FoxP3+) ratio was observed in injected and uninjected tumors after CLN-617monotherapy, and CLN-617 / Pembrolizumab combination therapy, supporting the mechanism of action of CLN-617.

[0211] Increased Granzyme B+ cells were detected in injected and uninjected tumors after CLN- 617 monotherapy, and CLN-617 / Pembrolizumab combination therapy. Granzyme B is a marker for cytolytic function. This data indicates that CLN-617 treatment activates cytolytic cells that are capable of killing tumor cells.

[0212] Increased M1 / M2 macrophage ratio was observed in injected and uninjected tumors after CLN-617 monotherapy, and CLN-617 / Pembrolizumab combination therapy. Ml (CD68+CD163- ) are proinflammatory macrophages and M2 (CD68+CD163+) are immunosuppressive macrophages. This data indicates that CLN-617 induces repolarization of the tumor microenvironment toward a more pro-inflammatory and anti-tumorigenic phenotype.

[0213] Increased PD-L1 expression was detected in injected and uninjected tumors after CLN-617 monotherapy. PD-L1 expression is known to be upregulated by IFNg, which is likely induced by CLN-617.

[0214] Increased CD11C+ dendritic cells were detected in injected and uninjected tumors after CLN-617 monotherapy, and CLN-617 / Pembrolizumab combination therapy. Dendritic cells are antigen presenting cells that are critical in the priming and activation of T cells.

[0215] The clinical biomarker data in Cohort 1 supports the mechanism of action and pharmacodynamic effect of CLN-617 in the tumor microenvironment and are in line with preclinical data.

[0216] Part 2 ( Dose Optimization )

[0217] Two dose levels at or close to MTD or MBED from Part 1 (Dose Escalation) will be selected for dose optimization. This decision will be made by SRC based on the totality of data in Part 1. Patients will receive CLN-617 IT injection weekly for the duration of Cycle 1 (Q1W), with pembrolizumab IV administration after the first dose of CLN-617. In cycles 2 and beyond both CLN-617 and pembrolizumab will be dosed Q3W. Pembrolizumab will be dosed 24h after CLN- 617 administration for Cycles 1-2, and then both study treatments will be dosed on the same day starting with Cycle 3 and beyond. Part 2 will investigate the safety, PK, PD, and initial anti-tumor activity of CLN-617 in combination with pembrolizumab enrolling patients with select advanced solid tumors with accessible injectable lesions to optimize the selected dose for further investigation. Each dose level will enroll up to ten patients. Patients will be treated until19progressive disease, intolerable toxicity, or up to 24 months of treatment whichever occurs first. According to the totality of data, SRC will determine a dose for dose expansion.

[0218] Part 3 (Dose Expansion)

[0219] Upon demonstrating an optimal dose with favorable safety and tolerability for the CLN- 617 IT injection in combination with pembrolizumab, the Part 3 Dose Expansion will be initiated.

[0220] Patients will receive CLN-617 IT injection weekly for the duration of Cycle 1 (Q1W), with pembrolizumab IV administration after the first dose of CLN-617. In cycles 2 and beyond both CLN-617 and pembrolizumab will be dosed Q3W. Pembrolizumab will be dosed 24h after CLN- 617 administration for Cycles 1-2, and then both study treatments will be dosed on the same day starting with Cycle 3 and beyond.

[0221] This part will investigate the safety and initial anti-tumor activity of pembrolizumab in combination with CLN-617, enrolling into the following two tumor- specific cohorts:

[0222] Cohort 1: CLN-617 + pembrolizumab in patients with advanced melanoma with accessible tumors for IT injection

[0223] Cohort 2: CLN-617 + pembrolizumab in patients with advanced HNSCC with accessible tumors for IT injection

[0224] Patients will be treated until progressive disease, intolerable toxicity, or up to 24 months of treatment, whichever occurs first.

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[0226] All features disclosed in the specification, including the claims, abstracts, drawings, and appendices, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, drawings, and appendices can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0227] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A method of treating cancer in a subject in need thereof, the method comprising administering a combination therapy which comprises a therapeutically effective amount of a checkpoint inhibitor and a therapeutically effective amount of an immunomodulatory fusion protein, the immunomodulatory fusion protein comprising: an interleukin 2 (IL-2); an interleukin (IL- 12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain.

2. The method of claim 1 , wherein the checkpoint inhibitor comprises an antagonist of PD- 1 , an antibody which binds PD-1, PD-L1, CTLA-4, LAG3, or TIM3 or an antigen binding fragment thereof.

3. The method of claim 2, wherein the antagonist of PD-1 is a humanized IgG4 anti-PD-1 monoclonal antibody.

4. The method of claim 2 or claim 3, wherein the antagonist of PD-1 is pembrolizumab.

5. The method of any one of the preceding claims, wherein the combination therapy comprises an additional checkpoint inhibitor.

6. The method of any one of the preceding claims, wherein the subject is receiving or has received chemotherapy, radiation, targeted cancer therapy, a cancer vaccine, and / or an immunomodulatory therapy.

7. The method of any one of the preceding claims, wherein the cancer is a solid tumor.

8. The method of claim 7, wherein the solid tumor is an advanced or metastatic solid tumor that tests positive for PD-L1 expression and is selected from the group consisting of bladder cancer, breast cancer, clear cell renal cell carcinoma (ccRCC), kidney cancer, head / neck squamous cell carcinoma, lung squamous cell carcinoma, malignant melanoma, non-small-cell lung cancer (NSCLC), squamous cell carcinoma (SCC), ovarian cancer, pancreatic cancer, prostate cancer,renal cell cancer, small-cell lung cancer (SCLC), triple negative breast cancer (TNBC), colorectal cancer, endometrial cancer, cervical cancer, sarcoma, and hepatocellular carcinoma (HCC).

9. The method of any one of the preceding claims, wherein the IL-2 comprises human IL-2 or human wild-type IL-2.

10. The method of any one of the preceding claims, wherein the IL-2 comprises at least about 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2.

11. The method of any one of the preceding claims, wherein the IL- 12 comprises human IL- 12 or human wild-type IL- 12.

12. The method of any one of the preceding claims, wherein the IL- 12 comprises at least about 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:

613. The method of any one of the preceding claims, wherein the albumin comprises a human albumin or human serum albumin.

14. The method of any one of the preceding claims, wherein the albumin comprises at least about 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs:16-18.

15. The method of any one of claims 1-14, wherein the albumin binding domain comprises at least about 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs:19- 22.

16. The method of any one of the preceding claims, wherein immunomodulatory fusion protein further comprises a glycine- serine (GS) repeat.

17. The method of any one of the preceding claims, wherein the LAIR1 has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14.

18. The method of any one of the preceding claims, wherein the LA1R2 has at least 80% identity to the amino acid sequence as set forth in SEQ ID NO: 15.

19. The method of any one of the preceding claims, wherein the immunomodulatory fusion protein comprises at least about 80% sequence identity to an amino acid sequence as set forth in SEQ ID NOs: 39, 45, 50, 51, 57, 63, 66, 69, or 73.

20. The method of any one of the preceding claims, wherein the combination therapy is administered via intratumoral, intraperitoneal, intranodal, pcritumoral, subcutaneous, or intravenous delivery.

21. The method of any one of the preceding claims, wherein the immunomodulatory fusion protein is administered simultaneously, concurrently, sequentially, successively, alternatively with or separately from the checkpoint inhibitor.

22. The method of any one of the preceding claims, wherein the combination therapy results in a synergistic reduction in tumor burden, tumor regression, and / or tumor development of said cancer.

23. The method of any one of the preceding claims, wherein the method does not result in cytokine release syndrome in the subject.

24. The method of any one of the preceding claims, wherein the subject does not experience grade 4 cytokine release syndrome.

25. A medicament comprising an immunomodulatory fusion protein for use in combination with a PD-1 antagonist for treating cancer in an individual, said immunomodulatory fusion protein comprising: an interleukin 2 (IL-2); an interleukin (IL- 12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain.

26. The medicament of claim 25, which further comprises a pharmaceutically acceptable excipient.

27. Use of an immunomodulatory fusion protein comprising: an interleukin 2 (IL-2); an interleukin (IL- 12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2,and albumin or an albumin binding domain in the manufacture of a medicament for treating a cancer in an individual when administered in combination with a PD- 1 antagonist.

28. A kit which comprises a first container, a second container and a package insert, wherein the first container comprises at least one dose of a medicament comprising an anti-PD-1 antagonist, the second container comprises at least one dose of a medicament comprising an immunomodulatory fusion protein comprising: an interleukin 2 (IL-2); an interleukin (IL- 12); a collagen binding domain selected from the group consisting of LAIR1 and LAIR2, and albumin or an albumin binding domain, and the package insert comprises instructions for treating an individual for cancer using the medicaments.

29. The kit of claim 28, wherein the instructions state that the medicaments are intended for use in treating an individual having a cancer that tests positive for PD-L1 expression by an immunohistochemical (IHC) assay.

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