WO2023154799A1

WO2023154799A1 - Combination immunotherapy for treating cancer

Info

Publication number: WO2023154799A1
Application number: PCT/US2023/062288
Authority: WO
Inventors: Md Masud ALAM; De YANG; Joost J. Oppenheim
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2022-02-14
Filing date: 2023-02-09
Publication date: 2023-08-17

Abstract

Methods are disclosed herein for treating a cancer in a subject. Embodiments of the methods include administering to the subject a therapeutically effective amount of a combination therapy comprising a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), and a checkpoint inhibitor. Optionally, the combination therapy administered to the subject can include a STING agonist (such as cGAMP or c-di-GMP). In some embodiments, the checkpoint inhibitor is a PD-L1 inhibitor, a PD-1 inhibitor, a TNFR-2 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the cancer is a colorectal cancer, a kidney cancer, or melanoma.

Description

COMBINATION IMMUNOTHERAPY FOR TREATING CANCER

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/309,826, filed February 14, 2022, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under project number Z01#: ZIA BC 009369 by the National Institutes of Health, National Cancer Institute. The United States Government has certain rights in the invention.

FIELD OF THE DISCLOSURE

This disclosure relates to anti-cancer therapy including the administration of a Toll-like receptor (TLR) 4 agonist, a TLR2/6 agonist, and an immune checkpoint inhibitor, specifically high mobility group nucleosome binding protein (HMGN)l, fibroblast stimulating lipopeptide (FSL)-l, and the checkpoint inhibitor.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (Name Sequences.xml; Size: 3,047 bytes; and Date of Creation: February 9, 2023) is herein incorporated by reference in its entirety.

SEQ ID NO: 1 is an exemplary nucleic acid sequence encoding a human HMGN1.

SEQ ID NO: 2 is the amino acid sequence of a human HMGN1.

BACKGROUND

Combination therapy is a treatment modality that combines two or more therapeutic agents for the treatment of cancer. The amalgamation of anti-cancer drugs enhances efficacy against neoplastic cells as compared to monotherapy because it targets several pathways in a characteristically synergistic or an additive manner. This approach is believed to reduce drug resistance and provide substantial therapeutic anti-cancer benefits, such as reducing tumor growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis.

Antibody blockade of immune checkpoints can significantly enhance anti-cancer immunity. In particular, the programmed death- 1 /pro grammed death ligand-1 (PD-1/PD-L1) interaction has been shown to suppress T cell responses in the cancer microenvironment. Antibody -mediated blockade of PD-L1 induced cancer regression and prolonged stabilization of disease in patients with advanced cancers, including melanoma. However, a need remains for more effective combination therapies for the treatment of cancer. SUMMARY OF THE DISCLOSURE

Disclosed herein is a combination therapy for treating a cancer in a subject that includes the use of a TLR4 agonist, a TLR2/6 agonist, and an immune checkpoint inhibitor.

In embodiments, the method of treating cancer in the subject includes administering to the subject a therapeutically effective amount of fibroblast stimulating lipopeptide (FSL)-l, a therapeutically effective amount of high mobility group nucleosome binding protein 1 (HMGN1), and a therapeutically effective amount of a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-L1 inhibitor, a PD-1 inhibitor, a tumor necrosis factor receptor 2 (TNFR-2) inhibitor, or a cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) inhibitor. In some embodiments, the cancer is colorectal cancer or kidney cancer.

In some embodiments, the methods also include administering to the subject a therapeutically effective amount of a cGAS/stimulator of interferon genes (STING) agonist, such as 2'3 '-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) or bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). In some embodiments, the cancer is melanoma.

In some embodiments, the therapeutic agents are administered to the subject intratumorally and/or intravenously.

In embodiments of the disclosed methods, administration of a combination of the disclosed therapeutic agents reduces cancer burden in a subject, increases survival of the subject, reduces the incidence of relapse of a cancer in the subject, induces maturation of dendritic cells in the subject, or a combination thereof.

Also disclosed are kits for use in any of the disclosed methods. In some embodiments, the kit includes FSL-1, HMGN1, and a checkpoint inhibitor, and optionally a STING agonist.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show phenotypic maturation and cytokine production in human monocyte derived dendritic cells (MoDCs) treated with HMGN1 or FSL-1. Human MoDCs at 5 x 10⁵/ml were stimulated with HMGN1 (Nl) or FSL-1 to measure the effect on expression of surface costimulatory molecules (gray area = sham matched; black area = activation) (FIGS. 1A-1B), and production of proinflammatory cytokines (mean ± standard deviation (SD); n = 3) (FIG. 1C) at 24 hours (h). Shown are the results of one experiment representative of two.

FIGS. 2A-2B show phenotypic maturation and cytokine production by human monocyte derived dendritic cells (MoDCs) treated with HMGN1 and FSL-1 together. Human MoDCs at 5 x 10⁵/ml were stimulated with (1) HMGN1, (2), FSL-1, or (3) HMGN1 and FSL-1 together to measure the effect on expression of surface costimulatory molecules (gray area = sham matched; black area = activation) (FIG. 2A), and production of proinflammatory cytokines (mean ± SD; n = 3) (FIG. 2B) at 24 h. Shown are the results of one experiment representative of two.

FIGS. 3A-3C show phenotypic maturation and cytokine production in mouse bone marrow derived dendritic cells (BMDCs) treated with HMGN1 or FSL-1. Mouse BMDCs at 5 x 10⁵/ml were stimulated with HMGN1 or FSL-1 to measure the effect on expression of surface costimulatory molecules (gray area = sham matched; black area = activation) (FIGS. 3A-3B), and production of proinflammatory cytokines (mean ± SD; n = 3) (FIG. 3C) at 24 h. Shown are the results of one experiment representative of two.

FIGS. 4A-4B show phenotypic maturation and cytokine production in mouse bone marrow derived dendritic cells (BMDCs) treated with HMGN1 and FSL-1 together. Mouse BMDCs at 5 x 10⁵/ml were stimulated with (1) HMGN1, (2), FSL-1, or (3) HMGN1 and FSL-1 together to measure the effect on expression of surface costimulatory molecules (gray area = sham matched; black area = activation) (FIG. 4A), and production of proinflammatory cytokines (mean ± SD; n = 3) (FIG. 4B) at 24 h. Shown are the results of one experiment representative of two.

FIGS. 5A-5C show CT26 colon cancer cell tumor-bearing mice treated with HMGN1, FSL-1, and a programmed death ligand 1 (PD-L1) inhibitor. CT26 tumor-bearing mice were treated intratumorally with (1) phosphate buffered saline (PBS,) (2) FSL-1, (3) HMGN1 and FSL-1, (4) FSL-1 and a PD-L1 inhibitor, or (5) HMGN1, FSL-1, and a PD-L1 inhibitor twice a week for two weeks. Mice were monitored for tumor growth (FIGS. 5A) and survival (FIG. 5B). Shown are the results of one experiment. CT26 colon cancer free mice were then inoculated subcutaneously with CT26 (2xl0⁵ / mouse) cells into the right flank, and 4T- 1 breast cancer cells (2xl0⁵1 mouse) cells into the left flank (FIG. 5C).

FIGS. 6A-6B show CT26 colon cancer cell tumor-bearing mice treated with HMGN1, FSL-1, and a CTLA-4 inhibitor. CT26 tumor-bearing mice were treated intratumorally with (1) PBS, or (2) HMGN1, FSL-1, and a CTLA-4 inhibitor together twice a week for two weeks. Mice were monitored for tumor growth (FIGS. 6A), and survival (FIG. 6B). Shown are the results of one experiment.

FIGS. 7A-7B show RENCA kidney cancer cell tumor-bearing mice treated with HMGN1, FSL-1, and a CTLA-4 inhibitor. RENCA tumor-bearing mice were treated intratumorally with (1) PBS, or (2) HMGN1, FSL-1, and a CTLA-4 inhibitor together twice a week for two weeks. Mice were monitored for tumor growth (FIGS. 7A), and survival (FIG. 7B). Shown are the results of one experiment.

FIGS. 8A-8D show functional maturation in MoDCs treated with HMGN1, FSL-1, and cGAMP. Human MoDCs at 5 x 10⁵/ml were stimulated with (1) HMGN1, (2) FSL-1, (3) cGAMP, or (4) HMGN1, FSL-1, and cGAMP together to measure the effect on expression of surface costimulatory molecules (gray area = sham matched; black area = activation) (FIG. 8A), and production of proinflammatory cytokines (mean ± SD; n = 3) (FIG. 8B) at 24 h. Shown are the results of one experiment representative of two. MoDCs treated with HMGN1, FSL-1, and cGAMP (each alone or together) were then co-cultured with allogenic CD4⁺ T cells at the ratios shown for 4 days and pulsed with tritiated thymidine ([³H]-TdR) for the last 18 h. CD4⁺ T cell proliferation was measured by [³H]-TdR incorporation (FIG. 8C). Supernatants from human CD4⁺ T cells co-cultured (CD4⁺ T:MoDCs = 50: 1) for 3 days with MoDCs treated with (1) HMGN1, (2) FSL-1, (3) cGAMP, or (4) HMGN1, FSL-1, and cGAMP together were quantitated for cytokine production (FIG. 8D). The average (mean ± SD) of two separate experiments is shown.

FIGS. 9A-9D show M3 melanoma cell tumor-bearing mice treated with HMGN1, FSL-1, a PD-L1 inhibitor, and cGAMP. M3 melanoma tumor-bearing mice were treated intratumorally with (1) PBS, (2) cGAMP, (3) HMGN1, FSL-1, and a PD-Ll inhibitor, or (4) HMGN1, FSL-1, a PD-L1 inhibitor, and cGAMP twice a week for two weeks. Mice were monitored for tumor growth (FIGS. 9A and 9B) and survival (FIG. 9B). M3 melanoma- free mice were then inoculated subcutaneously with M3 (2xl0⁵ / mouse) cells into the right flank, and Lewis Lung Carcinoma (LLC, 2xl0⁵ / mouse) cells into the left flank. The formation and growth of M3 melanomas and LLC carcinomas was monitored and plotted (FIG. 9D).

FIGS. 10A-10C show that treatment with HMGN1, FSL-1 and anti-PD-Ll promoted the infiltration of effector/memory CD8 T cells and upregulation of genes characteristic of Th 1 -polarization in CT26 tumor tissue. CT26-bearing Balb/C mice (n=5) were treated as described below. Forty-eight hours after the 3^rd treatment, the residual tumors were resected and halved. Half of the tumor was dissociated into single cell suspension, immunostained and analyzed by flow cytometry. (A) shows the gating strategy and plot of tumor of one representative mouse. (B) shows the average (mean ± SD) of each group. The other half of the tumor was used for the extraction of RNA and qPCR measurement of the expression (fold change) of CXCL9, CXCL10, IFNy, and TNFa in CT26 tumor tissue (C). Shown is the result of one out of three experiments. The statistical difference between the PBS and the treatment group was analyzed using a Student’s t-test: *p<0.05, **p<0.001, ***p<0.001, ****p<0.0001. [THIS IS FIG. 5 OF THE UPDATE.]

FIGS. 11A-11C. Treatment with Nl, FSL-1 and anti-PD-Ll induced the generation of anti-tumor immune responses in the tumor draining lymph nodes (dLNs). CT26-bearing Balb/C mice tn— 6) were treated as described below. Forty-eight hours after the 3^rd treatment, CT26 tumor- dLNs were harvested for flow cytometric analysis or measurement of CT26-specific cytotoxic CD8 T cells. (A) shows the gating strategy and plot of one representative dLN. (B) shows the average (means ± SD) of the percentage of CD4 Teffs (CD4⁺CD44^highCD62L ) cells and CD8 Teffs (CD8⁺CD44^highCD62L ) in dLNs. (C) shows the average (means ± SD) percentage of CT26-specific cytotoxic CD8 ⁺ T (CD8⁺ CD107⁺) cells in dLNs. Shown is the result of one out of three experiment. The statistical difference between PBS and treatment groups was analyzed using a Student’s t-test: *p<0.05, ***p<0.001, and ****p<0.0001. [THIS IS FIG. 6 OF THE UPDATE ]

FIGS. 12A-12B show that HMGN1 and FSL-1 synergistically induce cytokine production by human MoDCs and mouse BMDCs. (A) Human MoDCs at 5 x 10⁵/ml were stimulated with HMGN1 (ng/ml) and FSL-1 (ng/ml) by themselves or all together to measure their production of proinflammatory cytokines (pg/ml) such as TNFa and IL-12p70 (mean ± SD; n = 3) at 24 h. Shown are the results of one experiment representative of three. (B) Mouse BMDCs at 5 x 10⁵/ml were stimulated with HMGN1 (ng/ml) and FSL-1 (ng/ml) by themselves or all together to measure their production of proinflammatory cytokines (pg/ml) such as TNFa and IL-lf (mean ± SD; n = 3) at 24 h. Shown arc the results of one experiment representative of two.

DETAILED DESCRIPTION

Provided herein are methods of treating a cancer in a subject. The disclosed methods include administering a combination of therapeutic agents to the subject in need thereof. The therapeutic agents include a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), a checkpoint inhibitor (such as a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor, such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), and optionally a STING agonist (such as cGAMP or c-di-GMP).

It is disclosed herein that HMGN1 and FSL-1, and optionally cGAMP, synergistically induce phenotypic maturation and cytokine production by both human monocyte derived dendritic cells (MoDCs) and mouse bone marrow derived dendritic cells (BMDCs). It is disclosed herein that administration, such as intra-tumoral administration, of HMGN1, FSL-1, and a checkpoint inhibitor, such as a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor, suppressed the growth of cancers, such as colorectal and kidney cancers, in a dose-dependent manner. A synergistic effect was demonstrated in several types of cancers, using several checkpoint inhibitors. In addition, administration, such as intra-tumoral administration, of HMGN1, FSL-1, a checkpoint inhibitor (such as a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor), and a STING agonist (such as cGAMP or c-di-GMP), synergistically acted to suppress the growth of cancers, such as melanoma.

Therefore, in some embodiments, administration of a combination of the disclosed therapeutic agents reduces cancer burden in a subject, increases survival of the subject, reduces the incidence of relapse of a cancer in the subject, induces maturation of dendritic cells in the subject, or a combination thereof.

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Krebs et al (Eds.), Lewin ’s Genes XII, published by Jones & Bartlett Publishers, 2017; and Meyers et al. (Eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the” refer to both the singular as well as plural unless the context clearly indicates otherwise. Further, compositions of use in the methods herein can be used alone or in combination. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. The term “comprises” means “includes.” In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided: 2 '3 '-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP): A cyclic purine dinucleotide that has the formula C20H24N10O13P2 and functions as a high-affinity STING ligand. The structure is shown below:

About: Unless context indicates otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

Administer, Administering, Administration: As used herein, administering a therapeutic agent (e.g., a TLR4 agonist, a TLR2/6 agonist, a checkpoint inhibitor, and/or a STING agonist) to a subject means to apply, give, or bring the agent into contact with the animal, by any effective route. Administration can be accomplished by a variety of routes, such as, for example, intravenous, intratumoral, topical, oral, subcutaneous, transdermal, intrathecal, intramuscular, intraperitoneal, intranasal, and similar routes, or combinations thereof. Exemplary routes of administration are described herein.

Agent, Therapeutic Agent: A therapeutic agent includes treating agents, prophylactic agents, and replacement agents. A therapeutic agent may thus be any substance or any combination of substances that is useful for achieving an end or result, such as ameliorating a specific set of conditions in a subject with a disease or a disorder, for example, a substance or combination of substances (such as in a combination therapy for cancer treatment) useful for inhibiting cancer growth or metastasis in a subject. Agents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. Exemplary agents include TLR4 agonists (such as HMGN1), TLR2/6 agonists (such as FSL-1), checkpoint inhibitors (such as a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor), and STING agonists (such as cGAMP or c-di-GMP).

Antibody: A polypeptide ligand (such as an immunoglobulin, antigen-binding fragment, or derivative thereof) comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity. Antibodies are characterized by reacting specifically with the antigen in some demonstrable way. A therapeutic antibody, such as an immune checkpoint inhibitor antibody, such as an antibody that specifically binds PD-L1, an antibody that specifically binds PD-1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds

- 6 -

SUBSTITUTE SHEET ( RULE 26) TNFR-2, recognizes and binds to the antigen receptor to activate or inhibit a series of biological process for blocking cancer cell growth and/or triggering immune system.

Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and antigen binding fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, dsFv. Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and ds-scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2^nd ed., Springer- Verlag, 2010).

Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). Antibodies also include defucosylated forms of disclosed antibodies.

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (E) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the Vn region and the VL region are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles, and has some functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians

Antibody variable regions contain “framework” regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three- dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat etal. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia el al.. Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273,927-948, 1997; the “Chothia” numbering scheme), and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat and IMGT databases are maintained online.

A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies include humanized monoclonal antibodies. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP): A compound having the molecular formula C20H24N10O14P2 that stimulates the mammalian innate immune response by binding to the STING receptor, thereby converging with the cyclic GMP-AMP synthase (cGAS)-cyclic GMP-AMP (cGAMP) cytosolic DNA-surveillance pathway.

Cancer: A cancer is characterized by abnormal or uncontrolled cell growth (malignant cells). Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. “Metastatic disease” refers to cancer cells that have left the original cancer site and migrate to other parts of the body for example via the bloodstream or lymph system.

The “cancer burden” in a subject can be measured as the number, volume, and/or weight of one or more tumors. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is a cancer (and is referred to as “malignant”).

In some examples, the cancer is a hematological cancer, such as leukemias (including acute leukemias (such as l lq23 -positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) or chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia)), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. In specific non-limiting examples, the lymphoid malignancy can be adult T cell leukemia, cutaneous T cell lymphoma, anaplastic large cell lymphoma, Hodgkin’s lymphoma, or a diffuse large B cell lymphoma.

Examples of solid cancers, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer (e.g., colorectal carcinoma), lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and

- 8 -

SUBSTITUTE SHEET ( RULE 26) lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, kidney cancer (e.g., renal cell carcinoma), melanoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, bladder carcinoma, and CNS cancers (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoma can be a solid cancer in some presentations. In particular, non-limiting examples, a cancer is colorectal cancer, kidney cancer, or melanoma. cGAS/stimulator of interferon genes (STING) pathway: The STING pathway is the central cellular cytosolic double- stranded DNA (dsDNA) sensor, allowing innate immune to respond to infections, inflammation, and cancer. Both intrinsic and extrinsic self-DNA sensing can contribute to STING pathway activation. The STING pathway plays important roles in intrinsic anti-cancer immunity and is believed to be required for the therapeutic effects of immune checkpoint blockade. Mammalian cGAMP and bacterial c-di- GMP are second messengers that activate the innate immune STING pathway. Within this pathway, the binding of cGAS to dsDNA allosterically activates cGAS catalytic activity and leads to the production of the STING agonist, cGAMP. The STING1 protein is composed of a short cytosolic N-terminal segment, a four- span transmembrane domain, a connector region and a cytosolic ligand-binding domain (LBD) on which a C-terminal tail is appended. In the absence of a ligand, STING1 forms a domain-swapped homodimer, which on binding to cGAMP undergoes extensive conformational rearrangements that assist in side-by-side oligomerization of STING dimers. Oligomerization is further fostered by disulfide bridges spanning separate STING dimers as well as by palmitoylation of cysteine residues, C88 and C91. The oligomerized STING 1 dimers form the activated STING unit capable of initiating effector functions.

Chemotherapeutic agent: Any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. For example, chemotherapeutic agents can be useful for the treatment of a solid cancer, such as a sarcoma, carcinoma, lymphoma, colorectal or skin cancer. Particular examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In one embodiment, a chemotherapeutic agent is a radioactive compound. Other chemotherapeutic agents that can be used are provided in Sausville and Longo, Principles of Cancer Treatment, Chapter 69 in Harrison's Principles of Internal Medicine (20^th ed.), McGraw-Hill, 2018; Niederhuber el al., Cancer Pharmacology, Ch. 25 in Abeloff’ s Clinical Oncology (6^th ed.), Elsevier, 2019; Gullatte etal., Clinical Guide to Antineoplastic Therapy: A Chemotherapy Handbook (4^th ed.), Oncology Nursing Society, 2020; Chabner and Longo, Cancer Chemotherapy, Immunotherapy and Biotherapy: Principles and Practice (6th ed.), Lippincott Williams & Wilkins, 2018; Skeel, Handbook of Cancer Chemotherapy (9th ed.), Lippincott Williams & Wilkins, 2016. Combination chemotherapy is the administration of more than one chemotherapeutic agent to treat cancer.

Checkpoint inhibitor: Immune checkpoints are initiated by the binding of checkpoint proteins to their receptor, and thus can be readily inhibited by antibodies, small molecules, or recombinant forms of ligands or receptors. A “checkpoint inhibitor” reduces or blocks the binding of the checkpoint protein to its respective receptor. Examples of immune checkpoint inhibitor antibodies include antibodies that specifically bind CTLA-4, PD-1, PD-L1, PD-L2, TNFR-2, lymphocyte activating protein 3 (LAG-3), B and T lymphocyte associated protein (BTLA), B7H3 (also known as CD276), V-set domain containing T cell activation inhibitor 1 (VTCN1, also known as B7H4), T cell immunoglobulin and mucin domain-containing protein 3 (T1M3), or adenosine A2A receptor (A2aR). Examples of immune checkpoint inhibitors also include drugs such as cyclophosphamide, which can preferentially deplete tolerogenic CD8⁺ lymphoidresident dendritic cells, leading to diminished regulatory T cell suppression and enhanced effector T cell function.

Colorectal cancer: Adenocarcinomas of the colon and rectum make up 95% of all colorectal cancer cases. In the gastrointestinal tract, rectal and colon adenocarcinomas develop in the cells of the lining inside the large intestine. Other forms of colorectal cancer include gastrointestinal stromal tumors (GIST), lymphoma, carcinoids, Turcot syndrome, Peutz-Jeghers syndrome (PJS), familial colorectal cancer (FCC), juvenile polyposis coli. Less common forms of colorectal adenocarcinomas include mucinous adenocarcinoma and signet ring cell adenocarcinoma.

Combination therapy for cancer treatment: A treatment modality that combines two or more therapeutic agents for the treatment of cancer. A combination therapy for cancer treatment (also known herein as a “combination therapy”) may target several biological pathways in a characteristically synergistic or an additive manner to treat the cancer. The two or more (such as three or four) therapeutic agents of a combination therapy may comprise two or more different components administered substantially simultaneously or sequentially in any order, at two or more different times, or a combination thereof. A combination therapy disclosed herein may be useful for treating one type of cancer (such as melanoma) or two or more different types of cancers (such as kidney cancer and colorectal cancer).

Conservative variant: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody or protein, such as HMGN1, or an antibody that specifically binds PD-1, PD-L1, CTLA-4, or TNFR-2. As one example, a monoclonal antibody that specifically binds PD-1 can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the PD-1 poly peptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the variant retains activity. Non-conservative substitutions are those that reduce an activity of a protein. Conservative amino acid substitution tables providing functionally similar amino acids are known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Consist of: A therapeutic regimen that ‘consists of’ the use of a specific set of therapeutic agents for treatment of a particular disease or condition includes the use of only those agents for treatment of the disease or condition. However, other agents that are not used to treat the disease or condition can be administered to the subject. As a non-limiting example, a method for treating a cancer in a subject can consist of administration of a set of chemotherapeutic agents. That subject can be treated for other diseases or conditions, such as diabetes, and thus could also be administered a therapeutic agent such as insulin.

Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4): A member of the immunoglobulin superfamily, also known as CD152, that transmits an inhibitory signal to T cells. The protein contains a V domain, a transmembrane domain, and a cytoplasmic tail. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. CTLA-4 is a receptor that functions as an immune checkpoint and downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation. It acts as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. An exemplary protein sequence of CTLA-4 is provided in GENBANK® Accession No. NP_005205.2, December 21, 2021, and an exemplary nucleic acid encoding CTLA-4 is provided in GENBANK® Accession No. NM_005214.5, December 21, 2021.

Dendritic cell (DC): An antigen presenting cell that processes antigens and presents them to T cells and is thus capable of triggering an adaptive immune response. In vivo, dendritic cells are present in the skin, the nose, lungs, stomach, intestines, and in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages, DCs grow branched projections, called “dendrities.” Dendritic cells include conventional dendritic cells (eDCs), that are similar to monocytes, and plasmacytoid dendritic cells (pDCs). Monocyte-derived DCs (such as human monocyte-derived DCs, MoDCs) can be generated in vitro from peripheral blood mononuclear cells (PBMCs).

DC maturation is a process characterized by DC acquisition of a number of properties: antigen processing and presentation, migration, and T-cell co- stimulation. Triggering of TLRs (e.g., through interaction with a microbe-associated molecule) on DCs is thought to be important for their functional maturation to immunogenic DCs and the priming of naive T cells in response to infection, coupling innate and adaptive immunity. Activated, mature DCs provide the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 (“TCR/CD3”) complex and an antigenic peptide presented by a major histocompatibility complex (MHC) class I or II protein on the surface of APCs. The second type of signal, called a co- stimulatory signal, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals. This two-fold signaling can, therefore, result in a vigorous immune response. In most non-avian vertebrates, DCs arise from bone marrow-derived precursors. Immature DCs are found in the peripheral blood and cord blood and in the thymus. Additional immature populations may be present elsewhere.

Effective amount, therapeutically effective amount: The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent (such as one or more embodiments provided herein alone, in combination, or potentially in combination with other therapeutic agent(s)) that is sufficient to induce a desired biological result. That result may be amelioration or alleviation of the signs, symptoms, or causes of a disease (such as a reduced cancer burden in a subject), or any other desired alteration of a biological system. The effective amount can vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied. In some embodiments, an effective amount of a combination of therapeutic agents disclosed herein is an amount which, when administered to a subject, is sufficient to engender a detectable therapeutic response. Such a response may comprise, for instance, a reduced cancer burden, increased survival, and/or maturation of human MoDCs in a subject having cancer. Appropriate amounts in any given instance will be readily apparent or capable of determination by routine experimentation, such as administration of the therapeutic agent combination (such as in a combination therapy for cancer treatment) and observation of a cancer response in the subject.

In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor, such as reduce a tumor size and/or volume by at least 10%, at least 20%, al least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and/or reduce the number and/or size/volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size/volume/number prior to treatment.

Fibroblast stimulating lipopeptide 1 (FSL-1): A synthetic lipopeptide and TLR2/6 ligand derived from Mycoplasma salivarium. FSL-1 (also known as Pam2CGDPKHPKSF) is the N-terminal part of the 44 kDa lipoprotein LP44 of M. salivarium. FSL-1 induces TNF-a production in macrophages, upregulates proinflammatory cytokines, and activates the proinflammatory transcription factor NF-KB (Kurkjian et al. Sci Rep. 7: 17355, 2017).

High mobility group nucleosome binding protein 1 (HMGN1): HMGN1 has a combination of activities that potentially counter the mutagenic and immunosuppressive properties of cancers. HMGN 1 is a chromatin-binding nuclear protein and can also act as an extracellular alarmin. Alarmins are structurally diverse endogenous cytokine-like host defense signals, which rapidly alert host defenses and enhance both innate and adaptive immune responses and exhibit potent in vivo immunoadjuvant activity. Thus, HMGN1 acts as a chromatin modifier to regulate chromatin structure, gene expression and post-translational modification of core histones, all of which are factors that affect DNA repair and cancer progression. It also possesses chemotactic activities for immune cells and activates dendritic cell (DC) maturation by interacting with TLR4. It is known to have immunostimulating effects and has been shown to enhance Thl immune responses to antigens (Yang et al. 2012, J Exp Med. 209(1): 157-71 ; Yang et al. (2015) Immunotherapy 7(11): 1129-31). An exemplary HMGN1 amino acid sequence is provided in SEQ ID NO: 2 (GENBANK® Accession No. NP_004956, dated November 26, 2021, and UniProtKB Accession No. P05114.3, dated September 29, 2021, both incorporated herein by reference).

Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who has or is at risk of having a cancer. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as a cancer (such as a reduced cancer burden in a subject), after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease, such as improved survival of a subject having a cancer. Treatment may be assessed by objective or subjective parameters; including, but not limited to, the results of a physical examination, imaging, or a blood test. A “prophylactic” treatment is a treatment administered to a subject who docs not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology, such as to prevent the occurrence or recurrence of a cancer.

Kidney cancer: A form of cancer that primarily includes renal cell cancer, transitional cell cancer, and Wilms’ tumor. In renal cell cancer (also called renal cell adenocarcinoma), malignant cells are found in the lining of tubules in the kidney, and primarily include clear cell cancers, papillary cancers, and chromophobe renal cell cancers. Rare types of renal cell cancer include carcinoma of the collecting ducts and renal medullary carcinoma. Sometimes kidney cancers can contain more than one cell type. Transitional cell cancer can form in the renal pelvis, the ureter, or both. Wilms’ tumor is most common in children.

Melanoma: A form of cancer that originates in melanocytes. Melanocytes are found primarily in the skin but are also present in the bowel and eye. Melanoma in the skin includes superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna (melanoma). Any of the above types may produce melanin or can be amelanotic. Similarly, any subtype may show desmoplasia (dense fibrous reaction with neurotropism) which is a marker of aggressive behavior and a tendency to local recurrence. Other melanomas include clear cell sarcoma, mucosal melanoma, and uveal melanoma. Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in the embodiments of this disclosure are conventional. Remington: The Science and Practice of Pharmacy . (23^rd ed.) by Adeboye Adejare, Academic Press (2020), describes compositions and formulations suitable for pharmaceutical delivery of the therapeutic agents (such as HMGN-1, FSL-1, a checkpoint inhibitor, and/or cGAMP) herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Programmed death 1 (PD-1): An immune-inhibitory receptor expressed in activated T cells also known as CD279. PD-1 is a type I membrane protein of 288 amino acids, that is a member of the extended CD28/CTLA-4 family of T cell regulators. PD-1 is involved in the regulation of T-cell functions, including those of effector CD8⁺ T cells. PD-1 can also promote the differentiation of CD4⁺ T cells into T regulatory cells. PD-1 is expressed in many types of cancers including melanomas and plays a role in anti-cancer immunity. This protein is involved in safeguarding against autoimmunity; however, it can also contribute to the inhibition of effective anti-cancer and anti-microbial immunity. An exemplary PD-1 protein and mRNA sequence is provided in GENBANK® Accession No. NM_OO5O18.3, dated January 17, 2022, incorporated herein by reference.

Programmed death ligand 1 (PD-L1): A ligand that binds with the receptor PD-1, commonly found on T-cells, and acts to block T-cell activation. PD-L1 (also known as CD274) is a type I transmembrane protein that has immunoglobulin V-like and C-like domains. Interaction of this ligand with its receptor inhibits T-cell activation and cytokine production. During infection or inflammation of normal tissue, this interaction plays a role in preventing autoimmunity by maintaining homeostasis of the immune response. In cancer microenvironments, this interaction provides an immune escape for cancer cells through cytotoxic T-cell inactivation. Expression of this gene in cancer cells is prognostic in many types of human malignancies, including colon cancer and renal cell carcinoma. Exemplary PD-L1 protein and mRNA sequences are provided in GENBANK® Accession Nos. NP_054862.1, NP_001254635.1, NP_001300958, NM_014143.4, NM_001267706.2, NM_001314029.2, all dated January 17, 2022, and incorporated herein by reference.

Protein: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Protein” is used interchangeably with peptide or polypeptide, and is used herein to refer to a polymer of amino acid residues. “Protein” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or amide bond mimetic. A protein has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals, such as mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In an example, a subject is a human. In a particular example, the subject has a cancer. In an additional example, a subject is selected that is in need of inhibiting of growth of a cancer or metastasis. For example, the subject has been diagnosed with a colorectal cancer, a kidney cancer, or a melanoma, and is in need of treatment.

Toll-like receptor (TLR): Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that initiate the innate immune response by sensing conserved molecular patterns for early immune recognition of a pathogen. TLRs are expressed in innate immune cells such as dendritic cells and macrophages as well as non-immune cells such as fibroblast cells and epithelial cells (Kawasaki et al.. Front. Immunol, 5:1-8, 2014). Numerous roles for TLRs have been identified, such as recognition of self and non-self antigens: detection of invading pathogens; bridging the innate and adaptive immunity responses; and regulation of cytokine production, proliferation, and survival. Generally, TLRs are type I transmembrane proteins that contain three structural domains: a leucine-rich repeats (LRR) motif, a transmembrane domain, and a cytoplasmic Toll/IL-1 receptor (TIR) domain (Nie et al., Front. Immunol. 9:1- 19, 2018). The LRR motif is responsible for pathogen recognition, whereas the TIR domain interacts with signal transduction adaptors and initiates signaling.

TLRs can recognize molecules (“TLR ligands”) broadly shared by pathogens, known as pathogen- associated molecular patterns (PAMPs), and host endogenous damage-associated molecular pattern molecules (DAMPs). These TLR ligands are often TLR agonists that activate TLR signaling and are evolutionarily conserved. TLR agonists include pathogen-associated molecules, such as bacterial cellsurface lipopolysaccharides (LPS), lipoproteins, lipopeptides, and lipoarabinomannan; proteins, such as flagellin from bacterial flagella; double-stranded RNA of viruses; unmethylated CpG islands of bacterial and viral DNA; CpG islands in the eukaryotic DNA promoters; as well as other RNA and DNA molecules. This kind of recognition is multifarious, depending on the type of TLR. TLRs are largely classified into two subfamilies based on their localization, cell surface TLRs and intracellular TLRs. Cell surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, whereas intracellular TLRs are localized in the endosome and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13. Cell surface TLRs mainly recognize microbial membrane components such as lipids, lipoproteins, and proteins. Intracellular TLRs recognize nucleic acids derived from bacteria and viruses, and also recognize self-nucleic acids in disease conditions such as autoimmunity. TLR functions are mediated by subsequently initiated signaling pathways, resulting in the production of various cytokines and chemokines. TLR activation generally results in activation and phenotypic maturation of dendritic cells.

Toll-like receptor 4: TRL4 expressing cells include myeloid (erythrocytes, granulocytes, macrophages) rather than lymphoid (T-cells, B-cells, NK cells) cells, upon ligand binding at the cell surface, TLR4 receptors homodimerize through interactions between their intracellular TIR-domains, resulting in conformational changes in the molecule. The subsequent signaling process involves the recruitment of TIR-domain-containing adapter molecules to the cytoplasmic face of the TLR4 cluster via homophilic interactions between the TIR-domains. Four TIR-domain-containing adapter molecules belonging to two distinct pathways are known to mediate TLR4 signaling: Myeloid differentiation factor 88 (MyD88); MyD88-adapter-like (Mai) protein, also known as TIR-domain-containing adapter protein (TIRAP); TIR-domain-containing adapter inducing interferon-|3 (TRIF), also called TIR-domain-containing adapter molecule-1 (TICAM-1); and TRIF-related adapter molecule (TRAM), also called TIR-containing protein (TIRP), or TIR-containing adapter molecule-2 (TICAM-2). TLR4 requires all four of these adapters to mediate a comprehensive immune response (Vaure and Liu. Front Immunol. 5(316): 1-15. 2014). Exemplary TLR4 protein and mRNA sequences are provided in GENBANK® Accession Nos.

NP_612564.1, NP_612567.1, NP_003257.1, NM_138554.5, NM_138557.3, and NM_003266.4, all dated December 27, 2021, and incorporated herein by reference.

TLR4 initiates intracellular signaling by at least two major pathways: (i) the TIRAP-MyD88 pathway, which regulates early NF-KB activation and related inflammatory cytokine production, such as IL- 12; and (ii) the TRIF-TRAM pathway, which activates the interferon regulatory factor-3 (IRF3) transcription factor that effectuates the subsequent up-regulation of genes encoding type I interferons (IFNs) and co-stimulatory molecules. This TRIF-dependent pathway also activates TNF-a production and secretion. The subsequent binding of secreted TNF-a to its receptors leads to NF-KB activation. Thus, the TRIF-TRAM pathway is also responsible for the late phase NF-KB activation through IRF3 and TNF-a secretion. MyD88-independent signaling accounts for the majority of the lipopolysaccharide (LPS) response. The MyD88-independent pathway results in the induction of dendritic cell (DC) maturation (consequent to the expression of the genes encoding co-stimulatory molecules such as CD40, CD80, and CD86) and elevated expression of type-1 interferon genes and of IFN -regulated genes. This results in T cell activation, clonal expansion, and Thl polarization indirectly through stimulation and recruitment of antigen- presenting cells (APC) such as dendritic cells, macrophages, and monocytes.

Toll-like receptor 2/6 (TLR2/6): TLR2 is a TLR that heterodimerizes with TLR6 to recognize diacyl lipopeptides present in gram-positive and gram-negative bacteria. Ligand engagement of TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (z.e., nuclear translocation of NF-KB, and activation of MAPKs), resulting in production of proinflammatory cytokines (Mistry, etal., PNAS. 112(17)5455-5460, 2015). Exemplary TLR2 protein and mRNA sequences are provided in GENBANK® Accession Nos. AAH33756.1, dated October 7, 2003, and NM_001318787.2, NM_003264.5, NM_001318789.2, and NM_001318790.2, NM_001318791.2, NM_001318793.2, NM_001318795.2, and NM_001318796.2 all dated January 23, 2022, and incorporated herein by reference. Exemplary TLR6 protein and mRNA sequences are provided in GENBANK® Accession Nos.

NP_006059.2, NM_006068.5, and NM_001394553.1, all dated January 23, 2022, and incorporated herein by reference.

Tumor necrosis factor receptor (TNFR)- 2: A member of the tumor necrosis factor (TNF) receptor superfamily, also known as CD120b or TNF receptor superfamily member IB (TNFRSF1B). TNF- a signals via receptors TNFR-1 and TNFR-2. TNFR-1 contains an intracellular death domain and can activate either apoptotic or inflammatory pathways, whereas TNFR-2 binds TNF receptor-associated factors and can activate the canonical and noncanonical NF-KB pathway to control cell survival and proliferation. TNFR-2 and TNFR-1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c- IAP1 and C-IAP2, that have E3 ubiquitin ligase activity. The function of lAPs in TNF-receptor signaling is unknown; however, c-IAPl is thought to potentiate TNF-induced apoptosis by the ubiquitination and degradation of TNF-receptor-associated factor 2 (TRAF2), which mediates anti-apoptotic signals. Exemplary TNFR-2 protein and mRNA sequences are provided in GENBANK® Accession Nos. AAC50622.1, dated June 10, 2016, and NP_001057 and NM_001066.3, dated January 17, 2022, and incorporated by reference herein.

II. Methods of Treating Cancer in a Subject

Methods are provided herein for treating a cancer in a subject. In some embodiments, a therapeutically effective amount of a combination therapy comprising a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), and checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2) is administered to the subject that has a cancer, thereby treating the cancer in the subject. In some embodiments, the combination therapy administered to the subject also includes a therapeutically effective amount of a STING agonist (such as cGAMP or c-di-GMP). As shown herein, the administration of the disclosed combination therapies showed a surprisingly robust, synergistic therapeutic effect, resulting in treating or curing or eradicating established cancers. In some embodiments, administration of the combination therapies disclosed herein may also be used to reduce the incidence of relapse of a cancer.

In embodiments of the disclosed methods, administration of a therapeutically effective amount of a disclosed combination therapy to a subject reduces cancer burden in the subject, increases survival of the subject, reduces the incidence of relapse of a cancer in the subject, induces maturation of dendritic cells in the subject, or a combination thereof. The method can include selecting the subject with the cancer.

The methods of this disclosure may be useful in the treatment of a variety of cancers. Exemplary cancers that may be treated or prevented include thymoma, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, uterine cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid cancer, lymphoid and other hematopoietic cancers, Hodgkin lymphoma, B cell lymphoma, bronchial squamous cell cancer, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, pancreatic cancer, carcinoma, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In example embodiments, the cancer may be colon cancer, kidney cancer, liver cancer, skin cancer or melanoma, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, lung cancer, carcinoid, lymphoma or glioma. In exemplary embodiments, the cancer is thymoma, colorectal cancer, kidney cancer, or liver cancer.

In specific non-limiting examples, the cancer is a colorectal cancer, a kidney cancer, or a melanoma. In another specific, non-limiting examples, the colorectal cancer is an adenocarcinoma of the colon and/or rectum, gastrointestinal stromal tumors (GIST), lymphoma, carcinoids, Turcot syndrome, Peutz-Jeghers syndrome (PJS), familial colorectal cancer (FCC), juvenile polyposis coli, mucinous adenocarcinoma, or signet ring cell adenocarcinoma. In other specific, non-limiting examples, the kidney cancer is renal cell cancer (also known as renal cell adenocarcinoma, and including clear cell cancers, papillary cancers, and chromophobe renal cell cancers), transitional cell cancer, Wilms’ tumor, carcinoma of the collecting ducts, or renal medullary carcinoma. In additional specific, non-limiting examples, the melanoma is superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna, clear cell sarcoma, mucosal melanoma, or uveal melanoma.

Treatment of the conditions described herein are generally initiated after the development of a condition described herein, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer. For instance, treatment can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage III and stage IV cancers. Treatment prior to the development of the condition, such as treatment upon detecting dysplasia or an early (benign) precursor condition, is referred to herein as treatment of a subject that is “at risk” of developing the condition. In some embodiments, administration of a combination therapy can be performed during or after the occurrence of the conditions described herein.

Thus, a subject can be selected for treatment that has, or is at risk for developing a cancer, such as a cancer disclosed herein, such as, for example, a colorectal cancer, a kidney cancer, or a melanoma. Typical subjects intended for administration of the combination therapies disclosed herein include humans, as well as non-human primates and other animals. To identify relevant subjects, accepted screening methods are employed to determine risk factors associated with, and/or to diagnose, a targeted or suspected cancer (such as a colorectal cancer, a kidney cancer, or a melanoma) in a subject, or to determine the status of an existing cancer in the subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected cancer, as well as diagnostic methods, such as, but not limited to, various histopathological, morphological, and/or cytological analyses to identify or diagnose the targeted cancer. These and other routine methods allow the clinician to select patients in need of therapy. In accordance with these methods and principles, the combination therapies disclosed herein can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a followup, adjunct, or coordinate treatment regimen to other treatments.

In some embodiments, the subject has colorectal cancer. In other embodiments, the subject has kidney cancer. These methods can include administering to the subject a therapeutically effective amount of a combination therapy comprising a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), and a checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), thereby treating the colorectal cancer or the kidney cancer.

In one specific, non-limiting example, the subject has colorectal cancer, and the method includes administering to the subject a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds PD-1. In another specific, non-limiting example, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds CTLA-4 to the subject. In an additional nonlimiting example, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds PD-L1 to the subject. In a further non-limiting example, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds TNFR-2 to the subject.

In one specific, non-limiting example, the subject has kidney cancer, and the method includes administering to the subject a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds PD-1. In another specific, non-limiting example, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds CTLA-4 to the subject. In an addition non-limiting example, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds PD-L1 to the subject. In a further nonlimiting example, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, and an antibody that specifically binds TNFR-2 to the subject.

In other embodiments, the subject has melanoma. These methods include administering to the subject a therapeutically effective amount of a combination therapy comprising TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), a checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), and a STING agonist (such as cGAMP or c-di-GMP), thereby treating the melanoma in the subject. In some non-limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds PD-1, and cGAMP to the subject. In some non-limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds PD-L1, and cGAMP to the subject. In additional non-limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds CTLA-4, and cGAMP to the subject. In further nonlimiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds TNFR-2, and cGAMP to the subject.

In some non-limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds PD-1, and c-di-GMP to the subject. In some non-limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds PD-L1, and c-di-GMP to the subject. In more non- limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds CTLA-4, and c-di-GMP to the subject. In further non-limiting examples, the method includes administering a therapeutically effective amount of a combination therapy comprising HMGN1, FSL-1, an antibody that specifically binds TNFR-2, and c-di-GMP to the subject.

A variety of administration regimens are possible for each of the disclosed combination therapies. Administration with a therapeutically effective amount can be a single administration or multiple administrations. Administration can involve daily or multi-daily or less than daily (such as weekly, monthly, etc.) doses over a period of a few days to weeks or months, or even years. In particular nonlimiting examples, administration involves a once monthly dose, a once every three weeks dose, a once every two weeks dose, a weekly dose, a twice weekly dose, or a daily dose, or a combination thereof. The particular mode/manner of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (such as the subject, the disease, the disease state/severity involved, the particular administration, and whether the treatment is prophylactic). In some embodiments, additional chemotherapeutic agents are not administered to the subject. Thus, the method can consist, or consist essentially of, administration of the TLR4 agonist (such as HMGN1), the TLR2/6 agonist (such as FSL-1), the checkpoint inhibitor (such as the antibody that specifically binds PD-1, the antibody that specifically binds PD-L1, the antibody that specifically binds CTLA-4, or the antibody that specifically binds TNFR-2), and optionally the STING agonist (such as cGAMP or c-di-GMP).

More than one route, such as intratumoral, intravenous, intraperitoneal, intramuscular, subcutaneous, oral, or topical may be used for administration of the combination therapies, and particular routes may provide more immediate and more effective responses than other routes. Therapeutic agents of the disclosed combination therapies can be administered by the same or different routes. In some embodiments, the combination therapies are administered using any suitable route of administration, such as, for example, intravenous or intratumoral administration. In exemplary embodiments, the TLR4 agonist (such as HMGN1), TLR2/6 agonist (such as FSL-1), checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), and/or STING agonist (such as cGAMP or c-di-GMP) may be administered by intratumoral injection. Alternatively or additionally, the TLR4 agonist (such as HMGN1), TLR2/6 agonist (such as FSL-1), checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), and/or STING agonist (such as cGAMP or c-di-GMP) may be administered intravenously. In some embodiments, intratumoral administration, presumably based on better access to cancer antigens, may be more effective than systemic administration.

In some embodiments, a subject can be administered varying concentrations of each of the therapeutic agents of a given combination therapy, one or more times, at one or more different time intervals. The disclosed combination therapies include three or more components that are administered such that the effective time period of at least one component overlaps with the effective time period of at least one other component. In some embodiments, the effective time periods of all components administered overlap with each other. In an exemplary embodiment of a combination comprising three components, the effective time period of the first component administered may overlap with the effective time periods of the second and third components, but the effective time period of the second component independently may or may not overlap with that of the third component. In an exemplary embodiment of a combination comprising four components, the effective time period of the first component administered overlaps with the effective time periods of the second, third, and fourth components; the effective time period of the second component overlaps with those of the first and fourth components, but not that of the third component; and the effective time period of the fourth component overlaps with that of the second and third components only.

A combination therapy may comprise three or more, such as three or four individual components, or a combination may comprise three or more, such as three or four components and another separate component (or components) or composition(s) comprising the remaining component(s). In some embodiments, the three or more, such as three or four, components may be administered substantially simultaneously or sequentially in any order, at two or more different times, or a combination thereof.

In some embodiments, the checkpoint inhibitor (such as such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2) is administered intravenously to a subject having a cancer. In such embodiments, a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), and optionally a STING agonist (such as cGAMP or c-di-GMP) are administered, simultaneously with (/.<?., at the same time) or at another time than the checkpoint inhibitor. The TLR4 agonist (such as HMGN 1), the TLR2/6 agonist (such as FSL-1), and optionally the STING agonist (such as cGAMP or c-di-GMP) can be administered intratumorally to the subject.

Pharmaceutical compositions are of use in the disclosed method that include a therapeutically effective amount of a combination therapy comprising a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), a checkpoint inhibitor (such as such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), and/or a STING agonist (such as cGAMP or c-di-GMP). These pharmaceutical compositions can include any suitable carrier. For example, formulations suitable for intratumoral, intravenous, intramuscular, subcutaneous, intraperitoneal, or topical administration may comprise sterile aqueous solutions of the active components. Such formulations may be prepared by dissolving a therapeutic agent and/or additional active and/or inactive component(s) in water containing physiologically compatible substances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. A suitable carrier may be the same for some (such as two or more) or all of the therapeutic agents of the disclosed combination therapies, or may be different for all therapeutic agents.

In some embodiments, a therapeutic agent, such as HMGN1, can be administered by means of a nanoparticle vehicle. Nanoparticles (NPs) are synthetic particles with dimensions ranging from one to hundreds of nanometers comprising an inorganic core surrounded by an organic layer. Nanoparticles featuring inorganic cores such as gold, silica, superparamagnetic iron oxide (SPIO) are known. In cancer tissue, NPs extravasate from the leaky cancer vasculature to a higher degree than healthy tissue, and remain in the area by the enhanced permeability and retention (EPR) effect. Exemplary NPs suitable for use in the present embodiments are described in U.S. Patent Application Publication US-2019-0151466-A1, which is incorporated by reference herein in its entirety.

The dose of each therapeutic agent of a combination therapy administered to a subject should be sufficient to induce a beneficial therapeutic response in the subject over time, such as reducing a cancer burden in the subject, increasing survival of the subject, reducing the incidence of relapse of a cancer in the subject, inducing maturation of monocyte-derived dendritic cells in the subject, or a combination thereof. The beneficial therapeutic response may require one or more doses of one or more of the disclosed therapeutic agents, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500, or more doses, administered at the same or different times. The dose may vary from subject to subject or may be the same. An appropriate dose can be determined, for example, using routine experimentation. Generally, the administration of the disclosed combination therapies provides a robust, synergistic therapeutic effect for treating the cancer in the subject, for example as compared to the use of the therapeutic agents individually, or as compared to the use of only two of the agents. 1. TLR4 Agonists of Use in the Disclosed Methods

TLR4 agonists suitable for use in the present disclosure include, without limitation, HMGNl, bacterial lipopolysaccharide (LPS), mono-phosphoryl lipid A, CD138, a-crystallin A chain, P-defensin 2, endoplasmin, fibrinogen, fibronectin, heparan sulphate, HSP22, HSP72, HSP96, OxPAPC, resistin, S 100 proteins, surfactant protein A, synthetic mimetics of TLR4 agonist (including, for example, neoseptins), HMGB-1, granulysin, lactoferrin, and tenascin-C.

In exemplary embodiments, the TLR4 agonist is HMGNl. The High Mobility Group (HMG) family of chromosomal binding peptides are subdivided into three subfamilies, each of which has a characteristic functional sequence motif: HMGB (HMG-box motif), HMGN (nucleosomal binding domain), and HMGA (AT-hook motif). HMGN polypeptides include HMGNl (high mobility group nucleosome-binding protein 1; formerly known as HMG14), HMGN2, HMGN3a, HMGN3b, HMGN4, and Nsbpl(NBD-45).

An exemplary nucleic acid sequence encoding a human HMGN 1 gene is provided in GENBANK® Accession No. NC_000021.9x39349088-39342315, incorporated by reference herein in its entirety. An exemplary nucleic acid sequence encoding an HMGNl transcript (nucleic acids 172-474 of GENBANK® Accession No. NM_004965.7, incorporated by reference herein in its entirety) is provided below as SEQ ID NO: 1: ATGCCCAAGAGGAAGGTCAGCTCCGCCGAAGGCGCCGCCAAGGAAGAGCCCAAGAGGAGATCGGCGCGGTTG TCAGCTAAACCTCCTGCAAAAGTGGAAGCGAAGCCGAAAAAGGCAGCAGCGAAGGATAAATCTTCAGACAAA AAAGTGCAAACAAAAGGGAAAAGGGGAGCAAAGGGAAAACAGGCCGAAGTGGCTAACCAAGAAACTAAAGAA GACTTACCTGCGGAAAACGGGGAAACGAAGACTGAGGAGAGTCCAGCCTCTGATGAAGCAGGAGAGAAAGAA GCCAAGTCTGATTAA

An exemplary amino acid sequence of HMGNl (GENBANK® Accession No. NP_004956, dated November 26, 2021, and UniProtKB Accession No. P05114.3, dated September 29, 2021, both incorporated by reference herein, and disclosed in U.S. Patent 8,227,417, incorporated by reference herein) is provided below as SEQ ID NO: 2: MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKE DLPAENGETKTEESPASDEAGEKEAKSD

HMGN 1 has a combination of activities that potentially counter the mutagenic and immunosuppressive properties of cancers. HMGN l is a chromatin-binding nuclear protein and can also act as an extracellular alarmin. Alarmins are structurally diverse endogenous cytokine-like host defense signals, which rapidly alert host defenses and enhance both innate and adaptive immune responses and exhibit potent in vivo immunoadjuvant activity. Thus, HMGNl acts as a chromatin modifier to regulate chromatin structure, gene expression and post-translational modification of core histones, all of which are factors that affect DNA repair and cancer progression. It also imparts chemotactic capabilities on immune cells and activates dendritic cell (DC) maturation by interacting with TLR4. It is known to have immunostimulating effects and has been shown to enhance Thl immune responses to antigens (Yang et al. 2012, J Exp Med. 209(1): 157-71; Yang et al. (2015) Immunotherapy 7(11): 1129-31).

Thus, in some embodiments of the disclosed methods, a subject is administered an HMGNl protein that is least about 80%, 85%, 90%, 95%, 97%, 98%, or about 99% identical to the amino acid sequence of

- 23 -

SUBSTITUTE SHEET ( RULE 26) SEQ ID NO: 2. In particular embodiments, a subject is administered an HMGN 1 protein that is about 85% to about 100%, about 90% to 100%, or about 95% to about 100% identical to SEQ ID NO: 2. In other embodiments, the subject is administered an HMGN1 protein that is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the subject is administered an HGMN1 protein comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the HMGN1 protein comprises one or more amino acid modifications (i.e., substitutions, deletions, and/or additions), such as, for example, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions.

An HMGN1 fragment is also of use in the disclosed methods. An HMGN1 fragment may comprise any contiguous part of the HMGN 1 protein that retains a relevant biological activity of the protein (such as TLR4 agonist activity). Any given fragment of HMGN1 can be tested for such biological activity using standard methods (see, for example, U.S. Patent No. 8,227,417, which is incorporated herein by reference).

Exemplary HMGN1 proteins also include derivative proteins that can be modified by glycosylation, pegylation, phosphorylation, or any similar process that retains at least one biological function of the protein from which it was derived. HMGN1 proteins of use can also include one or more non-naturally occurring amino acids. For example, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into proteins. Non-classical amino acids include, but are not limited to, the D- isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon- Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-mcthyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). In other specific embodiments, branched versions of the HMGN1 protein are provided, such as by substituting one or more amino acids within the sequence with an amino acid or amino acid analog with a free side chain capable of forming a peptide bond with one or more amino acids (and thus capable of forming a "branch"). Cyclical proteins are also contemplated.

The half-life of proteins in human serum is dictated by several factors, including size, charge, proteolytic sensitivity, nature of their biology, and the turnover rate of proteins they bind. In further embodiments, a fusion protein can be used as the therapeutic molecule. Suitable fusions include any protein that can be used to increase half-life, such as conjugation to a high-density lipoprotein, transferrin, albumin, or an Fc domain.

Proteins can be obtained by methods known in the art. HMGN1 and fragments thereof also can be recombinantly produced using nucleic acids encoding them and standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4^th ed.), Cold Spring Harbor Press, N.Y. 2012; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2012. Further, these proteins can be isolated and/or purified from a natural source, e.g., a human. Methods of isolation and purification are well-known in the art. In this respect, HMGN1 (including a polypeptide comprising HMGN1 or a fragment or fragments thereof with TLR4 agonist activity) may be exogenous and may be synthetic, recombinant, or of natural origin. HMGN1 is also commercially available (e.g., R&D Systems Inc., Minneapolis, MN).

Also included are protein derivatives which are differentially modified during or after synthesis. In some examples, HMGN 1 (including a polypeptide comprising HMGN 1 or a fragment or fragments thereof with TLR4 agonist activity) is benzylated, acetylated, pegylated, glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, derivatized by known protecting/blocking groups, converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and ary Isulphonic acids, for example, p-toluenesulphonic acid. The disclosed methods can include administering two or more HMGN 1 polypeptides, any of which may be the same or different from one another. Furthermore, HMGN1, or a fragment or fragments thereof with TLR4 agonist activity, can be provided as part of a larger polypeptide construct. For instance, HMGN 1 or a fragment or fragments thereof can be provided as a fusion protein comprising an HMGN 1 polypeptide or a fragment or fragments thereof, along with other amino acid sequences or a nucleic acid encoding the same. By way of further illustration, the HMGN 1 polypeptide or the fragment or fragments thereof can be provided by two or more fragments of HMGN1 (e.g., different functional domains) with or without a linking amino acid sequence and/or flanking sequences. HMGN1 or a fragment or fragments thereof with TLR4 agonist activity also may be provided as part of a conjugate or nucleic acid(s) encoding the same. Conjugates, as well as methods of synthesizing conjugates in general, arc known in the art (See, for instance, Hudccz, F., Methods Mol. Biol. 298: 209-223, 2005; and Kirin et al., Inorg Chem. 44(15): 5405-5415, 2005).

Pharmaceutical compositions of use in the disclosed methods can include about 0.5% w/v to about 25% w/v, TLR4 agonist (such as HMGN1), or about 1% w/v to about 12% w/v TLR4 agonist (such as HMGN1), or about 12% w/v to about 25% w/v, or more, TLR4 agonist (such as HMGN1). In some embodiments, the pharmaceutical composition can include about 0.5%, 1%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% w/v, or more, TLR4 agonist (such as HMGN1). Exemplary pharmaceutical compositions include about 10% w/v TLR4 agonist (such as HMGN1).

In some embodiments, the subject is administered (for example, intravenously or intratumorally) about 0.3 mg/kg to about 15 mg/kg or more of a TLR4 agonist (such as HMGN1), such as about 0.3 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, or about 14 mg/kg to about 15 mg/kg of TLR4 agonist (such as HMGN1). In one specific non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg mg of a TLR4 agonist (such as HMGN1). In another specific, non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg of a TLR4 agonist (such as HMGN 1) in a single dose or in more than dose.

2. TLR 2/6 Agonists

In embodiments of the disclosed methods, a subject (such as a subject that has a cancer) is administered a therapeutically effective amount of a TLR2/6 agonist. TLR2 forms heterodimers with TLR1 and TLR6, and regulates host defense mechanisms against pathogens. Ligand binding to TLR2/6 activates the MyD88-dependent pathway (/.<?., nuclear translocation of NF-KB, and activation of MAPKs), resulting in production of proinflammatory cytokines (Mistry, et al., PNAS. 112( 17):5455 -5460, 2015).

TLR2/6 agonists suitable for use include, without limitation, FSL-1, macrophage-activated lipopeptide-2 (MALP-2), CBLB612 (a synthetic lipopeptide agonist of TLR2; Cleveland BioLabs; PROTECTAN®; disclosed in U.S. Patent No. 11/917,494), SV-283 (a synthetic peptide/small molecule agonist of TLR2; SapVax), OPN-305 (a humanised IgG4 monoclonal antibody against TLR2; also known as tomaralimab; Opsona Therapeutics; disclosed in PCT Publication No. WO2011/003925), synthetic triacylated and diacylated lipopeptides, Pam3Cys (tripalmitoyl-S -glyceryl cysteine), and S-[2,3- bis(palmitoyloxy)-(2RS)-propyl]-Npalmitoyl-(R)-cysteine, where “Pam3” is “tripalmitoyl-S-glyceryl.” Derivatives of Pam3Cys are also suitable TLR2/6 agonists, where derivatives include, but are not limited to: S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N- palmitoyl-(R)-Cys-(S)-Ser-(Lys)4 -hydroxytrihydrochloride; Pam3Cys-Scr-Scr-Asn-Ala; Pam3Cys-Scr-(Lys)4; Pam3Cys-Ala-Gly; Pam3Cys-Scr-Gly; Pam3Cys-Scr; Pam3Cys-OMe; Pam3Cys-OH; PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH, and the like. Other non-limiting examples of suitable TLR2/6 agonists are INNA-051, Pam2CSK4; Pam2CysSK4 (dipalmitoyl-S-glyceryl cysteine-serine-(lysine)4; or Pam2Cys-Ser-(Lys)4), which is a synthetic diacylated lipopeptide. A TLR2/6 agonist may be conjugated with one or more compounds or functional groups. Other synthetic TLR agonists include those described, e.g., in W02021/042171A1, Kellner et al., Biol. Chem. 373:1:51-5, 1992; Seifer et al., Biochem. J, 26:795-802, 1990; and Lee et al., J. Lipid Res., 44:479-486, 2003. In some embodiments, the TLR2/6 agonist is FSL-1.

Administering a therapeutically effective amount of a TLR2/6 agonist (such as FSL-1 or a fragment or fragments thereof with TLR2/6 agonist activity) to a subject may include administering a pharmaceutical composition including 0.5% w/v to about 25% w/v or more TLR2/6 agonist (such as FSL-1), or about 1% w/v to about 12% w/v TLR2/6 agonist (such as FSL-1), or about 12% w/v to about 25% w/v or more TLR2/6 agonist (such as FSL-1). In some embodiments, the combination may include about 0.5%, 1%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% w/v, or more TLR2/6 agonist (such as FSL-1). Exemplary combinations of therapeutic agents may include about 10% w/v TLR2/6 agonist (such as FSL-1).

In some embodiments, the subject is administered (for example, intravenously or intratumorally) about 0.3 mg/kg to about 15 mg/kg or more of a TLR2/6 agonist (such as FSL-1), such as about 0.3 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, or about 14 mg/kg to about 15 mg/kg of TLR2/6 agonist (such as FSL-1). In one specific non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg mg of a TLR2/6 agonist (such as FSL-1). In another specific, non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg of a TLR2/6 agonist (such as FSL-1) in a single dose or in more than dose.

3. Checkpoint inhibitors

Checkpoint inhibitors suitable for use in the present methods include, without limitation, agents that inhibit PD-1, PD-L1, CTLA-4, TNFR-2, lymphocyte activating protein 3 (LAG-3), B and T lymphocyte associated protein (BTLA), B7H3 (also known as CD276), V-set domain containing T cell activation inhibitor 1 (VTCN1, also known as B7H4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), or adenosine A2A receptor (A2aR). The checkpoint inhibitor can be a small molecule or chemical compound. The checkpoint inhibitor can be an antibody. In some non-limiting examples, the checkpoint inhibitor is an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, an antibody that specifically binds TNFR-2, an antibody that specifically binds LAG-3, an antibody that specifically binds BTLA, an antibody that specifically binds B7H3, an antibody that specifically binds VTCN1, an antibody that specifically binds TIM3, or an antibody that specifically binds A2aR. The checkpoint inhibitor can be a chemical compound, such as cyclophosphamide.

Checkpoint receptors encompass a specific subset of negative regulators that deliver inhibitory signals that dampen stimulatory signals and limit immune activation. Immune checkpoints refer to a plethora of pathways hardwired into the immune system that are crucial for maintaining self-tolerance (i.e., prevention of autoimmunity) and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize tissue damage. The expression of immune-checkpoint proteins is dysregulated by cancers as an important immune resistance mechanism. The inhibition of immune checkpoints facilitates anticancer immune response. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily inhibited by antibodies or modulated by recombinant forms of ligands or receptors. Blockade of immune checkpoints is a strategy to enhance the immune response against cancer cells. A number of immune checkpoint inhibitors are known in the art, e.g., Pardoll et al., Nature Reviews Cancer. 12:252-64, 2012; and Ding et al., Clinical and Developmental Immunology. 2012: 1-12. Examples of immune checkpoint inhibitors include antibodies that block immune checkpoints (e.g.. by targeting lymphocyte receptors or their ligands) or drug molecules that have a similar mechanism of action. Any checkpoint inhibitor is of use in the presently disclosed methods.

In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In other embodiments, the checkpoint inhibitor is a PD-L1 or PD-L2 inhibitor. PD-1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. Int Immunol. 8:765-75, 1996). Two ligands for PD-1, PD-L1 and PD- L2 have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. J Exp Med. 192:1027-34, 2000; Latchman et al. Nat Immunol. 2:261-8, 2001; Carter et al. Eur J Immunol. 32:634-43, 2002). PD-L1 is abundant in human cancers (Dong et al. J Mol Med. 81:281-7, 2003; Blank et al. Cancer Immunol Immunother. 54:307-314, 2005; Konishi et al. Clin Cancer Res. 10:5094, 2004). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with programmed death ligand (PD- L)1 or PD-L2.

In some examples, the checkpoint inhibitor can be an antibody (such as a monoclonal antibody) or antigen binding fragment thereof that binds to PD-1, PD-L1, or PD-L2. Antibodies, antibody fragments, and other inhibitors of PD-1, PD-L1 and PD-L2 are available in the art and may be used in the methods disclosed herein. For example, nivolumab (OPDIVO®), also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgGi monoclonal antibody which specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and PCT Publication No. W02006/121168. Cemiplimab (Regeneron/Sanofi; LIBTAYO®; REGN2810; disclosed in U.S. Patent No. 9,987,500 as H4H7798N) is a fully human antibody against PD-1. Pcmbrolizumab (Merck; KEYTRUDA®; disclosed in PCT Publication No. WO2008/156712) is a humanized IgG4 (S228P) antibody against PD-1. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD-1. Pidilizumab and other humanized monoclonal antibodies that specifically bind PD-1 are disclosed in PCT Publication No. W02009/101611. Lambrolizumab (also referred to as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Lambrolizumab and other humanized antibodies that specifically bind PD-1 are disclosed in U.S. Pat. No. 8,354,509 and PCT Publication No. W02009/114335. Atezolizumab (TECENTRIQ®; Genentech, Inc.; disclosed in U.S. Patent No. 8,217,149) is a fully humanized, engineered monoclonal antibody of IgGl isotype against PD-L1. Avelumab (Merck; MSB0010718C; BAVENCIO®; disclosed in U.S. Patent App. No. US2014341917 and PCT Publication Nos. WO2013/079174 and WO2016/137985) is a fully human monoclonal antibody of IgGl isotype against PD-L1. Durvalumab (AstraZeneca; IMFINZI®; disclosed in U.S. Patent No. 8,779,108) is a human IgGlK monoclonal antibody against PD-L1. MDPL3280A (Genentech/Roche) is a human Fc optimized IgGi monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Publication No. 2012/0039906. Other antibodies that specifically bind PD-L1 include YW243.55.S70 (heavy and light chain variable regions are shown in PCT Publication No. W02010/077634) and MDX- 1105 (also referred to as BMS-936559; disclosed in PCT Publication No. W02007/005874). AMP-224 (B7-DCIg; Amplimmune; disclosed in PCT Publication No. W02010/027827 and PCT Publication No. WO2011/066342) is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7- Hl. Other antibodies that specifically bind PD-1 include AMP-514 (Amplimmune), among others, e.g., antibodies that specifically bind PD-1 disclosed in U.S. Patent No. 8,609,089, U.S. Publication No. 2010/028330, and/or U.S. Publication No. 2012/0114649. Any of these PD-1 antagonists are of use in the methods disclosed herein. In some embodiments, the antibody that specifically binds PD-1 and the antibody that specifically binds PD-L1 are present in the same antibody molecule, e.g., as a bispecific or multispecific antibody molecule.

In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. The CTLA-4 inhibitor can be an antibody (such as a monoclonal antibody) or antigen binding fragment thereof that specifically binds CTLA-4. In some embodiments, the antibody that specifically binds CTLA-4 is ipilimumab (Bristol-Myers Squibb; also referred to as MDX-010 and MDX-101; YERVOY®; disclosed in U.S. Patents 7,605,238; 6,984,720; 5,811,097; 5,855,887; and 6051227), which fully human monoclonal IgGlK antibody against CTLA-4. In some embodiments, the antibody that specifically binds CTLA-4 is tremelimumab, which is a fully human monoclonal IgG2 antibody against CTLA-4 (Abgenix/Pfizer; formerly known as ticilimumab CP-675,206; disclosed in U.S. Patent Nos. 6,682,736; 5,811,097; 5,855,887; and 6051227). Other CTLA-4 inhibitors of use in the present disclosure are described in U.S. Application No. 17/482,138 and PCT Publication No. W02020092155. Any of these CTLA-4 inhibitors are of use in the methods disclosed herein.

In some embodiments, the checkpoint inhibitor is a TNFR-2 inhibitor. The TNFR-2 inhibitor can be an antibody (such as a monoclonal antibody) or antigen binding fragment thereof that specifically binds TNFR-2. Antibodies or antigen binding fragments that target TNFR-2 can be used in the treatment of various cancers (Yang et al. ImmunoTargets . 10:103-122, 2021; Fischer et al. Front Cell Dev Biol. 8:401, 2020). Several such antibodies globally affect activation of TNFR-2 by TNF. In some embodiments, the TNFR-2 inhibitor is infliximab (Janssen; REMICADE®; disclosed in U.S. Patent No. 2,261,630), a chimeric monoclonal antibody against TNF-a; adalimumab (Abbvie; HUMIRA®; disclosed in U.S. Patent No. 6,090,382; biosimilars include, for example, AMGEVITA®, IMRALDI®, HYRIMOZ®/HEFIYA®/HALIMATOZ®, HULIO®, IDACIO®, AMSPARITY®), a fully human antibody against TNF-a; golimumab (Janssen; SIMPONI®; disclosed in U.S. Patent 7,250,165), a fully humanized monoclonal IgGlK antibody against TNF-a; certolizumab-pegol (UCB; CIMZIA®; disclosed in U.S. Patent No. 7,977.464), a monoclonal antibody against TNF-a; etanercept (Amgen/Roche; ENBREL®; disclosed in U.S. Patent No. 8,063,182), a soluble TNFR-2 -Fc fusion protein that can inhibit; or biosimilars thereof. In some embodiments, the antibody that specifically binds TNFR-2 is E4 or E4F4, an antigen binding fragment thereof, or a conjugate thereof (see PCT Publication No. WO2018/213064, incorporated herein by reference). In other embodiments, the antibody that specifically binds TNFR-2 is TY 101 (see Jiang et al. Int Immunopharmacol. 101: 108345, 2021), a humanized antibody thereof, an antigen binding fragment thereof, or a conjugate thereof. Defucosylated forms of these molecules are also of use in the disclosed methods.

Additional TNFR-2 inhibitors of use in the present disclosure are described in PCT Publication Nos. W02020/089473, W02021/023098, and W02020/061210. Any of these TNFR-2 inhibitors are of use in the methods disclosed herein.

In some embodiments, a pharmaceutical composition of use in the disclosed methods includes about 0.5% w/v to about 25% w/v checkpoint inhibitor, or about 1% w/v to about 12% w/v checkpoint inhibitor, or about 12% w/v to about 25% w/v checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2). In some embodiments, a pharmaceutical composition of use includes about 0.5%, 1%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% w/v, or more checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2). Exemplary pharmaceutical compositions of use include about 10% w/v of the checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2).

Antibodies (such as monoclonal antibodies) and antigen binding fragments thereof that are administered intravenously can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution may then be added to an infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of Rituxan® in 1997. Antibody drugs can be administered by slow infusion or an IV push or bolus. In one example of administration by slow infusion, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.

In some embodiments, the subject is administered (for example, intravenously or intratumorally) about 0.3 mg/kg to about 15 mg/kg or more of a checkpoint inhibitor, such as about 0.3 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2mg/kg to about 3mg/kg, about 3mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, or about 14 mg/kg to about 15 mg/kg of a checkpoint inhibitor, such as a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor, such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2. In one specific non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg mg of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor. In another specific, non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a TNFR-2 inhibitor in a single dose or in more than dose.

4. STING Agonists

In embodiments of the disclosed methods, a subject (such as a subject that has a cancer) is administered a therapeutically effective amount of a STING agonist, such as in a combination therapy. The cGAS (cyclic GMP-AMP synthase)-STING (STING) pathway is a cytosolic DNA-sensing pathway that drives activation of type I IFN and other inflammatory cytokines in the host immune response, such as against cancers. Stimulator of interferon response cGAMP interactor 1 (STING 1, also known as STING), is an endoplasmic reticulum-sessile protein that serves as a signaling hub and adaptor protein in the STING pathway, receiving input from several PRRs, most of which sense ectopic DNA species in the cytosol. STING pathway signaling ensures the production of type I interferon (IFN) in response to invading DNA viruses, bacterial pathogens, as well as DNA leaking from mitochondria or the nucleus (e.g., in cells exposed to chemotherapy or radiotherapy). A type I IFN response is involved in the initiation of anticancer immune responses. (Amouzegar et al., Cancers. 13(11):2695, 2021; Le Naour et al., Oncolmmunol. 9(1):1777624, 2020)

STING can be activated by several cytoplasmic DNA sensors, including cyclic GMP-AMP synthase (CGAS), Z-DNA binding protein 1 (ZBP1, also known as DAI), DEAD-box helicase 41 (DDX41), intcrfcron-gamma inducible protein 16 (IFI16), LRR binding FLII interacting protein 1 (LRRFIP1), MRE11 homolog, double-strand break repair nuclease (MRE11), and possibly also protein kinase, DNA-activated, catalytic subunit (PRKDC, also known as DNA-PK). The accumulation of ectopic dsDNA in the cytosol activates the enzymatic function of CGAS to generate cyclic GMP-AMP (cGAMP), as well as other cyclic dinucleotides, which bind to and activate STING, triggering a signal transduction pathway that culminates in the initiation of interferon regulatory factor 3 (IRF3)- or NF-KB-dependent transcriptional programs.

STING agonists suitable for use in the present disclosure include, without limitation, cGAMP, c-di- GMP, flavone acetic acid (FAA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA, also known as ASA404 or vadimezan), ADU-S100, BMS-986301, E7766, GSK3745417, MK-1454, MK-2118, SB11285, BISTING (BI 1387446), GSK532, JNJ-4412, 3’3’-cyclic AIMP, ALG-031048, JNJ-6196, MSA-1, MSA-2, SNX281, SR-717, TAK676, TTI-10001, PC7A NP, cGAMP-NP, ONM500, XMT-2056, CRD-5500, exoSTING, SYNB1891, and STACT-TREX-1. Other exemplary STING agonists include Rp,Rp dithio 2', 3' c-di-AMP (e.g., Rp,Rp-dithio c-[A(2',5')pA(3',5')p]), or a cyclic dinucleotide analog thereof; a compound depicted in U.S. Patent Publication No. US2015/0056224 (e.g., a compound in FIG. 2c, e.g., compound 21 or compound 22); c-[G(2',5')pG(3',5')p], a dithio ribose O-substituted derivative thereof, or a compound depicted in FIG. 4 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806; c-[A(2’,5’)pA(3’,5')p] or a dithio ribose O-substituted derivative thereof, or is a compound depicted in FIG. 5 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806; c-[G(2',5')pA(3',5')p], or a dithio ribose O-substituted derivative thereof, or is a compound depicted in FIG. 5 of PCT Publication Nos. WO 2014/189805 and WO 2014/189806; 2'-O-propargyl-cyclic-[A(2',5')pA(3',5')p] (2'-O-propargyl-ML-CDA) or a compound depicted in FIG. 7 of PCT Publication No. WO 2014/189806. Other exemplary STING agonists are disclosed, e.g., in PCT Publication Nos. WO 2014/189805 and WO 2014/189806, and U.S. Publication No. 2015/0056225.

In some embodiments, a pharmaceutical composition of use in the disclosed methods includes about 0.5% w/v to about 25% w/v STING agonist, or about 1% to about 12% STING agonist, or about 12% w/v to about 25% w/v or more STING agonist (such as cGAMP or c-di-GMP). In some embodiments, the combination may include about 0.5%, 1%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% w/v, or more STING agonist (such as cGAMP or c-di-GMP). Exemplary combinations of therapeutic agents may include about 10% w/v STING agonist (such as cGAMP or c-di- GMP).

In some embodiments, the subject is administered (for example, intravenously or intratumorally) about 0.3 mg/kg to about 15 mg/kg or more of a STING agonist (such as cGAMP or c-di-GMP), such as about 0.3 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2mg/kg to about 3mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, or about 14 mg/kg to about 15 mg/kg of STING agonist (such as cGAMP or c-di-GMP). In one specific non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg mg of the STING agonist (such as cGAMP or c-di-GMP). In another specific, non-limiting example, the subject is administered (for example, intravenously or intratumorally) about 5 mg/kg of the STING agonist (such as cGAMP or c-di-GMP) in a single dose or in more than dose.

III. Kits

This disclosure also provides kits containing therapeutic agents (such as in a combination therapy) for use in the methods disclosed herein. In some embodiments, a kit may include a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), and a checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), such as for use in methods of treating a subject that has cancer, such as colorectal or kidney cancer. In another embodiment, a kit may include a TLR4 agonist (such as HMGN1), a TLR2/6 agonist (such as FSL-1), a checkpoint inhibitor (such as an antibody that specifically binds PD-1, an antibody that specifically binds PD-L1, an antibody that specifically binds CTLA-4, or an antibody that specifically binds TNFR-2), and a STING agonist (such as cGAMP or c-di- GMP), such as for use in methods of treating a subject that has cancer, such as melanoma.

The kit can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more therapeutic agent. In several embodiments the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label or package insert indicates that the composition is used for treating the particular condition, such as a specific type of cancer, for example, colon cancer, kidney cancer, or melanoma.

The label or package insert typically will further include instructions for use, for example, in a method of treating or preventing a cancer. The package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. The kits may additionally include buffers and other reagents routinely used for diluting or administering therapeutic agents.

EXAMPLES

The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified.

EXAMPLE 1

Materials and Methods

Mice and Reagents: Female wild-type C57BL/6, and Balb/c mice were obtained either by the animal production facility at NCI or Jackson laboratory.

Isolation and Purification of Cells: Human peripheral blood cell samples were obtained from healthy donors by leukopheresis. Mouse bone marrow derived hematopoietic progenitor cells were prepared from C57BL/6 wild type male mice by flushing from femur and tibia.

Measurement of Cytokine Levels in Culture Supernatants of Human MoDCs and Mouse Bone Marrow-derived Dendritic Cells (BMDCs): Multiple cytokines in the culture supernatants of human and mouse DCs were measured using V-PLEX (IL-ip, TNFa, or IL-12p70) and U-PLEX (IFNy, IL-5, IL-13, or IL17A) ultrasensitive plate assays.

Mixed Lymphocyte Reaction (MLR): Allogenic MLR was performed using treated human MoDCs co-cultured with purified allogenic CD4⁺ T cells at different ratios for 4 days at 37°C. Intratumoral treatment for CT26 colon tumors: Female mice (Balb/c, n=5, 8-12 weeks old) were subcutaneously injected with 0.1 ml PBS containing 2 x 10⁶/ml CT26 colon cancer cells into their right flank region. The appearance and size of tumors as well as mouse body weight were monitored twice weekly. The length (L) and width (W) of tumors were measured with a caliper. Tumor size was calculated using the formula: (LxW²)/2. When tumors reached approximately 7.5-8 mm in diameter, the tumor-bearing mice were treated with intratumoral (i.t.) injection of a CTLA-4 inhibitor (10 pg/0.05 ml), a PD-L1 inhibitor (10 pg/0.05 ml/mouse), HMGN1 (10 pg/0.05 ml), or FSL-1 (5 pg/0.05 ml), alone or in combination as specified, using PBS as a negative control. Cancer-free mice were re-challenged with CT26 colon cancer cells on the right flank and unrelated cancer cells on the contralateral flank, and tumor development and growth were monitored.

Intratumoral treatment for RENCA kidney tumors: Female mice (Balb/c, n=5, 8-12 weeks old) were subcutaneously injected with 0.1 ml PBS containing 0.5 x 10⁷/ml RENCA kidney cancer cells into their right flank region. The appearance and size of tumors as well as mouse body weight were monitored twice weekly. The length (L) and width (W) of tumors were measured with a caliper. Tumor size was calculated using the formula: (LxW²)/2. When tumors reached approximately -7.2 to 8 mm in diameter, the tumorbearing mice were treated with intratumoral (i.t.') injection of a CTLA-4 inhibitor (10 pg/0.05 ml), HMGN1 (10 pg/0.05 ml), or FSL-1 (5 pg/0.05 ml/mouse), alone or in combination as specified, using PBS as a negative control. Tumor formation and growth were monitored.

Intratumoral treatment for melanin producing M3 melanomas: Female mice (C57BL/6, n=5, 8-12 weeks old) were subcutaneously injected with 0.1 ml PBS containing 2 x 10⁶/ml M3 melanoma cells into their right flank region. The appearance and size of tumors as well as mouse body weight were monitored twice weekly. The length (L) and width (W) of tumors were measured with a caliper. Tumor size was calculated using the formula: (LxW²)/2. When tumors reached approximately -6.8 to 7 mm in diameter, the tumor-bearing mice were treated with intratumoral (i.t.) injection of a PD-L1 inhibitor (10 pg/0.05 ml), HMGN1 (10 pg/0.05 ml), FSL-1 (5 pg/0.05 ml), or cGAMP (5 pg/0.05 ml) alone or in combination as specified, using PBS as a negative control. Cancer-free mice were re-challenged with M3 melanoma cells on the right flank and unrelated cancer cells on the contralateral flank, and tumor development and growth were monitored.

EXAMPLE 2

HMGN1 and FSL-1 induce phenotypic maturation and cytokine production in human MoDCs Human MoDCs at 5 x 10⁵/ml were stimulated with HMGN1 or FSL-1 to measure the effect on expression of surface costimulatory molecules such as CD80, CD83, and CD86 (FIG. 1A), and production of proinflammatory cytokines such as TNFa and IL-12p70 (FIG. IB) at 24 h. HMGN1 and FSL-1 dose- dependently increased expression of surface costimulatory molecules and production of proinflammatory cytokines. Stimulating MoDCs with HMGN1 and FSL-1 had a synergistic effect on expression of surface costimulatory molecules (FIG. 2A) and production of inflammatory cytokines (FIG. 2B). The synergistic effect between HMGN1 and FSL-1 on MoDC production of TNFa and IL-12p70 can be observed at various combinations of concentrations (FIG. 12A).

EXAMPLE 3

HMGN1 and FSL-1 induce phenotypic maturation and TNFa production in mouse BMDCs

Mouse BMDCs at 5 x 10⁵/ml were stimulated with HMGN1 or FSL-1 to measure the effect on expression of surface costimulatory molecules such as CD80, CD83, CD86 and I-A\E (FIG. 3A), and production of proinflammatory cytokines such as TNFa (FIG. 3B) at 24 h. HMGN1 and FSL-1 dose- dependently increased expression of surface costimulatory molecules and production of proinflammatory cytokines. Stimulating BMDCs with HMGN1 and FSL-1 had a synergistic effect on expression of surface costimulatory molecules (FIG. 4A) and production of inflammatory cytokines (IL-1 and TNFa) (FIG. 4B). The synergistic effect of HMGN1 and FSL-1 on stimulating BMDC production of TNFa and IL- I p can be observed at various concentration combinations (FIG. 12B).

EXAMPLE 4

HMGN1 together with FSL-1 and a PD-L1 inhibitor cures mice with CT26 colon cancer cell tumors

CT26 colon cancer cells (2xl0⁵/ mouse) were inoculated subcutaneously into BALB/c mice and in 9 days grew into 7.5-8 mm diameter tumors. On day 9, CT26 tumors were treated intratumorally with (1) PBS, (2) FSL-1, (3) HMGN1 and FSL-1, (4) FSL-1 and a PD-Ll inhibitor, or (5) HMGN1, FSL-1, and a PD-L1 inhibitor twice a week for two weeks. Doses of 10 pg HMGN1 and the PD-L1 inhibitor per mouse and 5 pg of FSL-1 per mouse were injected into the tumor. Mice were monitored for tumor growth (FIGS. 5A and 5C) and survival (FIG. 5B). One out of five (1/5, 20%) CT26 colon tumor- bearing mice treated with either HMGN1 and FSL-1, or FSL-1 and the PD-L1 inhibitor, and 5/5 (100%) CT26 colon tumorbearing mice treated with HMGN1, FSL-1, and the PD-L1 inhibitor became long term survivors and were cancer free. CT26 colon cancer- free mice were then inoculated subcutaneously with CT26 cells (2xl0⁵1 mouse) into the right flank, and 4T-1 breast cancer cells (2xl0⁵ / mouse) into the left flank. The formation and growth of CT26 colon tumors and 4T-1 breast tumors were monitored (FIG. 5D). While 4T-1 tumors formed and increased in volume in all mice, CT26 tumors did not form.

Treatment of CT26 tumors with HMGN1, FSL-1 and anti-PD-Ll not only cured the tumors, but also promoted the generation of antitumor immune response, which was evidenced by the increase of effector/memory CD4 and CD8 T cells and expression of genes (CCL10, IFNg, and TNFa) characteristic of Thl response in the tumor tissue (FIG. 10) and elevation of effector/memory T cells and CT26-specific effector CTLs in the tumor draining lymph nodes (FIG. 11). EXAMPLE 5

HMGN1 together with FSL-1 and a CTLA-4 inhibitor cures mice with CT26 colon cancer cell tumors

CT26 colon cancer cells (2xl0⁵/ mouse) were inoculated subcutaneously into BALB/c mice and in 9 days grew into 7.1-7.2 mm diameter tumors. On day 9, CT26 tumors were treated intratumorally with (1) PBS, or (2) HMGN1, FSL-1, and the CTLA-4 inhibitor twice a week for two weeks. Doses of 10 pg HMGN1 and the CTLA-4 inhibitor per mouse and 5 pg of FSL-1 per mouse were injected into the tumor. Mice were monitored for tumor growth (FIGS 6A and 6C), and survival (FIG. 6B). Five out of five (5/5, 100%) CT26 colon tumor-bearing mice treated with HMGN1, FSL-1, and the CTLA-4 inhibitor became long term survivors and were cancer free.

EXAMPLE 6 HMGN1 together with FSL-1 and a CTLA-4 inhibitor cures mice with RENCA kidney cancer cell tumors

RENCA kidney cancer cells (5xl0⁵/ mouse) were inoculated subcutaneously into BALB/c mice and in 10 days grew into 7.2-8 mm diameter tumors. On day 10, RENCA tumors were treated intratumorally with (1) PBS, or (2) HMGN1, FSL-1, and the CTLA-4 inhibitor twice a week for two weeks. Doses of 10 pg HMGN 1 and the CTLA-4 inhibitor per mouse and 5 pg of FSL- 1 per mouse were injected into the tumor. Mice were monitored for tumor growth (FIGS 7A and 7C), and survival (FIG. 7B). Five out of five (5/5, 100%) RENCA kidney tumor-bearing mice treated with HMGN1, FSL-1, and the CTLA-4 inhibitor became long term survivors and were cancer free.

EXAMPLE 7

HMGN1, FSL-1, and cGAMP synergistically induce functional maturation of human MoDCs

Human MoDCs at 5 x 10⁵/ml were stimulated with (1) HMGN1, (2) FSL-1, (3) cGAMP, or (4) HMGN1, FSL-1, and cGAMP together, to measure the effect on expression of surface costimulatory molecules such as CD80, CD83, and CD86, and production of proinflammatory cytokines such as TNFa and IL-12p70 al 24 h. Stimulating MoDCs with HMGN1, FSL-1, and cGAMP together had a synergistic effect on expression of surface costimulatory molecules (FIG. 8A) and production of inflammatory cytokines (FIG. 8B). Human MoDCs treated with 31 ng/ml HMGN1, 2 ng/ml FSL-1, and 1 pg/ml cGAMP (each alone or together) were then co-cultured with allogenic CD4⁺ T cells at various ratios for 4 days and pulsed with [³H]-TdR for the last 18 h. CD4⁺ T cell proliferation was measured by [³H]-TdR incorporation (FIG. 8C). Supernatants from human CD4⁺ T cells co-cultured (CD4⁺ T:MoDCs = 50:1) for 3 days with MoDCs treated with (1) HMGN1, (2) FSL-1, (3) cGAMP, or (4) HMGN1, FSL-1, and cGAMP together were quantitated for cytokine production (FIG. 8D). Treatment with HMGN1, FSL-1, and cGAMP together increased production of each measured cytokine more than treatment with HMGN1, FSL-1, or cGAMP alone. EXAMPLE 8 HMGN1 together with FSL-1, a PD-L1 inhibitor, and cGAMP cures mice with melanin producing M3 melanoma

M3 melanoma cells (2xl0⁵/ mouse) were inoculated subcutaneously into C57BL/6 mice and in 13 days grew into ~6.8 to 7 mm diameter melanomas. On day 13, M3 melanoma cancers were treated intratumorally with (1) PBS, (2) cGAMP, (3) HMGN1, FSL-1, and a PD-L1 inhibitor, or (4) HMGN1, FSL- 1, the PD-L1 inhibitor, and cGAMP twice a week for two weeks. Doses of 10 pg HMGN1 and the PD-L1 inhibitor per mouse and 5 pg of cGAMP and FSL-1 per mouse were injected into the tumor. Mice were monitored for tumor growth (FIGS. 9A and 9C) and survival (FIG. 9B). Five out of five (5/5, 100%) M3 melanoma-bearing mice treated with HMGN1, FSL-1, the PD-L1 inhibitor, and cGAMP together became long term survivors and were cancer free. M3 melanoma-free mice were then inoculated subcutaneously with M3 (2xl0⁵ / mouse) cells into the right flank, and Lewis Lung Carcinoma (LLC, 2xl0⁵ / mouse) cells into the left flank. The formation and growth of M3 melanoma and LLC carcinoma were monitored (FIG. 9D). While LLC tumors formed and increased in volume in all mice, M3 tumors did not form.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of treating a cancer in a subject, comprising: administering to the subject a therapeutically effective amount of a combination therapy comprising a fibroblast stimulating lipopeptide (FSL)-l, a high mobility group nucleosome binding protein (HMGN)l, and a checkpoint inhibitor, thereby treating the cancer in the subject.

2. The method of claim 1, wherein the checkpoint inhibitor is a programmed death ligand (PD- L)1 inhibitor, a programmed death (PD)-l inhibitor, a tumor necrosis factor receptor (TNFR)-2 inhibitor, or a cytotoxic T-lymphocyte associated antigen (CTLA)-4 inhibitor.

3. The method of claim 2, wherein a) the PD-L1 inhibitor is an antibody that specifically binds PD-L1; b) the PD-1 inhibitor is an antibody that specifically binds PD-1; c) the CTLA-4 inhibitor is an antibody that specifically binds CTLA-4; or d) the TNFR-2 inhibitor is an antibody that specifically binds TNFR-2.

4. The method of any one of claims 1-3, wherein the cancer is colorectal cancer.

5. The method of any one of claims 1-3, wherein the cancer is kidney cancer.

6. The method of claim 4, wherein the checkpoint inhibitor is (a) an antibody that specifically binds PD-L1 or (b) an antibody that specifically binds CTLA-4.

7. The method of claim 5, wherein the checkpoint inhibitor is an antibody that specifically binds CTLA-4.

8. The method of any one of claims 1-7, wherein the combination therapy further comprises a cGAS/stimulator of interferon genes (STING) agonist.

9. The method of claim 8, wherein the STING agonist is 2'3 '-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) or bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP).

10. The method of claim 8 or claim 9, wherein the cancer is melanoma.

11. The method of claim 10, wherein the STING agonist is cGAMP, and the checkpoint inhibitor is an antibody that specifically binds PD-L1.

12. The method of any one of claims 1-11, wherein the subject is a human.

13. The method of any one of claims 1-12, wherein the FSL-1, the HMGN1, the checkpoint inhibitor, and/or the STING agonist are administered to the subject intratumorally and/or intravenously.

14. The method of any one of claims 1-13, wherein the method reduces cancer burden in the subject.

15. The method of any one of claims 1-14, wherein the method increases survival of the subject.

16. The method of any one of claims 1-15, wherein the method reduces the incidence of relapse of the cancer in the subject.

17. The method of any one of claims 1-16, wherein the method induces maturation of human monocyte dendritic cells in the subject.

18. The method of any one of claims 1-17, further comprising selecting the subject with the cancer prior to administering the combination therapy to the subject.

19. The method of claim 18, wherein the cancer is colorectal cancer or kidney cancer.

20. The method of claim 10, further comprising selecting the subject with the melanoma prior to administering the combination therapy to the subject.

21. A kit comprising a) FSL-1, HMGN1, and a checkpoint inhibitor, for use in the method of any one of claims 1-7 and 14-19; or b) FSL-1, HMGN1, a STING agonist, and a checkpoint inhibitor for use in the method of any one of claims 8-20.

22. The kit of claim 21, wherein the checkpoint inhibitor is a programmed death ligand (PD-L)l inhibitor, a programmed death (PD)-l inhibitor, a tumor necrosis factor receptor (TNFR)-2 inhibitor, or a cytotoxic T-lymphocyte associated antigen (CTLA)-4 inhibitor.

23. The kit of claim 22 wherein a) the PD-L1 inhibitor is an antibody that specifically binds PD-L1; b) the PD-1 inhibitor is an antibody that specifically binds PD-1; c) the CTLA-4 inhibitor is an antibody that specifically binds CTLA-4; or d) the TNFR-2 inhibitor is an antibody that specifically binds TNFR-2.