Methods of activating CXCR3 / CXCL10 pathways for treating cancers

Abstract

Described herein are methods of enhancing tumor clearance and inhibiting tumor recurrence comprising local administration of a recombinant CXCL10 protein to the tumor or an area surrounding the tumor. Also provided herein are methods of locally administering (e.g., intratumoral administration) a recombinant CXCL10 protein to treat a cancer, e.g., a cancerous tumor. Methods disclosed herein can be used in combination with one or more cancer therapies (e.g., chemotherapy, radiation therapy, immunotherapy (e.g., an immune checkpoint blockade agent, e.g., a PD-1 / PD-L1 inhibitor)) whose efficacy is limited by immunosuppression to enhance the efficacy of the immunotherapy.

Description

[0001] Attorney Docket No. 15670-0449WO1

[0002] METHODS OF ACTIVATING CXCR3 / CXCL10 PATHWAYS FOR TREATING CANCERS CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No.

[0004] 63 / 754,265, filed on February 5, 2025. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named 15670-0449_SL_ST26. The XML file, created on February 4, 2026, is 19,329 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0008] This invention was made with government support under DE033909. GM007752, GM136202, and CA023100 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0009] TECHNICAL FIELD

[0010] Described herein are methods of enhancing tumor clearance and inhibiting tumor recurrence comprising local administration of a recombinant CXCL10 protein to the tumor or an area surrounding the tumor. Also provided herein are methods of locally administering (e.g., intratumoral administration) a recombinant CXCL10 protein to treat a cancer, e.g., a cancerous tumor. Methods disclosed herein can be used in combination with one or more cancer therapies (e.g., chemotherapy, radiation therapy, immunotherapy (e.g., an immune checkpoint blockade agent, e.g., a PD-1 / PD-L1 inhibitor)) whose efficacy is limited by immunosuppression to enhance the efficacy of the immunotherapy.

[0011] BACKGROUND

[0012] Immunotherapy, including immune checkpoint blockade (ICB) agents, revolutionized cancer treatment by leveraging the host immune system to eliminateAttorney Docket No. 15670-0449WO1

[0013] tumors. However, despite their promise, ICB agents show favorable clinical outcomes for only -10-25% of patients. The failure of ICBs, as well as standard cancer treatments, is partly due to the ability of resistant tumors to limit the infiltration and activation of cytotoxic CD8+T and NK cells, which are essential for an effective response to immunotherapy .

[0014] SUMMARY

[0015] Immune-suppressive tumor microenvironments (TMEs) limit the impact of ICB agents in many cancers by restricting the infiltration and activation of CD8+T cells, CD4+T cells, and / or NK cells. The recruitment of T and NK cells into tumors is regulated by the chemokine receptor, CXCR3, in response to CXCL9 and 10, both of which are also important for the antitumoral functions of these cells. In order to exclude effector cells from the tumor microenvironment (TME), cancer cells utilize multiple strategies to reduce the expression of these ligands. Provided herein are methods of treatment that overcome reduced ligand expression in such tumors by, in part, locally administering CXCL10 to the TME.

[0016] Provided herein are methods of enhancing tumor clearance in a subject in need thereof comprising locally administering an effective amount of a recombinant CXCL10 protein to a tumor or an area surrounding the tumor.

[0017] Also provided herein are methods of treating a cancer comprising locally administering to a subject in need thereof an effective amount of a recombinant CXCL10 protein to a cancerous tumor or an area surrounding the cancerous tumor.

[0018] In some embodiments, the subject in need thereof has or is suspected of having at least one solid cancerous tumor. In some embodiments, the at least one solid cancerous tumor comprises a primary tumor, a metastatic tumor, or both. In some embodiments, the subject in need thereof has or is suspected of having at least one cancer selected from the group consisting of head and neck cancer, head and neck squamous cell carcinoma, colon cancer, liver cancer, gastric cancer, lung cancer, or melanoma.

[0019] In some embodiments, the local administration comprises administering the recombinant CXCL10 protein to a surgical margin following resection of the tumor. In some embodiments, the local administration comprises intratumoral injection of the recombinant CXCL10 protein.Attorney Docket No. 15670-0449WO1

[0020] In some embodiments, the recombinant CXCL10 protein is locally administered to the subject at least once. In some embodiments, the recombinant CXCL10 protein is locally administered to the subject once. In some embodiments, the recombinant CXCL10 protein is locally administered to the subject two or three times.

[0021] In some embodiments, the recombinant CXCL10 protein comprises about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3. In some embodiments, the amino acid sequence of the recombinant CXCL10 protein is SEQ ID NO: 3.

[0022] In some embodiments, the methods disclosed herein further comprise administering at least one cancer therapy to the subject in need thereof. In some embodiments, the cancer therapy is selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy. In some embodiments, the chemotherapy comprises administration of at least one chemotherapeutic (i.e., chemotherapy agent). In some embodiments, the at least one chemotherapeutic is selected from the group consisting of cisplatin, carboplatin, nedaplatin, satraplatin, picoplatin, phenanthriplatin, triplatin tetranitrate, gemcitabine, methotrexate, vinblastine, and adriamycin. In some embodiments, the immunotherapy comprises administration of at least one immune checkpoint blockade (ICB) agent. In some embodiments, the at least one ICB agent is an inhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA4), programmed cell death protein 1 (PD-1), PD-L1, LAG3, B7-H3, B7-H4, or TIM3. In some embodiments, the at least one ICB agent is a PD-1 / PD-L1 inhibitor. In some embodiments, the PD-1 / PD-L1 inhibitor is a small molecule. In some embodiments, the PD-1 / PD-L1 inhibitor is an antibody. In some embodiments, the at least one ICB agent is a PD-1 / PD-L1 inhibitor selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, pidilizumab, AMP-224, durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MED1-4736, MSB-0010718C. and MDX- 1105.

[0023] In some embodiments, the subject in need thereof has or is suspected of having a cancer that is resistant to PD-1 / PD-L1 inhibitors.

[0024] Provided herein are also pharmaceutical compositions comprising a recombinant CXCL10 protein and at least one pharmaceutically acceptable carrier. In some embodiments, the recombinant CXCL10 protein comprises about 95%. 96%.Attorney Docket No. 15670-0449WO1

[0025] 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3. In some embodiments, the amino acid sequence of the recombinant CXCL10 protein is SEQ ID NO: 3. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0026] Also provided herein are combination therapies for treating cancer, a combination therapy comprising intratumorally administering to a subject in need thereof a therapeutically effective amount of a recombinant CXCL10 protein and / or a pharmaceutical composition disclosed herein and a therapeutically effective amount of an immune checkpoint blockade (ICB) agent. In some embodiments, the ICB agent is systemically administered to the subject in need thereof. In some embodiments, the ICB agent is a PD-1 / PD-L1 inhibitor. In some embodiments, the ICB agent is an anti-PD-1 antibody or an anti-PD-Ll antibody.

[0027] Provided herein are also methods of making a recombinant CXCL10 protein. In some embodiments, a method of making a recombinant CXCL10 protein comprises: (i) preparing a vector encoding a fusion protein, wherein the fusion protein comprises, fromN terminus to C terminus, 6-His tag - ubiquitin - CXCL10; (ii) expressing the fusion protein in a host cell; (iii) harvesting the fusion protein from the host cell; (iv) purifying the harvested fusion protein by Ni-affmity chromatography; and (v) cleaving the 6-His tag from the CXCL10 with Usp2-cc to result in the recombinant CXCL10 protein. In some embodiments, the method of making a recombinant CXCL10 protein further comprises purifying the recombinant CXCL10 protein by reverse phase chromatography. In some embodiments, the method of making a recombinant CXCL10 protein can further comprise purifying the recombinant CXCL10 protein by reverse phase chromatography wherein resulting recombinant CXCL10 protein is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The term "about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative onlyAttorney Docket No. 15670-0449WO1

[0029] and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0030] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0031] DESCRIPTION OF DRAWINGS FIGS. 1A-1H: High Cxcr3 and CxcllO expression correlated with increased immune infiltration into tumors and improved outcomes of patients with HNSCC. (FIGS. 1A-1G) Transcriptomic data obtained and analyzed via cBioPortal (PanCancer Atlas, HNSCC cohort). Expression levels were based on whole tumor mRNA quantification. CXCR3hlgh / CXCL 10lllghcohorts (n = 128 and 132) and CXCL10lo'7CXCL10lowcohort (n = 131 and 126) represented the upper 25% and lower 25% threshold for Cxcr3 or CxcllO mRNA expression, respectively. (FIG. 1A) Overall survival of CXCR3hlghvs CXCR3lowcohorts to month 150, with hazard ratio of 0.5383 and p-value of 0.0016 (via Mantel-Cox and Mantel -Haenszel tests). (FIG. IB) Overall survival of CXCL10hlghvs CXCL10lowcohorts to month 150, with hazard ratio of 0.7330 and p-value of 0.1115 (via Mantel-Cox and Mantel-Haenszel tests). Correlation of Cxcr3 (FIG. 1C) and CxcllO (FIG. ID) with Cd8a mRNA expression (Spearman’s rank correlation). (FIG. IE) Relative Ifng and Gzmb mRNA expression of CXCL10highvs CXCL10lowcohorts. (FIG. IF) Differential expression of genes between CXCL10hlghvs CXCL10lowcohorts using Wilcoxon Rank Sum test. Log2 fold-change and P value (adjusted) cut-offs are 1 and 0.05, respectively. (FIG. 1G) AJCC tumor stage distribution of patients in CXCR3hlghand CXCR310" cohorts. (FIG. 1H) Immune enrichment scores for indicated immune cell subtype in CXCR3hlghand CXCR3lowcohorts.

[0032] FIGS.2A-2J: IT delivery of CXCL 10 suppressed tumor growth and reoccurrence in syngeneic models of HNSCC. (FIG.2A) Schematic detailing experimental setup and dosing schedule for 4MOSC1 and M0C1 models. 1.0 x 1064MOSC1 or 2.0 x 106MOC1 cells were implanted into the buccal space or flank, respectively, of C57BL / 6 mice on day 0. Mice received either 10 pg CXCL10 in 50 pL of PBS (per dose) or 50 pL of PBS (vehicle) on the days indicated (4). (FIG.2B)Attorney Docket No. 15670-0449WO1

[0033] Representative image of 4MOSC1 buccal tumors in vehicle vs CXCLIO-treated mice at day 30. Tumor boundaries were traced with a dotted line, while an asterisk (*) indicated complete tumor clearance. 4MOSC1 (FIG. 2C) and MOC1 (FIG. 2E) tumor kinetics of vehicle vs CXCLIO-treated mice (n = 9-10 per group). Survival proportions are shown on the right. 4MOSC1 (FIG. 2D) and MOC1 (FIG. 2F) tumor kinetics of tumor naive age-matched mice or previous complete responders to CXCL10 treatment after rechallenge with 1.0 x 1064MOSC1 or 2.0 x 106MOC1 cells 6 weeks post-tumor clearance. (FIGS. 2G-2H) Representative H&E (FIG. 2G) and IF (FIG. 2H) staining of 4MOSC1 tumors from vehicle vs CXCL10- treated mice at day 10. Scale bar, 800 pm. (FIGS.21-2 J) Tumor kinetics of WT or Cxcr3'f' mice receiving either vehicle or CXCL10 treatment. Differences between the tumor kinetics amongst experimental groups were analyzed by linear regression. Survival analysis was performed using the Kaplan-Meier method and log-rank tests. All data represent averages ± SEM, except where indicated.

[0034] FIGS. 3A-3I: CXCL10 remodeled the tumor immune microenvironment by enhancing infiltration of CD8+ T cells, CD4+ T cells, and NK cells. (FIG. 3A) 1.0 x 1064MOSC1 cells were implanted into the buccal space of C57BL / 6 mice on day 0. Tumors were treated on days 3, 6, and 9 with either vehicle or 10 pg of CXCL10 IT (per dose). mRNA from each tumor was isolated and comprehensive immune profiling was conducted using the NanoString nCounter® PanCancer Mouse Immune Profiling gene expression platform. Rosalind® software was used to analyze genes associated with the listed immune cells and each replicate was assigned a cell abundance score (n = 4 mice per group). (FIG.3B) Growth kinetics of 4MOSC1 tumors treated with either vehicle, CXCL10 alone, CXCL10 + anti-CD8, CXCL10 + anti-CD4, or CXCL10 + anti-NKl.l antibody. Volumes of individual tumors in mm3at day 20 are shown on the right. (FIG. 3C) Representative flow cytometry plots showing relative CD4+and CD8+cells amongst total CD3+cells. (FIG. 3D) Absolute number of live CD45+CD3+CD8+T cells, CD45+CD3+CD4+T cells, and CD45+NK1.1+ cells infiltrating vehicle or CXCLIO-treated 4MOSC1 tumors at days 7, 10, and 13. Shown is the average number of cells infiltrating per mm3of tumor (n = 3 mice per group; multiple unpaired Student’s t test; data are represented as mean ± SEM). (FIG. 3E) Representative immunostaining of CD8+ and CD4+ cells within the stroma and tumor parenchyma (PanCK) of vehicle and CXCLIO-treated tumors atAttorney Docket No. 15670-0449WO1

[0035] day 10. The scale bar represents 200 pm. (FIGS.3F-3G) Quantification of total CD8+T and CD4+T cells within the tumor stroma (FIG.3F) and tumor parenchyma (panCK) (FIG.3G); 77=4; 5-6 regions of interest (ROIs) screened per tumor; ROI designation of stroma vs tumor parenchyma based on PanCK expression. (FIG.3H) Representative immunostaining of FoxP3+ and CD4+cells within vehicle- and CXCLIO-treated 4MOSC1 tumors at day 10. Scale bar, 100 pm. (FIG.31) Quantification of percent FoxP3+cells amongst total CD4+cells within vehicle- and CXCLIO-treated 4MOSC1 tumors at day 10 (H=5; 2 ROIs screened per tumor).

[0036] FIGS.4A-4G: IT-CXCL10 enhanced the therapeutic efficiency of anti-PD-1 treatment to promote a durable anti-tumoral response. (FIG.4A) The frequency of PMN-MDSC and M-MDSC cells amongst total CD45+CD1 lb+cells (77=5). (FIG.4B) The frequency of PD-L1+cells amongst total PMN-MDSC and M-MDSC cells («=5). Representative flow cytometry' plots showing relative PD-L1+cells amongst total PMN-MDSC and M-MDSC cells are on the right. (FIG.4C) Schematic detailing experimental setup and dosing schedule for 4MOSC1 and MOC1 models. Mice received either 10 pg CXCL10 in 50 pL of PBS or 50 pL of PBS (vehicle) on the days indicated (A) and 200 pg of aPD-1 (5 mg / kg) IP every 3 days for 3 weeks initiated at time of vehicle or CXCL10 treatment. 4MOSC1 (FIG.4D) and MOC1 (FIG.4F) tumor kinetics of mice of indicated treatment groups (77= 10, 4MOSC1; 77=7-8, MOC1). Survival percentage of 4MOSC1 (FIG.4D) and MOC1 (FIG.4F) mice receiving indicated treatment is shown on the right. (FIG.4E, FIG.4G) The percentage of mice that fully cleared tumors (complete response) for indicated treatment group of matched duplicate experiments (the result for each of 3 independent experiments is graphed, with the exception of otPD-1 treatment group for MOC1, which only included in 2 independent experiments).

[0037] FIGS.5A-5I: CXCLIO-treated tumors exhibited an increased T cell-inflamed signature and enhanced memory while showing reduced indications ofT cell dysfunction, exhaustion, and angiogenesis. (FIG.5A) Differentially expressed genes in vehicle- vs CXCLIO-treated 4MOSC1 mice (77=4). Fold-change and P value (adjusted) cut-offs are 1.2 (1.5 bolded) and 0.05, respectively. (FIG.5B) Representative immunostaining of PanCK+, CD8+, CD4+, and GZMB+cells within the tumors of vehicle and CXCLIO-treated mice at day 10. Scale bar, 100 pm.

[0038] Percentage of CXCR3+cells (FIG.5C) or CD44+CD62L+cells (TcM-like) (FIG.5D)Attorney Docket No. 15670-0449WO1

[0039] amongst total CD45+CD3+CD8+T cells in 4MOSC1 tumors at day 10 (n=3-5).

[0040] (FIGS.5E-5F) Representative flow cytometry’ plots showing proportion of CD44+PD-lhlghcells amongst total CD45 CD3 CD8 T cells as well as quantification (FIG.5F) at day 10 (w=5). (FIG.5G) The proportion of PD-lhighTIM3+cells (terminally exhausted) amongst total CD45+CD3+CD8+T cells in 4MOSC1 tumors at day 10 (n=4-5). (FIG.5H) Quantification of total CD31+and LYVE-1+cells within 4MOSC1 tumor sections («=4 mice per group; 4 ROIs were screened per tumor). (FIG.51) Representative immunostaining of PanCK, CD3, and LYVE-1+cells of 4MOSC1 tumor sections of vehicle and CXCLIO-treated mice at day 10. Scale bar, 100 pm. (See FIG. 12A) for representative immunostaining at a larger scale).

[0041] FIGS.6A-6H: Tumor-draining lymph nodes (TdLNs) from CXCLIO-treated mice exhibited increased T cell proliferation and tumor antigen-specificity. (FIG.

[0042] 6A) Representative immunostaining of CD8+, CD4+, CDllc+, and Ki67+, cells within TdLN of vehicle and CXCLIO-treated mice at day 10. Scale bar, 100 pm. Germinal centers are indicated by A. (FIG.6B) Quantification of total CD8 . CD4+, and CD1 lc+cells per mm2within the TdLN (w=4, whole lymph node section screened).

[0043] (FIG.6C) Schematic showing the general structure and cell-specific regions within LNs. (FIG.6D) The proportion of CXCR3+CD3+cells amongst total CD45+cells in the TdLN of 4MOSC1 tumors at day 10 (w=4-5). (FIG.6E) The proportion of TCF1 Ki67 TOX TIM3 CD8 cells amongst total CD31cells in the TdLN of 4MOSC1 tumors at day 10 with (FIG.6F) representative flow cytometry plot (M=4-5) of data in (FIG.6E). (FIGS.6G-6H) Representative flow cytometry' plots showing proportion of OVA-H-2kB-Tetramer+cells amongst total CD45+CD3+CD8+T cells at day 10. Quantification on right (w=l 0).

[0044] FIG.7: Top 100 differentially expressed genes between CXCLl(flghand CXCLl(fmHNSCC tumors. Differential expression of genes between CXCL10hlghvs CXCL10lowcohorts using Wilcoxon Rank Sum test. Log2 fold-change and P value (adjusted) cut-offs are 1 and 0.05, respectively. Transcriptomic data was obtained and analyzed via cBioPortal (PanCancer Atlas, HNSCC cohort). Expression was based on mRNA levels (whole tumor). CXCR3hlgh / CXCL10hlghcohorts (n = 128 and 132) and CXCL10low / CXCL10lowcohort (n = 131 and 126) represent the upper 25% and lower 25% threshold for Cxcr3 or CxcllO mRNA expression respectively.Attorney Docket No. 15670-0449WO1

[0045] FIGS.8A-8F: Production and functional verification of CXCR3 ligands. Proportion of complete tumor clearance across independent experiments. (FIG.8A) Representative lane from SDS-PAGE gel demonstrating purity of CXCL10. (FIG. SB) Representative trace with mass-to-charge ratio of purified ligand (hCXCLIO) from intact protein mass spectrometry analysis. (FIG.8C) Approximate yield per liter of biomass processed for human and murine CXCR3 ligands. (FIG.8D) Percent of migrating hCXCR3+Jurkat cells in response to CXCL10 at varying concentrations.

[0046] (FIG.8E) Bystander BRET assay utilizing |3arr2-rLuc3 and membrane-bound rGFP-CAAX to measure beta-arrestin 2 (Parr2) recruitment in CXCR3+HEK293Ts after incubation in 100 nM of CXCL10. (FIG.8F) Proportion of mice exhibiting complete tumor clearance across multiple independent experiments. Note that there was no treatment group for anti-PD-1 alone included in experiment 20221015.

[0047] FIGS.9A-9C: NK cells isolated from CXCLIO-treated tumors exhibited increased activation. (FIG.9A) Representative immunostaining of PanCK+and NK1.1+cells within 4MOSC1 tumors of vehicle and CXCLIO-treated mice at day 10. Scale bar, 200 gm. (FIG.9B) Quantification of total NK1.1+ cells within the tumor stroma and tumor parenchyma (panCK) (n = 4 mice per group; 2 ROIs screened per tumor). (FIG.9C) The frequency of CD44+cells amongst total CD45+NK1.1+cells in the TME at day 7, 10, and 13 (n = 3-4 per group; unpaired Student's I test).

[0048] FIG. 10: Extended list of differentially expressed genes from tumors of vehicle and CXCLIO-treated mice. 1.0 x 1064MOSC1 cells were implanted into the buccal space of C57BL / 6 mice on day 0. Tumors injected on days 3, 6, and 9 with either vehicle (50 pL of PBS) or 10 pg of CXCL10 in 50 pL of PBS. mRNA from each tumor was isolated and quantified using the NanoString nCounter® PanCancer Mouse Immune Profiling gene expression platform. Rosalind® software was used to analyze differentially expressed genes (n = 4 per group). Differentially expressed genes in vehicle- vs CXCLIO-treated 4MOSC1 mice.

[0049] FIGS. 11A-11F: T cells isolated from CXCLIO-treated tumors exhibited unique phenotypic changes. (FIG. 11A) Proportion of OT-1 CD8+ T cells that were CXCR3+of the total CD45+CD3+CD44+CD8+cells following antigen stimulation and indicated treatment in vitro. (FIGS. 11B-11C) The frequency of CD44+CD62L’ cells or CD44+CD62L+cells (TcM-like) amongst total CD8+and CD4+cells in the TME at day 13 (n = 3 per group; unpaired Student’s t test). An increase in a TcM-likeAttorney Docket No. 15670-0449WO1

[0050] phenoty pe was not observed with CD4+T cells as was with CD8+T cells. (FIG. 11D) The frequency of PD-lhlghTOX+TIM3loKi6710cells amongst total CD8+cells in the TdLN at day 10 (n =4-5 per group; one-way Anova). (FIG. HE) Number of viable OT-1 CD8+ T cells after 48 h incubation with the indicated treatment. (FIG. HF) Proportion of OT-1 CD8+ T cells that were PD-1+of the total CD45+CD3+CD44+CD8+cells following antigen stimulation and indicated treatment in vitro.

[0051] FIGS. 12A-12C: CXCL10 treatment led to decreased heme- and lymphangiogenesis in vivo as well as unique phenotypic changes in tumor-infiltrating cells. (FIG. 12A) Representative immunostaining of PanCK, CD31, and LYVE-1+cells of 4MOSC1 tumor sections of vehicle and CXCL 10-treated mice at day 10. Scale bar, 200 pm. (FIG. 12B) The frequency of PD-LT cells amongst total CD45+CD1 lb+CD1 lc+cells in the TME at day 10 (n = 5 per group; unpaired Student’s t test). (FIG. 12C) The frequency of TCF TOX TIM3 CD84cells amongst total CD3+cells in the TME at day 10 (n = 5 per group; Kruskal-Wallis test).

[0052] FIG. 13: Summary of immune cell migration and enhanced function in response to increased tumor-localized CXCL10. Schematic depicts increased tumor-localized CXCL 10 mobilizing a self-reinforcing, immune-stimulatory' positive feedback loop that enhanced tumor antigen-specific T cell priming, effector function, and memory formation while limiting T cell dysfunction and exhaustion, thereby reshaping the TME towards a more antitumoral state.

[0053] DETAILED DESCRIPTION

[0054] Immunotherapy has revolutionized cancer treatment by leveraging the host immune system to eliminate tumors, offering options for patients with previously untreatable cancers. In particular, ICB agents, such as pembrolizumab and nivolumab, have exhibited success in treating some patients with recurrent or metastatic cancer (1). They disrupt PD-1 / PD-L1 signaling cascades that block the activation and proliferation of cytotoxic T cells, inhibit cytokine production, and accelerate CD8+T cell exhaustion (2, 3). However, favorable clinical outcomes vary across different cancers, with objective response rates limited to 33-40% in melanoma (4), 15-20% in non-small cell lung cancer (5), 22-25% in renal cell carcinoma (6), and 10-20% in head and neck squamous cell carcinoma (7, 8). TheAttorney Docket No. 15670-0449WO1

[0055] failure of these ICBs, as well as standard treatments, is partly due to the ability of resistant tumors to limit the infiltration and activation of cytotoxic CD8+T and NK cells (9, 10). CD8+T and NK cells release apoptosis-inducing cytotoxins like perforin and granzyme, as well as pro-inflammatory cytokines, interferon gamma (IFN-y) and tumor necrosis factor alpha (TNF-a), to eliminate cancer and are essential for an effective response to immunotherapy. Accordingly, the abundance of cytotoxic cells within tumor tissue following various treatment strategies is associated with a positive prognosis in several cancers (11, 12). However, exclusion of these cells from tumors can result from physical barriers like dense extracellular matrix deposition (13), immunosuppressive signaling from tumor and stromal cells (14), and dysfunctional vasculature that impairs effective immune cell infdtration (15). Further, the mere infiltration of cytotoxic cells into the tumor microenvironment (TME) does not guarantee positive outcomes. Immunosuppressive cytokines and chronic antigen stimulation can lead to the dysfunction and / or exhaustion of these cells, limiting their ability to control tumor growth (16). Additionally, the exclusion of dendritic cells (DCs) contributes to immune suppression by stunting the generation of T cells primed to tumor-specific antigens (TSAs) (17, 18). To broaden the success of immunotherapy, approaches are needed that override the obstruction of key anti-tumoral cells and promote the generation and maintenance of T cells that are reactive to TSAs.

[0056] The chemokine receptor CXCR3 is a key regulator of cytotoxic immune cell responses within solid tumors (19, 20). It responds to IFN-inducible ligands CXCL9, 10, and 11, and controls the migration, spatial distribution, and function of activated T and NK cells (19). CXCL9 and 10 are important for the anti -tumoral functions of CD8+T cells, type-1 helper (Thl) CD4+T cells, and NK cells (21), and reduced expression of either ligand contributes to immune exclusion within the TME (19, 22). Conversely, high expression of CXCL9, CXCL10, and CXCR3 is associated with improved responses to ICB treatment (21). CXCL10 has been shown to impact the differentiation of CD4+T cells into anti-tumoral Thl subtypes (23) and facilitates interactions between CD8+T cells and antigen-presenting DCs by fostering a pro-inflammatory niche (24).

[0057] The mechanisms by which CXCL10 shapes T cell phenoty pes within tumors include both direct effects from CXCR3 -dependent signaling and from indirectAttorney Docket No. 15670-0449WO1

[0058] modulation of broader immune cell networks. Direct effects were clear from in vitro experiments using CD8+T cells extracted from OT-1 transgenic mice which showed a reduction of markers of functional impairment following stimulation with CXCL10. As demonstrated herein, intratumoral (IT) augmentation of CXCL10 led to suppressed tumor grow th in murine models of cancer, and even complete tumor clearance in a substantial percentage of mice, by promoting the infiltration of cytotoxic CD8+T cells, CD4+T cells, and NK cells into the TME. However, IT-CXCL10 did more than recruit these cells; it enhanced T cell priming and activation, resulting in increased tumor antigen specificity, heightened cytotoxic activity7, and the establishment of durable immune memory that supported resistance to tumor regrowth.

[0059] In the present disclosure, IT delivery of recombinant CXCL10 led to the recruitment and activation of CD8+T, CD4+T and natural killer (NK) cells, and also the formation of effector and memory T cells and tumor antigen-specific CD8+T cells. The CD8+T cells also exhibited reduced indications of early T cell dysfunction following CXCLIO-treatment, which translated to higher anti-tumoral effector function of the T cells in CXCL10- vs vehicle treated mice. Without wishing to be bound by any particular theory7, the reduced dysfunction may be due to activation of Gi through CXCR3 which opposes Gs activation, a documented contributor to T cell dysfunction.

[0060] CXCR3 is rapidly downregulated upon ligand engagement (43, 61), and thus efforts to define the direct functional contribution of CXCR3 based on its surface expression can be complicated. Moreover, the presence of CXCR3 on cells did not prove a direct effect: for example, the reduction of TPEX cells in the TdLN of CXCR3 ' mice compared to WT mice suggests, but does not prove, a direct effect of the receptor on the induction of this T cell phenotype — it only showed that CXCR3 was needed. Nevertheless, despite these limitations — including reliance on flow cytometry7and bulk mRNA rather than single-cell sequencing to assign phenotypes to specific cell subsets — several hypotheses can still be proposed regarding the direct effects of CXCR3 activation. For example, as observed herein, CXCL 10 treatment resulted in a robust increase in CD4+T cells and notably, CXCR3 expression was preferentially high on Thl CD4+T cells following activation (52, 53). Some studies have shown that CXCL 10 directly induces the polarization of naive CD4+T cellsAttorney Docket No. 15670-0449WO1

[0061] towards immune-supporting Thl and Thl7 lineages (52, 53); thus these populations may represent a bulk of the CD4+T cells infiltrate in tumors of CXCL 10-treated mice. This premise was strengthened by the significant increase in IFN-y and TNFa related gene signatures, a reduction in the proportion of FoxP3+Tregs, as well as the overall increase in cytotoxic CD8 responses observed herein in CXCL 10-treated mice. Together, the data provided herein suggest that CXCL 10 not only acts as a chemoattractant of CXCR3-expressing cells but also influences their phenotypic fate.

[0062] The impact of CXCL 10 treatment was not limited to CXCR3-expressing immune cells, suggesting indirect mechanisms contributed to tumor growth inhibition. Consistent with the increase in TSA-T cells that accumulated in 4MOSC1 tumors in response to CXCL 10 treatment, eDCs were shown to play a critical role in the efficacy of the CXCL10 treatment. We also observed increased antigen-presenting CD1 lc+cells in TdLNs where they prime and activate CD8+and CD4+T cells, which also showed TdLN accumulation. This was striking because administration of CXCL 10 into tumors appears to induce the trafficking of both CXCR3+and non-CXCR3 -expressing cells between tumors and TdLNs. This dynamic interplay reflects a self-reinforcing, immune-stimulatory loop that enhanced antigen-specific T cell priming, infiltration, and effector function, reshaping the tumor microenvironment toward a more immunologically responsive state (see, e g., FIG. 13).

[0063] CXCL 10 not only affects immune cells — it acts on lymphatic and blood vessel endothelial cells that express CXCR3 (71). Herein, a dramatic reduction of both tumor angiogenesis and lymphangiogenesis from CXCL 10-treated mice was observed compared to control mice (FIG. 13). These data further define the multimodal mechanisms for the efficacy of CXCL 10 treatment, as it is well established that increased rates of angiogenesis in solid tumors is essential for the maintenance of tumor grow th, progression, and distal metastasis (15).

[0064] Data provided herein were consistent with prior studies using epigenetic modulators and vaccination via CXCL9 / CXCL10-engineered DCs. These studies demonstrated that amplification of CXCR3 signaling within the TME leads to improved responses to ICB in models of colorectal carcinoma, melanoma, and nonsmall cell lung cancer (20, 83). However, while promising, these methods, along with others (e.g., mRNA-based therapeutics), are less controlled with respect to CXCL10 levels and duration of treatment, which impact efficacy and results in undesiredAttorney Docket No. 15670-0449WO1

[0065] effects. In contrast, our intratumoral delivery of recombinant chemokine (e.g., CXCL10) provided a more immediate and controllable therapeutic approach that can be adjusted as needed, thereby reducing immunogenicity and off-target activity. The methods of IT delivery7as disclosed herein are superior to systemic delivery' which requires multiple doses at high concentrations to observe any redeemable effect. For example, ahCXCLIO-Fc was delivered systemically every other day for up to seven times, each at high concentrations (—50 - 200 pg), to observe a decrease in tumor growth (88). Furthermore, the treatment regimens with hCXCLIO-Fc were no more effective than delivery of anti-PD-1 alone (88), which is in direct contrast to the methods of the present disclosure in which IT delivery7of a recombinant CXCL10 protein alone was superior to anti-PD-1 treatment in regards to both complete tumor clearance and induced resistance to a rechallenge with additional cancer cells. As such, the methods provided herein offer more favorable treatment regimens by requiring fewer doses at lower concentrations of recombinant CXCL10 to be effective.

[0066] The present disclosure provides unexpected and promising results: demonstrating that IT delivery of recombinant CXCL10 alone resulted in significant tumor elimination, immune memory7against rechallenge with additional cancer cells and additionally enhanced the antitumoral efficacy of commonly used immunotherapies in a synergistic manner. To date, other studies showed that administration of CXCL10 alone had no significant therapeutic effect on primary7and distant tumors (87). Indeed, recombinant CXCL10 administration was only found to be somewhat effective when co-administered with a hydrogel comprising a dipeptidyl peptidase 4 (DPP4) inhibitor and a small molecule immune checkpoint blocker (87). The present disclosure provides methods of intratumoral delivery of recombinant CXCL10 alone that remains localized within the TME through glycosaminoglycan binding, which protects the recombinant CXCL10 from rapid proteolytic degradation and limits systemic dissemination, enabling sustained localized immune cell recruitment.

[0067] ICBs such as anti-PD-1 and anti-CTLA-4 are generally administered systemically and disrupt normal immune regulation and tolerance, sometimes resulting in immune-related adverse events (irAEs) such as dermatitis, colitis, thyroiditis, and pneumonitis in a significant proportion of patients (85). CombiningAttorney Docket No. 15670-0449WO1

[0068] CXCL 10-based treatments with ICBs may also enable anti -tumor efficacy to be achieved with a lower dose of the ICB, thereby minimizing well-known immune-related adverse events that are often associated with these treatments (85).

[0069] The present disclosure provides methods of IT-CXCL10 as an effective strategy' to enhance CXCR3-mediated immune cell recruitment while actively shaping T cell fate towards cytotoxic, durable, and non-exhausted states. Beyond direct effects on T cells, CXCL 10 coordinated a wider immune network that induced T cell priming and vascular remodeling to collectively constrain tumor growth. As such, local administration (e.g., IT delivery) of CXCL10 as disclosed herein provides a multimodal strategy' to overcome resistance to ICB immunotherapies and improve survival outcomes in cancer (e.g., HNSCC) patients.

[0070] C-X-C motif chemokine 10 (CXCL10)

[0071] C-X-C motif chemokine 10 (CXCL 10) is a pro-inflammatory cytokine involved in a wide variety of processes such as chemotaxis, differentiation, activation of peripheral immune cells, regulation of cell grow th, apoptosis, and modulation of angiostatic effects (Sidahmed AM et al., Cytokine. 2012 Aug;59(2):433-41; Angiolillo AL et al.. J Exp Med. 1995 Jul 1 ; 182(1): 155-62). CXCL10 binds to and activates the CXCR3 receptor, found most commonly on pro-inflammatory CD8+cytotoxic T (Tc) cells, CD4+type I helper T (Tnl) cells, and natural killer (NK) cells, and directs their selective migration to autoimmune sites and tumor sites (Brownell & Polyak, Clin Cancer Res. 2013 Mar 15;19(6):1347-52; Karin & Razon, Cytokine. 2018 Sep;109:24-28).

[0072] The present disclosure provides a recombinant CXCL 10 protein and methods of use thereof. In some embodiments, a recombinant CXCL 10 protein suitable for use herein can be CXCL 10, a variant of CXCL 10, or any isoform of CXCL 10. In some embodiments, a recombinant CXCL 10 protein suitable for use herein can comprise at least about or about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity' to the sequences provided in Table 1.

[0073] TABLE 1: CXCL10 Proteins

[0074]

[0075] Attorney Docket No. 15670-0449WO1

[0076]

[0077] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity’ between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty’ of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0078] In some embodiments, a recombinant CXCL10 protein suitable for use herein can comprise about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1. In some embodiments, a recombinant CXCL10 protein suitable for use herein can comprise about 95%. 96%. 97%. 98%. 99%. or 100% amino acid sequence identity’ to SEQ ID NO: 19. In some embodiments, a recombinant CXCL10 protein suitable for use herein comprises the amino acid sequence SEQ ID NO: 19. In some embodiments, a recombinant CXCL 10 protein suitable for use herein can comprise about 95%. 96%. 97%. 98%. 99%. or 100% amino acid sequence identity to SEQ ID NO: 2. In some embodiments, a recombinant CXCL 10 protein suitable for use herein can comprise about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 20. In someAttorney Docket No. 15670-0449WO1

[0079] embodiments, a recombinant CXCL10 protein suitable for use herein comprises the amino acid sequence SEQ ID NO: 20.

[0080] In some embodiments, a recombinant CXCL10 protein suitable for use herein can comprise at least about or about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity to IPLARTVRCNCIHIDDGPVRMRAIGKLEIIPASLSCPRVEIIATMKKN DEQRCLNPESKTIKNLMKAFSQKRSKRAPWS (SEQ ID NO: 3).

[0081] In some embodiments, a recombinant CXCL10 protein suitable for use herein can comprise about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity7to SEQ ID NO: 3. In some embodiments, a recombinant CXCL10 protein suitable for use herein comprises the amino acid sequence SEQ ID NO: 3.

[0082] The recombinant CXCL10 proteins contemplated herein can further comprise a tag. Protein tags are generally known in the art and can have a multitude of uses including (but not limited to) purification, detection, solubilization, localization, or protease protection. In some embodiments, a recombinant CXCL10 protein can be linked with one or more of the protein tags provided in Table 2. In some embodiments, the tag can be linked to the C-terminus of the recombinant CXCL10 protein. In some embodiments, the tag is not linked to the N-terminus of the recombinant CXCL10 protein. In some embodiments, a His tag (e.g., SEQ ID NO: 14) can be linked to the C-terminus of the recombinant CXCL10 protein.

[0083] TABLE 2: Protein Tags

[0084]

[0085] Attorney Docket No. 15670-0449WO1

[0086]

[0087] In some embodiments, at least one amino acid can be added to the N-terminus, C-terminus, or both, of the recombinant CXCL10 protein. In some embodiments, no more than one amino acid can be added to the N-terminus of the recombinant CXCL10 protein. In some embodiments, one or more amino acids can be added to the C-terminus of the recombinant CXCL10 protein to facilitate accurate determination of concentration without altering protein function. In some embodiments, a tryptophan-serine sequence can be added to the C-terminus of the recombinant CXCL10 protein to facilitate accurate determination of concentration without altering protein function.

[0088] Provided herein are methods of generating the recombinant CXCL10 proteins disclosed (e.g., a recombinant CXCL10 protein, a recombinant CXCL10 protein fused to at least one tag). Methods disclosed herein can include introducing a recombinant CXCL10 protein in a nucleic acid that encodes them into a cell (e.g., a target cell). In order to express a recombinant CXCL10 protein disclosed herein, a nucleic acid sequence encoding the CXCL10 protein can be subcloned into an expression vector that contains a promoter to direct transcription. In some embodiments, a nucleic acid sequence provided herein can encode for a CXCL10 protein with at least about or about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NOS: 1, 2, 3, 19 or 20. In someAttorney Docket No. 15670-0449WO1

[0089] embodiments, a nucleic acid sequence provided herein can encode for a CXCL10 fusion protein with at least about or about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NOS: 1, 2, 3, 19 or 20. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (3d ed. 2001); Kriegler, GENE TRANSFER AND EXPRESSION: A LABORATORY MANUAL (1990); and CU ENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the fusion proteins are available in, e.g., E. coli, Bacillus sp., and Salmonella (see, e.g., Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Standard transfection methods can be used herein to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, w hich are then purified using standard techniques (see, e.g.. Colley et al.. 1989, J. Biol. Chem., 264: 17619-22; GUIDE TO PROTEIN PURIFICATION, IN METHODS IN ENZYMOLOGY, vol. 182 (Deutscher, ed., 1990)).

[0090] Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacterial. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101 :347-362 (Wu et al., eds, 1983). Any of the known procedures for introducing foreign nucleotide sequences into host cells can be used in the present disclosure. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (3d ed. 2001); Kriegler, GENE TRANSFER AND EXPRESSION: A LABORATORY MANUAL (1990)).

[0091] In some embodiments, an E. coli codon-optimized variant of CXCL10 can be cloned into a vector (e.g., a pHUE vector) to produce a fusion protein comprising an N-terminal tag fused to CXCL10. In some embodiments, the fusion protein comprises a tag and a ubiquitin in addition to CXCL10. In some embodiments, the fusion protein comprises, from N terminus to C terminus, 6-His tag - ubiquitin -CXCL10. In some embodiments, the tag attached to the N-terminus of CXCL10 canAttorney Docket No. 15670-0449WO1

[0092] be removed by a deubiquitylating enzy me. In some embodiments, the tag attached to the N-terminus of CXCL10 is removed by Usp2-cc. In some embodiments, a CXCL10 fusion protein can be purified by chromatography. In some embodiments, a 6-His-ubiquitin-CXCL10 fusion protein is purified by Ni -affinity chromatography. Once the fusion protein (e.g., 6-His-ubiquitin-CXCL10) is purified, the CXCL10 protein can be cleaved from the N-terminal tag (e.g., 6-His) by a deubiquitylating enzyme (e.g., Usp2-cc). Following cleavage from the N-terminal 6-His tag, the CXCL10 protein can be loaded onto Ni-NTA resin. The resulting elution can then be subjected to reverse phase chromatography column (e.g., via a Cl 8 reverse phase chromatography column) to purify the recombinant CXCL10 protein. The purified recombinant CXCL10 protein can then be subjected to lyophilization.

[0093] In some embodiments, a recombinant CXCL10 protein is purified before use in the pharmaceutical composition and / or the methods disclosed herein. In some embodiments, a recombinant CXCL10 protein for use in the pharmaceutical composition and / or the methods disclosed herein is at least about or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, a recombinant CXCL10 protein for use in the pharmaceutical composition and / or the methods disclosed herein does not comprise a protein tag (e.g., the protein tag is removed prior to use). In some embodiments, a recombinant CXCL10 protein for use in the pharmaceutical composition and / or the methods disclosed herein comprises a protein tag.

[0094] In some embodiments, a recombinant CXCL10 protein for use in the pharmaceutical composition and / or the methods disclosed herein is not an antibody or antigen binding fragment thereof. In some embodiments, a recombinant CXCL10 protein for use in the pharmaceutical composition and / or the methods disclosed herein is not fused to with a Fc (fragment crystallizable) domain (including a mutant or variant Fc domain) or a fragment thereof.

[0095] Pharmaceutical Compositions and Methods of Administration

[0096] The methods described herein include the use of pharmaceutical compositions comprising or consisting of a recombinant CXCL10 protein disclosed herein, and / or compositions comprising a plurality of recombinant CXCL10 proteins disclosed herein.Attorney Docket No. 15670-0449WO1

[0097] Pharmaceutical compositions ty pically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for local delivery to a tumor or an area surrounding the tumor. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for local delivery7to a surgical margin following tumor resection. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for local delivery to the TME. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for delivery7into or on the surface of a tumor. In some embodiments, a pharmaceutical composition of the present disclosure can be injected directly into the tumor (e.g., intratumoral injection) with a needle (e.g., a Turner Biopsy Needle or a Chiba Biopsy Needle).

[0098] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for intratumoral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzy l alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity7such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The intratumoral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0099] Pharmaceutical compositions suitable for intratumoral injection use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intratumoral administration, suitable carriers include physiologicalAttorney Docket No. 15670-0449WO1

[0100] saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.

[0101] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a recombinant CXCL10 protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0102] In some embodiments, the recombinant CXCL10 protein disclosed can exist in a lyophilized formulation including the protein and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In some embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation can also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative. In some embodiments, the lyophilized recombinant CXCL10 protein may be constituted with an aqueous carrier. An aqueous carrier suitable for use herein is one which isAttorney Docket No. 15670-0449WO1

[0103] pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Nonlimiting examples of such diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution. In some embodiments, the lyophilized recombinant CXCL10 protein disclosed herein is reconstituted with a diluent prior to intratumoral (IT) delivery.

[0104] The location, number and frequency of each intratumoral injection of the compositions (e.g., recombinant CXCL10 protein) disclosed herein can be defined according to the specific therapeutic needs and cancer status of the subject in need of such treatment, with injections that may be given simultaneously or concurrently (during the same medical act) or sequentially (in one or more distinct medical acts, separated by hours, days, or weeks), in two, three, or more cycles of treatment. In some embodiments, the compositions disclosed herein are delivered to the tumor locally (e.g., by intratumoral injection) once. In some embodiments, the compositions disclosed herein are delivered to the tumor locally (e.g., by intratumoral injection) twice. In some embodiments, the compositions disclosed herein are delivered to the tumor locally (e.g., by intratumoral injection) three times. In some embodiments, the compositions (e.g., recombinant CXCL10 protein) disclosed herein are not delivered systemically.

[0105] Actual dosage levels of the recombinant CXCL10 protein in the pharmaceutical compositions disclosed herein can be varied so as to obtain an amount of CXCL10 which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. In some embodiments, compositions disclosed herein are administered to the tumor locally (e.g., by intratumoral injection) to deliver a dose of recombinant CXCL10 protein that ranges from about 1 ng to about 1 mg. In some embodiments, compositions disclosed herein are administered to the tumor locally (e.g.. by intratumoral injection) to deliver at least about or about I ng, 10 ng, 100 ng, 250 ng, 500 ng, 750 ng, 1 pg, 10 pg, 100 pg, 250 pg, 500 pg, 750 pg, or 1 mg recombinant CXCL10 protein.

[0106] In some embodiments, a pharmaceutical composition for local delivery to the tumor (e.g., by intratumoral injection) comprises a CXCL10 active ingredient and oneAttorney Docket No. 15670-0449WO1

[0107] or more excipients, wherein the CXCL10 active ingredient consists of a recombinant CXCL10 protein that comprises a protein tag. In some embodiments, a pharmaceutical composition for local delivery to the tumor (e.g., by intratumoral injection) comprises a CXCL10 active ingredient and one or more excipients, wherein the CXCL10 active ingredient consists of a recombinant CXCL10 protein that does not have a protein tag. In some embodiments, a pharmaceutical composition for local delivery to the tumor (e.g., by intratumoral injection) comprises a CXCL10 active ingredient and one or more excipients, wherein the CXCL10 active ingredient consists of a recombinant CXCL10 protein that is not an antibody or antigen binding fragment thereof. In some embodiments, a pharmaceutical composition for local delivery to the tumor (e.g., by intratumoral injection) comprises a CXCL10 active ingredient and one or more excipients, wherein the CXCL10 active ingredient consists of a recombinant CXCL10 protein that is not fused to with a Fc (fragment crystallizable) domain (including a mutant or variant Fc domain) or a fragment thereof.

[0108] A recombinant CXCL10 protein described herein can be administered according to the methods disclosed herein in combination with at least one additional cancer treatment. Combination therapies contemplated herein can be adapted to other cancer treatments that are administered (simultaneously or sequentially) using the same intratumoral route or alternatively, using other routes and means, including orally, subcutaneously, intradermally, intranasally, intravenously, intramuscularly, intrathecally, intranasally, intravesically, topically, and transdermally, which can improve the efficacy, safety, and / or clinical use of any of such treatments disclosed herein. Non-limiting examples of cancer treatments to be combined with the described compositions and methods for local administration (e.g., by intratumoral injection) of CXCL10 can include surgery, radiotherapy, chemotherapy, toxin therapy, cancer vaccination, laser therapy, phototherapy, immunotherapy, cryotherapy, and / or gene therapy.

[0109] In some embodiments, a recombinant CXCL10 protein described herein can be used in combination with at least one additional therapeutic agent to treat cancer. In some embodiments, a combination therapy disclosed herein can comprise administration of the recombinant CXCL10 protein by IT delivery and administration of at least one additional therapeutic agent by systemic delivery. In some embodiments, a combination therapy disclosed herein can comprise administration ofAttorney Docket No. 15670-0449WO1

[0110] the recombinant CXCL10 protein by IT delivery' and administration of at least one additional therapeutic agent by intravenous delivery. In some embodiments, a combination therapy disclosed herein can comprise administration of the recombinant CXCL10 protein and at least one additional therapeutic agent by IT delivery.

[0111] In some embodiments, a combination therapy disclosed herein can comprise administration of the recombinant CXCL10 protein by IT delivery and administration of at least one chemotherapeutic (i. e. , chemotherapy agent). Non-limiting examples of chemotherapeutics suitable for use herein can include cisplatin, carboplatin, nedaplatin, satraplatin, picoplatin, phenanthriplatin, triplatin tetranitrate, gemcitabine, methotrexate, vinblastine, adriamycin, or any combination thereof. In some embodiments, a combination therapy disclosed herein can comprise administration of the recombinant CXCL10 protein by IT delivery' and administration of radiation therapy. Radiation therapy and methods of administering thereof are known in the art and are suitable for use herein (see, e.g., Maani & Maani, Radiation Therapy.

[0112] [Updated 2022 Oct 24], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2025 Jan. Available from: ncbi.nlm.nih.gov / sites / books / NBK537036 / (accessed on February 3, 2026)).

[0113] In some embodiments, a combination therapy disclosed herein can comprise administration of the recombinant CXCL10 protein by IT delivery and administration of at least one immunotherapy for the treatment of cancer. As demonstrated herein, combination therapies comprising administration of recombinant CXCL10 protein by IT delivery' and at least one immunotherapy have a synergistic therapeutic effect on the tumor. In some embodiments, an immunotherapy that can be used as part of a combination therapy in treating cancer according to the methods disclosed herein can include at least one immune checkpoint blockade (ICB) agent. Exemplary ICB agents include those that inhibit one or more of (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD-1), (iii) PD-L1, (iv) LAG3, (v) B7-H3, (vi) B7-H4. and (vii) TIM3. In some embodiments, the ICB agent can be an antibody, an antigen binding fragment, an immunoadhesin, a fusion protein, or oligopeptide. In some embodiments, the ICB agent is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor suitable for use herein is selected from the group consisting of an anti-PDl antibody or an anti-PD-Ll antibody. In some embodiments, a PD-1 inhibitor suitable for use herein is selected from the group consisting ofAttorney Docket No. 15670-0449WO1

[0114] nivolumab (OPDIVO™, Bristol Myers Squibb, New York, New York), pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp, Kenilworth, NJ USA), cemiplimab (LIBTAYO®, Regeneron, Tarrytown, NY) or pidilizumab (CT-011). In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody such as durvalumab (IMFINZI®, Astrazeneca, Wilmington, DE), atezolizumab (TECENTRIQ®, Roche, Zurich, CH), or avelumab (BAVENCIO®, EMD Serono, Billerica, MA). In some embodiments, the PD-L1 inhibitor is chosen from YW243.55.S70, MPDL3280A. MED 1-4736, MSB-0010718C, or MDX-1105.

[0115] The pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration. In some embodiments, a kit provided herein can comprise a lyophilized recombinant CXCL10 protein and an aqueous carrier for constituting the lyophilized protein. In some embodiments, a kit provided herein can comprise a composition disclosed (e.g., a recombinant CXCL10 protein) and at least one immunotherapy for the treatment of cancer (e g., an ICB agent). A kit provided herein can comprise a plasmid encoding the recombinant CXCL10 protein disclosed herein and instructions for preparing and purifying the CXCL10 protein for use in the methods disclosed herein.

[0116] Methods of Treatment

[0117] Provided herein are methods of administering a recombinant CXCL10 protein or a composition comprising a recombinant CXCL10 protein to a subject having or suspected of having a cancer (e.g., a tumor). The terms “patient’" and “subject"’ and similar phrases can be used interchangeably herein and are intended to refer to subjects who are at risk for and / or have been diagnosed with one or more cancers. Preferably, the subject is human, but the methods can be used in other mammals, e.g., non-human veterinary subjects such as non-human primates, cats, dogs, horses, cows, goats, and rabbits. In some embodiments, a subject can have or can be suspected of having cancer, a tumor, or any combination thereof.Attorney Docket No. 15670-0449WO1

[0118] In some embodiments, a subject can have or can be suspected of having one or more primary’ tumors, one or more metastatic tumors such as solid tumors or any combination thereof. Solid cancerous tumors can form in various parts of the body, including but not limited to, breast, pancreatic, brain, mesothelioma, lung, thyroid, stomach, liver, kidney, ovarian, and prostate. Non-limiting examples of cancers derived from organs and / or tissues (i. e. , not blood cancers) that may be treated by the provided methods include: adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, colon cancer, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing’s tumor, glioma, hepatoma, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer, lung carcinoma, medullary carcinoma, medulloblastoma, neuroblastoma, oligodendroglioma, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, prostate cancer, rhabdomyosarcoma, renal cell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, testicular cancer, uterine cancer, and Wilm’s tumor. Preferably, a cancer to be treated by the provided methods is a head and neck cancer, head and neck squamous cell carcinoma, solid tumor, hematological tumor, colon cancer, liver cancer, gastric cancer, lung cancer, or melanoma.

[0119] As demonstrated herein, IT delivery of a recombinant CXCL10 protein overcame tumor-regulated immunosuppression to restore sensitivity’ to ICB immunotherapies in otherwise resistant tumors. In some embodiments, a subject can have or can be suspected of having a cancer that is resistant to at least one immunotherapy (e.g., an ICB agent). In some embodiments, a subject can have or can be suspected of having a cancer that is resistant to PD-1 / PD-L1 inhibition. Methods of assessing a subject for resistance to cancer immunotherapies are known in the art and are suitable for use herein (see, e g., Nowicki TS et al., Cancer J. 2018 Jan / Feb;24(l):47-53; Kluger HM et al., J Immunother Cancer. 2020

[0120] Mar;8(l):e000398).Attorney Docket No. 15670-0449WO1

[0121] The methods disclosed herein generally include locally administering a therapeutically effective amount of a recombinant CXCL10 protein to the tumor (via intratumorally delivery) or an area surrounding the tumor. As used herein the terms “administer,” “administering,” and “administration” are intended to mean introducing at least one agent (e.g., a recombinant CXCL10 protein) into a subject. In some embodiments, at least one agent (e.g., a recombinant CXCL10 protein) of the present disclosure is administered by local delivery to a tumor or an area surrounding the tumor. In some embodiments, at least one agent (e.g., a recombinant CXCL10 protein) of the present disclosure is administered to a surgical margin following tumor resection. In some embodiments, at least one agent (e.g., a recombinant CXCL10 protein) of the present disclosure is administered by IT injection. When administration is for the purpose of treatment, the agent (e.g., a recombinant CXCL10 protein) can be provided before, during, and / or after the onset of or progression of a symptom or sign of cancer. Detection of cancer symptoms and / or signs, and methods of diagnosing a cancer are known in the art and are suitable for use herein (see. e g., Kufe, D. W., Frei, E. Ill, Holland, J. F., Pollock, R. E., Weichselbaum, R. R., & Gansler, T. S. (Eds.). (2003). Cancer medicine 6: Review (6th ed.). American Cancer Society7; and U.S. Department of Health and Human Services, National Institutes of Health, & National Cancer Institute. (2012). Cancer: Causes, symptoms, signs, diagnosis, treatments, stages (Revised ed.). CreateSpace Independent Publishing Platform).

[0122] The methods provided by this disclosure can be used to treat an individual having any type of cancer (e.g., an individual diagnosed as having a cancer). As used herein, the terms “treatment” and “treating” can refer to obtaining a desired effect by use of the compositions according to the methods disclosed herein. The effect may be prophylactic in terms of completely or partially preventing a disease, disorder, or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease, disorder, and / or symptom attributable to the disease or disorder. Thus, the terms can cover any treatment of a disorder or disease in a subject, such as: (a) eliminating a tumor; (b) decreasing or ameliorating tumor growth; (c) preventing and / or decreasing the risk for cancer metastasis in a subject; (d) increasing survival rate of the subject; and / or (e) preventing recurrence of a cancer in a subject.

[0123] Administration of a therapeutically effective amount of a treatment described hereinAttorney Docket No. 15670-0449WO1

[0124] can result can reduce or eliminate tumor progression, reduce or inhibit metastasis, shrink tumors, induce tumor cell death, reduce tumor expansion, reduce lymphangiogenesis or angiogenesis lymphovascular invasion and / or reduce lymphogenous / hematogenous metastasis (e.g., reduce infiltration or metastasis of tumor cells into lymph nodes or distant organs). In some embodiments, methods disclosed herein can drive tumor elimination from a subject. In some embodiments, methods disclosed herein can inhibit recurrence of a cancer in a subject. In some embodiments, methods disclosed herein can enhance T cell effector function. In some embodiments, methods disclosed herein can increase the number of CD8+and CD4+T cells in tumor-draining lymph nodes (TdLNs) as compared to the number of these cells before treatment with CXCL10. In some embodiments, methods disclosed herein can prevent and / or ameliorate angiogenesis in the TME. In some embodiments, methods disclosed herein can prevent and / or ameliorate

[0125] ly mphangiogenesis in the TME.

[0126] Methods of combined IT administration of a recombinant CXCL10 protein with administration of a cancer therapy (e.g., chemotherapy and / or a radiation therapy) as disclosed herein can enhance the anti-cancer effects of the cancer therapy in a subject as compared to administration of the cancer therapy alone. In some embodiments, methods of combined IT administration of a recombinant CXCL10 protein with administration of a cancer therapy (e.g., chemotherapy and / or a radiation therapy) as disclosed herein can enhance tumor clearance in a subject as compared to that observed in a subj ect following administration of the cancer therapy alone. In some embodiments, methods of combined IT administration of a recombinant CXCL10 protein with administration of a cancer therapy (e.g., chemotherapy and / or a radiation therapy) as disclosed herein can reduce the risk of or prevent recurrence of a cancer in a subject as compared to that in a subject following administration of the cancer therapy alone. In some embodiments, methods of combined IT administration of a recombinant CXCL10 protein with administration of a cancer therapy (e g., chemotherapy and / or a radiation therapy) as disclosed herein can increase the survival rate in a subject as compared to the survival rate of a subject following administration of the cancer therapy alone.

[0127] Methods of combined IT administration of a recombinant CXCL10 protein with administration of an immunotherapy (e.g., an ICB agent) as disclosed herein canAttorney Docket No. 15670-0449WO1

[0128] enhance the anti -cancer effects of the immunotherapy in a subject as compared to administration of the immunotherapy alone. In some embodiments, methods of combined IT administration of a recombinant CXCL10 protein with administration of an immunotherapy (e.g., an ICB agent) as disclosed herein can enhance tumor clearance in a subject as compared to that observed in a subject following administration of the immunotherapy alone. In some embodiments, methods of combined IT administration of a recombinant CXCL10 protein with administration of an immunotherapy (e.g., an ICB agent) as disclosed herein can reduce the risk of or prevent recurrence of a cancer in a subject as compared to that in a subject following administration of the immunotherapy alone. In some embodiments, methods of combined IT administration of a recombinant CXCL10 protein with administration of an immunotherapy (e.g., an ICB agent) as disclosed herein can increase the survival rate in a subject as compared to the survival rate of a subject following administration of the immunotherapy alone.

[0129] EXAMPLES

[0130] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0131] Materials and Methods

[0132] The following materials and methods were used in the Examples below.

[0133] Study Design: To evaluate the impact of IT-CXCL10 treatment on tumor growth and immune infiltrate in mouse models of Head and Neck Squamous Cell Carcinoma (HNSCC), the following overall strategy was used. A previously described E. coli expression / purification (45) was used to produce a murine CXCL10 that contains a tr ptophan-serine sequence at the C-terminus to facilitate accurate determination of concentration without altering function. In brief, an E. coli codon-optimized variant of CXCL10 was cloned into a pHUE vector to produce a fusion protein containing (from N terminus to C terminus) N-terminal 6-His tag-ubiquitin-CXCL10. The N-terminal ubiquitin fusion system was developed as described herein as cleavage with Usp2-cc resulted in the native sequence (e.g., a recombinant CXCL10 protein). This was important because the exact nature of the N-terminal residues of CXCL10 affect their activity7. In contrast, CXCL10 was generally tolerant to modifications at its C-termini, so this region was chosen for the addition of non-Attorney Docket No. 15670-0449WO1

[0134] native residues for labeling. The 6-His-ubiquitin-CXCL10 fusion protein was purified by Ni-affinity chromatography, and the CXCL10 protein was cleaved from 6-His-ubiquitin using Usp2-cc, derivatized as required and loaded onto Ni-NTA resin prior to a final reverse phase chromatography and lyophilization. As ubiquitin elutes from a C 18 reverse phase chromatography column at a similar acetonitrile concentration as many chemokines (e g., CXCL10), the addition of a Ni-NTA column served to remove His-tagged ubiquitin and increase the purity of the final product.

[0135] For tumor implantation, 2 million MOC1 cells were injected into the right flanks or 1 million 4MOSC1 cells into the right buccal space of female C57BL / 6N mice (4-6 weeks of age). When average tumor volumes reached 10-20 mm3(-day 3) for 4MOSC1, or 40-60 mm3(-day 10) for MOC1, the mice were randomized into groups and treatment initiated. For IT treatment, 10 pg chemokine in 50 pl PBS or vehicle control (50 pl PBS) were injected directly into tumors. 4MOSC1 tumors were treated on days 3 and 6 post cell implantation, receiving a 3rddose on day 9 where indicated. MOC1 tumors were treated on days 10, 14, and 17. For experiments involving antibodies, mice were treated intraperitoneally (IP) with lOmg / kg anti-PD-1 antibody (clone J43) or indicated depletion antibody three times a week for 3 weeks. Tumor volumes were calculated as (width2)(length) / 2 (mm3). For survival experiments, mice were euthanized when the tumor burden exceeded 400-500 mm3. Otherwise, mice were euthanized and tumors dissected for flow cytometric analysis, bulk mRNA sequencing, or histologic evaluation at the indicated timepoints. The sample size for each experiment was selected based on power analysis of historical data and previous publications using the same tumor models (32, 44). Data collection was performed blind to the conditions of the experiments.

[0136] CXCL10 production and purification: A previously described E. coli expression / purification system involving chemokine fused to the C-terminus of a His-tagged ubiquitin variant (45) was modified and used herein to produce murine CXCL10 containing a tryptophan-serine sequence at the C-terminus to facilitate accurate determination of concentration without altering function. The His-tag enabled Ni -affinity chromatography purification of the detergent solubilized fusion protein, which was subsequently refolded in a redox buffer to form the correct disulfides. The ubiquitin facilitated cleavage of CXCL10 from ubiquitin by the deubiquitydating enzyme Usp2cc, leaving the native chemokine N-terminus that wasAttorney Docket No. 15670-0449WO1

[0137] required for WT activity' . A final purification step by reversed phase HPLC was used to isolate the desired protein with purity suitable for in vivo use and a yield of ~15 mg / L. The identity' of the final product was confirmed by mass spectrometry and the purity' was assessed via SDS-PAGE gel. The sequence of the final protein was as follows:

[0138] IPLARTVRCNCIHIDDGPVRMRAIGKLEIIPASLSCPRVEIIATMKKN DEQRCLNPESKTIKNLMKAFSQKRSKRAPWS (SEQ ID NO: 3).

[0139] Functionality' was assessed via cell migration with CXCR3+Jurkat cells and bioluminescent resonance energy transfer (BRET)-based G protein activation and -arrestin recruitment assays (86).

[0140] Animals: Animal studies using HNSCC tumor xenografts and orthotropic implantation studies were approved by the University' of California, San Diego IACUC, and adhered to all relevant ethical regulations for animal testing and research. Mice were housed at the Moores Cancer Center (University’ of California, San Diego) in a Micro-Isolator and individually ventilated cages supplied with acidified water. Mice were fed 5053 irradiated Picolab rodent diet 20 (LabDiet) in a temperature-controlled facility (~18 and 23°C, with 40-60% humidity ), with a 12 hour (h) light / 12 h dark cycle. Wildtype (WT) C57BL / 6N mice were from Charles River Labs. CXCR3" ’ mice (B6.129P2-Cxcr3tmlDgen / J), BATF3" ‘ mice (Batf3tmlKmm / J), and OT-1 mice (C57BL / 6-Tg(Tcra7c7'6)l lOOMjb / J, 003831) were from the Jackson Laboratory' (Bar Harbor, ME) and bred in-house.

[0141] 4MOSC1 andMOC cell lines: The 4MOSC1 cell line was generated in-house (32). M0C1 cells were provided as described (33). Authentication of cell lines was confirmed by multiplex STR profiling (Genetica DNA Laboratories, Inc. Burlington, NC). 4MOSC1 cells were grow n on culture dishes pre-coated with collagen (Coming, #354249) in Defined Keratinocyte-SFM medium (#10744019;

[0142] ThermoFisher) supplemented with 5 ng / mL EGF Recombinant Mouse Protein (#PMG8044; ThermoFisher), Defined Keratinocyte SFM Growth Supplement (#10784015; ThermoFisher), 50 pM Cholera Toxin (#C8052; Sigma-Aldrich), and 1% antibiotic / antimycotic solution (#BW17-603E; Fisher). MOC1 cells were cultured in 2:1 IMDM (#12440-046; ThermoFisher) and Hams Nutrient Mixture (#11765054; ThermoFisher) media supplemented with 5% fetal bovine serum (#Attorney Docket No. 15670-0449WO1

[0143] sh30071.03; HYCLONE), 1% antibiotic / antimycotic solution (#BW17-603E;

[0144] ThermoFisher), 5 mg / mL insulin (#10516; Sigma-Aldrich), 400ng / mL Hydrocortisone (# H0135; Sigma-Aldrich,), and 5 ng / mL EGF (#01-107; Millipore). All cells were cultured at 37°C / 5% CO2.

[0145] Flow cytometry: Established 4MOSC1 tumors were treated on days 3 and 6 with either vehicle or 10 pg of CXCL10 IT (per dose). Tumors were isolated, minced, and resuspended using the Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA), diluted into DMEM for subsequent processing with the gentleMACS Octo Dissociator. Digested tissues were passed through 70-pm strainers to produce single-cell suspensions. Samples were washed with PBS and processed for live / dead cell discrimination using Zombie viability stain (BioLegend, San Diego, CA). Cell suspensions were then washed with cell staining buffer (#420201; BioLegend) prior to staining with antibodies for 30 minutes (min) at 4°C, protected from light. Stained cells were washed and then fixed with BD cytofix for 20 min at 4°C, protected from light. In the case of intracellular staining, permeabilization was performed following surface staining by incubating the cells with intracellular targeted antibodies in fixation-permeabilization buffer (#88-8824-00; ThermoFisher) for 30 min at 4°C and protected from light. Samples were analyzed using either a BD LSR Fortessa™ X-20 or a Cytek® Aurora flow cytometer. Downstream analysis was performed using FlowJo™ version 10.8.1 (BD Life Sciences) software.

[0146] Tissue preparation and immunofluorescence (IF) staining for image acquisition: Tissues (buccal tumors and tumor draining lymph nodes) were harvested, fixed in zinc formalin fixative (Sigma- Aldrich) and sent to the Biorepository and Tissue Technology Shared Resources (BTTSR) at Moores Cancer Center (San Diego, CA) for paraffin embedding, sectioning, and IF staining. Histology samples were analyzed using QuPath 0.5.1 (Edinburgh, UK). Slides were scanned using a PhenoImager HT (Akoya Biosciences) at 40x resolution. H&E stained sections underwent brightfield whole slide scanning using an AT2 Aperio™ (Leica Biosystems) at 40x resolution. Image analyses were performed using the QuPath software using pixel classification of stained cells and positive cell counts.

[0147] The Cancer Genome Atlas (TCGA) analysis: The eBio Portal (cbioportal.org) was utilized to analyze transcriptomic data from the TCGA PanCancer Atlas data set for HNSCC (34). Hazard ratios were calculated from the log-rank test betweenAttorney Docket No. 15670-0449WO1

[0148] cohorts to determine the relationship between CXCR3 and CXCL10 on overall outcomes and inflammatory status in HNSCC patients. CXCR3hlgh / CXCL10hlghcohorts (n=128 and 132) and CXCL10low / CXCL10lowcohort (n=131 and 126) represent the upper 25% and lower 25% threshold for Cxcr3 or CxcllO mRNA expression respectively. Differentially expressed genes displayed in figures represent Log2 fold-change and P value (adjusted) cut-offs are 1 and 0.05, respectively between CXCL10hlghvs CXCL10lowcohorts using Wilcoxon Rank Sum test. Average mRNA gene expression amongst CXCL10hlghvs CXCL10lowcohorts were used to generate cell enrichment scores using the xCell Cell Enrichment (xcell.ucsf.edu) analysis platform (41).

[0149] NanoString nCounter® platform and data analysis with Rosalind®:

[0150] Established 4MOSC1 tumors were treated on days 3, 6, and 9 with either vehicle or 10 pg of CXCL10 IT (per dose). RNA was isolated by homogenization of whole tumors in TRIzol " (Invitrogen) followed by phenol: chloroform extraction and RNeasy Mini Kt based column purification with on-column DNase treatment (Qiagen). Hybndization of samples was performed at The Next Generation Sequencing Core (Salk Institute for Biological Studies, La Jolla, CA) according to the NanoString Hybridization Protocol for nCounter® XT CodeSet Gene Expression Assays. Samples were run on the nCounter® SPRINT Profiler with the nCounter® PanCancer Mouse Immune Profiling gene expression platform. Data was analyzed by Rosalind® (Rosalind®.bio) (San Diego, CA). Read distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step. Normalization, fold changes and p-values were calculated using criteria provided by NanoString. Rosalind® followed the nCounter® Advanced Analysis protocol of dividing counts within a lane by the geometric mean of the normalization probes from the same lane. Housekeeping probes used for normalization were selected based on the geNorm algorithm as implemented in the NormqPCR R library. Fold changes and pValues were calculated using the fast method as described in the nCounter® Advanced Analysis 2.0 User Manual. P-value adjustment was performed using the Benjamini -Hochberg method of estimating false discovery rates (FDR). Clustering of genes for the final heatmap of differentially expressed genes was done using the PAM (Partitioning Around Medoids) method using the fpc R library' that took into consideration the direction and type of all signals on a pathway (includingAttorney Docket No. 15670-0449WO1

[0151] the position, role and ty pe of every gene, etc). Hypergeometric distribution was used to analyze the enrichment of pathways, gene ontology, domain structure, and other ontologies. The top GO R library was used to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction.

[0152] Enrichment was calculated relative to a set of background genes relevant for the experiment. Cell abundance difference scores were generated using the Rosalind® Cell Type Profiling algorithm, which estimated relative cell abundance from NanoString data by averaging log2-normalized expression of curated marker genes, with positive or negative scores indicating enrichment in one group versus the other and opposite-signed values reflecting reciprocal comparisons rather than differences in magnitude. To ensure robustness, Rosalind® also provided a QC p-value based on marker gene consistency and specificity, with scores with p < 0.05 being considered highly statistically reliable.

[0153] Quantification of antigen-specific T cells in vivo: To detect TSA-T cells in the TME, 1 million 4MOSCl-LucOS cells (44) were implanted into the right buccal space of female C57B1 / 6 mice. When the average tumor volume reached 10-20 mm3, mice were randomized into groups and treatment was initiated. Treatments included 10 pg chemokine in 50 pl of PBS or 50 pl PBS (vehicle control) which were injected directly into tumors on days 3, 6, and 9. At days 10-15, tumors were isolated and processed to obtain a single-cell suspension as described above. Samples were then stained and analyzed via flow cytometry as detailed above. For antigen-specific T cell tetramer staining, Flex-T™ Biotin H-2 K(b) OVA Monomer (#280051;

[0154] BioLegend) paired with PE-streptavidin or APC-streptavidin (#405203 or 405207, respectively; BioLegend) were used according to manufacturers' instructions and as previously described (44).

[0155] T cell activation with CXCL10 in vitro: For T cell activation studies, a protocol was adapted from (70). Briefly, splenocytes were isolated from 4- to 6-week-old mice and mechanically disrupted. Red blood cells were lysed in red blood cell lysis buffer (BioLegend) according to manufacturer’s instructions. CD8+T cells were isolated with an EasySep™ CD8 isolation kit by / negative selection. For initial activation, naive CD8+T were cultured (0.5-1 million cells per mL) in 24-well plates with 50 U mL1IL-2 , 1 pg / mL of anti-mouse CD3 (#100340; BioLegend), and 2 pg / mL of anti-mouse CD28 (#102116; BioLegend) for 48 h. Cells were thenAttorney Docket No. 15670-0449WO1

[0156] collected, replated, and cultured in either 50 U / rnL IL-2 (PeproTech) alone, 50 U / mL IL-2 with 100 nM OVA257-264 peptide, or 50 U / mL IL-2 with OVA257-264 and CXCL10 (concentrations varying from 1-100 nM) for 24-48 h. Samples were then stained and analyzed via flow cytometry' as detailed in the main text.

[0157] Antibodies for therapeutic applications: The following antibodies were used: PD-1 antibody (clone J43, BE0033-2). isotype antibody (Armenian hamster IgG isotype control, BE0091), and CD8 depletion antibody (clone YTS 169.4, BE0117), CD4 depletion antibody (clone GK1.5, BE0003-1), and NK1.1 depletion antibody (clone PK136, BE0036) were obtained from Bio X Cell (Lebanon, NH).

[0158] Antibodies for flow cytometry: The following mouse antibodies and dilutions were used: CD45 (clone 30-FI I. 1:100). CD3 (clone 17A2. 1:200). CD8a (clone 53-6.7, 1:100), CD4 (clone RM4-4, 1:100), Slamf6 (clone 330AJ, 1:100), PD-1 (clone 29 F.1A12,1:1OO), CD44 (clone IM7, 1:100), CD 19 (clone 6D5, 1:100), CXCR3 (clone S18001A, 1:100), Tim3 (clone RMT3-23, 1:100), NK1.1 (clone PK136, 1:100), CD69 (clone H1.2F3, 1:100), CD62L (clone MEL-14, 1:100), Ly6C (clone HK.1.4, 1 : 100), CD 11 b (clone M 1 / 70, 1 : 100), CD 11 c (clone N418, 1 : 100), XCR1 (clone ZET, 1:100), CD64 (clone X54-5 / 7.1, 1:100), CD 103 (clone 2E7, 1:100), MHCII (clone M5 / 114.15.2, 1:200), CD80 (clone 16-10A1, 1:100), IFNy (clone XMG1.2, 1:100), granzyme B (clone GB11, 1:100), TNF-a (clone MP6-XT22, 1:100), LAG3 (clone C9B7W. l: 100). KLRGl (2F1 / KLRG1. 1:100). TCF1 (C63D9, 1:100), TOX (TXRX10, 1: 100), and Ki-67 (clone 16A8, 1 :100). Fluorochrome-conjugated antibodies were purchased from BD Biosciences (San Jose, CA) and BioLegend (San Diego, CA) unless otherwise indicated. Fluorochrome-conjugated antibodies were purchased from BD Biosciences (San Jose, CA) and BioLegend (San Diego, CA) unless otherwise indicated.

[0159] Antibodies for multiplex imaging: The following antibodies were used according to the BTTSR core’s protocol: CD4 (anti-mouse, Abeam), CD8 (antimouse, Invitrogen), CD 19 (anti-mouse, Cell Signaling). CD25 (anti-mouse, Cell Signaling), PanCK (anti-mouse. Dako), Granzyme B (anti -mouse, Invitrogen), CDllc (anti-mouse, Cell Signaling), CD80 (anti-mouse. Abeam), NK1.1 (anti-mouse, Cell Signaling), Ki67 (anti-mouse, GeneTex), LYVE-1 (anti-mouse, Invitrogen), and PD-1 (anti-mouse, Abeam).Attorney Docket No. 15670-0449WO1

[0160] Statistical analysis: Data analyses, variation estimation, and validation of test assumptions were conducted with GraphPad Prism version 10 statistical analysis program (GraphPad Software, Boston, MA) unless otherwise indicated. Differences between the tumor kinetics amongst experimental groups were analyzed using simple linear regression analysis, with solid lines representing mean tumor volumes and SEM at each timepoint and semi-transparent lines of corresponding color representing individual tumor volumes. The differences between experimental groups in cell density, response values, and tumor volume were analyzed using independent Atests or ANOVA (when >2 conditions). Survival analysis was performed using the Kaplan-Meier method and log-rank tests. The asterisks in each figure denote statistical significance, or ns for non-significant p > 0.05; *p < 0.05; **p < 0.01 ; and ***p < 0.001. All the data are reported as mean ± SEM, unless otherwise indicated. Experiments were independently repeated at least three times with similar results. Example 1: High CXCR3 and CXCL10 expression correlated with early tumor stage, increased immune infiltration, inflammatory gene signatures, and improved patient outcomes.

[0161] To determine the impact of CXCR3 and CXCL10 on outcomes and inflammatory status in HNSCC patients, we interrogated the Cancer Genome Atlas (TGCA, PanCancer Atlas) (34). Tumors with high CXCR3 and CXCL10 mRNA expression correlated with increased survival as characterized by hazard ratios (HR) less than 0.5 and 0.73, respectively (FIGS. 1A-1B). High CXCR3 and CXCL10 mRNA expression in HNSCC tumors also showed a positive correlation with CD8 antigen (CD8A) (FIGS. 1C-1D) indicating elevated levels of CD8+cells. Expression levels of both interferon gamma (IFNG) and granz me B (GZMB), indicative of inflammatory responses and cytotoxic activity, respectively, were significantly higher in the CXCL10hlghtumors (FIG. IE). IFN-y is a primary inducer of PD-L1 expression in cancer cells, rationalizing associations between IFNG expression or IFN-y-inducible gene signatures and positive clinical responses to PD-1 / PD-L1 blockade in HNSCC (35) and melanoma (36). Additional differentially expressed genes associated with interferon-mediated inflammatory’ responses in the CXCL10hlghvs CXCL10lowcohorts include CXCL9 and CXCL11, STAT1 / 2 (37), interferon regulatory factor 5 (IRF5) (38), and interferon-induced guanylate-binding proteinsAttorney Docket No. 15670-0449WO1

[0162] (GBP1 / 4 / 5) (39) (FIG. IF, FIG. 7). HNSCC lesions are often classified as early-stage (stage I— II) and late-stage (stage III-IV) based on tumor size and local invasion (40). A comparison of stage distribution amongst HNSCC tumors in CXCL10hlghvs CXCL10lowcohorts showed that 62.85% of tumors in the CXCL10lowcohort were graded in late-stage III-IV, whereas 44.87% of CXCL10hlghtumors were enriched in early-stage disease (FIG. 1G). These findings reinforced the role of CXCR3 / CXCL10 signaling in regulating tumor-immune suppression and disease progression.

[0163] To explore the association between immune infiltration and the CXCL10hlghvs CXCL10lo'vcohorts, cell enrichment profiles were also derived from TCGA gene expression data using the xCELL platform (41) (FIG. 1H). Elevated expression of CXCL10 in HNSCC patients was associated with over 2-fold increases in CD8 T cell subtypes, particularly antigen-experienced central (TCM) and effector (TEM) memory CD8+T cells. These cells can rapidly proliferate and differentiate into effector T cells, which enables a swift and robust immune response upon re-exposure to tumor antigen (42). CXCL10 expression also showed a strong correlation with NK cell (>40-fold) and Natural Killer T cell (NKT) (>4-fold) signatures (FIG. 1H). Notably, the data showed a correlation between high CXCL10 expression and broad DC gene signatures, including activated DCs (>2-fold), plasmacytoid DCs (pDCs, which express CXCR3) (>9-fold), and immature DCs (iDCs) (>500-fold) (FIG. 1H). DCs not only serve as the primary producers of CXCL10, but are also required for the presentation of tumor-derived antigens to CD4+and CD8+T cells and their priming and activation (18). These findings further indicate that increased CXCL10 expression had a strong association with anti-tumor immune signatures, particularly favoring the increased infiltration of cytotoxic cells and antigen-presenting DCs. Example 2: IT CXCL10 suppressed tumor growth and recurrence in syngeneic mouse models of HNSCC.

[0164] Given the correlation between positive outcomes in HNSCC patients and high expression of CXCL10, we investigated whether elevating tumor-localized CXCL10 levels by IT injection would promote tumor rejection in mouse models of HNSCC. Importantly, CXCL10 administered intratumorally (IT) was expected to be largely retained within the TME through glycosaminoglycan binding, which would extend itsAttorney Docket No. 15670-0449WO1

[0165] half-life by limiting proteolytic degradation and minimizing systemic dissemination, enabling sustained localized immune cell recruitment (43). For initial studies, we employed the 4MOSC1 model as tumors from this model share 98.9% similarity to human tobacco-associated HNSCC based on exome sequencing and mutational signatures, as well as similar immune infiltrates, and limited clinical responses to immunotherapy (32). 4MOSC1 tumors can be established orthotopically in the tongue or buccal space of mice, both of which are amenable to IT injection of CXCL10 (44). To produce murine CXCL10 in high yield (—13.5 mg / L) and purity (FIGS. 8A-8E), we modified a previously described E. coli expression / purification system (45).

[0166] 4MOSC1 tumors were established by injecting one million cells into the buccal space of 6-8 week-old C57BL / 6 mice. CXCL10 (10 pg) was administered intratumorally on days 3 and 6 post-tumor engraftment (FIG.2A). This treatment schedule was informed by prior observations that early lymphablation (days 3 and 6 post-tumor engraftment) eliminated responses to ICB and worsened overall survival by limiting antigen-specific T cell-driven immunity whereas late lymphablation (beyond day 11) did not impact the effectiveness of ICB (44). As shown in FIGS.2B and 2C, all mice treated with CXCL10 exhibited a significant reduction in tumor growth, with 30% (FIG. 2C; 24.4% of 4MOSC1 mice on average over all experiments, FIG. 8F) showing complete resolution and remaining tumor-free six weeks after clearance. Subsequent rechallenge with 4MOSC1 cells 6 weeks posttumor clearance did not result in tumor formation, indicating durable immunological memory (FIG. 2D)

[0167] We also used the MOC1 model, which like the 4MOSC1 model, exhibits a partial response to PD-1 / PD-L1 blockade that is comparable to patient outcomes (46). Following detection of MOC1 tumors in the flank at day 7 post-tumor engraftment, CXCL10 (10 pg) was administered on days 9, 13, and 16 (FIG. 2A). Consistent with the 4MOSC1 model, a significant suppression of tumor growth was observed in all CXCLIO-treated MOC1 tumors, with 22.2% (FIG. 2E; 21.9% of 4MOSC1 mice on average over all experiments, FIG. 8F) of the mice demonstrating a complete response and quickly clearing subsequent tumors upon rechallenge (FIG. 2F).

[0168] Although smaller, 4MOSC1 tumors from CXCLIO-treated mice exhibited HNSCCAttorney Docket No. 15670-0449WO1

[0169] histology similar to that of vehicle treated mice (FIGS. 2G-2H). Finally, we performed experiments in CXCR3-deficient (CXCR3 ) mice with the 4MOSC1 and MOC 1 models. For both models, mice failed to eliminate tumors post-CXCLIO treatment compared to wildtype (WT) controls (FIGS. 2I-2J).

[0170] Example 3: CXCL10 remodeled the tumor immune microenvironment by enhancing the infiltration of CD8+ T, CD4+ T, and NK cells.

[0171] Following activation, naive T cells upregulate CXCR3 expression, which remains persistent on effector CD8+T cells as well as type-1 helper (Thl) CD4+T cells that support CD8+T cell functions (47). CXCR3 is also highly expressed on innate NK cells and NKT cells, and is crucial for the localization of these first-line defenders to sites of inflammation and tumors (22, 48). Thus, we examined whether CXCL10 treatment would promote the infiltration of these cells into tumors. To explore CXCLIO-induced changes in the 4MOSC1 TME. we employed the nCounter® PanCancer Mouse Immune Profiling gene expression platform, which enabled broad characterization of immune cell populations and inflammatory status. Compared to vehicle treatment, CXCL10 treatment increased T and cytotoxic cell mRNA signatures, including signatures of CD8+T cells and NK cells (FIG. 3A). To investigate the dependence of the anti-tumoral response to CXCL10 on these immune subtypes, we treated 4MOSC1 tumor-bearing mice with CXCL10 and either CD8, NK1.1, or CD4-depleting antibodies. While a partial loss of CXCL10 response was observed with depletion of both CD4+and NK+cells, CD8+cell depletion resulted in the rapid and robust growth of tumors, highlighting the critical role that these cells play in the response to CXCL10 (FIG. 3B). Analysis by flow cytometry further validated these findings: tumors from CXCLIO-treated mice exhibited increased total CD8+T cell, CD4+T cell, and NK cell numbers as early as day 7, which persisted through day 10, compared to vehicle control (FIGS. 3C-3D).

[0172] The spatial distributions of tumor-infiltrating T cells can be a determinant of overall outcomes and are categorized into three phenotypic categories: immune-desert (lack of infiltration), immune-excluded (stromal infiltration without infiltration into the parenchyma), and immune-inflamed (infiltration into the tumor parenchyma) (49). In general, immune-inflamed tumors are associated with a more favorable overall prognosis and respond more positively to chemotherapy and ICB (50). We thereforeAttorney Docket No. 15670-0449WO1

[0173] investigated the abundance and spatial distribution of infiltrating immune cells within tumors via immunofluorescence (IF) staining. At day 10, tumors from CXCL10-treated mice showed substantial increases in infiltrating CD8+and CD4+T cells within the tumor parenchyma and surrounding stroma, suggesting an immune-infl amed tumor phenotype (FIG. 3E-3G). In contrast, most infiltrating NK cells were in the stroma of tumors from CXCLIO-treated mice, similar to the vehicle control mice (FIGS. 9A-9B). However, tumors from CXCLIO-treated mice also displayed a larger proportion of CD44+NK cells by flow cytometry at day 7, which persisted through day 13, compared to vehicle control (FIG. 9C). As increased CD44 expression on NK cells has been attributed to increased cytotoxic activity and IFN-y production (51), these data suggested that CXCL10 treatment induced these cells to acquire activation markers consistent with increased anti-tumoral functions.

[0174] CXCR3 expression is preferentially high on Thl CD4+ T cells following activation (52, 53); thus it is expected that this population makes up the bulk of infdtrating CD4+ T cell in tumors of CXCLIO-treated mice. However, CXCR3 is also expressed on regulatory T cells (Tregs), a subset of immunosuppressive CD4+T cells that ty pically promote tumor survival by limiting effector cell responses (54). To assess the proportion of tumor-infiltrating CD4+ cells that express forkhead box protein 3 (FoxP3), which serves as a crucial transcription factor for defining Treg subsets (54), we used IF. A reduced proportion of tumor-infiltrating FoxP31CD41cells were observed in CXCLIO-treated mice (FIGS. 3H-3I), suggesting that CXCLIO-treatment favors the recruitment of helper CD4+T cell subtypes.

[0175] Example 4: IT-CXCL10 enhanced the therapeutic efficiency of anti-PD-1 treatment to promote a durable anti-tumoral response.

[0176] When administered as a single agent, CXCL10 inhibited tumor grow th and resulted in complete tumor elimination in 24.4% of 4MOSC1 mice on average over all experiments, compared to 18.1% of mice with anti-PD-1 alone (FIG. 8F).

[0177] However, the remaining CXCLIO-treated mice developed resistance in subsequent weeks despite initially positive responses, resulting in relapse and tumor progression following the cessation of treatment. Tumor resistance frequently arises from the exploitation of immune checkpoints such as the PD-1 / PD-L1 pathway, which plays a central role in regulating T cell activation and exhaustion (2, 3). PD-1 isAttorney Docket No. 15670-0449WO1

[0178] predominately expressed on activated T and B lymphocytes, while its ligand PD-L1 is expressed by antigen-presenting cells, such as activated DCs and peripheral blood monocytes, as well as by cancer cells to evade immune detection (3). Notably, as for human HNSCC, 4MOSC1 tumors exhibit a high number of infiltrating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and monocytic myeloid-derived suppressor cells (M-MDSCs) that generally comprise the majority of PD-LlhlCD45+immune cells (32).

[0179] 4MOSC1 tumors from CXCLIO-treated mice contained a large percentage of PMN-MDSCs and M-MDSCs that were comparable to the vehicle-treated mice (FIG.

[0180] 4A) and likely contributed to the observed resistance. However, the PMN-MDSC and M-MDSC populations expressed higher levels of PD-L1 following CXCL10 treatment (FIG. 4B), suggesting that the anti -tumor efficacy following CXCL10 treatment might be boosted with the addition of PD-1 / PD-L1 blockade. To examine the effects of combined therapy in our HNSCC models, anti-PD-1 treatment was initiated at the start of the first IT-CXCL10 injection (FIG. 4C), resulting in an increase in tumor regression with 40% (FIGS. 4D-4E; 39.2% on average over all experiments, FIG. 8F) of the mice exhibiting complete and durable responses (>6 months) when compared to either single agent therapy.

[0181] MDSCs are also a major immunosuppressive cell population in MOC1 tumors (55). The benefit of combining CXCL10 with anti-PD-1 was therefore also explored in this model and resulted in 42.9% (FIGS. 4F-4G; 44.3% on average over all experiments, FIGS. 8F) of the mice showing complete tumor clearance and tumor-free survival for 6 months. This was a substantial improvement over treatments with either CXCL10 alone (28.6%, FIGS. 4F-4G; 21.9% on average over all experiments reported in this study, FIG. 8F) or anti-PD-1 alone (28.6%, FIGS. 4F-4G; 20.54% on average over all experiments reported in this study, FIG. 8F).

[0182] Example 5: CXCL10 promoted T cell-inflamed signatures, cytotoxic function, and memory formation in HNSCC tumors.

[0183] We next sought to determine if the robust increase in tumor-infiltrating T cells observed with CXCLIO-treatment was accompanied by broader transcriptional programs characteristic of T cell -inflamed tumors. Such signatures, encompassing coordinated expression of genes involved in T cell recruitment, activation,Attorney Docket No. 15670-0449WO1

[0184] cytotoxicity', and interferon signaling, have been strongly associated with productive anti-tumor immunity and improved responsiveness to immunotherapies (56). We therefore interrogated CXCL10- and vehicle-treated 4MOSC1 tumors via bulk mRNA profding, which showed that tumors from CXCLIO-treated mice not only displayed increased expression of T cell specific genes such as Cd3d, Cd3g, Cd7, and Cd8bl, but also many genes associated with T cell activation and cytotoxic function, notably Cd69. Ill8rl, Gzmk. Fas and Fas ligand (FIG. 5A, FIG. 10) (57-59). One of the most differentially expressed genes, Cd96. was highly expressed on effector and memory CD8+T cells as well as on CD4+T cells (particularly Thl cells), with expression being particularly elevated on antigen-experienced T cells and tumorinfiltrating lymphocytes (60). CXCL 10-treatment also amplified interferon-related gene signatures, exhibiting increased expression off / bg, Ifril2, and Cxcl9, CxcllO, and Cxclll (FIG. 5A), as well as interferon-stimulated genes (Isi44, Ifitl, Iflt2, Iflt3, and Isgl5) (FIG. 10). Altogether, increases in T cell-inflamed gene signatures observed with CXCL 10-treatment further supported the conclusion that CXCL 10 enhanced a transcriptional landscape consistent with heightened T cell infiltration, activation, and cytotoxic function. Consistent with this, IF staining revealed more extensive release of GZMB in proximity' to CD8+cells infiltrating the tumor parenchyma of CXC1 fl-treated mice (FIG. 5B).

[0185] CXCR3 is known to be rapidly downregulated upon ligand engagement and T cell activation, particularly^ in inflamed tissues such as tumors, which can obscure its detection at the protein level despite its functional involvement in T cell recruitment (43, 61), which we demonstrated in vitro (FIG. 11A). Consistent with this, the proportion of CXCR3+CD8+T cells within the TME of vehicle- vs CXCLIO-treated mice, although trending higher in CXCLIO-treated mice, was not significantly different and highly variable (FIG. 5C). Therefore, defining T cell subsets based on CXCR3 expression once they have reached the TME may not fully capture the functional relevance of the receptor due to its dynamic regulation.

[0186] Naive CD8+T cells undergo extensive phenotypic and functional diversification upon antigen enCounter®, giving rise to a spectrum of differentiated states including effector, memory, dysfunctional, progenitor-exhausted, and terminally exhausted populations (16, 42, 62). Early activation is marked by the upregulation of CD44 and downregulation of the circulatory marker CD62L, whichAttorney Docket No. 15670-0449WO1

[0187] reliably distinguishes antigen-experienced cells from naive counterparts but does not resolve the heterogeneity among these differentiated subsets. In addition to an increase in overall infiltration, flow cytometry revealed that a larger proportion of total CD4+and CD8+T cells were CD44+CD62L" by day 13 post-engraftment (FIGS.

[0188] 11B-11C), indicating that CXCL10 treatment favored the recruitment of activated antigen-experienced cells. However, additional phenotypic markers were required to define exact T cell lineage and functional states. We observed an increase in the proportion of CD44+CD62L+cells amongst the total CD8+T cells infiltrating tumors of CXCLIO-treated mice at day 10 that increased through day 13 (FIG. 5D). This indicated the emergence of a prominent population of CD8+T cells with a central memory (TcM)-like phenotype (63, 64) in the tumors of CXCLIO-treated mice. These TCM cells can self-renew and give rise to a pool of effector T cells, making them critical for long-term immunity (63). Further, this TcM-like population was significantly reduced in CXCR3' ' mice treated with CXCL10 compared to vehicle-treated WT control (FIG. 5D). suggesting that CXCR3 plays an important role in the maintenance of TcM-like CD8+T-cell populations within tumors.

[0189] Increased expression of genes involved in the function and maintenance of memory7T cell subsets was also revealed from bulk mRNA profiling (FIG.5A). Examples include Sell (controls CD62L expression), 117 r (controls CD 127, a marker highly expressed on TCM and TEM cells) (65), Bcl2 (critical for the persistence of memory T cells) (66), and Cxcr6 (controls retention of memory T cells) (67). Tumors from CXCLIO-treated mice also showed a marked increase in Il 10 gene expression (FIG. 5A) which is notable since a deficiency in IL- 10 signaling can lead to increased spontaneous tumor formation (68). IL- 10 also enhances IFN-y and granzyme production, facilitates antigen recognition, and is critical for the generation and maintenance of memory CD8+T cells (69).

[0190] Example 6: CXCR3 / CXCL10 signaling limited early indication of CD8+T cell dysfunction and exhaustion.

[0191] Chronic stimulation of TSA-T cells leads to the progressive loss of effector functions and upregulation of negative costimulatory markers associated with dysfunctional T cells (e.g., PD-lhlgh), abroad category- of hyporesponsive T cells, or cells that reach aterminally exhausted state (PD-lhlgh,TIM-3+) (16, 62). Thus, oneAttorney Docket No. 15670-0449WO1

[0192] might expect an increase in the expression of PD-1 and other inhibitory molecules (e.g., TIGIT and CTLA4) to be correlated with increased TSA-T cell activity in tumors. This was indeed observed in the tumors of CXCLIO-treated mice compared to a vehicle control via NanoString analysis of bulk tumor (FIG. 5A). However, a significant decrease in PD-1 expression compared to vehicle control was observed when probing only activated CD44+CD8+T cell populations (FIGS. 5E-5F). Thus, despite the overall increase in tumor-infiltrating cells in response to IT-CXCL10, a lower proportion of them were PD-lhlgh, suggesting that CXCL10 may inhibit the progression of activated T cells towards dysfunctional or exhausted states.

[0193] Importantly, while exhausted T cells represent a defined differentiation state with preserved proliferative potential (Ki67+), dysfunctional T cells reflect a more terminally impaired population defined by PD-llllghTOX+TIM310expression with diminished proliferative capacity and limited responsiveness to reinvigoration (16, 62). A decrease in the proportion of CD8+T cells displaying these markers of dysfunction was also observed in the TdLN of CXCLIO-treated mice (FIG. 11D). To evaluate if CXCL10 treatment alone reduced T cell dysfunction outside of the context of the TME, we conducted in vitro experiments with primary CD8+T cells from OT-1 transgenic mice whose TCRs are specific for the OVA peptide SIINFEKL (OVA257 264) (70). Upon stimulation with CXCL10, a decrease in PD-1 expression was observed in CD44 CD8 OT-1 T cell populations compared to cells activated with only SIINFEKL (FIGS. 11E-11F). These results suggested that CXCL10 limited T cell progression towards dysfunction even when antigen stimulation persists. While no change was observed with CXCL10 treatment alone, the addition of anti-PD-1 greatly reduced the proportion of terminally exhausted (PD-lhlghTIM3+) CD8+T cells in our 4MOSC1 models at day 10 (FIG. 5G). We also observed a significant increase in the proportion of terminally exhausted (PD-lhlghTIM3+) CD8+T cells in CXCR3 / _mice treated with CXCL10 (FIG. 5G), further suggesting that CXCR3 / CXCL10 signaling plays an important role in limiting dysfunction and exhaustion phenotypes in tumor responses.

[0194] Example 7: IT-CXCL10 reduced tumor angiogenesis and lymphangiogenesis.

[0195] CXCL10 has been shown to inhibit endothelial cell migration and proliferation associated with lymph and blood vessel development (15, 71). Consistent with this,Attorney Docket No. 15670-0449WO1

[0196] tumors from CXCLIO-treated mice showed downregulation of several genes that promote angiogenesis by regulating epithelial cell adhesion and vascular development, including DII4 (Delta ligand 4), Cdh5 (VE-cadherin), and Fw / '(Von Willebrand factor) (FIG. 5A) whose elevated expression is a prognostic biomarker of metastasis and recurrence in several human cancers (72, 73). IF-staining of 4MOSC1 tumor sections from CXCLIO-treated mice also showed decreased lymph and blood vessel density compared to tumors from vehicle-treated mice (FIGS. 5H-5I, FIG. 12A). This suggested that in addition to recruiting and activating T and NK cells, IT-CXCL10 may promote anti-tumoral effects and reduce metastasis by decreasing angiogenesis and lymphangiogenesis within the TME.

[0197] Example 8: IT-CXCL10 enhanced T cell proliferation and TSA-T cell formation in the TdLN.

[0198] Presentation of tumor antigens by DCs is a critical step in the activation of cytotoxic CD8+T cells and their ability to recognize TSAs (18). Following antigen uptake, DCs undergo maturation, marked by increased expression of costimulatory molecules such as CD80, CD86, and CD40, upregulation of PD-L1, secretion of proinflammatory cytokines, and migration to TdLNs via CCR7:CCL21 signaling (74). Conventional type 1 DCs (cDCls) also play a pivotal role in regulating the movement of activated T cells into the TME by secreting CXCL9 and CXCL10, indicating crosstalk between cDCls and T cells within tumors (75). Accordingly, we evaluated gene signatures related to DC function and observed a significant increase in those corresponding to costimulatory makers such as Cd.86. Cd48. and H2-M2. as well as Nlrc5, a key regulator of MHC-I, within the tumors of CXCLIO-treated mice compared to vehicle controls (FIG. 5A) (76). A larger proportion of DCs within the tumors of CXCLIO-treated mice also displayed increased PD-L1 (FIG. 12B).

[0199] Interferons upregulate MHC-I and II expression and stimulate antigen presentation (77). Thus, it is notable that tumors from CXCLIO-treated mice displayed increased interferon-related gene signatures (FIG.5A, FIG. 10). Additionally, there was an increase in Ccr7 alongside a marked decrease in Ccl21 expression within the TME following CXCL10 treatment, which may reflect an increase in DC trafficking to the TdLN (FIG. 5A). Consistent with the above, tumor rejection following CXCL10 treatment was lost in 4MOSC1 tumor-bearing BalCF ' mice, which are deficient inAttorney Docket No. 15670-0449WO1

[0200] eDCs (78) (FIG.3B). Together these data support the dependence of CXCL10-stimulated CD8+T cells on eDCs for priming to tumor-derived antigens.

[0201] TdLNs serve as hubs for antigen presentation by DCs to naive T cells, which induces rapid proliferation of TSA-T cells (79). Thus, a higher density of proliferating T cells within the TdLN is indicative of more robust T cell activation, expansion, and trafficking to the TME, and correlates with improved local and systemic immune surveillance for tumor-specific antigens (79, 80). Importantly, prior studies demonstrate that the CXCR3 axis is critical in the expansion of CD8+T cells in the TdLN following PD-1 blockade in the tumor microenvironment (20). As shown in FIGS. 6A-6C, IF staining of TdLNs from CXCLIO-treated mice showed a significant increase in both CD4+and CD8+T cells as well as antigen-presenting cells (CD1 lc+) within the paracortex region (T cell zone) compared to those from vehicle-treated mice. CD4+and CD8+T cells within the TdLN of CXCLIO-treated mice also displayed more robust proliferation, as indicated by increased Ki67 expression (FIG.

[0202] 6A). Notably, not only did a larger proportion of T cells within the TdLN of CXCLIO-treated tumors express CXCR3, consistent with successfully primed Thl and CD8 T cells, but CXCR3 expression increased further with the addition of anti-PD-1 therapy (FIG. 6D).

[0203] Progenitor exhausted T (TPEX) cells are a crucial subset of antigen-specific stem-like CD8 T cells, characterized by T-cell factor 1 (TCF1) expression, that reside in lymph nodes and possess self-renewal and proliferative abilities vital for maintaining sustained anti-tumor immunity, especially in response to checkpoint inhibitor immunotherapies (62, 80, 81). Prior studied show that ICB enhances TPEX abundance and proliferation in the TdLN, diminishes exhaustion phenotypes among tumor-infiltrating T cells, and improves survival outcomes in mouse tumor models (62, 82). Interestingly, we observed an increase in proliferating TPEX (Ki67+TCF1 TOX TIM3 ) CD8 T cells within the TdLN of mice treated with CXCL10 alone and in combination with anti-PD-1 compared to a vehicle control (FIG. 6E-6F). TPEX were reduced in the TdLN of CXCR3’ ‘ mice however (FIG. 6E-6F), highlighting a strong reliance on CXCR3 for the formation of these phenotypes. While a reservoir of TPEX cells was maintained in the TdLN, they also migrated into tumors, where they maintain stem-like properties and can differentiate into potent effector cells, supporting long-term anti-tumor immunity (62. 80, 81). Notably, aAttorney Docket No. 15670-0449WO1

[0204] trending increase in TPEX cells was observed in the TME following CXCL10-treatment at day 10 compared to a vehicle control, with a significant decrease in TPEX cells present in CXCR3 / _mice (FIG. 12C).

[0205] Given the observed increase in T cell proliferation and TPEX populations in the TdLN with CXCLIO-treatment, we examined whether CXCL10 would enhance the increased accumulation of CD8+T cells reactive to tumor-specific antigen (TSA-T cells) within the TME. To explore this, we employed the 4MOSCl-LucOS model in which mice develop 4MOSC1 tumors that express OVA peptide, and quantified the abundance of tumor-infiltrating CD8+TSA-T cells with the OVA-H-2Kb tetramer, as previously described (44). Tumors from CXCLIO-treated mice had a significantly higher proportion of TSA-T cells compared to control-treated mice (FIGS. 6G-6H).

[0206] These data suggested that CXCL10 treatment enhanced the recruitment and activation of cells that support cytotoxic function by favoring the formation and maintenance of tumor antigen-specific populations.

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[0299] OTHER EMBODIMENTS

[0300] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 15670-0449WO1WHAT IS CLAIMED IS:

1. A method of enhancing tumor clearance in a subject in need thereof comprising locally administering an effective amount of a recombinant CXCL10 protein to a tumor or an area surrounding the tumor.

2. A method of treating a cancer comprising locally administering to a subject in need thereof an effective amount of a recombinant CXCL10 protein to a cancerous tumor or an area surrounding the cancerous tumor.

3. The method of claim 1 or 2, wherein the subject in need thereof has or is suspected of having at least one solid cancerous tumor.

4. The method of claim 3, wherein the at least one solid cancerous tumor comprises a rimary tumor, a metastatic tumor, or both.

5. The method of any one of claims 1-4, wherein the subject in need thereof has or is suspected of having at least one cancer selected from the group consisting of head and neck cancer, head and neck squamous cell carcinoma, colon cancer, liver cancer, gastric cancer, lung cancer, or melanoma.

6. The method of any one of claims 1-5, wherein the local administration compnses administering the recombinant CXCL10 protein to a surgical margin following resection of the tumor.

7. The method of any one of claims 1-5, wherein the local administration comprises intratumoral injection of the recombinant CXCL10 protein.

8. The method of any one of claims 1-7, wherein the recombinant CXCL10 protein is locally administered to the subject at least once.Attorney Docket No. 15670-0449WO19. The method of claim 8, wherein the recombinant CXCL10 protein is locally administered to the subject two or three times.

10. The method of any one of claims 1-9, wherein the recombinant CXCL10 protein comprises about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3.

11. The method of claim 10, wherein the amino acid sequence of the recombinant CXCL10 protein is SEQ ID NO: 3.

12. The method of any one of claims 1-11, further comprising administering at least one cancer therapy to the subject in need thereof, wherein the cancer therapy is selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy.

13. The method of claim 12, wherein the immunotherapy comprises administration of at least one immune checkpoint blockade (ICB) agent.

14. The method of claim 13, wherein the at least one ICB agent is an inhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA4), programmed cell death protein 1 (PD-1), PD-L1. LAG3, B7-H3, B7-H4, or TIM3.

15. The method of claim 14, wherein the at least one ICB agent is a PD-1 / PD-L1 inhibitor selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, pidilizumab, AMP-224, durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MED1-4736, MSB-0010718C, and MDX-1105.

16. The method of any one of claims 12-15, wherein the subject in need thereof has or is suspected of having a cancer that is resistant to PD-1 / PD-L1 inhibitors.Attorney Docket No. 15670-0449WO117. A pharmaceutical composition comprising a recombinant CXCL10 protein and at least one pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein the recombinant CXCL10 protein comprises about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3.

19. The pharmaceutical composition of claim 18, wherein the amino acid sequence of the recombinant CXCL10 protein is SEQ ID NO: 3.

20. The pharmaceutical composition of any one of claims 17-19, wherein the pharmaceutical composition is formulated for intratumoral administration.

21. A combination therapy for treating cancer comprising intratumorally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of any one of claims 17-20 and a therapeutically effective amount of an immune checkpoint blockade (ICB) agent.

22. The combination therapy of claim 21, wherein the ICB agent is systemically administered to the subject in need thereof.

23. The combination therapy of claim 21 or 22, wherein the ICB agent is a PD- 1 / PD-L1 inhibitor, optionally wherein the ICB agent is an anti-PD-1 antibody or an anti-PD-Ll antibody.

24. A method of making a recombinant CXCL10 protein comprising:i. preparing a vector encoding a fusion protein, wherein the fusion protein comprises, from N terminus to C terminus, 6-His tag - ubiquitin - CXCL10;Attorney Docket No. 15670-0449WO1ii. expressing the fusion protein in a host cell:iii. harvesting the fusion protein from the host cell;iv. purifying the harvested fusion protein by Ni-affinity chromatography; andv. cleaving the 6-His tag from the CXCL10 with Usp2-cc to result in the recombinant CXCL 10 protein.

25. The method of claim 24, further comprising purify ing the recombinant CXCL10 protein by reverse phase chromatography wherein resulting recombinant CXCL10 protein is at least 80%. 85%. 90%. 95%. 96%. 97%. 98%. 99%. or 100% pure.

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