Methods and compounds for cancer treatment based upon antigen-agnostic interferon induction

Abstract

This invention provides methods, compositions, kits and diagnostics for treating cancer in a subject, which includes administering to the subject a cytokine composition comprising interleukin-17 (IL-17), and at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36. The cytokine composition is delivered to a tumor microenvironment (TME) using a targeted delivery system. The cytokine composition induces antigen- independent interferon-γ (IFN-γ) production by bystander immune cells in the TME, including at least CD8+ T cells, natural killer (NK) cells, or γδ T cells. At least one tumor response parameter is measured, which is selected from tumor size, tumor cell viability, immune-cell IFN-γ production, or tumor-associated immune activation markers. A dose or administration frequency of the cytokine composition is measured based on the measured tumor response parameter.

Description

Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 METHODS AND COMPOUNDS FOR CANCER TREATMENT BASED UPON ANTIGEN- AGNOSTIC INTERFERON INDUCTION STATEMENT REGARDING FEDERAEEY SPONSORED RESEARCH OR DEVEEOPMENT

[0001] This invention was made with government support under CA205965 awarded by the National Institutes of Health. The government has certain rights in the invention.FIEED OF THE INVENTION

[0002] This invention relates to treatments and compounds for cancer, and more particularly to mechanisms relative to tumor-specific T cells that kill tumors through interferon (IFN-y) production.BACKGROUND OF THE INVENTION

[0003] Tumor-specific T cells kill tumors through mechanisms that largely depend on IFN-y production. However, chronic antigen exposure makes tumor-specific T cell exhausted, and thus less able to kill and produce IFN-y in the tumor microenvironment (TME). It is desirable to enhance such mechanisms, and reduce negative effects associated therewith.SUMMARY OF THE INVENTION

[0004] This invention overcomes disadvantages of the prior art by providing methods and compositions for improving cancer treatment, including, increasing IFN-y from bystander immune cells in a tumor microenvironment (TME) though introduction of cytokines of predetermined composition, monitoring results in tumor cells associated with the TME, and modifying a level of the introduction in response to the monitoring.

[0005] In an illustrative embodiment, a method of treating cancer in a subject, comprising is provides. A cytokine composition comprising interleukin- 17 (IL- 17), and at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36, is administered to the subject. The cytokine composition is delivered to a tumor microenvironment (TME) using a targeted delivery system. The cytokine composition induces antigen-independent interferon-y (IFN-y) production by bystander immune cells in the TME, including at least CD8+T cells, natural killer (NK) cells, or y6 T cells. At least one tumor response parameter selected from tumor size, tumor cell viability, immune-cell IFN-y production, or tumor-associated immune activation markers is then measured. A dose orDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 administration frequency of the cytokine composition is adjusted based on the measured tumor response parameter. Illustratively, the cytokine composition comprises IL- 17. IL-2, and IL-33, and can further comprise IL-18. The cytokines can be delivered as nucleic acids encoding the cytokines, and / or the nucleic acids can comprise messenger RNA molecules encoding the cytokines. The cytokine composition can be delivered using lipid nanoparticles configured to accumulate within tumor tissue. The cytokine composition can alternately, or additionally be delivered by engineered immune cells expressing nucleic acids encoding the cytokines. A cytokine delivery system can comprise a targeting moiety7that binds a tumor-associated antigen. The targeting moiety7can comprise an antibody or antigen-binding fragment thereof. The cytokine composition can be targeted to immune cells expressing CD8, PD-1, or NKG2A. The cytokine composition can comprise an engineered IL-2 variant having reduced CD25 binding and increased CD122 binding. The cytokine composition can be administered by intratumoral injection, and / or in combination with an immune checkpoint blockade therapy. The immune checkpoint blockade therapy can target PD-1, PD-L1. PD-L2. CTLA-4, or LAG-3. The administration of the cytokine composition can increase anti-PD-L2 therapeutic activity within tumor tissue while maintaining lower anti-PD-L2 activity in non-tumor tissue.

[0006] In an illustrative embodiment, a cy tokine delivery sy stem for inducing IFN-y production in tumor-infiltrating immune cells is provided. It includes a carrier comprising lipid nanoparticles, engineered immune cells, viral vectors, or polymeric nanoparticles, and a payload comprising IL- 17 and at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36. The delivery7system can be configured to deliver the payload to a tumor microenvironment and induce antigen-independent IFN-y production in bystander immune cells. Illustratively, the carrier comprises lipid nanoparticles encapsulating mRNA encoding the at least one cytokine. A tumor-targeting moiety can bind a tumor-associated antigen, and / or can comprise an antibody against Her2 or another tumor-associated antigen. The carrier can comprise engineered immune cells expressing nucleic acids encoding the at least one cytokine.

[0007] In an illustrative embodiment, a kit for treating cancer is provided. A first container comprises IL- 17, and a second container comprises at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36. A delivery7formulation comprises lipid nanoparticles or nucleic acid vectors configured for delivery to a tumor microenvironment. Instructions for administering the cytokines are provided to induceDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 antigen-independent IFN-y production by tumor-infiltrating immune cells. Illustratively, the at least one cytokine is provided as mRNA molecules encoding the at least on cytokine. The kit can further comprise a tumor-targeting antibody or binding fragment. An immune checkpoint inhibitor can be selected from an anti-PD-1 antibody, anti-PD-L2 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, or anti-LAG-3 antibody. The instructions can specify intratumoral or tumor-targeted systemic administration.

[0008] In an illustrative embodiment, a pharmaceutical composition is provided. A delivery vehicle is selected from lipid nanoparticles, polymeric nanoparticles, viral vectors, or engineered immune cells. A payload comprising interleukin- 17 (IL-17) and at least one cytokine is selected from IL-2, IL-12, IL-18, IL-33, or IL-36, or nucleic acids encoding said cytokines. The delivery vehicle is configured to deliver the payload to a tumor microenvironment and induce interferon-y production in tumor-infiltrating immune cells.

[0009] In an illustrative embodiment, a method for treating cancer in a subject is provided. A biological sample is obtained from the subject, comprising tumor tissue or immune cells associated with a tumor microenvironment. In the biological sample, a biomarker is measured, which is selected from interferon-y (IFN-y) expression or programmed death ligand-2 (PD-L2) expression. Based on the measured biomarker, it is determined whether the subject is suitable for treatment with a cytokine composition comprising interleukin- 17 (IL- 17) and at least one additional cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36. The cytokine composition is administered to the subject. The cytokine composition induces antigen-independent interferon-y production in immune cells within the tumor microenvironment. Illustratively, the cancer comprises melanoma, and / or the biomarker comprises PD-L2 expression in tumor tissue. The subject can be an aged subject exhibiting chronic inflammation.

[0010] In an illustrative embodiment, a diagnostic method for identifying a subject suitable for treatment with a cytokine therapy is provided. A biological sample is obtained from a subject having cancer. Expression of a biomarker selected from interferon-y (IFN-y) or programmed death ligand-2 (PD-L2) is detected in the biological sample. It is then determined whether the subject is suitable for treatment with a cytokine composition comprising IL- 17 and at least one additional cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36, based on the detected biomarker expression.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0011] In an illustrative embodiment, a method of inducing antigen-independent interferon-y production in tumor-infiltrating immune cells in a tumor microenvironment is provided. It includes administering interleukin- 17 (IL-17) together with at least one cytokine capable of stimulating innate-like interferon-y production in T cells. The administration is then monitored and adjusted.

[0012] In an illustrative embodiment, a method of treating cancer is provided. It includes administering a cytokine composition comprising interleukin- 17 (IL-17) and one of either (a) interleukin-33 or (b) at least one additional cytokine. The administration is then monitored and adjusted. Illustratively, the composition is delivered to a tumor microenvironment using a targeted delivery vehicle that preferentially accumulates in tumor tissue.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The invention description below refers to the accompanying drawings, of which:

[0014] Fig. 1 is a diagram showing how PD-L2 signals differ markedly from PD1 / PD-L1, in which PD-L2 engages PD1 3.3-fold more avidly than PD-L1 and engages RGMB whereas PD1 and PD-L1 do not. RGMB binds multiple bone morphogenic proteins and neogenin. Thus, PD-L2 blockade outcomes differ markedly from PD1 / PD-L1 blockade;

[0015] Figs. 2A-2D are graphs showing how aPD-L2 treats aged B 16 and NrasQ61R-mutant melanomas, in which young (Figs. 2A or 2B) aged B16-challenged WT mice treated with aPDL, and young (Figs. 2C or 2D) aged NrasQ61R-mutant melanoma-challenged WT mice treated with aPD-L2. N= 9+ / group;

[0016] Fig. 3 is a graph showing that IFN-y is required for aPD-L2 efficacy, in which aged IFN-yKO mice challenged with B16 and treated as shown. N= 8+ / group;

[0017] Figs 4A-4D are graphs showing that IL-17 is required for aPD-L2 but not aPDl efficacy in aged, in which mice challenged with B16 and treated as shown, and N= 8+ / group;

[0018] Figs. 5A-5C are graphs showing aged host IL-17 modulates aPD-L2-mediated IFN-y effects, where in Fig. 5A IFN-y+CD8+ T cell prevalence, in Fig. 5B percell IFN-y production in CD8+ T cells, and in Fig. 5C CXCR3+IFN-y+CD8+ T cellDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 prevalence, and in which data from aged mice challenged SQ with Bl 6 and sacrificed when tumors -700 mm3for flow cytometry. N=7+ / group;

[0019] Fig. 6 is a graph showing aPD-L2 efficacy requires CD8+ T cells. Bl 6 challenged into aged WT and treated with aPD-L2 ± aCD8, tumor grow th. P;

[0020] Fig. 7 shows graphs of aPD-L2 efficacy against NCH1 melanoma is also IL-17-dependent, in which aged IL-17KO mice challenged with NCH1 SQ and treated with aPD-L2 or isot pe as described for Bl 6;

[0021] Figs. 8A-8E are graphs showing that y5 T cells are required for aPD-L2 efficacy and promote CD8+ T cell effector functions (Fig. 8A), and aged y5 TCRKO mice challenged with B16 and treated as shown, and flow cytometry of TIL in these mice (Figs.8B-8E);

[0022] Figs. 9A-9G are diagrams variously describing human y5 T cell dynamics in melanomas differ by host age, in which Fig. 9A shows UMAP projection of y5 T cells stratified into T d I and Ty517 subsets using IFNG and IL-17 A gene expression respectively, Fig. 9B shows differential gene expression of markers validating the classification of y5 T cells, Ty51 / Ty517 subsets and expression of PDCD1LG2 (PD-L2) in each subset, Fig. 9C shows overall prevalence of y5 T cell subsets stratified by age group (young < 67 years, old >67 years), Fig. 9D shows IL-17A w IFNG gene expression in Ty51 / Ty517 subsets stratified by patient age, Fig. 9E shows PDCD1LG2 gene expression in all y6 T cells and Ty51 / Ty§17 subsets stratified by patient age, Fig. 9F shows statistical analysis o!PDCDlLG2 gene expression in all y5 T cells and Ty51 / Ty517 subsets with a median age cutoff of 67 years (young < 67 years, old >67 years), and Fig.9G shows Gene Ontology (GO) analysis of pathway s enriched in tumor-infiltrating y5 T cells old versus young patients;

[0023] Figs. 10A-10D are graphs showing that aPDl elicits IL-17-independent IFN-y. Aged IL-17KO challenged with SQ B16, treated with aPDl. CD45+CD3+CD8+ or y5 T cells;

[0024] Fig. 11 is a pair of graphs showing that aPD-L2 treats young MB49 but treats aged better. WT mice challenged SQ with MB49 and treated as shown. N-10 / group;

[0025] Fig. 12 is a graph showing that exogenous IL- 17 controls young Bl 6 grow th and promotes aPD-L2 efficacy, in which young WT challenged with Bl 6 and treated with aPD-L2 ± rIL-17 1 pg / mouse daily on d-7 through d+11 ;Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0026] Fig. 13 is a graph showing that CD4+ T cell IL- 17 contributes to aPD-L2 efficacy, in which aged IL-17KO with transfer of 1 x 106age-matched naive WT CD4+ T cells IV 1 day before B16 challenge and treated with aPD-L2 as described. aIL-17R 25 pg given IP, day 18 (isotype n=6, aPD-L2 n=8);

[0027] Fig. 14 is a diagram showing NCH1 is highly metastatic, in which lung tissue shows metastatic tumor colonies in NCH1 via crystal violet stain, 40x light microscopy;

[0028] Fig. 15 is a diagram showing potential interactions between aPD-L2, cDCl, CD4+ T cells, CD8+ T cells and y6 T cells;

[0029] Figs. 16A and 16B are a pair of graphs showing IL- 17 signals differentially in young versus aged T cells, in which young and aged tumor-naive mice treated 4 days with recombinant IL-17, aPD-L2, both or controls for 4 days and sacrificed for study of spleen cells by flow cytometry gated, wherein Fig. 16A depicts CD45+CD3+CD8+CD4-cells, and Fig. 16B depicts CD45+CD3+CD8-CD4+ cells;

[0030] Figs. 17A and 17B are graphs showing immune checkpoint expression in the aged TME, in which flow cytometry of tumor-infiltrating cells in B16-bearing indicate mice;

[0031] Figs. 18A and 18B are graphs showing PD-L2-binding yeast clones show different blocking profiles, and in which Fig. 18 A shows flow cytometry plots of the naive yeast library and a clone binding to PD-L2, and Fig. 18B shows representative yeast clones that block both PD-1 and RGMB or RGMB only, and anti-mouse PD-L2 (clone TY25) serves as a positive control as it blocks both PD1 and RGMB binding interactions with PD-L2;

[0032] Fig. 19 is a flow diagram showing an exemplary method for inducing interferon-y production in tumor-infiltrating immune cells through administration of a cytokine composition and adjustment of treatment parameters;

[0033] Fig. 20 a diagram showing an exemplary cytokine delivery architecture showing delivery of cytokine-encoding nucleic acids to a tumor microenvironment using lipid nanoparticles;

[0034] Fig. 21 is a diagram showing an example of an immune signaling mechanism by which cytokine combinations activate bystander immune cells to produce interferon-y and mediate tumor cell killing;Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0035] Fig. 22 is a diagram showing treatment of melanoma in an aged host exhibiting chronic inflammation, in which cytokine therapy restores interferon-y production by immune cells and promotes melanoma tumor regression;

[0036] Fig. 23 is a diagram showing an exemplary combination therapy using cytokine compositions together with anti-PD-L2 checkpoint blockade to enhance tumorspecific immune responses and promote tumor cell killing;

[0037] Fig. 24 is a diagram showing an example of mechanistic signaling pathway by which cytokine combinations comprising IL- 17 and IL-33 stimulate immune cells in a tumor microenvironment to produce interferon-y (IFN-y) and promote tumor cell killing;

[0038] Fig. 25 is a flow diagram showing an exemplary workflow for biomarker-guided treatment selection, including detection of interferon-y (IFN-y) or PD-L2 expression and administration of cytokine therapy based on the detected biomarker;

[0039] Fig. 26 is a diagram show ing an example of a pharmacokinetic and tumortargeting model for cytokine therapy, showing delivery of cytokine compositions to tumor tissue and localized induction of IFN-y production within the tumor microenvironment;

[0040] Fig. 27 is a diagram showing an example of an integrated immunotherapy platform including biomarker-guided patient selection, cytokine therapy administration, immune checkpoint blockade, and tumor-specific immune activation leading to tumor regression;

[0041] Fig. 28 is a diagram showing an example of a clinical treatment protocol timeline for administering cytokine therapy, monitoring biomarkers, and evaluating tumor response in a subject receiving immunotherapy;

[0042] Fig. 29 is a diagram with a series of plots, based upon experimental results on splenocytes from young, naive BL6 mice, showing how IL- 17 elicits EOMES-associated IFN-y from young T cells in vitro

[0043] Fig. 30 is a diagram series of graphs, based upon experimental results, showing how aPD-L2 increases innate IFN-y in T cells of young naive BL6 mice in vitro in response to IL- 12 in an IL-17-dependent manner;

[0044] Figs. 31 A and 3 IB are diagrams of graphs, based upon experimental results from T cells of young, naive BL6 mice, how aCD134 + aCD137 elicit aPD-L2 efficacy in Bl 6 melanoma when challenged with an increase in innate IFN-y in T cells; andDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0045] Fig. 32 is a diagram of graphs, based upon experimental results from T cells of young, naive BL6 mice, showing that aCTLA4 improves aPD-L2 in an IL- 17-d epend ent manner after being challenged subcutaneously with B6 melanoma cells.DETAILED DESCRIPTION

[0046] I. Antigen-Agnostic Interferon (IFN)-y Induction

[0047] This description identifies a method to increase IFN-y from bystander immune cells in the TME that are not tumor-specific, and thus not exhausted. As the method capitalizes on the activated (but not exhausted) state of tumor-infiltrating T cells, systemic toxicity is mitigated. It is known that adaptive (antigen-specific) immune cells generate IFN-y by antigen recognition, whereas innate (antigen-independent) immune cells generate IFN-y in response to specific cytokines. It is also known that certain cytokines, notably IL-2, IL-12, IL-18, IL-33 and IL-36 in specific combinations can elicit innate behavior in adaptive immune cells, specifically meaning generating antigenindependent IFN-y from CD8+ T cells and other adaptive immune cells, but also increasing IFN-y innate immune cells such as NK and y5 T cells, to increase anti-tumor immunity further. For example, the specific combinations of IL-2+IL-33 and IL-18+IL-33 among other combinations elicits antigen-independent IFN-y from CD8+ T cells. It was found that IL- 17 very significantly increases CD8+ T cell IFN-y when combined with IL-2+IL-33 or IL-12+IL-33. IL- 17 also increases NK cell and y5 T cell IFN-y when used in combination with IL-2+IL-33 and IL-18+IL-33. As tumor antigen-specific T cells are generally exhausted whereas bystander, non-tumor specific T cells are not, inducing IFN-y in these bystander cells could improve cancer treatment efficacy.

[0048] Systemic delivery of these cytokines can induce toxicities that can be mitigated by delivery to the tumor microenvironment (TME) selectively. Such delivery can be in targeted lipid nanoparticles or in engineered T cells or other immune cells, such as B cells. Tumor targeting can be achieved by adding a tumor-specific engaging molecule such as an antibody to a tumor-specific antigen such as Her2.

[0049] Tumor targeting can also be achieved by coupling albumin or a tumorbinding fragment of albumin to the cytokine delivery moiety. Albumin coupling of the cytokine delivery moiety can also be achieved by adding an albumin targeting, moiety to the cytokine delivery system.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0050] Cytokine delivery directly to anti-tumor immune cells can also be used in this strategy such as targeting the cytokine moiety to CD8+ T cells, or y5 T cells, among other anti-tumor immune cells. Such immune cell targeting can be effected by targeting a cell-specific molecule, such as CD8, or a marker preferentially expressed by tumor microenvironmental cell such as PD1, or by molecules expressed bv activated immune cells such as NKG2A.

[0051] Cytokine combinations contemplated herein can include, but are not limited to, IL-17+IL-2+IL-33, IL-12+IL-33+IL-17 and others based on adding IL-17 to any combination using IL-2 or IL-12 plus IL-18, IL-33 or IL-36. IL-36 has 3 known isoforms, any of which is meant by “IL-36’' here.

[0052] In another example, IL-2 engineered to reduce CD25 binding, increase CD 122 binding or attenuate the strength of the IL-2 receptor signal strength will replace native IL-2.

[0053] In another example, any cytokine used for this purpose can be delivered as mRNA in any delivery system.

[0054] In another example, any combination of cytokines delivered in any fashion can be used to improve the efficacy of any cancer treatment known or thought to include IFN-y, including but not limited to adoptively transferred immune cells, immune checkpoint blockade (ICB — described further below), engineered cytokines, targeted small molecules, irradiation or cytotoxic chemotherapy.

[0055] In another example, direct inoculation of cytokines into a specific tumor(s) can be performed.

[0056] In another example, any cytokine can enhance a cancer vaccine by direct tumor injection, systemic delivery or admixture with the vaccine.

[0057] In some examples, IL-17 alone may be administered to enhance therapeutic efficacy.

[0058] II. Immune Checkpoint Blockade (ICB) Compounds and Methods

[0059] A. Overview

[0060] Immune checkpoint blockade (ICB) cancer immunotherapy has greatly improved cancer treatment, with FDA approval of nine monoclonal antibodies blocking the PD1, PD-L1, CTLA4 or Lag3 immune checkpoints with agents blocking many additional immune checkpoints in trials. While current ICB antibodies successfully treat diverse cancer types, under 15% of all cancers respond. Thus, identifying additionalDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 actionable ICB targets is warranted. Furthermore, although age is the single biggest risk factor for cancer development, pre-clinical studies testing ICB efficacy were initially done on young hosts.

[0061] B. PD-L2 Signals Differ Markedly from PD1 / PD-L1

[0062] In autoimmune kidney disease, both PD-L2 and PD-L1 protected against kidney pathology, loss of renal function and intrarenal lymphocyte infiltration. PD-L2 was protective by decreasing CD68+ cells while PD-L1 prevented infiltration of CD8+ T cells demonstrating functional differences in PD-L2 versus PD-L1 blockade. PD-L1 and PD-L2 have protective roles against type 1 diabetes and autoimmune encephalitis with PD-L2 having less pronounced regulatory effects versus PD-L1 although it can induce peripheral Tregs. In murine asthma, PD-L2 deficiency elicited airway hyperreactivity from increased NKT cell IL4 through decreased PD-L2+ dendritic cell suppression. In murine malaria, PD-L2 deficiency increased disease severity and death because PD-L2 expression was essential for establishing CD4+ T cell anti-malaria immunity.

[0063] PD-L1 and PD-L2 (also known as PDCD1LG2) expression are regulated distinctly and while PD-L1 is expressed in a variety of cell types, PD-L2 expression is more restricted, but highly expressed in cancers. Notably, PD-L2 affinity for PD1 is 3.3-fold greater versus PD-L1, suggesting it can outcompete PD-L1 for PD1 access. Further, PD-L2 exacts immunomodulatory and cancer survival signals through engaging bone morphogenic proteins and neogenin, whereas neither PD1 nor PD-L1 do (Fig. 1) meaning PD-L2 blockade affects distinct downstream pathways. Note that these studies capitalize on differences in PD-L2 and PD-L1 biology and immunity as well as distinct biology in their interaction with their shared receptor, PD1 and differential expression patterns.

[0064] C. PD-L2-mediated T Cell Inhibition

[0065] PD-L2 engages T cell PD1 for direct T cell inhibition through SHP2 recruitment to the TCR similar to PD-L1 engagement, although it is much more potent in this regard, as noted above. PD-L2 effects on other immune cells are little reported in cancer.

[0066] D. PD-L2 Signals in Cancer Immunotherapy

[0067] It is reported that aged, but not young PD-L2+ B cells inhibit subcutaneous (SQ) MC38 colon cancer growth. In young mice there is aPD-L2 synergy with aPD-Ll in SQ MC38, but aPD-L2 alone lacked efficacy. PD-L2 / RGMB interactions are effective ICB targets in a microbiome-dependent fashion in young hosts. A non-antibody PD-L2Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 neutralization molecule improved ovarian cancer immunity in mice. Chemotherapy-induced cancer cell senescence induces tumor cell PD-L2 expression that inhibits treatment efficacy and aPD-L2 improved chemotherapy efficacy. Tumor PD-L2 predicts metastases and poor survival in distinct human cancers and predicted aPDl response in head and neck cancers independent of tumor PD-L1, suggesting PD-L2 / PD1 interactions. Notably, PD-L2 appears to be an excellent ICB target that clearly differs from PD1 / PD-L1 interactions, but mechanisms and applications in cancer await further and clearer definitions.

[0068] E. Age Effects on ICB Immunotherapy

[0069] It is well-established that essentially all immunologic processes are altered with advancing age, usually for the worst. Notable examples include reduced vaccine efficacy and increased susceptibility to pathogens and autoimmunity. Nonetheless, a clear reduction in ICB cancer immunotherapy efficacy wi th age has yet to be unequivocally established. In the handful of available reports, ICB efficacy in young and aged patients receiving ICB for melanoma or non-small cell lung cancer are contradictory, with some studies suggesting age worsens, whereas others show it heightens ICB efficacy, an area that was recently reviewed. The results are significant, requiring further investigation.

[0070] F. IL -17 in Cancer

[0071] IL-17 biology is complex, including that there are six IL-17 family- members, 1L-17A-F. In this description, “IL-17?’ refers to 1L-17A unless otherwise specified, as a biologically active IL-17 family member based on known biologic activity. It can be produced by many immune cells but appears to primarily come from CD4+ and y5 T cells in cancers studied to date. Its role in cancers remains incompletely defined, being beneficial or detrimental to anti-cancer immunity depending on tumor type and context as detailed below. IL- 17 in melanomas and bladder cancer mediate both beneficial and detrimental effects based on current, published literature. Additional details in these regards will be introduced in relevant description to follow.

[0072] G. Significance

[0073] Although ICB immunotherapy with aPDl or aPD-Ll can be effective, most tumors do not respond. Although PD-L2 is an alternative PD1 ligand, its use as cancer ICB has received little attention. A significant PD-L2-driven pathology- has been identified, and shows novel aPD-L2 outcomes supporting needed treatment response biomarkers, thus suggesting specific treatment combinations. This description shows IL-Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 17 effects in tumor immunotherapy efficacy, including IL-17-mediated IFN-y induction that can be exploited in rational ICB treatment regimens. Insight on this mechanism could inform mechanisms for immune dysfunction in other scenarios including those that can improve ICB-unresponsive tumors. aPD-L2 mechanistic differences from aPDl are clinically exploitable. aPD-L2 ICB has not yet been tested clinically in humans, but this research helps define relevant contexts and drug combinations for initial human trials. Notably, emerging data suggest that there is not a clear human age effect for ICB, which is significant given the clear age-related reduction in other forms of immune-mediated outcomes such as vaccine efficacy. It is desirable to understand why ICB efficacy does not decline as readily as other immune-mediated processes to use the results herein to our advantage. If the aged benefit superiorly from selected ICB, it is desirable to understand mechanisms underlying such efficacy, to develop novel treatment strategies and help improve ICB efficacy for other patients.

[0074] H. Rigor and Biologic Variables

[0075] Males and females are studied in distinct cancer models in relevant orthotopic and metastatic settings. The method should validate pre-clinical mouse data with human tissues. Relevant controls are used for anti-tumor treatments. Immunoblots are done with relevant loading and antibody controls. Test on > 2 clones of engineered cells and / or use polyclonal cultures are performed herein to reduce clonal artefacts.

[0076] I. Rigor of Prior Literature

[0077] Most studies of cancer ICB against PD1 or PD-L1 do not account for potential PD-L2 signals. The precise mechanisms by which IL- 17 cytokine regulates cancer progression are far less well understood versus IFN-y. Much of the PD-L2 data in cancers are correlative with few mechanistic insights. Most studies of IL-17 / IFN-y interplay in cancer focus on Thl7 plasticity (relevant to CD4+ T cells) or Tcl7 (relevant to CD8+) without accounting for the many other immune cells that can produce IL-17, which are found in the aged. The recent report that IL- 17 can elicit IFN-y production in aPDl + aCTLA4 combination ICB therapy was effectively undertaken, and showed much human relevance, but did not provide a mechanism for IFN-y induction or test PD-L2 contributions. PD-L2 / RGMB interactions were recently reported to be an effective ICB target in a microbiome-dependent fashion in young hosts but did not address age or IL- 17 effects.

[0078] J. InnovationDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0079] aPD-L2 in cancer ICB is understudied with scant mechanistic insights. It is shown that aPD-L2 elicits IL- 17 that drives IFN-y production and elicits ICB efficacy in aged hosts with melanoma whereas oPD-L2 fails in young melanoma. It is clearly shown that distinct immune and mechanistic differences between aPDl ICB and aPD-L2 ICB, including by age. It is also shown that aPD-L2 requires y5 (and CD8+) T cells for efficacy in cutaneous melanomas. Studies herein include IL-17-producing non-Th!7 cells, including B cells, myeloid cells and y§ T cells. This description contemplates discerning aPD-L2 effects through PD1 versus RGMB blockade in these cancer ICB mechanisms based on strong experimental results and define host versus tumor PD-L2 contributions to aPD-L2 efficacy, which is unreported. Novel classes of aPD-L2 antibodies were generated for the experimental experiments herein. The demonstration that aPD-L2 is effective in aged, but not young, hosts with melanomas affords a highly useful model to understand mechanisms driving ICB failure and to explore age effects and treatment resistance in ICB efficacy.

[0080] K. Experimental results supporting AIM 1 identifying key IL- 17+ cells that induce IFN-y

[0081] 1. aPD-L2 treats aged, but not young mice bearing distinct melanomas.

[0082] The Experiments herein challenged young WT mice with B16 subcutaneously and treated with aPD-L2 (clone TY25 from BioXCell that blocks both PD1 and RGMB), but tumor growth was unaffected (Fig. 2A). Significantly, aPD-L2 significantly reduced Bl 6 tumor growth in aged WT mice (Fig. 2B). To exclude Bibspecific effects, a distinct PD-L1+PD-L2+ NrasQ61R-mutant NCH1 melanoma line was tested that was derived from a well-established autochthonous melanoma model, and found that aPD-L2 also significantly reduced its subcutaneous growth in aged but not young mice (Figs. 2C and 2D), indicating a strong age effect of aPD-L2 efficacy in two independent melanoma models. The experiments herein focused on Bl 6 melanoma for mechanistic studies due to limited age mouse inventory.

[0083] 2. IFN-y is Required for aPD-L2 Efficacy in B 16 in Aged Mice.

[0084] aPD-L2 ICB did not control B16 or improve survival in aged IFN-yKO mice (e.g., Fig. 3), consistent with a major mechanistic role for IFN-y in aPD-L2 efficacy as in other ty pes of ICB. As IFN-y-mediates important anti-pathogen and anti -tumor effects, aged IFN-yKO mice were carefully screened for tumors or infections. Only healthy-appearingDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 mice were used, which were also necropsied at experiment endpoint to confirm absence of pathology aside from transplanted tumors.

[0085] 3. aPD-L2 efficacy against B16 melanoma in aged hosts is also IL-17-dependent.

[0086] IL- 17 is reportedly often detrimental to anti -tumor immunity, including in Bl 6 melanoma in young mice. Aged IL-17KO mice were challenged with subcutaneous B16 and treated with aPD-L2, but strikingly, absence of host IL- 17 completely abrogated aPD-L2-mediated tumor growth control (Fig. 4A). As an independent confirmation of IL-17 effects, aged WT mice were challenged with B16 and treated with aPD-L2 plus IL- 17 neutralizing antibody, which also eliminated aPD-L2 efficacy (Fig. 4B). The results herein support an IL-17-dependent mechanism for aPD-L2 efficacy in subcutaneous B16 melanoma in aged hosts. To test if the IL-17 requirement for ICB efficacy was unique to aPD-L2, aged IL-17KO mice were challenged with subcutaneous Bl 6 and treated with aPDl. However, and notably, aPDl efficacy was maintained in aged IL-17KO mice (Figs.4C and 4D) with efficacy like aPDl efficacy reported in aged WT mice, distinguishing IL- 17 effects on PD-L2 versus PD1 blockade in aged hosts.

[0087] 4. aPD-L2 promotes IL-17-dependent IFN-y+ tumor-infiltrating cell prevalence and production in aged B16 melanoma.

[0088] Aged IL-17KO mice exhibited essentially no tumor-infiltrating immune cell IFN-y expression, like young WT and distinctly lower than aged WT by UMAP (not shown). To understand how IL-17 drives aPD-L2 efficacy, immune outcomes of aPD-L2 treatment in WT versus IL-17KO mice w ere investigated. Tumor IFN-y+CD8+ T cell content w as augmented by aPD-L2 in aged WT but not aged IL-17KO like young WT (Fig. 5 A), supporting that IL- 17 increases IFN-y+ content for aPD-L2 efficacy in aged hosts in this model.

[0089] To distinguish IFN-y production from IFN-y+ cell trafficking, it was first found that IL- 17 promoted aPD-L2-mediated per-cell IFN-y production by mean fluorescence intensity in CD8+ (trending in Fig. 5B) and CD4+ T cells (not shown) in aged WT but not aged IL-17KO or young WT. Cells expressing IFN-y-associated CXCR3 were significantly reduced in aged IL-17KO versus WT, including CD8+CXCR3+IFN-y+ (Fig. 5C) and CD4+CXCR3+IFN-y+ T cells (not shown) although aPD-L2 did not improve this effect. T cells expressing the CCR2 chemokine receptor associated with IL-17-driven chemotaxis were unaffected by IL-17KO (not shown). Tumor infiltration ofDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 proliferating CD4+CXCR3+IFN-y+ and CD8+CXCR3+IFN-y+ T cells was significantly higher in aged WT versus young WT and aged IL-17KO mice treated with aPD-L2 (not shown). The results herein accord with IL-17-induced IFN-y that promotes conventional IFN-y+ T cell trafficking through CXCR3. Generally, the results herein support a CD8+ T cell-mediated aPD-L2 efficacy mechanism as expected. For functional confirmation, the experiments depleted CD8+ T cells in aged B16-bearing mice and aPD-L2 efficacy was lost (Fig. 6).

[0090] 5. aPD-L2 efficacy is IL-17-dependent is a second melanoma model.

[0091] Aged IL-17KO mice were challenged with NCH1 and found that aPD-L2 was ineffective (Fig. 7).

[0092] 6. aPD-L2 efficacy also depends on y5 T cells in aged melanoma.

[0093] While the role of IFN-y+CD8+ T cells in tumor immune surveillance and efficacy of ICB agents is well-established, many researchers have also demonstrated that y3 T cells are important mediators of anti-tumor immunity, including through IFN-y. A functional role for y5 T cells was tested by challenging 5TCRKO mice (lacking all y5 T cells but retaining conventional CD4+ and CD8+ T cells) with B16 and treated with aPD-L2, which abrogated treatment efficacy (Fig. 8A).

[0094] 7. y8 T cells support tumor infiltrating CD8+ T cell function but not content.

[0095] As both CD8+ and y6 T cells were essential for aPD-L2 efficacy in aged B16-bearing mice, it was considered that one population could support function or content of the other. Absence of y3 T cells did not affect tumor-infiltrating CD8+ T cell prevalence (Fig. 8B), but significantly reduced their per-cell production of IFN-y, TNFa, and GzB (Figs. 8C-E). These findings suggest y§ T cells could help mediate aPD-L2 efficacy against subcutaneous Bl 6 melanoma in the aged host by promoting CD8+ T cell effector functions but not content.

[0096] 8. Human y5 T cells experience immune changes with age.

[0097] To understand age-related y5 T cell differences, publicly available human scRNA-seq data was mined and stratified y8 T cells by age and by distinct Ty51 and Ty317 subsets producing primarily IFN-y or IL-17 respectively (Figs. 9A and 9B). Ty517 prevalence trended higher in aged versus young (Fig. 9C), but expression of IFN-y and IL-17 in Ty51 and Ty517 populations, respectively did not differ notably based on host age (Fig. 9D). y5 T cell PD-L2 expression in Ty61 was significantly lower in aged and Ty517Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 PD-L2 expression trended to be lower in aged (Figs. 9E and 9F) supporting that y5 T cells might not be initial aPD-L2 targets or principal IL-17-producing cells for treatment efficacy. Antigen presenting pathways were increased in aged yo T cells (Fig. 9G), which can be a mechanism by which they increase CD8+ T cell functions, as yS T cells as antigen presenting cells has been reported including cross presenting tumor specific antigens.

[0098] 9. aPDl elicits IL-17-independent IFN-y in aged mice with melanoma.

[0099] aPDl increased IFN-y+CD8+ T cell and IFN-y+ y5 T cell prevalence and per-cell production in aged IL-17KO mice (Figs. 10A-10D) in sharp contrast to lack of these effects in aPD-L2-treated aged IL-17KO mice (Figs. 4, 5 A and 5B). Generally, the results herein distinguish the IL-17-independent aPD l mechanism from aPD-L2.

[0100] 10. Age-dependent aPD-L2 anti-tumor efficacy is not melanoma specific.

[0101] The experiments used heterotopic SQ MB bladder cancer challenge simply as a proof-of-concept, and not to understand bladder cancer biology, to find that aPD-L2 was effective in young and aged, although efficacy was significantly better in aged (Fig. 11, p=0.02 for aged versus young treatment effect, X2), demonstrating that aPD-L2 age-dependent efficacy is not melanoma-specific.

[0102] 11. Exogenous IL- 17 elicits aPD-L2 efficacy in young B16-bearing hosts.

[0103] A recent report showed that exogenous IL-17 improved aPDl plus aCTLA4 efficacy in Braf-mutated mouse melanoma models. Notably, exogenous IL-17 elicited aPD-L2 efficacy against Bl 6 (Fig. 12). Flow cytometry' of tumor-infiltrating lymphocytes found that efficacy correlated with immune cell trafficking through CXCR3 specifically in CD4+ and y8 T cells but not CD8+ T cells (not shown). The Tbet transcription factor drives antigen-specific CD8+ T cell IFN-y production. Tbet+CD8+ T cell prevalence significantly increased in mice given IL-17 plus aPD-L2 while prevalence of IFN-y+CD8+ T cells and their per-cell production of IFN-y remained unchanged (not shown). Further, prevalence of IFN-y+CD45+ and IFN-y+CXCR3+CD45+ immune cells, CD4+ T cells, and y3 T cells and their per-cell IFN-y production was also unchanged by aPD-L2 irrespective of concomitant exogenous IL-17. Together, these findings suggest an IL-17-driven efficacy program in B16 melanomas in young WT mice that can elicitDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 aPD-L2 efficacy, which can involve IFN-y at some point during treatment, not detected at endpoint here by flow cytometry.

[0104] 12. CD4+ T cell IL-17 mediates aPD-L2 efficacy in the aged melanoma TME.

[0105] As CD4+ T cells made significant IL- 17 and mediate IL-17-driven outcomes, age-matched WT CD4+ T cells were transferred into aged IL-17KO hosts, challenged B 16 subcutaneously the next day, and treated with aPD-L2. WT CD4+ T cell transfer, whose IL-17 production was validated by flow (not shown), elicited aPD-L2 efficacy. When the tumor curves had significantly separated on day 18 (p=0.024), alL-17 AR was administered, which abrogated treatment efficacy (Fig. 13). The results demonstrate that CD4+ T cells are a source of IL- 17 for aPD-L2 efficacy, but do not exclude other cell types.

[0106] 13. PD-L2 protein expression increases in human cells with age.

[0107] The experiments tested immune checkpoint expression versus age in PBMC from control subjects and cancer patients. PD-L2 increased in blood of controls on lin-DR+CD141+ conventional DC1 dendritic cells, but not on conventional DC2 dendritic cells, plasmacytoid dendritic cells, other myeloid cells, T cells or B cells, suggesting a relationship between age and PD-L2 expression on specific human immune cells.

[0108] 14. a-human PD-L2 elicits IL-17 in vitro.

[0109] In allogeneic mixed lymphocyte reactions of human dendritic cells and allogeneic PBMC, aPD-L2 clone 24F.10C12 elicited IL-17, whereas aPD-L2 clone MIH18 and aPDl (nivolumab) did not. Clone 24F.10C12 also elicited simultaneous IFN-y. which was significantly greater than that elicited by aPDl. No antibody elicited detectable IL-4, -10, -18, -21, -23 or -27.

[0110] 15. a-human PD-L2 improves tumor-specific tumor kill.

[0111] Cytotoxicity of human CD8+ T cells incubated with HLA-matched human tumor cells was significantly reduced by aPD-L2, showing that aPD-L2 could improve in vivo tumor cytotoxicity for efficacy.

[0112] 1 . NCH1 generates spontaneous multi-organ metastases.

[0113] The experiments detected lung (e.g., Fig. 14), draining LN, liver, brain and other organ metastases in NCH1 when cutaneous primary tumors were >500 mm3. The experiments validated live metastases by an established protocol and validated Trpl andDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 MITF expression by immunohistochemistry confirming them as melanomas (not shown), which is also an alternative method to quantify.

[0114] L. Hypothesis-aPD-L2 Induces CD4+ T Cell IL- 17 Through cDCl Engagement to Elicit Anti-melanoma IFN-y.

[0115] This mechanism appears to differ in distinct tumors, to be tested here. SIGNIFICANCE. ICB is effective but has limitations. The PD-L2 immune checkpoint has mechanistic differences that overcome aPD-Ll resistance and could combine well with other ICB or approaches. Although melanoma is often controlled by ICB, 40% of patients remain uncured, and principles can apply to other cancers as well.

[0116] 1. Model

[0117] cDC l appear a likely candidate as the initial aPD-L2 target. They can then help CD4+ T cells and / or y5 T cells. The CD4+ T cells can provide cytokines to CD8+ T cells and the yd T cells can amplify antigen presentation for CD8+ T cell activity (Fig. 15).

[0118] 2. AIM 1 Define cellular mechanisms for aPD-L2 melanoma efficacy.

[0119] (a) Mice — male and female BL6 mice are used for 2.5 to 6 months ("young") or 18 to 33 months old (“aged”), which are age and sex-matched for specific experiments, including adoptive cell transfer donors and recipients.

[0120] (b) Mice are challenged with 500,000 tumor cells in PBS (27g needle) for SQ tumors or 250,000 cells for IV unless otherwise specified. Growth is measured every 2 days with Vernier calipers.

[0121] (c) ICB treatments are exactly as reported and / or as in experimental results, all with relevant controls. In brief, aPD-L2 (clone TY25) 200 pg / mouse, aPDl or aPD-Ll (clone RMP1-14 or 10F.9G2. respectively) 200 pg / mouse. on days 7, 11, 15), all administered intraperitoneally or respective isotype controls. Mice will be monitored for toxicity by SGOT and albumin, IHC for organ damage and physical effects (e.g., ruffled fur). All experiments will always compare young to aged mice for age effects even if not explicitly stated.

[0122] (d) Immune factor depletions include aCD8 or aCD4 (conventional T cells, clones 2.43, GK1.5, respectively), anti- 5TCR (y5 T cells, clone UC7-13D5), aB220 (B cells, clone RA3-6B2), aIL-17 (clone eBio64DEC17), alFN-y (clone XMG1.2) and respective isotype controls.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0123] (e) Adoptive cell transfers are done retro-orbi tally with 2 x 105 to 2 x 106cells depending on cell type, availability, and efficacy for intended effects, the day before tumor challenge unless specified otherwise using age and sex-matched donors. Most studies allow for congenic CD45. One cell transfer to follow cells in vivo. Flow panels for transferred cells all gated on CD45+ include: CD3+CD4+CD8-5TCR-, CD3+CD4-CD8+5TCR-, and CD3+CD4-CD8-5TCR+ for CD4+. CD8+ and y5 T cells respectively; and CD3-NK1.1- B cells.

[0124] (f) Metastatic spread from orthotopic melanomas in lungs is by RT-qPCR for gplOO, tyr, trpl and trp2 and macroscopic tumor counts using hematoxylin + eosin staining with light microscopy as described.

[0125] (g) Tumor lines. In addition to B 16, the experiments will use YUMM1.1 and YUMM1.7 melanoma lines representing the common BrafV 600E mutation present in about 60% of human melanomas plus the common inactivation of Pten and Cdkn2a. The NCH1 line has the NrasQ61R mutation associated with sunbum-associated melanomas present in 15-20% of melanomas. All these lines generate spontaneous lung metastases from SQ challenge and other important features of human melanoma. YUMM and NCH1 lines are highly genetically human relevant. In all cases males and females were tested, and compared for outcomes in young versus aged hosts. Metastatic spread to TDLN and lungs is tested in each treatment or immune manipulation condition. In selected cases the experiments will use IV tumor challenge to understand additional mechanistic details regarding the immune basis for metastatic tumor control.

[0126] (h) Flow cytometry'. Tumors or draining lymph nodes (DLN) will be made into single-cell suspensions and studied by high-dimensional flow cytometry on a Cytek Aurora (40+ channel capacity) for T cell activation (e.g., CD69, CD25), central / effector memory (CD44 / CD62L), TRM (CD8+CD69+CD103+), effector functions (e.g., IFN-y, TNFa, GzB, perforin). Other immune cells include NK cells (CD3-NK1.1+), distinct myeloid cells subsets, notably CD3-CDllb+Grl+ MDSC and CD3-CDllb- CD1 lc+XCRl+ cDCl among other myeloid cells as has been extensively detailed.

[0127] (i) RNA-seq and ATAC-seq. Genomic and epigenetic changes at the bulk or single-cell and spatial level will be done using standard approaches from 10X Genomics, through the Dartmouth Sequencing Resource.

[0128] (j) Spatial studies. Experimental results suggest that interventions that alter aPD-L2 efficacy do not always change cell content or cytokine production, suggesting co-Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 localization and / or cell to cell communications (CCC) effects. These will initially be studied by multi-color IHC and confocal imaging. The experiments will also apply a discover^’ and validation phase-based approach leveraging multiple spatial transcriptomics platforms to assess cell-cell communication (CCC). In the discovery phase spatial transcriptomics can be performed using the Visium platform from lOx Genomics. While Visium does not achieve single cell resolution, it generates transcriptome-wide expression counts in a comprehensive and unbiased approach. A cell-type deconvolution algorithm using C-SIDE will be applied to identify cell-type specific gene expression profiles, and COMMOT will be used to identify ligand-receptor pairs mediating CCC, including action directionality and downstream signaling profiles association with CCC. To discern age, aPD-L2, IL- 17 and other effects, the experiments will use the differential CCC analysis module in COMMOT. From discover}' phase data, a probe set for spatial transcriptomics can be constructed with the multiplexed single-cell imaging 10X Technolog}' Xenium platform available at Dartmouth College, capable of measuring copy number and spatial distribution of up to 500 genes simultaneously. Notably, the probe set will include a collection of marker genes required to identify’ major cell types in these tissues, and custom probes targeted to identification of cell subpopulations as they are defined.COMMOT will be used to validate active ligand-receptor pairs and CCC expression profiles identified in the discovery phase. An independent method, MERINGUE, will also be applied to Visium data to validate discovery phase findings.

[0129] (k) Statistical considerations. The experiments will compare tumor growth trajectories and differences in immune cell composition and functions among groups using a repeated measures linear mixed model. The primary outcome is the logarithm of the variable, and the tested parameter is the genotype / time interaction. Ten aged mice / group achieves 90% power for 35% difference in SQ B16 growth in WT, day 17 with aPD-L2. This outcome implies an effect of loge (1-0.35) = -0.43, with o = 0.36 on the log-scale and standardized effect size = 1.2. One can assume log-linear tumor growth, a wi thin-subject correlation of 0.5, and a two-sided a=0.05. One can expect that sample sizes for sufficient power in specific tumors, treatments or age groups will be adjusted as additional data accrue in them.

[0130] Rationale. The functional data have identified CD4+ T cells as generating mechanistic IL-17 for aPD-L2 efficacy, and CD8+ and y5 T cells as cooperating for aPD-Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 L2 efficacy. Flow and bioinformatics suggest cDCl as an initial aPD-L2 target. Here will confirm cellular contributions and their interactions plus signaling mechanisms.

[0131] M. How do CD4+ T Cells Improve aPD-L2 Efficacy in Aged Melanoma?

[0132] Are CD4+ T cells mechanistically upstream of CD8+ T cells? The role of CD4+ T cells in aPD-L2 was seen in 2 experiments with similar results (e.g. Fig. 13). WT CD8+ T cell transfer failed to restore aPD-L2 efficacy in one parallel test of aged Bibbearing IL-17KO mice but needs repeat to confirm. If CD8+ T cells do not improve efficacy in IL-17KO mice, which suggests a need for CD4+ T cell IL- 17 for help. To confirm IL- 17 effects, CD4+ T cells can be transferred into aged IL-17KO as before, treat with aPD-L2 or isotype and test CD8+ T cell effector molecule status (e.g.. IFN-y, Perforin, GzB) and co-localization with CD4+ T cells and tumor cells. If CD8+ T cell transfer into aged IL-17KO restores aPD-L2 efficacy, that suggests a CD4+ T cell IL-17-independent mechanism, which can be IL- 17 induced by CD4+ T cells in another cell, such as a yd T cell. Flow' cytometry of tumor infiltrating immune cells under these conditions plus spatial imaging and scRNA-seq of immune cells plus CCC bioinformatics will address these issues. Mechanisms for IL-17-induced IFN-y are in AIM 2.

[0133] Are yd T cells downstream of CD4+ T cells? To continue work above, the experiments will challenge aged 5TCRKO mice lacking specifically yd T cells, inject WT versus IL-17KO CD4+ T cells, treat with aPD-L2 and if efficacy is seen, give odL-17RA as in Fig. 13 with similar analyses and follow without aIL-17RA to assess yd T cell functional status.

[0134] As tumor-infiltrating yd T cells are limiting and not expandable ex vivo as for human yd T cells, the experiments can FACS-sort them from B16-bearing dTCRKO or IL-17KO mice gated on CD45+CD3+dTCR+CD4-CD8-. It is reported that transfers of as few- as 20,000 yd T cells into SQ tumors is sufficient to establish important effects when injected into tumor base of dTCRKO hosts when ~50 mm3, establishing feasibility and the experiments herein test treatment with aPD-L2 or isotype as before. Initial estimates suggest the ability to obtain up to 50,000 yd T cells / tumor as they comprise up to 20% of tumor infiltrating cells. If efficacy is maintained in dTCRKO hosts, that suggests CD4+ T cell IL-17 is upstream of yd T cells. If efficacy is lost, other factors involved. In that case the experiments can challenge WT mice with CD4+ T cell depletion for aPD-L2 efficacy and immune consequences. The status of CD8+ T cells and other immune cells and CCC bioinformatics and co-localization studies will all be assessed as above. Contributions ofDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 other effector molecules and / or cells will be tested in parallel approaches. These experiments will establish how these three cells cooperate for aPD-L2 efficacy and define IL- 17 contributions.

[0135] Are CD4+ T cells activated by cDCl for aPD-L2 efficacy? The experiments will involve aging of cDCl -deficient IRF8A32 mice (Wash U. Tech Management) versus conditionally cDCl / 2 deficient Zbtb46DTR70 mice (Jax), challenge SQ with B16 and treat with aPD-L2 (and DT to deplete DCs is DTR mice). If efficacy is lost with cDCl deletion as expected, that implicates cDCl as early targets of aPD-L2. Otherwise, the CD1 IcDTR mice test if either cDCl or 2 are involved. Other upstream candidates will be tested with corresponding mice as data so dictate. To test if candidates act initially on CD4+ T cells, flow cytometry with DC deletions assesses immune consequences as above. Transfers of CD4+, CD8+ or y5 T cells from aged B16-bearing hosts into these tumor-bearing DCKO mice defines downstream DC targets for aPD-L2 efficacy. In vitro cell co-cultures will confirm effects and define deeper mechanistic details such as IL- 12 secretion from DCs.

[0136] Do other immune cells provide IL-17 for aPD-L2 efficacy? The CD4+ T cell efficacy effect is clear, but modest, suggesting other cell(s) could also participate. IL-17+B220+CD3-NK1.1- B cells are highly prevalent in the aged Bl 6 TME. It was shown that aged B7-DC+ (PD-L2+)B220+ B cells elicited IL- 17 and IFN-y from CD8+ T cells20 but did not define a mechanism. The experiments will deplete B cells from aged WT and test aPD-L2 efficacy against B16 and if an effect is seen. B cells defined as B220+CD3-NK1.1- will be tested for depletion, cytokine production, co-localizations and CCC. Aged B cell-deficient pMT mice will be used in confirmation and for adoptive cell transfers as above as data so dictate.

[0137] Do CD4+ T cells colocalize with cDCl, CD8+ or y5 T cells? CD8+ and y5 T cells are anti-tumor mediators that are defined as critical to aPD-L2 efficacy (Figs. 6, 8), and come activated through colocalization with IL-17+ cells. The experiments will obtain tissue sections from aged, WT B16-bearing mice treated with aPD-L2 for confocal imaging as described and assess these co-localizations with spatial scRNA-seq and bioinformatics for CCC data. Above depletion studies that reverse IFN-y production provide additional validation. Co-localized versus distant IFN-y induction are not mutually exclusive mechanisms, all of which will be tested here.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0138] N. What Cellular PD-L2 Signals Contribute to aPD-L2-mediated IL-17 Generation?

[0139] To define host versus tumor cell PD-L2 signal effects, the experiments made PD-L2KO B16 and will generate PD-L2KO NCH1 (PD-L2+) and PD-L2KO YUMM lines if they are PD-L2+ by CRISPR / Cas9 as for the prior tumor PD-L1 signal studies. Aged WT mice challenged with PD-L2KO B16 responded to aPD-L2, demonstrating that tumor PD-L2 is dispensable in this setting and implicating host (likely immune cell) PD-L2 requirements for aPD-L2 efficacy.

[0140] PD-L2KO versus CRISPR control tumor cells SQ into WT or PD-L2KO mice (B7-DCKO, Jax 017515) is challenged — as the experiment has previously used and tested aPD-L2 efficacy versus isotype control. If aPD-L2 efficacy is reduced in PD-L2KO mice as expected, adoptive transfers of specific PD-L2+ cells versus control PD-L2KO cells before tumor challenge and treatment will define the cell(s) required for treatment efficacy. The initial candidate PD-L2+ target of aPD-L2 is cDCl, which will be transferred IV as was successfully done to define IFNAR+ dendritic cell effects on cancer immunotherapy in previous work. Other cells to define are CD4+, CD8+ and y5 T cells. It was confirmed that all these candidate cells express PD-L2.

[0141] O. How do Cellular requirements for aPD-L2 Differ from Other ICB?

[0142] The data support that aPD-L2 is not simply an alternative means to block PD1. To support that thesis, the experiments will test whether aPDl or aPD-Ll alone are effective against BRAF-mutated YUMM lines of Nras-mutated NCH1 in parallel studies and test whether adding aPDl or aPD-Ll to aPD-L2 improves efficacy or alters cellular requirements in all models. A rigorous approach to PD-L2 / RGMB versus PD-L2 / PD1 is undertaken in AIM 2.

[0143] Expected outcomes. These studies will define the IL-17-producing cell(s) and PD-L2-expressing cells mediating aPD-L2 efficacy in melanomas, where aPD-L2 is effective in aged hosts by an IL-17-dependent mechanism. The experiments will use melanoma models with important human melanoma mutations and signal drivers and rigorously test young versus aged hosts to understand the age-driven factors behind age-related efficacy at the cellular level. Differential aPD-L2 effects, IL- 17 induction and IL-17-mediated IFN-y in primary versus metastatic (lung, TDLN) tumors will be defined and related to tumor control. The results validate PD-L2 and IL-17 contributions to aPD-L2 ICB efficacy in distinct tumors and TME. The results herein help define treatmentDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 response biomarkers and / or approaches to treating metastases, including IL-17-rich environments irrespective of age or tumor type. Effects of otPD-Ll or IL- 17 contributions based on tumor mutational status (BRAF vs Nras) are not expected to differ based on the research, and other published data. How PD-L2 blockade specifically differs from PD1 or PD-L1 blockade will be understood at the cellular level here, with additional mechanistic insights using the novel antibodies described in AIM 2.

[0144] Potential limitations and solutions. Experiments with aged, genetically altered mice are challenging due to lead time to age the mice, and availability of sufficient aged mouse numbers, but it has been amply demonstrated that these mice can be aged and coordinated for detailed mechanistic studies. Using neutralizing / blocking antibodies, adoptive cell transfers and other strategies in aged WT alleviates some of these logistical challenges. Full details of human relevance are also challenging as there are no clinical trials of aPD-L2 yet, but aPDl, aPD-Ll and aCTLA4 ICB approaches also started this way. Testing non-immune host PD-L2 contributions in melanomas to aPD-L2 efficacy is challenging as chimeras in aged mice suffer from poor bone marrow reconstitution and increased radiation toxicity’, but making the chimeras in younger mice (9-12 months old) as described, and then aging the chimeras is feasible if such studies are unexpectedly required.

[0145] P. Experimental Results Supporting AIM 2 on Signal Effect Studies

[0146] 1. Aged immune cells differ in IL- 17 response versus young.

[0147] The experiments treated naive young and aged mice in vivo with recombinant IL- 17, aPD-L2, both or controls and obtained spleens. The experiments analyzed them by flow cytometry to find that rIL-17 elicited significantly more IFN-y from aged versus young CD8+ and CD4+ T cells. aPD-L2 elicited less IFN-y from CD4+ T cells, but performed better in young (compare absolute and / or fold-changes, Figs. 16A and 16B).

[0148] Immune TME checkpoints. PD-L2 and RGMB were expressed on -4-5% of CD45+ B16 TME immune cells in young and aged with higher PD1 expression as reported and as additional examples, aPD-L2 significantly increased aged CD8+ T cell PD1 and RGMB (Figs. 17A and 17B). Many differences were found in immune checkpoint expression by age, host cytokines and aPD-L2, suggesting these increases could contribute to aPD-L2 efficacy, to be tested using specific, novel antibodies being developed.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0149] 2. PD1 and RGMB blocking interactions of aPD-L2 antibody fragments.

[0150] The aPD-L2 treatment clone TY25 used by the experiments is BE0112 from BioXCell, a non-depleting rat IgG2a antibody that blocks PD-L2 interaction with PD1 and RGMB. To define effects of blocking distinct PD-L2 receptors, the experiments generated antibodies that block PD-L2 interaction specifically with RGMB, PD1 or both. A human, scFv library (diversity ~ 1x109) was mined for antibody fragments that differentially block each and both interactions. Over repeated rounds of magnetic and flow cytometry -based selections, clones specific for PD-L2 were isolated and selectivity for full PD-L2 ligand block was tested. Clones blocking both PD1 and RGMB had decreased PD-L2 binding in the presence of PD1 or RGMB. Clones blocking only RGMB had decreased binding to PD-L2 in the presence of RGMB but not PD1 (Figs. 18A and 18B). A PD-L2 / PD1 exclusive blocker is in development.

[0151] Top candidates are cloned into mouse IgGl vectors and expressed in scFv-Fc by expiHEK293 or expiCHO transfection, purified by affinity chromatography, monomeric fraction isolated by size exclusion chromatography, and tested for recombinant PD-L2 and PD-L2+ cell line binding (ELISA, flow cytometry). PD-L2 affinity will be defined and competition with PD1 and RGMB confirmed by biolayer interferometry . Clones with best binding and blocking profiles will be expressed at larger scale with confirmed <0.1 EU / antibody dose to support in vivo testing.

[0152] Q. AIM 2 Define Molecular and Additional Cellular Mechanisms for aPD- L2 Efficacy

[0153] 1. Hypothesis: The inflamed aged TME elicits innate T cell behavior that augments IL-17-induced IFN-y to promote aPD-L2 efficacy in aged hosts.

[0154] 2. How does IL- 17 generate IFN-y?

[0155] AIM 1 RNA-seq data can be analyzed for IL-17-generating factors (e.g., increased IL-12, reduced IL4) and confirm leads with protein assay (Luminex) and immunoblots or IHC of tumor beds and / or implicated producer cells. Other considerations for IL-17-induced IFN-y, age effects and differences in distinct immune cells is differential IL-17R expression and / or signals. The IL-17R consists of five subunits (IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE) that homo- or heterodimerize into distinct receptor ty pes that differ by cell type and mediate distinct signals. IL-17RA and IL-17RC mediate IL-17A signals. Differential IL-17 sensitivity is supported by data in Fig. 16A and 16B. Age effects of IL-17R and IL- 17 signaling are unreported, which knowledge gapsDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 will be greatly filled here. IL- 17 elicits Stat3 as a good indicator of IL-17R signal strength, andNFkB, assessed by flow and immunoblots. IL- 17 elicitation of by antigenindependent IFN-y is reminiscent of innate behavior, which is exemplified by Eomes induction as opposed to Tbet induction by antigen in CD8+ T cells. These are not mutually exclusive outcomes and antigen-specific cells can also exhibit innate behavior. The experiments will assess Tbet versus Eomes induction in scRNA-seq data sets of tumor-bearing, aPD-L2-treated mice and follow with flow-based studies. Mechanisms for innate behavior by antigen specific T cells are unreported, and thus will have to be mined. Adoptive transfer of (initially non-age matched) 1 x 106pmel cells the day before tumor challenge is useful for initial study of tumor-antigen-specific cells, as the experiment ages pmel mice for age-matched studies in follow up. Using OVA+ tumors allows OT-I and OT-II cell studv for CD4+ T cell effects. As candidate mechanisms for IL-17-induced IFN-y are identified, they will be functionally verified (e.g., block increased IL-12 with aIL-12R if detected) in vitro and in vivo. If a secondary event, such as induced IL-12 is defined, the experiments will assess its receptor expression and test downstream signals in IFN-y+ producing cells in like fashion. As effector molecules are defined, the experiments will use molecular approaches to T cell engineering in vitro using transient nucleofections that last up to 5 days.

[0156] 3. Is IFN-y effective without IL-17?

[0157] It is unclear if IFN-y is sufficient for tumor control without IL-17. The experiments will challenge WT vs IL-17KO mice with B16 and treat with aPD-L2 ± rIFN-y. If IL- 17 is not required for IFN-y efficacy, replacing IFN-y in the absence of host IL- 17 should be effective. Immune analyses will define requirements for IL-17 in IFN-y if it is found that both cytokines are required.

[0158] 4. What factors generate IL-17-induced IFN-y in humans?

[0159] IL-17-elicited IFN-y was just reported in humans but mechanisms are undefined. The experiments will perform in vitro tests of IL-17-induced IFN-y using human PBMC analogous to mouse studies in the published in vitro human PBMC model. Downstream pSTAT3 or pSTATl tests IL-17 or IFN-y signal strength, respectively. It is predicted that IL- 17 will promote Tbet better in aged versus young CD8+ T cells and will assess if this effect is direct, transcriptional, requires accessory cell participation (e.g., dendritic cells as expected) and if through Thl7 plasticity versus other mechanisms and whether accessory cell or T cell PD-L2 versus other immune checkpoints are required, andDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 with approaches as for mouse cells described above and with additional models in development and / or described. Co-culturing young versus aged mouse cells with B16 (elicits aPD-L2-driven IL-17 in vivo in aged) and NCH1 and YUMM lines will help define tumor factors contributing to these TME differences or will be used to inform deeper mechanisms from in vivo leads generated from above studies or validate them in vitro.

[0160] 5. How does aPD-L2 elicit human IL-17?

[0161] IL-17 can induce IFN-y in non-cancer states, such as T cells in human coronary7artery' plaques, where IL- 17 promotes their IFN-y by transcriptional control. To test IL-17-driven IFN-y, aIL-17 to aPD-L2 are added to cultures at the outset. Flow cytometry of IL-17 versus IFN-y defines producer cells. If IL-17 neutralization reduces IFN-y, the experiments will sort candidate IL- 17+ cells from cultures prior to aPD-L2 and test for reduced IFN-y in their absence. Neutralization of IL6 or other Thl7-polarizing factors tests soluble factor contributions. In the current allogeneic MLR the experiments did not detect IL-23 but will assess it with aCD3 / aCD28-activated T cells, including adding additional cells such as monocytes as IL-23 sources to cultures. The dendritic cells in these assays were PD-L2+. To define dendritic cell PD-L2 contributions to IL-17, the experiments will use flow cy tometry -purified PD-L2+ versus PD-L2- dendritic cells and repeat the study. The experiments will sort any other candidate PD-L2+ cells as defined for their contributions. RNA-seq shows RGMB induction in these conditions. If confirmed by cytometry^ and immunoblots, aRGMB and using RGMB+Z- cells defines a role for it as for IL- 17. As needed, the experiments will use siRNA to deplete PD-L2 or other targets for mechanistic studies as described for the studies of myeloid cell B7H4 signals. Other culture systems (e.g. Thl7 polarized) will also be assessed if these approaches are inconclusive.

[0162] PBMC should be tested in human subjects of advancing ages to acquire additional details on immune cell checkpoint expression with age, and as available test tumor-infiltrating immune cell content in the melanoma cells, which is available in the extensive library for age-related immune checkpoint expression data.

[0163] 3. PD-L2 protein expression increases in mouse and human cells with age.

[0164] A publicly available single cell RNA sequencing (scRNAseq) dataset examining host age effects on the immune Bl 6 TME50 can be analyzed to determine whether specific immune populations differentially express PDCD1LG2 (PD-L2) in a hostDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 age-dependent manner. The experiments projected the scRNAseq dataset onto a UMAP and clustered immune cells by expression of their characteristic gene signatures as described in the original dataset. Intratumoural NK cells, neutrophils, plasmacytoid dendritic cells, and conventional type I dendritic cells (cDCl) expressed higher PDCD1LG2 in aged versus young mice suggesting a relationship between age and PD-L2 expression on specific murine immune cells. To test human translational relevance of initial findings, PDCD1LG2 (PD-L2) gene expression was examined by age by pooling three human melanoma bulk RNAseq datasets to find that PDCD1LG2 expression positively correlated with age, as in mice.

[0165] The experiments then analyzed three pooled human melanoma scRNAseq datasets to identify immune cell-specific changes in PDCD1LG2 expression stratified by host age. The experiments projected the datasets onto a UMAP and annotated individual cell subsets. To characterize myeloid subsets further, a previously published myeloid marker panel was used to examine specific cell subset PDCD1LG2 expression stratified by host age. It was found that myeloid subsets trended to exhibit higher PDCD1LG2 expression versus other immune subsets and identified PLA2G7.IMs, a subset of interstitial tumor associated macrophages that are immunosuppressive in cancer, and cDCl, as in mice, specifically expressed more PDCD1LG2 in aged versus young hosts. Intratumoural NK cell, and plasmacytoid dendritic cell PDCD1LG2 expression did not differ by age in human melanomas, highlighting species specificity of intratumoural PDCD1LG2 expression. Publicly available data from The Cancer Genome Atlas PanCancer database was mined, and it was determined that aged cancer patients, (>60 years), expressed higher PDCD1LG2, IL-17A (IL-17), and IFNG (IFN-y) than younger cancer patients (<60 years of age). To support the findings further, PBMC from normal subjects and cancer patients was studied. Notably, PDCD1LG2 expression increased on lineage-DR+CD141+ cDCl, but not on cDC2 dendritic cells, plasmacytoid dendritic cells, other myeloid cells, T cells or B cells (not shown), suggesting a relationship betw een age and P-L2 expression on specific human immune cells, including specifically cDCl, corroborating the scRNAseq analyses of human melanoma patients and aged mice. There w as insufficient data available to make clear conclusions regarding age effects on PD-L2 protein.

[0166] 4. What is the basis for age-related IL-17 signal differences?Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0167] Current data suggest that aPD-L2-mediated cell migration or proliferation are not major contributors to efficacy. Thus, effects can implicate age-dependent PD-L2 signals and / or IL-17 signals as was demonstrated in vitro (e.g., Figs. 16A and 16B).Further, the results herein are in tumor-naive mice. To assess IL-17 signals in the TME, the experiments will give young versus aged tumor-bearing mice exogenous IL- 17 as in Fig. 13 and assess immune cell IFN-y, Tbet and Eomes (elicit IFN-y), and Stat3 and NFkB (downstream of IL-17) signals in tumor and draining lymph nodes versus spleen (nontumor control), using all 3 melanoma lines. Flow cytometry can assess conventional and y5 T cells, NK cells, B cells and myeloid cells as in experimental results to assess for induced % positive cells. MFI (per-cell production) and proliferation by Ki67. Studies of how aPD-L2 generates IL- 17 and that generates IFN-y are in AIM 2. scRNA-seq data from AIM 1 will provide signal leads within cells and CCC tests effects between cells. As candidate targets are defined, the experiments will use molecular or pharmacologic approaches for validation, such as KO of a candidate IL- 17 -producing gene.

[0168] To assess PD-L2 signals, there is little reported about downstream intracellular effects, and thus, the research herein will initially focus on functional outcomes of aPD-L2 such as cell proliferation and effector molecule production (IFN-y, TNFa, IL-17, Perf, GzB). Assessing effects in spleen versus tumor and draining lymph nodes assess tumor environmental consequences. In vitro, the experiments will test if recombinant IL-17 alters aPD-L2 outcomes by age. For example, if IL-17 induces cDCl to generate IFN-y from CD8+ T cells or IL- 17 from CD4+ T cells better in aged versus young using co-culture and analytic conditions as described.

[0169] 5. How do y5 T cells promote CD8+ T cell functions?

[0170] Work in AIM 1 tests if y5 T cells act directly or indirectly on CD8+ T cells. The experiments will assess co-localization of y5 T cells with CD8+ cells versus other cells and assess if IFN-y is induced in co-localized cells including CD8+ T cells in aged WT mice bearing B16 tumors and treated with aPD-L2 or isotype. scRNA-seq on tumorinfiltrating immune cells in these cohorts plus CCC will define interactions. The experiments will assess y5 T cells in detail by cytometry included for production of beneficial IL-2, IFN-y and IL-17 versus suppressive regulatory y3 T cell features, e.g., FoxP3, CD39, CTLA-4. Bioinformatics from aged mice with Bl 6 did not disclose a clear lead, although antigen presenting function was suggested (Figs. 9A-9G). The results of experiments herein will be complimented with data to be generated on aPD-L2 effects. IfDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 antigen presenting capacity still emerges as the top pathway, the experiments will assess that by flow sorting TME y5 T cells from aged versus young and test their capacity to activate TME CD8+ T cells to proliferate, or make IFN-y GzB, and if a-P2-microglobulin (blocks class I) or aCDld (block canonical y5 T cell presentation) affects the outcome.

[0171] As more specific y5 T cell subsets (e g., PD-L2+) are defined, the experiments will attempt in vivo transfers of these versus testing effects through in vitro culture systems and use scRNA-seq to define treatment outcomes.

[0172] To distinguish CD8+ T cell mediated tumor kill versus killing by y3 T or other cells, cytotoxicity7will be tested in vitro. scRNA-seq and co-localization of y6 T or other candidate cells with tumor cells and assessing tumor kill (apoptosis) with confocal imaging as reported helps confirm if y5 T cells directly kill tumors in vivo. For CD8+ T cells, or any alternative cell implicated in tumor killing, the experiments will recover cells from tumors for in vitro tumor cytotoxicity assays which will also establish a role for IFN-y versus any other factor and will test PD-L2 effects directly by adding aPD-L2 to co-cultures at 10 pg / ml. In vivo confirmations include transfer of activated killer cells (isolated from tumors) and transfer into (3TCRKO or 6TCRKO recipients. Neutralizing IL-17 or IFN-y other implicated factors established the effector mechanism for improved tumor control, and flow cytometry and other studies of the TME as above define the downstream consequences.

[0173] 6. Expected results and interpretations.

[0174] These studies define molecular and additional cellular mechanisms of aPD-L2 efficacy in a TME and age-specific manner. It is expected that IL- 17 induces innate T cell behavior as one basis for IL-17-induced IFN-y, but additional mechanisms may exist. RGMB versus PD1 blockade effects of aPD-L2 should be defined, and distinguished from aPDl and oPD-Ll effects. This research will gain additional insights into effects on and contributions from y6 T cells, including if regulatory y5 T cells are reduced by aPD-L2.

[0175] Potential limitations and solutions. Human and mouse y6 T cells differ, but functions are similar. Other limitations are as in AIM 1.

[0176] AIM 3 Test rational PD-L2 blockade strategies and combinations based on mechanistic insights

[0177] Hypothesis: aPD-L2 efficacy will be augmented by increased T cell innate behavior, which can involve Treg depletion or inflammatory cytokines. Based on thisDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 hypothesis, it is expected that aCTLA4 to be improved based on its Treg depletion and inflammatory inducing properties. These effects can include RGMB signals.

[0178] 7. How does PD-L2 / PD1 differ from PD-L2 / RGMB signals and from PD1 / PD-L1 signals?

[0179] The experiments can employ the antibodies under development to assess RGMB versus PD1 contributions to PD-L2 blockade using all tumor models and in vitro assays. The experiments will compare effects to aPDL aPD-Ll and commercial TY25 aPD-L2 clone in like manner. The affinity matured antibodies able to be generated in sufficient quantity for in vivo work will be tested in young versus aged, primary versus metastatic tumors and in the distinct melanoma models as single agents, one antibody from each class (blocking RGMB. PD1 or both) with defined phenotypes will advance to in vivo testing.

[0180] 8. Does aPD-L2 improve aCTLA4 efficacy?

[0181] It was reported that aCTLA4 has little single-agent activity against aged mice with B16 melanoma. However, IL-17 improves aPDl+aCTLA4 efficacy against B16 (in young mice). As aPD-L2 can inhibit PD1 signals, IL- 17 can increase aPD-L2 / aCTLA4 therapy if the aPD-L2 inhibits PD1 signals, or through distinct mechanisms. It is hypothesized that aged adaptive T cells will acquire innate properties (cytokine-driven IFN-y) which can involve chronic inflammatory cytokine exposure or defective Treg functions, either of which could elicit innate behavior. Mouse aCTLA4 depletes Tregs, as does botensilimab, a human aCTLA4. Botensilimab is also more potent in Treg depletion versus commercial mouse aCTLA4 clones. It is, thus predicted that aCTLA4 will augment aPD-L2 based on enhanced innate behavior from CD8+ T cells, including bystander, non-tumor-specific cells. The experiments will test aCTLA4 (clone 9H10, BioXcel) versus the Fc-enhanced mouse aCTLA4DLE, the mouse version of botensilimab that is reported in all condition of age and tumor. Immune outcomes include IL- 17 and IFN-y generation, induction of tumor-specific immunity cDCl activation and yd T cell activation. Challenge into aged FoxP3DTR mice with a single DT-mediated Treg depletion as described tests if Treg depletion elicits aPD-L2 efficacy, based on eliciting innate T cell behavior, among other mechanisms to define.

[0182] An alternative means to elicit innate T cell behavior is aCD137+aCD134 which the experiments will test as an alternative to combine with aPD-L2. Combinations with aPDl / aPD-Ll were done in AIM 2 with TY25. Here the experiments tests if novelDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 aPD-L2 antibodies alter combination outcomes, and will define mechanisms. Given the IL- 17 rescue of young Bl 6 aPD-L2 response (Fig. 13), approaches to elicit innate behavior could improve O.PD-L2 efficacy in young hosts. Such data are translationally relevant to developing more effective ICB approaches.

[0183] R. Predicted results and interpretations.

[0184] These studies test rational treatment combinations based on mechanistic insights and define differential PD1 from RGMB blockade effects. It was previously reported that adoptive PD-L2+ B cell transfers improved anti-MC38 colon cancer immunotherapy in aged hosts. The experiments will test B cell transfers with aPD-L2 in melanoma models as they differ from MC38 colorectal cancer. If a specific B cell subset mediating aPD-L2 efficacy is defined as expected, it will be tested in a novel adoptive cell treatment combination. Insights into IL-17 requirements for aPDl / aCTLA4 combinations by age are useful as this combination is FDA-approved in distinct cancers, including melanoma. Potential limitations and solutions: Aged mice will always be limiting for the large numbers of conditions to test, but over time we have amply demonstrated that all such work can be accomplished. Some combinations can be omitted for lack of aged mice without compromise of overall scientific objectives.

[0185] III. Cytokine Delivery' Systems

[0186] In some embodiments, cytokine compositions described herein are delivered using targeted delivery systems configured to concentrate the cytokines in a tumor microenvironment (TME). Such delivery systems can include lipid nanoparticles, polymeric nanoparticles, viral vectors, liposomes, conjugated proteins, or engineered immune cells.

[0187] The delivery system can encapsulate cytokine proteins, nucleic acids encoding cytokines, or combinations thereof. In certain embodiments, messenger RNA molecules encoding cytokines such as IL-17, IL-2, IL-12, IL-18, IL-33, or IL-36 are encapsulated within lipid nanoparticles. After delivery' to the TME, host cells translate the mRNA to produce cytokines locally.

[0188] Targeting of the delivery system to tumor tissue may be achieved by incorporating tumor-binding ligands, including antibodies, antibody fragments, receptor ligands, peptides, or albumin-binding domains that promote accumulation within tumor tissue. In certain embodiments, the delivery sy stem comprises an antibody or antigen-Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 binding fragment specific for a tumor-associated antigen, including but not limited to Her2.

[0189] Alternatively, cytokines may be delivered using engineered immune cells, including T cells, B cells, dendritic cells, or NK cells genetically modified to express cytokine-encoding nucleic acids. Such cells can migrate to tumors and release cytokines locally within the tumor microenvironment.

[0190] Delivery systems described herein improve safety and therapeutic efficacy by limiting systemic exposure to cytokines while promoting local IFN-y induction in tumor-infiltrating immune cells.

[0191] IV. Therapeutic Kits

[0192] The invention also provides kits for performing the methods described herein. A kit may include multiple cytokines packaged separately or together, along with delivery reagents and instructions for use.

[0193] In one embodiment, a kit includes:

[0194] IL- 17 in a first container

[0195] a second cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36 in a second container

[0196] a delivery formulation such as lipid nanoparticles or nucleic acid vectors

[0197] In certain embodiments, the cytokines are provided as recombinant proteins or nucleic acids encoding the cytokines, including mRNA molecules.

[0198] The kit may further include targeting molecules, such as tumor-specific antibodies or immune-cell targeting ligands, enabling delivery to tumor cells or tumorinfiltrating immune cells.

[0199] The kit may also include immune checkpoint inhibitors to enable combination therapies with immune checkpoint blockade treatments.

[0200] Instructions included in the kit guide the user to administer the cytokine composition in a manner that induces antigen-independent IFN-y production in tumorinfiltrating immune cells, thereby enhancing anti-tumor immune responses.

[0201] V. Pharmaceutical Cytokine Compositions

[0202] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising cytokines that induce interferon-y (IFN-y) production in immune cells within a tumor microenvironment (TME). Such compositions may be used forDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 therapeutic treatment of cancer by enhancing anti-tumor immune responses mediated by tumor-infiltrating immune cells.

[0203] In some embodiments, the pharmaceutical composition comprises interleukin- 17 (IL-17) in combination with at least one additional cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36. The inventors have found that combinations including IL- 17 can synergistically stimulate antigen-independent IFN-y production by bystander immune cells present within tumor tissue, including but not limited to CD8+T cells, natural killer (NK) cells, y5 T cells, and CD4+T cells.

[0204] Without being bound by theory', cytokine combinations described herein may activate immune signaling pathways within tumor-infiltrating lymphocytes and other immune cells, thereby promoting local IFN-y production and enhanced anti-tumor immune activity within the tumor microenvironment.

[0205] V. Method for Inducing IFN- y

[0206] Referring now to Fig. 19, a flow diagram 1900 illustrates an example method for inducing interferon-y (IFN-y) production in tumor-infiltrating immune cells to improve cancer treatment outcomes. The method begins at step 1902, in which a cytokine composition is prepared. The cytokine composition may include IL- 17 and one or more additional cy tokines such as IL-2, IL-12, IL-18, IL-33, or IL-36. At step 1904, the cytokine composition is incorporated into a delivery system, such as lipid nanoparticles, viral vectors, polymeric carriers, or engineered immune cells. At step 1906, the cytokine composition is administered to a subject having a tumor, such that the cytokines are delivered to the tumor microenvironment (TME). At step 1908, the cytokines stimulate antigen-independent IFN-y production in bystander immune cells, including CD8+T cells, NK cells, and y5 T cells within the TME. At step 1910. tumor responses and immune activity are monitored, including measurement of IFN-y production, tumor cell viability, immune cell activation markers, or tumor size. At step 1912, treatment parameters are modified in response to the monitored results, including adjustment of cytokine dosage, delivery frequency, or cytokine composition. The method can then return to step 1906 for iterative treatment cycles until a desired therapeutic response is achieved.

[0207] A. Treatment of Melanoma

[0208] In certain embodiments, the cytokine compositions and methods described herein are used for treatment of melanoma, including metastatic melanoma. Melanoma tumors frequently exhibit immune-suppressive tumor microenvironments and exhaustedDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 tumor-specific T cells. These conditions limit the effectiveness of antigen-dependent immune responses. The inventors have found that cytokine combinations described herein can activate bystander immune cells within melanoma tumors, thereby inducing antigenindependent interferon-y production and enhancing anti-tumor immune activity.

[0209] In some embodiments, the methods described herein induce robust IFN-y production within melanoma tumors, leading to enhanced recruitment and activation of immune effector cells capable of destroying melanoma cells.

[0210] B. Treatment of Aged Subjects with Inflammat ory Immune Dysregulation

[0211] In certain embodiments, the compositions and methods described herein are particularly useful for treatment of cancer in aged subjects exhibiting chronic inflammation. Aging is frequently associated with a state of systemic inflammatory immune dysregulation, sometimes referred to as inflammaging, which can impair tumorspecific immune responses and reduce effectiveness of conventional immunotherapies. In some embodiments, the cytokine combinations described herein activate bystander immune cells that remain functionally responsive in aged hosts, thereby restoring effective immune responses within the tumor microenvironment. Also, in certain embodiments, the subject receiving treatment is an aged subject, such as a subject older than approximately 60 years, 65 years, or 70 years of age, and exhibits elevated inflammatory markers or chronic inflammatory immune activity.

[0212] The methods described herein can therefore provide enhanced therapeutic benefit in aged hosts having inflammatory immune conditions, including those in which tumor-specific T cell responses are impaired.

[0213] C. Tumor-Specific aPD-L2 Enhancement

[0214] It is contemplated that tumor-specific enhancement of anti-PD-L2 immune activity can occur herein. In certain embodiments, the cytokine compositions described herein enhance anti-PD-L2 mediated immune activity within tumor tissue. Programmed death ligand-2 (PD-L2) is an immune checkpoint molecule that interacts with the PD-1 receptor on immune cells and can suppress anti-tumor immune responses. In some embodiments, administration of the cytokine compositions described herein enhances the therapeutic activity' of anti-PD-L2 antibodies (aPD-L2).

[0215] Without being bound by theory', cytokine-mediated activation of bystander immune cells within the tumor microenvironment can:

[0216] 1. increase PD-L2 expression within tumor tissue, and / orDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0217] 2. increase sensitivity of tumor-infiltrating immune cells to PD-L2 blockade, and / or

[0218] 3. enhance IFN-y-mediated immune activation pathways that synergize with PD-L2 blockade.

[0219] In certain embodiments, enhancement of aPD-L2 activity occurs preferentially within tumor tissue, thereby increasing tumor-specific immune responses while minimizing systemic immune activation. In some embodiments, the cytokine compositions described herein are administered in combination with an anti-PD-L2 antibody, resulting in enhanced tumor-specific immune activation and improved anti-tumor responses. Note, particularly, that in certain embodiments, cytokine therapy increases anti-PD-L2-mediated immune activation preferentially within tumor tissue relative to non-tumor tissue.

[0220] VII. Cytokine Delivery Mechanism

[0221] Referring now to Fig. 20, a diagram 2000 illustrates an example architecture for delivery of cytokine compositions to a tumor microenvironment. A subject 2000 receives administration 2002 of a cytokine delivery system comprising lipid nanoparticles 2004. The lipid nanoparticles can encapsulate nucleic acids encoding cytokines, such as messenger RNA molecules encoding IL-17, IL-33, IL-2, IL-12, IL-18, or IL-36. Following administration, the lipid nanoparticles 2004 localize to tumor tissue 2006, where the nanoparticles release cytokine-encoding mRNA 2008 into local cells within the tumor microenvironment. Cells within the tumor microenvironment, including immune cells 2010, translate the mRNA and produce cytokines that stimulate interferon-y (IFN-y) production 2012. The increased IFN-y production promotes immune-mediated tumor cell killing 2014, thereby enhancing anti-tumor immune responses. Note that further deliver}’ systems can include, but are not limited to: exosomes, extracellular vesicles, polymeric micelles, antibody-cytokine fusion proteins, and / or hydrogel delivery', which can be implemented in a manner clear to those of skill.

[0222] VIII. Mechanistic Immune Pathway

[0223] Referring now to Fig. 21, a diagram illustrates an example immune signaling pathway 2100 through which cytokine compositions induce anti-tumor immune responses. Cytokine signaling 2102, including combinations of IL-17 and IL-33, optionally together with IL-2, IL-12, IL-18, or IL-36, activates immune signaling pathways within tumor-infiltrating immune cells. These cytokine signals promoteDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 activation of bystander immune cells 2104, including CD8+T cells, natural killer (NK) cells, and y5 T cells, even in the absence of tumor antigen recognition. Activated immune cells subsequently produce interferon-y 2106, which stimulates multiple anti -tumor immune mechanisms within the tumor microenvironment. In certain embodiments, cytokine-mediated immune activation enhances anti-PD-L2 immune activity 2108, including improved therapeutic responses to aPD-L2 antibodies within tumor tissue. The combined effects of cytokine signaling and immune checkpoint modulation promote immune-mediated tumor cell killing 2110, thereby improving therapeutic outcomes in cancer treatment.

[0224] VIII. Melanoma Treatment Considerations

[0225] Referring now to Fig. 22. a diagram 2200 illustrates an example therapeutic approach for treating melanoma in an aged host exhibiting chronic inflammatory immune dysregulation. In aged (or otherwise immunocompromised) subjects, the immune environment 2202 may exhibit chronic inflammatory signaling, sometimes referred to as inflammaging. This condition can include elevated inflammatory cytokines, immune dysregulation, and functional exhaustion of tumor-specific T cells. As a result, melanoma tumors within the tumor microenvironment 2204 may exhibit reduced interferon-y (IFN-y) production and impaired anti-tumor immune responses. To restore immune activity7, cytokine therapy 2206 may be administered to the subject. In certain embodiments, the cytokine therapy comprises IL-17 in combination with IL-33, optionally together with IL-2, IL-12, IL-18, or IL-36. These cytokines may be delivered using lipid nanoparticles, viral vectors, polymeric carriers, or engineered immune cells. The cytokine therapy activates bystander immune cells within the tumor microenvironment, leading to restoration of IFN-y production 2208. Immune cells activated by this process may include CD8+T cells, natural killer (NK) cells, and y5 T cells. Restored IFN-y production enhances anti -tumor immune responses 2210, including activation of immune cytotoxic pathways that target melanoma cell.

[0226] As a result of these immune responses, melanoma tumor regression 2212 may occur, demonstrating the therapeutic benefit of cytokine compositions described herein for treatment of melanoma in aged hosts having chronic inflammatory immune environments.

[0227] Note that dosage ranges and treatment schedules for the various embodiments herein can be varied on patient characteristics and the effectiveness ofDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 treatment (which can be determined on an ongoing basis). Baseline treatment protocols can be established via experimental trials using known and accepted techniques.

[0228] IX. Combining Cytokine and Checkpoint Inhibitor

[0229] Referring now to Fig. 23, a diagram illustrates an example therapeutic strategy 2300 that combines cytokine therapy with anti-PD-L2 immune checkpoint blockade to enhance tumor-specific immune responses. Within the tumor microenvironment 2302, tumor cells may express programmed death ligand-2 (PD-L2), which interacts with the PD-1 receptor on immune cells to suppress anti -tumor immune responses. Administration of cytokine therapy 2304, including combinations comprising IL-17 and IL-33, optionally together with IL-2, IL-12, IL-18, or IL-36, stimulates activation of bystander immune cells within the tumor microenvironment. In certain embodiments, an anti-PD-L2 antibody therapy (aPD-L2) 2306 is administered in combination with the cytokine composition. The aPD-L2 antibody blocks PD-L2 signaling and thereby restores immune cell activation that would otherwise be suppressed by PD-L2. The combination of cytokine-mediated immune activation and PD-L2 checkpoint blockade results in enhanced interferon-y (IFN-y) production 2308 by immune cells including CD8+T cells, natural killer (NK) cells, and y5 T cells.

[0230] In certain embodiments, the cytokine therapy enhances the tumor-specific activity of aPD-L2 antibodies, resulting in increased immune activation within tumor tissue relative to non-tumor tissue. The combined therapeutic effects promote immune-mediated tumor cell killing 2310, thereby improving anti -tumor immune responses and therapeutic outcomes.

[0231] X. Biomarker-Guided Patient Selection

[0232] In certain embodiments, the compositions and methods described herein can be implemented using biomarker-guided patient selection. Biomarkers useful for identifying subjects suitable for treatment include interferon-y (IFN-y) expression levels and PD-L2 expression levels within tumor tissue or immune cells associated with the tumor microenvironment. In some embodiments, tumor samples or immune cell samples are obtained from a subject and analyzed to determine baseline expression of IFN-y or PD-L2. Subjects exhibiting low IFN-y production or elevated PD-L2 expression within tumor tissue may be particularly suitable for treatment using the cytokine compositions described herein.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0233] Without being bound by theory, cytokine combinations comprising IL- 17 and IL-33 may enhance immune activation in such subjects by inducing antigenindependent IFN-y production by bystander immune cells within the tumor microenvironment. More particularly, use of biomarker results affords a vehicle for administering cytokine therapy in a manner generally described herein.

[0234] XL Further Diagnostic Methods

[0235] A. Detection of IFN-y or PD-L2

[0236] In certain embodiments, diagnostic methods are used to determine whether a subject is likely to respond to cytokine therapy. Detection of IFN-y or PD-L2 expression may be performed using techniques including: (a) immunohistochemistry (IHC), (b) ELISA assays, (c) flow cytometry, (d) RNA expression analysis, (e) quantitative PCR. and (f) next-generation sequencing.

[0237] In some embodiments, expression of PD-L2 within tumor tissue indicates an immune-suppressive tumor microenvironment that may respond favorably to combined cytokine therapy and anti-PD-L2 checkpoint blockade.

[0238] B. Further Diagnostic Kits

[0239] In certain embodiments, a diagnostic kit is provided for identifying subjects suitable for treatment with the cytokine compositions described herein. Such kits can include: (a) antibodies or probes for detecting IFN-y expression, (b) antibodies or probes for detecting PD-L2 expression, (c) reagents for performing immunohistochemistry or nucleic acid detection assays, (d) instructions for determining patient suitability for cytokine therapy.

[0240] In some embodiments, the diagnostic kit is used in conjunction with a therapeutic kit comprising the cytokine compositions described herein.

[0241] XII. IL- 17 / IL-33 Cytokine Signaling Pathway Inducing IFN-y

[0242] By way of further illustration of the mechanism operating herein, reference is made to Fig. 24, showing a conceptual diagram that illustrates an example signaling pathway 2400 through which cytokine compositions comprising IL- 17 and IL-33 stimulate immune responses within a tumor microenvironment.

[0243] A. Tumor Microenvironment

[0244] Within the tumor microenvironment 2402, tumor cells coexist with various immune cells including T lymphocytes, natural killer (NK) cells, macrophages, andDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 dendritic cells. In certain tumors, immune activity may be suppressed by inhibitory' signaling pathways or reduced interferon production.

[0245] B. Cytokine Administration

[0246] A cytokine composition 2404 comprising IL- 17 and IL-33 may be administered to the subject. In some embodiments, the cytokine composition further comprises one or more additional cytokines including IL-2, IL- 12, IL- 18. or IL-36.

[0247] In a manner clear to those of skill, such cytokine compositions can be delivered: (a) systemically, (b) intratumorally, (c) using lipid nanoparticles, (d) using viral vectors, and / or (e) using engineered immune cells.

[0248] C. Cytokine Receptor Activation

[0249] Following administration, IL- 17 and IL-33 bind their corresponding receptors on immune cells. For example: (a) IL- 17 binds the IL-17 receptor (IL-17R), and / or (b) IL-33 binds the ST2 receptor. Activation of these receptors is represented by element 2406.

[0250] D. Intracellular Signaling

[0251] Receptor engagement initiates intracellular signaling pathways 2408, including activation of: (a) NF-KB signaling pathways, (b) MAP kinase signaling cascades, and / or (c) transcription of pro-inflammatory genes. These signaling pathways promote immune activation and cytokine production.

[0252] E. Bystander Immune Cell Activation

[0253] The cytokine-mediated signaling stimulates bystander immune cells within the tumor microenvironment, represented by element 2410. Such cells can include: (a) CD8+T cells, (b) natural killer (NK) cells, (c) y5 T cells, and / or (d) other innate immune cells. Notably, in certain embodiments these cells can produce cytokines independent of antigen-specific recognition of tumor cells.

[0254] F. Induction of IFN-y

[0255] Activated immune cells produce interferon-y (IFN-y) as shown at 2412. The production of IFN-y may occur in an antigen-independent manner, thereby enabling activation of immune cells that may not directly recognize tumor antigens.

[0256] G. Tumor Cell Killing

[0257] The increased IFN-y production promotes immune-mediated tumor cell killing, represented by element 2414. In some embodiments, IFN-y enhances: (a) tumorDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 antigen presentation, (b) immune cell recruitment, and / or (c) cytotoxic activity of immune cells.

[0258] H. Synergy with Checkpoint Blockade

[0259] In certain embodiments, cytokine-mediated induction of IFN-y further enhances the therapeutic activity of immune checkpoint inhibitors, including antibodies targeting PD-1. PD-L1, or PD-L2.

[0260] XIII. Biomarker-Guided Cytokine Therapy

[0261] Referring now to Fig. 25, a workflow diagram illustrates an exemplary method / experiments 2500 for biomarker-guided cytokine therapy. A subject having cancer 2502 may first be identified as a potential candidate for treatment. A biological sample 2504 may be obtained from the subject. Such samples may include: (a) tumor biopsy tissue, (b) blood samples, (c) immune cell samples, and / or (d) circulating tumor cells. The biological sample is analyzed to detect one or more biomarkers 2506. In certain embodiments, the biomarkers include: (a) interferon-y (IFN-y) expression, (b) PD-L2 expression. Detection can be performed using: (a) immunohistochemistry’ (IHC). (b) ELISA assays, (c) nucleic acid amplification techniques, and / or (d) next-generation sequencing. Based on the biomarker measurements, the subject may be stratified 2508 to determine whether the subject is suitable for treatment with cytokine therapy.

[0262] Subjects meeting the biomarker criteria can, thus, receive cytokine therapy 2510 comprising IL-17 and IL-33, optionally together with one or more additional cytokines such as IL-2, IL-12, IL-18, or IL-36. Administration of the cytokine therapy’ stimulates immune activation 2512, including increased interferon-y production within the tumor microenvironment.

[0263] XIV. Cytokine Therapy Pharmacokinetics and Tumor Targeting

[0264] Referring to Fig. 26, a diagram 2600 illustrates an example pharmacokinetic and tumor-targeting pathway for cytokine therapy. A cytokine formulation 2602 may comprise IL-17 and IL-33, optionally combined with additional cytokines including IL-2, IL-12, IL-18, or IL-36.

[0265] In certain embodiments, the cytokines can be encapsulated in a delivery system, such as: (a) lipid nanoparticles, (b) polymeric nanoparticles, (c) viral vectors, and / or (d) extracellular vesicles.

[0266] The formulation can be administered to a subject 2604, for example via intravenous injection, intratumoral injection, or subcutaneous administration. FollowingDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 administration, the cytokine formulation circulates within the bloodstream 2606 and distributes throughout the body.

[0267] In some embodiments, the delivery system promotes preferential accumulation within tumor tissue, represented by element 2608. Once localized within the tumor microenvironment, the cytokines are released 2610, enabling interaction with immune cells present within the tumor. Activation of immune cells leads to local production of interferon-y 2612, which enhances anti -tumor immune responses. The resulting immune activity promotes tumor cell killing and tumor regression, represented by element 2614.

[0268] XV. Integrated Cytokine Immunotherapy Platform

[0269] Significantly. Fig. 27, described the integrated immunotherapy platform 2700 according to various embodiments. The platform 2700 combines biomarker-guided patient selection, cytokine therapy, and immune checkpoint blockade to enhance tumorspecific immune responses.

[0270] A. Patient Identification

[0271] A subject having cancer 2702 may be identified as a potential candidate for immunotherapy. In certain embodiments, the cancer can comprise melanoma, although other cancers can also be treated using the methods described herein.

[0272] B. Biomarker Analysis

[0273] Biomarker analysis 2704 can be performed on biological samples obtained from the subject. Suitable samples may include: (a) tumor biopsy samples, (b) blood samples, (c) immune cell samples. In certain embodiments, biomarkers include: (a) interferon-y (IFN-y) expression, and / or (b) PD-L2 expression.

[0274] C. Patient Stratification

[0275] Based on the biomarker analysis, the subject can be stratified 2706 to determine whether the subject is suitable for treatment using cytokine therapy described herein. Subjects exhibiting reduced IFN-y production or increased PD-L2 expression can be identified as particularly suitable candidates for treatment.

[0276] D. Cytokine Therapy Platform

[0277] Selected subjects can receive cytokine therapy 2708 as described variously herein, comprising IL-17 and / or IL-33. In certain embodiments, additional cytokines can also be administered, including: IL-2, IL-12, IL-18, and / or IL-36. The cytokine compositions can be delivered using delivery systems such as: (a) lipid nanoparticles, (b)Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 viral vectors, (c) engineered immune cells, and / or (d) other pharmaceutical delivery' platforms.

[0278] E. Immune Checkpoint Blockade

[0279] In some embodiments, cytokine therapy is administered together yvith immune checkpoint blockade therapy 2710. Checkpoint inhibitors can include antibodies targeting: PD-1, PD-L1, PD-L2, CTLA-4, and / or LAG-3. In certain embodiments, cytokine therapy enhances the tumor-specific activity of anti-PD-L2 antibodies.

[0280] F. Immune Activation

[0281] The combination therapy promotes immune activation within the tumor microenvironment 2712, including: (a) activation of bystander immune cells; (b) antigenindependent production of interferon-y; (c) enhanced immune recognition of tumor cells.

[0282] G. Tumor Regression

[0283] The increased interferon-y production promotes tumor cell killing and tumor regression 2714, thereby improving anti-tumor immune responses.

[0284] XVI. Clinical Treatment Protocol Timeline

[0285] Finally, referring to Fig. 28, a non-liming example of a possible clinical protocol 2800 is shown, including a treatment timeline 2801 for administering cytokine therapy and monitoring therapeutic responses in a subject with cancer, in accordance with the embodiments contemplated herein.

[0286] A. Baseline Assessment

[0287] Prior to treatment initiation, a baseline clinical assessment 2802 can be performed. Such an assessment may include: (a) imaging of tumor lesions, (b) biomarker testing, and / or (c) evaluation of patient eligibility for therapy. In certain embodiments, biomarker testing includes measuring interferon-y (IFN-y) levels or PD-L2 expression in tumor tissue or blood samples.

[0288] B. Cytokine Therapy Initiation

[0289] Following baseline evaluation, cytokine therapy 2804 can be initiated. In certain embodiments, the therapy comprises administration of: IL-17 and / or IL-33.Additional cytokines may optionally be administered, including: IL-2, IL- 12, IL- 18, and / or IL-36. The cytokine therapy can be administered weekly, biweekly, or according to another dosing schedule.

[0290] C. Checkpoint Blockade TherapyDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0291] In some embodiments, cytokine therapy is administered in combination with immune checkpoint blockade therapy 2806. Checkpoint inhibitors may include antibodies targeting: PD-1, PD-L1, PD-L2, CTLA-4, and / or LAG-3. In certain embodiments, cytokine therapy enhances the tumor-specific activity of anti-PD-L2 antibodies.

[0292] D. Biomarker Monitoring

[0293] During treatment, biomarkers may be periodically monitored 2808 to evaluate immune activation and treatment response. Biomarkers can include: (a) circulating IFN-y levels, (b) PD-L2 expression, and / or (c) immune cell activation markers. Monitoring can be performed using: (a) blood testing, (b) tumor biopsy, and / or (c) immunological assays.

[0294] E. Tumor Response Evaluation

[0295] Tumor response evaluation 2810 can be performed periodically during therapy. Assessment methods can include: (a) computed tomography (CT), magnetic resonance imaging (MRI), and / or positron emission tomography (PET) imaging. Tumor measurements can be used to determine: (a) tumor regression, (b) stable disease, and / or disease progression.

[0296] F. Treatment Adjustment

[0297] Based on biomarker monitoring and tumor response evaluation, the treatment regimen can be adjusted 2812. Adjustments can include: (a) modification of cytokine dose, (b) modification of checkpoint inhibitor therapy, and / or continuation or discontinuation of therapy.

[0298] XVII. Additional Experimental Results

[0299] Additional details are now provided. These experimental results are, more particularly, based upon experiments performed on young (<6 months), naive BL6 mice. Such experiments can be extrapolated to apply to other, more advanced mammalian studies, including eventual human trials.

[0300] Reference is made to Fig. 29. which shows IL- 17 elicits EOMES-associated IFN-y from young T cells in vitro. Spleen cells from young (<6 months) BL6 mouse were incubated in 96 well U-bottom plates for 96 hours in RPMI-1640 supplements with 10 mM HEPES, 10% fetal calf serum and antibiotic plus recombinant mouse IL-22.5 ng / ml + recombinant mouse IL-33 1 ng / ml, which was replenished every 2 days. Recombinant mouse IL- 17 was added at indicated concentrations in the last aboutDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 18 hours. Concentrations were analyzed by flow cytometry and Flo Jo software. IL- 17 elicits EOMES-associated IFN-y. To gain further insight into how IL- 17 elicits IFN-y in vitro cultures were used. In one example, splenocytes from young, naive BL6 mice were used, which were made into single cell suspensions for studies. T cells were conditioned to exhibit innate behavior from culture with IL-2 + IL-33 as reported, and found that IL- 17 dose-dependently increased IFN-y production, and also Eomes expression, which is a hallmark of innate behavior in adaprive immune cells.

[0301] With reference to Fig. 30, aPDL2 increases innate IFN-y in T cells in vitro in response to IL- 12 in an IL-17-dependent manner. T cells were negatively selected from young (<6 months) naive BL6 mice using a Miltenyi commercial kit and cultured with IL-2 + IL-33 as in Fig. 29 in 96 well U-bottom cell culture plates with either IL-17LO(10 ng / mL) or IL-17Hi (100 ng / mL) for 5 days, ± 1 ng / mL IL- 12 in the final ~18 hours on day 5 (top left and top right graphs). IFN-y detection in conditions stratified by IL-12 and / or aPDL2 in all T-cells or in central memory T cells (lower left and lower right graphs). Data generated from flow cytometry, statistics shown are by T-test and performed on a minimum of 3 technical replicates.

[0302] Because IL- 12 is a potent inducer of innate IFN-y, whether it could improve aPDL2-mediated IFN-y, and whether IL-17 could also contribute, was tested. Using the above-described in vitro culture system, it was found that IL- 12 potently augmented aPDL2-mediated IFN-y and that IL-17 further augment this effect.

[0303] To test if these innate conditioning conditions can be replicated in vivo, BL6 mice were challenged with B16 melanomas subcutaneously and used aCD134 + aCD137 as known to elicit innate behavior in adaptive cells. It was found that these agents elicited aPDL2 response (Fig. 31A and 31B), which are otherwise unresponsive to aPDL2. See for relevant teaching, Ontiveros CO, Garcia MG, Murray CE, Deng Y, Bai H, Tanner C, Leung B, Li X, Padron A, Reyes RM, Kancharla A, Soh KT, Krishnan S, Chand D, Balasubramanian A, Hegner C, Jakubzick CV, Gupta HB, Turk MJ, Sundrud M, Conejo-Garcia JR, Curiel TJ. Anti-PD-L2 immunotherapy is efficacious against melanoma in aged hosts through IL- 17 and IFNgamma signalling. Nature communications. 2025;16(l): 10176. Epub 20251119. doi: 10.1038 / s41467-025-65025-2. PubMed PMID: 41261166; PMCID: PMC12630879. Supporting the innate behavior, IFN-y was elicited from innate Eomes+Tbet+ CD8+ T cells (Fig. 3 IB, lower panels).Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019

[0304] Figs. 31A and 31B show aCD134 + aCD137 elicit aPDL2 efficacy in B16 melanoma challenge with an increase in innate IFN-y in T cells. Young BL6 hosts (<6 months) challenged subcutaneously with B16 melanoma 500,000 cell / flank and treated with aCD134 at 100 pg / mouse + aCD137 at 50 pg / mouse on days 5, 9, 13 ± aPDL2 on days 3, 7, 11, 15 at 200 pg / mouse versus respective, combined isotypes (iso). P values by 2-way ANOVA. 10 tumors / group. Cytometry data is from tumor infiltrating cells and tumor weights are all from at sacrifice on day 16. In the top two graphs and lower left graphs, the bar shade patterns match the legend (left bar in each graph is ISO, middle bar is aCD134 + aCD137, and right bar is aCD134 + aCD137 + aPD-L2). In the bottom, right graph, data is shown only from mice treated with all three (3) antibodies as indicated by individual legends. Flow data P values are from t-test. DN, double negative (for Eomes / T-bet), SP, single positive, DP, double positive (for Eomes / T-bet).

[0305] Fig. 32 shows that aCTLA4 improves aPDL2 in an IL-17-dependent manner. Young (<6 months old) BL6 mice were challenged subcutaneously with B6 melanoma cells 500,000 / flank and treated with anti-CTLA450 pg / mouse on day 7, aPDL2 on days 3, 7, 11, 15 at 200 pg / mouse. Anti-IL-17 given daily from days 3-17. N = 10 tumors / group. The full data are on the left. In the middle, and on the right specific treatments are broken out with their statistical analyses for clarity. P by ANOVA.Depletion of regulator}’ T cells elicits innate behavior. We tested aCTLA4 as it is known to deplete regulatory T cells. More particularly. Fig. 32 shows the totality of the experiment on the left graph. The other panels are details from the same experiment pulled out individually for clarity' and the specific statistical analyses. Anti-CTLA4 elicited aPDL2 efficacy in aPDL2-refractory Bl 6 tumors (Fig. 32, middle graph). Further, the effect was eliminated by anti-IL-17 (Fig. 32. right graph), showing IL- 17 dependence.

[0306] XVIII. Conclusion

[0307] It should be clear that the above-described methods and compositions can afford a variety of effective and advantageous bases for diagnosis and treatment of various cancerous conditions, including, but limited to, melanoma in various stages of progression, and with patients showing different age and other characteristics.

[0308] The foregoing has been a detailed description of illustrative embodiments of the invention. Various modifications and additions can be made without departing from the spirit and scope of this invention. Features of each of the various embodiments described above may be combined with features of other described embodiments asDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 appropriate in order to provide a multiplicity of feature combinations in associated new embodiments. Furthermore, while the foregoing describes a number of separate embodiments of the apparatus and method of the present invention, what has been described herein is merely illustrative of the application of the principles of the present invention. For example, where the term “substantially” or “approximately” is employed with respect to a given measurement, value or characteristic, it refers to a quantity that is within a normal operating range to achieve desired results, but that includes some variability due to inherent inaccuracy and error within the allowed tolerances of the system (e.g. 1-5 percent). Accordingly, this description is meant to be taken only by way of example, and not to otherwise limit the scope of this invention.

[0309] What is claimed is:

Claims

Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 CLAIMS1. A method of treating cancer in a subject, comprising:administering to the subject a cytokine composition comprising interleukin- 17 (IL-17) and at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36, the cytokine composition being delivered to a tumor microenvironment (TME) using a targeted delivery7system,wherein the cytokine composition induces antigen-independent interferon-y (IFN-y) production by bystander immune cells in the TME, including at least CD8+T cells, natural killer (NK) cells, or yd T cells;measuring at least one tumor response parameter selected from tumor size, tumor cell viability, immune-cell IFN-y production, or tumor-associated immune activation markers; andadjusting a dose or administration frequency of the cytokine composition based on the measured tumor response parameter.

2. The method as set forth in claim 1, wherein the cytokine composition comprises IL- 17, IL-2, and IL-33.

3. The method as set forth in claim 1, wherein the cytokine composition further comprises IL-18.

4. The method as set forth in claim 1, wherein the at least one cytokine is delivered as nucleic acids encoding the at least one cytokine.

5. The method as set forth in claim 4. wherein the nucleic acids comprise messenger RNA molecules encoding the at least one cytokine.

6. The method as set forth in claim 1, wherein the cytokine composition is delivered using lipid nanoparticles configured to accumulate within tumor tissue.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 7. The method as set forth in claim 1, wherein the cytokine composition is delivered by engineered immune cells expressing nucleic acids encoding the at least one cytokine.

8. The method as set forth in claim 1, wherein a delivery7system of the cytokine comprises a targeting moiety that binds a tumor-associated antigen.

9. The method as set forth in claim 8, wherein the targeting moiety7comprises an antibody or antigen-binding fragment thereof.

10. The method as set forth in claim 1, wherein the cytokine composition is targeted to immune cells expressing CD8. PD-1, orNKG2A.

11. The method as set forth in claim 1, wherein the cytokine composition further comprises an engineered IL-2 variant having reduced CD25 binding and increased CD122 binding.

12. The method as set forth in claim 1, wherein the cytokine composition is administered by intratumoral injection.

13. The method as set forth in claim 1. wherein the cytokine composition is administered in combination with an immune checkpoint blockade therapy.

14. The method as set forth in claim 13, wherein the immune checkpoint blockade therapy targets PD-1, PD-L1, PD-L2, CTLA-4, or LAG-3.

15. The method as set forth in claim 1, wherein administration of the cytokine composition increases anti-PD-L2 therapeutic activity within tumor tissue while maintaining lower anti-PD-L2 activity in non-tumor tissue.

16. A cytokine delivery' system for inducing IFN-y production in tumor-infiltrating immune cells, comprising:a carrier comprising lipid nanoparticles, engineered immune cells, viral vectors, or polymeric nanoparticles; andDocket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 a pay load comprising IL- 17 and at least one cytokine selected from IL-2, IL- 12, IL- 18, IL-33, or IL-36.wherein the delivery system is configured to deliver the payload to a tumor microenvironment and induce antigen-independent IFN-y production in bystander immune cells.

17. The delivery system as set forth in claim 16, wherein the carrier comprises lipid nanoparticles encapsulating mRNA encoding the at least one cytokine.

18. The delivery system as set forth in claim 16, further comprising a tumor-targeting moiety that binds a tumor-associated antigen.

19. The delivery system as set forth in claim 18, wherein the tumor-targeting moiety comprises an antibody against Her2 or another tumor-associated antigen.

20. The delivery system as set forth in claim 16, wherein the carrier comprises engineered immune cells expressing nucleic acids encoding the at least one cytokine.

21. A kit for treating cancer, comprising:a first container comprising IL- 17;a second container comprising at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36;a delivery formulation comprising lipid nanoparticles or nucleic acid vectors configured for delivery to a tumor microenvironment; andinstructions for administering the at least one cytokine to induce antigenindependent IFN-y production by tumor-infiltrating immune cells.

22. The kit as set forth in claim 21, wherein the at least one cytokine is provided as mRNA molecules encoding the at least one cytokine.

23. The kit as set forth in claim 22, wherein the kit further comprises a tumor-targeting antibody or binding fragment.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 24. The kit as set forth in claim 21, further comprising an immune checkpoint inhibitor selected from an anti-PDl antibody. anti-PD-1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, or anti-LAG-3 antibody.

25. The kit as set forth in claim 21, wherein the instructions specify intratumoral or tumor-targeted systemic administration.

26. A pharmaceutical composition comprising:a delivery vehicle selected from lipid nanoparticles, polymeric nanoparticles, viral vectors, or engineered immune cells; anda payload comprising interleukin- 17 (IL- 17) and at least one cytokine selected from IL-2, IL-12, IL-18, IL-33, or IL-36, or nucleic acids encoding the at least one cytokine,wherein the delivery vehicle is configured to deliver the payload to a tumor microenvironment and induce interferon-y production in tumor-infiltrating immune cells.

27. A method for treating cancer in a subj ect, comprising:obtaining a biological sample from the subject comprising tumor tissue or immune cells associated with a tumor microenvironment;measuring, in the biological sample, a biomarker selected frominterferon-y (IFN-y) expression or programmed death ligand-2 (PD-L2) expression;determining, based on the measured biomarker, that the subject is suitable for treatment with a cytokine composition comprising interleukin- 17 (IL-17) and at least one additional cytokine selected from IL-2, IL- 12. IL-18, IL-33, or IL-36; and administering the cytokine composition to the subject,wherein the cytokine composition induces antigen-independent interferon-y production in immune cells within the tumor microenvironment.

28. The method as set forth in claim 27, wherein the cancer comprises melanoma.

29. The method as set forth in claim 27, wherein the biomarker comprises PD-L2 expression in tumor tissue.Docket No.: 231 / 0022R Client Reference: 2024-052 / 2024-019 30. The method as set forth in claim 27, wherein the subject is an aged subject exhibiting chronic inflammation.

31. A diagnostic method for identifying a subject suitable for treatment with a cytokine therapy, comprising:obtaining a biological sample from a subject having cancer;detecting expression of a biomarker selected frominterferon-y (IFN-y) or programmed death ligand-2 (PD-L2) in the biological sample; and determining that the subj ect is suitable for treatment with a cytokine composition comprising IL-17 and at least one additional cytokine selected from IL-2. IL-12, IL-18, IL-33, or IL-36 based on the detected biomarker expression.

32. A method of inducing antigen-independent interferon-y production in tumorinfiltrating immune cells in a tumor microenvironment comprising:administering interleukin- 17 together with at least one cytokine capable of stimulating innate-like interferon-y production in T cells;monitoring the administering; andadjusting the administering based on the monitoring.

33. A method of treating cancer comprisingadministering a cytokine composition comprising interleukin- 17 and one of either (a) interleukin-33 or (b) at least one additional cytokine;monitoring the administering; andadjusting the administering based on the monitoring.

34. The method as set forth in claim 33, wherein the administering is delivered to a tumor microenvironment using a targeted delivery vehicle that preferentially accumulates in tumor tissue.

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