SYNERGY BETWEEN IFNalpha AND TLR3 LIGANDS IN SENSITIZING PD1-RESISTANT COLD TUMORS TO THERAPEUTIC EFFECTS OF PD-1 BLOCKADE

Abstract

Provided is a method for treating an individual for a programmed-death 1 (PD-1) immune checkpoint therapy resistant cancer. The method involves administering to the individual a combination of a Toll-like receptor 3 (TLR3) agonist and interferon-alpha (IFNα), followed by close in time administration of an anti-PD-1 immunotherapy.

Description

SYNERGY BETWEEN IFNa AND TLR3 LIGANDS IN SENSITIZING PD1- RESISTANT COLD TUMORS TO THERAPEUTIC EFFECTS OF PD-1 BLOCKADECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 471,175, filed June 5, 2023, the entire disclosure of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant no. 1P01CA234212 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Immune checkpoint inhibitors (ICIs) which block the interaction of T cell- expressed checkpoint molecules, such as programmed-death 1 (PD-1), with their ligands within tumor tissues (such as PD-L1 and PD-L2), have become standard of treatment in many advanced cancers and significantly extended survival in patients with previously hard-to-treat malignancies1'3, including metastatic, microsatellite instability-high / mismatch repairdeficient (MSI-H / dMMR) colorectal cancer (CRC)4. Since ICIs reactivate the immune system to specifically destroy cancer cells and promote long-lasting immune memory, responders often achieve durable tumor control5. However, the therapeutic benefit of ICIs is currently limited by the less than 25% of patients responding overall6, 7. Three major factors have been shown to predict the responding patients: high tumor mutational burden (TMB)8'10, presence of CD8+ tumor-infiltrating lymphocytes (TILs)11'17and intratumoral expression of PD-1 ligands18, 19. Although such factors can be interconnected with high TMB load driving Tumor infiltrating lymphocyte TIL infiltration and TIL infiltration promoting JFNy-driven induction of aPD-Ll- and PD-L2 on cancer cells, local stroma, and myeloid cells20, the relation between these factors and their relative values remain controversial21'26. Interestingly, a recent study demonstrated that artificial increase in TMB is not sufficient to enhance ICI responsiveness of mouse tumors27, raising a possibility that TIL content and PD1 sensitivity may be regulated at least partially independently from the TMB.

[0004] Several approaches the increase the numbers of tumor-infiltrating lymphocytes (TILs) have been proposed21, 28. Tumors lacking sufficient cytotoxic T lymphocytes (CTLs) have been classified as “cold” and minimally responsive to immune-based treatmentapproaches29, thus strategies to increase infiltration of CTLs into the tumor microenvironment (TME) have become increasingly sought. Recently, personalized mRNA vaccines demonstrated a dramatic ability of multi-epitope vaccination to enhance the clinical effectiveness of neoadjuvant PD1 blockade to prevent cancer recurrence30, demonstrating a promise of combinatorial approaches. However, there remains an ongoing and unmet need for combinatorial treatment of cancer, and particularly for treating PD1 -resistant “cold tumors”. The present disclosure is pertinent to this need.SUMMARY OF THE DISCLOSURE

[0005] The disclosure provides an approach to treating immunologically cold tumors. This approach promotes CTL infiltration using a chemokine modulatory (CKM) regimen that induces a pro-immune status in the “cold” TME31'42. CKM combines a type I interferon (IFN), which enhance TLR3 expression with toll-like receptor 3 (TLR3) ligands, such as poly-IC34'36, 41or rintatolimod41, a selective TLR3 ligand43, 44which does not activate NFkB, thus avoiding tumor necrosis factor alpha (TNFa) induction41, toxicity43, 44and NFkB / TNFa / PGE2-dependent suppressive aspects of inflammation41. In addition to reprograming of the stromal and myeloid component of the TME, rintatolimod induces TLR3-driven DC (dendritic cell) maturation and T cell stimulation43, 45, the desirable Demodulating function functions also amplified by the combination of TLR3 activation and IFNa34, 46, 47The disclosure accordingly provides a method for treating an individual for a programmed-death 1 (PD-1) immune checkpoint therapy resistant cancer. The method involves administering to the individual a combination of TLR3 agonist and interferon-alpha (IFNa), with or followed by close-in-time administration of an anti-PD-1 immunotherapy.

[0006] CKM preferentially activates tumor tissues (which show high baseline activation of NFkB, which is needed to sustain tumor viability, and acts as a co-activator for production of both CTL-attracting and Treg-attracting chemokines)34and the desirable CTL attraction34, 39, 41ex vivo, without or only marginal activation of healthy tissues, suggesting its potential to achieve local intratumoral effects, even after its systemic application.

[0007] In accordance with these in vitro predictions, our recently completed clinical study (NCT03599453) demonstrated sufficient tolerability of systemic CKM and its ability to reprogram the TME for selective CTL influx in triple negative breast cancer patients42. Despite these favorable immune effects, the subsequent application of PD1 blockade with an average delay of 1 week after CKM, showed only marginal clinical efficacy42, raising the need to evaluate the mechanisms involved in its effectiveness and identify the most effectivetiming of its application relative to ICI. This disclosure demonstrates that CKM works synergistically with PD-1 blockade, resulting in durable cures in mouse models of CRC, but, in contrast to the expectations resulting from previous work31'42, such synergy requires simultaneous or near simultaneous administration of CKM and anti-PD-1 (aPDl) components. Moreover, the disclosure demonstrates that, in addition to CXCR3- and CCR5- dependent CTL attraction to the TME, the effectiveness CKM + aPD-1 therapy involves conventional dendritic cells (cDCls) which are involved in the enhancement of systemic anticancer immunity protective of subsequent tumor re-challenge.BRIEF DESCRIPTION OF THE FIGURES

[0008] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0009] FIGS. 1A-1C display graphical representations of CKM inducing expression of CTL-attracting chemokines in vitro and systemic application promotes local expression of CTL-attractants and effector markers in the TME. As shown in FIG. 1 A, MC38 cells were treated in culture with 50 pg rintatolimod (Rint), 10,000 IU IFNa (interferon alpha - IFNa), or a combination (CKM). Control cells were cultured in DMEM-based media alone untreated (Untx). After a 24 incubation, cells were harvested and assayed for RNA message by real time quantitative polymerase chain reaction (RTqPCR). Transcripts (normalized to GAPDH) are shown. Statistical significance was determined by one way ANOVA with Tukey’s multiple comparisons; **** p < 0.0001; N = 3 / group. As shown in FIG. IB, mice were inoculated subcutaneously with MC38 cells. On day 17, mice were treated with one dose of CKM (50 pg rintatolimod and 10,000 IU IFNa) injected intraperitoneally (CKM) or not treated (Untx). Three days after treatment, tumors were excised and assayed for RNA message by RT-qPCR. Transcripts (normalized to GAPDH) are shown. Statistical significance was determined by unpaired t test; * p < 0.05; N = 6-9 / group. As shown in FIG. 1C, mice were inoculated subcutaneously with MC38 cells and treated intraperitoneally with PBS, IFNa, rintatolimod (RIN), or CKM with and without the addition of aPD-1. Survival was monitored. Statistical significance was determined by log-rank test; * p < 0.05, *** p < 0.001; N = 15-20 / group.

[0010] FIGS. 2A-2E display schematics and graphical representation of local and systemic administration of CKM in combination with PD-1 blockade that can abrogate tumor growth in mouse models of colorectal cancer. FIG. 2A displays a schematic of therapeuticschedule. Mice were inoculated with MC38 cells. Concomitant treatment represents three cycles consisting of two doses of CKM (50 pg rintatolimod and 10,000 IU IFNa) one day apart followed by 200 pg aPD-1 the next day. Sequential treatment represents two doses of CKM (50 pg rintatolimod and 10,000 IU IFNa) one day apart directly followed by three doses of aPD-1 (200 pg) one, five, and ten days after the second CKM dose. All treatment was administered intraperitoneally. Controls received intraperitoneal injections of PBS, CKM, or aPD-1 at the same dose or volume as the experimental groups. FIG. 2B displays results of mice inoculated with MC28 tumor cells intraperitoneally. Local treatment of early- stage tumors evaluated using intraperitoneal MC38 tumor and intraperitoneal treatment administration beginning on day 3. FIGS. 2C and 2E display results of systemic treatment of early-stage tumors evaluated using subcutaneous MC38 (FIG. 2C) and CT26 (FIG. 2E) tumors and intraperitoneal treatment administration beginning on day 3. FIG. 2D displays results of systemic treatment of late-stage tumors evaluated using subcutaneous MC38 tumors and intraperitoneal treatment administration beginning on day 8. As shown in FIGS. 2B - 2E, the statistical significance was determined by log-rank test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p > 0.0001; N = 5-10 / group.

[0011] FIGS. 3 A-3F display schematics and graphical representation of mice treated with CKM + aPD-1 that develop tumor-specific systemic CTL responses. MC38-bearing mice were treated with PBS (Untx) or CKM + aPD-1 (CKM + aPD-1, sequential) beginning on day 3. Spleen and lymph nodes were harvested 24 hours after the last injection. CD8- selected splenocytes and lymph node cells from untreated and CKM + aPD-1 -treated mice were cultured with 4T1 (Off Target) or MC38 cells. Following overnight incubation with 50 U / ml IL-2 (interleukin-2), samples were assayed for IFNy production (interferon gamma). FIG. 3A displays a schematic of spots used for quantification of FIG. 3B. FIGS. 3B-3C display the number of spots per 100,000 CD8+ cells. Statistical significance was determined by unpaired t test; * p < 0.05; N = 3 / group. Quantification of IFNy producing CD8-selected splenocytes (FIG. 3B) and lymph node cells (FIG. 3C). FIG. 3D displays a change in tumor size calculated by subtracting the tumor size on day 15 from the largest tumor size measured for each mouse and shown in correlation to the number of spots counted from the splenocytes and lymph node cells. FIG. 3E displays results of mice inoculated with 500,000 MC38 cells intraperitoneally and treated with sequential regimen consisting of represents two doses of CKM (50 pg rintatolimod and 10,000 IU IFNa) one day apart followed by three doses of aPD-1 (200 pg) one, five, and ten days after the second CKM dose. All treatment was administered intraperitoneally. Controls received intraperitoneal injections of PBS at thesame volume as the experimental group. At 90 days (indicated by arrow), surviving mice in the CKM + aPD-1 group were rechallenged with 1,000,000 MC38 cells and monitored for survival. No PBS-treated mice survived to the 90-day timepoint for rechallenge. N = 9 at time of rechallenge. FIG. 3F displays results of spleens from mice inoculated with subcutaneous MC38 and regressing tumors following systemic CKM + aPD-1 beginning on day 3 were harvested on after day 90. CD8+ splenocytes were cultured with media alone (Media), unrelated target cells (E0771), or MC38 cells and evaluated for IFNy production by ELISA. Statistical significance was determined by one-way ANOVA; ** p < 0.01, *** p < 0.001; N = 5 / group.

[0012] FIG. 4A-4F display schematic and graphical representations of therapeutic synergy between CKM and PD-1 blockade requiring their simultaneous administration or immediate pre-treatment with CKM: Loss of the synergy upon delayed application. Mice were inoculated subcutaneously with MC38 cells. As shown in FIG. 4A, beginning on day 3, mice were treated with CKM in combination with aPD-1 based on the schematics depicted. Mice experiencing a regression were defined as mice who displayed a tumor that was eliminated by treatment. Non-responders were defined by uncontrolled tumor growth following treatment. FIG. 4B displays standard concomitant schedule as shown in FIG. 2A- 2E. FIGS. 4C-4F display a standard sequential schedule as shown in FIG. 2D and modified to include aPD-1 immediately following CKM (FIG. 4C), three days after CKM (FIG. 4E), or five days after CKM (FIG. 4F). N=6-8 / group.

[0013] FIG. 5A-5D display graphical representations of the anti-tumor effects of CKM in combination with PD-1 blockade require intact immune system and endogenous Batf3 -containing eDCs. SCID (FIG. 5A), BATf3 knockout (KO) (FIG. 5B), CCR5 knockout (FIG. 5C), CXCR3 knockout (FIG. 5D) and C57BL / 6 wildtype (FIG. 5A-5D) mice were inoculated subcutaneously with MC38 cells and treated with CKM + aPD-1 or PBS control. All treatment was administered intraperitoneally. Statistical significance was determined by log-rank test; ** p < 0.01, *** p < 0.001, **** p > 0.0001; N = 5-10 / group.DETAILED DESCRIPTION OF THE DISCLOSURE

[0014] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0015] Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0016] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0017] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. The steps of the method described in the various examples and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an example, the method consists essentially of a combination of the steps of the methods disclosed herein. In another example, the method consists of such steps.

[0018] As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” or “approximately” it will be understood that the particular value forms another example. The term “about” and “approximately” in relation to a numerical value encompass variations of + / - 10%, + / - 5%, or + / - 1%.

[0019] Any result obtained by using a described combination as described herein can be compared to a suitable control. In examples, the control is a result obtained using only one of CKM or an anti-PDl agent. In examples the control comprises or consists of sequentially administering CDM and an anti-PDl agent, but wherein the anti-PDl agent is not administered until more than 3, 4, 5, 6, or more days after the CKM. In an example, an improved result can comprise a reduction in tumor volume using the described combination approach, relative to the control, or an inhibition of tumor growth, relative to the control, or sensitization to anti-PD-1 therapy relative to the control, or a synergistic anti -tumor response when using the described combination according to the described administration order and timing, and wherein the control does not result in a synergistic effect

[0020] In examples, the anti-PD-1 agent is pembrolizumab, nivolumab, or cemiplimab. In examples, the CKM comprises or consists of a combination of a TLR3agonist and an interferon (IFN) such as Type 1 IFN-alpha (IFNa). In examples, the TLR3 agonist comprises or consists of poly IC or a poly IC analog, such as rintatolimod which is sold under the tradename Ampligen®.

[0021] In examples, the described method uses a therapeutically effective amount of a described combination of described agents. The term “therapeutically effective amount” as used herein refers to an amount of an agent or combination of agents sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The amount required may vary based on its mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a dose, can be estimated initially either in cell culture assays or in animal models. An animal model can also be used to determine a suitable concentration range, and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, or to non-human animals. A precise dosage can be selected by in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of the combination of the described agents to achieve a desired effect. Additional factors which may be taken into account include the type or stage of cancer, the age, weight and gender of the patient, reaction sensitivities, and tolerance / response to therapy. In examples, a therapeutically effective amount is an amount that reduces one or more signs or symptoms of cancer, and / or reduces the severity of the cancer. A therapeutically effective amount may also inhibit or prevent the cancer relapse. In examples, a described combination administered according to the described sequence and time periods may result in using less of the anti-PD-1 agent than would be administered without the CKM combination. In examples, a described combination administered according to the described sequence and time periods may result in a greater than additive effect that if the CKM and the anti-PD-1 agent were administered sequentially but not in combination over a described time period. In examples, the described combination may be administered using the same, or different routes, including but not necessarily limited to intravenous administration.

[0022] In examples this disclosure demonstrates that use of both local or a systemic CKM combination, but not their individual components effectively sensitize PD-1 -resistant established MC38 or CT26 tumors to PD-1 blockade. CKM + aPD-1 combination promoted local influx of Batf3 -positive cDCls and CTLs, inhibited tumor growth and prolonged survival, inducing cures in 20-100% of the treated animals, depending on the tumor model, time of the onset of treatment (early- or late-stage disease), and the route of delivery (local orsystemic). CKM + aPD-1 -treated mice developed local and systemic tumor-specific CTL responses and resistance to tumor re-challenge.

[0023] In examples, and as described above, the described effectiveness of CKM comprises its administration at the time of, or directly before, PD1 blockade and is strongly reduced by 24-hour delay between CKM and PD1 blockade. Thus, in a representative examples, CKM is administered to a patient first, following the administration of the anti-PD- 1 agent. The CKM may be administered on the same day as the anti-PD-1 agent (provided CKM is administered first), or not more than one, two or three days after the CKM, and preferably within one day. In examples, the doses of CKM and the anti-PD-1 agent are both administered within a two-day period. In examples, the CKM is administered as a first dose, which may be followed by at least one additional CKM dose, after which the anti-PD-1 agent inhibitor is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours, including all times and ranges of time points there between, of the CKM. In examples, an individual may be treated with a described consecutive treatment for a period, followed by a period wherein neither CKM or the anti-PD-1 agent is administered, or only one of CKM or the anti-PD-1 is administered. In an example, such period may be 1-14 days. In an example, CKM is administered for three cycles, followed by CKM and an anti- PD-1 agent administered according to the described time period. After the initial one or several CKM administration, administration of the anti-PD-1 agent is performed according to a described time period, and may be repeated one or more times before the CKM is administered again.

[0024] As demonstrated by the description and figures of this disclosure, the CKM- driven intratumoral CTL accumulation and antitumor effects were abrogated in animals lacking Batf3 -positive cDCls, CD8+ T cells, or CXCR3 and CCR5, the CTL-expressed receptors for CXCL9 / CXCL10 and CCL5. The demonstration that both local and systemic CKM can promote the therapeutic effectiveness of PD1 blockade in nominally PD1 -resistant “cold tumors”, indicate that intratumoral CTL accumulation, rather than lack of sufficient antigenic targets is the dominant factor limiting the therapeutic effectiveness of ICI in mouse tumors. The unexpected observation that the effectiveness of the combination treatment requires close timing of the CKM component and the ICI component that supports the described improved treatment modalities including CKM and ICI and potentially, other forms of immunotherapy.

[0025] This disclosure demonstrates that the PD-1 blockade administered systemically at the same time or directly following CKM administration shows strongly enhanced antitumor effects. The present demonstration that tumor-bearing mice respond favorably to systemic CKM + aPD-1 treatment supports the use of systemic CKM widely to sensitizing tumors to immune-based therapies, even if tumors cannot be directly accessed. The demonstration that the effects of CKM require its administration at the same time or directly before aPD-1 treatment provides for a preferred implementation of the described approach.

[0026] Example 1

[0027] The following materials and methods were used to produce the results described in this disclosure.

[0028] Mice.

[0029] Female 6 - 8 week old C57BL / 6J (JAX stock #000664), BALB / cJ (JAX stock #000651), B6.129P2-Ccr5tmlKu7J (CCR5 KO; JAX stock #005427), and B6.129P2- Cxcr3tmlDgen / J (CXCR3 KO; JAX stock #005796) mice were purchased from The Jackson Laboratory and experiments began within 2 weeks of arrival. Female 6 - 8 week old C.B-Igh- lb!crTac-Prkdcscld / Ros (SCID) and NOD.Cg-PrkdcscldI12rgtmlwj1 / SzJ (NSG) mice were bred through the Roswell Park Comprehensive Cancer Center Comparative Oncology Shared Resource. All mice were maintained in specific pathogen-free conditions in a HEPA-filtered ventilated rack system at the Roswell Park Comprehensive Cancer Center Comparative Oncology Shared Resource. Mice were housed 5 or less per cage with Enrich-o’Cobs 14 (The Andersons) enrichment. Sterile pellet Teklad Global 2018SC Rodent Diet (Inotiv) and water were provided ad libitum. Mice were maintained on a 12-hour light / dark cycle at 24°C ± 2° (~70°F) and 50% average humidity (range 30-70%). All in vivo experiments were performed in accordance with institutional guidelines under protocols approved by the Institutional Animal Care and Use Committee at Roswell Park Comprehensive Cancer Center.

[0030] Cell lines.

[0031] MC38 (Kerafast) and CT26 (American Type Culture Collection) were obtained directly from the manufacturer and stored frozen in aliquots after <5 passages. Cells were cultured in RPMI-1640 with GlutaMAX™(Gibco) supplemented with 10% fetal bovine serum and other additives according to manufacturer recommendations. Prior to inoculation, cells were harvested at ~90% confluency at <10 passages from the original stock. For in vivo work, freshly harvested tumor cells were suspended in PBS and injected subcutaneously into the left flank or intraperitoneally. Subcutaneous injections of both CT26 and MC38 cellswere administered at 500,000 cells per 50 pl PBS for initial inoculation. Intraperitoneal injections of MC38 were given at 500,000 cells per 200 pl PBS. For rechallenge, mice surviving following treatment of intraperitoneal tumors were inoculated with 1,000,000 MC38 cells per 50 pl PBS subcutaneously.

[0032] Treatment Strategy.

[0033] Following subcutaneous tumor implantation, mice were monitored daily to identify palpable tumors at which time they were randomized into treatment groups. To model early stage CRC, treatments were administered beginning on Day 3. To model late stage CRC, treatments were administered to mice bearing palpable tumors at -10-14 days following inoculation (tumor volume —20-100 mm3). Rintatolimod (provided by AIM ImmunoTech Inc.) was administered in 50 pg doses, IFNa (Miltenyi Biotec) was administered in 10,000 IU doses, and aPD-1 (clone RMP1-14; Leinco Technologies, Inc.) was administered in 200 pg doses. For concomitant administration, three cycles consisting of two doses of CKM (50 pg rintatolimod and 10,000 IU IFNa) one day apart were given, followed by 200 pg aPD-1 the next day. For sequential treatment, two doses of CKM (50 pg rintatolimod and 10,000 IU IFNa) were given one day apart followed by three doses of aPD- 1 (200 pg) one, five, and ten days after the second CKM dose. When administering CKM or aPD-1 alone, doses were administered on the corresponding days these treatments were given in the combinatorial schedule of CKM + aPD-1. For mice receiving PBS control, 200 pl sterile PBS was administered on all days and treatment was administered to comparative groups of mice. When rintatolimod and IFNa were given together, they were administered in one intraperitoneal injection. All treatment was administered intraperitoneally in a volume of 200 pl. Tumors were measured with digital calipers and tumor volume was calculated in mm3using the formula (length*width*width) / 2. Following the initiation of treatment, tumors were measured at least twice a week until size reached 1000 mm3at which point measurements were taken daily. The endpoints for survival studies included when tumor volume reached >1500 mm3, mice became lethargic, or ascites developed prohibiting movement. Endpoints were noted when subcutaneous tumors reached 1500 mm3or the longest diameter reached 2 cm for subcutaneous tumors. For intraperitoneal tumors, endpoints were noted when the diameter or shape of the abdomen has clearly changed (abdominal distension). All mice were humanely euthanized at endpoints in accordance with the Institutional Animal Care and Use Committee at Roswell Park Comprehensive Cancer Center.

[0034] RT-qPCR.

[0035] The expression of mRNA was analyzed using TaqMan-based RT-qPCR. For in vitro analysis, MC38 cells were cultured and subsequently treated in DMEM media enriched with 10% FBS, HEPES, NEAA, sodium pyruvate, 1-glutamine, and antibiotics (penicillin streptomycin and gentamycin sulfate). For ex vivo analysis of splenocytes, spleens were excised from mice following treatment. Spleens were digested and red-blood cell lysed before incubation and treatment in AIM-V supplemented with antibiotics (penicillin streptomycin). Splenocytes were treated with 50 ug rintatolimod, 10,000 IU IFNa, or a combination of both. For ex vivo total TME analysis, tumors were excised from mice following treatment. All samples were processed in RLT buffer (Qiagen) and homogenizing tubes were used for digestion of tumors. Supernatants were collected and total RNA was extracted using RNeasy kit (Qiagen). For RNA, 250 ng per cDNA synthesis reaction was used to generate cDNA with a qScript cDNA Synthesis kit (QuantaBio) and the resulting cDNA was diluted 5X in DNase / RNase-free water for mRNA expression analysis. For ex vivo analysis of TILs and tumor cells, CD8+ cells were prior to processing in RLT buffer. TaqMan primers (Thermo Fisher Scientific) included: CD8a (MmOl 182107_gl), CCL5 (Mm01302427_ml), CCL22 (Mm00436439_ml), CCR5 (Mm01963251_sl), CXCL9 (Mm00434946_ml), CXCL10 (Mm99999072_ml), CXCR3 (Mm00438259_ml), FoxP3 (Mm00475162_ml), GZMB (Mm00442837_ml), and IFNy (MmOl 168134 ml).

[0036] ELiSPOT,

[0037] Splenic MC38-Ag specificCD8+T cells were quantified via IFNy production. Spleens were excised and single cell suspension was generated using a screen before ACK- lysis. CD8-selected splenocytes were incubated overnight with low-dose IL-2. MC38-Ag specificity was determined using a negative control cell line (4T1). Spots were counted to calculate total number of splenic Ag-specificCD8+T cells.

[0038] ELISA.

[0039] To measure IFNy production of splenicCD8+T cells, spleens were excised, and single cell suspension was generated using a screen before ACK lysis.CD8+splenocytes were incubated overnight with low-dose IL-2. Effector cells (CD8+splenocytes) and target cells (media, E0771, MC38) were incubated for 48 hours before ELISA. IFNy production was quantified using R&D Systems Mouse IFN-y ELISA kit (DY485).

[0040] Statistics.

[0041] One way ANOVA with Tukey’s post-test for multiple comparisons was used to compare readouts from four groups and unpaired t tests were used to compare readouts between two groups. All p values less than 0.05 are reported and indicated on the figures.Kaplan-Meier survival plots and log-rank tests were used to display and analyze the time from the beginning of treatment to death or the date of sacrifice. All tests were two-sided. All analyses were performed using GraphPad Prism version 10.

[0042] The following is a description of results obtained using the materials and methods described above.

[0043] Example 2

[0044] Combinatorial CKM selectively induces CTL-attracting chemokines in mouse TME in vitro and enhances intratumoral CTL-attractants and CTL markers in vivo upon its systemic application in tumor-bearing mice.

[0045] To determine the susceptibility of mouse CRC cells to CKM, we examined the impact of treatment on MC38 cells in culture. Cells were treated with IFNa, rintatolimod, or a combination of the two (CKM) to evaluate their synergy (FIG. 1 A). Expression of chemokines known to attract CTLs, including CCL5, CXCL9, and CXCL10 were significantly upregulated following treatment with CKM (FIG. 1 A). IFNa alone upregulated expression of CXCL9 to a similar extent as the combinatorial CKM but the induction of CCL5 and CXCL10 was strictly dependent on the double activation by both CKM components (FIG. 1 A), indicating that optimal TME modulation requires a combinatorial approach.

[0046] To test if similar changes can be also induced in mouse tumors in vivo, mice with established subcutaneous MC38 tumors were treated with one dose of intraperitoneal CKM. Tumors were harvested three days after treatment and mRNA expression was evaluated using RT-qPCR (FIG. IB). As predicted by our ex vivo data, systemic CKM enhanced local intratumoral expression of CTL-attracting chemokines CCL5 and CXCL10 with additional trend towards increased CXCL9 expression (FIG. IB). In addition, systemic CKM treatment promoted intratumoral increases of CTL markers: CD8a and granzyme B (FIG. IB).

[0047] Our previous work has shown that a combination approach, including both TLR-3 stimulation (i.e., poly:IC or rintatolimod) and type 1 IFN (i.e., IFNa), reprograms the TME of various human and mouse tumors34'36, 40, 41. We tested this concept in vivo and found slowed tumor growth (FIG. 1C) and longer survival (FIG. 1C) when applying both rintatolimod and IFNa. While the in vivo effects of CKM on mouse CRC were statistically significant, the impact was unimpressive. For synergistic action of both IFNa and rintatolimod in the induction of CTL attractants (FIG. 1 A), the therapeutic effects required combined application of rintatolimod and IFNa, in addition to PD1 blockade (FIG. 1C).

[0048] Example 3

[0049] Local and systemic administration of CKM converts nominally PD-1 -resistant tumors into PD1 -responsive ones: Synergy between IFNa and rintatolimod^

[0050] To test the efficacy of adding PD-1 blockade to CKM for treatment of mouse CRC, we compared two treatment schedules for combination of CKM and aPD-1. As shown in FIG. 2A, we developed a concomitant schedule where CKM was administered directly prior to aPD-1 for three cycles and a sequential schedule where CKM was administered first followed by three subsequent aPD-1 injections. In addition to evaluating the schedule for administrating CKM and aPD-1 in combination approaches, we also tested the effectiveness of local (FIG. 2B) and systemic (FIG. 2C-E) treatments by inoculating tumors via intraperitoneal or subcutaneous routes, respectively. Local treatment of early-stage MC38 tumors, where treatment is started three days after inoculation, resulted in complete abrogation of all mice treated by either concomitant or sequential schedules (FIG. 2B). When using the systemic model defined by early-stage subcutaneous tumors receiving intraperitoneal treatment, both concomitant and sequential schedules demonstrated statistically better efficacy than monotherapies (FIG. 2C). In this setting, concomitant drug administration did result in the optimal tumor control in 100% of tumor-bearing mice, while the sequential administration where the aPD-1 component was administered directly after the CKM resulted in only 70% effectiveness (FIG. 2C). We next explored the effects of CKM + aPD-1 on more established, late-stage MC38 tumors. When treating established tumors, both concomitant and sequential treatment cured approximately half of treated mice within 50 days of tumor inoculation (FIG. 2D).

[0051] Finally, to determine if the efficacy of CKM + aPD-1 can be extended from the relatively immunogenic MC38 cell line, most commonly used to optimize immune treatment approaches48'51(which showed partial responsiveness to PD1 alone), to fully PD1- resistant CRC, we tested the efficacy of the CKM + aPD-1 regimen on established CT26 CRC. We found that CKM + aPD-1 still uniformly delayed the progression and induced cures in 25% of the animals with CT26 tumors, which were fully resistant to PD1 blockade alone (FIG. 2E).

[0052] Example 4

[0053] CKM + aPD-1 treatment activates lymphoid tissues and induces systemic tumor-specific CTL responses.

[0054] We next evaluated production of IFNy from splenocytes and lymph nodes of mice treated with CKM + aPD-1 therapy. Organs were harvested 24 hours following the finaltreatment at which time treated mice were demonstrating tumor regression. CD8-selected splenocytes from treated and untreated MC38-bearing mice were cultured ex vivo with off target (4T1) cells, during with no IFNy production was observed (FIGS. 3A-B). However, when cultured in the presence of MC38 cells, CD8-selected splenocytes (FIGS. 3A-B) and lymph node cells (FIGS. 3C) from two of three treated mice demonstrated a notable increase in IFNy production. Importantly, the percentage (-67%) of responding mice corresponds with our observations of long-term responses from treating early-stage MC38 tumor with sequential administration of CKM + aPD-1 (FIGS. 2B). The mice which experienced the largest tumor regressions also had the highest production of IFNy (FIGS. 3D).

[0055] Unexpectedly, when rechallenged with double the number of the same MC38 tumor cell line, 66.7% of surviving mice rejected the original tumors (FIG. 3E), suggesting that, in addition to local attraction of CTLs, CKM may also promote the induction of tumorspecific systemic immunity. In addition, CD8+ splenocytes from long-term (over 90 day) survivors who experienced tumor regression following CKM + aPD-1 treatment exhibited MC38-antigen specific IFNy production (FIG. 3F). Taken together, these findings demonstrate that CKM + aPD-1 therapy can induce favorable immune changes which occur quickly after treatment but also result in long-lasting immunity.

[0056] Example 5

[0057] Transient character of the CKM-induced window of susceptibility to PD-1 blockade results in the need for concomitant or back-to-back treatment.

[0058] To test the duration of the CKM-induced window of sensitization of tumors to PD1 blockade, we compared the effectiveness of aPD-1 administered at the same time or at different timepoints after CKM completion of treatment (FIG. 4A). Unexpectedly, we found CKM-induced responsiveness to PD-1 blockade occurred almost immediately after treatment (FIGS. 4B-C). We saw more responding mice when aPD-1 was administered right after CKM (FIG. 4C) compared to when aPD-1 was administered one day after CKM (FIG. 4D). Similarly, using sequential administration, fewer mice experienced tumor regression when aPD-1 was delayed three or five days after CKM administration (FIGS. 4E-F). These data demonstrate that aPD-1 could effectively be administered on the same day as CKM, a strategy likely to ease clinical translation and compliance when designing subsequent clinical trials.

[0059] Example 6

[0060] Anti -turn or effects of CKM + aPD-1 combination require intact immune system, including endogenous Batf3 -containing eDCs, CCR5, and CXCRA

[0061] T cells and DCs play a pivotal role in regulating anti-tumor immune response52. We found no effect of CKM + aPD-1 treatment to MC38-bearing immunodeficient SCID53(FIG. 5 A) mice, indicating a therapeutic dependence on immune cell subsets, including T cells or NK (natural killer) cells. We found endogenous CD8a conventional DCs (eDCs) were required for the therapeutic efficacy of CKM + aPD-1 based on the lack of anti-tumor responsiveness in Batf3' ' mice55(FIG. 5B). Because cross-priming of CD8+ T cells requires Batf356, 57, these findings suggest that antigen cross-presentation by eDCs to CTLs represents a vital component of CKM + aPD-1 efficacy against mouse CRC.

[0062] To further elucidate the impact of CKM + aPD-1 on chemokine modulation, we investigated the role of two important chemokine receptors; CCR5, a ligand for CTL- attracting chemokine CCL558, and CXCR3, a ligand for CTL-attracting chemokines CXCL9 / 1059. We found that mice lacking CCR5 do not garner as robust of a response to CKM + aPD-1 therapy as WT C57BL / 6 mice, although, there is some efficacy of the treatment (FIG. 5C). However, mice lacking CXCR3, exhibit no response to CKM + aPD-1 therapy (FIG. 5D). Taken together, these findings indicate a minor role for the CCR5 / CCL5 interaction and a more prominent role of the CXCR3 / CXCL9 / 10 axis in the effects of this treatment.

[0063] It will be recognized from the foregoing examples and description that the present disclosure demonstrates that CKM, a combination therapy consisting of a TLR3 agonist (rintatolimod) and Type 1 IFN (IFNa) can induce CTL-attracting chemokines in murine CRC tumors. This treatment strategy is also associated with favorable immune changes in the TME, including an induction of CD8a, Granzyme B, and TNFa message. When PD-1 blockade is added to CKM, we show substantial tumor control and induction of anti-tumor immune responses. Both local and systemic administration of this combinatorial treatment regimen results in durable regression of MC38 tumors. Even late-stage tumors show a modest response (-50%) to the CKM + aPD-1 therapeutic regimen. In contrast to our original expectations resulting from our previous work31'42, which led to the design of our recent clinical trials in triple negative breast cancer patients (NCT03599453)42and colorectal cancer patients (NCT04119830) where PD1 blockade was started within days to weeks after completion of CKM treatment the currently provided and unexpected data show that the antitumor effectiveness of the combined CKM and aPD-1 therapy is dependent on the timing of its components and works best when aPD-1 was administered at the time of immediately after CKM (FIGS. 4A-4F).

[0064] The data also indicate that mice experiencing tumor regression following CKM + aPD-1 treatment can generate durable immunity. Treated mice cured of their tumor exhibit long-lasting capacity to upregulate immune factors, including CD8a, Granzyme B, IFNy, and TNFa. Further, splenocytes from treated and cured mice still upregulate CTL- attracting chemokines (CCL5, CXCL9, CXCL10) in response to CKM more than 90 days after tumor inoculation.

[0065] The disclosure demonstrates a requirement of two chemokine receptors for effective CKM + aPD-1 treatment. The described treatment combination shows a partial dependence on CCR5 (FIG. 5C), the ligand for CTL-attracting chemokine CCL558. CCR5 has been shown to be a necessary signaling component for macrophage-driven expression of inflammation in response to double-stranded RNA61, and since our treatment regimen contains rintatolimod, a double-stranded RNA which activates TLR344, it is not surprising that CCR5 deficiency impedes the effects of CKM + aPD-1 therapy. While CCL5 / CCR5 signaling does facilitate recruitment of CTLs to the TME, it has also been implicated in the recruitment of Tregs62and thus, its depletion could be, in part, combating immunosuppressive effects.

[0066] The disclosure shows a role for chemokine receptor CXCR3 (FIG. 5D), which serves as a ligand for T cell attracting chemokines CXCL9 / 10 / 11 and is prominently expressed on activated T and NK cells59. While CXCR3’s absence perturbs T cell trafficking into the TME, some T cells are able to infiltrate the tumor in mouse models and frequencies of T cells in the spleen and lymph node remain consistent with that in wild type tumorbearing mice67. Thus, the complete abrogation of CKM + aPD-1 efficacy in CXCR3 KO mice suggests a broader impact of CXCR3 in this model. CXCR3 has been shown to augment the ability of tissue-localizedCD8+T cells to find abnormal cells and enact effector functions in viral models68and to facilitate autoimmune reactions in murine graft-versus-host disease models69.

[0067] Our initial expectation of the combined CKM and aPD-1 therapy was that CKM-induced susceptibility to PD-1 blockade would occur optimally with a delay between CKM and aPD-1 administration, allowing time for T cell priming. However, our data suggests that the therapy regimen worked best when aPD-1 was administered immediately after CKM (FIGS. 4A-4F).

[0068] The disclosure demonstrates two efficacious schedules for drug delivery, a benefit that could be capitalized on during clinical trial design and implementation. This provides options to improve feasibility and compliance for patients receiving these therapiesin the clinical setting. Treatment administration following the described sequential approach would limit the number of visits a patient would need to make to a clinic for treatment. Further, the data showing that CKM sensitizes colorectal tumors to PD-1 blockade quickly, enables treating patients with CKM and aPD-1 in the same visit for colorectal cancers, as well as other cancers that are initially resistant to anti -PD-1 therapy.

[0069] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

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Claims

CLAIMS:

1. A method for treating an individual for a programmed-death 1 (PD-1) immune checkpoint therapy resistant cancer, the method comprising administering to the individual a combination of a Toll-like receptor 3 (TLR3) agonist and interferon-alpha (IFNa), followed by administration of an anti -PD-1 immunotherapy.

2. The method of claim 1, wherein the TLR agonist and the IFNa are administered to the individual simultaneously and the anti-PD-1 immunotherapy is administered not more than 24 hours after administration of the TLR agonist and the IFNa.

3. The method of claim 2, wherein the anti-PD-1 immunotherapy is administered not more than 12 hours after administration of the TLR3 agonist and the IFNa.

4. The method of claim 2, wherein the anti-PD-1 immunotherapy is administered not more than 6 hours after administration of the TLR3 agonist and the IFNa.

5. The method of claim 2, wherein the anti-PD-1 immunotherapy is administered not more than 3 hours after administration of the TLR3 agonist and the IFNa.

6. The method of claim 2, wherein the anti-PD-1 immunotherapy is administered not more than 1 hour after administration of the TLR3 agonist and the IFNa.

7. The method of any one of claims 1-6, wherein the TLR3 agonist is poly:IC or rintatolimod.

8. The method of claim 7, wherein the anti-PD-1 immunotherapy is pembrolizumab or nivolumab.

9. The method of claim 8, wherein the anti-PD-1 immunotherapy is the pembrolizumab.

10. The method of claim 9, wherein the pembrolizumab is administered to the individual not more than 1 hour after administration of rintatolimod and IFNa.

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