Polymer matrix delivery system for delivering cancer adjuvant and implant comprising same

Abstract

The present disclosure relates generally to cancer immunotherapies and, more particularly, to a polymer matrix delivery system for delivering a cancer adjuvant, especially for enhanced immunotherapy. More particularly, the disclosure relates to a polymer matrix delivery system for delivering a cancer adjuvant, comprising a biodegradable polymer; a cancer adjuvant; optionally, an immunomodulatory inorganic ion; and, a bulking agent, wherein the bulking agent is not a protein. An implant comprising the polymer matrix delivery system is also provided. A method of treating a solid tumor or the potential recurrence of a solid tumor comprising injecting the implant into the solid tumor or at a site of surgical resection of a solid tumor is also described.

Description

Docket No.30275 / 70597A / PC POLYMER MATRIX DELIVERY SYSTEM FOR DELIVERING CANCER ADJUVANT AND IMPLANT COMPRISING SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No.63 / 670,113, filed July 11, 2024, and entitled “A Polymer Matrix Delivery System for Delivering Cancer Adjuvant and Implant Comprising Same”, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates generally to cancer immunotherapies and, more particularly, to a polymer matrix delivery system for delivering a cancer adjuvant, especially for enhanced immunotherapy. BACKGROUND

[0003] Immunotherapies are a promising method of treatment for cancer that have the potential to eliminate local and metastatic tumors, promote systemic immune surveillance, establish long term immune memory, and mediate immune protection against tumor recurrence. The fundamental principle behind cancer immunotherapy is modulating and leveraging the host immune system to target and kill cancer cells. Immune checkpoint inhibitors (ICIs) are a class of immunotherapy drugs can signal immune cells to kill cancer cells. While these drugs have greatly revolutionized the field of cancer therapy, ICIs have shown to be effective in only 10-30% of patients [De Lorenzo, et al., Mechanisms of Primary and Acquired Resistance to Immune Checkpoint Inhibitors in Patients with Hepatocellular Carcinoma. Cancers (Basel), 2022.14(19)]. In this respect, many patients have “cold” tumor microenvironments (TMEs), in other words lack T-cell infiltration at the tumor site, thereby limiting the effectiveness of ICIs. For this reason, there has been interest in developing strategies for T-cell recruitment to the tumor site via inflammatory pathways. The cyclic guanosine monophosphate- adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway is a key regulator of innate immune sensing of cancer. STING is triggered when an intra-cellular protein detects DNA from either pathogens or dying tumor cells which causes the induction of a pro-inflammatory response, primarily dominated by type I interferons (IFNs). Type I IFNs promote the generation of cytotoxic T cell responses as well as type 1 T helper cell (Th1)-biased responses. Furthermore, type I IFNs promote the activation and functional maturation of dendritic cells (DCs), thereby facilitating antigen presentation to CD4+ T cells as well as antigen cross-presentation to CD8+ T cells [Motedayen Aval, et al., Challenges and Opportunities in the Clinical Development of STING Agonists for Cancer Immunotherapy. J Clin Med, 2020.9(10)]. DueDocket No.30275 / 70597A / PC to these immunomodulatory properties, the STING pathway has quickly escalated as a promising target for immunotherapy.

[0004] While preclinical studies of STING agonists have shown promising results, results from clinical studies have remained suboptimal. Clinical translation of STING agonists has been challenging due to poor pharmacokinetic and physiochemical properties of most CDNs. Many STING agonists are cyclic dinucleotides (CDNs) and due to their small molecular weight, these CDNs diffuse very quickly into systemic circulation upon administration and clear just as easily, limiting drug exposure in tumor tissues and potentially causing off-target toxicities. They are also susceptible to enzymatic degradation, and the negative charge of CDNs block their cell membrane penetration and cellular uptake.

[0005] There are currently more than 10 STING agonists in clinical development, a significant subsect of even more small molecule cancer adjuvants in development. However, moving these drug candidates from preclinical to clinical studies has been challenging due to poor physiochemical properties of these compounds which often have low stability, short half-life, and low cellular permeability [Le Naour, et al., Trial watch: STING agonists in cancer therapy. Oncoimmunology, 2020.9(1): 1777624]. Intracellular delivery of STING agonists is required for proper STING activation thus many STING agonists require localized delivery via an intratumoral injection [Motedayen Aval, et al., supra].

[0006] It is well-known that adherence rates for cancer treatments are typically low. Current STING agonist clinicals involve weekly or three times a month injections which translates to long term financial burden for the patient as well as reduced patient compliance and increased chance for chronic pain at the injection site. In addition, multiple intratumoral injections limit the scope of STING agonist-based therapies to readily accessible tumor types and introduce the risk of disrupting the tumor microenvironment (TME) and vascular network, potentially promoting cancer cell extravasation and metastases [Puts, et al., Factors influencing adherence to cancer treatment in older adults with cancer: a systematic review. Ann Oncol, 2014.25(3): p.564-577]. Additionally, the amount of STING agonist injected to tumor has been shown to dictate CD8+ T cell response, with high doses causing cytotoxicity [Sivick, et al., Magnitude of Therapeutic STING Activation Determines CD8(+) T Cell- Mediated Anti-tumor Immunity. Cell Rep, 2019.29(3): p.785-789]. SUMMARY OF THE INVENTION

[0007] A polymer matrix delivery system for delivering a cancer adjuvant, comprising a biodegradable polymer; a cancer adjuvant; optionally, an immunomodulatory inorganic ion; and, a bulking agent, wherein the bulking agent is not a protein.Docket No.30275 / 70597A / PC

[0008] An implant comprising the polymer matrix delivery system is also provided.

[0009] A method of treating a solid tumor or the potential recurrence of a solid tumor comprising injecting the implant into the solid tumor or at a site of surgical resection of a solid tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter, which is regarded as forming the present invention, the invention will be better understood from the following description taken in conjunction with the accompanying drawings.

[0011] Fig.1 illustrates a method for fabricating the exemplified CDA-Mn PLGA implants;

[0012] Fig.2 illustrates continuous CDA release with limited burst release from the exemplified 502H and 503H PLGA implants;

[0013] Fig.3 illustrates representative scanning electron images showing a lateral view and a circular cross-sectional view of an 503H PLGA exemplified milli-cylinder implant, while also showing continuous CDA and Mn2+ release from the exemplified implant;

[0014] Figs.4A and 4B both illustrate that implants loaded with a cyclic dinucleotide analogue retain the drug at the site of the injection for over seven days whereas the free soluble drug clears from the injection site after 24 hours;

[0015] Fig.5 illustrates the bioactivity of encapsulated c-di-AMP and manganese ion in PLGA implants in Raw-Dual Reporter cell lines at days 1, 3, and 5;

[0016] Figs.6A and 6B illustrate (A) BMDCs isolated from C57BL / 6 mice treated with the CDA and Mn2+ loaded PLGA implant relative to an untreated negative control and a free CDA and Mn2+ positive control and (B) release of IFN-^ and IL-6 from BMDCs on day 3, respectively;

[0017] Fig.7 illustrates the anti-tumor efficacy of the exemplified 503H CDA-Mn PLGA implant in a murine CT26 model;

[0018] Figs.8A-8D illustrate (A) a testing protocol, (B) the frequencies of CD4+ central memory T cells, CD8+ central memory T cells, and CD8+ effector memory T cells in the TME, (C) the ratios of M1 / M2 macrophages and the frequency of M2-like macrophages in the TME, and (D) the mean fluorescence intensity of CD80 on DCs in the TME, respectively;

[0019] Figs.9A-9F illustrate (A) a testing protocol, the frequencies of (B) CD4+ central memory T cells, (C) CD8+ central memory T cells, (D) ratio of M1 / M2 macrophages, (E)Docket No.30275 / 70597A / PC DCs, and (F) CD107+ NK cells in tumor-draining lymph nodes harvested on Day 22 and analyzed by flow cytometry; and.

[0020] Figs.10A-10G illustrate (A) a testing protocol, the frequencies of (B) CD4+ memory T cells and (C) CD8+ memory T cells, (D) the ratio of M1 / M2 macrophages, (E) CD80+ DCs and mean fluorescence intensity (MFI) of MHC-II in DCs, (F) MFI of CD107a, a degranulation marker in NK cells, and (G) the frequency of Ly6c+ MDSCs in spleens harvested at Day 22 and analyzed by flow cytometry. DETAILED DESCRIPTION

[0021] The invention provides a polymer matrix delivery system for delivering a cancer adjuvant, comprising a biodegradable polymer, a cancer adjuvant, particularly a cancer adjuvant capable of recruiting T-cells to a tumor microenvironment, optionally, an immunomodulatory inorganic ion, and a bulking agent, wherein the bulking agent is not a protein. In one preferred embodiment, the biodegradable polymer comprises poly(lactic-co- glycolic acid), the STING agonist comprises c-di-AMP, the immunomodulatory inorganic ion is present and comprises Mn2+, and the bulking agent comprises hydroxyethyl starch. Optionally, the polymeric matrix delivery system may include a porosigen and / or osmotic adjusting agent to increase the porosity of the particles. The disclosed polymer matrix delivery system represents an easily manufacturable and translational approach to cancer adjuvants capable of recruiting T-cells to tumor microenvironments, particularly via STING agonist-based therapy, by addressing key limitations associated with multiple injections, patient adherence as well as formulation and delivery challenges. Through sustained, localized drug release, the disclosed polymer matrix delivery system advantageously can improve patient outcomes, reduce treatment burdens, and expand the clinical scope of STING-targeted cancer immunotherapy.

[0022] The polymer matrix delivery system can advantageously improve the bioavailability and cellular uptake of cancer adjuvants, particularly cancer adjuvants that improve T-cell recruitment and function in the TME via inflammatory pathways that can improve innate immunity to cancer, such as STING agonists, as well as patient compliance. More specifically, the invention provides a polymer matrix delivery system for delivering a cancer adjuvant that can substantially replicate current clinical multi-dosage regimens with a single injection to improve patient adherence and thereby decrease the risk of metastasis as well as therapeutic cost. Moreover, the polymer matrix delivery system can facilitate delivery of other, additional immunotherapeutic agents. The polymer matrix delivery system for delivering a cancer adjuvant of the invention may be used post-tumor resection to prevent tumor reoccurrence and / or to treat solid tumor cancers including but not limited to breast,Docket No.30275 / 70597A / PC prostate, lung, and colorectal cancers. The polymer matrix system can be provided in the form of an implant, specifically, as an extruded implant, and injected into a tumor or at the site of resection.

[0023] Because release from the polymer matrix system can be controlled, degradation and dose-dumping of the encapsulated STING agonist can beneficially be reduced and / or even substantially eliminated. Advantageously, the polymer matrix delivery systems can continuously release the cancer adjuvant for extended periods, for example, for 3-4 weeks. Comparatively, as noted in the background section, when injected, the soluble cancer adjuvant drug clears quickly from circulation. Therefore, a single administration of the polymer matrix delivery system can advantageously demonstrates similar efficacy to the multi-dose intra-tumor injections of the soluble drug. As shown in the Example, CT26 tumor bearing mice, the tumors in the untreated mice (PBS negative control group) grew quickly whereas both the 4X intratumoral treatment with the soluble drug and the implant inhibited tumor growth at approximately the same rate, thereby showing similar efficacy.

[0024] Advantageously, the present disclosure provides a long-acting polymer matrix system as illustrated by the CDA-Mn²⁺ PLGA implant described in the Examples, a single- injection immunotherapy platform that co-delivers a cancer adjuvant such as a STING agonist and an immunomodulatory inorganic ion such as manganese ion, a nutritional transition metal known to amplify Type I interferons (IFNs). Beneficially, a simple solvent extrusion process can be used to provide the disclosed polymer matrix system such as the exemplified PLGA implant with high drug loading efficiency, continuous controlled drug release, and strong anti-tumor efficacy. As shown in the Example section, the anti-tumor effects of the exemplified CDA-Mn²⁺ PLGA implant are attributed to successful reprogramming of the TME and lymphoid tissues, leading to enhanced immune cell recruitment of T cells, DC activation, and M2-to-M1 macrophage repolarization, which collectively bolster innate and adaptive immune responses. Notably, the implant significantly increased memory T-cell populations, a key factor in long-term cancer immunity. This scalable, patient-centric approach provides a minimally invasive alternative to traditional STING-based therapy, which requires multiple dosing regimens.

[0025] Adherence to state-of-the-art STING agonist-based therapy is clinically challenging due to the requirement for multiple injections over an extended period, often administered by trained healthcare professionals via intratumoral injection. Dosing regimens being evaluated in current clinical trials involve three injections within a month or weekly injections for a nine- week cycle, with some protocols extending for up to two years to achieve therapeutic efficacy [X. Lu, et al., “Engineered PLGA microparticles for long-term, pulsatile release ofDocket No.30275 / 70597A / PC STING agonist for cancer immunotherapy”, Sci Transl Med 12 (2020); “Phase 1 Open-label, Multicenter Study of MK-1454 Administered by Intratumoral Injection as Monotherapy and in Combination With Pembrolizumab for Patients With Advanced / Metastatic Solid Tumors or Lymphomas, NCT03010176, 2021]. An alternative approach involves invasive drug delivery devices for sustained release [E. Minaei, et. al, “Enhancing pancreatic cancer immunotherapy: Leveraging localized delivery strategies through the use of implantable devices and scaffolds”, J Control Release 373 (2024) 145-160]. However, both strategies present significant drawbacks, including high financial costs, patient discomfort, and increased risks of injection site pain or metastasis. In sharp contrast, the disclosed polymer matrix delivery system offers a non-invasive injectable platform capable of controlled co- release of multiple immune-stimulating agents and can advantageously improve patient compliance, comfort, and affordability by reducing the dosing regimen to a single injection while maintaining therapeutic efficacy.

[0026] The disclosed polymer matrix delivery system may be well-suited for post-surgical maintenance therapy, where it can help prevent tumor recurrence. While surgical resection is an effective treatment for many cancers, long-term survival rates remain at approximately 50%, with tumor recurrence being the primary cause of mortality. Additionally, post-surgical immunosuppression can accelerate metastatic progression if residual tumor cells persist. Due to the risk of overstimulation and tumor promotion, immunotherapy is rarely administered during the immunosuppressive perioperative period. For this application, the disclosed polymer matrix delivery system, such as the exemplified CDA-Mn2+ PLGA implant, could be injected post-surgery so that multiple follow up visits to the clinic for treatments may not be needed. Due to controlled release, the implant can maintain immune- stimulating agents within physiological ranges which can potentially reverse immunosuppression and improve long term patient outcomes.

[0027] Immunosuppression has also been a crucial limitation of current checkpoint inhibitors in the clinic; thus, the disclosed polymer matrix delivery system, such as the exemplified CDA-Mn²⁺ PLGA implant, also has the potential to work synergistically with immune checkpoint therapy and make it more effective for patients with cold tumors.

[0028] Another promising application for the disclosed polymer matrix delivery system, such as the exemplified CDA-Mn²⁺ PLGA implant, is in treating deep-seated or hard-to-reach tumors. Current immune checkpoint inhibitors are primarily administered intravenously, which can lead to unwanted systemic side effects. For tumors located in major organs, intratumoral injections require CT or ultrasound guidance, necessitating multiple imaging sessions, which are both costly and inconvenient. By offering sustained drug release throughDocket No.30275 / 70597A / PC a single injection, the CDA-Mn²⁺ PLGA implant provides a practical alternative to frequent, image-guided injections. The disclosed approach is anticipated as being particularly useful for treating pancreatic cancer, where repeated intratumoral administration is particularly challenging.By improving targeted, localized delivery, the implant could expand the clinical applications of STING-targeted therapy to additional tumor types.

[0029] The polymer matrix delivery system for delivering a cancer adjuvant may include a biodegradable polymer chosen from one or more biodegradable polymers in the group of poly(lactides), poly(glycolides), poly(lactide-co-glycolides), poly(lactic-acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, poly(hydroxymethyl glycolide-co-lactide), polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates, poly(p-dioxanone), poly(alkylene oxalate)s, and polyurethanes. In a preferred embodiment, the biodegradable polymer comprises poly(lactic-co-glycolic acid) (PLGA), which advantageously can also serve as an adjuvant to stimulate the immune system. Moreover, PLGA can be tuned to control the release, which can reduce the number of injections, thereby increasing patient compliance and ease of treatment. The polymer matrix system may comprise at least about 65 wt.%, from about 70 wt.% to about 90 wt.%, or from about 75 wt.% to about 85 wt.%, for example, about 80 wt.% of the biodegradable polymer.

[0030] In various cases, the biodegradable polymer has a weight average molecular weight in the range of about 4 kDa to about 55 kDa, for example, about 4.5 kDa to about 50 kDa, about 5 kDa to about 45 kDa, about 7.5 kDa to about 40 kDa, and / or about 10 kDa to 35 kDa. The weight average molecular weight of the biodegradable polymer can be determined using methods known in the art, particularly gel permeation chromatography. When the biodegradable polymer is PLGA, the biodegreadable polymer can have a lactic acid content in the range of 50% to 100%, relative to the total amount of lactic acid and glycolic acid in the biodegradable polymer. Generally, when the biodegradable polymer is PLGA, the biodegradable polymer is an uncapped polymer.

[0031] The polymer matrix system may comprise up to about 4 wt.%, or from about 1 wt.% to about 4 wt.%, or from about 1.5 wt.% to about 3 wt.%, for example, about 2 wt.% of the cancer adjuvant. In one embodiment, the cancer adjuvant comprises a small molecule STING agonist. The cancer adjuvant is not limited to a particular type of STING agonist. In some embodiments, the STING agonist is one or more small molecule agonist of STING. In some embodiments, the small molecule agonists of STING are cyclic dinucleotides (CDNs). Cyclic dinucleotides include but are not limited to cGAMP, cdiAMP, cdiGMP, and cAIMP. Additional examples of cyclic purine dinucleotides are described in some detail in, e.g., U.S.Docket No.30275 / 70597A / PC Pat. Nos.7,709,458 and 7,592,326; WO2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett.18: 5631 (2008), each of which is hereby incorporated by reference for its disclosure of such CDNs. The STING agonist may also be chosen from 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone, 6,4′-dimethoxyflavone, 4′-methoxyflavone, 3′,6′- dihydroxyflavone, 7,2′-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, and / or any derivatives thereof. The small molecule agonist of STING may also comprise one or more of 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c-di-GMP Fluorinated, 2’3’- c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, and Gemcitabine.

[0032] In embodiments, one or more small molecule agonist of STING may be selectedDocket No.30275 / 70597A / PC, SB11285 (Spring Bank Pharmaceuticals), Gemcitabine (,c- c-cAIMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c- di-GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP, cGAMP, 2’3’-cGAMP, 2’2’-cGAMP, 3’3’- cGAMP, cGAM(PS)2, 2’3’-cGAM(PS)2(Rp / Sp), 2’2’-cGAM(PS)2, 2’3’-cGAM(PS)2, cGAMP Fluorinated, 3'3'-cGAMP Fluorinated, 2'3'-cGAMP Fluorinated, 2'2'-cGAMP Fluorinated, c-di- AMP, 2’3’-cdAMP, 2’2’-cdAMP, 3’3’-cdAMP, c-di-AM(PS)2, 2’3’-c-di-AM(PS)2 (Rp,Rp), 2’2’- c-di-AM(PS)2, 3’3’-c-di-AM(PS)2, c-di-AMP Fluorinated, 2’3’-cdAMP Fluorinated, 2’2’-cdAMP Fluorinated, 3’3’-cdAMP Fluorinated, cdGMP, 2’3’-cdGMP, 2’2’-cdGMP, 3’3’-cdGMP, c-di- GM(PS)2, 2’3’-c-di-GM(PS)2, 2’2’-c-di-GM(PS)2, 3’3’-c-di-GM(PS)2, cdGMP Fluorinated, 2’3’-cdGMP Fluorinated, 2’2’-cdGMP Fluorinated, 3’3’-cdGMP Fluorinated, cAIMP, 2’3’- cAIMP, 2’2’-cAIMP, 3’3’-cAIMP, cAIMP Difluor (3'3'-cAIMP Fluorinated, 2'3'-cAIMP Fluorinated, 2'2'-cAIMP Fluorinated, cAIM(PS)2 Difluor, 3’3’-cAIM(PS)2 Difluor (Rp / Sp), 2’3’- cAIM(PS)2 Difluor, 2’2’-cAIM(PS)2 Difluor, c-di-IMP, 2’3’-cdIMP, 2’2’-cdIMP, 3’3’-cdIMP, c- di-IM(PS)2, 2’3’-c-di-IM(PS)2, 2’2’-c-di-IM(PS)2, 3’3’-c-di-IM(PS)2, c-di-IMP Fluorinated, 2’3’- cdIMP Fluorinated, 2’2’-cdIMP Fluorinated, and 3’3’-cdIMP Fluorinated, and amidobenzimidazole (ABZI)-based compounds.

[0033] The polymer matrix delivery system for delivering a cancer adjuvant may further comprise an immunomodulatory inorganic ion including but not limited to one or moreDocket No.30275 / 70597A / PC inorganic ions chosen from the group of Zn2+, Mn2+, Fe2+, Fe3+, Cu2+, Ni2+, Co2+, Pb2+, Sn2+, Ru2+, Au2+, Mg2+, VO2+, Al3+, Co3+, Cr3+, Ga3+, Tl3+, Ln3+, MoO3+, Cu+, Au+, Tl+, Ag+, Hg2+, Pt2+, Pb2+, Hg2+, Cd2+, Pd2+, and Pt4+. By also delivering an immunomodulatory inorganic ion that is known to amplify innate immunity, such as manganese ion, relatively lower doses of STING agonists can advantageously be used while still achieving the same therapeutic efficacy. The polymer matrix system may comprise up to about 7 wt.%, or from about 1 wt.% to about 6 wt.%, or from about 2 wt.% to about 5 wt.%, for example, about 3.5 wt.% of a source (e.g., salt) for the inorganic ion.

[0034] The polymer matrix delivery system for delivering a cancer adjuvant comprises a bulking agent, typically, a water-soluble polymer, preferably a non-ionizable water-soluble polymer. The bulking agent must be biocompatible for intratumoral injection and should preferably be nonionic to prevent interactions with the drug and metal ion. The bulking agent is also useful for helping to provide the formulation with a viscosity suitable to cast implants, disperse the drug within polymer matrix (good drug distribution), high encapsulation efficiency, and ensure the target release kinetics. In one example, the bulking agent may comprise a polysaccharide. In one refinement, the bulking agent may comprise one or more water-soluble polymers chosen in the group of carboxymethyl cellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropyl starch, and hydroxyethyl starch. The polymer matrix system may comprise up to about 20 wt.%, or from about 5 wt.% to about 20 wt.%, or from about 12.5 wt.% to about about 17.5 wt.%, for example, about 14 wt.% of the water-soluble polymer.

[0035] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0036] The term “about” is used according to its ordinary meaning, for example, to mean approximately or around. In one embodiment, the term “about” means ±10% of a stated value or range of values. In another embodiment, the term “about” means ±5% of a stated value or range of values. A value or range described in combination with the term “about” expressly includes the specific value and / or range as well (e.g., for a value described as “about 40,” “40” is also expressly contemplated).

[0037] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, delivery system, or article that comprises a list of elements is notDocket No.30275 / 70597A / PC necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, an element A or B is satisfied by any one of the following: A is present and B is not present, A is not present and B is present, and both A and B are present.

[0038] In addition, use of the "a" or "an" are employed to describe elements and components of the embodiments herein. This is done merely for convenience and to give a general sense of the description. This description, and the claims that follow, should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

[0039] This detailed description is to be construed as examplary only and does not describe every possible embodiment, as describing every possible embodiment would be impractical, if not impossible. One could implement numerous alternate embodiments, using either current technology or technology developed after the filing date of this application. EXAMPLES Fabrication of Injectable PLGA Implants co-loaded with STING agonist and Manganese Ion:

[0040] Poly(D,L-lactide-co-glycolide) (PLGA) 50 / 50 implants were loaded with a commercially available STING agonist, c-di-AMP (CDA), and manganese ion by modification of a previously reported solvent-extrusion method (Figure 1) [Zhu, G., Mallery, S. & Schwendeman, S. Stabilization of proteins encapsulated in injectable poly(lactide-co-glycolide). Nature Biotechnology 18, 52–57 (2000)]. A suspension of the drug and anyadditional excipients (i.e. stabilizing agent / bulking agent / basic salts if required) in acetone- PLGA ( solution (50% wt / wt) is loaded in a syringe and extruded into silicone rubber tubing (0.8 mm i.d.) at approximately 0.1 ml / min. The solvent-extruded suspension is dried at room temperature (48 h) and then under vacuum at 45°C (72 h). If the particle side of the solid components is > 90 microns, they are micronized and sieved to < 90 microns in size before suspension in the acetone-PLGA solution.

[0041] In addition, a biocompatible water-soluble polymer bulking agent excipient, hydroxyethyl starch (EDMQ) was included at a specific level to facilitate continuous drug release as the highly potent CDA must have only a very low drug loading to avoid side effects. Briefly, RG502H or RG503H PLGA (Evonik) was dissolved in 50% w / v acetone. The solid components including hydroxyethyl starch, c-di-amp sodium salt (MedChemExpress), and ground manganese chloride tetrahydrate crystals (Sigma, <90 microns) were prepared as a solid mixture in a fixed ratio (Table 1). This solid mixture is incorporated into the polymer solution, extruded into silicon tubing (I.D. = 0.8 mm), and dried at room temperatureDocket No.30275 / 70597A / PC for 48 hours and under vacuum at 40°C for 72 hours. The tubing is removed with a razor to isolate the implant. Table 1: Composition of Injectable PLGA Implants Loaded with CDA & Manganese IonEvaluation of Drug Loading & Release Kinetics Study:

[0042] Implants were cut to 5mm in length, dissolved in acetone to extract CDA, resuspended in phosphate buffered saline (PBS), and analyzed by ultra-performance liquid chromatography (UPLC) to quantify drug loading. Extracted samples were also resuspended in 10% nitric acid and analyzed by ICP-MS to measure manganese ion loading. Release kinetic study was done in triplicates with 5mm implants in 1ml PBS with 0.02% Tween 80 at 37°C with analysis by UPLC for CDA and ICP-MS for manganese ion at selected timepoints of Day 1, 3, 7, 14, 21 and 28. Release media was replenished at each time point. In vivo Drug distribution analysis

[0043] To analyze in-vivo biodistribution of STING agonist, 2',3'-cGAMP-Cy5 was admixed with CDA (1:10, n / n) to prepare 2',3'-cGAMP-Cy5 + CDA + Mn2+PLGA implants, following the same fabrication procedure as mentioned previously. To quantify drug retention at the tumor site after intratumoral injection, 20 ^g 2',3'-cGAMP-Cy5-CDA in free form or in implants were injected into both sides of the rear flank of naïve BALB / c mice (n = 5 per group). Mice were imaged by IVIS® Lumina™ every day up to seven days with the excitation set to 640 nm and the emission wavelength set to 670 nm. Region of interest was kept the same amongst all mice, and the average radiant efficiency was calculated and normalized to day zero values. Reporter cell assays

[0044] 106RAW 264.7 dual IRF3 / NF-kB reporter cells were seeded in a 96-well plate. Cells were treated with either no treatment (negative control), free CDA (40 nmol) + Mn2+(10 nmol), or PLGA implant (40 nmol of CDA + 10 nmol of Mn2+) (n = 3). Supernatant was collected after 24 hours and media was replenished. IRF3 activity was quantified using QUANTILuc™ 4 Lucia / Gaussia Luciferase Detection Reagent (Invivogen), following manufacturer’s flash detection protocol. This process was repeated at 48 hours and 120 hours.Docket No.30275 / 70597A / PC Bone-marrow derived dendritic cell (BMDC) assays

[0045] BMDCs were prepared according to a previously described protocol [Lutz, et al., “An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow,” J Immunol Methods 223 (1999) 77-92]. Briefly, bone marrow was collected and plated in Petri dishes with culture media containing granulocyte–macrophage colony-stimulating factor (GM-CSF). The cell culture media were refreshed on days 3, 6 and 8. After 8 days of differentiation, BMDCs were collected and 106 BMDCs were seeded in a 24-well plate. BMDCs were incubated with either culture media as a no treatment negative control or with 40 nmol CDA + 10 nmol Mn2+ either in soluble form (from manganese chloride tetrahydrate, Sigma) or encapsulated in the PLGA implant. To observe for sustained proinflammatory cytokine response, supernatant was collected after 24 hours incubation at 37 °C under 5% CO2 and media was replenished. This was repeated after 72 hours, and the supernatants were submitted to Cancer Center Immunology Core of the University of Michigan for ELISA analysis. Separately, BMDCs were collected from the wells post incubation with the same treatments and timepoints and stained for flow cytometry analysis to observe for sustained DC activation. Briefly, cells were scrapped from the well, washed with fluorescence-activated cell sorting (FACS) buffer (1% BSA in PBS), stained for viability (Live / Dead fixable Near-IR, Invitrogen), treated with Fc blocker (anti-mouse CD16 / CD32 monoclonal antibody, Fisher Scientific), stained with surface antibodies for 30 minutes at room temperature, and fixed with 4% paraformaldehyde. The following antibodies were used for the surface staining: anti-CD80-BV421 (BioLegend), anti-CD86-BV605 (BD Horizon), anti-CD11c-FITC (Biolegend), anti-CD40-PE-Cy7 (BD Biosciences), anti-MHC-II-APC (BioLegend). The samples were run on a spectral flow cytometer (Cytek Aurora) and analyzed using Flowjo (v.10.10). Evaluating Anti-Tumor Efficacy in Murine CT26 Tumor Model:

[0046] To develop CT26 colon carcinoma murine tumor model, CT26 cells were cultured and 1.5×105cells in a volume of 100 ^L Hanks' Balanced Salt Solution was injected subcutaneously in the rear left flanks of female BALB / c mice aged 6-8 weeks. Once average tumors reached ~50 mm3, mice were administered intratumorally with either PBS (negative control); intratumoral injections of free 10 ^g CDA and 2.5 ^g Mn2+on days 10, 13, 16, and 19 as a positive control; or intratumoral injection of the 503H implant (containing 40 ^g CDA and 10 ^g Mn2+). Implants were inserted within the tumor using a 16G needle. Tumor size and survival were monitored every two days using a digital caliper. Mice were euthanized when tumor volume (length×width2× 0.5) exceeded 1500 mm3or if they developed unhealing ulcerations.Docket No.30275 / 70597A / PC In vivo immune profiling experiments

[0047] To observe for immune cell population changes in the TME and lymphatic system, the same experimental plan was carried out as the efficacy evaluation. However, on day 22 post tumor inoculation, tumors, tumor-draining lymph nodes (tdLNs), and spleen were harvested from the mice. To obtain single cell suspensions, tumors were cut into small pieces, digested with collagenase type IV (Sigma Aldrich, 1mg / ml) and DNAase (Sigma Aldrich, 0.1 mg / ml) for 30 minutes at 37^C, dissociated with a tissue homogenizer (gentleMACS™ Octo Dissociator), and filtered through a 70 ^m strainer. For spleen, tissue was filtered through a 70 ^m filter using FACS buffer, and ACK lysis buffer (Thermo Fisher Scientific) was added to lyse and wash away the red blood cells. For lymph nodes, tissue was simply stained through a 70^m filter using FACS buffer. All cells were then centrifuged and resuspended in FACS buffer and seeded into a 96-well plate at a seeding density typically around 2-5 million cells. These cells were then stained for viability (Live / Dead fixable Near-IR, Invitrogen), treated with Fc blocker (anti-mouse CD16 / CD32 monoclonal antibody, Fisher Scientific), and stained with surface antibodies for 30 minutes at room temperature, and fixed with 4% paraformaldehyde. The following surface antibodies were used: anti-CD107a-BUV395 (BD Horizon), anti-CD8-BUV496 (BD Horizon), anti-CD4- BUV805 (BD Horizon), anti-CD19-BV421(BioLegend), anti-CD80-Pacific Blue (BioLegend), anti-I-A / I-E-BV510 (BioLegend), anti-CD62L-BV570 (BioLegend), anti-F4 / 80-BV605 (BioLegend), anti-Ly6C-BV711 (BioLegend), anti-CD11c-BV785 (BioLegend), anti-CD45- FITC (BD Biosciences), anti-Ly6G-Spark Blue 550 (BioLegend), anti-CD11b-PerCP-Cy5.5 (BioLegend), anti-CD49b-PE (BioLegend), anti-CD44-PE-Cy5 (BioLegend), anti-NK1.1-Pe- Cy7 (BioLegend), anti-CD3-APC (BioLegend), and anti-CD206-Alex Fluor 700 (BioLegend). The samples were run on a spectral flow cytometer (Cytek Aurora) and analyzed using Flowjo (v.10.10). Statistical analysis

[0048] The results are expressed as means ± standard deviation (s.d.). One- or two-way ANOVA analysis, followed by Bonferroni’s multiple comparison post hoc test. Statistically significant differences were denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparisons without statistical significance were denoted as “ns”. Analyses of animal survival were performed using Kaplan−Meier survival analyses with Log-rank Mantel−Cox. All the animal studies were performed after randomization. Data collection and analysis were not performed blind to the conditions of the experiments. The data were approximately normally distributed, and variance was similar between groups. No samples were excluded from analysis. GraphPad Prism 10.0 (GraphPad Software) was used for statistical analyses.Docket No.30275 / 70597A / PC Results

[0049] With the hydroxyethyl starch content set at 14.3%, implants with a relatively high loading of CDA, 1.8% for the 502H implant and 1.7% for the 503H implant were made (Table 2). Our results demonstrate continuous release of CDA for 2 weeks for the 502H implant and 3-4 weeks for the 503H implant. Limited burst release was observed in both formulations (Figure 2). We further characterized the 503H implant and found that the manganese loading was also high (80% encapsulation efficiency) and that the manganese ion also released continuously for 3-4 weeks (Figure 3). We also collected scanning electron microscopy images to show the structure of these implants (Figure 3).

[0050] In naïve mice Balb / c mice, we found that implants loaded with cGAMP-cy5, an cyclic dinucleotide analogue of c-di-AMP with a fluorescent tag that has comparable molecular weight and physiochemical properties to c-di-AMP, can retain the drug at the site of the injection for over seven days as compared to the soluble drug clears from the injection site after 24 hours (Figures 4A, 4B). Specifically, IVIS fluorescence imaging at Day 0 showed high signal intensity at the injection site in the free drug group, but fluorescence rapidly decreased within 24 hr, indicating quick clearance from the injection site (Figure 4). In contrast, PLGA implants exhibited lower initial fluorescence at Day 0 but demonstrated prolonged drug retention, with fluorescence persisting at the injection site up to Day 7 (Figure 4). There was a ~4X increase in fluorescence on Day 7 with the PLGA implant group compared to the free drug group (Figure 4a). This supported that the PLGA implant retained the drug at the site of the injection for an extended period.

[0051] Bioactivity of encapsulated c-di-AMP and manganese ion in PLGA implants was evaluated in Raw-Dual reporter cell lines. IRF response was quantified using a luciferase assay on days 1, 3, and 5 and compared between 1) an untreated negative control, 2) free c-di-AMP. (40 nmol) + Mn2+(10 nmol) positive control, and 3) PLGA implant (40 nmol cy-di- AMP, 10 nmol Mn2+) treatment group. At each time point, media were replenished to mimic drug clearance in vivo. IRF activity increased by ~300-fold on Day 1 in both the soluble CDA+Mn²⁺ and PLGA implant groups, compared to untreated controls. However, by Days 3, IRF activity in the soluble drug-treated group dropped, whereas the activity in the PLGA implant group remained higher, resulting in ~3-fold increase in IRF activity, compared with free soluble group (Figure 5). This trend continued to Day 5, and we observed ~2-fold increase in IRF activity for the PLGA implant group, compared with the free soluble group (Figure 5). This suggests that CDA remained bioactive within the implant and can achieve extended cGAS-STING signaling, compared with free soluble injections.Docket No.30275 / 70597A / PC

[0052] Next, as illustrated in Figure 6A, BMDCs isolated from C57BL / 6 mice were treated with the CDA and Mn2+ loaded PLGA implant to evaluate the ability of the implant to activate DCs over three days as compared to an untreated negative control and free CDA and Mn2+ positive control. Media were replenished at each time point to mimic in vivo clearance. On day one, the free drug showed high expression of DC maturation markers and proinflammatory cytokines. On day three post treatment, the PLGA implant sustained increased maturation markers, including MHCII, CD80, and CD86, on BMDCs, compared to the free drug and untreated groups (Figure 6A). On day 3, the PLGA implant also promoted increased release of IFN-^ and IL-6 from BMDCs (Figure 6B).

[0053] In the CT26 murine model, we found that both the 502H and 503H implant inhibited tumor growth significantly compared to the PBS negative control group and that a single injection of implants performed comparably to a 4X intratumoral injection of the free CDA and manganese ion (Figure 7).

[0054] We also conducted immune profiling of the tumors, tumor draining lymph nodes, and spleen to observe changes in immune cell populations as a result of the treatment with the implant as compared to the soluble drugs and a negative untreated control. We found increases in memory T cells, M1 macrophages, and activated DCs & NK cells as well as decreases in MDSCs within various degrees within the three tissues (Figure 8-10). These results suggest comparable reprogramming of three tissues responsible for generating innate immunity and increased immunological memory with the implant.

[0055] Another key observation was increased natural killer (NK) cell activation in the CDA-Mn2+ PLGA implant group, as indicated by upregulation of CD107a, a degranulation marker, in both the tumor-draining lymph nodes (tdLNs) and spleen (Figure 9f, Figure 10f). In addition, the CDA-Mn²⁺ PLGA implant reduced the frequency of myeloid-derived suppressor cells (MDSCs) in the spleen (Figure 10g), which are known to promote immune suppression and tumor progression. These findings show that the CDA-Mn2+ PLGA implant also induced activation of NK cells while reducing immunosuppressive MDSCs.

[0056] Collectively, these results show that the CDA-Mn2+ PLGA implant induced activation and reprogramming of CD4+ and CD8+ T cells, myeloid cells, and NK cells to various degrees in the TME, tdLNs, and / or spleen, demonstrating strong immune-stimulating potential of the CDA-Mn2+ PLGA implant.

[0057] Drug distribution was evaluated in vivo to examine if the implant could prolong drug retention at the injection site, compared to the soluble CDA+Mn2+ drug combination (FIGS. 4A, 4B). PLGA implants were formulated with a CDA analogue of a similar molecular weight, cGAMP (MW: 674.42 g / mol) tagged with Cy5. cGAMP-Cy5 was mixed at a 1:10 ratio withDocket No.30275 / 70597A / PC CDA and encapsulated using the same solvent extrusion technique as described above. These cGAMP-Cy5-CDA PLGA implants were injected subcutaneously into naïve BALB / c mice, and their fluorescence signal in vivo was quantified using IVIS and compared to that from free cGAMP admixed 1:10 with CDA.

[0058] Table 2: Loading of CDA into 502H & 503H PLGA Implants

[0059] Table 3: Loading of CDA and Mn2+ into 503H CDA-Mn PLGA Implant

[0060] The cancer adjuvant (or other agent) content or loading is quantified as: Mass of therapeutic agent encapsulated in implant (or other delivery system) Total mass of implant (or other delivery system)) x 100.

[0061] The percentage encapsulation efficiency of the cancer adjuvant (or other agent) is calculated as: Mass of therapeutic agent encapsulated in in implant (or other delivery system) Total mass of therapeutic agent in loading solution) x 100.

[0062] Our findings show that the injectable PLGA implants comprising a cancer adjuvant, an immunomodulatory inorganic ion, and a bulking agent, wherein the bulking agent is not a protein, specifically, where the bulking agent is hydroxyethyl starch, can effectively load cancer adjuvants like CDA and immunomodulatory inorganic ions such as manganese ion to enhance cancer immunotherapy. The disclosed implants can achieve controlled release with tunable release kinetics and demonstrate similar efficacy as a multi-dose treatment, thereby providing a patient-centric solution that can promote patient comfort and adherence for out- patient care.

Claims

Docket No.30275 / 70597A / PC What is Claimed 1. A polymer matrix delivery system for delivering a cancer adjuvant, comprising: a biodegradable polymer; a cancer adjuvant capable of recruiting T-cells to a tumor microenvironment; optionally, an immunomodulatory inorganic ion; and, a bulking agent, wherein the bulking agent is not a protein.

2. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 1, wherein the biodegradable polymer is chosen from one or more biodegradable polymers in the group of poly(lactides), poly(glycolides), poly(lactide-co-glycolides), poly(lactic-acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, poly(hydroxymethyl glycolide-co-lactide), polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates, poly(p-dioxanone), poly(alkylene oxalate)s, and polyurethanes.

3. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 1, wherein the biodegradable polymer comprises poly(lactic-co-glycolic acid).

4. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 3, wherein the biodegradable polymer has a lactic acid content in the range of 50% to 100% and a molecular weight in the range of about 4 kDa to about 55 kDa.

5. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-4, wherein the biodegradable polymer is an uncapped polymer comprising free carboxyl groups at the end of the polymer.

6. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-5, wherein the cancer adjuvant comprises a small molecule STING agonist.

7. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 6, wherein the STING agonist is a cyclic dinucleotide.Docket No.30275 / 70597A / PC 8. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 6, wherein the STING agonist is chosen from one or more STING agonists in the group of 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone, 6,4′- dimethoxyflavone, 4′-methoxyflavone, 3′,6′-dihydroxyflavone, 7,2′-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, and any derivatives thereof.

9. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 6, wherein the STING agonist is chosen from one or more STING agonists in the group of 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c-di-GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, and Gemcitabine.

10. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-9, wherein the immunomodulatory inorganic ion is present and comprises one or more inorganic ions chosen in the group of Zn2+, Mn2+, Fe2+, Fe3+, Cu2+, Ni2+, Co2+, Pb2+, Sn2+, Ru2+, Au2+, Mg2+, VO2+, Al3+, Co3+, Cr3+, Ga3+, Tl3+, Ln3+, MoO3+, Cu+, Au+, Tl+, Ag+, Hg2+, Pt2+, Pb2+, Hg2+, Cd2+, Pd2+, and Pt4+.

11. The polymer matrix delivery system for delivering a cancer adjuvant according to claim 10, wherein the immunomodulatory inorganic ion comprises Mn2+ and the cancer adjuvant comprises a small molecule STING agonist.

12. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-11, wherein the bulking agent comprises a water-soluble polymer, preferably a non-ionizable water-soluble polymer.

13. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-12, wherein the bulking agent comprises a polysaccharide.

14. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-13, wherein the bulking agent comprises one or more water- soluble polymers chosen in the group of carboxymethyl cellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropyl starch, and hydroxyethyl starch.

15. The polymer matrix delivery system for delivering a cancer adjuvant according to any of claims 1-14, wherein the biodegradable polymer comprises acid- terminated poly(lactic-co-glycolic acid), the STING agonist comprises c-di-AMP, theDocket No.30275 / 70597A / PC immunomodulatory inorganic ion is present and comprises Mn2+, and the bulking agent comprises hydroxyethyl starch.

16. An implant comprising the polymer matrix delivery system according to any of claims 1-15.

17. A method of treating a solid tumor or the potential recurrence of a solid tumor comprising injecting the implant according to claim 16 into the solid tumor or at a site of surgical resection of a solid tumor.

18. The method according to claim 17, further comprising administering an immune checkpoint inhibitor.

19. The method according to claim 17 or 18, wherein the solid tumor is a pancreatic tumor.

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