Compositions of polymeric microdevices and their use in cancer immunotherapy

Abstract

Microparticle compositions and methods for the delivery and pulsatile release of one or more sting agonists and / or receptors have been developed. The compositions include polymeric microdevices formed from biodegradable and biocompatible polymers or copolymers thereof, comprising a shell and compartments, or distinct regions within the compartments, formed by additive processes such as micromolding, three-dimensional printing, and lithography. The compositions include microdevices that release individual doses of incorporated STING agonists and / or receptors in pulses at defined times, for example, up to several months after administration, with substantially no leakage between releases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 024,308, filed May 13, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention is directed generally to compositions for delivering therapeutic agents over a sustained period of time and methods of use thereof, particularly in cancer therapy. [Background technology]

[0003] Background of the Invention The advent of immune checkpoint blockade therapy (ICBT), with several drugs approved by the U.S. Food and Drug Administration (FDA), has had a profound impact on cancer treatment (Postow MA., et al., J Clin Oncol., 33(17):1974-82 (2015)). Despite great promise, the clinical benefit of ICBT remains limited by low response rates (Sharma P. and Allison JP., Science, 348:56-51 (2015)). Clinical trials have shown that patients who respond to ICBT have high levels of tumor-infiltrating lymphocytes (TILs) and exhibit a type I interferon (IFN)-producing gene signature, indicative of innate immune system activation (Harlin H., et al., Cancer Res., 69(7):3077-85 (2009); Gajewski TF., et al., Curr Opin Immunol., 25(2):268-76 (2013); and Galon J., et al., J Transl Med., 10:205 (2012)). Strategies to improve TIL infiltration and innate immune system activation have been proposed as combination therapies to further improve ICBT response rates.

[0004] Among the numerous innate immune pathways initiated by Toll-like receptors (TLRs), mitochondrial antiviral signaling proteins (MAVS), or P2X purinergic receptor 7, activation of stimulator of interferon genes (STING) shows great promise for expanding TILs and improving the antitumor efficacy of ICBT (Wang H., et al., Proc Natl Acad Sci USA, 114(7):1637-1642 (2017); Corrales L., et al., Cell Rep., 11(7):1018-30 (2015)). STING pathway activation is initiated by recognition of cytoplasmic DNA. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase senses cytoplasmic DNA to produce the second messenger cGAMP, which then binds to STING and triggers a signaling cascade via tank-binding kinase 1 (TBK1) / interferon regulatory factor 3 (IRF3) to produce type I IFNs and other cytokines (Chen Q., et al., Nat Immunol.,17(10):1142-9 (2016); Ablasser A., ​​et al., Nature,498(7454):380-4 (2013)). Compelling evidence has shown that intratumoral injection of STING agonists stimulates potent antitumor immunity in clinically relevant tumor models (Corrales L., et al., 2015; Shae D. et al., Nat Nanotechnol., 14(3):269-278 (2019); Fu J., et al., Sci Transl Med., 7(283):283ra52 (2015); Curran E., et al., Cell Rep., 15(11):2357-66 (2016); and Luo M. et al., Nat Nanotechnol., 12(7):648-654 (2017)). As a result, phase I clinical trials are underway using STING agonists alone or in combination with ICBT to treat patients with advanced solid tumors and lymphomas.

[0005] Dosing regimens for STING agonists in current clinical trials involve multiple intratumoral injections administered repeatedly over as long as two years to achieve therapeutic efficacy (e.g., three injections over a 28-day period, or one injection weekly for nine weeks per treatment cycle) (see, e.g., clinical trial identification numbers: NCT03010176 and NCT02675439 at http: / / www.clinicaltrials.gov). Such frequent administration over long periods of time can cause chronic injection pain, increase the risk of infection, and ultimately lead to poor adherence, especially if each dose requires a health care visit (Mathes T., et al, Cancer Epidemiol., 38(3):214-26 (2014); Claxton AJ., et al., Clin Ther., 23(8):1296-310 (2001); and Puts MT., et al., Ann Oncol., 25(2):307-15 (2014)). Adherence rates to cancer treatments are low, at approximately 52%, similar to levels reported for patients with other chronic diseases (approximately 50%) (Osterberg L and Blaschke T, N Engl J Med., 353(5):487-97 (2005); Puts MT., et al., Ann Oncol., 25(3):564-77 (2014)). Poor adherence can lead to treatment failure, resulting in a financial burden of approximately $100 billion annually in the United States alone (Tan H., et al., Adv Ther., 28(1):51-61 (2011)).Furthermore, multiple intratumoral injections also limit the scope of STING agonist-based therapies to easily accessible tumor types and risk perturbing the tumor microenvironment (TME) and vascular network, potentially leading to cancer cell leakage and metastasis (Hobson J., et al., Breast Cancer Res Treat., 139(2):391-401 (2013); Hansen NM., et al., Arch Surg., 139(6):634-9 (2004); and Estourgie SH., et al., Br J Surg., 91(10):1370-1 (2004)). Therefore, a delivery system that mimics current clinical dosing regimens within a single injection is an attractive solution for improving patient adherence, reducing the risk of metastasis and treatment costs, and expanding the scope of current STING agonist-based therapies.

[0006] There remains a need for drug delivery systems that improve patient compliance / adherence, minimize the number of injections given to patients, reduce the risk of metastasis and the cost of treatment, and extend the reach of current STING agonist-based therapies to less accessible tumors.

[0007] It is therefore an object of the present invention to provide a drug delivery system that simplifies multiple injection dosage regimens. It is a further object of the present invention to provide compositions and methods for cancer therapy, particularly immunotherapy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Postow MA., et al., J Clin Oncol., 33(17):1974-82 (2015) [Non-patent document 2] Sharma P. and Allison JP., Science, 348:56-51 (2015) [Non-patent document 3] Harlin H., et al., Cancer Res., 69(7):3077-85 (2009) [Non-patent document 4] Gajewski TF., et al., Curr Opin Immunol., 25(2):268-76 (2013) [Non-patent document 5] Galon J., et al., J Transl Med., 10:205 (2012) Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention Compositions and methods are provided for the delivery and pulsed release of one or more therapeutic and / or prophylactic agents to a site (e.g., a tumor). The compositions typically comprise microfabricated particles ("microdevices") that reside at the injection site and release the incorporated therapeutic agent(s) in a programmable sequence of pulses at predetermined time points that mimic multiple injections over a period of days to weeks.

[0010] In a preferred embodiment, the therapeutic agent stimulates an immune response, preferably an immune receptor binding agent such as a STING agonist, with a release profile designed to mimic repeated single-dose administration of the drug over a sustained period. Results show that a single intratumoral injection of such a STING agonist-loaded microdevice can elicit a potent local and systemic antitumor immune response, inhibiting tumor growth and metastasis and prolonging survival as effectively as multiple doses of a soluble STING agonist.

[0011] The microdevices have a biocompatible, biodegradable polymer shell and a compartment that encapsulates a therapeutic agent, such as a STING agonist. The shell can be formed from a biodegradable, biocompatible polymer or copolymer, such as poly(lactic acid), poly(glycolic acid), and copolymers thereof. In some embodiments, the polymer is poly(lactic-co-glycolic acid) (PLGA). The microdevices can be formed by micromolding, 3D printing, or stereolithography of the polymer, which can be used to form the microdevices into complex three-dimensional geometries.

[0012] Microdevices can have a variety of shapes. For example, microdevices can be box-shaped, such as rectangular prisms or cubes. In some embodiments, the microdevices have at least one exterior dimension between about 1 μm and 1000 μm, and / or the internal compartments have at least one dimension between about 1 μm and 800 μm. In certain exemplary embodiments, the sealed microdevices have exterior dimensions of 400×400×300 μm (length×width×height) and wall thicknesses of 100 μm in each dimension, and optionally an internal cavity of 200×200×100 μm (length×width×height), although alternative exterior and internal cavity dimensions are provided, as discussed in more detail below.

[0013] Therapeutic agents, e.g., STING agonists, can be released from the microdevices in vitro or in vivo over a defined period of time, such as about 1 day, about 4 days, about 8 days, about 11 days, about 15 days, about 18 days, about 97 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year. In certain embodiments, the release rate of the STING agonist is controlled by the number-average molecular weight of the polymer or copolymer, the weight-average molecular weight of the polymer or copolymer, the polydispersity index of the polymer or copolymer, the chain end functionality of the polymer or copolymer, the copolymer ratio, salt, polymer or copolymer blend ratio, shell thickness, and compartment matrix, or a combination thereof, as well as the inclusion of payloads and, if present, excipients.

[0014] In a preferred embodiment, the therapeutic agent is a STING agonist, which can be nucleic acid or small molecule.Preferably, the STING agonist is cyclic dinucleotide or acyclic dinucleotide.Exemplary STING agonists include cGAMP, DMXAA, MK-1454, MK-2118, E7766, MIW815 (ADU-S100), BMS-986301, GSK3745417, IMSA-101, SYNB1891l, SITX-799 and SB11285.

[0015] A population of microdevices can comprise a homogeneous plurality of microdevices and pharmaceutical compositions thereof. In some embodiments, the pharmaceutical composition comprises two or more populations of microdevices and a pharmaceutically acceptable buffer, carrier, diluent, or excipient. Each different population of microdevices releases the incorporated agent(s), preferably a STING agonist, at a different time period than the other populations in the pharmaceutical composition, thereby resulting in a pulsed release of the incorporated agent(s) over a sustained period.

[0016] The microdevices and compositions thereof can be used in various ways. For example, a method for delivering one or more agents, particularly STING agonists, to a subject can include administering to the subject a pharmaceutical composition comprising a microdevice, or a group, or two, three, four, or more groups of the microdevices. These may contain various therapeutic, preventive, and / or diagnostic agents, or combinations thereof, or ratios thereof, and can be released at different times or at the same time.

[0017] Similarly, methods of inducing or modulating an immune and / or inflammatory response in a subject are provided. Typically, such methods comprise administering to a subject, most preferably to a tumor where the microdevices are injected into a tumor, a pharmaceutical composition comprising, preferably a population of two, three, four or more microdevices, in an effective amount to induce an immune and / or inflammatory response, preferably over a sustained period of time.

[0018] Also described is a method for treating other types of cancer-related diseases or disorders.The method for treating cancer in a subject in need thereof can comprise administering to the subject an effective amount of any of the pharmaceutical compositions for treating cancer.In such a method, the therapeutic agent (for example, immune response stimulating therapeutic agent such as STING agonist) is preferably released from the microdevice in a pulsed manner at a specified period after administration.

[0019] In a preferred embodiment, the administered pharmaceutical composition contains two or more different populations of microdevices. For example, the composition can have three different populations of microdevices, where the first population releases the incorporated STING agonist about 4 days after administration, the second population releases the incorporated STING agonist about 8 days after administration, and the third population releases the incorporated STING agonist about 11 days after administration. Alternative pulsed release profiles can also be provided, for example, by adjusting the number-average molecular weight of the polymer or copolymer, the weight-average molecular weight of the polymer or copolymer, the polydispersity index of the polymer or copolymer, the chain end functionality of the polymer or copolymer, the copolymer ratio, salt, the blend ratio of the polymer or copolymer, the shell thickness, and the compartment matrix, or a combination thereof. Different populations of microdevices can contain the same or different STING agonists.

[0020] Generally, the compositions are administered (e.g., locally) in an amount effective to induce a local or systemic immune and / or inflammatory response, induce or enhance STING pathway activity, induce or enhance an interferon response, induce infiltration of the tumor microenvironment (e.g., by lymphocytes, basophils, macrophages, and / or dendritic cells), and / or reduce immunosuppression within the tumor microenvironment. Such an effect or response to administration can last for a variety of periods following administration, including from about 1 day to about 30 days, from about 21 days to about 28 days, from about 1 week to about 4 weeks, from about 1 month to about 6 months, or from about 6 months to about 1 year.

[0021] Administration of the microdevice composition can also reduce or prevent tumor recurrence and / or metastasis.

[0022] In some embodiments, particularly those in which the subject is afflicted with cancer, the composition is administered intratumorally. The composition may be administered as a single injection.

[0023] The treatment method may include further administering additional cancer therapy to the subject, such as, but not limited to, surgery, radiation therapy, chemotherapy, immunotherapy, cryotherapy, or gene therapy. For example, the subject may further be administered one or more STING agonists, one or more immune checkpoint blockers, or a combination thereof. The immune checkpoint blocker may include an antibody or an antigen-binding fragment thereof. Suitable antibodies or antigen-binding fragments thereof preferably include inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, LAG3, or a combination thereof.

[0024] In some embodiments, the subject has cancer such as melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal carcinoma, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell neoplasm, Wilms' tumor, astrocytoma, ependymoma, glioma, meningioma, medulloblastoma, neuroblastoma, or hepatocellular carcinoma. In some embodiments, the cancer is a solid tumor or is a lymphoma.

[0025] Compositions and methods for pulsed release over a sustained period are believed to be particularly advantageous for treating tumors that are difficult to reach with a single dose, especially where practitioners struggle to achieve the same sustained therapy using traditional methods of administering multiple doses of drug to the same area over the desired treatment period. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A pharmaceutical composition comprising a microdevice comprising a biocompatible, biodegradable polymer shell containing at least one discrete compartment therein, wherein the shell is made by an additive process; A pharmaceutical composition wherein the microdevice releases an immunoreceptor binding agent in an amount effective to elicit an immune response for one or more periods of time, the periods and / or times of release being the same or different. (Item 2) 2. The pharmaceutical composition of item 1, wherein the microdevice is formed by three-dimensional printing, micromolding, lithography, or a combination thereof. (Item 3) 3. The pharmaceutical composition of item 1 or 2, wherein the microdevice releases the drug at multiple times or time periods. (Item 4) 4. The pharmaceutical composition according to any one of items 1 to 3, wherein the release period is independently selected from about 1 day, about 4 days, about 8 days, about 11 days, about 15 days, about 18 days, about 97 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, and about 1 year. (Item 5) 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the release rate of the drug is controlled by the number average molecular weight of the polymer or copolymer, the weight average molecular weight of the polymer or copolymer, the polydispersity index of the polymer or copolymer, the chain end functionality of the polymer or copolymer, the copolymer ratio or a combination thereof, salt, polymer or copolymer blend ratio, the shell thickness and the compartment matrix. (Item 6) 6. The pharmaceutical composition according to any one of items 1 to 5, wherein the immunoreceptor binding agent is a STING agonist. (Item 7) 7. The pharmaceutical composition of item 6, wherein the STING agonist is a nucleic acid or a small molecule. (Item 8) 8. The pharmaceutical composition of item 7, wherein the STING agonist is a cyclic dinucleotide or an acyclic dinucleotide. (Item 9) 8. The pharmaceutical composition of item 7, wherein the STING agonist is selected from the group comprising cGAMP, DMXAA, MK-1454, MK-2118, E7766, MIW815 (ADU-S100), BMS-986301, GSK3745417, IMSA-101, SYNB1891l, SITX-799 and SB11285. (Item 10) 10. The pharmaceutical composition of any one of items 1 to 9, wherein the microdevice has at least one external dimension of about 1 μm to 1000 μm and / or the at least one compartment has a dimension of about 1 μm to 800 μm. (Item 11) 11. The pharmaceutical composition according to any one of items 1 to 10, wherein the microdevice has a rectangular parallelepiped or cubic shape. (Item 12) 12. The pharmaceutical composition of any one of items 1 to 11, wherein the microdevice shell, and optionally the boundaries of the one or more compartments, are formed from a biodegradable, biocompatible polymer, optionally wherein the polymer is selected from the group consisting of polyhydroxy acids, polyhydroxyalkanoates, and polyanhydrides. (Item 13) 13. The pharmaceutical composition according to item 12, wherein the polymer is poly(lactic acid), poly(glycolic acid) and / or copolymers thereof. (Item 14) 14. The pharmaceutical composition according to any one of items 1 to 13, wherein the polymer or copolymer forms the shell and / or compartments by three-dimensional printing. (Item 15) 14. The pharmaceutical composition according to any one of items 1 to 13, wherein the microdevice is formed by micromolding of the polymer. (Item 16) 14. The pharmaceutical composition according to any one of items 1 to 13, wherein the microdevice is formed by stereolithography of the polymer. (Item 17) 17. The pharmaceutical composition of any one of items 1 to 16, comprising different populations of microdevices, wherein a first population releases the agent at about 4 days after administration, a second population releases the agent at about 8 days after administration, and a third population releases the agent at about 11 days after administration. (Item 18) 18. The pharmaceutical composition according to any one of items 1 to 17, wherein the agent is a STING agonist. (Item 19) 19. The pharmaceutical composition of item 18, wherein the populations of microdevices comprise the same or different STING agonists. (Item 20) 20. The pharmaceutical composition according to any one of items 18 to 19, in a dosage for local administration in an amount effective to induce a local or systemic immune and / or inflammatory response in a subject. (Item 21) 21. The pharmaceutical composition of any one of items 18 to 20, in a dosage comprising an amount effective to induce or increase STING pathway activity in said subject when administered topically, intratumorally, subcutaneously, intramuscularly or peritoneally. (Item 22) 22. A method for localized delivery of one or more immune response inducing or enhancing agents to a subject for more than one period of time, the method comprising administering to the subject the pharmaceutical composition of any one of items 1 to 21. (Item 23) 23. The method according to item 22, comprising administering to the subject the pharmaceutical composition according to any one of items 1 to 21 in an effective amount to induce an immune and / or inflammatory response in or adjacent to the tumor. (Item 24) 24. The method of any one of items 22 to 23, wherein the composition is administered intratumorally. (Item 25) 25. The method of any one of items 22 to 24, wherein the composition is administered as a single injection. (Item 26) 26. The method of any one of items 22 to 25, wherein the composition is administered in an amount effective to induce or increase an interferon response in the subject. (Item 27) 27. The method of any one of items 22 to 26, wherein the composition is administered in an effective amount to induce infiltration of lymphocytes, basophils, macrophages and / or dendritic cells into the tumor microenvironment. (Item 28) 28. The method of any one of items 22 to 27, wherein the composition is administered in an amount effective to reduce immunosuppression within the tumor microenvironment. (Item 29) 29. The method of any one of items 22 to 28, wherein the induction or augmentation of the immune response, inflammatory response, STING pathway activity, induction or augmentation of the interferon response, tumor infiltration, and / or reduction in immunosuppression lasts for about 1 day to about 30 days, about 21 days to about 28 days, about 1 week to about 4 weeks, about 1 month to about 6 months, or about 6 months to about 1 year after administration. (Item 30) 30. The method of any one of items 22 to 29, wherein the administration reduces or prevents tumor recurrence and / or metastasis. (Item 31) 31. The method of any one of items 22 to 30, further comprising administering an additional cancer therapy to the subject. (Item 32) 32. The method of item 31, wherein the additional cancer therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, cryotherapy, or gene therapy. (Item 33) 33. The method of item 32, wherein the additional therapy is immunotherapy comprising administration of one or more STING agonists, one or more immune checkpoint blockers, or a combination thereof. (Item 34) 34. The method of claim 33, wherein the immune checkpoint blockade is an antibody or an antigen-binding fragment thereof, preferably, the antibody or antigen-binding fragment thereof is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, LAG3, or a combination thereof. (Item 35) 35. The method of any one of items 22 to 34, wherein the cancer is melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, testicular cancer, urothelial carcinoma, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal carcinoma, colorectal carcinoma, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell neoplasm, Wilms' tumor, astrocytoma, ependymoma, glioma, meningioma, medulloblastoma, neuroblastoma, or hepatocellular carcinoma. [Brief explanation of the drawings]

[0026] [Figure 1-1]Figures 1A-1E are schematic diagrams illustrating the design and fabrication of PLGA microdevices (PLGA-MPs). Figure 1A is a schematic diagram of a single-injection drug delivery platform for cancer immunotherapy. Different PLGA microdevices remain in the tumor after a single intratumoral injection and release incorporated STING agonists in pulses at distinct time points to promote tumor-infiltrating lymphocyte (TIL) infiltration. Figure 1B is a schematic diagram of the fabrication process for PLGA microdevices, which are prepared by loading the microdevice base with the cargo of interest and then sealing the base with a corresponding microdevice cap by briefly applying heat. Figures 1C-1D are representative SEM images of an empty microdevice base (Figure 1C) and a sealed microdevice (Figure 1D). Figure 1E is a representative high-resolution X-ray computed tomography image of a sealed microdevice encapsulating 3'3'-cGAMP. The red color represents dried 3'3'-cGAMP. [Figure 1-2] Same as above.

[0027] [Figure 2-1]Figures 2A–2L illustrate the quantification of PLGA-MP release rates. Figures 2A–2G show the cumulative in vitro release rates of AF647-dextran from PLGA microdevices, PLGA-1–PLGA-7, respectively (n = 6–8). Data represent the mean ± standard error. Figures 2H–2I show the cumulative in vitro release rates of pemetrexed (Figure 2H) and Cy5-labeled CpG DNA (Figure 2I) from PLGA-2 (n = 6–10). Data represent the mean ± standard error. Figures 2J–2K show the cumulative in vitro (Figure 2J) and in vivo (Figure 2K) release rates of AF647-dextran from PLGA-1, 2, and 3. PLGA-MP was administered subcutaneously (n = 6–8). Error bars represent the standard error of the mean (s.e.m.). Figure 2L is a graph showing the cumulative in vivo release rate of AF647-dextran-loaded PLGA-2 administered subcutaneously (n=8) or intratumorally in a B16F10 melanoma model (n=4) and a 4T1 breast cancer model (n=4). Error bars represent standard error. Figure 2M is a schematic diagram showing the treatment and sampling schedule of B16F10 tumor-bearing mice after intratumoral injection of AF647-loaded PLGA-1. Figure 2N is a graph showing the cumulative in vivo release of AF647 from PLGA-1 in B16F10 tumors (n=4). Figure 2O is a graph showing the serum AF647 concentration after intratumoral injection of AF647-loaded PLGA-1 (n=4). Error bars represent standard error. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above.

[0028] [Figure 3-1]Figure 3A is a graph showing the cumulative in vitro release of 3'3'-cGAMP from PLGA-1, 2, and 3 (n = 6-8). Error bars represent standard error. Figure 3B is a mass spectrum of 3'3'-cGAMP released from PLGA-2 on day 8, showing molecular ions [M+H]+ = 675.11, [M+Na]+ = 697.09, and [M+2Na]+ = 719.07. The microdevices were incubated in PBS at 37°C. Figure 3C is a bar graph showing the response of cGAMP incorporated into and released from PLGA-2 in an interferon regulatory factor (IRF) reporter cell line (n = 6). Error bars represent standard deviation. Statistical significance was calculated using one-way analysis of variance (ANOVA). Figure 3D is a schematic diagram of the treatment scheme for B16F10 and 4T1 tumor-bearing mice treated with a single injection of 3'3'-cGAMP-loaded PLGA-1, 2, and 3 on day 7, or four injections of soluble 3'3'-cGAMP on days 7, 11, 15, and 18 after tumor inoculation. Figures 3E-3F are graphs showing the mean tumor growth (Figure 3E) and Kaplan-Meier survival curves (Figure 3F) of B16F10 melanoma-bearing mice (n = 8 biologically independent samples) treated with various groups. The legend for Figure 3E is provided in Figure 3F. Figures 3G-3H are graphs showing the mean tumor growth curves (Figure 3G) and survival analysis (Figure 3H) of orthotopic 4T1 breast tumor-bearing mice (n = 8 biologically independent samples). Statistical significance was calculated by two-way ANOVA and Tukey's multiple comparison test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data represent the mean ± standard error. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above.

[0029] [Figure 4-1]Figures 4A-4I show analyses of immune responses and STING pathway activation. Figure 4A is a schematic diagram showing the treatment scheme of B16F10 tumor-bearing mice that received a single injection of cGAMP-S, cGAMP-loaded PLGA-1 and 2 on day 7, or three injections of soluble 3'3'-cGAMP on days 7, 11, and 15 after tumor inoculation. Tumors were dissected on day 16. Figures 4B-4C are bar graphs showing qPCR analysis of CXCL10 (Figure 4B) and IRF7 (Figure 4C) mRNA expression in tumors (n = 4). Data represent mean ± standard error. Figures 4D–4G are bar graphs showing the percentage of infiltrating lymphocytes, such as CD8+CD3+ T cells (Figure 4D), CD4+CD3+ T cells (Figure 4E), and NK1.1+CD3- NK cells (Figure 4F), as well as CD11b-CD11c+ dendritic cells, and myeloid cells, such as CD11b+F4 / 80+ macrophages, CDA-4I11b+F4 / 80-Ly6c+Ly6g+ neutrophils, CD11b+F4 / 80-Ly6c+Ly6g- monocytes, CD11b+Gr-1-CD200R3+ basophils, and CD11b+Gr-1-CD170+ eosinophils (Figure 4G), among all live cells in the TME (n = 4–5). Data represent the mean ± standard deviation (s.d.). Figure 4H shows representative flow cytometry histograms of DCs (CD86+CD11c+CD11b-) in tumors treated with different groups (n=4 to 5). Quantitative analysis is shown in the bar graphs on the right. Data represent mean ± standard deviation. Figure 4I shows representative flow cytometry histograms of M1 (CD86+CD11b+F4 / 80+) and M2 (CD206+CD11b+F4 / 80+) macrophages in tumors treated with different groups. The ratio of M1 / M2 macrophages was calculated and displayed on the right (n=4). Data represent mean ± standard deviation. Statistical significance was calculated by one-way ANOVA or Student's t-test when comparing multiple or two groups. Data were compared with the untreated group unless otherwise indicated. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above.

[0030] [Figure 5-1] Figure 5A is a graph showing quantitative analysis of IFNγ+ CD8+ T cells in serum collected on days 21 and 28 (n = 4-5, treatment scheme shown in Figure 3D). Untreated and 1xEP-treated mice did not survive on day 28. Data represent mean ± standard error. Figures 5B-5C are bar graphs showing the numbers of effective memory CL62L-CD44+CD4+CD3+ T cells (Figure 5B) and CL62L-CD44+CD8+CD3+ T cells (Figure 5C) in the TME on day 16 (treatment scheme shown in Figure 4A). Figure 5D is a schematic diagram of the treatment regimen for the contralateral B16F10 model. Tumors were inoculated into the right and left hind flanks of mice on days 0 and 2. The primary tumor (right side) was treated with a single intratumoral injection of cGAMP-S+cGAMP-MP and three intraperitoneal injections of anti-PD-1 antibody (ICB). Figures 5E-5F are graphs showing the average tumor growth curves of treated tumors (Figure 5E) and distant tumors (Figure 5F, n=8). Data represent mean ± standard error. Figure 5G is a schematic diagram of the treatment regimen for the metastatic 4T1 model. Figure 5H is a graph showing the number of metastatic foci on the lung surface after treatment (n=8). Figure 5I is a graph showing the percentage of tumor area within the total lung area after treatment (n=4 to 5). Statistical significance was calculated by t-test and two-way ANOVA: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0031] [Figure 6-1]Figures 6A-H demonstrate that a single injection of PLGA-MP induces systemic antitumor immunity and inhibits metastasis. Figure 6A is a schematic diagram of the treatment regimen in a surgically removed B16F10 model. Approximately 99% of the tumor mass was surgically removed 6 days after tumor inoculation. cGAMP-MP and cGAMP-S were deposited directly on the surgical bed. Figures 6B-6C are graphs showing the mean tumor growth curve (Figure 6B) and survival analysis (Figure 6C) of treated mice (n=8). Figures 6D-6E are graphs showing tumor growth (Figure 6D) and survival (Figure 6E) monitored over time in tumor-free mice treated with cGAMP-MP and 3x cGAMP-S (n=6) and rechallenged with B16F10 cells 60 days after tumor inoculation. Data represent mean ± standard error. Figure 6F is a schematic diagram of the treatment regimen for an orthotopic pancreatic tumor model. Figures 6G-6H show representative images (Figure 6G) and weight analysis (Figure 6H) of tumors isolated from the pancreas. Statistical significance was calculated by one-way or two-way ANOVA and Tukey's multiple comparison test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 6-2] Same as above. [Figure 6-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed Description of the Invention I. Definition As used herein, a "microdevice" refers to a microstructure having diverse or complex three-dimensional geometries that cannot be formed using standard techniques, such as emulsion or solvent evaporation techniques. A microdevice can have one or more internal compartments with an outer shell formed by solvent and / or thermal bonding of discrete powders or suspensions to form a desired shape and dimensions. A microdevice can have diverse compartment geometries, outer shell geometries, or diverse geometries of both the compartment and the outer shell. For example, the compartment and shell may have the same geometry, such as a cube-shaped compartment and a cube-shaped shell. The compartment and shell may have different geometries, such as a compartment being cubic while the shell is star-shaped or conical. A device can be formed by combining a "base" device and a cap. While described with reference to a "compartment," it is understood that there may be multiple compartments of the same or different dimensions and shapes.

[0033] A microdevice has microscale external dimensions, such as length, width, height, or diameter, with at least one dimension being less than 1 centimeter at most, and more preferably has a maximum diameter between 1 micrometer (μm) and 1000 μm. As used herein, the "diameter" of a non-spherical microdevice refers to the maximum linear distance between two points on the surface of the microdevice or between two points in a non-spherical compartment. When referring to multiple microdevices or multiple compartments, the diameter of the microdevice or compartment typically refers to the average diameter of the microdevice. The diameter of a microdevice or compartment can be measured using various techniques, including, but not limited to, optical microscopy or electron microscopy. The diameter of a microdevice can be measured by dynamic light scattering. In the case of spherical microparticles, "diameter" is used in its art-recognized definition.

[0034] As used herein, "base" or "bases" in the context of a microdevice refers to the base of the microdevice.

[0035] As used herein, "cap" or "caps" refers to a structure used to cap a base or bases. The cap may have any geometric shape, which may be the same as or different from the geometric shape of the base.

[0036] "Additive manufacturing" or "3D printing," as used herein, refers to the process of creating three-dimensional solid objects of virtually any shape from a digital model. 3D printing is accomplished using an additive process, where successive layers of material are built up in various shapes or thicknesses. In some embodiments, "3D printing" uses inks containing extruded or solvent-based polymers (e.g., PLGA, poly(L-lactide) ("PLLA"), etc.) that are jetted or extruded through a nozzle and solidified into the desired shape. The shape can be controlled in the x, y, and z directions.

[0037] "Micromolding," as used herein, generally refers to a process suitable for producing parts or devices on a microscale, or for producing parts or devices with properties or resistances on a microscale. Exemplary techniques include, but are not limited to, lithography.

[0038] The term "biocompatible," as used herein, refers to one or more materials that are not themselves toxic to a host (e.g., an animal or human) or that do not degrade (if they do degrade) at a rate that produces monomeric or oligomeric subunits or other by-products in toxic concentrations in the host.

[0039] The term "biodegradable," as used herein, means that the material will break down or decompose into its component subunits in the body as a function of hydrolysis and / or enzymatic degradation.

[0040] The term "homogeneous" when used in the context of a microdevice refers to a collection of two or more individual microdevices of the same type. For example, a homogeneous microdevice can have a uniform composition (e.g., formed from the same polymer or copolymer), structure (e.g., 3D geometry), drug (e.g., encapsulating the same therapeutic and / or prophylactic agent), and combinations thereof. When used in the context of a microdevice, "heterogeneous" means not homogeneous. For example, in some embodiments, a heterogeneous microdevice does not have a uniform composition (e.g., formed from the same polymer or copolymer), structure (e.g., 3D geometry), drug (encapsulating the same therapeutic and / or prophylactic agent), and combinations thereof.

[0041] As used herein, the term "agonist" refers to a molecule that binds to a receptor and activates the receptor to produce a biological response. Receptors can be activated by either endogenous or exogenous agonists. An "agonist" can be a full agonist, a partial agonist, or an inverse agonist.

[0042] "Immune response" as used herein typically refers to a response that induces, improves, or prolongs the activation or efficiency of innate and / or adaptive immunity. The immune response can be a specific response to an antigen, including a cancer antigen, or a vaccine, that generates immunity to current or future exposure in a host, such as a mammal.

[0043] "Hydrophilic" as used herein refers to a molecule that has a higher affinity for water than organic solvents, and therefore has a higher solubility. The hydrophilicity of a compound can be determined by measuring its partition coefficient between water (or aqueous buffer solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. If, after equilibration, a higher concentration of the compound exists in water than in organic solvent, the compound is considered hydrophilic.

[0044] "Hydrophobic" as used herein refers to a molecule that has a higher affinity for water than water and therefore has a higher solubility in organic solvents.The hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or aqueous buffer solution) and a water-immiscible organic solvent such as octanol, ethyl acetate, methylene chloride or methyl tert-butyl ether.If, after equilibration, a higher concentration of the compound exists in organic solvent than in water, the compound is considered to be hydrophobic.

[0045] "Treatment" or "treating" refers to administering a composition to a subject or system having an undesired condition (e.g., cancer). A condition can include one or more symptoms of a disease, pathological condition, or disorder. Treatment includes the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This includes active treatment, i.e., treatment specifically directed toward ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed toward eliminating the cause of an associated disease, pathological condition, or disorder. Furthermore, the term includes symptomatic treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the onset of an associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed toward ameliorating an associated disease, pathological condition, or disorder. It is understood that treatment is intended to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder, but does not necessarily require that cure, improvement, stabilization, or prevention actually occur. In some embodiments, treatment refers to administering a composition in an amount sufficient to reduce, alleviate, or improve one or more symptoms of the disorder, disease, or condition being treated. The effect of treatment can be measured or evaluated as described herein and as known in the art, as appropriate for the relevant disease, condition, or disorder. Such measurement and evaluation can be performed in qualitative and / or quantitative terms. Thus, for example, the characteristics or properties of a disease, pathological condition, or disorder, and / or the symptoms of a disease, pathological condition, or disorder can be reduced to any effect or any amount.

[0046] "Prevention" or "preventing" means administering a composition to a subject or system at risk of an undesired condition (e.g., cancer). A condition can include one or more symptoms of a disease, pathological condition, or disorder. A condition can also be a predisposition to a disease, pathological condition, or disorder. The effect of administering a composition to a subject can be the cessation of a particular symptom of the condition, the reduction or prevention of a symptom of the condition, the reduction in the severity of the condition, the complete elimination of the condition, the stabilization or delay of the occurrence or progression of a particular event or characteristic, or the reduction in the chance of a particular event or characteristic occurring.

[0047] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount sufficient to reduce or ameliorate one or more symptoms of the disorder, disease, or condition being treated, or otherwise achieve the desired pharmacological and / or physiological effect. Such an improvement merely requires a reduction or alteration, not necessarily elimination. The exact amount will vary depending on a variety of factors, including subject-dependent variables (e.g., age, immune health, weight, etc.), the disease or disorder being treated, and the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.

[0048] As used herein, the term "antibody" or "immunoglobulin" is used to include intact antibodies and their binding fragments. Typically, fragments compete with the intact antibody from which they are derived for specific binding to an antigen fragment and include individual heavy and light chains, Fab, Fab', F(ab')2, Fabc, and Fv. Fragments are produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes one or more immunoglobulin chains that are chemically conjugated to or expressed as fusion proteins with other proteins. The term "antibody" also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites.

[0049] The term "small molecule," as used herein, generally refers to an organic molecule having a molecular weight of less than about 2000 g / mol, less than about 1500 g / mol, or less than about 1000 g / mol. Small molecules are non-polymeric and / or non-oligomeric.

[0050] By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject together with a selected compound without causing any undesired biological effects or interacting in a deleterious manner with any other components of the pharmaceutical composition in which it is contained.

[0051] Use of the term "about" is intended to describe values ​​that are either above or below the stated value by approximately + / - 10%. In other embodiments, values ​​may range from values ​​that are either above or below the stated value by approximately + / - 5%.

[0052] II. Composition Polymeric microdevices and compositions thereof suitable for delivering one or more therapeutic and / or prophylactic agents that bind to receptors, particularly one or more STING agonists, have been developed for use as delivery systems that provide sustained and / or intermittent or pulsed release of the agents over a period of time with a single injection.

[0053] A. Polymer Microdevices Polymeric microdevices, and compositions and formulations containing such microdevices, can have a variety of three-dimensional geometries and contain one or more distinct internal cavities, such as compartments, which may contain therapeutic and / or prophylactic agents, such as STING agonists, as well as excipients or other inactive ingredients and controlled-release materials.

[0054] 1. Polymers The microdevices are formed from one or more polymers or copolymers. In preferred embodiments, the polymers are biocompatible and biodegradable. The microdevices can be made using hydrophobic polymers, hydrophobic polymers blended with hydrophilic polymers, amphiphilic polymers, or mixtures thereof.

[0055] Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides, hydrophilic polypeptides, poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine, polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO), poly(oxyethylated polyols), poly(olefinic alcohols), polyvinylpyrrolidone, poly(hydroxyalkylmethacrylamides), poly(hydroxyalkylmethacrylates), poly(saccharides), poly(hydroxy acids), poly(vinyl alcohols), and copolymers thereof.

[0056] Examples of hydrophobic polymers include polyhydroxy acids such as poly(lactic acid), poly(glycolic acid) and poly(lactic-co-glycolic acid), poly3-hydroxybutyrate or poly4-hydroxybutyrate, polycaprolactone, poly(orthoesters), polyanhydrides, poly(phosphazenes), polyhydroxyalkanoates such as poly(lactide-co-caprolactone), polycarbonates such as tyrosine polycarbonate, polyamides (including synthetic and natural polyamides), polypeptides and poly(amino acids), poly Examples of suitable hydrophobic polymers include esteramides, polyesters, poly(dioxanone), poly(alkylene alkylates), hydrophobic polyethers, polyurethanes, polyetheresters, polyacetals, polycyanoacrylates, polyacrylates, polymethyl methacrylates, polysiloxanes, poly(oxyethylene) / poly(oxypropylene) copolymers, polyketals, polyphosphates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(maleic acid), and copolymers thereof. In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In preferred embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic-co-glycolic acid) (PLGA).

[0057] Biodegradable polymers can include water-insoluble or poorly water-soluble polymers that are converted chemically or enzymatically into water-soluble materials in the body. Biodegradable polymers can include soluble polymers that are crosslinked with hydrolyzable crosslinking groups that render the crosslinked polymers insoluble or poorly soluble in water.

[0058] An amphiphilic polymer is a polymer that contains a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block can contain one or more of the above-mentioned hydrophobic polymers, or their derivatives or copolymers. The hydrophilic polymer block can contain one or more of the above-mentioned hydrophilic polymers, or their derivatives or copolymers.

[0059] In particularly preferred embodiments, the biodegradable polymer is a polyester or polyanhydride, such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid). Polyester homopolymers include glycolic acid units, referred to herein as "PGA," and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA," and caprolactone units, such as poly(ε-caprolactone), collectively referred to herein as "PCL," and copolymers containing lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA," characterized by the ratio of lactic acid to glycolic acid, as well as polyacrylates and their derivatives. Exemplary polymers also include copolymers of polyethylene glycol (PEG) with the above polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers." In certain embodiments, the PEG region is covalently attached to the polymer to obtain a "PEGylated polymer" via a cleavable linker.

[0060] Polymers can undergo phase changes based on physical or chemical changes in their environment. Exemplary environmental triggers that can change the physical or chemical characteristics of a polymer, such as solubility, degradation rate, crosslinking, and erosion rate, include changes in temperature, pH, and ionic strength.

[0061] The polymer may comprise or be a blend or copolymer of two or more polymers. The polymer may also contain other entities such as stabilizers, surfactants or lipids.

[0062] 2. Structure Microdevices can have complex three-dimensional geometries. Microdevices can be solid, layered, and / or contain compartments. Typically, microdevices include a shell / base having one or more internal compartments and caps that seal the compartments (see, e.g., FIG. 1B). The layers and / or compartments can contain drugs (see, e.g., FIG. 1B).

[0063] Microdevices can have geometric shapes including, but not limited to, cubes, rectangular prisms, stars, cylinders, square prisms, triangular prisms, pentagonal prisms, octahedrons, diamonds, ellipses, and spheres. The compartments of a microdevice can contain one or more distinct regions within one or more compartments, and have complex 3D geometries.

[0064] Microdevices typically have external dimensions, such as length, width, height or diameter, each between 50 micrometers (μm) and 1000 μm, 50 micrometers (μm) and 550 μm, 50 micrometers (μm) and 500 μm, 50 micrometers (μm) and 450 μm, 50 micrometers (μm) and 400 μm, between 50 μm and 350 μm, between 50 μm and 300 μm, between 50 μm and 250 μm, between 50 μm and 200 μm, between 50 μm and 150 μm, and between 50 μm and 100 μm. For example, the external dimensions of a rectangular parallelepiped shaped microdevice may be about 250 μm, about 300 μm, or about 400 μm in length, about 250 μm, about 300 μm, or about 400 μm in width, and about 250 μm, about 300 μm, or about 400 μm in height.

[0065] The compartments can each generally have microscale dimensions such as length, width, height, or diameter between 10 μm and 850 μm, between 10 μm and 800 μm, between 10 μm and 750 μm, between 10 μm and 700 μm, between 10 μm and 650 μm, between 10 μm and 600 μm, between 10 μm and 550 μm, between 10 μm and 500 μm, between 10 μm and 450 μm, between 10 μm and 400 μm, between 10 μm and 350 μm, between 10 μm and 300 μm, between 10 μm and 250 μm, between 10 μm and 200 μm, between 10 μm and 150 μm, between 10 μm and 100 μm, or between 10 μm and 50 μm.

[0066] Exemplary dimensions for cube- or rectangular-shaped compartments include length, width and height of about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, or about 130 μm, or about 140 μm, about 150 μm, about 200 μm, about 250 μm, or about 300 μm. For example, the dimensions of the rectangular parallelepiped shaped compartment may be a length of about 100 μm, about 150 μm, about 200 μm or about 250 μm, a width of about 100 μm, about 150 μm, about 200 μm or about 250 μm, and a height of about 100 μm, about 150 μm, about 200 μm or about 250 μm.

[0067] The experiments in the following examples utilized sealed microdevices with external dimensions of 400 x 400 x 300 μm (length x width x height), wall thicknesses of 100 μm in each dimension, and internal cavities of 200 x 200 x 100 μm (length x width x height). However, alternative external and internal cavity dimensions are also provided and can be independently selected by the practitioner to fine-tune microdevices with various wall thicknesses, as well as various external and / or internal cavity shapes and sizes, and combinations thereof.

[0068] 3. Microdevice Composition Various components of a microdevice, whether in a microdevice shell or in the walls of an internal compartment, may be formed from the same polymer composition, different polymers, and / or blends of two or more polymer compositions. For example, in a microdevice having a shell / base and a cap, the base may be formed from one polymer or copolymer, while the cap may be formed from a different polymer or copolymer. For example, the cap may be of the same polymer composition as that used to form the base, but with chemically modified ends. In another example, the cap may be formed from a different polymer than that used to form the shell / base by including different monomers, different degrees of polymerization, different copolymers with different copolymer ratios, different blends, or a combination thereof.

[0069] In one embodiment, the polymer forming the microdevice shell / base is PLGA, and the PLGA polymer forming the cap has modified ends, such as ester end-points, to aid in sealing the shell / base at lower sealing temperatures. Maintaining a lower sealing temperature minimizes stress on drugs incorporated into the compartments. PLGA caps with ester end-points also have increased hydrophobicity, which delays the onset of release of therapeutic and / or prophylactic agents contained within the compartments.

[0070] B. Therapeutic and Prophylactic Agents The microdevice may encapsulate one or more agents (e.g., therapeutic and / or prophylactic agents) that bind to and inhibit or activate the receptor. Typically, the agents reside in or are incorporated into a compartment of the microdevice. The agents contained in the microdevice may be proteins or peptides, sugars or carbohydrates, nucleic acids or oligonucleotides, lipids, small molecules (e.g., molecular weights of less than 2000 daltons, preferably less than 1500 daltons, more preferably 300-700 daltons), or combinations thereof.

[0071] immunomodulators In some preferred embodiments, the microdevices contain one or more immune receptor binding agents, such as STING agonists, as immunomodulatory therapeutic and / or prophylactic agents.

[0072] In some embodiments, the agent is not a vaccine antigen, an adjuvant, or a combination thereof.

[0073] STING agonists Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs) that activate the TBK1 / IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways, inducing robust type I interferon and proinflammatory cytokine responses. STING is encoded by the TMEM173 gene. Through various molecular mechanisms, STING acts as both a direct cytosolic DNA sensor (CDS) and an adaptor protein in type I interferon signaling. STING has been shown to activate the downstream transcription factors STAT6 and IRF3 via TBK1, which are responsible for antiviral and innate immune responses against intracellular pathogens.

[0074] STING resides in the endoplasmic reticulum, but in the presence of cytosolic DNA, the sensor cGAS binds to DNA and produces a cyclic dinucleotide. This dinucleotide binds to STING and promotes its aggregation and translocation from the ER through the Golgi to perinuclear sites. There, STING forms a complex with TBK1 and promotes its phosphorylation. Once phosphorylated, TBK1 phosphorylates the transcription factor IRF3, which dimerizes and translocates to the nucleus, where it activates the transcription of type I IFN and other innate immune genes.

[0075] STING induces antitumor CD8 T responses in mouse models of cancer. In the tumor microenvironment, T cells, endothelial cells, and fibroblasts stimulated with STING agonists ex vivo produce type I IFN (Corrales, et al., Cell Rep (2015) 11(7):1018-30). In contrast, tumor cells can inhibit STING pathway activation, potentially leading to immune evasion during carcinogenesis (He, et al., Cancer Lett (2017) 402:203-12; Xia, et al., Cancer Res (2016) 76(22):6747-59). Evidence indicates that activation of the STING pathway correlates with the induction of spontaneous antitumor T cell responses, including the expression of type I IFN genes (Chen, et al., Nat Immunol (2016) 17(10):1142-9; Barber, et al., Nat Rev Immunol (2015) 15(12):760-70; Woo, et al., Immunity (2014) 41(5):830-42). Furthermore, the host STING pathway is required for efficient DC-mediated cross-priming of tumor Ag-specific CD8+ T cells (Woo, et al., Immunity (2014) 41(5):830-42; Deng, et al., Immunity (2014) 41(5):843-52). Based on these results, direct pharmacological stimulation of the STING pathway has been explored as a cancer therapy.

[0076] Any STING agonist known in the art can be used in the present compositions and methods.STING agonist can be nucleic acid, protein, peptide, polymer or small molecule.STING agonist can be natural or synthetic.In some embodiments, STING agonist is hydrophilic.

[0077] Suitable STING agonists include cyclic dinucleotide (CDN) agonists or acyclic dinucleotide agonists.Cyclic purine dinucleotides can be used, such as, but not limited to, cGMP, cyclic di-GMP (c-di-GMP), cAMP, cyclic di-AMP (c-di-AMP), cyclic GMP-AMP (cGAMP), cyclic di-IMP (c-di-IMP), cyclic AMP-IMP (cAIMP) and any analogs thereof.CDNs can have 2'3', 2'5', 3'3' or 3'5' linkages, or any combination thereof, connecting cyclic dinucleotides.For example, 2'3'-cGAMP or 3'3'-cGAMP can be used.Cyclic purine dinucleotides can be modified by standard organic chemistry techniques to generate purine dinucleotide analogs. Suitable purine dinucleotides include, but are not limited to, adenine, guanine, inosine, hypoxanthine, xanthine, isoguanine, or any other suitable purine dinucleotide known in the art. Cyclic dinucleotides may be modified analogs. Any suitable modification known in the art may be used, including, but not limited to, phosphorothioate, biphosphorothioate, fluorinate, and difluorinate modifications.

[0078] In some embodiments, the cyclic dinucleotide is a compound of the following formula: [ka] The dinucleotides may include modified cyclic dinucleotides such as:

[0079] In further embodiments, R1 and R2 may independently be 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9-isoguanine.

[0080] Suitable STING agonists include stereoisomers of cyclic purine dinucleotides (e.g., substantially pure Rp, Rp or Rp, Sp diastereomers thereof). c-diAMP, c-diGMP, c-diIMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP, as well as their analogs, including but not limited to, phosphorothioate analogs, referred to herein as "thiophosphates," can be used. Phosphorothioates are variants of normal nucleotides in which one of the non-bridging oxygens is replaced by sulfur. Sulfurization of the internucleotide bond dramatically reduces the action of endonucleases and exonucleases, including 5'-to-3' and 3'-to-5' DNA POL1 exonuclease, nucleases S1 and P1, RNase, serum nuclease, and snake venom phosphodiesterase. Furthermore, the potential for crossing lipid bilayers is enhanced.

[0081] Phosphorothioate linkages are inherently chiral. Those skilled in the art will recognize that each phosphate in this structure can exist in either the R or S configuration. Thus, Rp,Rp, Sp,Sp, and Rp,Sp forms are possible. In each case, substantially pure Rp,Rp, and Rp,Sp diastereomers of these molecules are preferred.

[0082] Suitable cyclic purine dinucleotides also include 2'-O-substituted forms of CDNs, particularly CDN thiophosphates. Additional stability and bioavailability may be provided by substitution of the 2'-OH of the ribose moiety. Suitable substituents herein include, but are not limited to, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)R aa ), carboxyl (-C(O)OR aa ), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-OR aa ), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(R bb )(R cc )), Imino (=NR bb), amide (-C(O)N(R bb )(Rc C ) or -N(R bb )C(O)R aa ), azide (-N3), nitro (-NO2), cyano (-CN), carbamide (-OC(O)N(R bb )(R cc ) or -N(R bb )C(O)OR aa ), ureido(-N(R bb )C(O)-N(Rbb)(Rcc)), thioureido (-N(R bb )C(S)N(R bb )(R cc )), guanidinyl (-N(R bb )C(=NR bb )N(R bb )(R cc )), amidinyl (-C(=NR bb )N(R bb )(Rc C ) or -N(R bb )C(=NR bb )(R aa )), thiol (-SR bb ), sulfinyl (-S(O)R bb ), sulfonyl (-S(O)R b ) and sulfonamidyl (-S(O)N(R bb )(Rc C ) or -N(R bb )S(O)2R bb ) in which R aa , R bb and R cC are each independently H, an optionally linked chemical functionality, or further substituents such as H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic, and heteroarylalkyl. Suitable cyclic purine dinucleotides also include S-substituted forms of CDN, particularly CDN thiophosphates, which can advantageously provide prodrugs with improved bioavailability.

[0083] Acyclic dinucleotide agonists such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA; also known as vadimezan or ASA404) or any other acyclic dinucleotide agonist known in the art can also be used.

[0084] Exemplary STING agonists include, but are not limited to, STING agonist-1, ML RR-S2 CDA, ML RR-S2 c-diGMP, ML-RR-S2 cGAMP, 2'3'-c-diAM(PS)2, 2'3'-cGAMPdFHS, 3'3'-cGAMPdFSH, cAIMP, cAIM(PS)2, 3'3'-cAIMP, 3'3'-cAIMPdFSH, 2'2'-cGAMP, 2'3'-cGAM(PS)2,2'3'-cGsAsMP (bisphosphothioate analog of 2'3'-cGAMP), 3'3'-cGAMP, c- DiAMP, 2'3'-c-diAMP, 2'3'-c-diAM(PS)2, c-diGMP, 2'3'-c-diGMP, c-diIMP, c-diUMP, MK-2118, GSK3745417, TAK-676, CRD5500, SB11325, SB11396, TTI-10001, MAVU-104 (ENPP1 inhibitor), dispirodiketopiperazine (DSDP) (Antiviral research. 2017 Nov 1;147:37-46), benzo[b][1,4]thiazine-6-carboxamide (an indirect STING agonist), α-mangostin (a human STING-preferential agonist), benzamide and its analogs (see ACS Infect Dis 2019;5;1139-49), bicyclic benzamide and benzothiophene derivatives. Suitable STING agonists also include those disclosed in US2016 / 0287623, WO2019 / 183578, WO2019 / 069270, WO2019 / 069275, US9,695,212, US9,724,408, US10,450,341, WO2019 / 079261, WO2018 / 234805, WO2018 / 234808, WO2018 / 067423, and Ramanjulu JM., et al., Nature, 564(7736):439-443 (2018), which discloses amidobenzimidazole (ABZI) compounds as STING agonists, each of which is incorporated by reference in its entirety.

[0085] In a preferred embodiment, the STING agonist is selected from the group including cGAMP, DMXAA, MK-1454, MK-2118, E7766, MIW815 (ADU-S100), BMS-986301, GSK3745417, IMSA-101, SYNB1891 (E. coli), SITX-799 (Silicon Therapeutics) and SB11285.

[0086] The STING agonist can be functionalized with, for example, an ether, ester, or amide linkage, if desired. For example, DMXAA can be modified into a DMXAA ester, DMXAA ether, or DMXAA amide.

[0087] C. Additional medications The compositions and methods optionally include one or more additional therapeutic agents, which can be administered in particulate or soluble form in the same or a different pharmaceutical composition, using the same or a different delivery system, and can be delivered to a subject in need thereof at the same time or at a different time than the first therapeutic agent, e.g., a microdevice loaded with a STING agonist.

[0088] Thus, in some embodiments, the same or different microdevices are used to deliver one or more additional agents, particularly one or more active agents that prevent or treat one or more symptoms of a disease (e.g., cancer). The additional active agents may be co-loaded onto the same microdevice or onto microdevices in the same or different formulations.

[0089] Suitable additional therapeutic and / or prophylactic agents can be biomolecules such as enzymes, proteins, polypeptides, antibodies or fragments thereof, or nucleic acids (e.g., functional RNAs such as siRNA or miRNA), or small molecule agents (e.g., molecular weights of less than 2000 daltons, preferably less than 1500 daltons, more preferably 300-700 daltons), including organic, inorganic, and organometallic agents. The agents can be incorporated into compartments of the microdevice.

[0090] 1. Typical additional medications The microdevice may also include one or more therapeutic and / or prophylactic agents that are immunomodulatory agents. Representative additional agents include, but are not limited to, chemotherapeutic agents, immunomodulatory agents, including Toll-like receptor 9 (TLR9) agonists that stimulate immune responses (e.g., CpG DNA), and combinations thereof. The additional agents may be provided in the microdevice together with the STING agonist or by themselves.

[0091] 2. Immunomodulators The terms "immunomodulatory agent" and "immunotherapeutic agent" refer to active agents that induce a specific effect on the recipient's immune system. Immunomodulation can include suppressing, reducing, enhancing, prolonging, or stimulating one or more physiological processes of the innate or adaptive immune response compared to a control. Typically, immunomodulatory agents can modulate the immune microenvironment for a desired immunological response (e.g., increasing anti-tumor activity or increasing inflammatory activity at a site in need thereof) by targeting one or more immune cells or cell types at a target site, and therefore are not necessarily specific for any cancer type. In some embodiments, immunomodulatory agents specifically kill, inhibit the activity, or reduce the activity or amount of suppressive immune cells, such as tumor-associated macrophages, to increase anti-tumor responses at tumor sites. In some embodiments, immunomodulatory agents specifically increase the activity or amount of cytotoxic immune cells, such as CD8+ T cells, to increase anti-tumor responses at tumor sites.

[0092] 3. Chemotherapeutic agents In some embodiments, the additional therapeutic agent is any inhibitor that targets one or more of EGFR, ERBB2, VEGFR, Kit, PDGFR, ABL, SRC and mTOR.In some embodiments, the additional therapeutic agent is a tyrosine kinase inhibitor, such as a HER2 inhibitor or an EGFR tyrosine kinase inhibitor.Exemplary EGFR tyrosine kinase inhibitors include gefitinib, erlotinib, afatinib, dacomitinib and osimertinib.

[0093] The majority of chemotherapeutic agents can be classified as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Therefore, in some embodiments, chemotherapeutic agents that may be included include, but are not limited to, alkylating agents, antimetabolites, antimitotic agents, anthracyclines, cytotoxic antibiotics, topoisomerase inhibitors, and combinations thereof. Monoclonal antibodies and tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC® or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors), can also be used. Other suitable anticancer agents include bevacizumab (AVASTIN®) and rhuFAb. These include angiogenesis inhibitors, including antibodies against vascular endothelial growth factor (VEGF), such as V2 (ranibizumab, LUCENTIS®), other anti-VEGF compounds; thalidomide (THALOMID®) and its derivatives, such as lenalidomide (REVLIMID®); endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors, such as sunitinib (SUTENT®); tyrosine kinase inhibitors, such as sorafenib (NEXAVAR®), erlotinib (TARCEVA®), pazopanib, axitinib, and lapatinib; transforming growth factor-α or transforming growth factor-β inhibitors, and antibodies against the epidermal growth factor receptor, such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

[0094] Representative chemotherapeutic agents that may be used include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, daunorubici), lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, taxol and their derivatives, trastuzumab (HERCEPTIN®), cetuximab and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0095] 4. Immune checkpoint regulators Strategies to combine STING immunotherapy with other immunomodulatory agents are being explored. In mouse models of melanoma and colon cancer, enhancing STING activation by intratumoral injection of the cyclic dinucleotide GMP-AMP (cGAMP) potently enhanced antitumor CD8 T cell responses and led to growth control of the injected tumor and the contralateral tumor. The ability of cGAMP to induce antitumor immunity was further enhanced when combined with anti-programmed death-1 (PD-1) and anti-cytotoxic T-lymphocyte-associated 4 (CTLA-4) antibodies (Demaria, et al., Proc Natl Acad Sci USA (2015) 112(50):15408-13). In other studies, cyclic dinucleotides (CDNs) combined with anti-programmed death-L1-blocking antibodies induced much stronger antitumor effects than monotherapy in mouse models of squamous cell carcinoma and melanoma (Gadkaree, et al., Head Neck (2017) 39(6):1086-94; Wang, et al., Proc Natl Acad Sci USA (2017) 114(7):1637-42). Luo et al. showed that combining a STING-activating nanovaccine with an anti-PD1 antibody resulted in long-term antitumor memory in the TC-1 tumor model (Luo, et al., Nat Nanotechnol (2017) 12(7):648-54). Thus, in some embodiments, a STING agonist is combined with another immunomodulator, e.g., one using the same or a different mechanism, to enhance the immune response, preferably against cancer.

[0096] In preferred embodiments, the additional agent is an inhibitor of a checkpoint protein, such as a component of the PD-1 / PD-L1 axis or the CD28-CTLA-4 axis (e.g., a PD-1 antagonist, a PD-1 ligand antagonist, and a CTLA4 antagonist). Exemplary immune checkpoint inhibitors include pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PDL1 mAb), nivolumab (anti-PD1 mAb), tremelimumab (anti-CTLA4 mAb), avelumab (anti-PDL1 mAb), ipilimumab (anti-CTLA4 mAb), and RG7876 (CD40 agonist mAb), and others described in more detail below.

[0097] In some embodiments, the active agent is a PD-1 antagonist. While T cell activation typically depends on antigen-specific signals following contact of the T cell receptor (TCR) with antigenic peptides presented via the major histocompatibility complex (MHC), the extent of this response is controlled by positive and negative antigen-independent signals generated by various costimulatory molecules. The latter are generally members of the CD28 / B7 family. Conversely, programmed death-1 (PD-1) is a member of the CD28 family of receptors that, when induced on the T cell surface, leads to a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the strength and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, and include compounds or agents that either bind to and block a ligand of PD-1 without inducing inhibitory signaling by the PD-1 receptor, thereby preventing or inhibiting binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor.

[0098] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without inducing inhibitory signaling, and also binds to a ligand of the PD-1 receptor, reducing or inhibiting the ligand from causing signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and cause the transmission of inhibitory signals, fewer cells are compromised by the negative signals provided by PD-1 signaling, and a more robust immune response may be achieved.

[0099] PD-1 signaling is thought to be driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in the vicinity of a peptide antigen presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent simultaneous ligation of PD-1 and TCR on the T cell membrane surface are also useful PD-1 antagonists.

[0100] In preferred embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or prevents PD-1 receptor signaling by binding to a ligand of PD-1 or to PD-1 itself, particularly where simultaneous ligation of PD-1 with a TCR does not follow such binding, thereby resulting in inhibitory signaling by the PD-1 receptor.

[0101] Other PD-1 antagonists contemplated by the method include antibodies that bind to PD-1 or a ligand of PD-1, and other antibodies.

[0102] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications: PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196) PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168) PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708) PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771) PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286) PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875) PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174) PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874) PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533) Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0103] A specific example of an anti-PD-1 antibody is MDX-1106, a human anti-PD-1 antibody (see Kosak, US20070166281 (published July 19, 2007), paragraph 42), preferably administered at a dose of 3 mg / kg. Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following publications: PCT / US06 / 022423 (WO / 2006 / 133396, published December 14, 2006) PCT / US07 / 088851 (WO / 2008 / 083174, published July 10, 2008) US2006 / 0110383 (released May 25, 2006) Includes those listed in.

[0104] A specific example of an anti-B7-H1 antibody is MDX-1105 (WO / 2007 / 005874, published Jan. 11, 2007), a human anti-B7-H1 antibody.

[0105] Regarding anti-B7-DC antibodies, see U.S. Patent Nos. 7,411,051, 7,052,694, 7,390,888 and U.S. Published Application No. 2006 / 0099203.

[0106] The antibody may be a bispecific antibody comprising an antibody that binds to the PD-1 receptor cross-linked to an antibody that binds a ligand of PD-1, such as B7-H1. In some embodiments, the PD-1 binding moiety reduces or inhibits signaling through the PD-1 receptor.

[0107] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides (including homologs and variants thereof), and active fragments of any of the above, and fusion proteins incorporating any of the foregoing. In a preferred embodiment, the fusion protein comprises a soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0108] The PD-1 antagonist can also be a fragment of mammalian B7-H1, preferably derived from a mouse or primate, preferably a human, where the fragment binds to and blocks PD-1 but does not result in inhibitory signaling by PD-1. The fragment can also be part of a fusion protein, e.g., an Ig fusion protein.

[0109] Other useful polypeptide PD-1 antagonists include those that bind to the ligand of the PD-1 receptor. These include the PD-1 receptor protein or a soluble fragment thereof, which can bind to a PD-1 ligand, such as B7-H1 or B7-DC, preventing binding to the endogenous PD-1 receptor and thereby preventing inhibitory signaling. B7-H1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of the PD-1 protein containing mutations, such as the A99L mutation, that enhance binding to the natural ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or a soluble fragment thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor and thus prevent inhibitory signaling, is also useful.

[0110] PD-1 and B7-H1 antisense nucleic acids (both DNA and RNA) and siRNA molecules can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on the surface of T cells and the production of T cell ligands such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (for example, about 21 nucleotides in length, specific to the gene encoding PD-1 or PD-1 ligand, and this oligonucleotide can be easily purchased commercially) complexed with a carrier such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009)) can be easily taken up by cells expressing PD-1 and PD-1 ligand, reducing the expression of these receptors and ligands, thereby reducing the inhibitory signaling in T cells and activating T cells.

[0111] In some embodiments, the molecule is an agent that binds to an immune response-mediating molecule other than PD-1. In preferred embodiments, the molecule is an antagonist of CTLA4, e.g., an antagonistic anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies contemplated for use in the provided methods include those described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0112] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and can range from 0.1 to 100 mg / kg, with narrower ranges of 1 to 50 mg / kg being preferred, and 10 to 20 mg / kg being more preferred. Suitable doses for human subjects are 5-15 mg / kg, with 10 mg / kg of antibody (e.g., a human anti-PD-1 antibody such as MDX-1106) being most preferred.

[0113] Specific examples of anti-CTLA4 antibodies are the human anti-CTLA4 antibody ipilimumab, also known as MDX-010 or MDX-101, preferably administered at a dose of about 10 mg / kg, and the human anti-CTLA4 antibody tremelimumab, preferably administered at a dose of about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0114] In other embodiments, the antagonist is a small molecule.A series of small molecule organic compounds have been shown to bind to B7-1 ligand and inhibit its binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)).Such small molecule organic compounds can be administered alone or together with anti-CTLA4 antibody to reduce the inhibitory signal transduction of T cells.

[0115] III. Pharmaceutical Preparations The pharmaceutical composition comprises a microdevice containing one or more pharmaceutical agents and a pharmaceutically acceptable buffer, carrier, diluent or excipient. The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary agents, that facilitate the processing of the active agent and the microdevice into a pharmaceutically usable preparation.

[0116] Pharmaceutically acceptable excipients include compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, in accordance with guidelines from agencies such as the Food and Drug Administration, or that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable excipients include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof. These include suspending agents such as sterile water, phosphate buffered saline, saline, or non-aqueous solutions such as glycerol.

[0117] Appropriate formulation depends on the selected route of administration. In a preferred embodiment, the composition is formulated for local administration. In some embodiments, the composition is formulated for intratumoral, intramuscular or subcutaneous injection. Typically, the composition is formulated in sterile saline or buffer solution or methylcellulose solution for injection into the tissue to be treated. The composition can be lyophilized and stored in a single-use vial for rehydration immediately before use. Other suitable means for rehydration and administration known to those skilled in the art can be used.

[0118] Other representative excipients include solvents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) and may be administered to an individual without causing undesired biological side effects or undesired interactions.

[0119] Generally, pharmaceutically acceptable salts can be prepared by reacting the active agent in the form of a free acid or free base with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two (generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred). Pharmaceutically acceptable salts include salts of active agents derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by the reaction of a drug with a suitable organic ligand (e.g., quaternary ammonium salt). A list of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704.

[0120] Stabilizers / Excipients The agent incorporated into and / or delivered from the microdevice may be combined with one or more stabilizing excipients. Alternatively, the stabilizing excipient may be included in the polymer shell instead of in the compartment of the microdevice. In other embodiments, the stabilizing excipient is present in both the compartment of the microdevice and the polymer.

[0121] Stabilizing excipients can increase the structural stability of thermolabile and / or pH-sensitive drugs. Exemplary stabilizing agents include sugars, sugar alcohols, amino acids, vitamins, antioxidants, salts, buffering agents, polysaccharides, oils, and combinations thereof. Drugs that can benefit from stabilization include proteins, peptides, and nucleic acids.

[0122] Sugars are a typical group of stabilizers for proteins. Examples include simple sugars such as sorbitol, sucrose, fructose, mannitol, glucose, maltose, dextrose, and trehalose, as well as more complex sugars. See Alcock et al., Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass. Science Translational Medicine, 2(19):19-19ral2 (2010). Exemplary salts useful as stabilizing excipients, or stabilizing excipients, include magnesium chloride, calcium chloride, monosodium glutamate, potassium phosphate, and combinations thereof.

[0123] Exemplary buffering agents include MgCO3, CaCO3, Mg(OH)2, Al(OH)3, myristic acid, polymers such as poly-L-lysine, and combinations thereof. When incorporated into the microdevice, the buffering agent minimizes changes in the pH of the release medium. Al(OH)3 is a known adjuvant and can increase immunogenicity. Other agents suitable as stabilizer excipients include maltodextrin, methylcellulose, (hydroxypropyl)methylcellulose (HPMC), calcium heptagluconate, carboxymethylcellulose (CMC), silk, glycerol, alginate, ectoine, ubiquitin, gelatin, threonine, peptone, glycine, glutamine, serum albumin, and combinations thereof.

[0124] The stabilizing excipients may be used in any combination and in any amount effective to stabilize the drug against temperature, storage, humidity, pH, and oxidative damage. For example, the stabilizers sucrose, monosodium glutamate, and magnesium chloride may be used in an effective amount to stabilize the drug. The buffering agent aluminum hydroxide may be included with the stabilizer to control changes in environmental pH as the polymer decomposes or as the microdevice passes through the digestive tract.

[0125] The stability of the incorporated drug can be assessed during each step of the encapsulation and / or manufacturing process, during storage (25°C, room temperature, high humidity / high temperature conditions), under physiological conditions (pH 7.2, 37°C), and in vivo (animal models).

[0126] In some embodiments, the composition is administered locally, for example, by direct injection at the site to be treated. Typically, local administration results in a higher local concentration of the composition than can be achieved by systemic administration. In some embodiments, the composition is directly injected into a tumor (intratumoral injection). In some embodiments, the composition is injected or otherwise administered into vascular tissue at or adjacent to the site (e.g., tumor, surgical site).

[0127] A. Combinatorial Microdevice Formulation As introduced above, microdevices of the same or different polymer compositions and / or with the same or different drugs may be combined in one formulation. In some embodiments, microdevices encapsulating the same or different drugs are combined in a single formulation. In some embodiments, microdevices with different polymer compositions may be combined in a single formulation.

[0128] The release rate of a drug (e.g., a STING agonist) incorporated into a microdevice can be controlled by molecular weight (e.g., the number-average molecular weight of the polymer or copolymer, the weight-average molecular weight of the polymer or copolymer), the polydispersity index of the polymer or copolymer, the chain-end functionality of the polymer or copolymer, the ratio of the copolymers, or a combination thereof. In some embodiments, the release rate can be controlled by the shell composition, including 1) blending various ratios of PLA, PGA, or PLGA, and 2) blending various ratios of hydrophilic polymer, hydrophobic polymer, and salt. The release rate can also be controlled by the wall thickness of a surface-eroding polymer, such as a polyanhydride or polyorthoester. Thus, a formulation can contain a population of microdevices that are homogeneous with respect to their polymer composition (and thus properties) and / or incorporated drug. In some embodiments, a formulation includes two or more (e.g., 2, 3, 4, 5, or more) populations of microdevices that are heterogeneous with respect to their polymer composition (and thus properties) and / or incorporated drug. In some embodiments, the formulation contains two or more (eg, two, three, four, five, or more) different agents.

[0129] Formulations containing microdevices of the same polymer composition but encapsulating different drugs may be formulated to simultaneously deliver two or more different drugs as the polymer degrades. The formulations may be useful for combination therapy to co-deliver drugs with only a single administration.

[0130] The formulation containing microdevices with different polymer compositions but encapsulating the same drug can be formulated to provide two or more pulsed releases at two or more time points after a single administration.As shown in the examples, such formulations are useful for cancer therapy.Because the timing of the pulsed release of the incorporated drug can be adjusted, a single administration of such formulation can mimic the repeated administration of drugs or other drugs.

[0131] Formulations containing microdevices with different polymer compositions and encapsulating different agents can be formulated to provide two or more pulsed releases at two or more time points when the polymers of different compositions degrade to release different (e.g., two, three, four, five, or more) agents. Based on the composition of the microdevice, the formulation can release two or more agents in each pulsed release, or after a single administration, release only one type of agent in one release and another type of agent in a subsequent release.

[0132] These formulations may be useful in cancer therapy, vaccination, or therapy for autoimmune diseases.

[0133] IV. Methods for Fabricating Microdevices The method used to fabricate the microdevice should maintain drug stability both during processing and at body temperature, minimizing leakage after formation and administration. Sterilization after formulation can typically be achieved by a combination of sterile manufacturing conditions combined with methods such as gamma irradiation. Microdevices can be fabricated using any suitable technique known in the art, including, but not limited to, micromolding.

[0134] Typically, a method for making a microdevice includes micromolding a polymer base having compartments therein, inserting agents into the compartments, placing a polymer cap on the polymer base, and sealing the polymer cap to the polymer base. Micromolding the polymer base can include layer-by-layer sintering under microscopic alignment. Suitable methods for manufacturing microdevices are described in more detail below.

[0135] Microdevice compartments may be empty or may contain one or more drugs (e.g., solid or liquid drugs). Drugs are typically loaded into the compartments during the process of microdevice formation. Drugs may be injected into the channels during or after microdevice formation, for example, after shell / base formation. The volume of the void space of a compartment varies depending on the size of the compartment, the size of the microdevice, or both. Typically, the void space allows for drug loadings in the picogram (pg) to milligram (mg). Exemplary loadings include 10 pg to 1 mg, e.g., about 100 pg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 10 μg, 100 μg, and 1 mg. Suitable ranges include about 100 pg to 10 μg, 1 μg to 5 μg, 5 μg to 20 μg, 15 μg to 50 μg, and 50 μg to 150 μg.

[0136] Typically, microdevices containing compartments are loaded with drugs in a range of weight-to-weight (%w / w) concentrations from 1% to 90% w / w, 1% to 85% w / w, 1% to 80% w / w, 1% to 75% w / w, 1% to 70% w / w, 1% to 65% w / w, 1% to 60% w / w, and 1% to 5% w / w. For example, individual microdevices can contain approximately 2%, 4%, 8%, 5%, 13%, 19%, 20%, or 22% loading capacity for drug loading.

[0137] In some embodiments, the loading capacity of each microdevice is expressed as a percentage of the volume of the microdevice. The microdevice can have a loading capacity of 1% to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, or 1% to 5% for loading a drug. For example, the loading capacity of each microdevice can be about 8% to 10%, e.g., about 8.4%.

[0138] Exemplary loading capacities are approximately 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg or 10 μg of drug loading in each microdevice.

[0139] The solvent should be biocompatible, as some residues may be present in the polymer formulation. Acceptable solvent residues should meet Food and Drug Administration (FDA) guidelines. Typical polymer solvents include organic solvents such as chloroform, dichloromethane, tetrafluoroethylene, and acyl acetate. Drugs can be dissolved in aqueous or aqueous-miscible solvents such as acetone, ethanol, methanol, isopropyl alcohol, and mixtures thereof.

[0140] Micromolding Park et al., Biomed. Microdevices, 9:223-234 (2007) describe the use of micromolding to fabricate polymer microstructures with sophisticated designs. Micromolds were filled with polymer microdevices to produce multi-material microstructures with complex geometries and fabricated using mild processing conditions. These microdevices are typically prepared using an oil-water double emulsion system, spray drying, supercritical conditioning, and milling. In a preferred embodiment, the micromolds can be prepared by photolithographically fabricating a female master mold made from photoresist, molding a male master structure from polydimethylsiloxane (PDMS) from the female master mold, and molding a female replica mold from PDMS from the male master structure. Polymer microdevices can be micromolded using temperature / compression methods and / or from solvents.

[0141] Polymer microdevices ranging in size from 1 to 30 μm can be fabricated from PLA, PGA, and PLGA using spray-drying and emulsion techniques. These polymer microdevices are filled into PDMS micromolds at room temperature and then fused or bonded together, for example, by ultrasonically welding the microdevices together within the mold while maintaining the voids inherent in their packing structure. Using mild processing conditions, molds can be filled with solid polymer microdevices instead of molten polymer to replicate microstructures with complex geometries and composed of multiple materials. Microdevices can easily flow into the micromold cavities at room temperature and low pressure, facilitating the fabrication of microstructures with high aspect ratios. Furthermore, polymer microdevices can incorporate chemical compounds such as drugs, and molds can be filled in successive layers to accommodate compositions of multiple materials. After the mold is filled, the microdevices within the mold can be welded to form the final microstructure using plastic welding methods, including thermal and ultrasonic welding, as well as solvent- and gas-based welding.

[0142] These same techniques can be used to prepare microdevice compositions having the polymeric materials and conditions necessary to exhibit narrow time-series release of incorporated drugs at specific times after administration.

[0143] Stamp assembly of polymer layers (SEAL) Microdevices can be produced using stamp assembly of polymer layers (SEAL). See McHugh KJ., et al., Science, 357(6356):1138-1142 (2017). The SEAL method produces an array of compartment-shell polymer devices. First, a prefabricated silicone mold is used to melt-press a polymer of choice, such as PLGA. The mold is then transferred to another substrate, where it is peeled off, leaving behind an array of polymer bases. These are then filled with any drugs or other agents using an inkjet piezoelectric nozzle and then dried. A cap is then aligned with the base device and sealed. The resulting array of compartment-shell microdevices is then removed from the base and stored until use.

[0144] 1. Mold In some embodiments, the molds are formed as follows: Two or more silicon molds with complementary patterns are etched using standard microfabrication techniques. Polydimethylsiloxane (PDMS) is then cured on the surface of each silicon wafer to create an inverted elastomeric mold. The polymer is then heated and pressed into the PDMS mold to create the desired layered microstructure component.

[0145] The initial layers are then peeled off from their respective surfaces, such as glass, using thermally assisted microtransfer molding. Subsequent layers of the final structure are then assembled using a layer-by-layer sintering process under microscopic alignment to generate large arrays of microstructures. This process leverages elements from existing technologies, including laminate fabrication, microfabrication-based surface patterning, and thermal bonding of PLGA, to create polymeric microdevices with well-defined geometries.

[0146] 2. Layer-by-layer alignment and sintering To ensure high-fidelity microdevice fabrication, a technique is used to align the layers with high precision during sintering. In some embodiments, this approach allows for simultaneous alignment and thermal bonding using a photomask aligner (MA4, Karl Suss, Sunnyvale, CA) retrofitted with a Peltier heater, temperature controller, relay, and voltage source. The mask holder vacuum is applied to hold the glass slide containing the first microstructure layer facing downward, while the next layer, still in the PDMS mold, is held on the wafer chuck. After optically aligning adjacent features using the mask aligner's microscope and alignment knobs, the layers are brought into contact and heated to just above the glass transition temperature of the polymer for up to 3 minutes. During this time, the sealing process is continuously monitored by observing the disappearance of the optical diffraction pattern.

[0147] When the two layers come into contact, a small air gap between them generates diffraction that is eliminated when the heated polymer flows and closes the gap. After the sample cools to room temperature, the PDMS micromold containing the second layer is peeled off, yielding a multilayered microstructure. Individual microdevices are then removed from the glass slide.

[0148] 3. Filling and capping After filling the shell / base of the micromolded microdevice, it is sealed using a BioJet Ultra inkjet piezoelectric nozzle, which can rapidly dispense picoliter volumes of drugs or other agents into the microdevice compartments. To seal the filled device, a cap mold is aligned, sealed with the shell / base, and peeled away. The resulting array of compartment-shell devices is then removed from the base and stored until use.

[0149] 4. Scrum Elimination In some cases, the polymer used to fill the micromold forms a "scrum" on top, which should be removed before capping.

[0150] V. How to use Methods of using the microdevices and compositions or formulations thereof are also described. In some embodiments, the microdevices containing one or more agents are used to treat cancer. In other embodiments, the microdevices containing one or more agents are used to treat immune diseases. In other embodiments, the microdevices containing one or more agents are used to treat diseases or disorders in which modulation of the STING pathway is beneficial. The method typically includes administering to a subject in need thereof an effective amount of a composition containing a microdevice and one or more active agents to modulate an immune and / or inflammatory response, for example, to reduce immunosuppression within the tumor microenvironment and / or to enhance an anti-tumor response. The composition is preferably administered locally, including, but not limited to, by subcutaneous, intratumoral, and intramuscular injection. In some embodiments, upon release from the microdevice, the incorporated agent can be distributed systemically.

[0151] In some embodiments, the method comprises locally administering to a subject in need thereof an effective amount of a composition comprising a microdevice and one or more active agents to induce a local or systemic immune and / or inflammatory response in the subject, induce or enhance STING pathway activity in the subject, induce or enhance type I IFN secretion and / or an interferon response in the subject, induce infiltration of the tumor microenvironment (e.g., by lymphocytes, basophils, macrophages and / or dendritic cells), and / or reduce immunosuppression within the tumor microenvironment.

[0152] In some embodiments, the subject to be treated is a human. In some embodiments, the composition is administered by injection using a standard needle and completely decomposes over time (and therefore does not require removal). These properties are believed to be advantageous for patient compliance.

[0153] All of the methods described can include a step of identifying and selecting a subject in need of treatment or who would benefit from administration with the composition.

[0154] A. Treatment Method 1. Cancer treatment In a preferred embodiment, the composition is used in a method for treating cancer. Preferably, the composition is administered locally (e.g., via injection directly into a target site such as a tumor). Such a method includes administering to a subject (e.g., via intratumoral injection) an effective amount of the composition to treat and / or alleviate one or more symptoms associated with cancer. Typically, the method induces immunogenicity and / or induces or enhances an anti-tumor immune response.

[0155] In some embodiments of the method of treating cancer in a subject, the method comprises administering to the subject a therapeutically effective amount of a composition, wherein the composition upregulates a STING-mediated immune response in the subject, thereby increasing tumor targeting of the subject's immune system. The composition is administered intratumorally to the subject.

[0156] Similarly, a method for preventing metastasis of cancer in a subject is provided herein.The method comprises administering a therapeutically effective amount of a composition to a subject to prevent one or more tumors at one location in the subject from promoting the growth of one or more tumors at another location in the subject.In some embodiments, the composition is administered intratumorally to a first tumor at one location to prevent metastasis of one or more tumors at a second location.

[0157] Administration of the composition can reduce cancer cell proliferation or survival, increase apoptosis within the tumor, and / or reduce tumor burden in the subject. In some embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., tumor growth in a corresponding subject after administration of a free STING agonist, or tumor growth in an untreated subject).

[0158] In some embodiments, the subject being treated has been diagnosed with stage I, stage II, stage III, or stage IV cancer.

[0159] Cancer being treated Cancer is a disease of genetic instability that allows cancer cells to acquire hallmark features proposed by Hanahan and Weinberg, including (i) self-sufficiency in growth signals, (ii) insensitivity to anti-growth signals, (iii) evasion of apoptosis, (iv) persistent angiogenesis, (v) tissue invasion and metastasis, (vi) unlimited replicative potential, (vii) reprogramming of energy metabolism, and (viii) evasion of immune destruction (Cell., 144:646-674, (2011)).

[0160] Tumors that can be treated by the method are classified according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the epithelial lining and glands of the skin or internal organs. Sarcomas, which occur less frequently, originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can appear in multiple organs or tissues of the body to establish cancer.

[0161] The method can be used to treat solid tumors. The term "solid tumor" refers to an abnormal mass of tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form the tumor. Examples of solid tumors include, but are not limited to, sarcoma, carcinoma, and lymphoma. Examples of solid tumor cancers include, but are not limited to, cancer of the colon, breast, stomach, ovary, lung, cervix, melanoma, kidney, prostate, lymphoma, neuroblastoma, pancreas, and bladder. Exemplary cancers that may be treated include, but are not limited to, cancer of the lung, bone, pancreas, skin, head, neck, uterus, ovary, stomach, colon, breast, esophagus, small intestine, bowel, endocrine system, thyroid, parathyroid, adrenal gland, urethra, prostate, penis, testicle, ureter, bladder, kidney, or liver; rectal cancer; cancer of the anal region; fallopian tube, endometrial, cervical, vaginal, vulva, renal pelvis, renal cell cancer; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma ; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemangioma; hepatocytoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary CNS lymphoma; CNS neoplasms; spinal axis tumors; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brain stem glioma; pituitary adenoma; bronchial adenoma; chondroitin hamartoma; mesothelioma; Hodgkin's disease, or a combination of one or more of the above cancers.

[0162] In some preferred embodiments, the cancer / tumor to be treated is a cancer / tumor that is difficult to reach or that is not well suited to multiple invasive procedures or injections (e.g., pancreatic cancer, brain cancer such as glioma or glioblastoma multiforme). In preferred embodiments, the cancer is melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, testicular cancer, urothelial carcinoma, bladder cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colon cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell neoplasm, Wilms' tumor, or hepatocellular carcinoma.

[0163] More specific examples of cancer include, but are not limited to, leukemias, such as, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroid leukemia, and myelodysplastic syndromes; chronic leukemias, such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease; smoldering multiple myeloma, non-secretory myeloma, osteomalacia, plasma cell myeloma, and the like. leukemia, multiple myeloma including solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; including but not limited to: osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, glioma, glioma, glioma, sarcoma ... Sarcomas of bone and connective tissue such as membrane sarcomas; brain tumors, including but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary cerebral lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal gland cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; papillary or follicular thyroid carcinoma, thyroid cancer, such as medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, VIPoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors; pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer, including but not limited to intraocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma;vulvar cancer, including but not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancer, including but not limited to squamous carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasma cell neoplasm, verrucous carcinoma, and oat cell (small cell) carcinoma; adenocarcinoma, including but not limited to adenocarcinoma, fungus (polypoid), ulcerative, superficial spreading, widespread spreading, intracranial malignant lymphoma, fatty liver liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, including but not limited to adenocarcinoma; bile duct cancer, including but not limited to papillary, nodular, and diffuse; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancer, including but not limited to germinal tumor, seminoma, undifferentiated, classic (typical), spermatocyte, non-seminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor); prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer oral cancer, including but not limited to squamous cell carcinoma; basal cell carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and warts; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentigo melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adenocarcinoma, adrenal nephroma, fibrosarcoma, transitional cell carcinoma (of the renal pelvis and / or ureter); Wilms' tumor;Bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma, is discussed. For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America.

[0164] 2. Other Uses Because STING agonists regulate innate immune responses, they can be used as a therapeutic strategy for the treatment or prevention of other diseases, such as viral infections. For example, STING agonists have been shown to stimulate the immune response of influenza vaccines (Wang J., et al., Science, 367(6480). pii: eaau0810. (2020)). Wang et al. reported that the use of cGAMP as an adjuvant enhanced influenza vaccine-induced humoral immune responses and CD8 expression in mice by simulating the early stages of viral infection without concurrent excessive inflammation. + It has been shown that STING agonists significantly enhance T cell immune responses, and therefore compositions incorporating one or more STING agonists can be used as vaccine adjuvants.

[0165] A method for treating an infectious disease (e.g., influenza) in a subject in need thereof comprises administering to the subject an effective amount of any of the compositions. In some embodiments, administration of the compositions can upregulate a STING-mediated immune response (e.g., type I interferon) in the subject. Methods have been developed for vaccinating a subject against an infectious agent by administering to the subject an effective amount of any of the compositions, and for improving or enhancing a humoral and / or cellular immune response to an antigen in a subject in need thereof by administering to the subject an effective amount of any of the compositions. Vaccine-induced humoral and / or cellular (e.g., CD8 + Methods for improving or enhancing an immune response (e.g., T cell) include administering to a subject an effective amount of any of the compositions (e.g., a pharmaceutical composition comprising a microdevice encapsulating one or more STING agonists). Preferably, the vaccine is an influenza (i.e., flu) vaccine.

[0166] B. Effective Dose The dosage and administration regimen depend on the severity and location of the disorder or injury and / or the method of administration and are known to those skilled in the art. An effective amount or therapeutically effective amount can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or otherwise provide a desired pharmacological and / or physiological effect, for example, to reduce, inhibit, or reverse one or more of the underlying pathophysiological mechanisms underlying a disease or disorder, such as cancer. For example, a therapeutically effective amount of a microdevice composition used to treat cancer is typically sufficient to reduce or alleviate one or more symptoms of cancer in a subject. In some embodiments, the subject is a mammal, most preferably a human.

[0167] Symptoms of cancer can be physical, such as tumor burden, or biological, such as cancer cell proliferation. Thus, the amount of the composition can be effective, for example, to kill tumor cells or inhibit tumor cell proliferation or metastasis. Preferably, a composition comprising a microdevice encapsulating one or more active agents, such as an immunomodulatory agent (e.g., a STING agonist), is preferentially delivered locally to tumor tissue and its surrounding cells, such as cancerous cells or immune cells associated with tumor tissue (e.g., tumor microenvironment), for example, by intratumoral injection. In some embodiments, the agent does not target or otherwise regulate the activity or amount of healthy cells not present in or associated with tumor tissue, or targets or otherwise regulates them at a lower level than cancer or cancer-associated cells. In this way, by-products and other side effects associated with the composition are reduced, and preferably, directly or indirectly, result in the death of cancer cells. In some embodiments, the therapeutic and / or diagnostic agent directly or indirectly reduces cancer cell migration, angiogenesis, immune evasion, radiation resistance, or a combination thereof. In some embodiments, the agent directly or indirectly induces changes in the cancer cells themselves or their microenvironment that reduce immunosuppression or induce activation of an immune response against the cancer cells. For example, in some embodiments, the composition is administered in an amount effective to enhance and / or prolong the activation, proliferation and / or function of T cells (i.e., increase tumor-specific proliferation of T cells, enhance cytokine production by T cells, stimulate differentiation, stimulate T cell effector function, and / or promote T cell survival).

[0168] In some in vivo methods, the composition is administered to a subject in a therapeutically effective amount to reduce tumor size. In some embodiments, an effective amount of the composition is used to remit cancer and / or maintain cancer in remission. Similarly, an effective amount of the composition is provided to reduce or stop the proliferation of cancer stem cells.

[0169] The amount of the administered composition can be expressed as an amount effective to achieve a desired effect in the recipient. For example, in a preferred embodiment, the microdevice containing one or more STING agonists is administered in an amount effective to induce / enhance the expression of interferon-stimulated genes, inhibit or reduce cancer cell proliferation and / or survival, inhibit tumor growth, reduce tumor size, reduce tumor burden, induce / enhance the infiltration of tumors or tumor microenvironments by lymphocytes (e.g., CD8+ and / or CD4+ T cells), natural killer cells, dendritic cells, basophils and / or macrophages, improve response to immune checkpoint blockade, induce immunological memory that protects against tumor re-challenge, and / or improve survival rates of cancer patients.

[0170] The actual effective amount of the composition may vary depending on factors including the specific active agent administered, the specific composition formulated, the mode of administration, and the age, weight, condition, and route of administration of the subject being treated, as well as the disease or disorder (e.g., and the type, stage, and location of the cancer / tumor being treated). Thus, it is not possible to specify an exact amount for every therapeutic composition. However, an appropriate amount can be determined by those skilled in the art using only routine experimentation, given the teachings herein. For example, effective dosages and schedules for administering therapeutic agents may be determined empirically, and making such determinations is within the skill of the art. The dosage range for administering the composition is large enough to affect the desired response. For example, the dosage range for administering the composition is large enough to, for example, cause a decrease in cell proliferation or viability in target cancer cells, or reduce tumor burden.

[0171] The dosage should not be so large as to cause adverse side effects such as undesired cross-reactions, anaphylactic reactions, etc. The dosage can be adjusted by an individual physician in the event of any adverse indications. It is also understood that the effective dosage of the composition used for treatment can be increased or decreased over the course of a particular treatment. The change in dosage can be obtained and made clear from the results of diagnostic assays.

[0172] In certain embodiments, a subject is administered a composition having one or more therapeutic and / or diagnostic agents (e.g., a STING agonist) at about micrograms, milligrams, μg / kg, micrograms / kg / min, mg / kg / min, micrograms / kg / hr, or mg / kg / hr, or any range derivable therein. Exemplary dosages of STING agonists include 1 μg to 3 mg, such as about 1 μg to 10 μg, 10 μg to 50 μg, 50 μg to 100 μg, 100 μg to 500 μg, 500 μg to 1 mg, 1 mg to 2 mg, and 1 mg to 3 mg. Preferred ranges include about 100 μg to 500 μg, 500 μg to 1 mg.

[0173] Preferred administration and release schedules using STING agonist compositions and methods for treating cancer are discussed in more detail below and illustrated in the Examples. However, in general, the timing and frequency of administration can be adjusted to balance the efficacy of a given treatment. Preferably, the method involves a single administration. However, in some embodiments, two or more administrations can be used, depending, for example, on the length of time for which treatment is desired. Exemplary administration frequencies include single and multiple administrations, such as hourly, daily, weekly, monthly, or yearly administration, or every other day, two days, three days, four days, five days, or six days. In some embodiments, the dosage is administered about once or twice every week, every two weeks, three weeks, or four weeks. In some embodiments, the dosage is administered about once or twice every month, every two months, three months, four months, five months, or six months.

[0174] It is understood by those skilled in the art that the dosing regimen can be any length of time sufficient to treat the disease or disorder in a subject.In some embodiments, the regimen comprises one or more cycles of treatment, followed by a rest period (e.g., drug-free).The rest period can be 1, 2, 3, 4, 5, 6 or 7 days; or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.

[0175] The microdevices allow for controlled release of incorporated drugs. In particular, injectable formulations of polymeric microdevices release encapsulated drugs two or more times, within a short period of time, and without leakage of the drug from the microdevice between releases.

[0176] Controlled release can be achieved by incorporating microdevices of different polymer compositions (i.e., two or more different populations of microdevices) into a single formulation. Controlled release can also be achieved through the geometric design of the microdevice. For example, in compartmentalized microdevices, the drug is encapsulated within the compartment, where it is stable and protected from the external environment. The drug is also prevented from leaking out of the microdevice until the microdevice shell is degraded. Once the shell is degraded, release is rapid (e.g., for PLGA microdevices, complete drug release can be achieved within hours to days in vitro or in vivo). Rapid release can be characterized as the release of substantially all of the encapsulated drug within a short period of time, such as within one hour, several hours, one day, or one week. Unlike solvent evaporation microencapsulation, in which the drug is incorporated into microspheres with a polymer, the formulation provides a decoupling of device loading and release kinetics.

[0177] The microdevices also exhibit no measurable leakage of drug between bursts of release. No measurable leakage can be characterized as less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 0.5% of the total amount of drug released prior to the burst release. No measurable leakage can be characterized as an undetectable amount of drug released prior to the burst release.

[0178] The formulations exhibit a rapid release followed by a material-dependent delay. The delay in drug release depends on the shell thickness of the compartment-shell device, the shell polymer composition, the geometry of the compartment-shell device, and other factors.

[0179] By combining microdevices with different polymer compositions, compartment-shell geometries, or microdevice sizes, the pulsed release of drugs can be adjusted to a desired controlled release regimen.This dosing regimen advantageously mimics and simplifies the administration schedule that typically requires repeated drug administration.The typical dosing schedule for STING agonists in clinical trials is: (a) intratumoral administration on days 1, 8, and 15 of each 21-day cycle during cycles 1, 2, and 3, then on day 1 of each 21-day cycle during cycle 4 and up to 35 additional cycles (up to a maximum of approximately 2 years) (see ClinicalTrials.gov Identifier: NCT03010176); (b) intratumoral administration on day 1 of each 21-day cycle for a maximum of 35 cycles, then on days 1 and 8 of each 21-day cycle for two cycles, then on day 1 of each 21-day cycle for a maximum of 35 cycles, then on days 1, 8, and 15 of each 21-day cycle for two cycles, then on day 1 of each 21-day cycle for a maximum of 35 cycles (see ClinicalTrials.gov Identifier: NCT03010176); (c) intratumoral administration on days 1, 8, and 15 of each 28-day cycle at a starting dose of 50 micrograms (see ClinicalTrials.gov identifier: NCT02675439); (d) intratumoral administration on days 1 and 8 of each 21-day cycle at a starting dose of 200 micrograms (see ClinicalTrials.gov identifier: NCT02675439); (e) intramuscular or intratumoral administration on days 1 and 22 of cycle 1 (see ClinicalTrials.gov identifier: NCT03956680); (f) IV infusion (0.3-14 μg / Kg SB11285; see ClinicalTrials.gov Identifier: NCT04096638) on days 1, 8, 15, and 22 of repeated 28-day cycles in escalating doses; and (g) IV infusion (0.3-3.0 SB11285 μg / Kg; see ClinicalTrials.gov Identifier: NCT04096638) on days 1, 8, 15, and 22 of repeated 28-day cycles in escalating doses.

[0180] Thus, in some embodiments of the method, the microdevice formulation is administered locally (e.g., intratumorally) to achieve a pulsed release of the agent (e.g., a STING agonist), where the release of the agent mimics any of the administration regimens described above, but requires fewer, preferably a single local administration of the microdevice composition compared to administration of a soluble drug.

[0181] C. Combination Therapies and Procedures The compositions can be administered alone or in combination with one or more conventional therapies, for example, conventional cancer therapies. In some embodiments, the conventional therapy includes administering one or more compositions in combination with one or more additional active agents. Combination therapy can include administering active agents together in the same mixture or in separate mixtures. Thus, in some embodiments, the pharmaceutical composition includes two, three, or more active agents. The additional active agents can have the same or different mechanisms of action. In some embodiments, the combination provides an additive effect in the treatment of cancer. In some embodiments, the combination provides a more than additive effect in the treatment of a disease or disorder.

[0182] The additional therapy or procedure may be administered simultaneously or sequentially with the administration of the microdevice composition, hi some embodiments, the additional therapy is administered between drug cycles or during drug holidays that are part of the administration regimen of the composition.

[0183] Combination therapy can be achieved by using a single pharmaceutical composition containing the therapeutic agents, or by administering two or more separate compositions at the same or different times. Multiple therapies may be given in any order and may precede or follow other treatments by intervals ranging from minutes to weeks. In embodiments in which other agents are applied separately, it is preferable to administer the therapies within a time frame that still allows the agents to exert their beneficial combined effect on the patient. In such cases, it is contemplated that both modalities may be administered within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. However, in some situations, it may be desirable to extend the duration of treatment significantly, in which cases from several days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) lapse between each administration.

[0184] In some embodiments, the additional therapy or procedure is surgery, radiation therapy, chemotherapy, immunotherapy, cryotherapy, or gene therapy.

[0185] Immunotherapy includes, but is not limited to, the administration of one or more STING agonists, one or more immune checkpoint blockers, or a combination thereof. Exemplary immune checkpoint blockers include, but are not limited to, antibodies or antigen-binding fragments thereof, such as antibodies or antigen-binding fragments thereof that are inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, LAG3, or combinations thereof, such as pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PDL1 mAb), nivolumab (anti-PD1 mAb), tremelimumab (anti-CTLA4 mAb), avelumab (anti-PDL1 mAb), ipilimumab (anti-CTLA4 mAb), and RG7876 (CD40 agonist mAb), and any of those specifically introduced above or otherwise mentioned herein.

[0186] In some embodiments, the compositions and methods are used prior to or in conjunction with immunotherapy, such as adoptive T cell therapy and / or cancer vaccines.

[0187] Suitable additional therapeutic agents for use in combination therapy include conventional cancer treatments such as chemotherapeutic agents, cytokines and chemokines, including, but not limited to, any of those specifically introduced above or otherwise mentioned herein.

[0188] In some embodiments, the compositions and methods are used prior to or in conjunction with surgical resection of a tumor, e.g., to prevent primary tumor metastasis, hi some embodiments, the compositions and methods are used to enhance the body's own anti-tumor immune function.

[0189] VI. Kit Microdevices and formulations and other materials can be packaged together in any suitable combination as a kit useful for carrying out or supporting the performance of a method.The components of a given kit are useful when they are designed and adapted for use together in a method.For example, a kit that is packaged with one or more doses for injection into a subject can include a pre-measured dose of a microdevice formulation in a sterile needle, an ampoule, a tube, a container, or other suitable container.The kit can also include instructions for dosage and administration regimen.

[0190] The invention will be further understood by reference to the following non-limiting examples. [Example]

[0191] The examples demonstrate the development and use of a multi-dose drug delivery platform by fabricating polylactic-co-glycolic acid (PLGA), an FDA-approved commercially available polymer, into cubic microdevices (Figure 1A). Unlike commonly used topical drug delivery materials such as hydrogels or microdevices produced using double emulsion-solvent evaporation techniques, which exhibit sustained drug release kinetics (see Kamaly N., et al., Chem Rev., 116(4):2602-63(2016); Li J. and Mooney DJ, Nat Rev Mater., 1(12). pii: 16071 (2016); Lin CC. and Anseth KS., Pharm Res., 26(3):631-43 (2009); Wang H. and Mooney DJ, Nat Mater., 17(9):761-772 (2018)), these microfabricated PLGA microdevices (PLGA-MPs) release individual doses of incorporated STING agonists in a pulsatile manner for up to several months, with virtually no leakage. The examples show that in several tumor models, a single injection containing multiple populations of STING agonist-loaded PLGA-MP inhibits tumor growth and improves survival as effectively as multiple injections of a soluble STING agonist. The ability to combine multiple doses into a single-injection PLGA-MP also reduces metastasis, expanding the potential for applying current STING agonist-based therapies to hard-to-reach tumors. This is the first demonstration of an injectable, fully degradable drug delivery platform that can mimic the kinetics of multiple injections for effective cancer immunotherapy.

[0192] Example 1 Fabrication of PLGA microdevices with different release kinetics. material and method Microdevice Manufacturing PLGA was purchased from Evonik (Germany) and PolySciTech (West Lafayette, IN). PLGA microdevices were fabricated by the stamp assembly of polymer layers (SEAL) process (McHugh KJ., et al., Science, 357(6356):1138-1142 (2017)). Photomasks with microscale patterns for the base and cap were fabricated using Front Range Photomask (Palmer Lake, CO). Positive master molds for the base and cap of the microdevice were fabricated by SU-8 lithography on a silicon wafer. Next, a mixture of PDMS base and curing agent (Sylgard 184, Dow Corning, Midland, Michigan) was poured into the silicon master mold after high vacuum for 1 hour. Next, a glass slide with two cover glasses on either end was pressed into the silicon mold while curing in an oven at 150°C for 2 hours to obtain a thin PDMS mold. The resulting PDMS mold was then used as a negative mold to press the desired microdevice. PLGA film was prepared by solvent casting 60% wt / vol PLGA in an acetone solution. The PLGA film thickness was approximately 1650-1750 μm. To mold the cap of the microdevice, a small piece of PLGA film was placed between the PDMS cap mold and a Teflon® film and covered with a glass slide. The microdevice was then compressed in a vacuum oven at 120°C for 2 hours, using a pair of spring-loaded clamps. The PLGA film melted and poured into the PDMS cap mold while heated. The device was then cooled to room temperature and separated to obtain the PLGA cap within the PDMS mold. To mold the base of the microdevice, the above process was repeated, but without the Teflon® film. Briefly, the PLGA base was separated from the PDMS base mold and attached to a cover slide.The desired cargo was loaded into PLGA microdevices using a BioJet Ultra picoliter dispenser (Biodot, CA). Aqueous solutions of the cargo were dispensed in multiple 15-drop cycles of 180–200 pL droplets. The loaded microdevices were then aligned and sintered with the corresponding PLGA caps using a photomask aligner (MA4, Karl SUSS, Sunnyvale, CA) retrofitted with a Peltier heater for simultaneous alignment and sealing. The sealed microdevices were then separated from the glass slide using a razor blade. SEM images were collected using a JSM-5600LV SEM (JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV. High-resolution X-ray CT images were collected at the Biotechnology Resource Center of Cornell University.

[0193] result Using soft lithography techniques, we fabricated arrays of cubic PLGA microdevices with fully enclosed cavities for drug loading (Figure 1B). Briefly, PLGA was heated and pressed into a polydimethylsiloxane (PDMS) mold to form a microdevice base with an internal cavity (200 × 200 × 100 μm length × width × height), corresponding to a volume of 4 nL. Aqueous drugs or model drugs were then loaded into the base using a piezoelectric dispenser capable of dispensing volumes on the order of 100 picoliters (pL). The water component of the drug solution evaporated rapidly due to its small volume, providing space for loading additional cargo. To achieve maximum cargo loading, multiple loading and drying cycles were used. The loaded microdevices were then aligned with PLGA caps embedded in the PDMS mold and sealed by heating above the glass transition temperature of PLGA (approximately 50 °C). The sealed microdevices had external dimensions of 400 × 400 × 300 μm (length × width × height) and wall thicknesses of 100 μm in each dimension. The loading capacity of each microdevice was 8.4% by volume. Scanning electron microscopy (SEM), high-resolution X-ray computed tomography (CT), and optical images demonstrated that the microdevices could be fabricated in large arrays (more than 300 per array) with high fidelity (Figures 1C-1D).

[0194] Example 2 PLGA microdevices exhibit similar release kinetics in vitro and in vivo. material and method Microdevice Manufacturing PLGA microdevices were fabricated as described in Example 1.

[0195] in vitro release kinetics To test in vitro release kinetics, the following drugs were obtained from the indicated suppliers: AF647-dextran (Life Technologies, Carlsbad, CA), 3'3'-cGAMP (Invivogen, San Diego, CA), Cy5-CpG DNA (5'-TCC ATG ACG TTC CTG ACG TT-Cy5-3', IDT Inc. Coralville, Iowa), and pemetrexed (Sigma-Aldrich, MO). PLGA microdevices were individually loaded with 1 μg of AF647-dextran, 2 μg of 3'3-cGAMP, 1 μg of Cy5-CpG, or 2 μg of pemetrexed, respectively. To determine the cargo loading in the microdevices, the loaded microdevices were individually suspended in 200 μL of PBS buffer, vortexed for 15 seconds, and centrifuged at 14,000 rcf for 1 minute. The supernatants were then analyzed by a microplate reader (AF647, AF647-dextran, Cy5-CpG), HPLC (pemetrexed), and Nanodrop™ (3'3'-cGAMP, absorbance at 260 nm). Results were quantified using a standard curve of serial dilutions of the stock solution. The filled microdevices were then sealed with the corresponding PLGA caps. Each microdevice was placed in a 0.5 mL microcentrifuge tube (Eppendorf, Hamburg, Germany) in 200 μL of PBS (pH = 6.84) and incubated at 37 °C on an orbital shaker. The supernatant from each tube was collected at the designated time points. The supernatants from the AF-647-dextran and Cy5-CpG groups were analyzed by a microplate reader (Tecan Infinite M200 spectrophotometer, excitation / emission = 640 / 680 nm). The supernatants of 3'3'-cGAMP and pemetrexed were analyzed by HPLC (Alliance HPLC system, Waters Co., MA, USA) using a C-18 column (Acclaim™ PolarAdvantage II, 3 μm, 4.6 × 150 mm) and a photodiode detector at 260 nm for 3'3'-cGAMP and 254 nm for pemetrexed. Water and acetonitrile were used as the mobile phase.Results were quantified using a standard curve of serial dilutions of the stock solution and normalized to the total cumulative release (n = 6–8). The actual release date for each PLGA was determined as the day when more than 50% of the cargo was released.

[0196] In vivo release kinetics One PLGA microdevice encapsulating AF647-dextran (1 μg) was loaded onto the tip of an 18-gauge Monoject filter needle (Covidien, Dublin, Ireland) in approximately 20 μl of 15 mg / ml methylcellulose (MC, Sigma-Aldrich), used as a viscosity enhancer. The microdevices were then injected subcutaneously into the left and hind flanks (one microdevice per side) of hairless mice (SKH1-E) or intratumorally into tumor-bearing mice. Mice were imaged every 1–2 days using a PerkinElmer Spectrum In Vivo Imaging System (excitation / emission = 640 / 700 nm, IVIS, Hopkinton, MA).

[0197] The cumulative release was normalized to the maximum and minimum values ​​of total fluorescence in the region of interest, which correspond to the complete release and background signal of a particular microdevice, respectively. Because fluorescence decreased after release due to biological clearance, the value after the maximum signal was set to 100% in Figure 2. The release timing was determined as the day when the fluorescence reached half of its final maximum above background.

[0198] To assess the amount of released cargo in tumors, 10 microdevices loaded with AF647 (0.5 μg AF647 per microdevice) were intratumorally injected into B16F10 tumor-bearing mice on day 0. Control tumor growth was treated with intratumoral administration of free cGAMP on days 0 and 4. Four mice were euthanized, and tumors and serum were isolated daily until day 7. Tumors were homogenized in PBS buffer. Unreleased AF647-PLGA-1 was physically disrupted to release AF647 during homogenization. Supernatants and serum samples containing unreleased AF647 were analyzed by a microplate reader (excitation / emission = 640 / 680 nm). Micro-CT analysis of microdevice distribution

[0199] During the fabrication of PLGA-1 films, PLGA-1 was doped with 5% phosphotungstic acid (PTA) to enhance contrast for microCT imaging. PTA-doped PLGA-1 was injected intratumorally into B16F10 tumor-bearing mice. The mice were then euthanized 1 hour after injection. The tumors were isolated and imaged using a Bruker Skyscan 1276 microCT imaging system. Reconstructed images were analyzed using MicroView.

[0200] result To test the release kinetics, PLGA microdevices with different polymer properties (Table 1) were loaded with a fluorescently labeled macromolecule, Alexa Fluor 647-labeled dextran (AF647-dextran). These microdevices were sealed with corresponding caps and incubated in phosphate-buffered saline (PBS, pH = 6.84) at 37 °C to mimic the acidic TME. The PLGA microdevices released AF647-dextran in pulses at approximately 1 ± 0, 4 ± 0, 8 ± 0, 11 ± 1, 15 ± 1, 18 ± 1, and 97 ± 2 days without detectable leakage prior to release (Table 1; Figures 2A–2G). To mimic a four-dose regimen with 3 to 4 days between doses, which has been shown to effectively inhibit tumor growth in animal models (Corrales L., et al., 2015), we selected microdevices that release AF647-dextran on days 4 (PLGA-1), 8 (PLGA-2), and 11 (PLGA-3) for further testing (Figure 2J). To verify the in vivo release kinetics, AF647-dextran-loaded PLGA-1, 2, and 3 microdevices were subcutaneously injected into hairless mice. AF647-dextran release was monitored by in vivo fluorescence imaging (IVIS). The released AF647-dextran showed a >100-fold increase in fluorescence intensity compared to the incorporated AF647-dextran, due to the self-quenching effect of the fluorophore upon drying or at very high local concentrations. The microdevices released AF647-dextran in vivo with release times similar to those in vitro (Figure 2K). The mean in vivo release times for PLGA-1, 2, and 3 were 3.9 ± 1.1, 8.1 ± 1.5, and 11.5 ± 1.4 days, respectively.

[0201] [Table 1]

[0202] The molecular weight of PLGA was measured by tetrahydrofuran gel permeation chromatography with a light scattering detector (Malvern, UK). Data represent the mean ± standard deviation (n = 6 to 8). Mn, Mw, and PDI represent the number-average molecular weight, weight-average molecular weight, and polydispersity index, respectively.

[0203] Next, we evaluated the influence of various TMEs on release kinetics. AF647-dextran-loaded PLGA-2 was intratumorally injected into B16F10 or 4T1 tumor-bearing mice. Release kinetics were then monitored daily by IVIS imaging. PLGA-2 showed consistent release kinetics in both the tumor and subcutaneous environments (Figure 2L). To test the distribution of microdevices within the tumor, PLGA-1 was doped with 5% phosphotungstic acid (PTA) and the tumor was imaged using micro-CT. The microdevices were successfully injected into the tumor and aggregated at the injection site due to their low mobility in the restricted environment. To further demonstrate that PLGA-MP released all of its incorporated cargo during the release window, AF647-loaded PLGA-1 microdevices were fabricated and intratumorally injected into B16F10 tumor-bearing mice. AF647 is a small hydrophilic molecule with a MW of 753.9, similar to the molecular weight of cGAMP (MW 675.1). Free AF647 was rapidly cleared from tumors after intratumoral injection (>95% within 2.5 h). The amount of unreleased AF647 was measured daily in the tumor, and the amount of released AF647 was back-calculated (Figure 2M). As shown in Figure 2N, PLGA-1 completely released AF647 in tumors between days 3 and 6. Some of the released AF647 also diffused into the bloodstream, as evidenced by the increased serum AF647 concentrations from days 3 to 6 (Figure 2O). These data demonstrated that PLGA-MPs released all of their incorporated cargo into tumors at the expected time points.

[0204] Example 3 A single injection of the cGAMP-loaded microdevice effectively inhibited tumor growth. material and method Microdevice Manufacturing PLGA microdevices were fabricated as described in Example 1.

[0205] Release kinetics Release kinetics was evaluated as described in Example 2. For sustained release systems, a fast dextran hydrogel kit (part number TURE2-1KT), a fast PVA hydrogel kit (part number TRUE4-1KT), and a 3D collagen kit (part number ECM675) were purchased from Millipore Sigma. 40 μg of cGAMP was loaded into 40 μL of hydrogel according to the manufacturer's instructions. To test the in vitro release rate, the cGAMP-loaded hydrogel was incubated on an orbital shaker at 37°C. The supernatant from each centrifuge tube was collected at predetermined time points and analyzed by Nanodrop™.

[0206] Biological activity of released 3'3'-cGAMP To assess the activity of 3'3'-cGAMP after microdevice fabrication, cGAMP-loaded PLGA-2 was placed in PBS buffer and mechanically disrupted with a scalpel to release the incorporated cargo. To assess the activity of cGAMP after release, cGAMP-loaded PLGA-2 was incubated in PBS buffer at 37°C on an orbital shaker. The supernatant was collected in the release window and quantified using Nanodrop™. 5 x 10 RAW-Lucia™ ISG cells were seeded into a 96-well plate. Serial dilutions of the cGAMP stock solution, dissolved cGAMP after microdevice fabrication, and released cGAMP were incubated with the cells for 24 hours before adding the QUANTI-Luc™ solution. The plate was then analyzed using a microplate reader. Results were quantified using a standard curve of serial dilutions of the stock solution.

[0207] Animals and cell lines All animal procedures were approved by the Massachusetts Institute of Technology Committee on Animal Care. Six- to eight-week-old female SKH1-E, C57BL / 6, and BALB / c mice were purchased from Charles River Laboratories Inc. The mouse breast cancer cell line 4T1 and melanoma cell line B16F10 were purchased from the American Type Culture Collection. The RAW-Lucia™ ISG cell line was purchased from InvivoGen Inc. KPC (LSL-KrasG12D / +; LSL-Trp53R172H / +; Pdx-1-Cre). Pancreatic cancer cells were kindly provided by Dr. Serguei Kozlov (Frederick National Laboratory of Cancer Research). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, B16F10), DMEM / F12 (KPC), and RPMI 1640 (4T1) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units / mL), and streptomycin (100 μg / mL) at 37°C and 5% CO. RAW-Lucia™ ISG cells were cultured in DMEM supplemented with 2 mM L-glutamine, 10% FBS, 100 μg / mL Normocin™, and 200 μg / mL Zeocin™.

[0208] Treatment of B16F10 and 4T1 tumors 2×10 5 4T1 cells or 2 x 10 5 B16F10 cells were subcutaneously injected into the right posterior flank of BALB / c or female C57BL / 6 mice, respectively. For the orthotopic 4T1 model, 2 × 10 54T1 cells were injected into the mammary fat pad. Seven days after tumor injection, B16F10 tumor-bearing mice were divided into six experimental groups (n = 8 per group): no treatment, 1x empty microdevice (EP), 1x cGAMP-S + EP, 1x collagen gel, 4x cGAMP-S, and 1x cGAMP-S + cGAMP-MP (Figure 3D). For the 4x cGAMP-S group, mice were intratumorally administered 10 µg of cGAMP in 50 µL of MC solution (cGAMP-S) on days 0, 4, 8, and 11, reproducing four doses of soluble cGAMP. The total dose of cGAMP was 40 µg per mouse throughout the treatment period. In the 1xcGAMP-S+cGAMP-MP group, mice received a single intratumoral injection of a mixture of 10 μg of cGAMP-S, five PLGA-1 microdevices containing 10 μg of cGAMP, five PLGA-2 microdevices containing 10 μg of cGAMP, and five PLGA-3 microdevices containing 10 μg of cGAMP (total of 40 μg) in 50 μL of MC solution via an 18G filter needle. In the 1xEP and 1xcGAMP-S+EP groups, five empty PLGA-1, PLGA-2, and PLGA-3 microdevices, each containing or not containing 40 μg of cGAMP-S, were intratumorally injected in 50 μL of MC solution. Mice from the untreated group received an intratumoral injection of 50 μL of MC solution on day 7 after tumor inoculation. In the subcutaneous and orthotopic 4T1 models, tumor-bearing mice were subjected to untreated, 4x cGAMP-S, and 1x cGAMP-S + cGAMP-MP treatments on day 7 after tumor inoculation (n = 8 for each group). Tumor size was measured with a digital caliper every other day starting on day 7 after tumor inoculation. Tumor volume was calculated using the following formula: length (mm) x width. 2 (mm) × 0.5. If the animal showed signs of illness or the tumor size reached 1500 mm 3 If the rats exceeded this limit, they were euthanized.

[0209] statistical analysis All statistical analyses were performed using the GraphPad Prism software package (PRISM 8.0.2; GraphPad Software, USA). Biological replicates were used in all experiments unless otherwise stated. Survival benefit was determined using the log-rank test. All experimental results were presented as mean ± standard deviation or mean ± standard error. One-way and two-way analysis of variance (ANOVA) were used for multiple comparisons.

[0210] result STING agonist-loaded PLGA-1, 2, and 3 were fabricated with a drug loading of 2 μg per microdevice. 3'3'-cGAMP, a linkage isomer of the naturally occurring 2'3'-cGAMP, was used here because of its improved stability against ecto-nucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), which primarily hydrolyzes cGAMP (Kato K. et al., Nat Commun., 9(1):4424(2018)). 3'3'-cGAMP was released in a pulsed manner from PLGA-1, 2, and 3 in vitro at approximately the same time as fluorescent molecules (Figure 3A). The stability of the incorporated cGAMP under physiological conditions is important for the retention of bioactivity upon release. To test the stability of 3'3'-cGAMP in the microdevices, cGAMP-loaded PLGA-2 microdevices were incubated in PBS at 37°C, and the structural integrity of cGAMP in the supernatant was analyzed over time by liquid chromatography-mass spectrometry (LC-MS). The released cGAMP exhibited the same elution time and molecular mass as standard 3'3'-cGAMP (Figure 3B). The bioactivity of the released cGAMP was also tested using an interferon regulatory factor (IRF) reporter cell line (RAW-Lucia™ ISG cells). cGAMP released from PLGA-2 maintained over 95% bioactivity (Figure 3C). Collectively, these data demonstrated that incorporated cGAMP remained stable and could be completely released from PLGA microdevices (Wu J., et al., Science, 339(6121):826-30 (2013)).

[0211] To determine whether a single injection of several timed-release populations of cGAMP-loaded PLGA-MPs could stimulate antitumor immunity comparable to multiple injections of soluble cGAMP (cGAMP-S), mice bearing poorly immunogenic B16F10 melanoma tumors were intratumorally treated with: 1) a single injection of cGAMP-S (10 μg) combined with cGAMP-loaded PLGA-1, 2, and 3 microdevices (cGAMP-MPs, 10 μg of cGAMP per formulation), mimicking four doses; 2) four injections of cGAMP-S (10 μg of cGAMP per injection) administered at multiple time points corresponding to PLGA release (Figure 3D); 3) a single intratumoral injection of empty PLGA-1, 2, and 3 microdevices (EP); and 4) a single intratumoral injection of high-dose cGAMP-S (40 μg) and EP. Untreated mice served as negative controls.

[0212] To compare the therapeutic efficacy of PLGA-MP with other sustained-release systems, we fabricated three sustained-release formulations, including dextran hydrogel, polyvinyl alcohol (PVA) hydrogel, and collagen hydrogel. In vitro release kinetics studies showed that >99% of cGAMP was released from all three hydrogel formulations within 24 hours, consistent with previously reported sustained-release systems for cGAMP (Leach DG., et al., Biomaterials, 163:67-75 (2018); Junkins RD. et al., J Control Release., 270:1-13 (2018)). The collagen gel, which exhibited the slowest release rate of the gels tested, was loaded with 40 μg of cGAMP and administered intratumorally to tumor-bearing mice as a control.

[0213] Tumors grew rapidly in both the untreated and EP-treated groups, and all mice died within 21 days, indicating that PLGA microdevices alone did not inhibit tumor growth (Figures 3E and 3F). A single injection of high-dose cGAMP-S (40 μg) and EP demonstrated antitumor effects at early time points but failed to achieve sustained tumor inhibition. Survival time was slightly extended from 21 days in untreated mice to 25 days, indicating that multiple doses were required for effective tumor inhibition. Collagen gel did not demonstrate superior tumor inhibition or survival compared with cGAMP-S plus EP. In contrast, a single injection of cGAMP-S with cGAMP-MP significantly inhibited tumor growth and extended animal survival, with no statistical difference compared with four injections of an equivalent dose of cGAMP-S. Similar results were observed after the same treatment in both orthotopic (Figures 3G and 3H) and subcutaneous triple-negative breast cancer models (4T1). The systemic interleukin-6 (IL-6) response of mice bearing orthotopic 4T1 tumors was assessed from day 1 to day 7. IL-6 levels were elevated in both the cGAMP-MP and 4 × cGAMP-S treatment groups, indicating successful release of cGAMP from PLGA-MP into the tumor and bloodstream.

[0214] Example 4 A single injection of cGAMP-MP stimulated potent antitumor immunity through STING pathway activation. material and method Western blot and quantitative polymerase chain reaction (qPCR) B16F10 tumor-bearing mice were divided into four experimental groups (n = 8): untreated, 1x EP, 3x cGAMP-S, and 1x cGAMP-S + cGAMP-MP (Figure 4A). Tumors were harvested 16 days after tumor inoculation and cut into 50-100 mg pieces in 1.5 mL microcentrifuge tubes. Tumors were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), homogenized, and centrifuged at 20130 rcf for 10 min. Protein content in the supernatant was quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, USA). Equal amounts of protein (20 μg) were separated on a 4-15% gradient SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and electrotransferred to a nitrocellulose membrane. The membrane was then blocked with 5% milk in tris-buffered saline supplemented with 0.05% Tween® 20 and further incubated overnight at 4°C with GAPDH monoclonal antibody (Invitrogen, CA, Catalog No. MA5-27912), phosphorylated TBK1 / NAK (Ser172) (D52C2) rabbit mAb (Cell Signaling Technology, Catalog No. 5483S), or phosphorylated IRF-3 (S396) rabbit mAb (Cell Signaling Technology, Catalog No. 4947S). The membrane was then incubated with goat anti-rabbit IgG (H+L) secondary antibody, HRP (Invitrogen, Catalog No. TG266717) at room temperature for 1 hour. Protein bands were visualized by chemiluminescence using ECL Western blotting substrate (Thermo Fisher Scientific, MA).

[0215] For q-PCR experiments, total RNA was extracted from tumors using the RNeasy Kit (Qiagen, Inc.) according to the manufacturer's protocol. Total RNA was then reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA). The resulting cDNA was amplified using TaqMan Gene Expression Master mix (Thermo Fisher Scientific, MA) in a 384-well LightCycler 480 (Roche, Venlo, The Netherlands). The primers used were IRF7 (Thermo Fisher, assay ID: Mm00516793_g1), CXCL10 (Thermo Fisher, assay ID: Mm00445235_m1), and GAPDH (Thermo Fisher, assay ID: Mm99999915_g1). Samples were analyzed in triplicate.

[0216] Flow cytometry To stain cell surface markers for flow cytometry analysis, cells were pretreated with anti-CD16 / 32-Fc blocker (Biolegend, catalog no. 101319) and stained with fluorophore-conjugated antibody solutions at the manufacturer's recommended dilutions for 1 hour on ice. To stain intracellular markers, such as IFN-γ, cells were prestimulated with a cell stimulation cocktail (eBioscience, catalog no. 00-4970-93) for 4–6 hours, fixed and permeabilized using a fixation / permeabilization solution kit (BD, catalog no. 554714), and then stained with both anti-IFN-γ and other surface antibodies.The antibodies used for flow cytometry studies were anti-CD86-BUV395 (BD, Cat. No. 564199), anti-CD45-BUV737 (BD, Cat. No. 564880), anti-TCRβ-BV421 (Biolegend, Cat. No. 109229), anti-NK1.1-BV605 (Biolegend, Cat. No. 108739), anti-NK1.1-BV605 (Biolegend, Cat. No. 108739), anti-CD8a-FITC (BD, Cat. No. 553030), anti-CD4-PerCP / Cy5.5 (BD, Cat. No. 550954), and anti-CD62L-P. E (Biolegend, Catalog No. 104407), anti-CD19-PE / Cy7 (eBioscience, Catalog No. 25-0193-81), anti-CD3-PE / 594 (Biolegend, Catalog No. 100245), anti-FOXP3-APC (eBioscience, Catalog No. 17-5773-80), anti-CD11b-AF700 (Biolegend, Catalog No. 201222), anti-CD8a-BV421 (Biolegend, Catalog No. 100737), anti-Ly6g-BV510 (Biolegend, Catalog No. 127633), anti-Siglec F-BV605 (BD, Catalog No. 740388), anti-MHC II-BV786 (BD, Catalog No. 743875), anti-Ly6c-AF488 (Biolegend, Catalog No. 128021), anti-CD11c-PerCP / Cy5.5 (Biolegend, Catalog No. 117327), anti-CD206-PE (Biolegend, Catalog No. 141705), anti-CD197-PE / 594 (Biolegend, Catalog No. 120121), anti-F4 / 80-PE / Cy7 (Biolegend, Catalog No. 123113), anti-CD200R3-APC (Biolegend, Catalog No. 142207), anti-CD11b-AF700 (Biolegend, Catalog No. 101222), and viability dye eFluor 780 (eBiosciecne, catalog number 65-0865-14). Flow cytometry data were acquired on an LSR Fortessa cell analyzer (BD) and analyzed using FlowJo software.

[0217] result Next, we examined the activation of the STING pathway and antitumor immunity within the TME of B16F10 melanoma tumors. A combination of soluble cGAMP and cGAMP-loaded PLGA-1 and -2 microdevices was injected intratumorally on day 0, mimicking a total of three doses (Figure 4A). cGAMP-MP substantially inhibited tumor growth, consistent with the findings of tumor inhibition. One day after the third cGAMP-S injection, tumors were isolated and analyzed by Western blot and quantitative polymerase chain reaction (qPCR). cGAMP-MP enhanced messenger RNA (mRNA) levels of the interferon-stimulated genes (ISGs) CXCL10 (6.8-fold over untreated) and IRF7 (58.5-fold over untreated), comparable to those in 3x cGAMP-S-treated mice (7.2-fold and 66.5-fold enhancements of CXCL10 and IRF7, respectively; Figures 4B-4C). Furthermore, tumors treated with cGAMP-MP and 3×cGAMP-S showed high expression levels of phosphorylated TBK1 (p-TBK1) and phosphorylated IRF3 (p-IRF3). Untreated tumors and tumors treated with EP showed no detectable expression of p-TBK1 or p-IRF3. These data demonstrate that cGAMP-MP successfully activated the STING pathway and induced ISG production at levels similar to multiple injections (Burdette DL., et al., Nature, 478(7370):515-8 (2011); Corrales L., et al., J Clin Invest., 126(7):2404-11 (2016)). In contrast, empty microdevices did not induce the production of p-TBK1, p-IRF3, or ISGs.

[0218] Activation of the STING pathway in the TME has been shown to promote lymphocyte infiltration, a key mediator of effective cancer immunotherapy (Cheng N., et al., JCI Insight, 3(22). pii: 120638 (2018)). Flow cytometry analysis of tumors showed that 3×cGAMP-S and cGAMP-MP increased TILs by approximately 23.5- and 17.6-fold compared with the untreated group. Among these TILs, tumor-infiltrating CD8+ and CD4+ T cells were substantially increased by 24.4- and 23.6-fold in the 3×cGAMP-S-treated group and 16.2- and 22.1-fold in the cGAMP-MP-treated group, respectively (Figures 4D-4E). The abundance of CD8+ and CD4+ T cells in the cGAMP-S-treated group was slightly higher than that in the cGAMP-MP-treated group, but the difference was not statistically significant. Treatment with 3×cGAMP-S and cGAMP-MP also demonstrated a 1.2-fold and 1.5-fold improvement in the CD8+ / CD4+ T cell ratio, a commonly reported positive prognostic indicator of immunotherapy (Rudqvist NP., et al., Cancer Immunol Res.,6(2):139-150 (2018); Shae D. et al., 2019). Consistent with this enriched CD8+ T cell infiltration and enhanced antitumor activity, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) demonstrated a higher abundance of apoptotic cells in the cGAMP-MP and 3×cGAMP-S treatment groups. Furthermore, 3×cGAMP-S and cGAMP-MP increased infiltrating natural killer (NK) cells, another important group of cytotoxic lymphocytes that form the adaptive immune response (Figure 4F), and were found to be effective in spontaneous STING-mediated defense against B16F10 tumors (Marcus A., et al., Immunity, 49(4):754-763.e4 (2018)). Alternatively, the group receiving empty microdevices did not increase TILs in the TME, confirming their inability to activate the STING pathway. No differences in regulatory T cells were observed across all groups.

[0219] Next, we evaluated changes in dendritic cell (DC) and myeloid cell composition in the B16F10 melanoma TME after treatment. Both 3×cGAMP-S and cGAMP-MP promoted the influx of DCs (CD11b-CD11c+), basophils (CD11b+Gr-1-CD200R3+), monocytes (CD11b+F4 / 80-Ly6c+Ly6g-), and macrophages (CD11b+F4 / 80+), potentially generating an innate inflammatory niche that primes adaptive immunity (Figure 4G; Iwasaki A. and Medzhitov R., Nat Immunol., 16(4):343-53 (2015)). In contrast, empty microdevices did not increase myeloid cell populations, suggesting the low immunogenicity of PLGA. Furthermore, surface expression of CD86, a maturation marker overexpressed on the surface of activated tumor-infiltrating DCs (Han T., et al., J. Immunother., 32(4):399-407 (2009)), was increased by 1.7-fold and 1.5-fold in the 3×cGAMP-S and cGAMP-MP treated groups (Figure 4H).

[0220] DC maturation combined with enriched TILs and enhanced intratumoral ISGs suggests potential activation of adaptive immunity (Bose D., Int J Mol Sci.,18(11) (2017); Vatner RE., Mol Immunol.,110:13-23 (2019)). Next, we evaluated another important function associated with cGAMP: the polarization of macrophages within the TME (Ohkuri T. Cancer Immunol Immunother.,66(6):705-716 (2017)). After three doses of soluble cGAMP or one dose of cGAMP-MP, we observed a repolarization of intratumoral M2-like macrophages to an M1-like phenotype, consistent with previous studies of STING agonist-treated tumors, suggesting a reduced immunosuppressive TME. Notably, cGAMP-MP consistently downregulated the canonical M2 surface marker (CD206) and upregulated the M1 surface marker (CD86, Figure 4I). Quantitative analysis showed that cGAMP-MP induced an approximately two-fold greater M1 / M2 ratio than the 3xcGAMP-S treatment group. Empty microdevices slightly increased the M1 / M2 ratio compared with the untreated group, but this was not statistically significant. These data suggest that loading PLGA-MPs with cGAMP may promote M1-like polarization, possibly due to acidic degradation products of PLGA, which have been shown to stimulate proinflammatory macrophages (Nilsson B., et al., Mol Immunol., 44(1-3):82-94 (2007); Amini AR., et al., J Long Term Eff Med Implants., 21(2):93-122 (2011); Ceonzo K., et al., Tissue Eng., 12(2):301-8 (2006)). Further studies of macrophage polarization kinetics are needed to fully elucidate the function of PLGA-MPs on macrophage polarization in the TME.

[0221] Example 5 A single injection of cGAMP-MP elicits potent systemic antitumor immunity. material and method Treatment of contralateral B16F10 tumors 2 x 10 on day 0 5 B16F10 cells were injected subcutaneously into the right posterior flank of female C57BL / 6 mice. Another 2 × 10 5 B16F10 cells were subcutaneously injected into the left rear flank on day 2 to mimic metastatic tumors. Seven days after primary tumor inoculation, B16F10 tumor-bearing mice were divided into four experimental groups (n = 8 per group): untreated, cGAMP-MP, anti-PD1, and cGAMP-MP + anti-PD1. cGAMP-MP (10 μg cGAMP-S, five PLGA-1 microdevices containing 10 μg cGAMP, and five PLGA-2 microdevices containing 10 μg cGAMP) in 50 μL of MC solution was intratumorally injected into the primary tumor (right side). For the anti-PD1 and cGAMP-MP + anti-PD1 treatment groups, 100 μg of anti-PD1 antibody (Biolegend, catalog no. 114114) was injected intraperitoneally on days 7, 10, and 14 after primary tumor inoculation (Figure 5D). The distal tumor (left side) did not receive any treatment. Tumor size was measured with a digital caliper every other day starting on day 7 after tumor inoculation. Tumor volume was calculated using the following formula: length (mm) x width. 2 (mm) x 0.5. If the animal showed signs of illness or the tumor size on either side exceeded 1500 mm 3 If the rats exceeded this limit, they were euthanized.

[0222] Treatment of metastatic 4T1 model 2×10 54T1 cells were injected into the mammary fat pad. Seven days after injection, tumor-bearing mice were divided into three experimental groups: untreated, 3× cGAMP-S, and 1× cGAMP-S + cGAMP-MP (10 μg cGAMP-S, five PLGA-1 microdevices containing 10 μg cGAMP, and five PLGA-2 microdevices containing 10 μg cGAMP). Primary tumors were surgically removed on day 18 to prolong survival. Mice were euthanized on day 34. Lung tissue was stained with India ink and fixed in Fekete's solution. Metastatic foci in the lungs were counted under a microscope. Unstained lung tissue was fixed in formalin and stained with H&E. Quantification of metastatic tumor cells in H&E-stained sections was performed using an Aperio ImageScope with a calibrated positive pixel counting algorithm. Briefly, the input hue value of the positive pixel counting algorithm was adjusted to positively select normal lung tissue in the red to orange range, while tumors were negatively selected in purple. The area percentage of tumor metastases per total lung area was calculated by negative counts (purple) / total counts (purple, orange, and red) × 100%. Three H&E sections per lung at different depths were analyzed and averaged to obtain the percentage of tumor on the lung of one mouse. Four or five mice were analyzed for each group.

[0223] Immunofluorescence staining Tumor sections (5 μm) were fixed with 4% paraformaldehyde, blocked with 3% bovine serum albumin, and permeabilized with 0.1% Triton X-100 in PBS. Next, tumor sections were incubated with anti-CD8 alpha antibody (1:200, Abcam, catalog no. ab217344) overnight at 4°C and goat anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (1:1000, Abcam, catalog no. ab150077) for 1 hour at room temperature. Apoptotic tumor cells were stained using an in situ cell death detection kit (Roche) according to the manufacturer's instructions. Images were acquired with a Nikon A1R Ultra-Fast Spectral Scanning Confocal Microscope (Shinagawa, Tokyo).

[0224] result To test whether STING activation in the TME induces systemic antitumor immunity, mouse sera were collected 21 and 28 days after tumor inoculation from an antitumor efficacy study in a B16F10 melanoma model (Figure 3D) and analyzed by flow cytometry. Treatment with cGAMP-MP and 4×cGAMP-S resulted in a 5.1-fold and 4.9-fold increase in serum IFNγ+ CD8+ T cells on day 21 compared with the untreated group (Figure 5A). The number of IFNγ+ CD8+ T cells remained at the same level on day 28, demonstrating a long-lasting systemic immune response. Furthermore, cGAMP-MP also increased the number of memory CD62L- CD44+ CD4+ T cells (approximately 6.2-fold higher than the untreated group) and CD62L- CD44+ CD8+ T cells (approximately 5.4-fold higher than the untreated group) in the TME (Figures 5B-5C). Taken together, a single injection of cGAMP-MP generates long-lived systemic antitumor immunity and local immune memory, potentially preventing tumor recurrence and metastasis.

[0225] Next, we tested whether cGAMP-MP could inhibit the growth of distant tumors using a contralateral B16F10 tumor model. Primary tumors were treated with a single intratumoral injection of cGAMP-S, cGAMP-loaded PLGA-1, or PLGA-2 on days 0, 4, and 8, respectively, for a total of three doses. Distal tumors received no treatment (Figure 5D). cGAMP-MP significantly inhibited the growth of both primary and distant tumors compared with the untreated group, thereby demonstrating potent systemic antitumor immunity (Figures 5E and 5F).

[0226] To evaluate whether cGAMP-MP can improve the antitumor efficacy of immune checkpoint blockade (ICB) therapy, we tested the combination of cGAMP-MP and anti-programmed death 1 in the same contralateral B16F10 tumor model. Indeed, the combination of cGAMP-MP and ICB showed greater inhibition of primary and distant tumor growth than either therapy alone (Figures 5E and 5F), demonstrating that combining cGAMP-MP and ICB may enhance the therapeutic effect.

[0227] Next, we used an orthotopic 4T1 model to test the effect of cGAMP-MP on inhibiting metastasis. Primary tumors were treated with a single injection of cGAMP-MP on day 7 after tumor inoculation or multiple injections of cGAMP-S on days 7, 11, and 15 after tumor inoculation. Primary tumors were surgically removed on day 18 to extend the survival period necessary to allow metastasis development. Lungs were then isolated and metastasis analyzed on day 34. Treatment with cGAMP-MP and 3×cGAMP-S significantly reduced the number of metastatic foci on the lung surface (Figure 5H) and the relative tumor area in the lung compared with the untreated group (Figure 5I). cGAMP-MP also showed a greater ability to reduce the percentage of metastatic tumor cells in the lung compared with 3×cGAMP-S (Figure 5I), suggesting the benefit of a single injection in reducing metastasis.

[0228] Example 6 A single injection of cGAMP-MP inhibits tumor recurrence and metastasis, facilitating the treatment of hard-to-reach tumors. material and method Treatment of surgically removed B16F10 tumors 2×10 5B16F10 cells were subcutaneously injected into the right posterior flank of female C57BL / 6 mice. Six days after tumor inoculation, B16F10 tumor-bearing mice were randomly divided into four experimental groups (n = 8 per group): untreated, 1x cGAMP-S + EP, 3x cGAMP-S, and 1x cGAMP-S + cGAMP-MP. Approximately 99% of the tumor volume was surgically removed, leaving approximately 1% residual tumor to mimic residual microtumors. Upon tumor removal, 50 μL of cGAMP-MP (10 μg cGAMP-S, five PLGA-1 microdevices containing 10 μg cGAMP, and five PLGA-2 microdevices containing 10 μg cGAMP) or 1× cGAMP-S+EP (10 μg cGAMP-S, five empty PLGA-1 and PLGA-2 microdevices) in MC solution was applied directly to the surgical bed from a micropipette. For the 3× cGAMP-S treatment group, 10 μg of cGAMP-S was applied to the surgical bed after surgery, followed by intratumoral injection of 10 μg of cGAMP on days 4 and 8 after surgery (Figure 6A). The wound was closed using an autoclip-on-clip system. Tumor size was measured every other day starting on day 7 after tumor inoculation using a digital caliper. For rechallenge experiments, 2×10 5 B16F10 cells were injected subcutaneously into the left rear flank of treated mice that achieved a complete response. Tumor size was measured every other day with a digital caliper.

[0229] Treatment of orthotopic pancreatic tumor models A small incision was made to expose the spleen and pancreas of a female C57BL / 6 mouse. 5 × 10 cells were cultured in 50 μL of PBS and Matrigel (1:1 mixture by volume). 5KPC cells were injected into the tail of the pancreas. cGAMP-MP (10 μg cGAMP-S, five PLGA-1 microdevices containing 10 μg cGAMP, and five PLGA-2 microdevices containing 10 μg cGAMP) or 1×cGAMP-S+EP (30 μg cGAMP-S, five empty PLGA-1 and PLGA-2 microdevices) in 50 μL of MC solution was also injected into the tail of the pancreas. The wound was then closed using an autoclip-on-clip system. 25 days after tumor inoculation, the mice were euthanized. Tumors were isolated and weighed. Lung metastasis was assessed by H&E staining of lung sections.

[0230] Biodegradation of PLGA-MP Five empty PLGA-2 microdevices were subcutaneously injected into the posterior flank of SKH1-E mice. Mice were euthanized 2, 8, and 30 days after injection. Skin and subcutaneous tissues were collected and fixed in formalin-free fixative (Sigma-Aldrich) for 24 hours. The tissues were then embedded in paraffin, cut into 5 μm tissue sections, stained with H&E, and imaged using an Aperio AT2 slide scanner (Leica Biosystems, Buffalo Grove, IL).

[0231] result In clinical practice, patients often develop recurrent tumors after surgery due to the presence of residual microtumors and circulating tumor cells (Demicheli R., et al., Ann Oncol., 19(11):1821-8 (2008); Alieva M., et al., Clin Exp Metastasis, 35(4):319-331 (2018); Al-Sahaf O., et al., Ann Surg., 252(6):1037-43 (2010)). To further expand the clinical application of cGAMP-MP, we employed a surgical tumor resection model to evaluate the efficacy of cGAMP-MP in inhibiting tumor recurrence (Wang C., et al., Nat Biomed Eng., 1(2017), doi:10.1038 / s41551-016-0011; Chen Q., et al., Nat Nanotechnol., 14(1):89-97 (2019)). Six days after tumor inoculation, approximately 99% of the B16F10 tumor was surgically removed. Next, a combination of soluble cGAMP, cGAMP-loaded PLGA-1, and PLGA-2 was directly deposited onto the new surgical bed to achieve a total of three doses on days 0, 4, and 8, respectively (Figure 6A). Improved tumor inhibition (Figure 6B) and increased survival (Figure 6C) were observed in mice treated with cGAMP-MP. The tumor recurrence rates in both the cGAMP-MP and 3×cGAMP-S treated groups were 25%, significantly lower than the untreated group (100%), whereas a single administration of cGAMP-S+EP had limited efficacy, resulting in a high recurrence rate of 87.5%. Six of eight cGAMP-MP treated mice were tumor-free and survived for more than 60 days after inoculation (Figure 6C). These tumor-free mice were then rechallenged by subcutaneous injection of B16F10 cells. Tumor growth was significantly slower in cGAMP-MP and 3×cGAMP-S treated mice than in naive mice (Figure 6D). Survival analysis also showed that survival time was prolonged in the treated groups (Figure 6E), suggesting that cGAMP-MP and 3×cGAMP-S conferred protective immunity.

[0232] The therapeutic efficacy of cGAMP-MP was further tested in a pancreatic cancer allograft model (KPC model). A combination of soluble cGAMP, cGAMP-loaded PLGA-1, and PLGA-2 was injected into the pancreas after tumor inoculation to achieve a total of three doses (10 μg per dose) on days 0, 4, and 8, respectively (Figure 6F). Multiple intratumoral injections of soluble cGAMP are very challenging in such difficult-to-reach tumors. Therefore, a single injection of high-dose cGAMP-S (30 μg) containing EP was performed on day 0. Untreated mice were used as a negative control. Tumor growth and metastasis were then analyzed 25 days after treatment. cGAMP-MP significantly inhibited primary tumor growth in the pancreas and metastasis to the lungs compared with the untreated group (Figures 6G–6H and data not shown). In contrast, high-dose cGAMP-S containing EP showed no benefit on tumor growth or metastasis. Collectively, these data demonstrate that cGAMP-MP is useful in hard-to-reach tumors and suggest that effective treatment requires multiple doses over extended time points.

[0233] Toxicity analysis Due to its biodegradability and biocompatibility, PLGA is used in numerous FDA-approved medical devices (Makadia HK. and Siegel SJ., Polymers (Basel)., 3(3):1377-1397 (2011)). Flow cytometry analysis of immune cells in the TME demonstrated that intratumorally administered empty PLGA-MP induced minimal inflammation in situ (Figures 4D-4H). Furthermore, for all in vivo studies, no weight loss or behavioral changes were observed in any of the animals throughout the treatment period (not shown). Hematoxylin and eosin (H&E) staining of histological sections of major organs (heart, liver, spleen, lungs, and kidneys) showed no obvious changes in morphology (not shown). The biodegradability of PLGA-MP was further tested by subcutaneously injecting empty PLGA-2 into immunocompetent mice, and H&E staining of skin tissue was performed at 2, 8, and 30 days after injection. On day 2, the PLGA-MPs exhibited a cubic morphology under the skin, with few lymphocytes and leukocytes around the microdevices, suggesting minimal inflammation. The microdevices then transformed into ellipsoidal shapes due to PLGA hydrolysis, with a decrease in immune cells immediately around the release window on day 8, consistent with flow cytometry analysis. None of the microdevices were observed remaining in the mice at day 30, indicating complete degradation and clearance. Collectively, these data demonstrate that microfabricated PLGA-MPs exhibit minimal toxicity and can be completely degraded and cleared in vivo.

[0234] Adherence to current STING agonist-based therapies is challenging due to the need for frequent injections over a long period of time and for each injection to be administered by a trained medical professional. Low patient adherence poses serious challenges, leading to treatment failure and significant economic costs (Osterberg L., et al., 2005; Brown MT and Bussell JK., Mayo Clin Proc., 86(4):304-14 (2011)). Frequent injections for cancer treatment also cause significant strain on patients' daily lives (Haithcox S., et al., BMC Nurs., 2(1):2 (2003)). Current research efforts have primarily focused on improving the cellular uptake and tumor-targeting efficacy of STING agonists after systemic administration (Shae D. et al., 2019; Cheng N., et al., 2018; Koshy ST., et al., Adv Biosyst., 1(1-2). pii: 1600013 (2017)). This example demonstrates a unique approach to improving the overall efficacy of STING agonist therapy by ensuring patients receive all required doses at the correct time.

[0235] To replace multiple intratumoral injections of soluble STING agonists, PLGA-MP can remain within tumors and release biologically active cargo at predetermined times. Micro-CT analysis showed that these microdevices aggregate at the tumor injection site due to their low mobility. LC-MS and in vitro cellular analysis showed that cGAMP maintained >95% bioactivity after release from PLGA-MP. As demonstrated in the Examples, the release kinetics of PLGA-MP was independent of the released cargo (AF647, AF647-dextran, and STING agonist, Figures 2A, 2B, 2E, 2F, and 3A) and the in vivo microenvironment (subcutaneous, B16F10, and 4T1 tumors, Figure 2C). These observations are consistent with PLGA degradation being primarily hydrolytically driven. Enzymatic activity has negligible impact on PLGA degradation (Brown MT, et al., 2011). Although the slightly acidic tumor microenvironment did not accelerate the release of low-MW PLGA (Figure 2C), it may affect the long-term release of PLGA-MP due to acid-catalyzed hydrolysis of PLGA. Therefore, in vitro release studies in an acidic environment can be used to estimate the release kinetics in tumors (Figure 2A).

[0236] Pulsatile release over a long period of time is typically achieved by implantable drug delivery devices, which require invasive surgery for administration and removal (Farra R., et al., Sci Transl Med., 4(122):122ra21 (2012)). One advantage of PLGA-MP is that it can be injected using a standard needle and completely degrades over time, thus improving patient compliance. Other injectable long-term drug-release systems, such as emulsion-based microdevices (including PLGA formulations) or hydrogels, often exhibit an initial phase of burst release followed by a second phase of sustained release of hydrophilic drugs (Formiga FR., et al., J Control Release.,147(1):30-7 (2010); Shahani K. and Panyam J., J Pharm Sci., 100(7):2599-609 (2011)). Such release kinetics result in a high initial dose that can cause toxic side effects. Furthermore, achieving sustained release of small molecule / hydrophilic drugs, such as STING agonists, over a period of several weeks is extremely challenging. Drug encapsulation efficiency is also relatively low for emulsion-based microdevices (Yeo Y. and Park K., Arch Pharm Res., 27(1):1-12 (2004)). PLGA microdevices can achieve essentially 100% drug encapsulation efficiency and exhibit multiple burst release events combined for up to several months (see Figures 2A-2G). This example demonstrates that low-MW PLGA can achieve release times within the treatment schedule previously reported using cGAMP in animal models (Figure 2). By adjusting the PLGA MW, chain-end functional groups, and copolymer ratio, MP release kinetics can encompass pulsatile release over several months or even a year. Therefore, customizable doses can be administered by physically mixing PLGA-MPs with different release profiles in a single injection.

[0237] As shown in the Examples, the antitumor efficacy of a single dose of PLGA-MP was comparable to that of multiple injections of cGAMP solution in multiple mouse models (Figures 3E to 3H). PLGA-MP-treated B16F10 tumors consistently exhibited high levels of ISGs and phosphorylated TBK-1 and IRF-3 proteins 16 days after treatment, suggesting successful activation of the STING pathway by a series of cGAMP pulses. PLGA-MP induced an immunogenic TME, as demonstrated by a significant increase in tumor-infiltrating CD8+ T cells, NK cells, and DCs, and a shift in macrophage phenotype from M2 to M1 (Figure 4). Increased numbers of memory T cells and circulating IFNγ+ CD8+ T cells were observed in the tumor, which contributed to the inhibition of distant tumor growth (Figures 5E and 5F), reduced metastasis (Figures 5H and 5I), and protective immunity against rechallenge (Figures 6D and 6E). Neither cGAMP-MP nor 3×cGAMP-S-treated mice completely rejected tumor rechallenge. This observation is consistent with reports that repeated intratumoral injections of STING agonists attenuated systemic T cell responses (Sivick KE., et al., Cell Rep., 25(11):3074-3085.e5 (2018)). Nevertheless, multiple doses demonstrated superior tumor-inhibitory effects compared with a single dose of soluble STING agonist. PLGA-MP showed no apparent toxicity and could be completely degraded, as supported by body weight and histological analyses, respectively. Collectively, these results demonstrated the efficacy and safety of PLGA-MP, recapitulating multiple soluble injections.

[0238] Current STING agonist therapies in clinical trials focus on easily accessible tumors. Intratumoral injection of therapeutic agents into major organs in clinical settings is usually achieved under CT or ultrasound guidance (Aznar MA., et al., J Immunol., 198(1):31-39. (2017)). Therefore, applying STING agonist therapy to difficult-to-reach tumors is challenging due to the complexity and high economic costs of multiple imaging-guided injections. This example demonstrates that cGAMP-MP can be administered to orthotopic pancreatic tumors and effectively inhibit tumor growth and metastasis with a single injection (Figures 6G and 6H). Therefore, cGAMP-MP may be beneficial not only for easily accessible tumors (e.g., melanoma) but also for cancers in other major organs. Furthermore, cGAMP-MP can also be used after surgical resection of tumors that are not amenable to multiple intratumoral injections to prevent tumor recurrence (Figures 6A to 6E). It is contemplated that cGAMP-MP will enable broadening the scope of STING agonist-based therapies.

[0239] To maximize the number of doses achievable in a single injection, the drug loading of MPs can be increased. One way to increase drug loading is to reduce the wall thickness of the microdevice while maintaining the external dimensions. For example, reducing the wall thickness from 100 μm to 50 μm increases drug loading by 450%. Using thinner-walled microdevices, 10 μg of cGAMP was loaded onto each microdevice. The dose of STING agonist in some current clinical trials is 100 μg / injection, requiring approximately 10 microdevices to accommodate the same dose. The volume of 10 microdevices is 4.8 × 10 -4 cm 3 The total volume of PLGA-MP for 20 doses is 9.6 x 10 -3 cm 3 and this is 1 cm 3The PLGA-MPs comprise less than 1% of the tumor volume. The size and geometry of the PLGA-MPs can also be optimized to increase drug loading and / or allow injection using smaller needles. The manufacturing process for PLGA-MPs uses a combination of photolithography, soft lithography, and ultralow volume dispensing technology.

[0240] In summary, by engineering polymers such as PLGA into compartmental shell microstructures, we have developed a fully degradable delivery system for STING agonists that can improve patient adherence and reduce economic costs by eliminating repeated injections and physician visits, reduce the risk of metastasis, and result in better efficacy of STING agonist-based cancer immunotherapy. PLGA-MPs extend the reach of STING agonist-based therapy to hard-to-reach tumors and serve as adjuvant therapy to prevent tumor recurrence after surgery. This platform is compatible with any hydrophilic drug (e.g., pemetrexed and CpG DNA; see Figures 2H–2I) and even delivers different drugs at different times for synergistic cancer therapy.

[0241] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A pharmaceutical composition comprising a population of microdevices, each microdevice comprising a biocompatible, biodegradable polymer shell containing at least one discrete compartment therein comprising one or more immunoreceptor binding agents, said shell being fabricated by an additive process; each of the microdevices in the population has at least one external dimension between 50 micrometers (μm) and 1000 μm; wherein the population of microdevices releases immunoreceptor binding agents in an amount effective to elicit an immune response against one or more diseases or disorders for one or more time periods, the duration and / or time of release being the same or different for two or more time periods; The pharmaceutical composition, wherein the one or more immunoreceptor binding agents are stimulator of interferon genes (STING) agonists.

2. The pharmaceutical composition of claim 1, wherein each microdevice in the population of microdevices comprises a base and a cap.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the population of microdevices releases the one or more immunoreceptor binding agents at multiple times or time periods.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the release rate of the one or more immunoreceptor binding agents is controlled by the number average molecular weight of the polymers or copolymers in the population of microdevices, the weight average molecular weight of the polymers or copolymers, the polydispersity index of the polymers or copolymers, the chain end functionality of the polymers or copolymers, the ratio of copolymers in the population of microdevices, or a combination thereof, the salt blend ratio, the polymer or copolymer blend ratio in the population of microdevices, the shell thickness and the compartment matrix.

5. The pharmaceutical composition of claim 4, wherein the STING agonist is a nucleic acid or a small molecule.

6. 6. The pharmaceutical composition of claim 5, wherein the STING agonist is selected from the group consisting of cGAMP, DMXAA, MK-1454, MK-2118, E7766, MIW815 (ADU-S100), BMS-986301, GSK3745417, IMSA-101, SYNB18911, SITX-799, and SB11285.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the biocompatible, biodegradable polymer shell, and optionally the boundaries of the one or more compartments, are formed from a biodegradable, biocompatible polymer, optionally wherein the polymer is selected from the group consisting of polyhydroxy acids, polyhydroxyalkanoates, and polyanhydrides, and copolymers comprising one or more of these polymers.

8. 8. The pharmaceutical composition of claim 7, wherein the polymer is poly(lactic acid), poly(glycolic acid) and / or a copolymer comprising one or more of these polymers.

9. 9. The pharmaceutical composition of any one of claims 1-8, wherein the population of microdevices comprises distinct populations of microdevices, a first population releasing the one or more immunoreceptor binding agents about 4 days after administration, a second population releasing the one or more immunoreceptor binding agents about 8 days after administration, and a third population releasing the one or more immunoreceptor binding agents about 11 days after administration.

10. A pharmaceutical composition described in any one of claims 1 to 9, wherein the composition is in a dosage for local administration in an effective amount to induce a local or systemic immune and / or inflammatory response to a disease or disorder in a subject.

11. 11. The pharmaceutical composition of any one of claims 1 to 10 for localized delivery of one or more immune response inducing or enhancing agents to a subject for more than one period of time.

12. The pharmaceutical composition described in claim 11, characterized in that the pharmaceutical composition is administered to the subject in an effective amount to induce an immune and / or inflammatory response in or adjacent to the tumor.

13. 13. The pharmaceutical composition according to claim 11 or 12, characterized in that the composition is administered as a single injection.

14. The pharmaceutical composition according to any one of claims 11 to 13, characterized in that the composition is administered in an amount effective to induce or increase an interferon response in the subject.

15. The pharmaceutical composition according to any one of claims 11 to 14, characterized in that the composition is administered in an amount effective to induce infiltration of lymphocytes, basophils, macrophages and / or dendritic cells into the tumor microenvironment.

16. The pharmaceutical composition according to any one of claims 12 to 15, wherein the pharmaceutical composition is administered to the subject in combination with an additional cancer therapy, wherein the additional cancer therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, cryotherapy, or gene therapy.

17. 17. The pharmaceutical composition of claim 16, wherein the additional therapy is an immunotherapy comprising administration of one or more STING agonists, one or more immune checkpoint blockers, or a combination thereof.

18. 18. The pharmaceutical composition of claim 17, wherein the immune checkpoint blockade is an antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, LAG3, or a combination thereof.

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