Silk fibroin-based microneedles and uses thereof

Abstract

To provide microneedles and microneedle devices comprising silk fibroin-based microneedle tips for sustained dermal delivery of anti-cancer agents and / or immunomodulatory agents, and methods of making and using the same.SOLUTION: A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles comprises a first microneedle comprising an anti-cancer agent; and a second microneedle comprising an immunomodulatory agent, wherein the first and / or second microneedle comprises silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin), and wherein the microneedle device is configured to deliver the anti-cancer agent and the immunomodulatory agent to a subject.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 62 / 912,832, filed October 9, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure generally relates to silk fibroin-based microneedles configured to release a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, to treat a subject having a disease or disorder, e.g., cancer. [Background technology]

[0003] Drug delivery to the skin for local or systemic effects is extremely challenging due to the high effective barrier properties of the stratum corneum, the outermost layer of the skin. Microneedle devices include submillimeter needles designed to be minimally invasive, bypass the stratum corneum, and access the skin's microcirculation to achieve local and / or systemic delivery of therapeutic agents via the transdermal route. However, conventional microneedle designs and materials are associated with various limitations that impair their fabrication and limit their performance (see, for example, Donnelly et al. Drug Deliv. 17(4):187-207, 2010). Specifically, it is difficult to design microneedles that have both sufficient mechanical strength to penetrate the stratum corneum and the ability to incorporate and subsequently release effective amounts of therapeutic agents. Improved microneedle design and fabrication processes are needed. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the recognition that silk fibroin has suitable properties for use in the fabrication of microneedles, including all-aqueous processing, mechanical strength, biocompatibility, and the ability to stabilize and control the release of various therapeutic agents from silk-based matrices. Furthermore, the present disclosure is based, at least in part, on the recognition that local administration of therapeutic agents, e.g., anticancer agents and / or immunomodulatory agents, to the location of a tumor can result in inhibition of tumor growth at or near the site of administration and can also result in a systemic immune response to eliminate the tumor at a distant site. Furthermore, certain diseases, such as cancer, are associated with tumor-specific antigens (e.g., neoantigens) that are poorly and / or ineffectively presented to a subject's immune system by antigen-presenting cells (APCs), e.g., due to an immunosuppressive tumor microenvironment (TME). Without wishing to be bound by theory, the present disclosure provides silk fibroin-based microneedles and silk fibroin-based microneedle devices that may be used to alter the tumor microenvironment, for example, by administering an effective amount of an anti-cancer drug, an immunomodulatory agent, or a combination thereof, thereby promoting the presentation of tumor-specific antigens (e.g., neoantigens) to APCs in the subject's body and promoting the development of robust and durable immunity (e.g., cancer immunity) against the tumor-specific antigens. In some embodiments, the silk fibroin-based microneedles and silk fibroin-based microneedle devices may be used to deliver patient-specific neoantigens (e.g., cancer vaccines) to achieve enhanced immunity in a subject via sustained release of a therapeutic agent, for example, an anti-cancer drug and / or an immunomodulatory agent.

[0005] The present disclosure provides silk fibroin-based microneedles and silk fibroin-based microneedle devices that are configured to incorporate and subsequently release (e.g., administer) an effective amount of a therapeutic agent or combination of therapeutic agents to a subject (e.g., a human subject). In some embodiments, the silk fibroin-based microneedles and silk fibroin-based microneedle devices comprise an anti-cancer agent, an immunomodulatory agent, or a combination thereof. In some embodiments, the microneedles disclosed herein may be used in combination with a second therapeutic agent or procedure, such as a cancer therapy (e.g., one or more of an anti-cancer agent, immunotherapy, photodynamic therapy (PDT), surgery, and / or radiation).

[0006] The disclosed silk fibroin-based microneedles may be configured to release a therapeutic agent or combination of therapeutic agents (e.g., anticancer agents, immunomodulatory agents, or combinations thereof) according to a variety of release kinetics, such as burst release (e.g., immediate or rapid dissolution of the microneedle, typically within minutes, when applied to a biological barrier, e.g., skin, mucosal surface, tumor, oral cavity, or buccal cavity), and / or sustained release. Examples of sustained release include, but are not limited to, zero-order release (e.g., the release rate is independent of the concentration of therapeutic agent in the dosage form, e.g., the release rate is approximately constant over a period of time, e.g., a constant amount of therapeutic agent is cleared per unit time), first-order release (e.g., the release rate is a function of the amount of therapeutic agent remaining in the dosage form, e.g., a constant ratio, e.g., percentage, of drug cleared per unit time), and second-order release (e.g., doubling the concentration of therapeutic agent in the dosage form will quadruple the release rate). In some embodiments, a portion of the microneedles are configured for a first type of release, e.g., burst release, and another portion of the microneedles are configured for a second type of release, e.g., sustained release. Furthermore, release (e.g., administration) of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the silk fibroin-based microneedles described herein may be facilitated by diffusing the therapeutic agent from the microneedle or portion thereof; by degrading (e.g., protease-mediated degradation) the microneedle or portion thereof; and / or by dissolving the microneedle or portion thereof.

[0007] The disclosed silk fibroin-based microneedles may be configured to have sufficient mechanical properties (e.g., strength) and suitable geometries (e.g., tip sharpness, tip included angle, length, needle-to-needle spacing) to penetrate biological barriers (e.g., skin, tumor, tissue, cell membrane, mucosal surface, oral cavity, or buccal cavity) to achieve local and / or systemic delivery of a therapeutic agent or combination of therapeutic agents (e.g., anti-cancer agent, immunomodulatory agent, or combination thereof) to a subject. In some embodiments, the microneedles or devices described herein are configured to administer a therapeutic agent (e.g., anti-cancer agent, immunomodulatory agent, or combination thereof) to the location of a tumor (e.g., intratumorally and / or peritumorally). In some embodiments, the microneedles or devices described herein are configured to administer a therapeutic agent (e.g., anti-cancer agent, immunomodulatory agent, or combination thereof) to the location of a skin lesion.

[0008] In some embodiments, local administration of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) to the site of a tumor and / or skin lesion via silk fibroin-based microneedles or silk fibroin-based microneedle devices described herein may result in an anti-cancer effect (e.g., inhibition of tumor growth) at or near the site of administration and, if desired, a systemic anti-cancer effect (e.g., immune response) to eliminate the tumor at a distant site.

[0009] In some embodiments, the silk fibroin-based microneedles and silk fibroin-based microneedle devices are administered in combination with a standard of care treatment (e.g., a steroid drug) selected from surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy, and / or radiation therapy, as appropriate. For example, for cancer or skin conditions.

[0010] Methods of making and using the microneedles for treating a disease, such as cancer, in a subject are also disclosed. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein, which equivalents are intended to be encompassed by embodiment (E) below.

[0011] E1. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, said plurality of microneedles comprising: a first microneedle containing an anticancer drug; and a second microneedle containing an immunomodulatory agent; Optionally, a third microneedle containing an anti-cancer drug and / or an immunomodulatory agent. Including, A microneedle device, wherein the first and / or second microneedles comprise silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin) and are configured to deliver anti-cancer drugs and immunomodulatory agents to a subject.

[0012] E2. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles are: Two or more microneedles containing an anti-cancer drug, A microneedle device, wherein two or more microneedles comprise silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin) and are configured to deliver an anti-cancer drug to a subject.

[0013] E3. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles are: two or more microneedles containing an immune modulating agent Including, A microneedle device, wherein two or more microneedles comprise silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin) and are configured to deliver an immunomodulatory agent to a subject, and optionally, the immunomodulatory agent enhances the immune response against cancer.

[0014] E4. The first and / or second microneedle of the plurality of microneedles is (i) a base (e.g., a dissolvable base); (ii) a silk fibroin chip (e.g., an implantable silk fibroin chip) comprising silk fibroin applied to a base; and (iii) (Optional) Backing applied to the base 10. The microneedle device of any one of the preceding embodiments, comprising:

[0015] E5. A first microneedle containing an anticancer drug; and a second microneedle comprising an immunomodulatory agent; Optionally, a third microneedle containing an anti-cancer drug and / or an immunomodulatory agent. Including, A plurality of microneedles, wherein the first and / or second microneedles comprise silk fibroin, for example, regenerated silk fibroin and / or recombinant silk fibroin.

[0016] E6. A first microneedle containing an anticancer drug; and A second microneedle containing an anti-cancer drug Including, A plurality of microneedles, wherein the first and / or second microneedles comprise silk fibroin, for example, regenerated silk fibroin and / or recombinant silk fibroin.

[0017] E7. A first microneedle comprising an immunomodulatory agent; and a second microneedle containing an immunomodulatory agent; Including, A plurality of microneedles, wherein the first and / or second microneedles comprise silk fibroin, for example, regenerated silk fibroin and / or recombinant silk fibroin.

[0018] E8. The microneedle device of embodiment E4, wherein the silk fibroin tip comprises an anti-cancer agent and / or an immunomodulatory agent. E9. The microneedle device of embodiment E4, wherein the base comprises an anti-cancer agent and / or an immunomodulatory agent.

[0019] E10. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the microneedles are configured to penetrate a biological barrier (e.g., skin).

[0020] E11. The microneedle device of embodiment E4 or E8, wherein the microneedle is configured to embed a silk fibroin tip into a biological barrier (e.g., skin) of a subject.

[0021] E12. The microneedle device of any one of embodiments E1-E4 or E8-E11, wherein the microneedle device is configured to achieve local and / or systemic delivery (e.g., release) of an anti-cancer agent and / or an immunomodulatory agent to a subject.

[0022] E13. The microneedle device of embodiments E1-E4 or E8-E12, wherein the microneedle device is configured to deliver an effective amount of an anti-cancer agent and / or an immunomodulatory agent to a subject.

[0023] E14. The microneedle device of embodiment E4 or E8, wherein the silk fibroin tip comprises regenerated silk fibroin and / or recombinant silk fibroin. E15. The microneedle device or plurality of microneedles of any one of the preceding embodiments, configured to deliver (e.g., release) two or more anti-cancer drugs (e.g., three or more, four or more, or five or more anti-cancer drugs).

[0024] E16. The microneedle device or plurality of microneedles of any one of the preceding embodiments, configured to deliver (e.g., release) two or more immunomodulatory agents (e.g., three or more, four or more, or five or more immunomodulatory agents).

[0025] E17. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein said plurality of microneedles comprises at least one additional microneedle, wherein the additional microneedle comprises the same first anticancer drug.

[0026] E18. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the plurality of microneedles comprises at least one additional microneedle, wherein the additional microneedle comprises an anti-cancer drug different from the first anti-cancer drug (a "second anti-cancer drug").

[0027] E19. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein said plurality of microneedles comprises at least one additional microneedle, wherein the additional microneedle comprises the same first immune modulating agent.

[0028] E20. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein said plurality of microneedles comprises at least one additional microneedle, wherein the additional microneedle comprises an immunomodulating agent different from the first immunomodulating agent (a "second immunomodulating agent").

[0029] E21. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the plurality of microneedles comprises additional silk fibroin-based microneedles comprising a second anti-cancer drug.

[0030] E22. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the plurality of microneedles comprises additional silk fibroin-based microneedles comprising a second immunomodulatory agent.

[0031] E23. The microneedle device or plurality of microneedles of any one of the preceding embodiments, comprising a plurality of said first, second, and / or additional microneedles.

[0032] E24. The microneedle device or plurality of microneedles of any one of embodiments E1, E2, E4-E6, or E8-E23, wherein the anti-cancer agent is selected from one or more of a small molecule (e.g., a chemotherapy drug), a biologic (e.g., an antibody), a viral cancer therapeutic, a nanomedicine, and a nucleic acid molecule (e.g., DNA and / or RNA).

[0033] E25. The microneedle device or plurality of microneedles of any one of embodiments E1, E2, E4-E6, or E8-E24, wherein the anti-cancer agent is not a chemotherapeutic nucleotide.

[0034] E26. The microneedle device or plurality of microneedles of any one of embodiments E1, E2, E4-E6, or E8-E25, wherein the anti-cancer agent is mRNA, and optionally the mRNA encodes an anti-cancer agent and / or an immunomodulatory agent, and optionally the mRNA encodes a checkpoint inhibitor, a TLR agonist, a STING agonist, a RIG agonist, a cancer vaccine, a targeted therapy, and / or a cytokine.

[0035] E27. Anticancer drugs include gemcitabine (GEMZAR®), vemurafenib (ZELBORAF®), dabrafenib (TAFINLAR®), trametinib (MEKINIST®), doxorubicin (ADRIAMYCIN®), encorafenib (BRAFTOVI®), cobimetinib (COTELLIC®), binimetinib (MEKTOVI®), dacarbazine (DTIC), temozolomide (TEMODAR®), ipilimumab (YERVOY®), pembrolizumab (KEYTRUDA®), The microneedle device or plurality of microneedles of any one of embodiments E1, E2, E4-E6, or E8-E26, wherein the microneedle device or plurality of microneedles is selected from one or more of nivolumab (OPDIVO®), aldesleukin (Proleukin®), recombinant interferon alfa-2b (IntronA), pegylated interferon alfa-2b (PEG-Intron / Sylatron), oxaliplatin, and talimogene laherparepvec (IMLYGIC®).

[0036] E28. The microneedle device or plurality of microneedles of any one of embodiments E1, E3-E5, or E7-E27, wherein the immunomodulatory agent is selected from a checkpoint inhibitor, a Toll-like receptor (TLR) agonist, a STING agonist, a RIG agonist, a cancer vaccine, and a cytokine.

[0037] E29. The microneedle device or plurality of microneedles of embodiment E28, wherein the checkpoint inhibitor inhibits a checkpoint molecule selected from CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, and A2aR.

[0038] E30. The microneedle device or plurality of microneedles of embodiment E28 or E29, wherein the checkpoint inhibitor is a PD-1 inhibitor. E31. The microneedle device or plurality of microneedles of embodiment E28 or E29, wherein the checkpoint inhibitor is a CTLA4 inhibitor.

[0039] E32. The microneedle device or plurality of microneedles of embodiment E28, wherein the TLR agonist is selected from a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-10 agonist, a TLR-1 / 2 agonist, a TLR-2 / 6 agonist, or a TLR-7 / 8 agonist.

[0040] E33. The microneedle device or plurality of microneedles of embodiment E28 or E32, wherein the TLR agonist is a TLR-7 agonist. E34. The microneedle device or plurality of microneedles of embodiment E28 or E32, wherein the TLR agonist is a TLR-9 agonist (e.g., an unmethylated CG dinucleotide (CpG ODN).

[0041] E35. The microneedle device or plurality of microneedles of embodiment E28, wherein the STING agonist is a cyclic dinucleotide, such as a cyclic dinucleotide comprising a purine or pyrimidine nucleobase (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobase), optionally bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP).

[0042] E36. The microneedle device or plurality of microneedles of embodiment E28, wherein the cytokine is GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ.

[0043] E37. The microneedle device or plurality of microneedles of embodiment E28, wherein the cytokine is GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, or TNFβ.

[0044] E38. The microneedle device or plurality of microneedles of embodiment E28 or E36, wherein the cytokine is IL-2. E39. The microneedle device or plurality of microneedles of embodiment E28 or E36, wherein the cytokine is IL-12.

[0045] E40. The microneedle device or plurality of microneedles of embodiment E28 or E36, wherein the cytokine is IL-15. E41. The microneedle device or plurality of microneedles of embodiment E28 or E37, wherein the cytokine is IL-18.

[0046] E42. The microneedle device or plurality of microneedles of any one of embodiments E28, E36, or E37, wherein the cytokine is a genetically engineered cytokine.

[0047] E43. The microneedle device or plurality of microneedles of any one of embodiments E28, E36, or E37, wherein the cytokine is an engineered interleukin (e.g., engineered IL-2 or IL-18).

[0048] E44. The microneedle device or plurality of microneedles of any one of embodiments E28, E36, or E37, wherein the cytokine is engineered interleukin-2.

[0049] E45. The microneedle device or plurality of microneedles of embodiment E28 or E37, wherein the cytokine is engineered interleukin-18. E46. The microneedle device or plurality of microneedles of any one of embodiments E28, E36, or E37, wherein the cytokine is a decoy-resistant interleukin.

[0050] E47. The microneedle device or plurality of microneedles of embodiment E28 or E37, wherein the cytokine is decoy-resistant interleukin-18. E48. The microneedle device or plurality of microneedles of embodiment E28 or E36, wherein the cytokine is GM-CSF.

[0051] E49. Anti-PD1 antibody and / or anti-CTLA4 antibody, (i) IL-2; (ii) IL-12; (iii) IL-15; (iv) gemcitabine (GEMZAR®); (v) vemurafenib (ZELBORAF®); (vi) dabrafenib (TAFINLAR®); (vii) trametinib (MEKINIST®); (viii) doxorubicin (ADRIAMYCIN®); (ix) c-di-GMP; (x) mRNA; (xi) TLR-9 agonists (e.g., unmethylated CG dinucleotides (CpG ODNs)); (xii) oxaliplatin; and (xiii) GM-CSF 10. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the microneedle device or plurality of microneedles is configured for administration in combination with one or more of:

[0052] E50. Anti-PD1 antibody and / or anti-CTLA4 antibody, (i) IL-2; (ii) IL-12; (iii) IL-15; (iv) IL-18 (v) gemcitabine (GEMZAR®); (vi) vemurafenib (ZELBORAF®); (vii) dabrafenib (TAFINLAR®); (viii) trametinib (MEKINIST®); (ix) doxorubicin (ADRIAMYCIN®); (x) c-di-GMP; (xi) mRNA; (xii) TLR-9 agonists (e.g., unmethylated CG dinucleotides (CpG ODNs)); (xiii) oxaliplatin; and (xiv)GM-CSF 10. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the microneedle device or plurality of microneedles is configured for administration in combination with one or more of:

[0053] E51. The microneedle device or plurality of microneedles of embodiment E28 or E37, configured to administer an anti-PD1 antibody and / or an anti-CTLA4 antibody in combination with one or more of IL-2, IL-12, or IL-18.

[0054] E52. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the microneedle device or plurality of microneedles is configured to administer an anti-PD1 antibody and / or an anti-CTLA4 antibody in combination with IL-2 and IL-12.

[0055] E53. The microneedle device or plurality of microneedles of any one of the preceding embodiments, wherein the microneedle device or plurality of microneedles is configured to administer an anti-PD1 antibody and / or an anti-CTLA4 antibody in combination with IL-2 and a TLR-9 agonist (e.g., an unmethylated CG dinucleotide (CpG ODN)).

[0056] E54. The microneedle device or plurality of microneedles of any one of the preceding embodiments configured to administer IL-2 and c-di-GMP, and optionally IL-12.

[0057] E55. The microneedle device or plurality of microneedles of any one of the preceding embodiments configured to administer IL-12 and c-di-GMP, and optionally IL-2.

[0058] E56. The method of any one of the preceding embodiments, wherein the method is configured to administer a cancer vaccine, and optionally the cancer vaccine comprises a tumor antigen, e.g., a neoantigen. dollar device or multiple microneedles.

[0059] E57. (i) a base (e.g., a dissolvable base); (ii) an implantable silk fibroin chip comprising silk fibroin applied or adhered to a base; and (iii) (Optional) Backing applied to the base Including, configured to implant the silk fibroin chip into a biological barrier (e.g., skin) of a subject, e.g., a human subject; the silk fibroin chip comprises silk fibroin, e.g., regenerated silk fibroin and / or recombinant silk fibroin; The microneedle, wherein the silk fibroin tip further comprises an anti-cancer drug in an amount sufficient to induce an anti-cancer response.

[0060] E58. A microneedle device or a plurality of microneedles comprising the microneedles of embodiment E57, optionally wherein at least two microneedles of the plurality comprise the same or different anticancer drugs.

[0061] E59.(i) Bases (e.g., soluble bases); (ii) an implantable silk fibroin chip comprising silk fibroin applied to a base; and (iii) (Optional) Backing applied to the base Including, configured to implant the silk fibroin chip into a biological barrier (e.g., skin) of a subject, e.g., a human subject; the silk fibroin chip comprises silk fibroin, e.g., regenerated silk fibroin and / or recombinant silk fibroin; The microneedle, wherein the silk fibroin tip further comprises an immunomodulatory agent in an amount sufficient to stimulate and / or suppress the immune system.

[0062] E60. The microneedle device or plurality of microneedles of any one of embodiments E1, E3-E5, E7-E56, or E59, optionally wherein at least two microneedles of the plurality comprise the same immunomodulatory agent or different immunomodulatory agents.

[0063] E61. A microneedle device comprising a plurality of silk fibroin-based microneedles, wherein said plurality of microneedles comprises one or more microneedles according to any one of the preceding embodiments.

[0064] E62. The microneedle device, plurality of microneedles, or microneedle of any one of the preceding embodiments, wherein the device, plurality, or microneedle is configured for sustained release of an anti-cancer agent and / or an immunomodulatory agent.

[0065] E63. The microneedle device, plurality of microneedles, or microneedles of embodiment E62, wherein the sustained release comprises a substantially continuous low dose administration of the anti-cancer agent and / or immune modulating agent.

[0066] E64. The sustained release comprises continuous administration of greater than about 0% to about 100% of the total amount of anticancer drug and / or immunomodulatory agent present on the silk fibroin chip. The microneedle device, plurality of microneedles, or microneedle of embodiment E62 or E63, comprising:

[0067] E65. The microneedle device, plurality of microneedles, or microneedles of any one of E62-E64, wherein the sustained release is over a period comprising at least about 3 days (e.g., about 3, 4, 5, 6, 7 days or more, e.g., between about 5 and about 10 days, e.g., between about 7 and about 15 days, e.g., between about 1 and about 2 weeks, between about 1 and about 3 weeks, or between about 2 and about 4 weeks, e.g., between about 1 and about 3 months, e.g., between about 2 and about 4 months, e.g., between about 3 and about 6 months).

[0068] E66. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E62-E65, wherein the sustained release is for a period of between about 2 days and about 28 days.

[0069] E67. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E62-E66, wherein the sustained release is for a period of between about 5 days and about 21 days.

[0070] E68. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to release an effective amount of an anti-cancer drug and / or immune modulating agent to enhance exposure of a subject's immune system to cancer-associated neo-antigens (e.g., neo-antigens released following tumor cell lysis), thereby inducing and / or expanding immune effector cells, e.g., T cells, specific for the neo-antigens.

[0071] E69. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to release an effective amount of an anti-cancer drug and / or an immunomodulatory agent to induce T cell activation and / or overcome immunosuppression in the tumor microenvironment.

[0072] E70. The microneedle device, plurality of microneedles, or microneedle of any one of the preceding embodiments, wherein the device, plurality, or microneedle is configured for burst release of the anti-cancer agent and / or immune modulating agent.

[0073] E71. The microneedle device, plurality of microneedles, or microneedles of embodiment E70, wherein the burst release comprises a rapid administration of an anti-cancer agent and / or an immune modulating agent.

[0074] E72. The microneedle device, plurality of microneedles, or microneedles of embodiment E70 or E71, wherein the burst release comprises rapid administration of greater than 0% to about 100% of the total amount of anticancer drug and / or immunomodulatory agent present on the silk fibroin chip.

[0075] E73. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E70-E72, wherein the burst release is over a period comprising at least about 1 hour (e.g., about 1 to about 30 minutes, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 24 hours).

[0076] E74. Any of embodiments E12, E15, E16, or E62-E73, wherein the release of the anticancer drug occurs at substantially the same rate (e.g., simultaneously) as the release of the immunomodulatory agent. 10. The microneedle device, the plurality of microneedles, or the microneedle according to any one of the preceding claims.

[0077] E75. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E12, E15, E16, or E62-E74, wherein release of the anti-cancer drug occurs at a different rate than release of the immunomodulatory agent, such that the anti-cancer drug is released substantially before or substantially after the immunomodulatory agent is released.

[0078] E76. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to be applied (e.g., administered) to a biological barrier selected from a skin layer, a cell membrane, a mucosal surface, the oral cavity, or the buccal cavity.

[0079] E77. The microneedle device, plurality of microneedles, or microneedle of any one of the preceding embodiments, configured to be applied (e.g., administered) to a tumor (e.g., a metastatic tumor).

[0080] E78. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to be applied (e.g., administered) to the location of a tumor after tumor resection, e.g., to induce an immune response against the tumor and / or to remove any cancer cells left behind after resection.

[0081] E79. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to be applied (e.g., administered) to the location of a tumor prior to resection of the tumor, e.g., to induce an immune response against the tumor and / or to remove any cancer cells left behind after resection.

[0082] E80. The microneedle device, plurality of microneedles, or microneedle of any one of the preceding embodiments, configured to be applied (e.g., administered) to the skin.

[0083] E81. The microneedle device, plurality of microneedles, or microneedle of any one of the preceding embodiments, configured to be applied (e.g., administered) to an eye.

[0084] E82. The microneedle device, plurality of microneedles, or microneedle of any one of the preceding embodiments, configured to be applied (e.g., administered) to the oral cavity.

[0085] E83. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to be applied (e.g., administered) intratumorally.

[0086] E84. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to be applied (e.g., administered) around a tumor.

[0087] E85. The microneedle device, plurality of microneedles, or microneedles of any one of the preceding embodiments, configured to be applied (e.g., administered) to or proximal to a skin lesion (e.g., a skin lesion associated with cancer or a precancerous condition).

[0088] E86. By local and / or systemic delivery (e.g., release) (i) inhibition of tumor growth at or near the site of administration; (ii) eliciting a local immune response to eliminate tumors at or near the site of administration; (iii) an increase in activated immune effector cells (e.g., T cells) in the tumor microenvironment; (iv) reduction of local immunosuppressive cells (e.g. regulatory T cells (Tregs)); (v) elicitation of a systemic immune response to eliminate tumors at distant sites; (vi) immunological memory against cancer or precancerous conditions; and / or (vii) an immune response to tumor antigens, e.g., neoantigens; and / or (viii) preventing and / or inhibiting cancer recurrence (e.g., cancer relapse) The microneedle device of embodiment E12, wherein

[0089] E87. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4 or E57-E59, wherein the backing is selected from a solid support, such as a paper-based material, a plastic material, a polymeric material, or a polyester-based material (e.g., Whatman 903 paper, polymeric tape, plastic tape, adhesive-backed polyester tape, or other medical tape).

[0090] E88. Bases (e.g., soluble bases) (i) polysaccharides (e.g., dextran); (ii) disaccharides (e.g., sucrose, maltose, and trehalose); (iii) polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and hyaluronate); (iv) proteins (e.g., gelatin); (v) plasticizers (e.g., glycerol, propanediol); and (vi) surfactants (e.g., octylphenol ethoxylates (e.g., Triton-X), polysorbates, poloxamers, and / or polyethoxylated alcohols); The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E9, E57-E59, or E87, comprising two or more of:

[0091] E89. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E9, E57-E59, E87, or E88, wherein the base comprises one or more of gelatin, dextran, glycerol, polyethylene glycol (PEG) (e.g., including low molecular weight PEG), sucrose, trehalose, maltose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, methylcellulose, and / or surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, e.g., P188, and / or polyethoxylated alcohols), and optionally the microneedles are configured for sustained release and / or burst release.

[0092] E90. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E9, E57-E59, or E87-E89, wherein the base comprises dextran, sucrose, glycerol, and a surfactant, and is optionally configured for sustained release.

[0093] E91. Constructed for sustained release, (i) between about 20% and about 40%, e.g., 30%, 70 kDa dextran; (ii) between about 5% and about 15%, e.g., about 10%, sucrose; (iii) between about 0.5% and about 2.5%, e.g., about 1%, glycerol; and (iv) between about 0.001% and about 1%, e.g., about 0.01% Triton-X (Optionally, this is the solution used for casting and / or the base composition that is dried and solidified). The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E62 to E67, E89, or E90, including

[0094] E92. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E88-E91, wherein the molecular weight of the dextran is between about 30 kD and about 600 kDa.

[0095] E93. The microneedle device, the plurality of microneedles, or the microneedle of any one of embodiments E88-E92, wherein the dextran is derived from Leuconostoc mesenteroides.

[0096] E94. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, E9, E57-E59, E87, or E88-E93, wherein the base comprises polyvinyl alcohol (PVA) and sucrose and is optionally configured for burst release.

[0097] E95. Configured for burst emission: (i) about 15% to about 20%, e.g., about 18%, PVA; (ii) about 25% to about 75% sucrose, e.g., about 50% sucrose; (iii) about 25% to about 75% PVA, e.g., about 50% PVA; and (iv) about 15% to about 20%, e.g., about 18%, sucrose (Optionally, this is the solution used for casting and / or the base composition that is dried and solidified). The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E70-E72, E89, or E94, including:

[0098] E96. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9, E57-E59, E87, or E88-E95, wherein the base does not comprise poly(acrylic acid) (PAA).

[0099] E97.Silk fibroin chips (i) disaccharides (e.g., sucrose, maltose, and trehalose); (ii) polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate); (iii) amino acids (e.g., threonine); (iv) plasticizers (e.g., glycerol, propanediol); and (v) buffer solution (e.g., PBS) The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, E9-E96, comprising two or more of:

[0100] E98. The silk fibroin chip of any of embodiments E4, E8, E9-E97, wherein the silk fibroin chip comprises an excipient. The microneedle device, the plurality of microneedles, or the microneedle described in any one of the above.

[0101] E99. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, E9-E98, wherein the silk fibroin tip comprises one or more of carboxymethylcellulose (CMC), sucrose, and threonine.

[0102] E100. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9-E99, wherein the silk fibroin tip comprises a buffer solution, optionally phosphate buffered saline (PBS).

[0103] E101. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E70-E72, E89, E94, or E95, wherein the silk fibroin tip is configured for burst release and comprises between about 2% and about 8% sucrose (e.g., about 5% sucrose).

[0104] E102. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E70-E72, E89, E94, E95, or E101, wherein the silk fibroin tip is configured for burst release and comprises about 0.5% to about 3% w / v CMC (e.g., about 1% CMC).

[0105] E103. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E70-E72, E89, E94, E95, E101, or E102, wherein the silk fibroin tip is configured for burst release and comprises about 50 mM to about 100 mM of an amino acid, such as threonine (e.g., about 75 mM threonine).

[0106] E104. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) silk fibroin, or silk fibroin having a molecular weight distribution according to Figure 5, or, for example, an amount of silk fibroin between about 20 μg and about 245 μg per 121 microneedle array.

[0107] E105. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) of a 10MB silk fibroin solution, or a silk fibroin solution according to FIG. 5.

[0108] E106. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) of a 60MB silk fibroin solution, or a silk fibroin solution according to Figure 5, such as a 100 kDa to 200 kDa (e.g., about 153 kDa) silk fibroin solution.

[0109] E107. Silk fibroin chips are prepared by dissolving 120MB silk fibroin at about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v). A microneedle device, a plurality of microneedles, or a microneedle described in any one of embodiments E4, E8, or E9 to E103, comprising a solution, or a silk fibroin solution according to FIG. 5, for example, a silk fibroin solution of 70 kDa to 150 kDa (e.g., about 100 kDa).

[0110] E108. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 180MB silk fibroin solution, or a silk fibroin solution according to Figure 5, for example, a 36 kDa to 100 kDa (e.g., about 71 kDa) silk fibroin solution.

[0111] E109. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 480MB silk fibroin solution, or a silk fibroin solution according to Figure 5, for example, a 1 kDa to 60 kDa (e.g., about 16 kDa) silk fibroin solution.

[0112] E110. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E62-E67, E89, E90, or E91, wherein the silk fibroin tip is configured for sustained release and comprises between about 1% and about 10% w / v of a 60MB silk fibroin solution.

[0113] E111. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E62-E67, E89, E90, or E91, wherein the silk fibroin tip is configured for sustained release and comprises between about 1% and about 10% w / v of a 60MB silk fibroin solution.

[0114] E112. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E62-E67, E89, E90, or E91, wherein the silk fibroin tip is configured for sustained release and comprises between about 1% and about 10% w / v of a 120MB silk fibroin solution.

[0115] E113. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E62-E67, E89, E90, or E91, wherein the silk fibroin tip is configured for sustained release and comprises between about 1% and about 10% w / v of a 180MB silk fibroin solution.

[0116] E114. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E62-E67, E89, E90, or E91, wherein the silk fibroin tip is configured for sustained release and comprises between about 1% and about 10% w / v of a 480MB silk fibroin solution.

[0117] E115. The microneedle device, the plurality of microneedles, or the microneedle of any one of the preceding embodiments, comprising substantially only dissolvable material, e.g., substantially only polymer-based and / or sugar-based material.

[0118] E116. The microneedle device of any one of the preceding embodiments, configured to be applied to the location of a tumor, e.g., after resection, and to dissolve completely, leaving it in place. microneedles, multiple microneedles, or microneedles.

[0119] E117. The microneedle device, plurality of microneedles, or microneedles of any one of embodiments E4, E8, or E9-E114, wherein the microneedle is configured to embed the silk fibroin tip into a biological barrier of a subject to a depth of between about 100 μm and about 1 mm (e.g., a maximum penetration depth of the distal portion of the tip).

[0120] E118. The microneedle device, the plurality of microneedles, or the microneedle of any one of the preceding embodiments, wherein the length of the microneedle is between about 350 μm and about 1500 μm.

[0121] E119. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9-E114, or E117, wherein the height of the silk fibroin tip may extend to approximately half the total length of the microneedle.

[0122] E120. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9-E114, or E117, wherein the silk fibroin tip height is between about 75 μm and about 475 μm.

[0123] E121. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9-E114, or E117, wherein the silk fibroin tip comprises a tip radius of between about 0.5 μm and about 100 μm.

[0124] E122. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9-E114, or E117, wherein the silk fibroin tip comprises a tip radius of between about 5 μm and about 10 μm.

[0125] E123. The microneedle device, plurality of microneedles, or microneedle of any one of embodiments E4, E8, E9-E114, or E117, wherein the silk fibroin tip comprises an angle of between about 5 degrees and about 45 degrees.

[0126] E124. A method for treating and / or inducing an immune response against cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) to a subject a site of cancer (e.g., a metastatic tumor) with any one of the microneedle devices or plurality of microneedles of embodiments E1-E56, E58, or E60-E123, such that: (i) lysis of cancer cells, e.g., tumor cells, to release and / or expose cancer-associated antigens (e.g., neoantigens) to the subject's immune system; (ii) presentation of cancer-associated antigens (e.g., neoantigens) complexed with major histocompatibility complexes (MHC) by antigen-presenting cells (APCs) on their surface to immune system cells (e.g., accessory cells, e.g., B cells, dendritic cells, etc.); (iii) recognition of the presented cancer-associated antigen (e.g., neoantigen) by immune effector cells, e.g., T cells and / or NK cells; (iv) activation and / or expansion of immune effector cells, e.g., T cells and / or NK cells, specific for the presented cancer-associated antigen (e.g., neoantigen) in the subject; and (v) enhancing, e.g., stimulating or upregulating, the immune response of immune effector cells, e.g., T cells and / or NK cells, that promote killing and / or inhibiting the growth or proliferation of target cells expressing a cancer-associated antigen (e.g., a neoantigen) in a subject. A method comprising the steps of:

[0127] E125. The method of embodiment E124, wherein the cancer expresses multiple antigens (e.g., neoantigens), but the tumor microenvironment prevents activation of immune effector cells, e.g., T cells, that recognize them.

[0128] E126. The method of embodiment E124 or E125, wherein an enhanced, e.g., stimulated or upregulated, immune response against target cells expressing the cancer-associated antigen (e.g., neoantigen) is achieved at or near the site of administration.

[0129] E127. The method of any one of embodiments E124-E126, wherein the enhanced, e.g., stimulated or upregulated, immune response against target cells expressing a cancer-associated antigen (e.g., a neoantigen) is a systemic immune response (e.g., a broad-based response) to eliminate tumors at distant sites.

[0130] E128. A method for treating and / or inducing an immune response against cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) to a subject a site of cancer (e.g., a metastatic tumor) with a microneedle device or plurality of microneedles of any one of embodiments E1-E56, E58, or E60-E123.

[0131] E129. A method for treating cancer (e.g., metastatic cancer) or a precancerous condition (e.g., a precancerous skin condition), comprising contacting (e.g., administering) a tumor (e.g., a metastatic tumor) or lesion (e.g., a skin lesion) in a subject with a microneedle device or a plurality of microneedles described in any one of embodiments E1-E56, E58, or E60-E123.

[0132] E130. A method for preventing cancer recurrence, comprising contacting (e.g., administering) to a tumor (e.g., on the skin, e.g., a metastatic tumor) in a subject with a microneedle device or a plurality of microneedles described in any one of embodiments E1-E56, E58, or E60-E123.

[0133] E131. A method for treating cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) a microneedle device or plurality of microneedles described in any one of embodiments E1-E56, E58, or E60-E123 to a subject at or proximal to a site of surgical resection.

[0134] E132. A method for treating cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) a microneedle device or a plurality of microneedles described in any one of embodiments E1-E56, E58, or E60-E123 to a location of a tumor (e.g., a metastatic tumor) in a subject, such that: (i) a local immune response and / or local cytotoxicity against the cancer (e.g., killing of cancer cells at or proximal to the location of the applied microneedle device, as evidenced by, e.g., a reduction in local tumor size and / or local tumor burden); and / or (ii) a distal immune response and / or distal cytotoxicity against the cancer (e.g., killing of cancer cells distal to the location of the applied microneedle patch, as evidenced by, e.g., a reduction in distal tumor size and / or tumor burden); A method comprising the step of inducing

[0135] E133. By contacting (e.g., administering) (i) inhibition of tumor growth at or near the site of administration; (ii) eliciting a local immune response to eliminate tumors at or near the site of administration; (iii) an increase in activated immune effector cells (e.g., T cells) in the tumor microenvironment; (iv) reduction of local immunosuppressive cells (e.g. regulatory T cells (Tregs)); (v) elicitation of a systemic immune response to eliminate tumors at distant sites; (vi) immune memory against cancer or precancerous conditions; (vii) an immune response to tumor antigens, e.g., neoantigens; and / or (viii) preventing and / or inhibiting cancer recurrence (e.g., cancer relapse) The method of any one of embodiments E124 to E132, wherein one or more of:

[0136] E134. The method of embodiment E133, wherein immunological memory results in elimination of the recurrent tumor at or near the site of administration and / or elimination of the recurrent tumor at a distant site, optionally if the recurrent tumor appears within about 1 to about 6 months after administration, and optionally if the recurrent tumor appears within about 1 to about 5 years after administration.

[0137] E135. The method of embodiment E133 or E134, wherein immunological memory prevents tumor recurrence. E136. The method of any one of embodiments E133-E135, wherein the immunological memory prevents cancer recurrence within the first 5 years after the initial administration of the microneedles.

[0138] E137. The method of any one of embodiments E124-E136, wherein the cancer is a metastatic cancer. E138. The method of any one of embodiments E124-E137, wherein the cancer is a relapsing cancer.

[0139] E139. The method of any one of embodiments E124-E138, wherein the cancer is a refractory cancer. E140. Cancers include: anal cancer; basal cell carcinoma; bladder cancer; bone cancer; brain tumor; breast cancer; cervical cancer; colorectal cancer; endometrial cancer; esophageal cancer; gastrointestinal stromal tumor; gestational trophoblastic disease; head and neck cancer; Hodgkin's lymphoma; Kaposi's sarcoma; kidney (renal cell) cancer; leukemia; liver cancer; lung cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; multicentric Castleman's disease; multiple myeloma and other plasma cell neoplasms; myeloproliferative neoplasms; neuroblastoma; non-Hodgkin's lymphoma The method of any one of embodiments E124 to E139, wherein the cancer is selected from: tumor; ovarian, fallopian tube, or primary peritoneal cancer; pancreatic cancer; penile cancer; pheochromocytoma and paraganglioma; prostate cancer; retinoblastoma; rhabdomyosarcoma; skin cancer; squamous cell carcinoma; soft tissue sarcoma; any solid tumor in the body; stomach (gastric) cancer; testicular cancer; thyroid cancer; vaginal cancer; vulvar cancer; and Wilms' tumor and other childhood kidney cancers.

[0140] E141. The method of any one of embodiments E124 to E140, wherein the cancer is melanoma. E142. The method of any one of embodiments E124 to E140, wherein the cancer is basal cell carcinoma.

[0141] E143. The method of any one of embodiments E124 to E140, wherein the cancer is squamous cell carcinoma. E144. The method of any one of embodiments E124 to E140, wherein the cancer is Merkel cell carcinoma.

[0142] E145. The method of any one of embodiments E124 to E140, wherein the cancer is breast cancer. E146. The method of any one of embodiments E124-E140, wherein the cancer is associated with skin lesions and / or tumors.

[0143] E147. The method of any one of embodiments E124 to E140, wherein the tumor is a metastatic tumor. E148. The method of any one of embodiments E124-E140, wherein the tumor is accessible without surgery.

[0144] E149. The method of any one of embodiments E124-E140, wherein the tumor is accessible by surgery. E150. The method of any one of embodiments E124-E140, wherein the tumor is present on the skin.

[0145] E151. The method of any one of embodiments E124-E140, wherein the tumor is present in the eye. E152. The method of any one of embodiments E129 or E133-E151, wherein the precancerous condition is a precancerous skin condition optionally selected from actinic keratosis (AK), lentigo maligna, leukoplakia, and Bowen's disease.

[0146] E153. The method of any one of embodiments E124-E152, wherein the contacting (eg, administering) is performed intratumorally. E154. The method of any one of embodiments E124-E152, wherein the contacting (eg, administering) is performed peritumor.

[0147] E155. The method of any one of embodiments E124-E152, wherein the contacting (eg, administering) occurs prior to surgical resection. E156. The method of any one of embodiments E124-E152, wherein the contacting (eg, administering) occurs after surgical resection.

[0148] E157. The method of any one of embodiments E124-E152, wherein the contacting (eg, administering) is performed simultaneously with surgery and / or biopsy taking. E158. The method of any one of embodiments E124 to E152, wherein the contacting (e.g., administering) is performed in combination with standard of care treatment (e.g., for cancer or a precancerous condition) selected from surgery, chemotherapy, immunotherapy, targeted therapy, hormonal therapy, and / or radiation therapy, as appropriate.

[0149] E159. The method of embodiment E158, wherein a standard of care treatment is administered before, after, or simultaneously with the microneedle device. E160. The method of any one of embodiments E124-E159, wherein the subject is a human subject.

[0150] E161. A method for manufacturing a microneedle device, comprising: providing a mold including a mold body having an array of needle cavities having a predetermined shape, e.g., pyramidal and / or conical needle cavities formed therein; filling the tip of the needle cavity with a composition comprising silk fibroin, an anti-cancer drug, and / or an immunomodulatory agent solution; The filled tip of the needle cavity was dried to produce a silk fibroin tip. Optionally, annealing the tip of the needle; Filling the needle cavity of the mold with a base (e.g., a dissolvable base) solution; drying the base solution to form a base layer for the silk fibroin tip; and (optionally) applying a backing to the base layer to create a microneedle device. A method comprising:

[0151] E162. A method for manufacturing a microneedle device, comprising: providing a mold including a mold body having an array of needle cavities having a predetermined shape, e.g., pyramidal and / or conical needle cavities formed therein; filling the tip of the needle cavity with a composition comprising a solution of silk fibroin and a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or both); drying the filled tip of the needle cavity to produce a silk fibroin tip, and optionally annealing the silk fibroin tip; further filling the needle cavity of the mold with a first base (e.g., dissolvable base) solution; drying the first base solution to form a first base layer; (Optionally) forming one or more additional base layers by adding one or more additional base solutions to the first base layer and drying the additional base solutions, where the additional base solutions are optionally different from the first base solution; applying a backing (if necessary) to the base layer (e.g., the first base layer or one or more additional base layers), thereby producing a microneedle device; A method comprising:

[0152] E163. The method of embodiment E161 or E162, wherein the base solution (eg, the first base solution, the one or more additional base solutions, or both) comprises a melt liquid.

[0153] E164. The method of embodiment E161 or E162, wherein the base solution (eg, the first base solution, the one or more additional base solutions, or both) comprises a slurry.

[0154] E165. The method of any one of embodiments E161-E163, wherein filling (eg, into a mold or needle cavity) comprises filling with a base solution comprising a molten liquid.

[0155] E166. The method of any one of embodiments E161-E165, wherein filling (eg, into a mold or needle cavity) comprises filling with a base solution comprising a slurry.

[0156] E167. The method of any one of embodiments E161-E166, further comprising solidifying the base layer (e.g., the first base layer, the one or more additional base layers, or both) using a chemical reaction (e.g., after filling).

[0157] E168. The method of any one of embodiments E161-E167, further comprising, if desired, removing the microneedle device from the mold before applying the backing.

[0158] E169. The method of any one of embodiments E161-E168, wherein the microneedle device is removed by bending the mold away from the microneedle device.

[0159] E170. The method of any one of embodiments E161 to E169, further comprising packaging the microneedle device in a container with low water vapor transmission rate and a desiccant to maintain a relative humidity inside the package of between about 0% and about 50% (e.g., between about 0% and 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, or between about 40% and 50%, e.g., about 25%).

[0160] E171. The method of any one of embodiments E161-E170, wherein the silk fibroin, anti-cancer drug, and / or immunomodulatory agent solution is dispensed into each needle cavity of the mold via nanoliter printing.

[0161] E172. The method of any one of embodiments E161-E171, wherein filling the tips of the needle cavities comprises dispensing a solution, such as a silk fibroin, anti-cancer drug, and / or immunomodulatory agent solution, into each needle cavity.

[0162] E173. The method of any one of embodiments E161-E172, wherein drying the filled tip of the needle cavity comprises a primary drying step and a secondary drying step.

[0163] E174. The method of any one of embodiments E161 to E173, wherein drying the base (e.g., dissolvable base) solution comprises centrifuging the mold at 3900 rpm for 2 minutes to finish the needle cavity containing 50 μL of base solution.

[0164] E175. Embodiments E161-E1, in which the filling of the base is performed by nanoliter (nL) dispensing (e.g., nanoliter printing). 74. The method according to any one of claims 1 to 74.

[0165] E176. The method of any one of embodiments E161-E175, further comprising the step of annealing after filling the tip of the needle cavity (eg, before filling the base).

[0166] E177. The method of any one of embodiments E161-E176, further comprising the step of annealing in water after filling the tip of the needle cavity (eg, before filling the base).

[0167] E178. The method of any one of embodiments E161-E177, wherein the backing layer comprises one of a paper backing layer and an adhesive plastic tape. E179. The method of any one of embodiments E161-E178, wherein the backing layer comprises an adhesive-coated plastic tape.

[0168] E180. The method of any one of embodiments E161-E179, wherein the backing layer comprises a porous layer. E181. The method of any one of embodiments E161-E180, wherein the backing layer comprises one or more adhesives selected from the group consisting of acrylic, acrylate, cyanoacrylate, silicone, polyurethane, and synthetic rubber.

[0169] E182. The method of any one of embodiments E161-E181, wherein the backing layer comprises an adhesive that can be cured by irradiation with light. E183. A microneedle device, a plurality of microneedles, a microneedle, or a component thereof, containing about 0.5 μg to about 500 μg of silk fibrions (e.g., about 0.5 μg to about 5 μg, or about 1 μg to about 10 μg, or about 5 μg to about 15 μg, or about 10 μg to about 20 μg, or about 15 μg to about 25 μg, or about 20 μg to about 30 μg, or about 25 μg to about 35 μg, or about 30 μg to about 40 μg, or about 35 μg to about 45 μg, or about 40 μg to about 50 μg, or about 45 μg to about 55 μg, or about 50 μg to about 60 μg, or about 55 μg to about 65 μg, or about 60 μg to about 70 μg, or about 65 μg to about 75 μg, or about 70 μg to about 80 μg). The microneedle device, plurality of microneedles, microneedles, or method of any one of embodiments E1-E182, comprising silk fibrions in an amount of from about 80 μg to about 80 μg, or from about 75 μg to about 85 μg, or from about 80 μg to about 90 μg, or from about 85 μg to about 95 μg, or from about 90 μg to about 100 μg, or from about 95 μg to about 150 μg, or from about 125 μg to about 175 μg, or from about 150 μg to about 200 μg, or from about 225 μg to about 275 μg, or from about 250 μg to about 300 μg, or from about 325 μg to about 375 μg, or from about 350 μg to about 400 μg, or from about 425 μg to about 475 μg, or from about 450 μg to about 500 μg of silk fibrions.

[0170] E184. The microneedle device, plurality of microneedles, microneedle, or method of any one of embodiments E1-E183, wherein the microneedle device, plurality of microneedles, microneedle, or component thereof comprises silk fibrions in an amount of about 1% to about 75% by weight silk fibrions (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight silk fibrions). [Brief explanation of the drawings]

[0171] [Figure 1]1A-1D are schematic diagrams of a microneedle manufacturing process according to examples of the present disclosure. [Figure 2] 1 is a series of microneedles configured to release various therapeutic agents via sustained release or via burst (also referred to as "bolus") release, according to examples of the present disclosure. The leftmost microneedle is configured to release an anti-cancer agent, such as a chemotherapeutic agent, over a two-week period. The middle microneedle is configured to release an immune modulating agent, such as a checkpoint inhibitor (e.g., an anti-PD1 antibody), over a two-week period. The rightmost microneedle is configured to rapidly release an immune modulating agent, such as a cytokine (e.g., IL-2), over a short period of time, e.g., several minutes. [Figure 3] FIG. 1 shows a completed microneedle device having an array of microneedles applied to a backing or "handle" layer according to an embodiment of the present disclosure. [Figure 4] 1 illustrates a microneedle device according to an example of the present disclosure, comprising a plurality of microneedles having sufficient mechanical properties (e.g., strength) and suitable geometries (e.g., tip sharpness, tip point angle, length, and inter-needle spacing) to penetrate a biological barrier (e.g., skin) to achieve local and / or systemic delivery of a therapeutic agent or combination of therapeutic agents (e.g., anti-cancer agents, immunomodulatory agents, or combinations thereof) to a subject. [Figure 5] FIG. 1 shows various molecular weight profiles of silk fibroin solutions useful for fabricating microneedles as described herein. [Figure 6A] FIG. 1 shows the dosing regimens used to compare bolus intratumoral (IT) doses with daily IT doses of IL-2 or gemcitabine in an exemplary mouse model. [Figure 6B] FIG. 1 is a graph depicting tumor volume over time in mice treated with either bolus IL-2 (IT) or daily IL-2 (IT). [Figure 6C]Graph comparing survival in mice treated with either bolus (IT) or daily IL-2 (IT). [Figure 6D] 1 is a graph depicting tumor volume over time in mice treated with either bolus or daily gemcitabine (IT). [Figure 6E] 1 is a graph depicting survival in mice treated with either bolus (IT) or daily gemcitabine (IT). [Figure 7A] 7A-7B show the dosage regimens used to compare bolus intratumoral (IT) gemcitabine (FIG. 7A) with daily IT gemcitabine (FIG. 7B) in an exemplary mouse model. [Figure 7B] 7A-7B show the dosage regimens used to compare bolus intratumoral (IT) gemcitabine (FIG. 7A) with daily IT gemcitabine (FIG. 7B) in an exemplary mouse model. [Figure 7C] 1 is a graph depicting tumor volume over time in mice treated with either bolus (IT) or daily (IT) gemcitabine. [Figure 7D] 1 is a graph depicting survival in mice treated with either bolus (IT) or daily (IT) gemcitabine. [Figure 8] 1 is a graph depicting the stability of IL-2 in exemplary silk formulations over 14 days at 4° C., room temperature (RT), or 37° C., as determined by % IL-2 recovery. DETAILED DESCRIPTION OF THE INVENTION

[0172] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0173] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0174] The term "about," when referring to a measurable value, e.g., amount, duration over time, etc., is meant to encompass a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variation is appropriate, for example, for performing the disclosed methods.

[0175] The terms "combination" or "in combination with" are not intended to suggest that therapies or therapeutic agents may be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope described herein. Therapeutic agents in a combination may be administered simultaneously with, prior to, or after one or more other additional therapies or therapeutic agents. Therapeutic agents or therapeutic protocols may be administered in any order. Generally, each agent will be administered at a dose and / or time schedule determined for that agent. It will further be understood that additional therapeutic agents utilized in the combination may be administered together in a single composition or separately in separate compositions. Generally, it is expected that additional therapeutic agents utilized in a combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in the combination will be lower than those utilized individually.

[0176] The terms "inhibition," "inhibitor," or "antagonist" include a decrease in a particular parameter, e.g., the activity of a given molecule, and include, for example, immune checkpoint inhibitors. For example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more inhibition of activity, e.g., PD-1 or PD-L1 activity, is encompassed by the term; thus, inhibition need not be 100%.

[0177] The terms "activation," "activator," or "agonist" include an increase in a particular parameter, e.g., the activity of a given molecule, including, e.g., a costimulatory molecule. For example, an increase in activity, e.g., costimulatory activity, of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more is encompassed by the term.

[0178] The terms "anti-tumor effect" and "anti-cancer effect" are used interchangeably and refer to a biological effect that can be demonstrated by various means, including, but not limited to, a reduction in tumor or cancer volume, a reduction in the number of tumor or cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor or cancer cell proliferation, a reduction in tumor or cancer cell survival, prevention of recurrence, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" or "anti-cancer effect" may also be demonstrated by the ability of the microneedles of the present disclosure in preventing the appearance of a tumor or cancer in the first place and / or the recurrence or relapse of cancer. An "anti-tumor effect" or "anti-cancer effect" may also be demonstrated by the ability of the microneedles of the present disclosure in generating an immune response against cancer, for example, an immune response against a cancer-associated antigen (e.g., a neoantigen).

[0179] As used herein, "tumor antigen" or, equivalently, "cancer antigen" includes any molecule present on or associated with a cancer, e.g., a cancer cell or tumor microenvironment, that can elicit an immune response. As used herein, "immune cell antigen" includes any molecule present on or associated with an immune cell that can elicit an immune response. In some embodiments, tumor antigen refers to a neoantigen (e.g., an antigen such as a peptide resulting from a somatic mutation in a tumor that, unlike the wild-type antigen, may be specific to each tumor and / or subject).

[0180] As used herein, the term "anti-cancer agent" refers to a therapy and / or drug that can induce an anti-tumor and / or anti-cancer effect. In some embodiments, the anti-cancer effect includes, but is not limited to, a decrease in tumor or cancer volume, a decrease in the number of tumor or cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in tumor or cancer cell proliferation, a decrease in tumor or cancer cell survival, prevention of relapse, or an improvement in various physiological symptoms associated with a cancerous condition.

[0181] As used herein, "increase" or "decrease" in measured value is typically compared to baseline value unless otherwise specified.For example, the increase or decrease in measured value may be compared to the baseline level of the measured value expected for a healthy subject.Alternatively, the increase or decrease in measured value may be compared to a previous time point for the same subject, for example, before treatment.In some cases, the increase or decrease in measured value may be compared to a previous time point for the same subject, for example, during the course of treatment.

[0182] As used herein, the term "immunomodulatory agent" refers to a therapy and / or drug that can modulate (e.g., increase and / or decrease), enhance, induce, stimulate, suppress, reduce, or upregulate one or more aspects of the immune response, e.g., in a subject with cancer. For example, an immunomodulatory agent can enhance or promote immune attack of target cells, e.g., cancer cells, and / or enhance immune function. In some embodiments, an immunomodulatory agent administered as described herein, e.g., by a microneedle or device described herein, can enhance a subject's immune response to cancer.

[0183] As used herein, the term "cancer" is meant to include all types of cancerous growths or tumorigenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of cancerous disorders include, but are not limited to, solid tumors, blood cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas) of various organ systems, such as those affecting the liver, lung, breast, lymphatic system, gastrointestinal (e.g., colon), genitourinary (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant forms of most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. Malignant squamous cell carcinomas include those affecting the lungs, esophagus, skin, head and neck region, oral cavity, anus, and cervix. Metastatic lesions of the aforementioned cancers may also be treated or prevented using the methods and microneedles of the present disclosure.

[0184] The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0185] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, e.g., an accessory cell (e.g., B cell, dendritic cell, etc.), that displays foreign antigens complexed with major histocompatibility complexes (MHC) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0186] As used herein, "immune cell" refers to any of a variety of cells that function in the immune system, protecting against, for example, infectious diseases and foreign agents. In embodiments, the term includes white blood cells, such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate white blood cells include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate white blood cells identify and eliminate pathogens by attacking large pathogens through contact or by engulfing and then killing the microorganisms, and are mediators in the activation of the adaptive immune response. Cells of the adaptive immune system are specialized types of white blood cells called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, while T cells are involved in cell-mediated immune responses. The term "immune cell" includes immune effector cells.

[0187] "Immune effector cells" or "effector cells," as these terms are used herein, refer to cells that are involved in an immune response, for example, promoting an immune effector response. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0188] An "immune effector" or "effector" "function" or "response," as these terms are used herein, refers to a molecule that enhances or inhibits immune attack of a target cell, for example. It refers to the function or response of immune effector cells that promotes.For example, immune effector function or response refers to the property of T cell or NK cell that promotes killing or inhibiting the growth or proliferation of target cell.For T cell, primary stimulation and costimulation are examples of immune effector function or response.

[0189] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be cytolytic activity or helper activity, including, for example, the secretion of cytokines.

[0190] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a disorder, e.g., a proliferative disorder, or an improvement in one or more symptoms (preferably one or more discernible symptoms) of the disorder resulting from the administration of one or more therapies. In specific embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, e.g., tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of the progression of a proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0191] The terms "polypeptide," "peptide," and "protein" (when used in a single chain) are used interchangeably herein to refer to amino acid polymers of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation, e.g., conjugated with a labeling component. Polypeptides may be isolated from natural sources, produced by recombinant technology from eukaryotic or prokaryotic hosts, or the product of synthetic procedures.

[0192] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be either single-stranded or double-stranded, and, if single-stranded, may be the coding strand or non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, either not occurring in nature or linked to another polynucleotide in a non-natural arrangement.

[0193] The term "substantially identical," as used herein in the context of nucleotide sequences, refers to a first nucleic acid sequence that includes a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or that encode a common structural polypeptide domain or a common functional polypeptide activity, e.g., a first nucleic acid sequence that has at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence. It refers to a nucleotide sequence, such as the sequences provided herein.

[0194] The term "variant" refers to a polypeptide having substantially the same amino acid sequence as, or encoded by a substantially identical nucleotide sequence to, a reference amino acid sequence. In some embodiments, the variant is a functional variant.

[0195] The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially identical to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and that can possess one or more activities of the reference amino acid sequence.

[0196] The term "cytokine" (e.g., GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ) includes full-length, fragments, or variants, e.g., functional variants, of naturally occurring cytokines (including fragments and functional variants thereof that have at least 10%, 30%, 50%, or 80% of the activity, e.g., immunomodulatory activity, of the naturally occurring cytokine). In some embodiments, the cytokine has an amino acid sequence that is substantially identical (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a naturally occurring cytokine, or that is encoded by a nucleotide sequence that is substantially identical (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a naturally occurring nucleotide sequence encoding the cytokine. In some embodiments, as understood in context, the cytokine further comprises a receptor domain, e.g., a cytokine receptor domain (e.g., IL-15 / IL-15R).

[0197] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result.

[0198] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, remission, or eradication of a disease state (e.g., cancer). The term "prophylaxis," as used herein, means the prevention or protective treatment of a disease or condition (e.g., cancer).

[0199] "Refractory," as used herein, refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to as a resistant cancer.

[0200] "Relapse" or "relapse," as used herein, refers to the return or reappearance of a disease (e.g., cancer) or signs and symptoms of a disease, such as cancer, after a period of improvement or response, e.g., after a pretreatment therapy, e.g., cancer therapy. The initial period of response may involve a decrease in the level of cancer cells below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may involve a rise in the level of cancer cells above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of some cancers, reappearance may involve, for example, the reappearance of a tumor after a response. In some embodiments, a response (e.g., complete or partial response) may involve the absence of detectable tumor or detectable MRD (minimal residual disease). In some embodiments In, the initial period of response lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0201] Ranges: Throughout this disclosure, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to include all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, a description of a range, e.g., 1 to 6, should be considered to include specifically disclosed subranges, e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range, e.g., 95-99% identity, includes those with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges, e.g., 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

[0202] As used herein in some embodiments in connection with cancer treatment, "adjuvant" refers to additional cancer treatment given after the initial treatment to reduce the risk that the cancer will recur. Adjuvant therapy can be, for example, chemotherapy, radiation therapy, hormone therapy, targeted therapy, or biological therapy.

[0203] As used herein in some embodiments in connection with vaccine delivery, an "adjuvant" refers to a substance that can aid or amplify the cascade of immunological events, ultimately increasing an immunological response, including cellular and / or humoral immune responses, such as the body's overall response to an antigen. Non-limiting examples of adjuvants include aluminum (e.g., aluminum gel and / or aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), lipids (e.g., squalene, monophosphoryl lipid A (MPL)), AS03 (e.g., an adjuvant comprising D,L-alpha-tocopherol (vitamin E), squalene, and polysorbate 80), AS04 (e.g., an adjuvant comprising a combination of aluminum hydroxide and MPL), and MF59® (e.g., an adjuvant comprising squalene).

[0204] As used herein, the term "backing" refers to a material suitable for bonding and / or adhering to a component of a microneedle. In some embodiments, the backing material is suitable for bonding and / or adhering to the dissolvable base of a microneedle described herein.

[0205] As used herein, the term "base" refers to a layer that forms the base of the microneedle (e.g., serves as a support for a distal silk tip loaded with an anti-cancer drug, an immunomodulatory agent, or a combination thereof) and / or may also serve as a layer connecting adjacent microneedles to form a continuous microneedle array or microneedle patch. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the base dissolves after application to a biological barrier, e.g., skin, tumor, tissue, cell membrane, mucosal surface, oral cavity, or buccal cavity.

[0206] As used herein, the term "dose" refers to a dose that is sufficient to induce an anti-cancer response and / or an immune response (e.g., humoral and / or cellular immune response) in an organism. By "anti-cancer agent" is meant the amount of anti-cancer agent and / or immunomodulatory agent administered (e.g., as a microneedle as described herein) to a target tissue.

[0207] As used herein, "standard dose" refers to the amount of an anti-cancer agent and / or immune modulator administered in a typical human dose, e.g., as approved for sale by a national or international regulatory authority (e.g., USFDA, EMEA).

[0208] As used herein, a "split dose" refers to a dosage that includes a fraction of a total dose (e.g., a standard dose) of an anti-cancer agent and / or immunomodulatory agent administered (e.g., as a microneedle) to induce an anti-cancer and / or immune response in an organism. In some embodiments, the amount of the anti-cancer agent and / or immunomodulatory agent administered in a split dose is 1 / X or less of the total dose (e.g., a standard dose) of the anti-cancer agent and / or immunomodulatory agent administered, where X is any number, for example, X is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more.

[0209] As used herein, the term "gelatin" refers to a water-soluble protein derived from collagen. In some embodiments, the term "gelatin" refers to a sterile, non-pyrogenic protein preparation (e.g., fraction) produced by partial acid hydrolysis (type A gelatin) or partial alkaline hydrolysis (type B gelatin) of animal collagen, typically derived from bovine, porcine, and fish sources. Gelatin can be obtained in a variety of molecular weight ranges. Recombinant sources of gelatin may also be used.

[0210] As used herein, the term "polyethylene glycol (PEG)" refers to an oligomer or polymer of ethylene oxide. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE). The structure of PEG is H—(O—CH—CH) n It is usually shown as -OH.

[0211] As used interchangeably herein, the term "sustained-release silk fibroin tip" refers to the distal end, e.g., tip, of a microneedle that can penetrate a biological barrier of a subject, e.g., the skin, a mucosal surface, a tumor, the oral cavity, or the buccal cavity, and deposit within the biological barrier, a skin layer (e.g., the dermis). In embodiments, the tip comprises silk fibroin protein in an amount sufficient to sustain-release a therapeutic agent, e.g., an anti-cancer agent and / or an immunomodulatory agent, over an extended period of time, e.g., at least about 1 day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, e.g., about 4 days to about 30 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., about 2-12 months).

[0212] As used herein, the term "microneedle" refers to a structure having at least two, more typically three, components, e.g., layers, for transporting or delivering a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, through a biological barrier, e.g., skin, tissue, tumor, or cell membrane. In some embodiments, the microneedle comprises a base (e.g., a dissolvable base described herein), a tip (e.g., an implantable tip described herein), and, optionally, a backing material. In embodiments, the dimensions of the microneedle are between about 350 μm and about 1500 μm (e.g., between about 350 μm and about 1500 μm, e.g., about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, or a combination thereof). In some embodiments, the microneedles have a height of about 950 μm, about 1000 μm, about 1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1250 μm, about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about 1500 μm). In some embodiments, the microneedles are fabricated to have any dimension and / or geometry that allows for the placement of a silk fibroin tip, e.g., an implantable sustained release tip, into the dermal layer of the skin at a depth of between about 100 μm and about 900 μm (e.g., at a depth of about 800 μm) to release, e.g., controlled-release or sustained-release, an anti-cancer agent, an immunomodulatory agent, or a combination thereof.

[0213] As used herein, the terms "microneedle patch" and "microneedle array" refer to a device comprising a plurality of microneedles, e.g., silk fibroin-based microneedles, arranged, e.g., randomly or in a predetermined pattern, e.g., an array. In some embodiments, the microneedle patch or microneedle array of the present disclosure may comprise silk fibrion in an amount of about 0.5 μg to about 500 μg of silk fibrion. In some embodiments, the microneedle patch or microneedle array of the present disclosure provides a microneedle patch or microneedle array having a concentration of from about 0.5 μg to about 5 μg, or from about 1 μg to about 10 μg, or from about 5 μg to about 15 μg, or from about 10 μg to about 20 μg, or from about 15 μg to about 25 μg, or from about 20 μg to about 30 μg, or from about 25 μg to about 35 μg, or from about 30 μg to about 40 μg, or from about 35 μg to about 45 μg, or from about 40 μg to about 50 μg, or from about 45 μg to about 55 μg, or from about 50 μg to about 60 μg, or from about 55 μg to about 65 μg, or from about 60 μg to about 70 μg, or about 65 μg The silk fibrion may be present in an amount of from about 75 μg to about 80 μg, or from about 75 μg to about 85 μg, or from about 80 μg to about 90 μg, or from about 85 μg to about 95 μg, or from about 90 μg to about 100 μg, or from about 95 μg to about 150 μg, or from about 125 μg to about 175 μg, or from about 150 μg to about 200 μg, or from about 225 μg to about 275 μg, or from about 250 μg to about 300 μg, or from about 325 μg to about 375 μg, or from about 350 μg to about 400 μg, or from about 425 μg to about 475 μg, or from about 450 μg to about 500 μg. In some embodiments, the microneedle patch or microneedle array of the present disclosure may comprise silk fibrions in an amount of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions.In some embodiments, the microneedle patch or microneedle array of the present disclosure may comprise silk fibrions in an amount of up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions. In some embodiments, the microneedle patch or microneedle array of the present disclosure may comprise silk fibrions in an amount of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions.

[0214] In some embodiments, the microneedle patch or microneedle array of the present disclosure comprises about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% silk fibrions by weight. In some embodiments, the microneedle patch or microneedle array of the present disclosure comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 2 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions. In some embodiments, the microneedle patch or microneedle array of the present disclosure comprises up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions. In some embodiments, the microneedle patch or microneedle array of the present disclosure comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions.

[0215] As used herein, the term "silk fibroin" includes silkworm fibroin and insect or spider silk proteins. Either type of silk fibroin may be used in accordance with various aspects described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an environmentally friendly, renewable resource. For example, silk fibroin used in microneedles (e.g., silk fibroin tips, e.g., implantable controlled-release or sustained-release tips of microneedles) may be obtained by removing sericin from B. mori cocoons. In some embodiments, the silk fibroin is regenerated silk fibroin, e.g., silk fibroin obtained after extracting sericin from B. mori cocoons and performing additional processing, e.g., via a boiling step. Organic silkworm cocoons are also commercially available. However, many different silks exist and may be used, including spider silk (e.g., obtained from Nephila clavipes), transgenic silk, recombinant and / or engineered silk, e.g., silk from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants (see, e.g., WO 97 / 08315; U.S. Pat. No. 5,245,012), and variants thereof.

[0216] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In certain embodiments of the aspects described herein, the subject is a mammal (e.g., a primate, e.g., a human). The subject may be male or female. In certain embodiments, the subject is a mammal. The mammal may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Additionally, the methods and formulations described herein may be used to treat domestic animals and / or pets. In some embodiments, the term "subject" is intended to include organisms in which an immune response can be elicited (e.g., mammals, e.g., humans).

[0217] As used herein, the terms "release" and "controlled or sustained release" refer to the release of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof (e.g., from a microneedle, microneedle device, formulation, composition, article, device, or formulation described herein) over a period of time, e.g., for at least about 1 to about 28 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days or more, e.g., between about 4 days and about 14 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., between about 1 month and about 3 months). For example, from a silk fibroin-based microneedle tip described herein. In some embodiments, controlled or sustained release of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or combination thereof, by a microneedle, microneedle device, formulation, composition, article, device, or formulation described herein over a period of about 1 to about 14 days, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, may result in, for example, an anti-cancer and / or immune response in a subject. In some embodiments, controlled or sustained release of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or combination thereof, by a microneedle, microneedle device, formulation, composition, article, device, or formulation described herein over a period of about 1 to about 4 weeks, e.g., about 1, 2, 3, or 4 weeks, may result in, for example, an anti-cancer and / or immune response in a subject. In some embodiments, formulations and preparations comprising silk fibroin and a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, have controlled- or sustained-release properties (e.g., formulated and / or configured to release a therapeutic agent to, e.g., the skin of a subject, over a period of 1, 5, 10, 15, 30, or 45 minutes, or at least these periods; 1, 2, 3, 4, 5, 10, or 24 hours, or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at least these periods; 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or at least these periods; 1, 2, 3, 4, 5 years, or at least these periods or more). In some embodiments, the controlled- or sustained-release is via burst release. In some embodiments, the microneedles and microneedle devices described herein may be configured for sustained release of an anti-cancer agent, such as a chemotherapeutic agent, for about 28 days or more.

[0218] As used herein, the terms "therapeutic agent" and "active agent" are art-recognized terms and refer to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Various forms of therapeutic agents may be used that can be released from the microneedles described herein into adjacent tissues or bodily fluids upon administration to a subject.

[0219] Various embodiments of the compositions and methods herein are described in further detail below. Additional definitions are set forth throughout the specification. explanation Provided herein are silk fibroin-based microneedles and silk fibroin-based microneedle devices (e.g., microneedle patches) that are configured to incorporate and subsequently release (e.g., administer) an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) in a subject (e.g., into and / or through a biological barrier, e.g., the skin, of the subject). Use of silk fibroin-based microneedles and silk fibroin-based microneedle devices (e.g., microneedle patches) may result in anti-cancer effects and / or immunity (e.g., long-term broad-spectrum immunity to cancer-associated antigens, e.g., neoantigens) to cancer in patients.

[0220] Without wishing to be bound by theory, administration of the microneedles or microneedle devices disclosed herein to an application site on a subject (e.g., the site of abnormal cells, e.g., a tumor or lesion) results in the release of an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) into the subject, thereby eliciting a local therapeutic effect (e.g., a local anti-cancer effect) at or near the administration site. In addition to this local therapeutic effect, the microneedles or microneedle devices disclosed herein may also be used to treat cancer. Administration of the device also results in a systemic therapeutic effect (e.g., a systemic anti-cancer effect) at a distant site (e.g., a distant site of abnormal cells, e.g., a distant tumor or distant lesion) that has characteristics similar to the site of application.

[0221] In some embodiments, the microneedles or microneedle devices disclosed herein may be administered to a subject to provide sustained release of an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) in the subject over a predefined period of time, e.g., 4 to 15 days. In some embodiments, the microneedles or microneedle devices disclosed herein may be administered to a subject to provide sustained release of an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) in the subject over a predefined period of time, such as at least 1, 5, 10, 15, 30, 45 minutes; 1, 2, 3, 4, 5, 10, 24 hours, or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8 weeks, or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or at least these periods; or 1, 2, 3, 4, 5 years, or at least these periods or more.

[0222] In some embodiments, the microneedles or microneedle devices disclosed herein may be administered to a subject to provide a burst release of an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) into the subject over a predefined period of time, e.g., about 24 hours or less. In some embodiments, the microneedles or microneedle devices disclosed herein may be administered to a subject to provide a burst release of an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) into the subject over a predefined period of time, e.g., at least 1, 5, 10, 15, 30, 45 minutes; or 1, 2, 3, 4, 5, 10, 24 hours, or at least these periods.

[0223] In some embodiments, the microneedles or microneedle devices disclosed herein may be administered in combination with a second therapeutic agent or procedure. Accordingly, disclosed herein are manufacturing processes for making improved silk fibroin-based microneedles and devices containing same for use in treating disease in a subject. Silk fibroin-based microneedles In some embodiments, the present disclosure provides silk fibroin-based microneedles and microneedle devices (e.g., microneedle patches) for transporting and releasing an effective amount of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, into and / or across a biological barrier (e.g., skin, tumor, mucosa, tissue, such as organ tissue and muscle tissue, buccal cavity, oral cavity, or cell membrane).

[0224] Thus, the silk fibroin-based microneedles and microneedle devices disclosed herein may be configured to have a variety of mechanical properties (e.g., strength), designs and geometries (e.g., needle shape and sharpness), and release kinetics (e.g., sustained release and / or burst release) to enable the administration of an effective amount of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, to a subject to treat, for example, a disease or disorder, e.g., cancer and / or a skin condition.

[0225] Mechanical properties Microneedles, including silk fibroin-based microneedles, as disclosed herein The needle may be designed to be inserted into the skin without breaking. In some embodiments, microneedle insertion is achieved by using a needle with a sharp tip and sufficient length to overcome surface deflections of the biological barrier (e.g., skin) prior to insertion. In some embodiments, the integrity of the microneedle during insertion may be achieved by minimizing the required insertion force, e.g., by using a sharp-tipped needle, maximizing mechanical strength, and / or optimizing the needle diameter (see, e.g., Park et al. J. Korean Phys. Soc. 56(4):1223-1227, 2010). Thus, in some embodiments, the mechanical properties of the silk fibroin-based microneedle are optimized (e.g., by adjusting the concentrations of various formulation components, including silk fibroin crystallinity, as disclosed herein) to avoid sudden microneedle failure due to buckling and enable successful penetration and insertion of the microneedle into a biological barrier (e.g., skin, tumor, tissue, cell membrane, mucosal surface, oral cavity, or buccal cavity). In some embodiments, the microneedles disclosed herein may be configured to have a profile with a length-to-equivalent diameter aspect ratio of less than 4:1 and / or have mechanical strength characterized by a Young's modulus greater than 500 MPa and a fracture stress greater than 10 MPa. In some embodiments, the microneedles have a tip angle of 15 degrees and / or an aspect ratio of about 4:1. In some embodiments, the base formulation has a flex modulus of about 1000 to about 1500 MPa and a fracture stress of about 15 to about 30 MPa. In some embodiments, the microneedles disclosed herein may be configured to have a profile with a length-to-equivalent diameter aspect ratio of less than 2:1. In some embodiments, the microneedles have a tip angle of about 5 degrees to about 50 degrees. For example, the microneedles may have a tip angle of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 degrees. In some embodiments, the microneedles have a tip angle of 30 degrees.In some embodiments, the microneedles have a tip angle of 30 degrees and / or an aspect ratio of about 1.87: 1. In some embodiments, the base formulation has a flexural modulus of about 50 to about 1500 MPa (e.g., about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 560, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 MPa) and / or a flexural stress at break of about 1 to about 30 MPa (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 MPa).

[0226] Microneedle Design In some embodiments, the present disclosure provides silk fibroin-based microneedles and devices comprising the same, having various design configurations. The silk fibroin-based microneedles disclosed herein may be of any shape and / or geometry suitable for use in penetrating biological barriers (e.g., skin, tumors, tissues, cell membranes, mucosal surfaces, the oral cavity, or the buccal cavity) to enable the release, for example, of sustained and / or burst release, of therapeutic agents (e.g., anticancer agents, immunomodulatory agents, or combinations thereof) within a subject. Non-limiting examples of the shape and / or geometry of the microneedles include cylindrical, wedge, conical, pyramidal, and / or irregular shapes, or any combination thereof.

[0227] In some embodiments, the silk fibroin-based microneedles are comprised of dissolvable and / or degradable microneedles. In some embodiments, the dissolvable and / or degradable (e.g., resorbable) microneedles of the present disclosure encapsulate a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) in a formulation, such as a silk fibroin-based formulation, that dissolves and / or degrades upon entry into a subject (e.g., skin). In some embodiments, the degradable microneedles Release of the therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the microneedle is by protease-mediated degradation. In some embodiments, only a portion (e.g., the tip, e.g., the silk fibroin tip) of the silk fibroin-based microneedle is configured to be dissolvable and / or degradable. In some embodiments, substantially all of the silk fibroin-based microneedle is dissolvable and / or degradable. In some embodiments, release of the therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the dissolvable and / or degradable (e.g., resorbable) microneedle is by diffusion-controlled release through the microneedle material.

[0228] In some embodiments, the silk fibroin-based microneedles are solid microneedles. In some embodiments, the solid microneedles of the present disclosure are designed as a two-part system. In some embodiments, a microneedle device comprising silk fibroin-based solid microneedles is first applied to the skin to create microscopic wells just deep enough to penetrate the outermost layer of a biological barrier (e.g., skin), and then to administer a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) via a transdermal patch. In some embodiments, the release of the therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the solid microneedles is by diffusion through the microneedle material and / or degradation of the microneedle material, e.g., protease-mediated degradation. In some embodiments where the solid microneedles degrade, the solid microneedles are typically referred to as dissolvable or resorbable microneedles.

[0229] In some embodiments, the silk fibroin-based microneedles are hollow microneedles. In some embodiments, the hollow microneedles of the present disclosure comprise a reservoir that delivers a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) directly into the site of application (e.g., a biological barrier, e.g., the skin).

[0230] In some embodiments, the silk fibroin-based microneedles are coated microneedles. In some embodiments, a therapeutic agent (e.g., an anticancer agent, an immunomodulatory agent, or a combination thereof) is applied directly to a portion (e.g., the surface) of the microneedle. In some embodiments, the coated microneedles are also coated with a surfactant (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer (e.g., P188, and / or polyethoxylated alcohol) and / or a thickener to ensure proper delivery of the therapeutic agent.

[0231] In some embodiments, the silk fibroin-based microneedles of the present disclosure may include (1) a backing material layer (optionally); (2) a base layer (e.g., a dissolvable base); and (3) a silk fibroin tip layer. For example, the microneedles described herein may include a backing material (optionally) applied to a dissolvable base layer that supports a distal silk fibroin tip comprising silk fibroin, and a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof).

[0232] In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain silk fibrions in an amount of about 0.5 μg to about 500 μg of silk fibrions. In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain silk fibrions in an amount of about 0.5 μg to about 5 μg, or about 1 μg to about 10 μg, or about 5 μg to about 15 μg, or about 10 μg to about 20 μg, or about 15 μg to about 25 μg, or about 20 μg to about 30 μg, or about 25 μg to about 35 μg, or about 30 μg to about 40 μg, or about 35 μg to about 45 μg, or about 40 μg to about 50 μg, or about 45 μg to about 5 5 μg, or about 50 μg to about 60 μg, or about 55 μg to about 65 μg, or about 60 μg to about 70 μg, or about 65 μg to about 75 μg, or about 70 μg to about 80 μg, or about 75 μg to about 85 μg, or about 80 μg to about 90 μg, or about 85 μg to about 95 μg, or about 90 μg to about 100 μg, or about 95 μg to about 150 μg g, or about 125 μg to about 175 μg, or about 150 μg to about 200 μg, or about 225 μg to about 275 μg, or about 250 μg to about 300 μg, or about 325 μg to about 375 μg, or about 350 μg to about 400 μg, or about 425 μg to about 475 μg, or about 450 μg to about 500 μg. In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise silk fibrions in an amount of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions. In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise silk fibrions in an amount of up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions. In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise silk fibrions in an amount of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions.

[0233] In some embodiments, the silk fibroin-based microneedles of the present disclosure comprise about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% silk fibrions by weight. In some embodiments, the silk fibroin-based microneedles of the present disclosure comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% silk fibrions by weight. In some embodiments, the silk fibroin-based microneedles of the present disclosure comprise up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions. In some embodiments, the silk fibroin-based microneedles of the present disclosure comprise up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions.

[0234] backing Exemplary backing materials that may be used in the manufacture of microneedles of the present disclosure include, but are not limited to, solid supports such as paper-based materials, plastic materials, polymeric materials, or polyester-based materials (e.g., Whatman 903 paper, polymeric tape, plastic tape, adhesive-backed polyester tape, or other suitable tape). In some embodiments, the backing comprises Whatman 903 paper. In some embodiments, the backing comprises polyester tape. In some embodiments, the polyester tape comprises adhesive-backed polyester tape. In some embodiments, the backing material may be coated (e.g., on at least one side) with an adhesive suitable for bonding and / or adhering to the dissolvable base of the microneedles described herein. In some embodiments, the backing may extend beyond the microneedle array. In some embodiments, an adhesive coating on the backing The array may be suitable for adhesion to the skin of a subject to hold the array in place.

[0235] The backing material used in the microneedles of the present disclosure may have various properties, including, but not limited to, the ability to bond and / or adhere to the dissolvable base layer and allow for release. The backing material may be strong enough to maintain the integrity of the patch, for example, if the dissolvable base layer has a crack or cut. The backing material may be flexible enough to conform to an uneven surface, such as the skin surface. In particular, the backing may be flexible enough to remain in place for a period of time, for example, after the patch is applied (e.g., pressed) to the skin. The backing may include and / or consist of a non-dissolvable material so that the backing maintains its integrity after application of the patch to the skin surface and during removal of the patch from the skin surface.

[0236] In some embodiments, the backing layer comprises an adhesive-coated plastic tape, a porous material, and / or an adhesive. In some embodiments, the adhesive is selected from the group consisting of acrylic, acrylate, cyanoacrylate, silicone, polyurethane, and synthetic rubber. In some embodiments, the adhesive comprises a material that can be cured by irradiation with light.

[0237] The backing may have any dimensions suitable for application to a target biological barrier, e.g., the skin surface. In some embodiments, the dimensions of the backing include a circular shape. In some embodiments, the dimensions of the backing include a rectangular shape (e.g., a square, or a rectangular strip). The backing may have rounded corners (e.g., a square or rectangle with rounded corners). The backing may further include an extension, e.g., for use as a "handle" (see, e.g., FIG. 3).

[0238] The backing may have any suitable dimensions, e.g., to accommodate the microneedle array and / or further conform to the intended application site. For example, the backing may have a diameter of about 5 mm to about 50 mm, e.g., about 6 mm to about 40 mm, about 8 mm to about 35 mm, about 10 mm to about 30 mm, about 11 mm to about 25 mm, about 12 mm to about 24 mm, about 10 mm to about 20 mm, or about 10 to about 15 mm. In some embodiments, the backing has a diameter of about 5 mm or greater, e.g., about 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, or greater.

[0239] In some embodiments, the backing is circular with a diameter of between about 8 mm and about 16 mm, e.g., about 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, or 16 mm. In some embodiments, the backing is square with dimensions of about 8 mm x 8 mm, about 9 mm x 9 mm, about 10 mm x 10 mm, about 11 mm x 11 mm, about 12 mm x 12 mm, about 13 mm x 13 mm, about 14 mm x 14 mm, about 15 mm x 15 mm, or about 16 mm x 16 mm. In some embodiments, the backing is a rectangle (e.g., a rectangular strip) having a width of about 8 mm or more (e.g., a width of about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm or more) and a length that is greater than the width, for example, a length of about 10 mm or more (e.g., a length of about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm or more).

[0240] In some embodiments, the dimensions of the backing may be a circle with a diameter of 12 mm. In some embodiments, the dimensions of the backing extend beyond the edges of the 12 mm x 12 mm patch. The largest may be a 12 mm wide strip with a 12 mm long "handle" section (see, e.g., FIG. 3). In some embodiments, a 12 mm square polyester tape may be used with an extended "handle" that is approximately 12 mm square (see, e.g., FIG. 3). In some embodiments, the backing may be even larger, e.g., about 25 mm square with rounded corners if desired. In some embodiments, the backing may be circular with a diameter of about 25 mm. In some embodiments, the area of ​​the backing that extends beyond the array can help hold the patch on the skin using a biocompatible skin adhesive.

[0241] Dissolvable base The base layer (e.g., a dissolvable base layer) forms the base of the needle (e.g., serves as a support for the distal silk fibroin tip, which can be loaded with a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof). The base layer (e.g., a dissolvable base layer) may also serve as a layer that connects adjacent needles to form a microneedle array or patch.

[0242] In some embodiments, the base layer (e.g., a dissolvable base layer) comprises a material that can dissolve in a subject, for example, within the intended wear time (e.g., about 5 minutes). In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the dissolvable base layer is dissolved within the intended wear time (e.g., about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes or more) after application to a biological barrier (e.g., skin) of a subject.

[0243] The material used in the manufacture of the dissolvable base can be strong enough to allow the microneedles to penetrate the skin, and can also be sturdy enough (e.g., not excessively brittle) to allow the microneedles to be released during manufacture. The dissolvable base material is suitable for daily handling, may not be severely damaged, and can retain its mechanical properties between release and application (e.g., is not hygroscopic enough to melt due to ambient humidity). The dissolvable base layer material can be non-toxic and non-reactive at the dosage used in the patch. In some embodiments, the dissolvable base layer includes a water-soluble component.

[0244] Non-limiting examples of materials that may be used to manufacture a base layer (e.g., a dissolvable base layer) include polysaccharides, disaccharides, polymers, proteins, plasticizers, and / or surfactants. In some embodiments, the base layer (e.g., a dissolvable base layer) includes one or more (e.g., two or more, three or more, four or more, five or more, or all) of polysaccharides (e.g., dextran); disaccharides (e.g., sucrose, maltose, and trehalose); polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate); proteins (e.g., gelatin, fibroin); plasticizers (e.g., glycerol, propanediol); and surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, and / or polyethoxylated alcohol).

[0245] The base layer disclosed herein may comprise a polysaccharide, disaccharide, polymer, protein, plasticizer, and / or surfactant at a concentration of between about 0.001% and about 75% (e.g., between about 0.001% and about 1%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%). In some cases, the dry solids basis may comprise up to about 10% of the polysaccharide, disaccharide, polymer, protein, plasticizer, and / or surfactant. The dry solids base may contain polysaccharides, disaccharides, polymers, proteins, plasticizers, and / or surfactants at a concentration of about 0.001%. In some cases, the dry solids base may contain surfactants at a concentration of about 0.001%.

[0246] In some embodiments, the base layer (e.g., a dissolvable base layer) is configured for burst and / or sustained release of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof). In some embodiments, the base layer (e.g., a dissolvable base layer) comprises one or more (e.g., two or more, three or more, four or more, five or more, or six or more) of gelatin, dextran, glycerol, polyethylene glycol (PEG), sucrose, trehalose, maltose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, methylcellulose, and / or surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, and / or polyethoxylated alcohol), and, optionally, the microneedles are configured for sustained and / or burst release.

[0247] In some embodiments, the base layer (e.g., a dissolvable base layer) comprises one or more (e.g., two or more, three or more, or four or more) of dextran, sucrose, glycerol, and surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, and / or polyethoxylated alcohol) and is configured for sustained release as needed.

[0248] In some embodiments, the base layer (eg, a dissolvable base layer) comprises polyvinyl alcohol (PVA) and sucrose and is configured for on-demand burst release.

[0249] In some embodiments, the base layer (e.g., a dissolvable base layer) comprises dextran. In some embodiments, the dextran may have a molecular weight between about 30 kDa and about 600 kDa. In some embodiments, the dextran has a molecular weight of about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, or about 600 kDa. In some embodiments, a mixture of different dextrans, e.g., a mixture of dextrans having various molecular weights, may be used. In some embodiments, the dextran may be obtained and / or derived from various bacterial sources, including, but not limited to, Leuconostoc mesenteroides.

[0250] In some embodiments, the base layer (e.g., the dissolvable base layer) does not include poly(acrylic acid) (PAA). In some embodiments, the dissolvable base layer, as described herein, has improved biocompatibility compared to, for example, a dissolvable base layer comprising poly(acrylic acid) (PAA). In some embodiments, the dissolvable base layer material may result in a reduced inflammatory response and / or reduced tissue necrosis. In some embodiments, the dissolvable base layer material is not PAA and induces a reduced inflammatory response and / or reduced tissue necrosis compared to PAA. In some embodiments, the dissolvable base layer material has a pH similar to that of the biological barrier into which it will be dissolved, e.g., a pH of about 4.0 to about 8.0, e.g., about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

[0251] In other embodiments, the base layer comprises silk fibroin and / or a therapeutic agent. The base layer (e.g., a dissolvable base layer) may comprise microneedles and / or microneedles. The therapeutic agent may comprise less than 98% (e.g., less than about 98%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the total amount (e.g., dose) of therapeutic agent loaded into the device.

[0252] In some embodiments, the base layer does not contain detectable amounts of silk fibroin and / or therapeutic agent, for example, in some embodiments, the base layer is formulated to limit and / or reduce the amount of leakage (e.g., diffusion) of therapeutic agent from the silk fibroin tip into the base layer, for example, compared to base layer formulations known in the art, for example, base layer formulations comprising PAA. In some embodiments, the amount of limited and / or reduced leakage (e.g., diffusion) of therapeutic agent from the silk fibroin chip can be determined, for example, as compared to a base layer formulation comprising PAA, after manufacture and storage (e.g., storage at about 4°C (e.g., refrigerated), about 25°C (e.g., room temperature), about 37°C (e.g., body temperature), about 45°C, and / or about 50°C) for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days; about 1 week, about 2 weeks, or about 3 weeks; about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months; or about 1 year or more.

[0253] In some embodiments, the dissolvable base comprises between about 10% and about 70% gelatin (e.g., hydrolyzed gelatin) (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% gelatin).

[0254] In some embodiments, the dissolvable base comprises between about 10% and about 70% plasticizer, such as glycerol (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% glycerol). In some embodiments, the plasticizer is added to reduce brittleness. For example, the brittleness of a dissolvable base layer comprising a plasticizer can be reduced by about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 200%, about 300%, or more, relative to a dissolvable base layer substantially free of plasticizer.

[0255] In some embodiments, the dissolvable base comprises between about 0.001% and about 5% of a surfactant described herein, such as a polysorbate (e.g., between about 0.001% and about 1%, or between about 1% and about 5% of a surfactant). In some embodiments, the surfactant is added to aid in processing. In some embodiments, the surfactant is added as a plasticizer.

[0256] In some embodiments, the dissolvable base comprises between about 1% and about 70% polyethylene glycol (PEG) (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% PEG).

[0257] In some embodiments, the dissolvable base comprises between about 1% and about 35% sucrose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% sucrose).

[0258] In some embodiments, the dissolvable base has a CMC of between about 1% and about 35% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%). %, about 15%, about 20%, about 25%, about 30%, or about 35% CMC).

[0259] In some embodiments, the dissolvable base comprises between about 10% and about 70% PVP (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% PVP).

[0260] In some embodiments, the dissolvable base comprises between about 1% and about 35% PVA (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% PVA).

[0261] In some embodiments, the dissolvable base comprises between about 1% and about 75% hyaluronate (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% hyaluronate).

[0262] In some embodiments, the dissolvable base comprises between about 1% and about 75% maltose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% maltose).

[0263] In some embodiments, the dissolvable base comprises between about 1% and about 75% methylcellulose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% methylcellulose).

[0264] In some embodiments, the dissolvable base layer may comprise 40% w / v hydrolyzed gelatin and 10% w / v sucrose in deionized (DI) water. Optionally, the base layer may comprise 1% low-viscosity carboxymethylcellulose (CMC), which may reduce brittleness. In some embodiments, the dissolvable base layer may comprise up to 50% w / v 10 kD MW polyvinylpyrrolidone (PVP) in DI water; up to 20% 87% hydrolyzed polyvinyl alcohol (PVA) at 13 kD MW in DI water; or up to 10% CMC in DI water. 30% PVP and 10% PVA; 37% PVP, 5% PVA, and 15% sucrose; or various other ratios of PVP, PVA, and sucrose combinations may also be suitable for use in manufacturing the dissolvable base layer.

[0265] The dissolvable base layer may be of any suitable dimension, size, or shape. For example, the dissolvable base layer may have a dimension, size, or shape suitable for accommodating the microneedle array and / or for further conforming to the intended application site. In some embodiments, the shape of the dissolvable base layer comprises a circle. In some embodiments, the shape of the dissolvable base layer comprises a rectangle (e.g., a square, or a rectangular strip). The dissolvable base layer may have rounded corners (e.g., a square or rectangle with rounded corners) or sharp corners (e.g., a square or rectangle with sharp corners).

[0266] In some embodiments, the dissolvable base layer has a thickness of between about 5 mm and about 50 mm, e.g., between about 6 mm and about 40 mm, between about 8 mm and about 35 mm, or between about 10 mm and about 30 mm. In some embodiments, the dissolvable base layer has a diameter of about 5 mm or more, e.g., about 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm or more.

[0267] In some embodiments, the dissolvable base layer is circular with a diameter of between about 8 mm and about 16 mm, e.g., about 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, or 16 mm. In some embodiments, the dissolvable base layer is square with dimensions of about 8 mm x 8 mm, about 9 mm x 9 mm, about 10 mm x 10 mm, about 11 mm x 11 mm, about 12 mm x 12 mm, about 13 mm x 13 mm, about 14 mm x 14 mm, about 15 mm x 15 mm, or about 16 mm x 16 mm. In some embodiments, the dissolvable base layer is a rectangle (rectangular strip) having a width of about 8 mm or more (e.g., a width of about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm or more) and a length that is greater than the width, for example, a length of about 10 mm or more (e.g., a length of about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm or more).

[0268] The thickness of the dissolvable base layer may be adjusted according to the particular microneedle device and / or the intended application of the microneedle device. In some embodiments, the thickness of the dissolvable base layer is between about 0.5 and 1 mm, e.g., between about 0.55 and 0.95 mm, between about 0.60 and 0.90 mm, between about 0.65 and 0.85 mm, or between about 0.70 and 0.80 mm. In some embodiments, the thickness of the dissolvable base layer is about 0.50 mm or more, e.g., about 0.55 mm, about 0.60 mm, about 0.65 mm, about 0.70 mm, about 0.75 mm, about 0.80 mm, about 0.85 mm, about 0.90 mm, about 0.95 mm or more.

[0269] The dissolvable base layer may comprise one or more layers, for example, by using more than one base layer solution in preparing the microneedle device using the methods described herein. In some embodiments, the dissolvable base layer comprises a single layer. In some embodiments, the dissolvable base layer comprises two layers. In some embodiments, the dissolvable base layer comprises three layers. In some embodiments, the dissolvable base layer comprises four or more layers. In some embodiments, the dissolvable base layer comprises more than one layer, where one or more of the layers are different, for example, when each base layer is formed using a different composition of one or more of the base layer solutions. In some embodiments, the dissolvable base layer comprises more than one layer, where each layer is different.

[0270] In some embodiments, the dissolvable base layer is approximately 12 mm square and 0.75 mm thick. In some embodiments, the dissolvable base layer may cover the entire patch (e.g., a microneedle patch). In some embodiments, the dimensions of the base layer may be a circle with a diameter of 12 mm or a square of 12 x 12 mm.

[0271] Silk fibroin chip The methods provided herein may be used to fabricate silk fibroin chips of any size, such as silk fibroin-based implantable sustained-release and / or burst-release chips, which may be configured to contain and release an effective amount of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof).

[0272] In some embodiments, the silk fibroin chip has dimensions ranging from about 75 μm to about 800 μm in height / length (e.g., about 75, about 100 μm, about 125 μm, about 150 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 550 μm, about 550 μm to about 600 μm, about 600 μm to about 650 μm, about 650 μm to about 700 μm, about 700 μm to about 750 μm, about 750 μm to about 800 μm).

[0273] In some embodiments, the silk fibroin chip, e.g., an implantable chip, may have a diameter of any size based on, for example, the type of biological barrier (e.g., skin layer, tumor, tissue, cell membrane, mucosal surface, oral cavity, or buccal cavity) intended to be penetrated by the chip. In some embodiments, the silk fibroin chip has a tip radius of about 10 μm or less (e.g., between about 1 μm and about 10 μm, e.g., about 1 μm or less, about 2 μm or less, about 3 μm or less, about 4 μm or less, about 5 μm or less, about 6 μm or less, about 7 μm or less, about 8 μm or less, about 9 μm or less, or about 10 μm or less). In embodiments, the tip may have a dimension (e.g., diameter) in the range of about 50 nm to about 50 μm (e.g., about 50 nm to about 250 nm, about 250 nm to about 500 nm, about 500 to about 750 nm, about 750 nm to about 1 μm, about 1 μm to about 5 μm, about 5 μm to about 10 μm, about 10 μm to about 15 μm, about 15 μm to about 20 μm, about 20 μm to about 25 μm, about 25 μm to about 30 μm, about 30 μm to about 35 μm, about 35 μm to about 40 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm). It can be appreciated that there are no fundamental limitations that prevent tips from having even smaller diameters (e.g., limitations of silk replica casting have been demonstrated at resolutions of tens of nanometers, see, e.g., Perry et al., 20 Adv. Mat. 3070 (2008)).

[0274] In some embodiments, the sharpness of a silk fibroin tip, e.g., an implantable sustained release tip, is described herein in terms of tip radius. The molds used in the manufacture of the microneedles described herein are designed to have a tip radius of about 0.5 μm to about 10 μm (e.g., about 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm). In some embodiments, the tip radius is about 20 μm to about 25 μm (e.g., about 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, or 25 μm). Without being bound by theory, it can be understood that a blunt needle may require more force to penetrate the epidermis. In embodiments, other dimensions of the silk fibroin tip, e.g., implantable sustained release tip, may be controlled by the mold shape and fill volume. In some embodiments, the silk fibroin tip, e.g., implantable sustained release tip, may have a tip angle of between about 5 degrees and about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 degrees). In some embodiments, the silk fibroin tip, e.g., an implantable sustained release tip, may have a tip angle of between about 15 degrees and 45 degrees (e.g., about 15 degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19 degrees, about 20 degrees, about 21 degrees, about 22 degrees, about 23 degrees, about 24 degrees, about 25 degrees, about 26 degrees, about 27 degrees, about 28 degrees, about 29 degrees, about 30 degrees, about 31 degrees, about 32 degrees, about 33 degrees, about 34 degrees, about 35 degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39 degrees, about 40 degrees, about 41 degrees, about 42 degrees, about 43 degrees, about 44 degrees, or about 45 degrees).

[0275] In embodiments, the height of a silk fibroin tip, e.g., an implantable sustained release tip, depends on the formulation and fill volume (e.g., fill volume or droplet dispense volume), which can affect surface tension and drying rate. In some embodiments, the height of the tip may extend up to half the total length of the microneedle. In some embodiments, the height of the silk fibroin tip, e.g., implantable sustained release tip, is between about 75 μm and about 475 μm (e.g., about 75, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 375 μm, about 400 μm, about 425 μm, or about 475 μm). In some embodiments, a portion of the tip includes a thin "shell"-like layer approximately between about 5-10 μm thick (e.g., about 5, 6, 7, 8, 9, or 10 μm thick). In some embodiments, silk fibroin chips, e.g., implantable sustained release chips, may dry into more solid constructs with minimal "shells" and may be close to 150 μm in height (e.g., between about 50 μm and about 200 μm) and greater than 50 μm in thickness (e.g., between about 25 μm and about 75 μm).

[0276] Additionally, the microneedles of the present disclosure may utilize techniques known in the art (e.g., active agents, such as dyes and sensors) developed to functionalize silk fibroin, for example. See, for example, U.S. Patent No. 6,287,340, Bioengineered anterior cruciate ligament; WO 2004 / 000915, Silk Biomaterials & Methods of Use Thereof; WO 2004 / 001103, Silk Biomaterials & Methods of Use Thereof; WO 2004 / 062697, Silk Fibroin Materials & Use Thereof; WO 2005 / 000483, Method for Forming Inorganic Coatings; WO 2005 / 012606, Concentrated Aqueous Silk Fibroin Solution & Use Thereof; WO 2011 / 005381, Vortex-Induced Silk Fibroin Gelation for Encapsulation & Delivery; WO 2005 / 123114, Silk-Based Drug Delivery System; WO 2006 / 076711, Fibrous Protein Fusions & Uses Thereof in the Formation of Advanced Organic / Inorganic Composite Materials; U.S. Patent Application Publication No. 2007 / 0212730, Covalently Immobilized Protein Gradients In Three-Dimensional Porous Scaffolds; WO 2006 / 042287, Method for Producing Biomaterial Scaffolds; WO 2007 / 016524, Method for Stepwise Deposition of Silk Fibroin Coatings; WO 2008 / 085904, Biodegradable Electronic Devices; WO 2008 / 118133, Silk Microspheres for Encapsulation & Controlled Release; WO 2008 / 108838, Microfluidic Devices & Methods for Fabricating Same; WO 2008 / 127404, Nanopatterned Biopolymer Device & Method of Manufacturing Same; WO 2008 / 118211, Biopolymer Photonic Crystals & Method of Manufacturing Same; WO 2008 / 127402, Biopolymer Sensor & Method of Manufacturing Same; WO 2008 / 127403, Biopolymer Optofluidic Device & Method of Manufacturing the Same; WO 2008 / 127401, Biopolymer Optical Wave Guide & Method of Manufacturing the Same turing Same; WO 2008 / 140562, Biopolymer Sensor & Method of Manufacturing Same; WO 2008 / 127405, Microfluidic Device with Cylindrical Microchannel & Method for Fabricating Same; WO 2008 / 106485, Tissue-Engineered Silk Organs; WO 2008 / 140562, Electroactive Biopolymer Optical & Electro-Optical Devices & Method of Manufacturing Same; WO 2008 / 150861, Method for Silk Fibroin Gelation Using Sonication; WO 2007 / 103442, Biocompatible Scaffolds & Adipose-Derived Stem Cells; WO 2009 / 155397, Edible Holographic Silk Products; WO 2009 / 100280, 3-Dimensional Silk Hydroxyapatite See Compositions; WO 2009 / 061823, Fabrication of Silk Fibroin Photonic Structures by Nanocontact Imprinting; WO 2009 / 126689, System & Method for Making Biomaterial Structures.

[0277] In various embodiments, the silk fibroin-based microneedle tip may further comprise at least one additional therapeutic agent, which may be dispersed throughout the microneedle or may form at least a portion of the microneedle tip. In some embodiments, the additional therapeutic agent is useful in the treatment of diseases and / or disorders described herein, such as cancer. Optionally, the silk fibroin-based microneedle tip may further comprise an excipient and / or adjuvant as described herein.

[0278] In embodiments, the microneedle tip may be fabricated from silk fibroin and may contain a therapeutic agent described herein (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof). In some embodiments, the tip may be designed to be placed in the dermal layer of the skin (e.g., not in the subcutaneous space) because the population of professional antigen-presenting cells in the dermis is much greater than in the subcutaneous space. In humans, the thickness of the dermis ranges from approximately 1000 to 2000 μm (e.g., approximately 1 to 2 mm), depending on the location and the patient's age and health. In rodents, the dermis is much thinner (e.g., approximately 100 to 300 μm in mice and approximately 800 to 1200 μm in rats). Without wishing to be bound by theory, a 650 μm-tall microneedle tip, e.g., an implantable sustained-release tip, may be placed at a depth of between approximately 100 μm and approximately 600 μm to achieve controlled or sustained release of a therapeutic agent described herein.

[0279] Without being bound by theory, the molecular weight of the silk fibroin solution used in the fabrication of the microneedles described herein may function as a control factor to modulate the release of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the chip. In some embodiments, a high molecular weight silk fibroin solution may facilitate a delayed controlled or sustained release (e.g., reducing the initial burst amount (e.g., the amount released on day 0) by at least about 10%, followed by release of additional antigen over at least the next about 4 days). In some embodiments, the controlled or sustained release of the anti-cancer agent, immunomodulatory agent, or a combination thereof from the chip is maintained for at least about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, e.g., between about 4 days and about 15 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1 In some embodiments, the release occurs over a period of about 1 week to about 2 weeks.

[0280] In embodiments, the silk fibroin solution used in the manufacture of the microneedles described herein may be a low molecular weight silk fibroin composition comprising a population of silk fibroin fragments having various molecular weights, characterized in that no more than 15% of the total number of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of silk fibroin fragments in the population have a molecular weight within a specified range, wherein the specified range is between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa. In other words, the silk fibroin solution used in the manufacture of the microneedles described herein may contain a population of silk fibroin fragments having various molecular weights, wherein no more than 15% of the total moles of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and wherein at least 50% of the total moles of silk fibroin fragments in the population have a molecular weight within a specified range, wherein the specified range is between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa (see, e.g., International Publication No. WO 2014 / 145002, herein incorporated by reference).

[0281] Exemplary silk fibroin (e.g., regenerated silk fibroin) solutions may have a variety of molecular weight profiles, as determined, for example, by size exclusion chromatography (SEC) (see, e.g., FIG. 5). In some embodiments, silk fibroin solutions may be prepared, for example, according to established methods. In some embodiments, portions of cocoons from the silkworm, Bombyx mori, were first boiled in 0.02 M Na2CO3 to remove sericin proteins present in untreated native silk and then analyzed by SEC. In some embodiments, the silk fibroin compositions are prepared by heating the silkworm, Bombyx mori, cocoons at atmospheric boiling temperature for about 480 minutes or less, e.g., less than 480 minutes, less than 400 minutes, less than 300 minutes, less than 200 minutes, less than 180 minutes, less than 120 minutes, less than 100 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, or even less. In one embodiment, the silk fibroin composition may be a composition or mixture produced by removing gum from cocoons from silkworms (Monocotyledonous silkworms) in aqueous sodium carbonate at ambient boiling temperature for about 480 minutes or less, e.g., less than 480 minutes, less than 400 minutes, less than 300 minutes, less than 200 minutes, less than 180 minutes, less than 120 minutes, less than 100 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, or even less.

[0282] In some embodiments, the silk fibroin solution may be a 10 minute boiled (10MB), 60 minute boiled (60MB), 120 minute boiled (120MB), 180 minute boiled (180MB), or 480 minute boiled (480MB) silk fibroin solution (see, e.g., FIG. 5). In some embodiments, a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, may be formulated as a 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 10MB silk fibroin solution. In some embodiments, a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, may be formulated as a 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 60MB silk fibroin solution. In some embodiments, a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, may be formulated as a 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) solution of 120MB silk fibroin. In some embodiments, a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, may be formulated as a 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) solution of 180MB silk fibroin. In some embodiments, a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, may be formulated as a 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) solution of 480MB silk fibroin.

[0283] In some embodiments, the silk fibroin chip, eg, an implantable sustained release chip, may comprise silk fibrions in an amount of about 0.5 μg to about 500 μg of silk fibrions. In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, has a mass of about 0.5 μg to about 5 μg, or about 1 μg to about 10 μg, or about 5 μg to about 15 μg, or about 10 μg to about 20 μg, or about 15 μg to about 25 μg, or about 20 μg to about 30 μg, or about 25 μg to about 35 μg, or about 30 μg to about 40 μg, or about 35 μg to about 45 μg, or about 40 μg to about 50 μg, or about 45 μg to about 55 μg, or about 50 μg to about 60 μg, or about 55 μg to about 65 μg, or about 60 μg to about 70 μg, or about 65 μg The silk fibrion may be present in an amount of from about 75 μg to about 80 μg, or from about 75 μg to about 85 μg, or from about 80 μg to about 90 μg, or from about 85 μg to about 95 μg, or from about 90 μg to about 100 μg, or from about 95 μg to about 150 μg, or from about 125 μg to about 175 μg, or from about 150 μg to about 200 μg, or from about 225 μg to about 275 μg, or from about 250 μg to about 300 μg, or from about 325 μg to about 375 μg, or from about 350 μg to about 400 μg, or from about 425 μg to about 475 μg, or from about 450 μg to about 500 μg. In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, may comprise silk fibrions in an amount of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions.In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, may contain silk fibrions in an amount of up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions. In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, may contain silk fibrions in an amount of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrions.

[0284] In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, comprises about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% by weight of silk fibrions. In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions. In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, comprises up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions. In some embodiments, the silk fibroin chip, e.g., an implantable sustained release chip, comprises up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions.

[0285] Without being bound by theory, the primary tunable feature of silk fibroin chips, e.g., implantable sustained-release chips, is their crystallinity, as measured by beta-sheet content (inter- and intramolecular beta-sheets). This affects the solubility of the silk chip matrix and its ability to retain antigens. With increased beta-sheet content, the chip also becomes mechanically stronger. The release profile of a particular therapeutic agent (e.g., an anticancer agent and / or an immunomodulator) is achieved through modulation of the crystallinity and diffusivity of the silk matrix. This is achieved through both the silk input material and formulation, as well as post-treatments to increase crystallinity (e.g., water annealing, methanol / solvent annealing). In some embodiments, silk fibroin chips, e.g., implantable controlled-release or sustained-release microneedle chips, comprise a beta-sheet content of between about 10% and about 60% (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%), based on, for example, a "crystallinity index," e.g., a "crystallinity index" known in the art. In some embodiments, silk fibroin tips, for example, implantable controlled-release or sustained-release microneedle tips, may be formulated as particles (eg, microparticles and / or nanoparticles).

[0286] In another aspect, the present disclosure features microneedles (e.g., silk fibroin-based microneedles) that can stabilize a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or both) disposed on the microneedle, due in part to, for example, the thermostabilizing properties of the microneedle composition (e.g., the thermostabilizing properties of the silk fibroin composition). In some embodiments, the anti-cancer agent, the immunomodulatory agent, or both (e.g., an anti-cancer agent or an immunomodulatory agent described herein) are stabilized by the microneedles or microneedle devices described herein. Without wishing to be bound by theory, the ability of the microneedles or microneedle devices of the present disclosure to stabilize a therapeutic agent can facilitate storage of the microneedle device, for example, to prevent loss of biological activity of the therapeutic agent during storage. Furthermore, the biological activity of the therapeutic agent can be maintained after administration of the microneedle or microneedle device. For example, a therapeutic agent can be stabilized within the silk fibroin tip after administration with a microneedle or microneedle device to prevent loss of biological activity, for example, during release (e.g., controlled release) at body temperature.

[0287] In some embodiments, the anti-cancer agent and / or immunomodulating agent retains at least 50% (e.g., about 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or more) of its original biological activity after storage at, for example, room temperature (e.g., about 25° C.) for a period of two weeks or more. In some embodiments, the anti-cancer agent and / or immunomodulating agent retains at least 70%, 80%, or 90% of its original biological activity after storage at about 25° C. for at least about two weeks (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks). In some embodiments, the anti-cancer agent and / or immunomodulating agent retains at least 60%, 70%, or 80% of its original biological activity after storage at about 37° C. for at least about 2 weeks (about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks). In some embodiments, the anti-cancer agent and / or immunomodulating agent retains at least 50%, 60%, or 70% of its original biological activity after storage at about 45° C. for at least about 2 weeks (about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks).

[0288] In some embodiments, the silk fibroin-based microneedles of the present disclosure stabilize cytokines. For example, silk fibroin can stabilize cytokines for extended periods of time (e.g., 1, 2, 3, 4, 5, The silk fibroin-based microneedles may stabilize cytokines (e.g., interleukins, e.g., IL-2) during storage at various temperatures (e.g., storage at 4°C, room temperature (e.g., 25°C), or 37°C), including for 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, 1, 2, 3, or 4 weeks or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, or 1, 2, 3, or 4 years or more). In some embodiments, the silk fibroin-based microneedles stabilize interleukins. In some embodiments, the silk fibroin-based microneedles stabilize interleukin-2 (IL-2). In some embodiments, the silk fibroin-based microneedles stabilize IL-2 for at least 14 days at 4°C. In some embodiments, the silk fibroin-based microneedles stabilize IL-2 for at least 14 days at room temperature (about 25°C). In some embodiments, the silk fibroin-based microneedles stabilize IL-2 for at least 14 days at body temperature (about 37° C.).

[0289] Microneedle dimensions In some embodiments, the present disclosure provides silk fibroin-based microneedles and devices containing same having a variety of dimensions and geometries.

[0290] In embodiments, the length of the silk fibroin-based microneedles can be made long enough to allow delivery (e.g., implantation) of silk fibroin chips containing therapeutic agents (e.g., anti-cancer agents, immunomodulatory agents, or combinations thereof) to a desired depth within a biological barrier (e.g., skin) to, e.g., induce a cancer response, e.g., induce an immune response.

[0291] In some embodiments, the biological barrier is a tumor and / or a skin lesion. In some embodiments, the length of the silk fibroin-based microneedles may be between about 350 μm and about 1500 μm (e.g., about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, about 1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1250 μm, about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about 1500 μm).

[0292] In some embodiments, the length of the silk fibroin-based microneedles is sufficient to allow delivery to the epidermis (eg, about 10 μm to 120 μm below the skin surface). In some embodiments, the length of the silk fibroin-based microneedles is sufficient to allow delivery to the dermis (eg, about 60 μm to about 2.1 mm below the skin surface).

[0293] In some embodiments, the length of the silk fibroin-based microneedles is sufficient to allow delivery to the eye (e.g., about 10 μm to 120 μm below the ocular surface). In some embodiments, the length of the silk fibroin-based microneedles is sufficient to allow delivery to the tumor (e.g., about 10 μm to 120 μm below the tumor surface).

[0294] In some embodiments, the microneedle is configured to embed the silk fibroin tip into the biological barrier of the subject to a depth of between about 100 μm and about 600 μm (e.g., maximum penetration depth of the distal portion of the tip). In some embodiments, the length of the microneedle is between about 350 μm and about 1500 μm. In some embodiments, the height of the silk fibroin tip may extend to approximately half the total length of the microneedle. In some embodiments, the height of the silk fibroin tip is between about 75 μm and about 475 μm. In some embodiments, the silk fibroin tip has a tip depth of between about 0.5 μm and about 25 μm. In some embodiments, the silk fibroin tip comprises a tip radius of between about 5 μm and about 10 μm, In some embodiments, the silk fibroin tip comprises an angle of between about 5 degrees and about 45 degrees.

[0295] In some embodiments, the silk fibroin tip, e.g., an implantable tip, may have a diameter of any size, for example, based on the type of biological barrier (e.g., skin layer) intended to be penetrated by the tip. In some embodiments, the silk fibroin tip has a tip radius of about 10 μm or less (e.g., between about 1 μm and about 10 μm, e.g., about 1 μm or less, about 2 μm or less, about 3 μm or less, about 4 μm or less, about 5 μm or less, about 6 μm or less, about 7 μm or less, about 8 μm or less, about 9 μm or less, or about 10 μm or less). In embodiments, the tip may have a dimension (e.g., diameter) in the range of about 50 nm to about 50 μm (e.g., about 50 nm to about 250 nm, about 250 nm to about 500 nm, about 500 to about 750 nm, about 750 nm to about 1 μm, about 1 μm to about 5 μm, about 5 μm to about 10 μm, about 10 μm to about 15 μm, about 15 μm to about 20 μm, about 20 μm to about 25 μm, about 25 μm to about 30 μm, about 30 μm to about 35 μm, about 35 μm to about 40 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm). It can be appreciated that there are no fundamental limitations that prevent tips from having even smaller diameters (e.g., limitations of silk replica casting have been demonstrated at resolutions of tens of nanometers, see, e.g., Perry et al., 20 Adv. Mat. 3070 (2008)).

[0296] In some embodiments, the sharpness of a silk fibroin tip, e.g., an implantable sustained release tip, is described herein in terms of tip radius. The molds used in the manufacture of the microneedles described herein are designed to have a tip radius of about 0.5 μm to about 10 μm (e.g., about 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm). In some embodiments, the tip radius is about 20 μm to about 25 μm (e.g., about 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, or 25 μm). Without being bound by theory, it can be understood that a blunt needle may require more force to penetrate the epidermis. In embodiments, other dimensions of the silk fibroin tip, e.g., implantable sustained release tip, may be controlled by the mold shape and fill volume. In some embodiments, the silk fibroin tip, e.g., implantable sustained release tip, may have a tip angle of between about 5 degrees and about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 degrees). In some embodiments, the tip may have a point angle of between about 15 degrees and 45 degrees (e.g., about 15 degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19 degrees, about 20 degrees, about 21 degrees, about 22 degrees, about 23 degrees, about 24 degrees, about 25 degrees, about 26 degrees, about 27 degrees, about 28 degrees, about 29 degrees, about 30 degrees, about 31 degrees, about 32 degrees, about 33 degrees, about 34 degrees, about 35 degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39 degrees, about 40 degrees, about 41 degrees, about 42 degrees, about 43 degrees, about 44 degrees, or about 45 degrees).

[0297] Without wishing to be bound by theory, one of skill in the art can adjust the length of silk fibroin-based microneedles with respect to numerous factors, including, but not limited to, tissue thickness, e.g., skin thickness, (e.g., depending on age, sex, location in the body, species of subject (e.g., human), drug delivery profile, diffusion characteristics of the therapeutic agent (e.g., ionic charge and / or molecular weight, and / or shape of the therapeutic agent), or any combination thereof).

[0298] However, without wishing to be bound by theory, it is believed that with microneedles of approximately 650 μm height, the silk fibroin tip can penetrate approximately 100 μm into the dermis layer of the skin. In some embodiments, the microneedles may be positioned (e.g., embedded) at a depth of between about 500 μm and about 600 μm to achieve release (e.g., burst release or sustained release) of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the silk fibroin chip. In some embodiments, the microneedles may be about 800 μm in height (e.g., between about 500 μm and 1200 μm in height).

[0299] Silk fibroin-based microneedle devices for combination therapy In some embodiments, the silk fibroin-based microneedle device (e.g., a microneedle patch) of the present disclosure may include multiple microneedles disclosed herein. The silk fibroin-based microneedle device (e.g., a microneedle patch) may include multiple microneedles of the same type. Alternatively, the microneedle device may include multiple microneedles of different types.

[0300] For example, a silk fibroin-based microneedle device may include multiple microneedles containing anti-cancer drugs. In some embodiments, a silk fibroin-based microneedle device may include multiple microneedles containing a combination of anti-cancer drugs (e.g., two, three, four, five, six, or more anti-cancer drugs). In the case of microneedles configured to deliver a combination of anti-cancer drugs (e.g., two, three, four, five, six, or more anti-cancer drugs), the microneedle device may include, for example, multiple identical microneedles containing the same combination (e.g., all) of anti-cancer drugs; or multiple different microneedles containing different anti-cancer drugs or different combinations of anti-cancer drugs. In some embodiments, multiple microneedles contain one anti-cancer drug. In some embodiments, multiple microneedles contain two or more anti-cancer drugs (e.g., two, three, four, five, six, or more anti-cancer drugs).

[0301] In some embodiments, the silk fibroin-based microneedle device may comprise multiple microneedles comprising an immunomodulatory agent. In some embodiments, the silk fibroin-based microneedle device may comprise multiple microneedles comprising a combination of immunomodulatory agents (e.g., two, three, four, five, six, or more anti-cancer agents). For microneedles configured to deliver a combination of immunomodulatory agents (e.g., two, three, four, five, six, or more immunomodulatory agents), the microneedle device may comprise, for example, multiple identical microneedles comprising the same combination (e.g., all) of immunomodulatory agents; or multiple different microneedles comprising different immunomodulatory agents or different combinations of immunomodulatory agents. In some embodiments, the multiple microneedles comprise one immunomodulatory agent. In some embodiments, the multiple microneedles comprise two or more immunomodulatory agents (e.g., two, three, four, five, six, or more immunomodulatory agents).

[0302] In some embodiments, the silk fibroin-based microneedle device may include a plurality of microneedles containing a combination of an anti-cancer drug and an immunomodulatory agent. For microneedles configured to deliver a combination of an anti-cancer drug and an immunomodulatory agent, the microneedle device may include, for example, a plurality of identical microneedles containing the same combination of anti-cancer drug and immunomodulatory agent; or a plurality of different microneedles containing different combinations of anti-cancer drug and immunomodulatory agent.

[0303] In some embodiments, with respect to microneedles configured to deliver a combination of an anti-cancer drug and an immunomodulatory agent, the microneedle device may include multiple microneedles containing one anti-cancer drug; multiple microneedles containing two or more anti-cancer drugs (e.g., two, three, multiple microneedles comprising one immunomodulating agent; multiple microneedles comprising two or more immunomodulating agents (e.g., two, three, four, five, six or more immunomodulating agents); and / or multiple microneedles comprising both anti-cancer agents (e.g., one, two, three, four, five, six or more anti-cancer agents) and immunomodulating agents (e.g., one, two, three, four, five, six or more immunomodulating agents).

[0304] In some embodiments, the plurality of microneedles may be arranged in a random or predetermined pattern to form the microneedles or patches described herein. The patches may include a carrier, backing, or "handle" layer attached to the back of the base (see, e.g., Figure 3). This layer can provide structural support and area by which the patch can be handled and manipulated without disturbing the array of needles.

[0305] The microneedle devices (e.g., microneedle patches) described herein may be designed to accommodate various numbers of microneedles. In some embodiments, the microneedle device comprises at least 50 microneedles, such as at least about 60, about 64, about 70, about 80, about 81, about 90, about 100, about 110, about 121, about 144, about 150, about 169, about 175, about 196, about 200, about 225, about 250, about 256, about 289, about 300, or more microneedles. In some embodiments, the microneedle device (e.g., a microneedle patch) comprises between about 50 and 500 microneedles, e.g., between about 50 and 400 microneedles, between about 75 and about 300 microneedles, between about 100 and 200 microneedles, or between about 100 and 150 microneedles. The microneedles may be arranged in a grid, e.g., a square grid. In some embodiments, the microneedles are arranged in an 8x8 grid, a 9x9 grid, a 10x10 grid, an 11x11 grid, a 12x12 grid, a 13x13 grid, a 14x14 grid, a 15x15 grid, a 16x16 grid, or a 17x17 grid. In some embodiments, the microneedles are arranged in an 11x11 grid. In some embodiments, the microneedle device has a pitch of between about 0.5 mm and 1.0 mm, for example, about 0.55 mm, 0.60 mm, 0.65 mm, 0.70 mm, 0.75 mm, 0.80 mm, 0.85 mm, 0.95 mm, or 1 mm. In some embodiments, the microneedle device has a pitch of about 0.75 mm.

[0306] In some embodiments, the microneedle device comprises approximately 121 needles in an 11x11 square grid with an approximately 0.75 mm pitch. In some embodiments, the microneedle device (e.g., a microneedle patch) may comprise approximately 121 needles in an 11x11 square grid with an approximately 0.75 mm pitch. In some embodiments, the microneedle device comprises individual needles that are approximately 0.65 mm long, have a base diameter of approximately 0.35 mm, and are conical with a tip angle of approximately 30°. In some embodiments, the microneedle device comprises individual needles with silk fibroin tips, which are sharp enough to penetrate biological barriers (e.g., skin). In some embodiments, the microneedle device comprises individual needles with silk fibroin tips, which should ideally have a radius of bend of 0.01 mm or less. Exemplary microneedles and devices of the present disclosure are illustrated in Figures 2-4.

[0307] Release kinetics The disclosed silk fibroin-based microneedles may be configured to release a therapeutic agent or combination of therapeutic agents (e.g., anti-cancer agents, immunomodulatory agents, or combinations thereof) according to a variety of release kinetics.

[0308] In some embodiments, the silk fibroin-based microneedles may be configured to release an effective amount of an anti-cancer drug and / or an immunomodulatory agent to induce an anti-cancer response in a subject. In certain embodiments, the release of the anti-cancer drug and / or immunomodulatory agent enhances the exposure of the subject's immune system to neoantigens associated with the cancer, resulting in the induction, activation, and / or expansion of immune effector cells, e.g., T cells, specific for the neoantigen in the subject. The release of the anti-cancer drug and / or immunomodulatory agent can promote the development of long-term immunity against cancer in the subject.

[0309] In some embodiments, the silk fibroin-based microneedles may be configured to release a therapeutic agent or combination of therapeutic agents (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) via a burst release. In some embodiments, the burst release comprises a rapid administration of an anti-cancer agent and / or an immunomodulatory agent to a subject. The burst release may comprise a rapid administration of greater than 0% to about 100% (e.g., about 1 to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 100%) of the total amount of anti-cancer agent and / or immunomodulatory agent present on the silk fibroin chip. In some embodiments, the burst release is over a period of at least about 1 hour (e.g., about 1 to about 30 minutes, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 24 hours).

[0310] In some embodiments, silk fibroin-based microneedles may be configured to release a therapeutic agent or combination of therapeutic agents (e.g., anti-cancer agents, immunomodulatory agents, or combinations thereof) via sustained release. Examples of sustained release include, but are not limited to, zero-order release, first-order release, and second-order release. In some embodiments, zero-order release is a release rate that is independent of the concentration of therapeutic agent in the dosage form (e.g., microneedles). In some embodiments, zero-order release is a release of therapeutic agent over approximately a constant time period (e.g., a constant amount of therapeutic agent released per unit time). In some embodiments, first-order release is a release rate that is a function of the amount of therapeutic agent remaining in the dosage form (e.g., microneedles). In some embodiments, first-order release is a constant ratio, e.g., percentage, of therapeutic agent released from the dosage form (e.g., microneedles) per unit time. In some embodiments, second-order release is one in which doubling the concentration of therapeutic agent results in a quadrupling of the release rate in the dosage form. In some embodiments, sustained release comprises substantially continuous low-dose administration of the anticancer agent and / or immunomodulatory agent. The sustained release may comprise continuous administration of greater than about 0% to about 100% (e.g., about 1 to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 100%) of the total amount of anticancer agent and / or immunomodulatory agent present on the silk fibroin chip. In some embodiments, sustained release is over a period of at least about 3 days (e.g., about 3, 4, 5, 6, 7 days or more, e.g., between about 5 and about 10 days, e.g., between about 7 and about 15 days, e.g., between about 1 and about 2 weeks, between about 1 and about 3 weeks, or between about 2 and about 4 weeks, e.g., between about 1 and about 3 months, e.g., between about 2 and about 4 months, e.g., between about 3 and about 6 months). In certain embodiments, sustained release is over a period of about 7 days to about 15 days.

[0311] In some embodiments, release (e.g., administration) of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the silk fibroin-based microneedles described herein may be facilitated by diffusion of the therapeutic agent from the microneedle or a portion thereof.

[0312] In some embodiments, release (e.g., administration) of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the silk fibroin-based microneedles described herein may be facilitated by degradation (e.g., protease-mediated degradation) of the microneedle or a portion thereof.

[0313] In some embodiments, release (e.g., administration) of a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof) from the silk fibroin-based microneedles described herein may be facilitated by dissolving the microneedle or a portion thereof.

[0314] Without wishing to be bound by theory, release of the anti-cancer drug may occur at substantially the same rate (e.g., simultaneously) as release of the immunomodulatory agent, hi other embodiments, release of the anti-cancer drug may occur at a different rate than the release rate of the immunomodulatory agent, such that the anti-cancer drug is released substantially before or substantially after the release of the immunomodulatory agent.

[0315] therapeutic agent In some embodiments, the present disclosure provides silk fibroin-based microneedles comprising a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof). The present disclosure also provides combination treatment methods for administering the microneedles disclosed herein with an additional therapeutic agent. Non-limiting examples of therapeutic agents that may be incorporated into the silk fibroin-based microneedles of the present disclosure and / or administered as a combination therapy with the fibroin-based microneedles of the present disclosure are disclosed below.

[0316] anticancer drugs In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with a low-molecular-weight or small-molecular-weight chemotherapeutic agent. Exemplary low-molecular-weight or small-molecular-weight chemotherapeutic agents include, but are not limited to, 13-cis-retinoic acid (isotretinoin, ACCUTANE®), 2-CdA (2-chlorodeoxyadenosine, cladribine, LEUSTATIN™), 5-azacytidine (azacytidine, VIDAZA®), 5-fluorouracil (5-FU, fluorouracil, ADRUCIL®), 6-mercaptopurine (6-MP, mercaptopurine, PURINETHOL®), 6-TG (6-thioguanine, thioguanine, THIOGUANINE), and 6-methyl-2-methyl-1,3-dihydro-1,3-dihydro-2 ... TABLOID®), Abraxane (paclitaxel protein-bound), actinomycin-D (dactinomycin, COSMEGEN®), alitretinoin (PANRETIN®), all-trans retinoic acid (ATRA, tretinoin, VESANOID®), altretamine (hexamethylmelamine, HMM, HEXALEN®), amethopterin (methotrexate, methotrexate sodium, MTX, TREXALL™, RHEUMATREX®), amifostine (ETHYOL®), arabinosylcytosine (Ara-C, cytarabine, CYTOSAR-U®) , arsenic trioxide (TRISENOX®), asparaginase (Erwinia L-asparaginase, L-asparaginase, ELSPAR®, KIDROLASE®), BCNU (carmustine, BiCNU®), bendamustine (TREANDA®), bexarotene (TARGRETIN®), bleomycin (BLENOXANE®), busulfan (BUSULFEX®, MYLERAN®), leucovorin calcium (citrovorum factor, folinic acid, leucovorin), camptothecin-11 (CPT-11, irinotecan, CAMPTOSAR®), trademark), capecitabine (XELODA®), carboplatin (PARAPLATIN®), carmustine wafers (prolifeprospan 20 with carmustine implants, GLIADEL® wafers), CCI-779 (temsirolimus, TORISEL®), CCNU (lomustine, CeeNU), CDDP (cisplatin, PLATINOL®, PLATINOL-AQ®), chlorambucil (leukeran), cyclophosphamide (CYTO XAN®, NEOSAR®), dacarbazine (DIC, DTIC, imidazole carboxamide, DTIC-DOME®), daunomycin (daunorubicin, daunorubicin hydrochloride, rubidomycin hydrochloride, CERUBIDINE®), decitabine (DACOGEN®), dexrazoxane (ZINECARD®), DHAD (mitoxantrone, NOVANTRONE®), docetaxel (Taxotere®), doxorubicin (ADRIAMYCIN®, UBEX®), epirubicin (ELLENCE™), eribulin (HALAVEN®), estramustine (EMCYT®), etoposide (VP-16, etoposide phosphate, TOPOSAR®, VEPESID®, ETOPOPHOS®), floxuridine (FUDR®), fludarabine (FLUDARA®), fluorouracil (cream) (CARAC™, EFUDEX®, FLUOROPLEX®), gemcitabine (GEMZ AR®), hydroxyurea (HYDREA®, DROXIA™, MYLOCEL™), idarubicin (IDAMYCIN®), ifosfamide (IFEX®), ixabepilone (IXEMPRA™), LCR (leulocristine, vincristine, VCR, ONCOVIN®, VINCASARPFS®), L-PAM (L-sarcolysin, melphalan, phenylalanine mustard, ALKERAN®), mechlorethamine (mechlorethamine hydrochloride, mustine,Nitrogen mustard, MUSTARGEN®), mesna (MESNEX™), mitomycin (mitomycin-C, MTC, MUTAMYCIN®), nelarabine (ARRANON®), oxaliplatin (ELOXATIN™), paclitaxel (Taxol®, ONXAL™), pegaspargase (PEG-L-asparaginase, ONCOSPAR®), pemetrexed (ALIMTA®), pentostatin (NIPENT®), procarbazine (MATULANE®), streptomycin (STP®), thiazolinone (TH ... These include Zosyn (ZANOSAR®), temozolomide (TEMODAR®), teniposide (VM-26, VUMON®), TESPA (thiophosphoamide, thiotepa, TSPA, THIOPLEX®), topotecan (HYCAMTIN®), vinblastine (vinblastine sulfate, vincaleukoblastine, VLB, ALKABAN-AQ®, VELBAN®), vinorelbine (vinorelbine tartrate, NAVELBINE®), and vorinostat (ZOLINZA®).

[0317] In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with an FDA-approved targeted therapy. For example, for the treatment of melanoma, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with binimetinib (MEKTOVI®), cobimetinib (COTELLIC®), dabrafenib (TAFINLAR®), encorafenib (BRAFTOVI®), trametinib (MEKINIST®), and / or vemurafenib (ZELBORAF®).

[0318] In some embodiments, for the treatment of basal cell carcinoma, the silk fibroin-based The microneedles may contain and / or be administered in combination with sonidegib (ODOMZO®) and / or vismodegib (Erivedge®).

[0319] In some embodiments, for the treatment of breast cancer, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with abemaciclib (VERZENIO®), alpelisib (PIQRAY®), olaparib (LYNPARZA®), palbociclib (IBRANCE®), ribociclib (KISQALI®), and / or talazoparib (TALZENNA®).

[0320] In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with a biological agent.Biological agents useful in the treatment of cancer are known in the art, and the silk fibroin-based microneedles described herein may be administered in combination with, for example, such known biological agents. For example, the FDA has approved the following biologics for the treatment of breast cancer: HERCEPTIN® (trastuzumab, Genentech Inc., South San Francisco, CA; a humanized monoclonal antibody with antitumor activity in HER2-positive breast cancer); FASLODEX® (fulvestrant, AstraZeneca Pharmaceuticals, LP, Wilmington, DE; an estrogen receptor antagonist used to treat breast cancer); ARIMIDEX® (anastrozole, AstraZeneca Pharmaceuticals, LP; a nonsteroidal aromatase inhibitor that blocks aromatase, an enzyme necessary to produce estrogen); AROMASIN® (exemestane, Pfizer Inc., New York, NY; an irreversible steroidal aromatase inactivator used in the treatment of breast cancer); FEMARA® (letrozole, Novartis) Pharmaceuticals, East Hanover, NJ; a nonsteroidal aromatase inhibitor approved by the FDA to treat breast cancer; and NOLVADEX® (tamoxifen, AstraZeneca Pharmaceuticals, LP; a nonsteroidal anti-estrogen approved by the FDA to treat breast cancer). Other biologics that may be combined with the silk fibroin-based microneedles of the present disclosure include AVASTIN® (bevacizumab, Genentech Inc.; the first FDA-approved therapy designed to inhibit angiogenesis); and ZEVALIN® (ibritumomab tiuxetan, Biogen Idec, Cambridge, Massachusetts; a radiolabeled monoclonal antibody recently approved to treat B-cell lymphoma.

[0321] Additionally, the FDA has approved the following biologics for treating colorectal cancer: AVASTIN®; ERBITUX® (cetuximab, ImClone Systems Inc., New York, NY, and Bristol-Myers Squibb, New York, NY; a monoclonal antibody against the epidermal growth factor receptor (EGFR); GLEEVEC® (imatinib mesylate; a protein kinase inhibitor); and ERGAMISOL® (levamisole hydrochloride, Janssen Pharmaceutical Products, LP, Titusville, NJ; an immune modulator approved by the FDA in 1990 for adjuvant treatment in combination with 5-fluorouracil after surgical resection in patients with Dukes' stage C colon cancer).

[0322] For the treatment of lung cancer, exemplary biologics include TARCEVA® ) (erlotinib HCL, OSI Pharmaceuticals Inc., Melville, NY; a small molecule designed to target the human epidermal growth factor receptor 1 (HER1) pathway).

[0323] For the treatment of multiple myeloma, an exemplary biologic is VELCADE® Velcade (bortezomib, Millennium Pharmaceuticals, Cambridge, Massachusetts; a proteasome inhibitor). Additional biologics include THALIDOMID® (thalidomide, Clegene Corporation, Warren, New Jersey; an immunomodulatory agent believed to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells and anti-angiogenesis).

[0324] Additional exemplary cancer therapeutic antibodies include, but are not limited to, 3F8, abagovomab, adecatumumab, adotrastuzumab emtansine (KADCYLA®), afutuzumab, alacizumab pegol, alemtuzumab (CAMPATH®, MABCAMPATH®), altumomab pentete (HYBRI-CEAKER®), anatumomab mafenatox, anrukinzumab (IMA-638), apolizumab, arcitumomab (CEA-SCAN®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), bavituximab, bectumomab (LYMPHOSCAN®), belimumab (BENLYSTA®, LYMPHOSTAT-B®), besilesomab (SCINTIMUN®) , bevacizumab (AVASTIN®), bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide (PROSTASCINT®), catumaxomab (REMOVAB®), CC49, cemiplimab-rwlc (LIBTAYO®), cetuximab (C225, ERBITUX®), sitatuzumab vogatoxin (citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, denosumab (PROLIA®), detumomab, ecromeximab, edrecolomab (PANOREX®), elotuzumab , epitumomab cituxetan, epratuzumab ), ertumaxomab (REXOMUN®), etaracizumab etaracizumab, farletuzumab, figitumumab, fresolim Mab, galiximab, gemtuzumab ozogamicin (MYLOTARG®), girentuximab, glenbatumumab vedotin, ibritumomab (ibritumomab tiuxetan, ZEVALIN®), igovomab (INDIMACIS-125®), intetumumab, inotuzumab Mab ozogamicin, ipilimumab, iratumumab, labetuzumab (CEA-CIDE®), lexatumumab, lintuzumab, lucatumumab, lumiri Ximab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, nacolomab butafenatox (nacolomab tafenatox), naptumomab estafenatox ), necitumumab, nimotuzumab (THERACIM®, THERALOC®), nofetumomab merpentan (VERLUM A®), ofatumumab (ARZERRA®), olaratumab, oportuzumab monatox, oregovomab (OVAREX®), trademark), panitumumab (VECTIBIX®), pemtumomab (THERAGYN®), pertuzumab (OMNITARG®), pintumomab, pritumumab, ramucirumab, ranibizumab Lucentis (registered trademark), rilotumumab, rituximab (MABTHERA (registered trademark), RITUXAN (registered trademark), robatumumab, satumab pendetide, sibrotuzumab, siltz ximab, sontuzumab, tacatuzumab tetraxetan (AFP-CIDE®), taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tositumomab (BEXX AR®), trastuzumab (HERCEPTIN®), trastuzumab and hyaluronidase-oysk (HERCEPTINHYLECTA®), tremelimumab, tucotuzumab celmoleukin, bevacizumab lutuzumab, volociximab, votumumab (HUMASPECT®), zalutumumab (HUMAX-EGFR®) and zanolimumab (HUMAX-CD4®). do.

[0325] In other embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with viral cancer therapeutics. Exemplary viral cancer therapeutics include, but are not limited to, vaccinia virus (vvDD-CDSR), carcinoembryonic antigen-expressing measles virus, recombinant vaccinia virus (TK-deleted and GM-CSF), Seneca Valley virus-001, Newcastle virus, Coxsackievirus A21, GL-ONC1, recombinant modified vaccinia Ankara vaccine expressing EBNA1 C-terminus / LMP2 chimeric protein, carcinoembryonic antigen-expressing measles virus, G207 oncolytic virus, p53-expressing modified vaccinia virus Ankara vaccine, OncoVEX GM-CSF-modified herpes simplex type 1 virus, fowlpox virus vaccine vector, recombinant vaccinia prostate-specific antigen vaccine, human papillomavirus 16 / 18 L1 virus-like particle / AS04 vaccine, and MVA-EBNA1 / LMP2Inj.Vaccines, quadrivalent HPV vaccine, quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine (GARDASIL®), recombinant fowlpox-CEA(6D) / TRICOM vaccine; recombinant vaccinia-CEA(6D)-TRICOM vaccine, recombinant modified vaccinia Ankara-5T4 vaccine, recombinant fowlpox-TRICOM vaccine, oncolytic herpesvirus NV1020, HPV L1 VLP vaccine V504, bivalent human papillomavirus (types 16 and 18) vaccine (CERVARIX®), herpes simplex virus HF10, Ad5CMV-p53 gene, recombinant vaccinia DF3 / MUC1 vaccine, recombinant vaccinia-MUC-1 vaccine, recombinant vaccinia-TRICOM vaccine, ALVAC MART-1 vaccine, human preproenkephalin (NP2)-expressing replication-deficient herpes simplex virus type I (HSV-1) vector, wild-type reovirus, reovirus type 3 Dearing (REOLYSIN®), oncolytic virus HSV1716, recombinant modified vaccinia Ankara (MVA)-based vaccine encoding Epstein-Barr virus target antigen, recombinant fowlpox-prostate-specific antigen vaccine, recombinant vaccinia prostate-specific antigen vaccine, recombinant vaccinia-B7.1 vaccine, rAd-p53 gene, Ad5-delta24RGD, HPV vaccine 580299, JX-594 (thymidine kinase-deleted vaccinia virus and GM-CSF), HPV-16 / 18 L1 / AS04, fowlpox virus vaccine vector, vaccinia-tyrosinase vaccine, MEDI-517 HPV-16 / 18 VLP Examples include AS04 vaccine, herpes simplex virus TK99UN, HspE7, adenoviral vector containing thymidine kinase FP253 / fludarabine, ALVAC(2) melanoma multi-antigen therapeutic vaccine, ALVAC-hB7.1, canarypox-hIL-12 melanoma vaccine, Ad-REIC / Dkk-3, rAd-IFN SCH721015, TIL-Ad-INFg, Ad-ISF35, and Coxsackievirus A21 (CVA21, CAVATAK®). In other embodiments, the silk fibroin of the present disclosure. The in-based microneedles may contain and / or be administered in combination with talimogene laherparepvec (IMLYGIC®), an FDA-approved treatment for melanoma.

[0326] In other embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with a neoantigen vaccine. In certain embodiments, the neoantigen vaccine may be prepared, for example, as described in Schumacher et al. Science. 348(6230):69-74, 2015, the entire contents of which are incorporated herein by reference. In some embodiments, the microneedles disclosed herein may be used in methods for identifying neoantigens and / or in methods for preparing neoantigen vaccines. Without wishing to be bound by theory, cancer neoantigens derived from random somatic mutations in tumor tissue represent an attractive type of target for cancer immunotherapy, including cancer vaccines. Vaccination against tumor-specific neoantigens minimizes the potential induction of central and peripheral tolerance and the risk of autoimmunity (see, e.g., Guo et al. Frontiers in Immunology. Vol. 9 Article 1499, 2018, the entire contents of which are incorporated herein by reference). In certain embodiments, application of the microneedles of the present disclosure to a tumor can expose the subject's immune system to tumor-specific neoantigens, resulting in vaccination and the development of immunity against cancers bearing the same or similar tumor-specific neoantigens.

[0327] In other embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with nanomedicines. Exemplary cancer nanomedicines include, but are not limited to, Abraxane® (paclitaxel-conjugated albumin nanoparticles), CRLX101 (camptothecin (CPT) conjugated to a linear cyclodextrin-based polymer), CRLX288 (docetaxel conjugated to the biodegradable polymer poly(lactic-co-glycolic acid)), cytarabine liposomes (liposomal Ara-C, DEPOCYT™), daunorubicin liposomes (DAUNOXOME®), doxorubicin liposomes (DOXIL®, CAELYX®), encapsulated daunorubicin citrate liposomes (DAUNOXOME®), and PEG-anti-VEGF aptamer (MACUGEN®).

[0328] In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with paclitaxel or paclitaxel formulations, such as Taxol®, protein-bound paclitaxel (e.g., Abraxane®). Exemplary paclitaxel formulations include, but are not limited to, nanoparticle albumin-bound paclitaxel (Abraxane®, marketed by Abraxis Bioscience), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, Taxoplexin, marketed by Protarga), polyglutamic acid-bound paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX, marketed by CellTherapeutic), tumor-activated prodrug (TAP), ANG105 (Angiopep-2 conjugated to three molecules of paclitaxel, marketed by ImmunoGen), paclitaxel-EC-1 (paclitaxel conjugated to the erbB2-recognizing peptide EC-1; Li et al. al., Biopolymers (2007) 87:225-230), and glucose-conjugated paclitaxel (e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate (see, e.g., Liu et al., Bioorganic & Medicinal Chemistry Letters (2007) 17:617-620).

[0329] Exemplary RNAi and antisense RNA agents for treating cancer include, but are not limited to, CALAA-01, siG12DLODER (local drug EluteR), and ALN-VSP02.

[0330] Other cancer therapeutic agents include, but are not limited to, cytokines (e.g., aldesleukin (IL-2, interleukin-2, PROLEUKIN®), alpha interferons (IFN-alpha, interferon alpha, INTRON® A (interferon alpha-2b), ROFERON-A® (interferon alpha-2a)), epoetin alpha (PROCRIT®), filgrastim (G-CSF, granulocyte-colony stimulating factor, NEUPOGEN®), GM-CSF (granulocyte-macrophage colony-stimulating factor, sargramostim, LEUKINE™), IL-11 (interleukin-11, oprelvekin, NEUMEGA®), interferon interferon alfa-2b (PEG conjugate) (PEG-interferon, PEG-INTRON™), and pegfilgrastim (NEULASTA™), hormonal therapies (e.g., aminoglutethimide (CYTADREN®), anastrozole (ARIMIDEX®), bicalutamide (CASODEX®), exemestane (AROMASIN®), fluoxymesterone (HALOTESTIN®), flutamide (EULEXIN®), fulvestrant (FASLODEX®), goserelin (ZOLADEX®), letrozole (FEMARA®), leuprolide (ELIGARD™, LUPRON®, LUPRON®), DEPOT®, VIADUR®), megestrol (megestrol acetate, MEGACE®), nilutamide (ANANDRON®, NILANDRON®), ocretide (ocretide acetate, SANDOSTATIN®, SANDOSTATIN®),LAR®), raloxifene (EVISTA®), romiplostim (NPLATE®), tamoxifen (NOVALDEX®), and toremifene (FARESTON®), phospholipase A2 inhibitors (e.g., anagrelide (AGRYLIN®), biological response modifiers (e.g., BCG (THERACYS®, TICE®), and darbepoetin alfa (ARANESP®)), targeted therapies (e.g., bortezomib (VELCADE®), dasatinib (SPRYCEL™), denileukin diftitox (ONTAK®), erlotinib (TARCEVA®), ), everolimus (AFINITOR®), gefitinib (IRESSA®), imatinib mesylate (STI-571, GLEEVEC™), lapatinib (TYKERB®), sorafenib (NEXAVAR®), and SU11248 (sunitinib, SUTENT®), immunomodulatory and antiangiogenic agents (e.g., CC-5013 (lenalidomide, REVLIMID®), and thalidomide (THALOMID®), glucocorticosteroids (e.g., cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, ALA-CORT®, HYDROCORT®), ACETATE®, hydrocortone phosphate, LANACORT®, SOLU-CORTEF®), Decadron (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, DEXASONE®, DIODEX®, HEXADROL®, MAXIDEX®), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, DURALONE®, MEDRALONE®, MEDROL®, M-PREDNISOL®, SOLU-MEDROL®), prednisolone (DELTA-CORTEF®, ORAPRED®, PEDIAPRED®, PRELONE®), and prednisone (DELTASONE®, LIQUID PRED®, METICORTEN®, ORASONE®), and bisphosphonates (e.g., pamidronate (AREDIA®), and zoledronic acid (ZOMETA®)).

[0331] In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor). Exemplary tyrosine kinase inhibitors include, but are not limited to, epidermal growth factor (EGF) pathway inhibitors (e.g., epidermal growth factor receptor (EGFR) inhibitors), vascular endothelial growth factor (VEGF) pathway inhibitors (e.g., antibodies against VEGF, VEGF traps, vascular endothelial growth factor receptor (VEGFR) inhibitors (e.g., VEGFR-1 inhibitors, VEGFR-2 inhibitors, VEGFR-3 inhibitors)), platelet-derived growth factor (PDGF) pathway inhibitors (e.g., platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., PDGFR-β inhibitors)), RAF-1 inhibitors, KIT inhibitors, and RET inhibitors. In some embodiments, the anticancer agent used in combination with the AHCM agent is axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Irmanobufenib), rituximab (TARCEVA®), rituximab (Irmanobufenib ... nib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®), SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF1120 (VARGATEF®), AP24534, JNJ-264833 27, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, AEE788, AG-490, AST-6, BMS-599626, CUDC-101, PD153035, pelitinib (EKB-569), vandetanib (zactima), WZ3146, WZ4002, WZ8040, ABT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951 (tivozanib), axitinib , BAY73-4506 (regorafenib), brivanib alaninate (BMS-582664), brivanib (BMS-540215), cediranib (AZD2171), CHIR-258 (dovitinib), CP673451, CYC116, E7080, Ki8751, masitinib (AB1010), MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, pazopanib hydrochloride, PD173074, sorafenib tosylate (Bay43-9006), SU5402, TSU-68 (SU6668), vatalanib,XL880 (GSK1363089, EXEL-2880). The tyrosine kinase inhibitor is selected from sunitinib, erlotinib, gefitinib, or sorafenib, hi one embodiment, the tyrosine kinase inhibitor is sunitinib.

[0332] In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with one of an anti-angiogenic agent or a vascular targeting or vascular disrupting agent. Exemplary anti-angiogenic agents include, but are not limited to, vascular endothelial growth factor (VEGF) inhibitors (e.g., anti-VEGF antibodies (e.g., bevacizumab); VEGF receptor inhibitors (e.g., itraconazole); inhibitors of cell proliferation and / or endothelial cell migration (e.g., carboxyamidotriazole, TNP-470); and angiogenesis stimulator inhibitors (e.g., suramin), among others. Vascular targeting agents (VTA) or vascular disrupting agents (VDA) are designed to damage the vasculature (blood vessels) of cancer tumors, resulting in central necrosis (see, e.g., Thorpe, PE (2004) Clin. Cancer 2004). Res. Vol. 10:415-427). The VTA may be a small molecule. Exemplary small molecule VTAs include, but are not limited to, microtubule-destabilizing drugs (e.g., combretastatin A-4 disodium phosphate (CA4P), ZD6126, AVE8062, Oxi4503); and vadimezan (ASA404).

[0333] In certain embodiments, the anti-cancer agents described herein may be formulated as sustained release particles.

[0334] immune modulators Checkpoint inhibitors In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with an immune checkpoint inhibitor. For example, in certain embodiments, silk fibroin-based microneedles may be used to locally administer a therapeutic agent (e.g., an anti-cancer agent and / or an immunomodulatory agent) to a tumor in a subject in combination with systemic administration (e.g., by injection) of a checkpoint inhibitor (e.g., an anti-PD1 antibody).

[0335] In embodiments, immune checkpoint inhibitors inhibit checkpoint molecules. Exemplary checkpoint molecules include, but are not limited to, CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, and A2aR. See, e.g., Pardoll. Nat. Rev. Cancer 12.4 (2012): 252-64, which is incorporated herein by reference.

[0336] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, for example, an anti-PD-1 antibody, for example, nivolumab, pembrolizumab or pidilizumab. Nivolumab (also known as MDX-1106, MDX-1106-04, ONO-4538 or BMS-936558) is a fully human IgG4 monoclonal antibody that specifically inhibits PD1. For example, see U.S. Patent No. 8,008,449 and International Publication No. 2006 / 121168. Pembrolizumab (also known as lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1 (see, e.g., Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, U.S. Patent No. 8,354,509, and WO 2009 / 114335). 11 or CureTech) is a humanized IgG1k monoclonal antibody that binds to PD1 (see, e.g., WO 2009 / 101611). In one embodiment, the PD-1 inhibitor is an antibody molecule having a sequence substantially identical to or similar to, e.g., at least 85%, 90%, 95% or more identical to, the sequence of nivolumab, pembrolizumab, or pidilizumab. Additional anti-PD1 antibodies, such as AMP514 (Amplimmune), are described, e.g., in U.S. Patent Nos. 8,609,089, 2010028330, and / or 20120114649.

[0337] In some embodiments, the PD-1 inhibitor is an immunoadhesin, e.g., an immunoadhesin comprising the extracellular / PD-1-binding portion of a PD-1 ligand (e.g., PD-L1 or PD-L2) fused to a constant region (e.g., the Fc region of an immunoglobulin). In embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg, e.g., as described in WO 2011 / 066342 and WO 2010 / 027827), a PD-L2Fc fusion soluble receptor that blocks the interaction between B7-H1 and PD-1.

[0338] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, such as an antibody molecule. In some embodiments, the PD-L1 inhibitor is YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105. In some embodiments, the anti-PD-L1 antibody is MSB0010718C (also referred to as A09-246-2; Merck Serono), which is a monoclonal antibody that binds to PD-L1. Exemplary humanized anti-PD-L1 antibodies are described, for example, in WO 2013 / 079174. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody, such as YW243.55.S70. The YW243.55.S70 antibody is described, for example, in WO 2010 / 077634. In one embodiment, the PD-L1 inhibitor is MDX-1105 (also referred to as BMS-936559), which is described, for example, in WO 2007 / 005874. In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche), which is a human Fc-optimized IgG1 monoclonal antibody against PD-L1. See, for example, U.S. Patent No. 7,943,743 and U.S. Patent Application Publication No. 20120039906. In one embodiment, the PD-L1 inhibitor is an antibody molecule having a sequence substantially identical or similar to that of YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105, e.g., at least 85%, 90%, 95% or more identical.

[0339] In embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor, such as AMP-224 (a PD-L2Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1. See, e.g., WO 2010 / 027827 and WO 2011 / 066342).

[0340] In one embodiment, the immune checkpoint inhibitor is a LAG-3 inhibitor, such as an anti-LAG-3 antibody molecule. In one embodiment, the anti-LAG-3 antibody is BMS-986016 (also referred to as BMS986016; Bristol-Myers Squibb). BMS-986016 and other humanized anti-LAG-3 antibodies are described, for example, in U.S. Patent No. 2011 / 0150892, WO 2010 / 019570, and WO 2014 / 008218.

[0341] In embodiments, the immune checkpoint inhibitor is a TIM-3 inhibitor, e.g., an anti-TIM3 antibody molecule, and is described, for example, in U.S. Pat. No. 8,552,156, WO 2011 / 155607, EP 2581113, and U.S. Patent Application Publication No. 2014 / 04472 It is described in No. 8.

[0342] In embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, for example, an anti-CTLA-4 antibody molecule. Exemplary anti-CTLA4 antibodies include tremelimumab (an IgG2 monoclonal antibody from Pfizer, formerly known as ticilimumab, CP-675,206); and ipilimumab (also known as MDX-010, CAS number 477202-00-9). Other exemplary anti-CTLA-4 antibodies are described, for example, in U.S. Patent No. 5,811,097.

[0343] TLR agonists In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with a Toll-like receptor (TLR) agonist.

[0344] TLRs are a family of pattern recognition receptors (TLRs) originally identified as sensors of the innate immune system that recognize microbial pathogens. In humans, TLRs include TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, ​​TLR-7, TLR-8, TLR-9, and TLR-10. TLR-1, -2, -4, -5, and -6 are expressed on the cell surface, while TLR-3, -7 / 8, and -9 are expressed in the ER compartment. Human dendritic cell subsets can be identified based on distinct TLR expression patterns. The myeloid or "conventional" subset of human dendritic cells expresses TLRs 1-8, while the plasmacytoid subset of dendritic cells expresses only TLR-7 and TLR-9. Ligand binding to TLRs triggers a cascade of intracellular signaling pathways that induce the production of factors involved in inflammation and immunity. Upon stimulation, the myeloid and plasmacytoid subsets of human dendritic cells lead to antigen-specific CD4+ and CD8+ T cell priming and NK cell and T cell activation, respectively.

[0345] In some embodiments, the TLR agonist is selected from one or more of a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-10 agonist, a TLR-1 / 2 agonist, a TLR-2 / 6 agonist, or a TLR-7 / 8 agonist. In one embodiment, the TLR agonist is a TLR7 agonist.

[0346] In some embodiments, the TLR agonist is imiquimod or 3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine. Imiquimod or 3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine can bind to and activate TLR-7 and / or TLR-8.

[0347] In some embodiments, the TLR agonist is 852A. 852A is disclosed, for example, in Inglefield et al. J Interferon Cytokine Res. 2008;28(4):253-63. 852A can bind to and activate TLR-7 and / or TLR-8.

[0348] In some embodiments, the TLR agonist is Bacille Calmette-Guerin (BCG), which can bind to and activate TLR-9. In some embodiments, the TLR agonist is EMD120108. EMD120108 is a synthetic oligonucleotide comprising phosphorothioate oligodeoxynucleotides. EMD1201081 can bind to and activate TLR-9, initiating immune signaling pathways, activating B cells, and inducing cytokine production in helper T cells, for example, in monocytes / macrophages, plasmacytoid dendritic cells (DCs), and B cells.

[0349] In some embodiments, the TLR agonist is IMO-2055. IMO-2055 is a synthetic oligonucleotide containing unmethylated CpG dinucleotides. By mimicking the unmethylated CpG sequence in bacterial DNA, IMO-2055 can bind to and activate TLR-9, for example, in monocytes / macrophages, plasmacytoid dendritic cells (DCs), and B cells, initiating immune signaling pathways, activating B cells and DCs, and inducing cytokine production by helper T cells.

[0350] Other exemplary TLR agonists that may be used in combination include, for example, TLR-1 / 2 agonists (e.g., Pam3Cys), TLR-2 agonists (e.g., CFA, MALP2, Pam2Cys, FSL-1, or Hib-OMPC), TLR-3 agonists (e.g., polyribosinic:polyribocytidylic acid (Po ly I:C), polyadenosine-polyuridylic acid (poly AU), poly-L-lysine and carboxymethylcellulose-stabilized polyinosine-polycytidylic acid (Hiltonol®), TLR-4 agonists (e.g., monophosphoryl lipid A (MPL), LPS, sialyl-Tn (STn)), TLR-5 agonists (e.g., bacterial flagellin), TLR-7 agonists (e.g., imiquimod), TLR-7 / 8 agonists (e.g., resiquimod or loxoribine), and TLR-9 agonists (e.g., unmethylated CpG dinucleotides (CpG-ODN)).

[0351] In another embodiment, the TLR agonist is used in combination with a GITR agonist, for example, as described in WO 2004 / 060319 and WO 2014 / 012479.

[0352] STING agonists In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with a STING agonist.

[0353] In some embodiments, the STING agonist is a cyclic dinucleotide, for example, a cyclic dinucleotide comprising a purine or pyrimidine nucleobase (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobase). In some embodiments, the nucleobases of the cyclic dinucleotide comprise the same nucleobase or different nucleobases.

[0354] In some embodiments, the STING agonist comprises an adenosine or guanosine nucleobase. In some embodiments, the STING agonist comprises one adenosine nucleobase and one guanosine nucleobase. In some embodiments, the STING agonist comprises two adenosine nucleobases or two guanosine nucleobases.

[0355] In some embodiments, the STING agonist comprises a modified cyclic dinucleotide, e.g., comprising a modified nucleobase, a modified ribose, or a modified phosphate linkage. In some embodiments, the modified cyclic dinucleotide comprises a modified phosphate linkage, e.g., a thiophosphate.

[0356] In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) with a 2',5' or 3',5' phosphate bond. In some embodiments, the STING agonist comprises an Rp or Sp stereoisomer around the phosphate bond. These include cyclic dinucleotides with modified chemistry (eg, modified cyclic dinucleotides).

[0357] In some embodiments, the STING agonist is Rp,Rp-dithio 2',3'c-di-AMP (e.g., Rp,Rp-dithio c-[A(2',5')pA(3',5')p]) or a cyclic dinucleotide analog thereof. In some embodiments, the STING agonist is a compound illustrated in U.S. Patent Application Publication No. 2015 / 0056224 (e.g., the compound in Figure 2c, e.g., Compound 21 or Compound 22). In some embodiments, the STING agonist is c-[G(2',5')pG(3',5')p], its dithioribose O-substituted derivative, or a compound illustrated in Figure 4 of WO 2014 / 189805 and WO 2014 / 189806. In some embodiments, the STING agonist is c-[A(2',5')pA(3',5')p] or a dithioribose O-substituted derivative thereof, or a compound depicted in Figure 5 of WO 2014 / 189805 and WO 2014 / 189806. In some embodiments, the STING agonist is c-[G(2',5')pA(3',5')p] or a dithioribose O-substituted derivative thereof, or a compound depicted in Figure 5 of WO 2014 / 189805 and WO 2014 / 189806. In some embodiments, the STING agonist is 2'-O-propargyl-cyclic-[A(2',5')pA(3',5')p](2'-O-propargyl-ML-CDA) or the compound depicted in Figure 7 of WO 2014 / 189806.

[0358] Other exemplary STING agonists are disclosed, for example, in International Publication Nos. 2014 / 189805 and 2014 / 189806, and U.S. Patent Application Publication No. 2015 / 0056225.

[0359] RIG agonists In some embodiments, the silk fibroin-based microneedles of the present disclosure may comprise and / or be administered in combination with an RIG agonist (i.e., an agonist of retinoic acid-inducible gene I (RIG-I), encoded by the gene DDX58). Exemplary RIG agonists are described in Elion et al. Oncotarget. 9(48):29007-29017, 2018, which is incorporated herein by reference in its entirety.

[0360] cytokines In some embodiments, the silk fibroin-based microneedles of the present disclosure may contain and / or be administered in combination with cytokines.

[0361] Cytokines are generally polypeptides that affect cellular activity, for example, through signal transduction pathways. Therefore, cytokines are useful and may be associated with receptor-mediated signaling, transmitting signals from the outside of the cell membrane to modulate intracellular responses. Cytokines are proteinaceous signaling compounds that mediate immune responses. They regulate many different cellular functions, including proliferation, differentiation, and cell survival / apoptosis; cytokines are also involved in several pathophysiological processes, including viral infections and autoimmune diseases. Cytokines are synthesized by various cells of both the innate immune system (monocytes, macrophages, dendritic cells) and the adaptive immune system (T and B cells) under various stimuli. Cytokines can be classified into two groups: pro-inflammatory and anti-inflammatory. Pro-inflammatory cytokines, including IFNγ, IL-1, IL-6, and TNF-alpha, are primarily derived from innate immune cells and Th1 cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL-13, and IL-5, are synthesized by Th2 immune cells.

[0362] Thus, in some embodiments, the cytokine molecule is an interleukin or a variant thereof, e.g., a functional variant thereof. In some embodiments, the interleukin is a pro-inflammatory interleukin. In some embodiments, the interleukin is selected from interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-7 (IL-7), or interferon gamma. In some embodiments, the cytokine molecule is a pro-inflammatory cytokine. In certain embodiments, the cytokine molecule is IL-18.

[0363] The cytokine may be wild-type (e.g., wild-type recombinant) or genetically engineered (e.g., to introduce one or more mutations). In some embodiments, the cytokine is a genetically engineered cytokine, such as a genetically engineered interleukin. In some embodiments, the genetically engineered cytokine contains one or more mutations, e.g., to confer different biological properties relative to the wild-type variant. For example, the cytokine molecule may be a genetically engineered "decoy-resistant" interleukin (e.g., decoy-resistant IL-18) as described by Zhou et al. (Nature (2020) 583:609-614; incorporated herein by reference in its entirety). In some embodiments, the cytokine is a genetically engineered interleukin (e.g., genetically engineered IL-2 or genetically engineered IL-18).

[0364] In some embodiments, the cytokine is genetically engineered to improve one or more pharmacokinetic properties. In some embodiments, the genetically engineered cytokine includes a cytokine fused to another molecule (e.g., an antibody) to, for example, increase the serum half-life of the cytokine. In some embodiments, the cytokine is fused to a long-half-life protein or protein domain (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion). In some embodiments, the cytokine is fused to an inert polypeptide (e.g., XTEN or recombinant PEG (rPEG); homoamino acid polymer (HAP; e.g., by HAPylation); proline-alanine-serine polymer (PAS; e.g., by PASylation); or elastin-like peptide (ELP; e.g., by ELPylation)). In some embodiments, the cytokine is conjugated to repeating chemical moieties (e.g., a polymer, e.g., PEG by PEGylation; or hyaluronic acid) to, for example, increase the hydrodynamic radius of the cytokine. In some embodiments, the negative charge of the cytokine is increased, for example, by polysialylation of the cytokine or by fusing a negatively charged, highly sialylated peptide (e.g., carboxy-terminal peptide [CTP; chorionic gonadotropin (CG) β-chain]) to the cytokine. In some embodiments, the cytokine is non-covalently bound to a long half-life protein, for example, HSA, human IgG, or transferrin. In some embodiments, the cytokine is chemically conjugated to a long half-life protein, for example, human IgG, Fc portion, or HSA. Methods for preparing and using fusion proteins and similar modified proteins, for example, to extend half-life, have been described (see, for example, Strohl et al. BioDrugs (2015) 29:215-239; and references cited therein).

[0365] Genetically engineered cytokines (e.g., genetically engineered interleukins) may be generated by known methods, for example, by directed evolution techniques (see, e.g., Zhou et al., supra). Examples of genetically engineered cytokines (e.g., genetically engineered interleukins) have been described (e.g., WO 2012 / 107417; WO 2009 / 061853; U.S. Pat. No. 9,580,486; Minsahwi et al.Front Immunol.2020(11);1794;Tang et al. Cytokine X (2019) 100001; Mitra et al. Immunity (2015) 42:826-838; Casadesus et al. OncoImmunology (2020) 9:1770565; Zhou et al., supra; each of which is incorporated by reference in its entirety.

[0366] Without wishing to be bound by theory, an engineered cytokine (e.g., a "decoy-resistant" interleukin, e.g., decoy-resistant IL-18) may have beneficial properties compared to its wild-type counterpart. By way of example, decoy-resistant IL-18 may maintain signaling potential while remaining less susceptible to inhibition by IL-18 binding protein (IL-18BP). In some embodiments, the engineered cytokine has improved stability (e.g., improved temperature-dependent stability, pH-dependent stability, or both) compared to the wild-type cytokine. In some embodiments, the engineered cytokine has an improved serum half-life compared to the wild-type cytokine. In some embodiments, the engineered cytokine has an altered affinity (e.g., enhanced affinity) for a receptor different from the wild-type cytokine. In some embodiments, the engineered cytokine has reduced toxicity relative to the wild-type cytokine.

[0367] In certain embodiments, the cytokine is a single-chain cytokine. In certain embodiments, the cytokine is a multi-chain cytokine (e.g., the cytokine comprises two or more (e.g., two) polypeptide chains). An exemplary multi-chain cytokine is IL-12.

[0368] Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. In one embodiment, the cytokine of the multiproperty or multifunctional polypeptide is a cytokine selected from the group of GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β, and TGF-β. In one embodiment, the cytokine of the multiproperty or multifunctional polypeptide is a cytokine selected from the group consisting of GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β, and TGF-β. In one embodiment, the cytokine of the multiproperty or multifunctional polypeptide is a cytokine selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IFN-α, and IFN-γ. In one embodiment, the cytokine of the multiproperty or multifunctional polypeptide is a cytokine selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. In certain embodiments, the cytokine is mutated to remove N- and / or O-glycosylation sites. Elimination of glycosylation can increase the homogeneity of the product available in recombinant production.

[0369] In one embodiment, the cytokine is IL-2. In certain embodiments, the IL-2 cytokine induces one or more of the following cellular responses selected from the group consisting of proliferation in activated T lymphocytes, differentiation in activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in natural killer (NK) cells, differentiation in NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) anti-tumor cytotoxicity. It is possible.

[0370] In another embodiment, the cytokine is IL-12. In certain embodiments, the IL-12 cytokine is a single-chain IL-12 cytokine. In one embodiment, the IL-12 cytokine can induce one or more of the following cellular responses: proliferation in NK cells, differentiation in NK cells, proliferation in T cells, and differentiation in T cells.

[0371] In another embodiment, the cytokine is IL-10. In another specific embodiment, the IL-10 cytokine is a monomeric IL-10 cytokine. In one embodiment, the IL-10 cytokine can induce one or more of the following cellular responses selected from the group consisting of inhibiting cytokine secretion, inhibiting antigen presentation by antigen-presenting cells, reducing oxygen radical release, and inhibiting T cell proliferation.

[0372] In certain embodiments, the IL-15 cytokine is a mutant IL-15 cytokine with reduced binding affinity to the α-subunit of the IL-15 receptor. Without wishing to be bound by theory, mutant IL-15 polypeptides with reduced binding to the α-subunit of the IL-15 receptor have a reduced ability to bind to fibroblasts throughout the body and have improved pharmacokinetics and toxicity profiles compared to wild-type IL-15 polypeptides. In one embodiment, the IL-15 cytokine is capable of inducing one or more cellular responses selected from the group consisting of proliferation in activated T lymphocytes, differentiation in activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in natural killer (NK) cells, differentiation in NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) anti-tumor cytotoxicity.

[0373] In another embodiment, the cytokine is interleukin-18 (IL-18). In one embodiment, the cytokine is an IL-18 variant polypeptide. In one embodiment, the IL-18 cytokine is decoy-resistant IL-18 (see, e.g., U.S. Patent Application Publication No. 2019 / 0070262; and Zhou et al., supra, each of which is incorporated herein by reference in its entirety). In one embodiment, the IL-18 cytokine can induce one or more cellular responses selected from the group consisting of proliferation in NK cells, differentiation in NK cells, proliferation in T cells, differentiation in T cells, and simulation of lymphocytes (e.g., innate lymphocytes). Without wishing to be bound by theory, IL-18 can be used as an effective immunotherapeutic agent and is well tolerated in humans (see, e.g., Robertson et al. Clin. Cancer Res. (2006) 12:4265-4273). In some embodiments, IL-18 (e.g., decoy-resistant IL-18) increases the population of precursor T cells (e.g., CD8+ T cells) that express a transcription factor (e.g., Tcf1) with anti-tumor function. In some embodiments, IL-18 (e.g., decoy-resistant IL-18) promotes the differentiation of T cells toward a highly active, multifunctional effector phenotype. In some embodiments, IL-18 (e.g., decoy-resistant IL-18) reduces the prevalence of exhausted CD8+ T cells (e.g., those expressing exhausted TOX transcriptional regulators). In some embodiments, IL-18 (e.g., decoy-resistant IL-18) enhances the activity and / or maturation of NK cells.

[0374] In some embodiments, the cytokine is decoy-resistant interleukin IL-18. In some embodiments, the cytokine is an IL-18 variant polypeptide, wherein the IL-18 variant polypeptide specifically binds to the IL-18 receptor (IL-18R) compared to wild-type (WT) IL-18, and the IL-18 variant polypeptide exhibits at least and exhibits significantly reduced binding to IL-18 binding protein (IL-18BP). In some embodiments, the cytokine is an IL-18 variant polypeptide that includes at least one mutation selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X compared to a wild-type IL-18 sequence, e.g., SEQ ID NO: 30 of U.S. Patent No. 2019 / 0070262. In some embodiments, the cytokine is selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G59A, M60K, M60Q, M60R, M60L, E77D, E77E, E77F, E77G, E77F, E77H ... , Q103E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H. In some embodiments, the cytokine is an IL-18 variant polypeptide that includes at least six mutations selected from Y1X, L5X, K8X, M51X, K53X, 555X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X compared to a wild-type IL-18 sequence, e.g., SEQ ID NO: 30 of U.S. Patent No. 2019 / 0070262.In some embodiments, the cytokine is an IL-18 variant polypeptide that includes mutations at positions M51, K53, Q56D110, and N111 compared to a wild-type IL-18 sequence, e.g., SEQ ID NO: 30 of US 2019 / 0070262. In some embodiments, the cytokine is an IL-18 variant polypeptide sequence that includes the following five mutations compared to a wild-type IL-18 sequence, e.g., SEQ ID NO: 30 of U.S. Patent No. 2019 / 0070262: (i) M51E, M51R, M51K, M51T, M51D, or M51N; (ii) K53G, K53S, K53T, or K53R; (iii) Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K; (iv) D110S, D110N, D110G, D110K, D110H, D110Q, or D110E; and (v) N111G, N111R, N111S, N111D, N111H, or N111Y. In some embodiments, the cytokine is an IL-18 variant polypeptide that further comprises mutations at positions P57 and M60 compared to a wild-type IL-18 sequence, e.g., SEQ ID NO: 30 of US 2019 / 0070262. In some embodiments, the cytokine comprises the following seven mutations compared to a wild-type IL-18 sequence, e.g., SEQ ID NO: 30 of US 2019 / 0070262: (i) M51E, M51R, M51K, M51T, M51D, or M51N; (ii) K53G, K53S, K53T, or K53R; (iii) Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or (iv) D110S, D110N, D110G, D110K, D110H, D110Q, or D110E; (v) N111G, N111R, N111S, N111D, N111H, or N111Y; (vi) P57A, P57L, P57G, or P57K; and (vii) M60L, M60R, M60K, or M600.In some embodiments, the cytokine is an IL-18 variant polypeptide sequence comprising a polypeptide sequence described in U.S. Patent No. 2019 / 0070262 (e.g., SEQ ID NOs: 34-59, 60-72, 73-91, 191-193, or one of fragments thereof).

[0375] Without wishing to be bound by theory, IL-18 variant proteins (e.g., decoy-resistant IL-18) can avoid potential attenuation of activity due to the presence of IL-18 binding proteins, such as IL-18BP. Without wishing to be bound by theory, IL-18 variant proteins (e.g., decoy-resistant IL-18) can avoid IL-18BP, further promoting innate anti-tumor immunity by stimulating NK cell activity and / or enhancing NK cell maturation, and exerting anti-tumor immunity against tumors that are resistant to standard immune checkpoint blockade therapy (e.g., anti-PD-1 and anti-CLTA-4) due to loss of MHC class I surface expression.

[0376] Mutant cytokine molecules useful as effector moieties may be prepared by deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes may be verified, for example, by sequencing. Substitutions or insertions may involve natural and unnatural amino acid residues. Amino acid modifications include well-known methods such as chemical modification, for example, adding or removing glycosylation sites, or attaching carbohydrates.

[0377] In one embodiment, the cytokine is GM-CSF. In certain embodiments, the GM-CSF cytokine can induce proliferation and / or differentiation in granulocytes, monocytes, or dendritic cells.

[0378] In one embodiment, the cytokine is IFN-α. In certain embodiments, the IFN-α cytokine can induce one or more cellular responses selected from the group consisting of inhibiting viral replication in virus-infected cells and upregulating the expression of major histocompatibility complex I (MHCI). In another specific embodiment, the IFN-α cytokine can inhibit proliferation in tumor cells. In one embodiment, the cytokine, particularly a single-chain cytokine, is IFNγ. In certain embodiments, the IFN-γ cytokine can induce one or more cellular responses selected from the group consisting of increasing macrophage activity, increasing the expression of MHC molecules, and increasing NK cell activity.

[0379] In one embodiment, the cytokine, particularly the single chain cytokine, is IL-7. In certain embodiments, the IL-7 cytokine is capable of inducing proliferation of T and / or B lymphocytes.

[0380] In one embodiment, the cytokine is IL-8. In certain embodiments, the IL-8 cytokine can induce chemotaxis in neutrophils. In one embodiment, the cytokine, particularly the single-chain cytokine, is MIP-1α. In certain embodiments, the MIP-1α cytokine can induce chemotaxis in monocytes and T lymphocyte cells. In one embodiment, the cytokine is MIP-1β. In certain embodiments, the MIP-1β cytokine can induce chemotaxis in monocytes and T lymphocyte cells. In one embodiment, the cytokine is TGF-β. In certain embodiments, the TGF-β cytokine can induce one or more cellular responses selected from the group consisting of chemotaxis in monocytes, chemotaxis in macrophages, upregulation of IL-1 expression in activated macrophages, and upregulation of IgA expression in activated B cells.

[0381] In some embodiments, the microneedle or combination treatment disclosed herein comprises a cytokine. In embodiments, the cytokine may be a full-length cytokine, a fragment or a polypeptide. a variant; a cytokine receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule against a cytokine receptor (e.g., an agonist antibody).

[0382] In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18, IL-7, IL-21, or interferon gamma, or a fragment or variant thereof, or a combination of any of the foregoing cytokines. The cytokine molecule may be a monomer or a dimer. In embodiments, the cytokine molecule may further comprise a cytokine receptor dimerization domain.

[0383] In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, for example, an antibody molecule (eg, an agonist antibody) against a cytokine receptor selected from IL-15Ra or IL-21R.

[0384] Other immune modulating agents include, but are not limited to, cancer vaccines, eg, cancer vaccines that include viral cancer therapeutics, and / or tumor antigens, eg, neoantigens.

[0385] Active ingredient formulation At least one therapeutic agent disclosed herein may be incorporated into various formulations, compositions, articles, devices, and / or preparations for administration, for example, to achieve controlled release and / or sustained release. More specifically, at least one therapeutic agent may be formulated into a formulation, composition, article, device, and / or preparation by combining with a suitable pharmaceutically acceptable carrier or diluent, and may be formulated into a semi-solid, solid, or liquid format preparation. In some embodiments, the formulations, compositions, articles, devices, and / or preparations described herein include silk fibroin. Exemplary formulations, compositions, articles, devices, and / or preparations include microneedles (e.g., microneedle devices, e.g., microneedle patches, e.g., as described herein), implantable devices (e.g., pumps, e.g., subcutaneous pumps), injectable formulations, depots, gels (e.g., hydrogels), implants, and particles (e.g., microparticles and / or nanoparticles). Thus, administration of the compositions may be achieved in a variety of ways, including intradermal, intramuscular, transdermal, subcutaneous, or intravenous administration. Additionally, the formulations, compositions, articles, devices, and / or preparations may be formulated and / or administered to achieve controlled and / or sustained release of the therapeutic agent.

[0386] In some embodiments, the therapeutic agent is administered for, e.g., substantially continuously, over a period of 1, 5, 10, 15, 30, or 45 minutes or at least these periods; 1, 2, 3, 4, 5, 10, or 24 hours or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or at least these periods; 1, 2, 3, 4, 5, 6, 7, or 8 weeks or at least these periods; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months or at least these periods; or 1, 2, 3, 4, 5 years or more or at least these periods. In one embodiment, the therapeutic agent, e.g., an anticancer agent, an immunomodulatory agent, or a combination thereof, is administered as a controlled-release or sustained-release formulation, dosage form, or device. In one embodiment, the therapeutic agent, e.g., an anticancer agent, an immunomodulatory agent, or a combination thereof, is administered as a burst-release formulation, dosage form, or device. In certain embodiments, the therapeutic agent is formulated for continuous delivery, e.g., intradermally, intramuscularly, and / or intravenously. In some embodiments, the composition or device for controlled or sustained release of the therapeutic agent is administered using a microneedle (e.g., a microneedle device, e.g., a microneedle pump). The therapeutic agent may be administered via an implantable device, such as a pump (e.g., a subcutaneous pump), an implant, an injectable formulation, a depot, a gel (e.g., a hydrogel), an implant, or a particle (e.g., a microparticle and / or nanoparticle). In one embodiment, the therapeutic agent is a silk fibroin-based microneedle controlled- or sustained-release dosage form or formulation (e.g., a microneedle described herein). In one embodiment, the therapeutic agent is administered via an implantable device, such as a pump (e.g., a subcutaneous pump), an implant, an implantable tip of a microneedle, or a depot. Delivery methods may be optimized so that a dose (e.g., a standard dose) of a therapeutic agent described herein is administered and / or maintained in a subject for a predetermined period of time (e.g., 1, 5, 10, 15, 30, 45 minutes; 1, 2, 3, 4, 5, 10, 24 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; 1, 2, 3, 4, 5, 6, 7, 8 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; 1, 2, 3, 4, 5 years or at least longer). Substantially sustained or sustained release of a therapeutic agent may be used to prevent or treat a disease and / or disorder, such as cancer, over a period of hours, days, weeks, months, or years.

[0387] The present disclosure provides, in some embodiments, formulations, compositions, articles, devices, and / or preparations that may be formulated and / or configured for controlled or sustained release of a therapeutic agent in an amount (e.g., dosage) and / or for a period of time sufficient to produce an immune response (e.g., a cellular immune response and / or a humoral immune response) against an antigen (e.g., a tumor-specific antigen, e.g., a neoantigen) in a subject.

[0388] In some embodiments, the formulations, compositions, articles, devices, and / or preparations of the present disclosure may be formulated and / or configured for controlled or sustained release of at least a therapeutic agent in an amount (e.g., dosage) and / or for a period of time sufficient to result in immunity (e.g., cancer immunity) in a subject.

[0389] Delivery or formulations that provide substantially continuous or sustained release of a therapeutic agent may be used to prevent or treat a disease or disorder, such as cancer, over a period of hours, days, weeks, months, or years.

[0390] In some embodiments, a therapeutic agent described herein may be added to a silk fibroin solution, for example, prior to forming a silk fibroin microneedle or microneedle device described herein. In embodiments, the silk fibroin solution may be mixed with a therapeutic agent and then used in the fabrication of an implantable microneedle tip, for example, by a filling and / or casting, drying, and / or annealing process, to produce a microneedle having any of the desired material properties described herein.

[0391] Without being bound by theory, the ratio of silk fibroin to therapeutic agent in the silk fibroin tip of the microneedle, e.g., the implantable tip, influences their release. In some embodiments, increasing the concentration of silk in the tip aids in delayed release and / or better retention of the therapeutic agent within the tip. Any concentration of silk can be used as long as it is printable and has sufficient mechanical strength to penetrate the skin.

[0392] In some embodiments, silk fibroin may be used at a concentration ranging from about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) in the manufacture of microneedles or components thereof described herein. In some embodiments, silk fibroin may be used at a concentration ranging from about 1% w / v to about 30% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) in the manufacture of microneedles or components thereof described herein. , about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% w / v).

[0393] In some embodiments, silk fibroin may be used in an amount of about 0.5 μg to about 500 μg of silk fibrion in the manufacture of microneedles or components thereof described herein. In some embodiments, silk fibroin may be used in an amount of about 0.5 μg to about 5 μg, or about 1 μg to about 10 μg, or about 5 μg to about 15 μg, or about 10 μg to about 20 μg, or about 15 μg to about 25 μg, or about 20 μg to about 30 μg, or about 25 μg to about 35 μg, or about 30 μg to about 40 μg, or about 35 μg to about 45 μg, or about 40 μg to about 50 μg, or about 45 μg to about 55 μg, or about 50 μg to about 60 μg, or about 55 μg to about 65 μg, or about 60 μg to about 70 μg or about 65 μg to about 75 μg, or about 70 μg to about 80 μg, or about 75 μg to about 85 μg, or about 80 μg to about 90 μg, or about 85 μg to about 95 μg, or about 90 μg to about 100 μg, or about 95 μg to about 150 μg, or about 125 μg to about 175 μg, or about 150 μg to about 200 μg, or about 225 μg to about 275 μg, or about 250 μg to about 300 μg, or about 325 μg to about 375 μg, or about 350 μg to about 400 μg, or about 425 μg to about 475 μg, or about 450 μg to about 500 μg of silk fibrions may be used. In some embodiments, silk fibroin may be used in the manufacture of microneedles or components thereof described herein in an amount of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrion.In some embodiments, silk fibroin may be used in the manufacture of microneedles or components thereof described herein in amounts of up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrion. In some embodiments, silk fibroin may be used in the manufacture of the microneedles or components thereof described herein in an amount of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg of silk fibrion. In some embodiments, silk fibroin may be used in the manufacture of the microneedles or components thereof described herein in an amount of about 2.42 μg to 242 μg.

[0394] In some embodiments, silk fibroin may be used in the manufacture of microneedles or components thereof described herein in an amount of about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% by weight of silk fibrions. In some embodiments, silk fibroin may be used in the manufacture of microneedles or components thereof described herein in an amount of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions. In some embodiments, silk fibroin is used in the manufacture of microneedles or components thereof described herein in an amount of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% silk fibrion by weight. In some embodiments, silk fibroin may be used in the manufacture of microneedles or components thereof described herein in an amount of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrion.

[0395] Exemplary Excipients Additionally, the formulations, compositions, articles, devices, and / or preparations may be formulated with common excipients, diluents, or carriers for administration by intradermal, intramuscular, transdermal, subcutaneous, or intravenous routes. In some embodiments, the formulations, compositions, articles, devices, and / or preparations may be administered transdermally, for example, and may be formulated as controlled- or sustained-release dosage forms, etc. The formulations, compositions, articles, devices, and / or preparations described herein may be administered alone, in combination with each other, or used in combination with other known therapeutic agents.

[0396] Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences (1985). Additionally, for a review of methods for drug delivery, see Langer (1990) Science 249:1527-1533. The formulations, compositions, articles, devices, and / or preparations described herein can be manufactured in a manner known to those skilled in the art, for example, by mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The following methods and excipients are merely exemplary and in no way limiting.

[0397] The silk fibroin formulations used in the manufacture of the microneedles described herein may contain excipients. In certain embodiments, the inclusion of an excipient may be for the purpose of improving the stability of the incorporated therapeutic agent, e.g., an anticancer agent, an immunomodulatory agent, or a combination thereof, increasing the porosity of the silk matrix and the diffusibility of the therapeutic agent, e.g., an anticancer agent, an immunomodulatory agent, or other combination thereof, from the formulation, composition, article, device, preparation, and / or microneedle, e.g., the microneedle tip, and / or increasing the crystallinity / beta-sheet content of the silk matrix to render the silk material insoluble.

[0398] Exemplary excipients include sugars or sugar alcohols (e.g., sucrose, trehalose, sorbitol, mannitol, or combinations thereof), divalent cations (e.g., Ca 2+ , Mg 2+ , Mn 2+ , and Cu 2+ ), surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbates, poloxamers, and / or polyethoxylated alcohols), polyols (e.g., glycerol), glycols (e.g., propylene glycol, PEG), and / or buffers. In some embodiments, the concentration of excipients can be used to modify the porosity of the matrix; for example, sucrose is the most common excipient used for this purpose. Excipients may also be added to support silk self-assembly into an ordered beta-sheet secondary structure; such excipients generally participate in hydrogen bonding or charge interactions with the silk to achieve this effect. Non-limiting examples of excipients that can be used to support silk self-assembly into an ordered beta-sheet secondary structure include sodium glutamate (e.g., L-glutamic acid), lysine, sugar alcohols (e.g., sorbitol and / or glycerol), and solvents (e.g., DMSO, methanol, and / or ethanol).

[0399] In some embodiments, the sugar or sugar alcohol is, for example, less than 70% (w / v), less than 60% (w / v), less than 50% (w / v), less than 40% (w / v), less than 30% (w / v), less than 50% (w / v), less than 60% (w / v), less than 70% (w / v), less than 60% (w / v), less than 50% (w / v), less than 40% (w / v), less than 30% (w / v), less than 5 ... Sucrose present in an amount of less than 0% (w / v), less than 20% (w / v), less than 10% (w / v), less than 9% (w / v), less than 8% (w / v), less than 7% (w / v), less than 6% (w / v), or 5% (w / v) or less.

[0400] In some embodiments, the sugar or sugar alcohol is sucrose, present in an amount of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just prior to drying.

[0401] In some embodiments, the sugar or sugar alcohol is trehalose, present in an amount of, for example, about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), just prior to drying.

[0402] In some embodiments, the sugar or sugar alcohol is sorbitol, present in an amount of, for example, about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), just prior to drying.

[0403] In some embodiments, the sugar or sugar alcohol is glycerol, present in an amount of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just prior to drying.

[0404] In some embodiments, the surfactant (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, and / or polyethoxylated alcohol) is present in an amount of about 0.005% (w / v) to about 1% (w / v), about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), e.g., just prior to drying.

[0405] In some embodiments, the polyol (e.g., glycerol) is present in an amount of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), e.g., just prior to drying.

[0406] In some embodiments, the glycol (e.g., propylene glycol, e.g., PEG) is present in an amount of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% (w / v) to about 5% (w / v), or about 2.4% (w / v), about 2.5%, or about 5% (w / v), e.g., just prior to drying.

[0407] In some embodiments, the therapeutic agent preparation further comprises a divalent cation. In some embodiments, the divalent cation is Ca 2+ , Mg 2+ , Mn 2+ , and Cu 2+ A group consisting of In some embodiments, the divalent cation is present in the preparation in an amount of 0.1 mM to 100 mM, e.g., immediately prior to drying. In some embodiments, the divalent cation is present in the preparation in an amount of 0.1 mM to 100 mM, e.g., immediately prior to drying. In some embodiments, the divalent cation is present in the preparation in an amount of 10 mM to 100 mM, e.g., immediately prior to drying. -7 Molar ~ 10 -4 In some embodiments, the divalent cation is present in the preparation in a molar amount of 10 immediately prior to drying. -10 moles ~ 2 x 10 -3 It is present in the preparation in molar amounts.

[0408] In some embodiments, the therapeutic agent further comprises poly(lactic-co-glycolic acid) (PGLA). In some embodiments, the therapeutic agent preparation further comprises a buffer, e.g., immediately prior to drying. In some embodiments, the buffer has a buffering capacity of pH 3 to pH 8, pH 4 to pH 7.5, or pH 5 to pH 7. In some embodiments, the buffer is selected from the group consisting of PBS, HEPES, and CP buffer. In some embodiments, the buffer is present in the preparation in an amount of 0.1 mM to 100 mM, e.g., immediately prior to drying. In some embodiments, the buffer is present in the preparation in an amount of 10 mM to 100 mM per standard dose of the therapeutic agent (e.g., anti-cancer agent, immunomodulatory agent, viral immunogen, or combination thereof). -7 Molar ~ 10 -4 In some embodiments, the buffer is present in a 10 molar amount immediately prior to drying. -10 moles ~ 2 x 10 -3 Present in molar amounts.

[0409] Furthermore, the therapeutic agent can also be formulated as a depot, gel, or hydrogel preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic agent can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt.

[0410] In one embodiment, the therapeutic agent is administered via an implantable infusion device, e.g., a pump (e.g., a subcutaneous pump), implant, or depot. Implantable infusion devices typically include a housing containing a liquid reservoir that can be filled percutaneously with a hypodermic needle that penetrates an infusion port septum. The medication reservoir is generally connected via an internal fluid path to a device outlet port for delivering the liquid through a catheter to a patient's body site. A typical infusion device also includes a controller and a fluid transfer mechanism, e.g., a pump or valve, for moving the liquid from the reservoir, through the internal fluid path, to the device outlet port.

[0411] In some embodiments, therapeutic agents may be packaged and / or formulated as particles, e.g., microparticles and / or nanoparticles. Typically, nanoparticles have a diameter of 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150, or 200 nm, or between 200 nm and 1,000 nm, e.g., 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150, or 200, or between 20, 30, or 50, and 400 nm. Smaller particles tend to be cleared from the system more rapidly. Therapeutic agents, including those described herein, may be encapsulated within or linked to nanoparticles, e.g., covalently bonded to or otherwise attached to nanoparticles.

[0412] Liquid or oil-based nanoparticles, such as liposomes and solid lipid nanoparticles, can be used to deliver therapeutic agents, such as anti-cancer agents, immunomodulatory agents, or combinations thereof, as described herein.Solid lipid nanoparticles for the delivery of therapeutic agents have been described (see, for example, Serpe et al. (2004) Eur.J.Pharm.Bioparm.58:673-680, and Lu et al. (2006) Eur.J.Pharm.Sci.28:86-95).Polymer-based nanoparticles, such as PLG PLGA-based nanoparticles can be used to deliver the drugs described herein. They rely on a biodegradable scaffold with a therapeutic agent (with or without covalent attachment to the polymer) inserted into the polymer matrix. PLGA has been widely used in polymeric nanoparticles (see, for example, Hu et al. (2009) J. Control. Release 134:55-61; Cheng et al. (2007) Biomaterials 28:869-876; and Chan et al. (2009) Biomaterials 30:1627-1634). PEGylated PLGA-based nanoparticles can also be used to deliver therapeutic agents (see, for example, Danhhier et al. (2009) J. Control. Release 133:11-17; Gryparis et al. (2007) Eur. J. Pharm. Biopharm. 67:1-8). Metal-based nanoparticles, e.g., gold-based nanoparticles, can also be used to deliver therapeutic agents. Protein-based nanoparticles, e.g., albumin-based nanoparticles, can be used to deliver the therapeutic agents described herein. In some embodiments, the therapeutic agent can be bound to nanoparticles of human albumin.

[0413] A wide range of nanoparticles are known in the art. Exemplary approaches include those described in International Publication Nos. 2010 / 005726, 2010 / 005723, 2010 / 005721, 2008 / 121949, 2010 / 075072, 2010 / 068866, 2010 / 005740, 2006 / 014626, U.S. Patent Nos. 7,820,788 and 7,780,984, the contents of which are incorporated herein by reference in their entirety.

[0414] Dosage Any dosage (e.g., standard doses and / or divided doses) of a therapeutic agent capable of eliciting a therapeutic response in a subject (e.g., an anti-cancer response, e.g., an immune response) when administered by the microneedles of the present disclosure may be used in accordance with the methods described herein.

[0415] Without wishing to be bound by theory, the total dosage (e.g., a standard dose) of a therapeutic agent to be administered by the microneedles described herein may be divided among multiple microneedles (e.g., within a patch) such that the microneedle tip can contain less than about 1% of the total dosage (e.g., in an array comprising about 121 microneedles), or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more of the total dosage.

[0416] In some embodiments, the dosage of the therapeutic agent loaded into the microneedle patch may be manipulated through the concentration of the therapeutic agent in the formulation solution forming the needle tip, the volume of solution dispensed into the tip of each needle, and the total number of needles. In some embodiments, the former two are more convenient means of varying the dose. The dosage released into a subject is related to the placement efficiency (the portion of the needle tip that is left behind in the biological barrier, e.g., the skin, after removing the patch) and the time-dependent release profile and residence time of the tip within the subject. Due to the continuous shedding of skin from the epidermis, the depth of placement within the skin is related to the length of residence time. As a result, it is desirable to maximize the penetration depth of the needle tip (to the limit defined by the depth of pain receptors within the skin, e.g., between about 100 μm and about 600 μm) and to have the antigen spatially concentrated toward the needle tip.

[0417] The formulations, compositions, articles, devices, and compositions described herein, including silk fibroin chip formulations, The device, and / or preparation is designed to not only release, e.g., sustain release, of the therapeutic agent over the period of retention of the tip in the dermis, but also to maintain stability of the therapeutic agent during this period (e.g., at least about 1-2 weeks). In some embodiments, for example, approximately 95-100% of the total dosage incorporated into the formulations, compositions, articles, devices, preparations, and / or microneedles described herein may be expected to be available for delivery into a subject, e.g., into a subject's tissue, e.g., skin, tumor, mucosa, organ tissue, buccal cavity, tissue, or cell membrane. Without being bound by theory, successful placement of the microneedles into the skin is at least about 50% of the array, and may be as high as 100% (e.g., upon application, at least about 50%, 60%, 70%, 80%, 90% or more (e.g., 100%) of the total number of microneedles in the array are successfully placed, e.g., within the skin, for controlled or sustained release of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof). In some embodiments, a portion of the antigen may not be released from the silk tip during placement.

[0418] Method for manufacturing microneedles A schematic illustrating an exemplary method of fabricating microneedles of the present disclosure is shown in Figure 1. Machine vision-guided dispensing of precise nanoliter (nL) volumes of silk fibroin solution into individual needle cavities allows different dosages and formulations to be incorporated into the releasable tip of a microneedle device (e.g., a microneedle array or patch). An exemplary microneedle device (e.g., a microneedle array or patch) includes an 11 x 11 conical array. It should be understood that a microneedle device may include needle cavities fabricated in an array with various numbers of cavities and orientations to achieve a desired result.

[0419] Mold manufacturing In some embodiments, the mold is used in the manufacture of a microneedle device. As will be discussed in more detail below, the sterilized mold is used to generate a microneedle device having an array of releasable tips incorporating a therapeutic agent-silk formulation (e.g., an anti-cancer agent-silk formulation, an immunomodulatory agent-silk formulation, an antigen-silk formulation, or a combination thereof).

[0420] For example, silicone (DOW Corning Sylgard® 184) resin may be cast against a positive master having the intended geometry of the microneedle array. Once the silicone has hardened, it can be removed from the master. The master may then be reused for multiple silicone castings. Throughout the manufacturing process (e.g., during casting), the silicone mold may be inspected for defects. If necessary, the silicone mold may be sterilized, for example, by autoclaving. In one embodiment, the mold includes a mold body having an array of needle cavities formed therein.

[0421] In some embodiments, other types of silicone and / or other materials and processes may be used to manufacture the mold. For example, liquid silicone injection molding and thermoplastic elastomer injection molding may be used. Without wishing to be bound by theory, it can be understood that the key requirement is that the mold material be soft and flexible (e.g., having a Shore hardness of about 50A) and have low adhesion to silk and other materials used in the construction of the patch.

[0422] Chip filling The chip formulation, consisting of silk fibroin in aqueous solution, a therapeutic agent (e.g., an anti-cancer agent, an immunomodulator, an antigen, or a combination thereof), and possibly other excipients, is used to The solution is dispensed into each needle cavity of the mold via a nozzle print. Currently, this is performed on a laboratory scale using a machine vision-guided automated dispensing system, such as the BioDot Biojet Elite™ AD3400 dispensing system, although systems with similar capabilities manufactured by other suppliers may also be used. In some embodiments, the working volume of the dispenser (e.g., a BioDot™ dispenser) used to fill the tips is sealed and maintained at a high relative humidity (RH), e.g., 60% or higher, e.g., to slow drying of the formulation and / or to prevent the accumulation of dried solids on the dispensing nozzle. In certain embodiments, the working volume of a BioDot™ dispenser is sealed and maintained at 60% relative humidity (RH) to slow drying of the formulation and to prevent the accumulation of dried solids on the dispensing nozzle.

[0423] The molds are placed in a fixture that holds them in place on the processing platform of the BioDot™ dispenser. The machine uses a camera to image each mold, and machine vision algorithms identify the exact location and orientation of the array of needle cavities in each mold. This location is used to direct subsequent dispensing steps. The filled molds are inspected using a stereomicroscope for filling defects, such as misaligned dispenses or large air bubbles in the liquid.

[0424] Primary drying In some embodiments, the filled mold is secured and dried, for example, in an enclosure that maintains a desired ambient humidity. In certain embodiments, the filled mold is secured and dried in a mechanical enclosure for about 7 minutes. In some embodiments, silk fibroin solubility may be modulated by manipulating the drying time. Without wishing to be bound by theory, during drying, the silk structure may shift further to beta sheets, making it less soluble (e.g., insoluble); this effect can be enhanced by drying more slowly and / or by incubating at high humidity, for example, from about 10% to about 100% RH.

[0425] After drying, the dispensing process described above may be repeated. In some embodiments, the mold is transferred to a chamber with near-saturated humidity and incubated overnight to slowly dry the chip. During this time, the silk structure shifts further to beta sheets and becomes less soluble (e.g., insoluble) (annealing).

[0426] Secondary drying In some embodiments, the mold is transferred to a room temperature and relative humidity (RH) controlled chamber at about 10% to about 25% humidity and maintained overnight (about 14 hours) to complete drying, which is the "secondary" drying step.

[0427] Water Annealing In some embodiments, the mold (e.g., the mold containing the dried silk fibroin chips) is transferred to a vacuum desiccator that also contains about 500 mL of deionized water (DIW). The desiccator is closed and a vacuum is applied using the laboratory's main vacuum line for about 5 minutes. After 5 minutes, the outlet valve of the desiccator is closed and the mold is placed in an incubator at 37°C for 4 hours. After 4 hours, the desiccator is evacuated and the mold is returned to a chamber at 25% RH and room temperature.

[0428] Drying after annealing The mold may be maintained at about 10% to about 25% RH for at least 4 hours or up to overnight before subsequent steps.

[0429] Base layer filling The dissolvable base layer may be formed by filling a mold with a base solution described herein. In some embodiments, the base solution comprises 40% w / v hydrolyzed gelatin and 10% w / v sucrose in DIW. In some embodiments, the base solution comprises 30% dextran 70 kDa, 10% sucrose, 1% glycerol, and 0.01% Triton-X100. The base layer may be filled in any suitable manner. For example, first, a suitable volume of base solution for the mold (e.g., 150 μL) may be evenly spread across the entire mold using a pipette. The mold may then be centrifuged (e.g., at 3900 rpm for up to about 2 minutes). The mold may be inspected, and if any needle cavities remain unfilled, the filling and centrifugation process may be repeated. The mold may be further "finished" with a suitable amount of base solution (e.g., 50 μL of base solution). In some embodiments, centrifugal filling may be used. In some embodiments, the base is filled into the mold cavity in the same manner as the tip by using vision-guided droplet dispensing.

[0430] In some embodiments, more than one base layer may be applied. For example, a first base layer may be formed according to the method outlined above. Then, for example, after the first base layer is dried, the process may be repeated to add one or more additional base layers. The one or more additional base layers may be applied using the same base layer solution as used for the first base layer, or one or more different base layer solutions (e.g., one or more base layer solutions described herein) may be used as needed. In some embodiments, the base solution is a molten liquid or a slurry. In some embodiments, the base layer is solidified (e.g., after filling) using a chemical reaction.

[0431] Drying the base The filled mold may be returned to the chamber at about 10% to about 25% RH and allowed to dry for at least overnight and up to 3 days.

[0432] Applying a Backing Patches used to produce controlled or sustained release of therapeutic agents and / or improve immunogenicity (see, e.g., the Examples) have a paper backing layer; however, subsequent developments have shown that adhesive plastic tape can perform well as a backing layer.

[0433] In some embodiments, the paper backing process is as follows: The dried base layer is partially rewetted with 10-30 μL of DIW, spreading it over the entire surface using a pipette. Whatman 903 paper is punched into a 12 mm diameter circle. The paper circle is gently pressed onto the wet surface of the base layer. The wet base layer is partially immersed in the paper. The mold with the backing is returned to a 25% RH chamber and allowed to dry for at least 4 hours before use. In some embodiments, the backing (e.g., a backing containing an adhesive) is cured by light irradiation.

[0434] Adhesive Tape Process Cut a strip of adhesive-backed polyester tape (e.g., 3M® magic™ tape) approximately 12 mm wide and 25 mm long. Align one end of the tape with the patch and gently press it onto the surface of the base layer. Fold the free end of the tape over on itself to form a non-adhesive "handle."

[0435] Demolding Remove the patch from the mold before use. Gradually bend the flexible mold to make it more rigid. The mold is then released from the patch and the patch is removed from the mold. The patch is inspected for defects, such as missing or broken needles.

[0436] packaging In the studies described above, the patches were used immediately after demolding and were not packaged. If long-term storage is required, the assembled patches may be packaged in a container with low water vapor transmission rate (e.g., a glass vial or a thermoformed plastic tray made of a low MVTR material and a foil-lined, heat-sealed lid) with a desiccant to maintain a relative humidity inside the package of between about 0% and about 50% (e.g., between about 0% and 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, or between about 40% and 50%, e.g., about 25%).

[0437] Therapeutic applications In one aspect, the present disclosure provides methods for delivering (e.g., administering) an effective amount of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, across a biological barrier (e.g., a skin layer, a cell membrane, a mucosal surface, the oral cavity, a skin lesion, a tumor, or the buccal cavity).

[0438] In one aspect, the present disclosure provides a method for treating, preventing, and / or ameliorating a disease and / or disorder in a subject, such as cancer and / or a skin condition in a subject. In some embodiments, delivery is by intratumoral delivery. However, in some embodiments, microneedles may be applied in proximity to a tumor (e.g., peritumoral delivery). In some embodiments, microneedle administration is as a primary therapy, for example, for unresectable tumors. In some embodiments, microneedle administration is as a neoadjuvant to shrink a tumor (e.g., before primary treatment, e.g., surgery to remove the tumor). In some embodiments, microneedle administration is as an adjuvant to reduce the risk of cancer recurrence (e.g., after tumor resection).

[0439] In one aspect, the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject a microneedle of the present disclosure, such that the cancer is treated in the subject. Exemplary cancers treatable by the microneedles of the present disclosure are known in the art and disclosed herein.

[0440] In one aspect, the present disclosure relates to a method of treating a skin condition in a subject, the method comprising administering to the subject a microneedle of the present disclosure, such that cancer is treated in the subject. Exemplary skin conditions treatable by the microneedles of the present disclosure are known in the art and disclosed herein.

[0441] In one aspect, the present disclosure relates to methods of eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, hi some embodiments, the anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in the subject comprises a reduction in tumor or cancer volume, a reduction in the number of tumor or cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor or cancer cell proliferation, a reduction in tumor or cancer cell survival, prevention of relapse, and / or an improvement in various physiological symptoms associated with a cancerous condition.

[0442] In some embodiments, the present disclosure provides a method for eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, thereby reducing tumor or cancer volume in the subject by at least about 5%, e.g., compared to a baseline value. %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or greater reduction is obtained.

[0443] In some embodiments, the present disclosure relates to methods of eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, thereby resulting in at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more reduction in the number of tumor or cancer cells in the subject, e.g., as compared to a baseline value.

[0444] In some embodiments, the present disclosure relates to methods of eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, thereby resulting in at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more reduction in the number of metastases in the subject, e.g., as compared to baseline values.

[0445] In some embodiments, the present disclosure relates to methods of eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, and extending the subject's lifespan by at least about 15, 30, 60, 90, 120, 180, or 360 days.

[0446] In some embodiments, the present disclosure relates to methods of eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, thereby resulting in at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more reduction in tumor cell proliferation or cancer cell proliferation, e.g., compared to baseline values.

[0447] In some embodiments, the present disclosure relates to methods of eliciting an anti-cancer and / or immune response (e.g., a local and / or systemic immune response) in a subject in need thereof, thereby resulting in at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more decrease in tumor cell viability or cancer cell viability, e.g., compared to baseline values.

[0448] In one aspect, the present disclosure provides methods for treating diseases associated with expression of tumor-specific antigens (e.g., neoantigens). In one aspect, the present disclosure relates to methods for inhibiting the growth of tumors and / or skin lesions, for example, tumors and / or skin lesions that express tumor-specific antigens (e.g., neoantigens). In certain embodiments, the tumor to be treated may be associated with a cancer-specific or tumor-specific antigen that may not be present in normal cells. However, without wishing to be bound by theory, the neoantigen may not be presented sufficiently or efficiently to the subject's immune system, for example, due to an immunosuppressive tumor microenvironment (TME). In some embodiments, the methods described herein can improve presentation of the neoantigen to the subject's immune system and promote the development of a robust and long-lasting immune response (e.g., an anti-cancer response, e.g., cancer immunity) in the subject. In certain embodiments, the methods described herein can result in the ablation of tumors at or near the location of initial microneedle application, and can also result in the ablation of tumors in the subject, for example, wherever cancer cells expressing the neoantigen are present. This results in the removal of tumors at distant sites within the body.

[0449] The present disclosure provides silk fibroin-based microneedles and silk fibroin-based microneedle devices for treating and / or eliciting an immune response against cancer (e.g., metastatic cancer) and / or skin conditions (e.g., skin conditions). In some embodiments, the methods disclosed herein involve contacting (e.g., administering) a microneedle device or plurality of microneedles comprising an anti-cancer agent, an immunomodulatory agent, or a combination thereof to a site or lesion (e.g., skin lesion) of a cancer (e.g., metastatic tumor) in a subject, thereby (i) lysis of cancer cells, e.g., tumor cells, to release and / or expose cancer-associated antigens (e.g., neoantigens) to the subject's immune system; (ii) presentation of cancer-associated antigens (e.g., neoantigens) complexed with major histocompatibility complexes (MHC) by antigen-presenting cells (APCs) to immune system cells (e.g., accessory cells, e.g., B cells, dendritic cells, etc.). (iii) recognition of the presented cancer-associated antigen (e.g., neoantigen) by immune effector cells, e.g., T cells and / or NK cells; (iv) activation and / or expansion of immune effector cells, e.g., T cells and / or NK cells, specific for the presented cancer-associated antigen (e.g., neoantigen) in the subject; and (v) enhancement, e.g., stimulation or upregulation, of an immune response of immune effector cells, e.g., T cells and / or NK cells, that promotes killing and / or inhibition of growth or proliferation of target cells expressing the cancer-associated antigen (e.g., neoantigen) in the subject.

[0450] In one aspect, the present disclosure relates to methods of releasing and / or exposing tumor-specific antigens (e.g., neoantigens) to a subject's immune system to elicit an anti-cancer and / or immune response. In one aspect, the present disclosure relates to a method of inducing cancer immunity in a subject, optionally wherein the cancer immunity is directed against a tumor-specific antigen (e.g., a neoantigen).

[0451] In some embodiments of any of the methods described herein, administration of microneedles results in activation of immune cells in response to tumor-specific antigens (e.g., neoantigens) exposed and / or released from the tumor as a result of treatment with the microneedles (e.g., microneedles comprising an anti-cancer agent and an immunomodulatory agent, and / or a combination thereof). The activated immune cells then target cancer cells expressing the tumor-specific antigens (e.g., neoantigens), thereby inhibiting cancer growth and / or proliferation. Optionally, the targeted cancer cells are present at or near the site of microneedle administration. Optionally, the targeted cancer cells are located at a distal site.

[0452] In one aspect, the present disclosure relates to a method of inducing an anti-cancer response in a subject. In some embodiments, the method includes administering a microneedle of the present disclosure to a subject, e.g., to induce an immune response against a disease associated with tumor-specific antigen (e.g., neoantigen) expression. In various aspects of any of the methods described herein, the immunity (e.g., cancer immunity) persists in the subject for a period of time after administration of the microneedle. For example, immunity (e.g., cancer immunity) may persist in a subject for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 2 years, about 3 years, about 4 years, or about 5 years after administration of the microneedles to the subject.

[0453] In one embodiment, an effective amount of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, is delivered to the location of the tumor after tumor resection, e.g., to induce an immune response against the tumor and / or to remove any cancer cells left behind after resection. Methods for administering (e.g., administering) are provided herein.

[0454] In one aspect, provided herein are methods for delivering (e.g., administering) an effective amount of a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof, to the location of a tumor prior to resecting the tumor, e.g., to induce an immune response against the tumor and / or to remove any cancer cells left behind after resection.

[0455] Such methods may include providing microneedles containing a therapeutic agent, such as an anti-cancer agent described herein, an immunomodulatory agent, or a combination thereof. For example, such methods may include providing at least one microneedle or at least one microneedle device described herein, wherein the microneedle or microneedle device comprises a silk fibroin-based tip having a therapeutic agent, e.g., an anti-cancer agent, an immunomodulatory agent, or a combination thereof; penetrating the microneedle or microneedle device into a biological barrier (e.g., skin, e.g., a tumor); and releasing an effective amount of the therapeutic agent from the silk fibroin tip over a period of time, e.g., at least about 1 day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, e.g., between about 4 days and about 14 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, e.g., 1, 2, 3, 4, 5 years or more).

[0456] Combination Therapy The microneedles disclosed herein may be used in combination with a second therapeutic agent or procedure.

[0457] In embodiments, the microneedles and the second therapeutic agent or procedure disclosed herein are administered / performed after a subject is diagnosed with cancer, for example, before the cancer has disappeared from the subject. In embodiments, the microneedles and the second therapeutic agent or procedure are administered / performed concomitantly or simultaneously. For example, delivery of one treatment is still occurring when delivery of the second begins, for example, there is overlap in the administration of the treatments. In other embodiments, the microneedles and the second therapeutic agent or procedure are administered / performed sequentially. For example, delivery of one treatment is stopped before delivery of the other treatment begins.

[0458] In embodiments, the combination therapy may result in a more effective treatment than monotherapy using either agent alone. In embodiments, the combination of the first and second treatments is more effective (e.g., leads to a greater reduction in symptoms and / or cancer cells) than the first or second treatment alone. In embodiments, the combination therapy allows for the use of lower doses of the first or second treatment compared to the doses of the first or second treatment normally required to achieve a similar effect when administered as monotherapy. In embodiments, the combination therapy has a partially additive effect, a fully additive effect, or a greater-than-additive effect.

[0459] In one embodiment, the microneedle molecules are administered in combination with a therapy, e.g., a cancer therapy (e.g., one or more of an anti-cancer agent, immunotherapy, photodynamic therapy (PDT), surgery, and / or radiation). The terms "chemotherapy," "chemotherapeutic agent," and "anti-cancer agent" are used interchangeably herein. The administration of the microneedle and the therapy, e.g., a cancer therapy, may be sequential or concomitant (with or without overlap). The administration of the microneedle may be continuous or intermittent during the course of treatment (e.g., cancer therapy). Certain therapies described herein may be used to treat cancer and non-cancerous diseases. For example, the effectiveness of a therapeutic agent can be assessed in cancerous and non-cancerous conditions using the methods and compositions described herein. It may be strengthened.

[0460] Patient selection In some embodiments of any of the methods of treating a subject or compositions for use disclosed herein, the subject has a disorder, eg, cancer.

[0461] "Cancer," as used herein, may encompass all types of tumorigenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues, or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, e.g., stages of cancer invasiveness / severity. In embodiments, cancer includes relapsing and / or resistant cancers. The terms "cancer" and "tumor" may be used interchangeably. For example, both terms encompass solid and liquid tumors. As used herein, the terms "cancer" or "tumor" include pre-cancers, as well as malignant cancers and tumors. Examples of various cancers are described herein, and include, but are not limited to, anal cancer; basal cell carcinoma; bladder cancer; bone cancer; brain cancer; breast cancer; cervical cancer; colorectal cancer; endometrial cancer; esophageal cancer; gastrointestinal cancer (e.g., gastrointestinal stromal tumor); gestational trophoblastic disease; head and neck cancer; Hodgkin's lymphoma; Kaposi's sarcoma; kidney (renal cell) cancer; leukemia; liver cancer; lung cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; multicentric Castleman disease; multiple myeloma, and Other plasma cell neoplasms; myeloproliferative neoplasms; neuroblastoma; non-Hodgkin's lymphoma; ovarian, fallopian tube, or primary peritoneal cancer; pancreatic cancer; penile cancer; pheochromocytoma and paraganglioma; prostate cancer; retinoblastoma; rhabdomyosarcoma; skin cancer; squamous cell carcinoma; soft tissue sarcoma; any solid tumor in the body; stomach (gastric) cancer; testicular cancer; thyroid cancer; vaginal cancer; vulvar cancer; and Wilms' tumor and other childhood kidney cancers.

[0462] In some embodiments, the cancer is melanoma. In some embodiments, the cancer is basal cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is Merkel cell carcinoma. In some embodiments, the cancer is breast cancer.

[0463] In some embodiments of any of the methods of treating a subject or compositions for use disclosed herein, the subject has a skin condition. Examples of various skin conditions are described herein and include, but are not limited to, actinic keratosis (AK), lentigo maligna, leukoplakia, and Bowen's disease.

[0464] Exemplary Kits In certain embodiments, the present disclosure relates to a package or kit comprising the microneedles described herein. In some embodiments, the present disclosure relates to a package or kit comprising the therapeutic agent described herein. In some embodiments, the kit may further comprise an additional therapeutic agent for combination therapy with the microneedles. In some embodiments, the kit may further comprise an antiseptic (e.g., alcohol swabs). In some embodiments, the kit may further comprise instructions (e.g., instructions useful for applying or administering the microneedle devices described herein). In some embodiments, such packages and kits described herein can be used for vaccination purposes, e.g., to achieve broad-spectrum immunity in a subject, as described herein. In some embodiments, such packages and kits described herein can be used for cancer treatment or prevention purposes, e.g., to treat or prevent cancer in a subject, as described herein.

[0465] vaccine The microneedles and methods described herein can also be used in the delivery of vaccines to subjects in need thereof.With regard to vaccine delivery, the present disclosure is based at least in part on the discovery that by modulating the kinetics of antigen presentation through controlled and / or sustained release compositions and devices (e.g., microneedles, e.g., silk-based microneedles and microneedle devices), including vaccines as described herein, such as viral vaccines, for example, influenza vaccines, it is possible to induce stronger and / or longer-lasting immune responses (e.g., stronger and / or longer-lasting cellular and humoral immune responses) in subjects, for example, when compared with administering a single dose or bolus of vaccine.In some embodiments, the controlled or sustained release of vaccines as disclosed herein can be used to achieve broad-spectrum immunity in subjects.

[0466] In some embodiments, the microneedles and microneedle devices described herein demonstrate controlled or sustained release of a vaccine (e.g., an influenza vaccine) for at least about 1 to 2 weeks (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), which results in one or more of improved immunogenicity, an enhanced immune response, and / or broad-spectrum immunity.

[0467] In certain embodiments, the microneedles of the present disclosure may be configured to achieve controlled or sustained release of a vaccine, antigen, and / or immunogen (e.g., influenza vaccine) as described herein. Without wishing to be bound by theory, administration of the microneedles disclosed herein containing a vaccine may induce the development of broad-spectrum immunity to the virus in a subject.

[0468] Non-limiting examples of vaccines for use in the microneedles and microneedle devices (e.g., microneedle patches) described herein may include commercial vaccines such as seasonal vaccines, pandemic vaccines, and / or universal vaccines; egg-based vaccines, cell culture-based vaccines; recombinant vaccines; live attenuated, inactivated whole virus, split virion, and / or protein subunit vaccines; and adjuvanted vaccines.

[0469] As used herein, the term "virus" refers to an infectious agent composed of nucleic acid encapsidated in protein. Such infectious agents are incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery). The viral genome may be single-stranded (ss) or double-stranded (ds) RNA or DNA, and may or may not use reverse transcriptase (RT). Furthermore, ssRNA viruses can be either sense (+) or antisense (-). Exemplary viruses include, but are not limited to, dsDNA viruses (e.g., adenovirus, herpesvirus, poxvirus), ssDNA viruses (e.g., parvovirus), dsRNA viruses (e.g., reovirus), (+)ssRNA viruses (e.g., picornavirus, togavirus), (-)ssRNA viruses (e.g., orthomyxovirus, rhabdovirus), ssRNA-RT viruses, i.e., (+)sense RNA viruses with a DNA intermediate in their life cycle (e.g., retrovirus), and dsDNA-RT viruses (e.g., hepadnavirus). In some embodiments, viruses can also include wild-type (natural) viruses, killed viruses, live attenuated viruses, modified viruses, recombinant viruses, or any combination thereof. An exemplary retrovirus is human immunodeficiency virus (HIV). Other examples of viruses include enveloped viruses, respiratory syncytial viruses, non-enveloped viruses (e.g., human papillomavirus), and the like. Examples of viruses that infect bacteria include, but are not limited to, human papillomavirus (HPV), bacteriophages, recombinant viruses, and viral vectors. The term "bacteriophage" as used herein refers to a virus that infects bacteria.

[0470] By way of example, various commercially available influenza viruses that can be incorporated into the microneedles of the present disclosure are listed below. Additionally, influenza vaccines comprising mRNA, DNA, viral vectors, and / or virus-like particles (VLPs) are suitable for use in the microneedles and microneedle devices (e.g., microneedle patches) described herein. In some embodiments, the influenza vaccine may target matrix protein 1, matrix protein 2 (M2e), and / or nucleoprotein (NP) of the influenza virus.

[0471] [Table 1] [Example]

[0472] The present disclosure will now be described in further detail with reference to the following experimental examples. These examples are Examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present disclosure should not be construed as limited to the examples below, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0473] Example 1. In vivo evaluation of the efficacy of silk-based microneedle administration of anti-cancer and immunomodulatory agents for the treatment of solid tumors / cancers An in vivo mouse melanoma model can be used to evaluate the efficacy of sustained release of multiple therapeutic agents (e.g., cytokines, checkpoint inhibitors, chemotherapeutic drugs, mRNA encoding anti-cancer drugs, or a combination of two, three, or all four) in their ability to control the tumor microenvironment. While melanoma is the model used in these experiments, it is expected that this platform will be used more broadly in other cancers, including solid tumors.

[0474] Mouse melanoma cells (B16-F10) are injected into the flank (right side) of mice (C57B1 / 6) and tumors are allowed to form. Drugs are administered intratumorally or peritumorally (i.e., intradermally or subcutaneously) as either: 1. Single bolus, 2. A series of divided injections where the total dose given over a time frame is equal to a single bolus. These injections can be given daily or every other day.

[0475] 3. Silk microneedle patches formulated with one or more therapeutic agents. Therapeutic agents to be investigated in these experiments (i.e., as single agents and in combination) may include, but are not limited to: Anticancer drugs, for example: Chemotherapy drugs: gemcitabine, doxorubicin, oxaliplatin, dacarbazine, temozolomide Targeted therapy: vemurafenib, tablafenib, trametinib Others: mRNA encoding anticancer drugs Immunomodulators, for example: Cytokines: IL-2, IL-12, IL-15, GM-CSF ○Immunomodulators: CpG, c-di-GMP Checkpoint inhibitors: anti-PD1, anti-PD-L1, anti-CTLA4 Other: mRNAs encoding cytokines and other immunoregulatory molecules The duration of sustained release (daily injections or silk microneedles) will be investigated and optimized (approximately 2-28 days). Furthermore, multiple cycles of sustained release are believed to be optimal for tumor elimination. To test this, the drug will be administered via either a single bolus or sustained release (daily injections or silk microneedles) over a period of approximately 2-28 days. For a period of time (approximately 5-14 days), the animals will not receive treatment, followed by a second round of drug administration identical to the first round.

[0476] Tumor growth is measured two to three times weekly. Animals are euthanized upon reaching humane endpoints. These endpoints include weight loss of more than 15%, a body condition score of less than 1, and tumor growth of 1 cm. 3 Humane endpoints include tumor volume greater than 100 μg, tumor length (defined as the longest dimension) greater than 1.5 cm, or tumor ulceration / necrosis. These humane endpoints apply to all in vivo experiments using melanoma models.

[0477] Sustained release of therapeutic agents can result in inhibition of tumor growth (compared to traditional single bolus injections) or total elimination of tumors. These experiments identify optimal dosing kinetics, timing, and route of administration. Importantly, it is important to identify drugs or drug combinations that have an effect on tumor growth. The goal is to unselect and identify these agents. The ability of these agents to enhance abscopal antibody and memory responses will be tested (experiments detailed below).

[0478] Intratumoral administration of IL-2 or gemcitabine in B16-F10 mice Following the methods outlined above, daily intratumoral administration was compared with a single intratumoral bolus dose of IL-2 or gemcitabine using the B16-F10 mouse melanoma tumor model. For this study, two cycles of treatment were performed, including bolus administration on days 7 and 21, and daily administration on days 7-11 and 21-25 (see Figure 6A). On day 0, mice were inoculated with B16-F10 cells via subcutaneous administration. On days 4 and 9, mice received a dose of anti-PD1 mAb (100 μg per dose) via intraperitoneal injection. Data were pooled from two experiments with 15 mice per group.

[0479] First cycle: On day 7, mice in the "single bolus dose" group received a single bolus dose of IL-2 (5 μg) or gemcitabine (475 μg). Mice in the "daily dose" group received daily doses (intratumor) of IL-2 (1 μg) or gemcitabine (95 μg) on ​​days 7 through 11 (5 total doses). Each daily dose was a divided dose equal to approximately 1 / 5 of the single bolus dose.

[0480] Second cycle: On day 21, mice in the "single bolus dose" group received a single bolus dose of IL-2 (5 μg) or gemcitabine (475 μg). Mice in the "daily dose" group received daily doses (intratumor) of IL-2 (1 μg) or gemcitabine (95 μg) from days 21 to 25 (5 total doses). Each daily dose was a divided dose equal to approximately 1 / 5 of the single bolus dose.

[0481] B16-F10 mice receiving daily doses of IL-2 had lower tumor burden compared to their bolus-dose counterparts, as determined by tumor volume over time (see Figure 6B; mean + SEM). Mice receiving daily doses of IL-2 also had better survival than mice receiving bolus doses of IL-2, as demonstrated by the Kaplan-Meier curves shown in Figure 6C.

[0482] Similarly, B16-F10 mice treated with a daily intratumoral dose of gemcitabine exhibited lower tumor burden compared to comparable mice given bolus gemcitabine, as determined by tumor volume over time (see Figure 6D; mean + SEM). Mice given daily intratumoral gemcitabine had higher survival compared to the bolus group, as shown by the Kaplan-Meier curves provided in Figure 6E.

[0483] Intratumoral administration of gemcitabine in CT26 mice Daily intratumoral administration compared with a single intratumoral bolus dose was further investigated using gemcitabine in a murine CT26 colon carcinoma model (see Figures 7A-7B). On day 0, mice were inoculated with subcutaneous administration of CT26 cells. On days 4 and 9, mice received doses of anti-PD1 mAb (100 μg per dose) via intraperitoneal injection. Data were pooled from two experiments with 10 mice per group.

[0484] CT26 mice in the "single bolus dose" group received a single bolus dose of gemcitabine (475 μg) (intratumorally) on day 7 and four doses of saline (intratumorally) for the next four days (Figure 7A). CT26 mice in the "daily dose" group received daily doses of gemcitabine (95 μg) (intratumorally) from days 7 to 11 (five total doses) (Figure 7B). Each daily dose was a divided dose equal to approximately 1 / 5 of the single bolus dose. Analysis of tumor volume over time showed that mice in the daily dose group had significantly larger tumor volumes compared to the single bolus group. The overall survival of mice in the daily dose group was also higher than that in the bolus dose group, as evidenced by the Kaplan-Meier curves provided in Figure 7D.

[0485] Example 2. In vivo evaluation of the abscopal effect in distant tumors To determine whether sustained release produces an abscopal effect, a phenomenon in which local treatment of a tumor can inhibit further growth of or promote the elimination of distant tumors, tumors are induced in both flanks (right and left) of mice using B16-F10 melanoma cells. Once tumors develop, drug(s) are administered to the right tumor as follows: 1. Single bolus 2. A series of split-dose injections 3. Silk microneedle patch formulated with drug(s).

[0486] The growth of both tumors is measured two to three times per week over time. Once the human endpoint is reached, the animals are euthanized. If the sustained release of the drug(s) promotes an abscopal effect, the left (untreated) tumor should be significantly smaller than the tumor in the single-bolus-treated animal.

[0487] Example 3. In vivo assessment of the ability to establish a memory response leading to future tumor rejection To determine whether a good immunological memory response is established upon primary tumor treatment, tumors are induced in a single flank (right side) of mice using B16-F10 melanoma cells. The drug(s) are administered as follows: 1. Single bolus 2. A series of split-dose injections 3. Silk microneedle patch formulated with drug(s).

[0488] Tumor size is measured over time. After an extended period (approximately 60-90 days), cells are injected into the opposite (left) flank from the flank of the initial tumor. The size of the new tumor is followed over time. If the sus...

Claims

1. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, the plurality of microneedles comprising: a first microneedle containing an anticancer drug; and A second microneedle containing an immunomodulatory agent. Including, A microneedle device, wherein the first and / or second microneedles comprise silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin), and wherein the microneedle device is configured to deliver anti-cancer drugs and immunomodulatory agents to a subject.

2. the first and / or second microneedles of the plurality of microneedles are (i) a base (e.g., a dissolvable base); (ii) a silk fibroin chip (e.g., an implantable silk fibroin chip) comprising silk fibroin applied to a base; and (iii) an (optional) backing applied to the base; The microneedle device of claim 1 , comprising:

3. a first microneedle containing an anticancer drug; and A second microneedle containing an immunomodulatory agent. Including, the first and / or second microneedles comprise silk fibroin, e.g., regenerated silk fibroin and / or recombinant silk fibroin; Multiple microneedles.

4. The microneedle device of claim 2 , wherein the silk fibroin tip comprises an anti-cancer drug and / or an immunomodulatory drug.

5. The microneedle device of claim 2 , wherein the base comprises an anti-cancer drug and / or an immunomodulatory drug.

6. 6. The microneedle device or plurality of microneedles of claim 1 , wherein the microneedles are configured to penetrate a biological barrier (e.g., skin).

7. 7. The microneedle device or plurality of microneedles of claim 1, further comprising a third microneedle, optionally comprising an anti-cancer drug and / or an immunomodulatory agent.

8. 8. The microneedle device or plurality of microneedles of claim 1 , configured to deliver (e.g., release) two or more anti-cancer drugs (e.g., three or more, four or more, or five or more anti-cancer drugs).

9. 9. The microneedle device or plurality of microneedles of claim 8, wherein two or more anticancer drugs are present in the same microneedle.

10. 9. The microneedle device or plurality of microneedles of claim 8, wherein two or more anticancer drugs are present in different microneedles.

11. 11. The microneedle device or plurality of microneedles of any one of claims 1 to 10, configured to deliver (e.g., release) two or more immunomodulatory agents (e.g., three or more, four or more, or five or more immunomodulatory agents).

12. 12. The microneedle device or plurality of microneedles of claim 11, wherein two or more immune modulating agents are present in the same microneedle.

13. 12. The microneedle device or plurality of microneedles of claim 11, wherein two or more immune modulating agents are present in different microneedles.

14. 14. The microneedle device or plurality of microneedles of claim 1, wherein the anti-cancer drug and the immunomodulatory agent are present in the same microneedle.

15. 15. The microneedle device or plurality of microneedles of any one of claims 1 to 14, wherein the anti-cancer drug and the immunomodulatory agent are present in different microneedles.

16. 16. The microneedle device or plurality of microneedles of any one of claims 1 to 15, wherein the anti-cancer agent is selected from one or more of a small molecule (e.g., a chemotherapy drug), a biological agent (e.g., an antibody), a viral cancer therapeutic, a nanomedicine, and a nucleic acid molecule (e.g., DNA and / or RNA).

17. 17. The microneedle device or plurality of microneedles of any one of claims 1 to 16, wherein the anti-cancer agent is mRNA, and optionally the mRNA encodes an anti-cancer agent and / or an immunomodulatory agent, and optionally the mRNA encodes a checkpoint inhibitor, a TLR agonist, a STING agonist, a RIG agonist, a cancer vaccine, a targeted therapy, and / or a cytokine.

18. 18. The microneedle device or plurality of microneedles of any one of claims 1 to 17, wherein the immunomodulatory agent is selected from a checkpoint inhibitor, a Toll-like receptor (TLR) agonist, a STING agonist, a RIG agonist, a cancer vaccine, and a cytokine.

19. 19. The microneedle device or plurality of microneedles of claim 18, wherein the checkpoint inhibitor inhibits a checkpoint molecule selected from CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, and A2aR.

20. 19. The microneedle device or plurality of microneedles of claim 17 or 18, wherein the TLR agonist is selected from a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-10 agonist, a TLR-1 / 2 agonist, a TLR-2 / 6 agonist, or a TLR-7 / 8 agonist.

21. The STING agonist may be a cyclic dinucleotide, e.g., a purine or pyrimidine nucleic acid.

19. The microneedle device or plurality of microneedles of claim 17 or 18, which is a cyclic dinucleotide comprising a base (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobase), optionally bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP).

22. 19. The microneedle device or plurality of microneedles of claim 17 or 18, wherein the cytokine is GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, or TNFβ.

23. an anti-PD1 antibody and / or an anti-CTLA4 antibody, (i) IL-2; (ii) IL-12; (iii) IL-15; (iv) IL-18; (v) gemcitabine (GEMZAR®); (vi) vemurafenib (ZELBORAF®); (vii) dabrafenib (TAFINLAR®); (viii) trametinib (MEKINIST®); (ix) doxorubicin (ADRIAMYCIN®); (x) c-di-GMP; (xi) mRNA; (xii) TLR-9 agonists (e.g., unmethylated CG dinucleotides (CpG ODN)); (xiii) oxaliplatin; and (xiv) GM-CSF 23. The microneedle device or plurality of microneedles of any one of claims 1 to 22, configured for administration in combination with one or more of:

24. 24. The microneedle device or plurality of microneedles of any one of claims 1 to 23, wherein the device or devices are configured for sustained release of the anti-cancer agent and / or immune modulating agent, wherein the sustained release is over a period of time comprising at least about 2 days (e.g., about 2, 3, 4, 5, 6, 7 days or more, such as between about 5 and about 10 days, for example, between about 7 and about 15 days, such as between about 1 and about 2 weeks, between about 1 and about 3 weeks, or between about 2 and about 4 weeks, such as between about 1 and about 3 months, for example, between about 2 and about 4 months, for example, between about 3 and about 6 months).

25. 25. The microneedle device or plurality of microneedles of any one of claims 1 to 24, wherein the device or devices are configured for burst release of the anti-cancer agent and / or immunomodulatory agent, the burst release being over a period of time including at least about 1 hour (e.g., from about 1 to about 30 minutes, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 24 hours).

26. 26. The microneedle device or plurality of microneedles of any one of claims 8 to 15, 24 or 25, wherein release of the anti-cancer drug occurs at a different rate than release of the immunomodulatory agent, such that the anti-cancer drug is released substantially before or substantially after release of the immunomodulatory agent.

27. Applied to biological barriers selected from skin layers, cell membranes, mucosal surfaces, oral cavity, or buccal cavity ( 27. The microneedle device or plurality of microneedles of any one of claims 1 to 26, configured for administration.

28. 28. The microneedle device or plurality of microneedles of any one of claims 1 to 27, configured to be applied (e.g., administered) to a tumor (e.g., a metastatic tumor).

29. 29. The microneedle device or plurality of microneedles of any one of claims 1 to 28, configured to be applied (e.g., administered) to the location of a tumor after tumor resection, for example to induce an immune response against the tumor and / or to remove any cancer cells left behind after resection.

30. 30. The microneedle device or plurality of microneedles of any one of claims 1 to 29, configured to be applied (e.g., administered) to the location of a tumor prior to tumor resection, for example to induce an immune response against the tumor and / or to remove any cancer cells left behind after resection.

31. 31. The microneedle device or plurality of microneedles of any one of claims 1 to 30, configured to be applied (e.g., administered) intratumorally.

32. 32. The microneedle device or plurality of microneedles of any one of claims 1 to 31 configured for peritumoral application (e.g., administration).

33. 33. The microneedle device or plurality of microneedles of any one of claims 1 to 32, configured to be applied (e.g., administered) to or proximal to a skin lesion (e.g., a skin lesion associated with cancer or a precancerous condition).

34. By local and / or systemic delivery (e.g., release), (i) inhibition of tumor growth at or near the site of administration; (ii) eliciting a local immune response to eliminate tumors at or near the site of administration; (iii) an increase in activated immune effector cells (e.g., T cells) in the tumor microenvironment; (iv) reduction of local immunosuppressive cells (e.g., regulatory T cells (Tregs)); (v) eliciting a systemic immune response to eliminate tumors at distant sites; (vi) immunological memory against cancer or a precancerous condition; and / or (vii) an immune response to a tumor antigen, e.g., a neoantigen; and / or (viii) preventing and / or inhibiting cancer recurrence (e.g., cancer relapse) 34. The microneedle device or plurality of microneedles according to any one of claims 1 to 33,

35. 3. The microneedle device or plurality of microneedles of claim 2, wherein the backing is selected from a solid support, such as a paper-based material, a plastic material, a polymeric material, or a polyester-based material (e.g., Whatman 903 paper, polymeric tape, plastic tape, adhesive-backed polyester tape, or other medical tape).

36. The base (e.g., a dissolvable base) (i) polysaccharides (e.g., dextran); (ii) disaccharides (e.g., sucrose, maltose, and trehalose); (iii) polymers (e.g., methylcellulose, polyethylene glycol (PEG)); carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and hyaluronate); (iv) proteins (e.g., gelatin); (v) plasticizers (e.g., glycerol, propanediol); and (vi) surfactants (e.g., octylphenol ethoxylates (e.g., Triton-X), polysorbates, poloxamers, and / or polyethoxylated alcohols); 36. The microneedle device or plurality of microneedles of claim 2, 5, or 35, comprising two or more of:

37. 37. The microneedle device or plurality of microneedles of any one of claims 2, 5, 35 or 36, wherein the base comprises one or more of gelatin, dextran, glycerol, polyethylene glycol (PEG) (including, for example, low molecular weight PEG), sucrose, trehalose, maltose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, methylcellulose, and / or surfactants (for example, octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, e.g., P188, and / or polyethoxylated alcohols), and optionally the microneedles are configured for sustained release and / or burst release.

38. Silk fibroin chips (i) disaccharides (e.g., sucrose, maltose, and trehalose); (ii) polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate); (iii) an amino acid (e.g., threonine); (iv) plasticizers (e.g., glycerol, propanediol); and (v) a buffer solution (e.g., PBS) 5. The microneedle device or plurality of microneedles of claim 2 or 4, comprising two or more of:

39. 39. The microneedle device or plurality of microneedles of claim 2, 4, or 38, wherein the silk fibroin tip comprises an excipient.

40. 40. The microneedle device or plurality of microneedles of claim 2, 4, 38, or 39, wherein the silk fibroin tip comprises one or more of carboxymethyl cellulose (CMC), sucrose, and threonine.

41. 41. The microneedle device or plurality of microneedles of any one of claims 2, 4, or 38 to 40, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 60MB silk fibroin solution, or a silk fibroin solution according to Figure 5, for example, a 100 kDa to 200 kDa (e.g., about 153 kDa) silk fibroin solution.

42. 42. The microneedle device or plurality of microneedles of any one of claims 2, 4, or 38 to 41, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 180MB silk fibroin solution, or a silk fibroin solution according to Figure 5, for example, a 36 kDa to 100 kDa (e.g., about 71 kDa) silk fibroin solution.

43. From about 0.5 μg to about 500 μg of silk fibrions, optionally from about 0.5 μg to about 5 μg, or from about 1 μg to about 10 μg, or from about 5 μg to about 15 μg, or from about 10 μg to about 20 μg, or from about 15 μg to about 25 μg, or from about 20 μg to about 30 μg, or from about 25 μg to about 35 μg, or from about 30 μg to about 40 μg, or from about 35 μg to about 45 μg, or from about 40 μg to about 50 μg, or from about 45 μg to about 55 μg, or from about 50 μg to about 60 μg, or from about 55 μg to about 65 μg, or from about 60 μg to about 70 μg, or from about 65 μg to about 75 μg, or from about 70 μg to about 80 μg, or from about 75 μg to about 43. The microneedle device or plurality of microneedles of any one of claims 2, 4, or 38 to 42, comprising silk fibrions in an amount of about 85 μg, or about 80 μg to about 90 μg, or about 85 μg to about 95 μg, or about 90 μg to about 100 μg, or about 95 μg to about 150 μg, or about 125 μg to about 175 μg, or about 150 μg to about 200 μg, or about 225 μg to about 275 μg, or about 250 μg to about 300 μg, or about 325 μg to about 375 μg, or about 350 μg to about 400 μg, or about 425 μg to about 475 μg, or about 450 μg to about 500 μg of silk fibrions.

44. 44. The microneedle device or plurality of microneedles of any one of claims 2, 4, or 38 to 43, comprising silk fibrions in an amount of about 1% to about 75% by weight of silk fibrions, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight of silk fibrions.

45. 45. The microneedle device or plurality of microneedles of any one of claims 2, 4, or 38 to 44, wherein the microneedles are configured to embed the silk fibroin tip into a biological barrier of a subject to a depth of between about 100 μm and about 1 mm (e.g., a maximum penetration depth of the distal portion of the tip).

46. 50. A method for treating and / or inducing an immune response against cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) to a subject a site of cancer (e.g., a metastatic tumor) with a microneedle device or plurality of microneedles according to any one of claims 1 to 45, such that: (i) lysis of cancer cells, e.g., tumor cells, to release and / or expose cancer-associated antigens (e.g., neoantigens) to the subject's immune system; (ii) presentation of cancer-associated antigens (e.g., neoantigens) complexed with major histocompatibility complexes (MHCs) on their surface by antigen-presenting cells (APCs) to cells of the immune system (e.g., accessory cells, e.g., B cells, dendritic cells, etc.); (iii) recognition of presented cancer-associated antigens (e.g., neoantigens) by immune effector cells, e.g., T cells and / or NK cells; (iv) activation and / or expansion of immune effector cells, e.g., T cells and / or NK cells, specific for the presented cancer-associated antigen (e.g., neoantigen) in the subject; and (v) enhancing, e.g., stimulating or upregulating, the immune response of immune effector cells, e.g., T cells and / or NK cells, that promote killing and / or inhibiting the growth or proliferation of target cells expressing a cancer-associated antigen (e.g., a neoantigen) in a subject. A method comprising the steps of:

47. 46. ​​A method for treating and / or inducing an immune response against cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) a microneedle device or plurality of microneedles according to any one of claims 1 to 45 to a site of cancer (e.g., metastatic tumor) and / or skin disorder (e.g., skin lesion) in a subject. method.

48. 46. ​​A method for treating cancer (e.g., metastatic cancer) or a precancerous condition (e.g., a precancerous skin condition), comprising contacting (e.g., administering) a microneedle device or plurality of microneedles described in any one of claims 1 to 45 to a tumor (e.g., a metastatic tumor) or lesion (e.g., a skin lesion) in a subject.

49. 39. A method for treating cancer (e.g., metastatic cancer), comprising contacting (e.g., administering) a microneedle device or plurality of microneedles according to any one of claims 1 to 38 to the location of a tumor (e.g., a metastatic tumor) in a subject, such that: (i) a local immune response and / or local cytotoxicity against the cancer (e.g., killing of cancer cells at or proximal to the location of the applied microneedle device, as evidenced by, e.g., a reduction in local tumor size and / or local tumor burden); and / or (ii) a distal immune response and / or distal cytotoxicity against the cancer (e.g., as evidenced by the killing of cancer cells at a location distal to the location of the applied microneedle patch, e.g., a reduction in distal tumor size and / or tumor burden); The method includes the step of inducing

50. Cancers include: anal cancer; basal cell carcinoma; bladder cancer; bone cancer; brain tumor; breast cancer; cervical cancer; colorectal cancer; endometrial cancer; esophageal cancer; gastrointestinal stromal tumor; gestational trophoblastic disease; head and neck cancer; Hodgkin's lymphoma; Kaposi's sarcoma; kidney (renal cell) cancer; leukemia; liver cancer; lung cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; multicentric Castleman disease; multiple myeloma and other plasma cell neoplasms; myeloproliferative neoplasms; neuroblastoma; and non-Hodgkin's lymphoma.

50. The method of any one of claims 46 to 49, wherein the cancer is selected from: ovarian, fallopian tube, or primary peritoneal cancer; pancreatic cancer; penile cancer; pheochromocytoma and paraganglioma; prostate cancer; retinoblastoma; rhabdomyosarcoma; skin cancer; squamous cell carcinoma; soft tissue sarcoma; any solid tumor in the body; stomach (gastric) cancer; testicular cancer; thyroid cancer; vaginal cancer; vulvar cancer; and Wilms' tumor and other childhood kidney cancers.

51. 51. The method of any one of claims 46 to 50, wherein the cancer is melanoma.

52. 51. The method of any one of claims 46 to 50, wherein the cancer is basal cell carcinoma.

53. 51. The method of any one of claims 46 to 50, wherein the cancer is squamous cell carcinoma.

54. 51. The method of any one of claims 46 to 50, wherein the cancer is Merkel cell carcinoma.

55. 51. The method of any one of claims 46 to 50, wherein the cancer is breast cancer.

56. 49. The method of claim 48, wherein the precancerous condition is a precancerous skin condition optionally selected from actinic keratosis (AK), lentigo maligna, leukoplakia, and Bowen's disease.

57. 57. The method of any one of claims 46 to 56, wherein the contacting (e.g., administering) occurs intratumorally.

58. 57. The method of any one of claims 46 to 56, wherein the contacting (e.g., administering) occurs around the tumor.

59. 57. Any of claims 46 to 56, wherein the contacting (e.g., administering) occurs prior to surgical resection.

10. The method according to claim 1.

60. 57. The method of any one of claims 46 to 56, wherein the contacting (e.g., administering) occurs after surgical resection.

61. 61. The method of any one of claims 46 to 60, wherein the contacting (e.g., administering) is performed in combination with a standard of care treatment (e.g., for cancer or a precancerous condition) optionally selected from surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy, and / or radiation therapy.

62. 62. The method of claim 61, wherein standard of care treatment is administered before, after, or simultaneously with the microneedle device.

63. 63. The method of any one of claims 46 to 62, wherein the subject is a human subject.

64. 1. A method of manufacturing a microneedle device, comprising: providing a mold including a mold body having an array of needle cavities having a predetermined shape, e.g., pyramidal and / or conical needle cavities formed therein; filling the tip of the needle cavity with a composition comprising a solution of silk fibroin and a therapeutic agent (e.g., an anti-cancer agent, an immunomodulatory agent, or both); drying the filled tip of the needle cavity to produce a silk fibroin tip, and optionally annealing the silk fibroin tip; further filling the needle cavity of the mold with a first base (e.g., dissolvable base) solution; drying the first base solution to form a first base layer; (Optionally) forming one or more additional base layers by adding one or more additional base solutions to the first base layer and drying the additional base solutions, where the additional base solutions are optionally different from the first base solution; applying a backing (if necessary) to the base layer (e.g., the first base layer or one or more additional base layers) to produce a microneedle device; A method comprising:

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