Particles for expansion and activation of immune cells

Abstract

Compositions, kits, methods of making and methods of use comprising a particle comprising at least three agonists that respectively bind to Cluster of Differentiation 3 (CD3), 4-1BB (aka CD137), and Interleukin 2 Receptor (IL-2R).

Description

Attorney Docket No. UTSDP4503WO- 1001386307TITLE PARTICLES FOR EXPANSION AND ACTIVATION OF IMMUNE CELLS CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 776,836, filed March 24, 2025, the contents of which are hereby incorporated by reference in their entirety.BACKGROUND1. Field

[0002] The present disclosure relates to immunoregulation and cancer immunotherapy. 2. Discussion of Related Art

[0003] Tumor infiltrating lymphocyte (TIL) therapy is highly effective in melanoma and holds great potential for improving cancer immunotherapy in other cancers. A key limitation for expansion of TIL therapy to other cancer types is the complex process required to activate and expand TILs.

[0004] Immunostimulatory proteins, such as antibodies and cytokines, can elicit potent antitumor immunity in a variety of tumor models, often via activation of T cell and natural killer (NK) cell responses. Clinical use of immunomodulatory agonists has been hindered by doselimiting inflammatory toxicities that often accompany systemic administration, for example, intravenous anti-4-1 BB has been shown to elicit liver inflammatory damage, systemic inflammatory cytokine release, hematologic abnormalities, and disruptions in lymphocyte trafficking, whereas systemic anti-CD3 administration can include cytokine release syndrome (CRS), and whereas systemic IL-2 therapy has also been associated with potentially lethal side effects such as capillary leak syndrome. Although local injections may reduce systemic exposure to potent immuno-agonists, these approaches are inherently dose-limited, as soluble proteins rapidly reach the systemic circulation following intra- or peritumoral injection, via lymphatic drainage or direct entry into leaky tumor vasculature. Furthermore, concurrent activation and / or inhibition of more than one T cell pathway may not be achieved with the use of soluble agents, which may not be spatially co-localized following administration.

[0005] There is a strong need for refining strategies to enable TIL immunotherapy.SUMMARY

[0006] Solutions to address at least some of the above challenges are presented. Methods and compositions are disclosed herein to directly activate and expand TILs in vivo, thereby302413766 - 1 -Attorney Docket No. UTSDP4503WO- 1001386307eliminating the standard complex TIL production processes, while minimizing toxicities associated with systemic administration of potent immunostimulatory molecules. A multifunctional nanoparticle (NP) platform is disclosed with multiple co-stimulatory molecules. As shown herein, the compositions and methods are suitable for in vivo stimulation of TILs, such as T cells, and these TILs can subsequently provide a systemic immunotherapy responses against disease, such as cancer.

[0007] In some aspects, provided herein are particles comprising: an agonist of cluster of differentiation 3 (CD3); an agonist of 4-1 BB (CD137); and an agonist of interleukin 2 (IL-2) receptor (IL-2R); wherein the agonist of CD3, the agonist of 4-1 BB, and the agonist of IL-2R are bound to a core of the particle. In some aspects, the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprises a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody. In some aspects, the agonist of CD3 comprises a CD3 ligand. In some aspects, the CD3 ligand comprises CD3L1 and / or a CD3 activating fragment thereof. In some aspects, the agonist of 4-1 BB comprises a 4-1 BB ligand. In some aspects, the 4-1 BB ligand comprises 4-1 BB Ligand (4-1 BBL) and / or a 4-1 BB activating fragment thereof. In some aspects, the agonist of IL-2R comprises an IL-2R ligand. In some aspects, the IL-2R ligand comprises IL-2 and / or an IL-2R activating fragment thereof. In some aspects, the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprises the antibody or the active fragment of an antibody. In some aspects, an active fragment of an antibody comprises a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a scFV fragment, a (Fab')2 fragment, or any combination thereof.

[0008] In some aspects, provided herein are particles wherein the hydrodynamic diameter of the particle is from 50 to 500 nm, or 50 to 300 nm, or 100 to 200 nm. In some aspects, a particle comprises at least a 5%, 10%, 15%, 20%, or 25% greater concentration of agonist of 4-1 BB relative to the agonist of CD3, the agonist of IL-2R, or both the agonist of CD3 and the agonist of IL-2R. In some aspects, a particle comprises a ratio of agonist of 4-1 BB to agonist of CD3 (4-1 BB: CD3) that is or is about 1.5: 1 to 2: 1 , or is or is about 2: 1 to 4:3, or is or is about 3:2, or is or is about 15:9. In some aspects, a particle comprises a ratio of agonist of 4-1BB to agonist of IL-2R (4-1 BB: IL-2R) that is or is about 1.5:1 to 2:1, or is or is about 2:1 to 4:3, or is or is about 3:2, or is or is about 15:10. In some aspects, a particle comprises a ratio of agonist of CD3 to agonist of IL-2R (CD3: IL-2R) that is or is about 1:10 to 10:1, or is or is about 1:5 to 5: 1 , or is or is about 2: 1 to 1 :2, or is or is about 0.8: 1 to 1 :0.8, or is or is about 0.9:1, or is or is about 1:1. In some aspects, a particle comprises a ratio of agonist of 4-1BB to agonist of CD3 to agonist of IL-2R (4-1 BB: CD3: IL-2R) that is or is about 2:1:1 to 4:3:3, or is or is about 3:2:2, or is or is about 15:9:10. In some aspects, a particle comprises a concentration of, at least, or302413766 - 2 -Attorney Docket No. UTSDP4503WO- 1001386307about 30, 40, 50, 60, 70, 80, 90, or 100 g of total combined agonist of CD3, agonist of 4-1BB, and agonist of IL-2R per 1000 pg of particle.

[0009] In some aspects, provide herein are particles wherein at least one of the agonists of CD3, agonist of 4-1BB, and agonist of IL-2R are conjugated to the particle by click chemistry. In some aspects, the agonist of CD3, agonist of 4-1BB, and agonist of IL-2R are conjugated to the particle by click chemistry. In some aspects, the agonist of CD3, the agonist of 4-1 BB, and the agonist of IL-2R are not conjugated directly to another one of the agonist of CD3, the agonist of 4-1 BB, and the agonist of IL-2R.

[0010] In some aspects, provided herein are particles comprising PEG-PLGA particle cores at least 50 kDa in mass. In some aspects, PEG-PLGA particle cores comprise a 1:10 to 10:1, or 1:5 to 5:1, or 1:3 to 3:1, or 1:2 to 2:1, or 1:1, or 1:2, wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some aspects, PEG-PLGA particle cores comprise a 1:1 or a 1:2 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some aspects, the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R are humanized and / or human. In some aspects, particles provided herein are not a lipid nanoparticle.

[0011] Also provided herein, in some aspects, are compositions comprising a population of particles. In some aspects, a polydispersity index (PDI) of a composition is equal to or less than 0.07, 0.065, 0.06, 0.055, 0.5, 0.45, 0.4, 0.35, or 0.3. In some aspects, a composition comprises a pharmaceutically acceptable carrier.

[0012] Also provided herein are kits comprising particles and / or compositions disclosed herein. Also provided herein are medicaments comprising particles and / or compositions disclosed herein. Also provided are methods of use of compositions and / or particles described herein for the production of a medicament for activating and / or inducing a tumor infiltrating lymphocyte (TIL) cell response. In some aspects, provided herein are compositions and / or particles for use in the production of a medicament for treatment of cancer. In some aspects, TILs may comprise one or more types of cells including T cells (e.g., CD8+ T cells and / or CD4+ T cells), natural killer (NK) cells, dendritic cells, macrophages, and / or B cells. In some aspects, a TIL comprises a T cell.

[0013] Also provided herein, in some aspects, are methods of activating a TIL cell, comprising contacting the TIL cell with the particle and / or composition disclosed herein. In some aspects, provided herein are methods of inducing a TIL cell immune response, comprising contacting the TIL with a particle and / or composition disclosed herein. In some aspects, provided are methods of inducing a TIL cell immune response in a subject in need thereof, comprising administering to the subject an effective amount of a particle and / or composition disclosed302413766 - 3 -Attorney Docket No. UTSDP4503WO- 1001386307herein. In some aspects, provided herein are methods of activating TIL cells in a subject in need thereof, comprising administering to the subject an effective amount of a particle and / or composition disclosed herein. In some aspects, provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the particle and / or composition disclosed herein. In some aspects, a TIL comprises a T cell.

[0014] In some aspects, provided herein are methods of treating cancer, wherein the cancer comprises breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, uterine cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, liver cancer, skin cancer, blood cancer, or any combination thereof. In some aspects, the cancer comprises solid tumors. In some aspects, a subject has been diagnosed with cancer. In some aspects, particles and / or compositions provided herein are administered via a route comprising intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intrathecally, intraventricularly, intraorbitally, intranasally, implantation, inhalation, intratumorally, or any combination thereof. In some aspects, a particle and / or composition is administered intratumorally. In some aspects, methods of treating cancer further comprise a step of administering to the subject an effective amount of a chemotherapeutic agent and / or radiation therapy. In some aspects, a subject is a mammal. In some aspects, a mammal is a human.

[0015] Also provided herein, in some aspects, are methods of making particles and / or compositions disclosed herein, wherein the method comprises combining purified functionalized PEG-PLGA particle cores at least 50 kDa in mass with functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of I L-2R. In some aspects, functionalized PEG-PLGA particle cores are functionalized for click chemistry, and the functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R are functionalized for click chemistry. In some aspects, the functionalized PEG-PLGA particle cores are azide-functionalized PEG-PLGA particle cores, and the functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R are DBCO-functionalized. In some aspects, functionalized PEG-PLGA particles comprise a 1:10 to 10:1, or 1:5 to 5:1, or 1:3 to 3:1, or 1:2 to 2:1, or 1:3 to 1:1.5, or 1:2, or 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some aspects, functionalized PEG-PLGA particles comprise a 1:2 or 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some aspects, the average degree of functionalization is 10-20 for functionalized agonist of 4-1 BB, 5-15 for functionalized agonist of CD3, and 5-15 for functionalized agonist of IL-2R. In some aspects, the average degree of302413766 - 4 -Attorney Docket No. UTSDP4503WO- 1001386307functionalization is or is about 15 for functionalized 4-1 BB agonists, is or is about 9 for functionalized CD3 agonists, and is or is about 10 for functionalized IL-2R agonists. In some aspects, the target degree of bioconjugation is, or is about 30, 40, 50, 60, 70, 80, 90, or 100 pg of total combined agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R per 1000 pg of particle.

[0016] Certain aspects of the present invention are characterized through the following enumerated, non-limiting, embodiments.

[0017] Embodiment 1 is a particle comprising: an agonist of cluster of differentiation 3 (CD3); an agonist of 4-1BB (CD137); and an agonist of interleukin 2 (IL-2) receptor (IL-2R); wherein the agonist of CD3, the agonist of 4-1 BB, and the agonist of IL-2R are bound to a core of the particle.

[0018] Embodiment 2 is the particle of Embodiment 1, wherein the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprises a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody.

[0019] Embodiment s is the particle of Embodiment 2, wherein the active fragment of an antibody comprises a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a scFV fragment, a (Fab')2 fragment, or any combination thereof.

[0020] Embodiment 4 is the particle of Embodiment 1 or Embodiment 2, wherein the agonist of CD3 comprises a CD3 ligand.

[0021] Embodiment 5 is the particle of Embodiment 4, wherein the CD3 ligand comprises CD3L1 and / or a CD3 activating fragment thereof.

[0022] Embodiment 6 is the particle of Embodiment 1 or Embodiment 2, wherein the agonist of 4-1 BB comprises a 4-1 BB ligand.

[0023] Embodiment 7 is the particle of Embodiment 6, wherein the 4-1 BB ligand comprises 4-1 BB Ligand (4-1 BBL) and / or a 4-1 BB activating fragment thereof.

[0024] Embodiment 8 is the particle of Embodiment 1 or Embodiment 2, wherein the agonist of IL-2R comprises an IL-2R ligand.

[0025] Embodiment 9 is the particle of Embodiment 8, wherein the IL-2R ligand comprises IL-2 and / or an IL-2R activating fragment thereof.

[0026] Embodiment 10 is the particle of Embodiment 2, wherein the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprises the antibody or the active fragment of an antibody.302413766 - 5 -Attorney Docket No. UTSDP4503WO- 1001386307

[0027] Embodiment 11 is the particle of Embodiment 10, wherein the active fragment of an antibody comprises a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a scFV fragment, a (Fab')2 fragment, or any combination thereof.

[0028] Embodiment 12 is the particle of any one of Embodiments 1-11, wherein the hydrodynamic diameter of the particle is from 50 to 500 nm, or 50 to 300 nm, or 100 to 200 nm.

[0029] Embodiment 13 is the particle of any one of Embodiments 1-12, comprising at least a 5%, 10%, 15%, 20%, or 25% greater concentration of agonist of 4-1BB relative to the agonist of CD3, the agonist of IL-2R, or both the agonist of CD3 and the agonist of IL-2R.

[0030] Embodiment 14 is the particle of any one of Embodiments 1-13, wherein the ratio of agonist of 4-1BB to agonist of CD3 (4-1BB:CD3) is or is about 1.5:1 to 2:1, or is or is about 2:1 to 4:3, or is or is about 3:2, or is or is about 15:9.

[0031] Embodiment 15 is the particle of any one of Embodiments 1-14, wherein the ratio of agonist of 4-1BB to agonist of IL-2R (4-1 BB: IL-2R) is or is about 1.5:1 to 2:1, or is or is about 2:1 to 4:3, or is or is about 3:2, or is or is about 15:10.

[0032] Embodiment 16 is the particle of any one of Embodiments 1-15, wherein the ratio of agonist of CD3 to agonist of IL-2R (CD3:IL2R) is or is about 1:10 to 10:1, or is or is about 1:5 to 5:1, or is or is about 2:1 to 1:2, or is or is about 0.8:1 to 1:0.8, or is or is about 0.9:1, or is or is about 1:1.

[0033] Embodiment 17 is the particle of any one of Embodiments 1-16, wherein the ratio of agonist of 4-1 BB to agonist of CD3 to agonist of IL-2R (4-1BB:CD3:IL2R) is or is about 2:1:1 to 4:3:3, or is or is about 3:2:2, or is or is about 15:9:10.

[0034] Embodiment 18 is the particle of any one of Embodiments 1-17, wherein there is or is about 30, 40, 50, 60, 70, 80, 90, or 100 pg of total combined agonist of CD3, agonist of 4-1BB, and agonist of IL-2R per 1000 pg of particle.

[0035] Embodiment 19 is the particle of any one of Embodiments 1-18, wherein at least one of the agonists of CD3, agonist of 4-1 BB, and agonist of IL-2R are conjugated to the particle by click chemistry.

[0036] Embodiment 20 is the particle of any one of Embodiments 1-19, wherein the agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R are conjugated to the particle by click chemistry.302413766 - 6 -Attorney Docket No. UTSDP4503WO- 1001386307

[0037] Embodiment 21 is the particle of any one of Embodiments 1-19, where the agonist of CD3, the agonist of 4-1BB, and the agonist of IL-2R are not conjugated directly to another one of the agonist of CD3, the agonist of 4-1BB, and the agonist of IL-2R.

[0038] Embodiment 22 is the particle of any one of Embodiments 1-21, wherein the particle comprises PEG-PLGA particle cores at least 50 kDa in mass.

[0039] Embodiment 23 is the particle of Embodiment 22, wherein the PEG-PLGA particle cores comprise a 1:10 to 10:1, or 1:5 to 5:1, or 1:3 to 3:1, or 1:2 to 2:1, or 1:1, or 1:2, wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K).

[0040] Embodiment 24 is the particle of Embodiment 22 or Embodiment 23, wherein the PEG-PLGA particle cores comprise a 1:1 or a 1:2 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K).

[0041] Embodiment 25 is the particle of any one of Embodiments 1-24, wherein the agonist of CD3, the agonist of 4-1BB, and / or the agonist of IL-2R are humanized and / or human.

[0042] Embodiment 26 is the particle of any one of Embodiments 1-25, wherein the particle is not a lipid nanoparticle.

[0043] Embodiment 27 is a composition comprising a population of particles of any one of Embodiments 1-26.

[0044] Embodiment 28 is the composition of Embodiment 27, wherein a polydispersity index (PDI) of the composition is equal to or less than 0.07, 0.065, 0.06, 0.055, 0.5, 0.45, 0.4, 0.35, or 0.3.

[0045] Embodiment 29 is the composition of Embodiment 27 or Embodiment 28, further comprising cells.

[0046] Embodiment 30 is the composition of Embodiment 29, wherein the cells comprise immune cells and / or tumor infiltrating lymphocytes.

[0047] Embodiment 31 is the composition of any one of Embodiments 27-30, further comprising a pharmaceutically acceptable carrier.

[0048] Embodiment 32 is a kit comprising the composition of any one of Embodiments 27-31.

[0049] Embodiment 33 is a use of the composition of any one of Embodiments 27-31 in the production of a medicament for activating and / or inducing a Tumor infiltrating lymphocyte (TIL) cell response.

[0050] Embodiment 34 is the use of Embodiment 33, wherein the TIL comprises a T cell.302413766 - 7 -Attorney Docket No. UTSDP4503WO- 1001386307

[0051] Embodiment 35 is a use of the composition of any one of Embodiments 27-31 in the production of a medicament for treatment of cancer.

[0052] Embodiment 36 is a method of activating a TIL cell, comprising contacting the TIL cell with the particle or composition of any one of Embodiments 1-31.

[0053] Embodiment 37 is the method of Embodiment 36, wherein the TIL comprises a T cell.

[0054] Embodiment 38 is a method of inducing a TIL cell immune response, comprising contacting the TIL with the particle or composition of any one of Embodiments 1-31.

[0055] Embodiment 39 is the method of Embodiment 38, wherein the TIL comprises a T cell.

[0056] Embodiment 40 is a method of inducing a TIL cell immune response in a subject in need thereof, comprising administering to the subject an effective amount of the particle or composition of any one of Embodiments 1-31.

[0057] Embodiment 41 is the method of Embodiment 40, wherein the TIL comprises a T cell.

[0058] Embodiment 42 is a method of activating TIL cells in a subject in need thereof, comprising administering to the subject an effective amount of the particle or composition of any one of Embodiments 1-31.

[0059] Embodiment 43 is the method of Embodiment 42, wherein the TIL comprises a T cell.

[0060] Embodiment 44 is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the particle or composition of any one of Embodiments 1-31.

[0061] Embodiment 45 is the method of Embodiment 44, wherein the TIL comprises a T cell.

[0062] Embodiment 46 is the method of Embodiment 44, wherein the cancer comprises breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, uterine cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, liver cancer, skin cancer, blood cancer, or any combination thereof.

[0063] Embodiment 47 is the method of Embodiment 44, wherein the cancer comprises solid tumors.

[0064] Embodiment 48 is the method of any one of Embodiments 44-47, wherein the subject has been diagnosed with cancer.

[0065] Embodiment 49 is the method of any one of Embodiments 40-48, wherein the particle is administered via a route comprising intravenously, intramuscularly, subcutaneously,302413766 - 8 -Attorney Docket No. UTSDP4503WO- 1001386307topically, orally, transdermally, intraperitoneally, intrathecally, intraventricularly, intraorbitally, intranasally, implantation, inhalation, intratumorally, or any combination thereof.

[0066] Embodiment 50 is the method of Embodiment 49, wherein the particle is administered intratumorally.

[0067] Embodiment 51 is the method of any one of Embodiments 40-50, further comprising a step of administering to the subject an effective amount of a chemotherapeutic agent and / or radiation therapy.

[0068] Embodiment 52 is the method of any one of Embodiments 40-51, wherein the subject is a mammal.

[0069] Embodiment 53 is the method of Embodiment 52, wherein the mammal is a human.

[0070] Embodiment 54 is a method of making the particle of any one of Embodiments 1-26, the method comprising combining purified functionalized PEG-PLGA particle cores at least 50 kDa in mass with functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R.

[0071] Embodiment 55 is the method of Embodiment 54, wherein the functionalized PEG-PLGA particle cores are functionalized for click chemistry, and the functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R are functionalized for click chemistry.

[0072] Embodiment 56 is the method of Embodiment 54 or Embodiment 55, wherein the functionalized PEG-PLGA particle cores are azide-functionalized PEG-PLGA particle cores, and the functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R are DBCO-functionalized.

[0073] Embodiment 57 is the method of any one of Embodiments 54-56, wherein the functionalized PEG-PLGA particles comprise a 1:10 to 10:1, or 1:5 to 5:1, or 1:3 to 3:1, or 1:2 to 2:1, or 1:3 to 1:1.5, or 1:2, or 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K).

[0074] Embodiment 58 is the method of Embodiment 57, wherein the functionalized PEG-PLGA particles comprise a 1:2 or 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K).

[0075] Embodiment 59 is the method of any one of Embodiments 54-58, wherein the average degree of functionalization is 10-20 for functionalized agonist of 4-1 BB, 5-15 for functionalized agonist of CD3, and 5-15 for functionalized agonist of IL-2R.302413766 - 9 -Attorney Docket No. UTSDP4503WO- 1001386307

[0076] Embodiment 60 is the method of any one of Embodiments 54-59, wherein the average degree of functionalization is or is about 15 for functionalized 4-1 BB agonists, is or is about 9 for functionalized CD3 agonists, and is or is about 10 for functionalized IL-2R agonists.

[0077] Embodiment 61 is the method of any one of Embodiments 54-60, wherein the target degree of bioconjugation is, or is about 30, 40, 50, 60, 70, 80, 90, or 100 g of total combined agonist of CD3, agonist of 4-1BB, and agonist of IL-2R per 1000 pg of particle.

[0078] Embodiment 62 is the method, use, or kit of any one of Embodiments 32-61, wherein the TILs are from breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, uterine cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, liver cancer, skin cancer, blood cancer, or any combination thereof.

[0079] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (3rd ed. 2006); The Cambridge Dictionary of Science and Technology (Walker ed., 1990); The Glossary of Genetics, 5th Ed., R. Rieger et al. (2008), The HarperCollins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0080] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989); Ausubel etal. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0081] Unless the context indicates otherwise, it is specifically intended that the various features of described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0082] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a”302413766 - 10 -Attorney Docket No. UTSDP4503WO- 1001386307is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0083] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface.

[0084] The terms “comprising,” “including,” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including,” and “having” mean to include, but not necessarily be limited to the things so described.

[0085] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B, or C” or “A, B, and / or C” mean any of the following: “A,” “B,” or “C”; “A, and B”; “A, and C”; “B and C”; “A, B, and C.” An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.

[0086] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included. As used herein, the phrase “consisting essentially of” limits the scope to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the subject matter.

[0087] The term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose,302413766 - 11 -Attorney Docket No. UTSDP4503WO- 1001386307temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0088] As used herein, “treatment,” “therapy,” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition.

[0089] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and / or degree of onset or severity in the absence of intervention.

[0090] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The term “therapeutically effective amount,” as used herein, means an amount of a particle or composition disclosed herein that ameliorates, attenuates, or eliminates one or more symptoms of cancer or prevents or delays the onset of one or more symptoms of cancer as defined herein.

[0091] As used herein, “individual,” “subject,” “host,” and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject can be a human. In other embodiments, the subject can be a human in need of treating a cancer.

[0092] The terms “antibody” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to antigen, including but not limited to Fab, Fv, single chain Fv (scFv), and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins including an antigen-binding portion of an antibody and a non-antibody protein. The antibodies can in some embodiments be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies can in some embodiments be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. Also encompassed by the terms are Fab',302413766 - 12 -Attorney Docket No. UTSDP4503WO- 1001386307Fv, F(ab')2, and other antibody fragments that retain specific binding to antigen (e.g., any antibody fragment that comprises at least one paratope).

[0093] Antibodies can exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bi-functional (i.e., bi-specific) hybrid antibodies (see e.g., Lanzavecchia et al., 1987) and in single chains (see e.g., Huston et al., 1988 and Bird et al., 1988, each of which is incorporated herein by reference in its entirety). See generally, Hood et al., 1984, and Hunkapiller & Hood, 1986. The phrase “detection molecule” is used herein in its broadest sense to include any molecule that can bind with sufficient specificity to a biomarker to allow for detection of the particular biomarker. To allow for detection can mean to determine the presence or absence of the particular biomarker member and, in some embodiments, can mean to determine the amount of the particular biomarker. Detection molecules can include antibodies, antibody fragments, and nucleic acid sequences.

[0094] All publications, patent applications, patents, accession numbers and other references mentioned herein are incorporated by reference herein in their entirety.

[0095] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.302413766 - 13 -Attorney Docket No. UTSDP4503WO- 1001386307BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. Aspects of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0097] FIGS. 1A-1B provide an exemplary schema for producing and testing particles described herein. FIG. 1A depicts a schema for producing exemplary functionalized nanoparticles comprising three agonists of Tumor Infiltrating Lymphocytes (TILs): a CD3 agonist (anti-CD3), a 4-1 BB agonist (anti-4-1 BB), and an IL-2 receptor (IL-2R) agonist (IL-2), particles comprising agonists of these three targets are referenced as “TIL-NPs”. FIG. 1B depicts a schema for a proposed mechanism of action for the effects of TIL-NPs on tumors in vivo.

[0098] FIGS. 2A-2B show the characterization of populations of exemplary particles before and after conjugation with co-stimulatory molecules. FIG. 2A is a graph displaying the size (x axis, in nanometers) distribution as a percentage (y axis) of particle cores (NP) before and TIL-NPs after conjugation with co-stimulatory molecules, and a chart showing the average size and polydispersity index (PDI) for the populations. FIG. 2B are a series of results for in vitro FACS binding assays of TIL-NPs, either left unstained (left column) or stained (right column) with Dylight 594 labeled CD3 and Dylight 650 labeled 4-1 BB (top row), Af488 labeled IL-2 and Dylight 594 labeled CD3 (middle row), or AF488 labeled IL-2 and Dylight 650 labeled 4-1 BB (bottom row).

[0099] FIGS. 3A-3B show how TILs contacted with TIL-NPs displayed improved cytotoxicity against murine colon adenocarcinoma (MC38) cells and increased IFN-y secretion levels relative to controls. FIG. 3A is a series of charts showing viability (y axis) of MC38 cancer cells alone, MC38 cells after in vitro treatment with TILs derived from tumors from MC38 bearing mice, or MC38 cells after in vitro treatment with TILs derived from tumors from MC38 bearing mice with free antibodies against CD3 and 4-1 BB with IL-2 (“free antibodies”) or with TIL-NPs prepared as described in FIG. 1A. The results showed that TILs administered with TIL-NPs had significantly increased cytotoxicity against cancer cells relative to controls, as accessed by Measure Cell Viability (MTS) assays and shown at Target: effector (E:T) ratios of 1:2, 1:5, 1:10, and 1:15. FIG. 3B is a series of charts displaying IFN-y concentrations in the supernatants of MC38 cells or co-culture of MC38 and TILs following various treatments as described in FIG 3A, as determined by ELISA assays. The results showed that supernatants of co-cultures comprising TILs and TIL-NPs had significantly increased levels of IFN-y relative302413766 - 14 -Attorney Docket No. UTSDP4503WO- 1001386307to controls, shown at E:T ratios of 1:2, 1:5, 1:10, and 1:15. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0100] FIGS. 4A-4C show how TIL-NP administration to bilateral tumor model mice resulted in significantly decreased tumor growth in injected tumors (right side) and in untreated secondary tumor sites (left side) relative to controls. FIG. 4A displays graphs of tumor growth (y axis) over time for the treated primary tumor (inoculated at the right flank) for MC38 bearing mice, where the mice were either left untreated (control), treated with free antibodies against CD3 and 4-1 BB with IL-2 (“free antibodies”), or treated with TIL-NPs. FIG. 4B displays graphs of tumor growth (y axis) over time for the untreated secondary tumor (inoculated at the left flank), treated as described in FIG. 4A. FIG. 4C displays the survival curve of MC38 subcutaneous tumor-bearing mice treated as described in FIG. 4A. ****p<0.001. Together these results showed that TIL-NPs displayed significantly improved primary and secondary tumor control, and significantly improved subject survival relative to the combination of agonists administered freely.

[0101] FIG. 5 shows human TIL expansion using kidney cancer (RCC) tumors. In the 786-0 groups, the bars represent, from left to right, 768-0, 786-O+TIL-NPs, 786-O+TILs, 786-O+TILs+Free antibodies, and 786-O+TILs+TIL-NPs. In the ACHN groups, the bars represent, from left to right, ACHN, ACHN+TIL-NPs, ACHN+TILs, ACHN+TILs+Free antibodies, and ACHN+TILs+TIL-NPs. ns: none significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0102] FIGS. 6A-6C, FIG. 6A shows tumor inoculation and treatment schedule. FIG. 6B shows tumor growth curves of right flank tumor (treatment side). FIG. 6C shows tumor growth curves of left flank (non-treatment side). **p<0.01

[0103] FIG. 7 shows representative immunohistochemical images of human CD8+T-cell infiltration in the tumor tissue.DETAILED DESCRIPTION

[0104] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0105] The current disclosure describes the utilization of nanoparticles that can deliver multiple agonists of TILs, such as T cells, simultaneously to the cell surface. In some embodiments the current disclosure relates to compositions and approaches that combine302413766 - 15 -Attorney Docket No. UTSDP4503WO- 1001386307multiple TIL agonists, such as at least three TIL agonists, in one particle to increase immune activation. While not being bound by theory, it is thought that therapeutic efficacy of immune cell activating agents may be improved by temporally and spatially controlling the activation of multiple cell signaling pathways, such as pathways associated with CD3, 4-1 BB, and IL-2R signaling, through proximity based activation. This approach is not limited to antibody mediated agonism, but also includes other means for perpetuating a signaling response, such as through ligand (or active fragment thereof) binding to the cognate receptor.I. Particles

[0106] Provided herein, amongst other things, are particles comprising three or more T cell agonists conjugated to their surface. In some embodiments, particles comprise biocompatible cores. In some embodiments, a particle comprises three different agonists that each bind a different protein receptor on a T cell surface. In some embodiments, a particle comprises three different agonists that each bind a different protein receptor on a T cell surface, and one or more additional agonist and / or antagonist of an immune cell signaling pathway. In some embodiments, a particle can comprise equal to or more than three or more than four (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) different targeting agents, including agonists and antagonists, that each bind a different protein receptor on a T cell surface.

[0107] In some embodiments, the receptors on the T cell surface can be CD3, 4-1 BB, IL-2R, LFA-1, CD2, ICOS, CD28, CTLA-4, PD-1, HVEM, AITR, CD40L, CD27, CD30, 0X40, TOR, BTLA, DR3, GITR, SLAM, 2B4, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1, B7-1, B7-H1, and any combination thereof, as well as any other receptor on a T cell surface that is now known or later identified. (See, e.g., Chen and Flies. “Molecular mechanisms of T cell co-stimulation and co-inhibition” Nat. Rev. Immunol., 2013 Apr. 13(4): 227-242. doi:10.1038 / nri3405.).

[0108] In some embodiments, a particle comprises an agonist of cluster of differentiation 3 (CD3); an agonist of 4-1 BB (aka CD137); and an agonist of interleukin 2 (IL-2) receptor (IL-2R); wherein the agonist of CD3, the agonist of 4-1 BB, and the agonist of IL-2R are bound to a core of the particle. In some embodiments, the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprise a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody.

[0109] In some embodiments, at least one of the agonists is an antibody or active fragment thereof. In some embodiments, at least two of the agonists are antibodies or active fragments thereof. In some embodiments, at least three of the agonists are antibodies or active fragments thereof. In some embodiments, at least one of the agonists is a ligand for a target receptor, or active fragment thereof. In some embodiments, at least two of the agonists are ligands for at302413766 - 16 -Attorney Docket No. UTSDP4503WO- 1001386307least two target receptors, or active fragments thereof. In some embodiments, at least three of the agonists are ligands for at least three target receptors, or active fragments thereof. In some embodiments, at least two agonists are antibodies or active fragments thereof, and at least one agonist is a ligand for a target receptor, or active fragment thereof. In some embodiments a particle comprises at least one agonist of CD3, wherein the agonist comprises an antibody targeted to CD3, or an active fragment thereof; at least one agonist of 4-1 BB, wherein the agonist comprises an antibody targeted to 4-1 BB, or an active fragment thereof; and at least one agonist of IL-2R, wherein the agonist comprises IL-2, or an active fragment thereof.

[0110] In some embodiments of the present invention, the antibody or active fragment is selected from the group consisting of a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a single chain variable fragment (scFV) fragment, a (Fab')2 fragment, and any combination thereof.

[0111] In some embodiments, a particle disclosed herein comprises 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg, 42 pg, 43 pg, 44 pg, 45 pg, 46 pg, 47 pg, 48 pg, 49 pg, 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 91 pg, 92 pg, 93 pg, 94 pg, 95 pg, 96 pg, 97 pg, 98 pg, 99 pg, 100 pg, or any value derivable therein, total combined agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R per 1000 pg of particle. In some embodiments, a population of particles provided herein comprises a target degree of bioconjugation that is, is at least, or is about 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg, or any number therebetween or range derivable thereof, of total combined agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R per 1000 pg of particle.

[0112] In some embodiments, a particle provided herein comprises a hydrodynamic diameter of from 50 to 500 nm, or 50 to 300 nm, or 100 to 200 nm. In some embodiments, a particle provided herein comprises a hydrodynamic diameter of, of greater than, of less than, or of about 2 nm, 4 nm, 6 nm, 8 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 26 nm, 28 nm, 30 nm, 32 nm, 34 nm, 36 nm, 38 nm, 40 nm, 42 nm, 44 nm, 46 nm, 48 nm, 50 nm, 52 nm, 54 nm, 56 nm, 58 nm, 60 nm, 62 nm, 64 nm, 66 nm, 68 nm, 70 nm, 72 nm, 74 nm, 76 nm, 78 nm, 80 nm, 82 nm, 84 nm, 86 nm, 88 nm, 90 nm, 92 nm, 94 nm, 96 nm, 98 nm, 100 nm, 102 nm, 104 nm, 106 nm, 108 nm, 110 nm, 112 nm, 114 nm, 116 nm, 118 nm, 120 nm, 122 nm, 124 nm, 126 nm, 128 nm, 130 nm, 132 nm, 134 nm, 136 nm, 138 nm, 140 nm, 142302413766 - 17 -Attorney Docket No. UTSDP4503WO- 1001386307nm, 144 nm, 146 nm, 148 nm, 150 nm, 152 nm, 154 nm, 156 nm, 158 nm, 160 nm, 162 nm, 164 nm, 166 nm, 168 nm, 170 nm, 172 nm, 174 nm, 176 nm, 178 nm, 180 nm, 182 nm, 184 nm, 186 nm, 188 nm, 190 nm, 192 nm, 194 nm, 196 nm, 198 nm, 200 nm, 202 nm, 204 nm, 206 nm, 208 nm, 210 nm, 212 nm, 214 nm, 216 nm, 218 nm, 220 nm, 222 nm, 224 nm, 226 nm, 228 nm, 230 nm, 232 nm, 234 nm, 236 nm, 238 nm, 240 nm, 242 nm, 244 nm, 246 nm, 248 nm, 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 262 nm, 264 nm, 266 nm, 268 nm, 270 nm, 272 nm, 274 nm, 276 nm, 278 nm, 280 nm, 282 nm, 284 nm, 286 nm, 288 nm, 290 nm, 292 nm, 294 nm, 296 nm, 298 nm, 300 nm, 302 nm, 304 nm, 306 nm, 308 nm, 310 nm, 312 nm, 314 nm, 316 nm, 318 nm, 320 nm, 322 nm, 324 nm, 326 nm, 328 nm, 330 nm, 332 nm, 334 nm, 336 nm, 338 nm, 340 nm, 342 nm, 344 nm, 346 nm, 348 nm, 350 nm, 352 nm, 354 nm, 356 nm, 358 nm, 360 nm, 362 nm, 364 nm, 366 nm, 368 nm, 370 nm, 372 nm, 374 nm, 376 nm, 378 nm, 380 nm, 382 nm, 384 nm, 386 nm, 388 nm, 390 nm, 392 nm, 394 nm, 396 nm, 398 nm, 400 nm, 402 nm, 404 nm, 406 nm, 408 nm, 410 nm, 412 nm, 414 nm, 416 nm, 418 nm, 420 nm, 422 nm, 424 nm, 426 nm, 428 nm, 430 nm, 432 nm, 434 nm, 436 nm, 438 nm, 440 nm, 442 nm, 444 nm, 446 nm, 448 nm, 450 nm, 452 nm, 454 nm, 456 nm, 458 nm, 460 nm, 462 nm, 464 nm, 466 nm, 468 nm, 470 nm, 472 nm, 474 nm, 476 nm, 478 nm, 480 nm, 482 nm, 484 nm, 486 nm, 488 nm, 490 nm, 492 nm, 494 nm, 496 nm, 498 nm, 500 nm, or any number therebetween or range derivable thereof.

[0113] In some embodiments, a population of particles provided herein comprises a polydispersity index (PDI) of, less than, or about 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.05, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.06, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.07, or any number therebetween or range derivable thereof. In some embodiments, a population of particles provided herein comprises a polydispersity index (PDI) of, less than, or about 0.07, 0.065, 0.06, 0.055, 0.5, 0.45, 0.4, 0.35, or 0.3.

[0114] In some embodiments, a particle does not comprise a lipid core. In some embodiments, a particle may be comprised within a lipid or liposome for sustained release.A. Particle Cores

[0115] In some aspects, provided herein are particles comprising a solid core. In some aspects, a particle comprises a functionalized particle core. In some aspects the particle core is, is at least, is at most, or is about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa,302413766 - 18 -Attorney Docket No. UTSDP4503WO- 100138630731 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, 57 kDa, 58 kDa, 59 kDa, 60 kDa, 61 kDa, 62 kDa, 63 kDa, 64 kDa, 65 kDa, 66 kDa, 67 kDa, 68 kDa, 69 kDa, 70 kDa, 71 kDa, 72 kDa, 73 kDa, 74 kDa, 75 kDa, 76 kDa, 77 kDa, 78 kDa, 79 kDa, 80 kDa, 81 kDa, 82 kDa, 83 kDa, 84 kDa, 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, 95 kDa, 96 kDa, 97 kDa, 98 kDa, 99 kDa, 100 kDa, 101 kDa, 102 kDa, 103 kDa, 104 kDa, 105 kDa, 106 kDa, 107 kDa, 108 kDa, 109 kDa, 110 kDa, 111 kDa, 112 kDa, 113 kDa, 114 kDa, 115 kDa, 116 kDa, 117 kDa, 118 kDa, 119 kDa, 120 kDa, 121 kDa, 122 kDa, 123 kDa, 124 kDa, 125 kDa, 126 kDa, 127 kDa, 128 kDa, 129 kDa, 130 kDa, 131 kDa, 132 kDa, 133 kDa, 134 kDa, 135 kDa, 136 kDa, 137 kDa, 138 kDa, 139 kDa, 140 kDa, 141 kDa, 142 kDa, 143 kDa, 144 kDa, 145 kDa, 146 kDa, 147 kDa, 148 kDa, 149 kDa, 150 kDa, 151 kDa, 152 kDa, 153 kDa, 154 kDa, 155 kDa, 156 kDa, 157 kDa, 158 kDa, 159 kDa, 160 kDa, 161 kDa, 162 kDa, 163 kDa, 164 kDa, 165 kDa, 166 kDa, 167 kDa, 168 kDa, 169 kDa, 170 kDa, 171 kDa, 172 kDa, 173 kDa, 174 kDa, 175 kDa, 176 kDa, 177 kDa, 178 kDa, 179 kDa, 180 kDa, 181 kDa, 182 kDa, 183 kDa, 184 kDa, 185 kDa, 186 kDa, 187 kDa, 188 kDa, 189 kDa, 190 kDa, 191 kDa, 192 kDa, 193 kDa, 194 kDa, 195 kDa, 196 kDa, 197 kDa, 198 kDa, 199 kDa, 200 kDa, or any number therebetween or range derivable thereof, in mass. In some embodiments, a particle comprises a functionalized core that is, is at least, is at most, or is about 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa in mass. In some embodiments, a particle comprises a functionalized core that is at least 50 kDa in mass.

[0116] In some embodiments, the core comprises a polymer. In some embodiments the core comprises a pharmaceutically acceptable polymer. In some embodiments, the core comprises, consists of, or consists essentially of PLGA, PLA, chitosan, lipids, silica nanoparticles, gold nanoparticles, dendrimers, or combinations thereof. In some embodiments, the core comprises, consists of, or consists essentially of PLGA and / or PLA. In some embodiments, the core is utilized to provide an advantage in drug delivery, controlled release, biocompatibility, and / or targeted therapy. In some embodiments a polymer is used to bind one or more of the agonist to the core. In some embodiments, one or more of the agonist are bound to each other through a polymer directly (e.g., the core is not, or is not only, an intermediate component of the binding between the agonists). In some embodiments, none of the agonists are bound to each other directly (e.g., the core is an intermediate component in the binding between the bound agonists). In some embodiments, a functionalized core comprises functionalized PEG-PLGA. In some embodiments, the functionalized core is functionalized using / for click chemistry. In some embodiments, functionalization of a core includes azide mediated functionalization. In some embodiments, the functionalized core is302413766 - 19 -Attorney Docket No. UTSDP4503WO- 1001386307purified, for example through size exclusion, following functionalization and prior to conjugation to one or more agonists.

[0117] In some embodiments, functionalized PEG-PLGA comprises N3-PEG(5K)-PLGA (30K). In some embodiments, functionalized PEG-PLGA comprises methoxy-PEG(2K)-PLGA (20K). In some embodiments, functionalized PEG-PLGA comprises a mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some embodiments, a particle comprising functionalized PEG-PLGA comprises a ratio of 1:10 to 10:1, or 1:5 to 5:1, or 1:3 to 3:1, or 1:2 to 2:1, or 1:3 to 1:1.5, or 1:2, or 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K) or any ratio therebetween or thereof. In some embodiments, a particle comprising functionalized PEG-PLGA comprises about a 1:2 to 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some embodiments, a particle comprising functionalized PEG-PLGA comprises about a 1:2 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K). In some embodiments, a particle comprising functionalized PEG-PLGA comprises about a 1:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K).B. CD3 Agonists

[0118] CD3 (cluster of differentiation 3) is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3E chains. These chains associate with the T-cell receptor (TCR) and the CD3-zeta (^-chain) to generate an activation signal in T lymphocytes.

[0119] The CD3 complex is a signaling-machinery-orchestrating subunit of the T cell receptor. Its function inherently makes it a target accessible for signal modulation and redirection. CD3 subunits are nonvariant subunits of the T cell receptor complex and are present on all T cells — both CD4+ and CD8+ T cells and a- and y-b T cells — making them a readily accessible, pan-T cell target. The multimeric nature of the CD3 complex — 6 chains of 3 different types of CD3 proteins, 10 ITAMs of 6 different types as well as their modular arrangement as hetero and homodimers — provides a degree of modularity and potentially several foci of therapeutic intervention where the CD3 agonistic signal can be fine-tuned and customized to exhibit a wide range and scope of regulatory and modulatory effects on the T cell activation axis.

[0120] In some embodiments, a CD3 agonist comprises a monoclonal antibody, or an active fragment thereof, targeting CD3. In some embodiments, a CD3 agonist comprises OKT3 or a modified version or active fragment thereof. In some embodiments, a CD3 agonist comprises 145-2C11 or a modified version (e.g., humanized version, etc.) or active fragment thereof. In some embodiments, a CD3 agonist comprises G4.18 or a modified version (e.g., humanized302413766 - 20 -Attorney Docket No. UTSDP4503WO- 1001386307version, etc.) or active fragment thereof. In some embodiments, a CD3 agonist comprises Teplizumab or a modified version or active fragment thereof. In some embodiments, a CD3 agonist comprises Otelixizumab or a modified version or active fragment thereof. In some embodiments, a CD3 agonist comprises Foralumab or a modified version or active fragment thereof. In some embodiments, a CD3 agonist comprises Muromonab-CD3 (OKT3 variant) or a modified version or active fragment thereof. In some embodiments, a CD3 agonist comprises Visilizumab or a modified version or active fragment thereof. In some embodiments, a CD3 agonist comprises CD3-specific DNA and / or RNA aptamer. In some embodiments, a CD3 agonist comprises a CD3 ligand 1 (CD3L1) or a modified version or a CD3 activating fragment thereof. In some embodiments, a CD3 agonist comprises a bispecific and / or multispecific antibody specific for CD3 and one or more additional targets. In some embodiments, selection of agonist provides a particular immunomodulatory (e.g., immune-stimulating property) for therapeutic application.

[0121] In some embodiments, an agonist of CD3 comprises, consists essentially of, or consists of a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody. In some embodiments, an agonist of CD3 comprises, consists essentially of, or consist of a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a scFV fragment, a (Fab')2 fragment, or any combination thereof. In some embodiments, an agonist of CD3 comprises, consists essentially of, or consists of a CD3 ligand. In some embodiments, a CD3 ligand comprises, consists essentially of, or consists of CD3L1 and / or a CD3 activating fragment thereof.

[0122] In some embodiments, an agonist of CD3 is conjugated to a particle by click chemistry.

[0123] In some embodiments, a particle comprises, comprises at least, comprises at most, or comprises about 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg, 42 pg, 43 pg, 44 pg, 45 pg, 46 pg, 47 pg, 48 pg, 49 pg, 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 91 pg, 92 pg, 93 pg, 94 pg, 95 pg, 96 pg, 97 pg, 98 pg, 99 pg, 100 pg, or any number therebetween or range derivable thereof, agonist of CD3 per 1000 pg of particle.

[0124] In some embodiments, a particle comprises a lower level of CD3 agonist conjugated to the particle relative to agonist of 4-1BB conjugated to the particle.302413766 - 21 -Attorney Docket No. UTSDP4503WO- 1001386307

[0125] In some embodiments, a particle comprises a ratio of, of at least, of at most, or of about 1:10to 10:1, or 1:5to 5:1, or2:1 to 1:2, or0.8:1 to 1:0.8, or0.9:1, or 1 :1 , or any range derivable thereof or therebetween, of agonist of CD3 to agonist of IL-2R (CD3 agonist: IL-2R agonist).C. 4-1 BB Agonists

[0126] 4-1 BB (also known as TNFRSF9 and CD137) is a member of the tumor necrosis factor (TNF) receptor family, and is a type 1 transmembrane protein, expressed on surfaces of leukocytes and non-immune cells. 4-1 BB is a costimulatory molecule functioning to stimulate T cell proliferation, dendritic cell maturation, and promotion of B cell antibody secretion. As a T cell co-stimulator, T cell receptor (TCR) and CD28 signaling causes expression of 4-1BB on T cell membranes. When 4-1 BB then reacts with the 4-1 BB ligand, it leads to 4-1 BB upregulation (positive feedback cycle). When 4-1 BB interacts with its ligand, it leads to T cell cytokine production and T cell proliferation, among other signaling pathway responses.

[0127] In some embodiments, a 4-1 BB agonist comprises a monoclonal antibody targeting 4-1BB, or an active fragment thereof. In some embodiments, a 4-1 BB agonist comprises Urelumab, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises Utomilumab, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises ELI101, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises AGEN2373, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises ADG206, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises HOT-1030, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises ADG106, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises ATOR-1017, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises PE0116, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises CTX-471, ora modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises LVGN6051, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises STA551, or a modified version or active fragment thereof. In some embodiments, a 4-1 BB agonist comprises 4-1 BBL, or a modified version or 4-1 BB binding fragment thereof. In some embodiments, a 4-1 BB agonist comprises 4-1 BB-specific DNA and / or RNA aptamer. In some embodiments, a 4-1 BB agonist comprises a bispecific and / or multispecific antibody specific for 4-1 BB and one or more additional targets. In some embodiments, selection of agonist provides a particular immunomodulatory (e.g., immune-stimulating) property for therapeutic application.302413766 - 22 -Attorney Docket No. UTSDP4503WO- 1001386307

[0128] In some embodiments, an agonist of 4-1 BB comprises, consists essentially of, or consists of a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody. In some embodiments, an agonist of 4-1 BB comprises, consists essentially of, or consist of a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a scFV fragment, a (Fab')2 fragment, or any combination thereof. In some embodiments, an agonist of 4-1 BB comprises, consists essentially of, or consists of a 4-1 BB ligand. In some embodiments, a 4-1 BB ligand comprises, consists essentially of, or consists of 4-1 BBL and / or a 4-1 BB activating fragment thereof.

[0129] In some embodiments, a particle provided herein comprises a greater concentration of 4-1 BB agonist relative to the agonist of CD3 and / or the agonist of IL-2R. In some embodiments, a particle comprises least a 5%, 10%, 15%, 20%, or 25%, or any number therebetween or range thereof, greater concentration of agonist of 4-1 BB relative to the agonist of CD3, the agonist of IL-2R, or both the agonist of CD3 and the agonist of IL-2R.

[0130] In some embodiments, a particle comprises a ratio of, at least, at most, or about 1.5:1 to 2: 1 , or 2: 1 to 4:3, or 3:2, or 15:9, or any range therebetween, of agonist of 4-1 BB to agonist of CD3 (4-1 BB agonist: CD3 agonist).

[0131] In some embodiments, a particle comprises a ratio of, at least, at most, or about 1.5:1 to 2:1, or2:1 to4:3, or3:2, or 15:10, or any range therebetween, of agonist of 4-1 BB to agonist of IL-2R (4-1 BB agonist: IL-2R agonist).

[0132] In some embodiments, a particle comprises a ratio of, of at least, of at most, or of about 2:1:1 to 4:3:3, or 3:2:2, or 15:9:1, or any range therebetween, of agonist of 4-1 BB to agonist of CD3 to agonist of IL-2R (4-1 BB agonist: CD3 agonist: IL-2R agonist).

[0133] In some embodiments, a particle comprises or comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%,302413766 - 23 -Attorney Docket No. UTSDP4503WO- 1001386307180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, or any value derivable thereof or therebetween or range thereof, greater concentration of agonist of 4-1 BB relative to the agonist of CD3, the agonist of IL-2R, or both the agonist of CD3 and the agonist of IL-2R.

[0134] In some embodiments, a particle comprises, comprises at least, comprises at most, or comprises about 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg, 42 pg, 43 pg, 44 pg, 45 pg, 46 pg, 47 pg, 48 pg, 49 pg, 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 91 pg, 92 pg, 93 pg, 94 pg, 95 pg, 96 pg, 97 pg, 98 pg, 99 pg, 100 pg, or any number therebetween or range derivable thereof, agonist of 4-1 BB per 1000 pg of particle.

[0135] In some embodiments, an agonist of 4-1 BB is conjugated to a particle by click chemistry.D. IL-2R Agonists

[0136] lnterleukin-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds and responds to the cytokine IL-2. IL-2 and IL-2R have been implicated as key components of the immune system, particularly via their direct effects on T cells.

[0137] In some embodiments, an IL-2R agonist comprises a monoclonal antibody targeting IL-2R, or an active fragment thereof. In some embodiments, an IL-2R agonist comprises IL-2 or a modified version or IL-2R binding fragment thereof. In some embodiments, a IL-2R agonist comprises an IL-2R-specific DNA and / or RNA aptamer. In some embodiments, an IL-2R agonist comprises IL-2 variants (for example but not limited to, Aldesleukin). In some embodiments, an IL-2R agonist comprises I L-2 / anti-IL-2 complexes. In some embodiments, an IL-2R agonist comprises engineered IL-2 muteins. In some embodiments, an IL-2R agonist comprises IL-2R-targeting bispecific and / or multispecific antibodies, In some embodiments, an IL-2R agonist comprises a bispecific and / or multispecific antibody specific for IL-2R and one or more additional targets. In some embodiments, selection of agonist provides a particular immunomodulatory property (e.g., immuno-stimulatory) for therapeutic application.

[0138] In some embodiments, an agonist of IL-2R comprises, consists essentially of, or consists of a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody. In some embodiments, an agonist of IL-2R comprises, consists essentially of, or302413766 - 24 -Attorney Docket No. UTSDP4503WO- 1001386307consist of a monoclonal antibody, a Fab fragment, a Fab'-SH fragment, a FV fragment, a scFV fragment, a (Fab')2 fragment, or any combination thereof. In some embodiments, an agonist of IL-2R comprises, consists essentially of, or consists of a IL-2R ligand. In some embodiments, a IL-2R ligand comprises, consists essentially of, or consists of IL-2 and / or an IL-2R activating fragment thereof.

[0139] In some embodiments, a particle comprises, comprises at least, comprises at most, or comprises about 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg, 42 pg, 43 pg, 44 pg, 45 pg, 46 pg, 47 pg, 48 pg, 49 pg, 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 91 pg, 92 pg, 93 pg, 94 pg, 95 pg, 96 pg, 97 pg, 98 pg, 99 pg, 100 pg, or any number therebetween or range derivable thereof, agonist of IL-2R per 1000 pg of particle.

[0140] In some embodiments, an agonist of IL-2R is conjugated to a particle by click chemistry.E. Methods of Making and Particle Characteristics

[0141] Also provided herein are methods of making particles described herein. In some embodiments particles comprise a core conjugated to an agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R, wherein the conjugation is mediated using click chemistry. In some embodiments, an agonist of CD3, an agonist of 4-1 BB, and an agonist of IL-2R are not conjugated directly to another one of the agonist of CD3, the agonist of 4-1 BB, and the agonist of IL-2R.

[0142] In some embodiments, functionalized particle cores (e.g., PEG-PLGA cores) are functionalized for click chemistry, and one or more of the agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R are functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R functionalized for click chemistry. In some embodiments, functionalized particle cores (e.g., PEG-PLGA cores) are azide-functionalized PEG-PLGA particle cores, and the functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R are DBCO-functionalized.

[0143] In some embodiments, the average degree of functionalization of 4-1BB agonist is or is 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, 28, 29, or 30, or any range derivable therein. In some embodiments, the average degree of functionalization of 4-1BB agonist is or is about 10-20, or is or is about 15.302413766 - 25 -Attorney Docket No. UTSDP4503WO- 1001386307

[0144] In some embodiments, the average degree of functionalization of CD3 agonist is or is 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, 28, 29, or 30, or any range derivable therein. In some embodiments, the average degree of functionalization of CD3 agonist is or is about 5-15, or is or is about 10, or is or is about 9.

[0145] In some embodiments, the average degree of functionalization of IL-2R agonist is or is 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, 28, 29, or 30, or any range derivable therein. In some embodiments, the average degree of functionalization of IL-2R agonist is or is about 5-15, or is or is about 10.

[0146] In some embodiments, the average degree of functionalization is or is about 10-20 for functionalized agonist of 4-1 BB, 5-15 for functionalized agonist of CD3, and 5-15 for functionalized agonist of IL-2R. In some embodiments, the average degree of functionalization is or is about 15 for functionalized 4-1 BB agonists, is or is about 9 for functionalized CD3 agonists, and is or is about 10 for functionalized IL-2R agonists.

[0147] In some embodiments, functionalized particle cores are purified, for example as through use of an Amicon Ultra- 15 Centrifugal Filter Unit, prior to conjugation to an agonist. In some embodiments, functionalized CD3 agonists, 4-1 BB agonists, and IL-2R agonists are conjugated to the azide-functionalized PEG-PLGA NPs through stain-promoted azide-alkyne cycloaddition. In some embodiments, the stain-promoted azide-alkyne cycloaddition is performed at 1-10 °C, or at 4 °C, for 10-24 h or 15-18 h (in the dark) to form co-stimulatory molecule functionalized particles (e.g., exemplary “TIL-NPs”).II. Methods of treating

[0148] In some aspects, the current disclosure encompasses methods for treating and / or preventing a disease or disorder, the method comprising administering a composition disclosed herein.

[0149] In some embodiments, the present disclosure provides methods of activating a T cell, comprising contacting the T cell with a particle or composition of the disclosure, under conditions whereby each different agonist can bind their respective target protein receptor on the same T cell surface.

[0150] In some embodiments, the present disclosure provides methods of inducing a T cell immune response, comprising contacting the T cell with a particle or composition of the disclosure, under conditions whereby each different agonist can bind their respective target protein receptor on the same T cell surface.

[0151] In some embodiments, the present disclosure provides methods of inducing a T cell immune response in a subject in need thereof, comprising administering to the subject an302413766 - 26 -Attorney Docket No. UTSDP4503WO- 1001386307effective amount of a particle or composition of the disclosure, under conditions whereby each different agonist can bind their respective target protein receptor on the same T cell surface.

[0152] In some embodiments, the present disclosure provides methods of activating T cells in a subject in need thereof, comprising administering to the subject an effective amount of a particle or composition of the disclosure, under conditions whereby each different agonist can bind their respective target protein receptor on the same T cell surface.

[0153] In some embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a particle or composition of the disclosure, under conditions whereby each different agonist can bind their respective target protein receptor on the same T cell surface.

[0154] In some aspects, the amount of a particle or composition of the disclosure administered is, is greater than, is less than, or is between 0.001 g to 100 g per dose, or any number therebetween or any range thereof. In some aspects, the amount of the particle or composition of the disclosure is at least, at most, or exactly 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5051, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 g, or any number therebetween or any range thereof. In some aspects, the particle or composition of the disclosure is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 dose(s), or any number therebetween or any range thereof. When more than one dose is administered, the doses may be about 1 , 4, 8, 12, or 24 hours or 1 , 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.A. Cancer

[0155] In some aspects, the current disclosure encompasses a method of treating, preventing, reducing the symptoms of, or delaying the onset of a cancer in a subject in need thereof, the method comprising administering particles and / or compositions disclosed herein to the subject in need thereof.

[0156] “Subject” means any animal, but is preferably a mammal, such as, for example, a human, monkey, mouse, rabbit or rat. In some aspects, the subject is a mammal who has, or is suspected of having a cancer. In some aspects, the subject has, or is suspected of having a lung tumor, primary brain tumors, stomach cancer, colon cancer, pancreatic cancer, carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia, cervical302413766 - 27 -Attorney Docket No. UTSDP4503WO- 1001386307cancer, endometrial cancer, adrenal cortical cancer, or prostate cancer. In some aspects, the cancer is a solid tumor. In some aspects, the cancer corresponds to a sarcoma selected from chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma; a melanoma selected from acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma; a carcinoma selected from acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidernoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney,302413766 - 28 -Attorney Docket No. UTSDP4503WO- 1001386307reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma viflosum; a leukemia acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia; or other cancers selected from Hodgkin's Disease, NonHodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, or prostate cancer.

[0157] In some embodiments, the provided herein are methods of treating cancer in a subject in need thereof, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, uterine cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, liver cancer, skin cancer, blood cancer, and any combination thereof.

[0158] In some aspects, the present disclosure provides methods of treating sarcomas, carcinomas, and lymphomas. In some aspects, the present disclosure provides methods for treating solid cancers, such as but not limited to breast cancer, liver cancer, kidney cancer,302413766 - 29 -Attorney Docket No. UTSDP4503WO- 1001386307prostate cancer, lung cancer, bladder cancer, thyroid cancer, stomach cancer, anal cancer, gallbladder cancer, ovarian cancer, soft-tissue sarcoma, skin cancer, esophageal cancer, and / or colon cancer. In some aspects, the present disclosure provides methods for treating primary tumors and metastasis.

[0159] In some embodiments, the present disclosure provides methods of treating cancer in a subject (e.g., a subject in need thereof), wherein the subject has been diagnosed with cancer.

[0160] In some aspects, particles and compositions of the disclosure may be used as an adjuvant cancer treatment. One or more additional cancer therapies may be administered simultaneously or staggered with the compositions and particles utilized in the disclosed methods.

[0161] In some embodiments, the present disclosure provides methods of treating cancer in a subject (e.g., a subject in need thereof), wherein the particle or composition is administered via a route selected from the group comprising intratumorally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intrathecally, intraventricularly, intraorbitally, intranasally, by implantation, by inhalation, or any combination thereof.Additional Anti-Cancer Therapies

[0162] The methods may comprise or further comprise administration of an additional anticancer therapy or agent. The compositions may comprise or further comprise an additional anti-cancer therapy or agent. The methods and / or compositions may exclude an additional anti-cancer therapy.

[0163] Immunostimulators - The anti-cancer therapy or agent may be an immunostimulator. The term “immunostimulator” as used herein refers to a compound that can stimulate an immune response in a subject, and may include an adjuvant. In some aspects, an immunostimulator is an agent that does not constitute a specific antigen, but can boost the strength and longevity of an immune response to an antigen. Such immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.), liposomes and liposomal formulations such as AS01, synthesized302413766 - 30 -Attorney Docket No. UTSDP4503WO- 1001386307or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments.

[0164] The additional anti-cancer therapy may comprise an agonist for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and / or combinations thereof. The additional anti-cancer therapy may comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited immunostimulators comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381, U.S. Published Patent Application 2010 / 0075995, or WO 2010 / 018132; immunostimulatory DNA; or immunostimulatory RNA. In some aspects, the additional anti-cancer therapies also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA, poly l:C or poly I : poly C12U (available as Ampligen.RTM., both poly l:C and poly l:polyC12U being known as TLR3 stimulants), and / or those disclosed in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280 A2. In some aspects, an additional anti-cancer therapy may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some aspects, additional therapies may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725.

[0165] In some aspects, additional anti-cancer therapies may be proinflam matory stimuli released from necrotic cells (e.g., urate crystals). In some aspects, additional anti-cancer therapies may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some aspects, additional anti-cancer therapies may be activated components of immune complexes. Additional anti-cancer therapies also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some aspects, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some aspects,302413766 - 31 -Attorney Docket No. UTSDP4503WO- 1001386307immunostimulators are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. In some aspects, the cytokine receptor agonist is a small molecule, antibody, fusion protein, ligand, or aptamer.

[0166] Immunotherapies - in some aspects, the additional anti-cancer therapy comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies are known in the art, and some are described below.

[0167] Inhibition of co-stimulatory molecules - in some aspects, the immunotherapy comprises an inhibitor of a co-stimulatory molecule. In some aspects, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1 BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0168] Dendritic cell therapy - in some aspects, an additional anti-cancer therapy may comprise dendritic cells. Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0169] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0170] Dendritic cells can also be activated in vivo by making tumor cells express GM-CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.302413766 - 32 -Attorney Docket No. UTSDP4503WO- 1001386307

[0171] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (e.g., a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0172] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

[0173] CAR-T cell therapy - in some aspects, an additional anti-cancer therapy may comprise a chimeric antigen receptor (CAR). CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0174] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0175] Cytokine therapy - in some aspects, an additional anti-cancer therapy may comprise cytokines. Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.302413766 - 33 -Attorney Docket No. UTSDP4503WO- 1001386307

[0176] Interferons are produced by the immune system. They are usually involved in anti-viral response, but also have use for cancer. They fall in three groups: type I (IFNa and I FN p) , type II (IFNy) and type III (IFNA).

[0177] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0178] Adoptive T-cell therapy - in some aspects, an additional anti-cancer therapy may comprise adoptive T cell therapy. Adoptive T cell therapy is a form of passive immunization by the transfusion of T-cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically, they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0179] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens, or through exposure and / or coadministration with compositions described herein.

[0180] Checkpoint Inhibitors and Combination Treatment - in some aspects, an additional anti-cancer immunotherapy comprises immune checkpoint inhibitors. Certain aspects are further described below.

[0181] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Additional anti-cancer therapies of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0182] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.302413766 - 34 -Attorney Docket No. UTSDP4503WO- 1001386307

[0183] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0184] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some aspects, the PDL1 inhibitor comprises AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional anti-cancer PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0185] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHlgM12B7.

[0186] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with302413766 - 35 -Attorney Docket No. UTSDP4503WO- 1001386307and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0187] Another immune checkpoint that can be targeted in the methods provided herein as an additional anti-cancer therapy is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell costimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTI-A-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTI_A-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0188] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0189] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675.206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.302413766 - 36 -Attorney Docket No. UTSDP4503WO- 1001386307

[0190] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0191] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99%, or any number therebetween or range derivable thereof, variable region amino acid sequence identity with the above-mentioned antibodies.

[0192] Oncolytic virus - in some aspects, an additional anti-cancer therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy.

[0193] Polysaccharides - in some aspects, an additional anti-cancer therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.

[0194] Neoantigens - in some aspects, an additional anti-cancer therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.

[0195] Chemotherapies - in some aspects, an additional anti-cancer therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs302413766 - 37 -Attorney Docket No. UTSDP4503WO- 1001386307(e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0196] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2 to about 20 mg / m2, or any number therebetween or range thereof, for 5 days every three weeks for a total of three courses being contemplated in certain aspects.

[0197] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain aspects, appropriate intravenous doses for an adult include about 60 mg / m2 to about 75 mg / m2 at about 21 -day intervals or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.

[0198] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to302413766 - 38 -Attorney Docket No. UTSDP4503WO- 1001386307about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0199] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-Fll) and floxuridine (fluorodeoxyuridine; FudR). 5-Fll may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-Fll dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0200] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0201] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent, or any number therebetween or range thereof. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.

[0202] Targeted Therapies - in some aspects, an additional anti-cancer therapy comprises a targeted therapy. Targeted therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules (“molecular targets”) that are involved in the growth, progression, and / or spread of cancer. Targeted cancer therapies are sometimes called “molecularly targeted drugs,” “molecularly targeted therapies,” “precision medicines,” or similar names. Non-limiting examples of targeted therapies include hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, toxin delivery molecules, and the like. In particular aspects, the302413766 - 39 -Attorney Docket No. UTSDP4503WO- 1001386307targeted therapy may be a poly ADP ribose polymerase (PARP) inhibitor (e.g., niraparib). PARP (e.g., PARP-1 and / or PARP-2) inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15, INO-1001, PJ34, 3-aminobenzamide, 4-amino-1,8- naphthalimide, 6(5H)-phenanthridinone, benzamide, NLI1025).

[0203] Hormone Therapy - in some aspects, an additional anti-cancer therapy comprises hormone therapy. In some aspects, one or more anticancer therapies may be hormonal therapy, Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LLIPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0204] Radiotherapy - in some aspects, an additional anti-cancer therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0205] In some aspects, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 Gy, or any number therebetween or any range thereof. In some aspects, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 dose(s), or any number therebetween or any range thereof. When more than one dose is administered, the doses may be about 1 , 4, 8, 12, or24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0206] In some aspects, the amount of IR may be presented as a total dose of IR, which is then administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of I R is at least, at most, or about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,302413766 - 40 -Attorney Docket No. UTSDP4503WO- 100138630759, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150, or any number therebetween or any range thereof. In some aspects, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy, or any number therebetween or any range thereof. In some aspects, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered, or any number therebetween or any range thereof. In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any number therebetween or any range thereof, fractionated doses are administered per day. In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or any number therebetween or any range thereof, fractionated doses are administered per week.

[0207] Surgery - in some aspects, an additional anti-cancer therapy may comprise surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0208] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months, or any number therebetween or any range thereof. These treatments may be of varying dosages as well.III. Compositions and Kits

[0209] In certain embodiments, the disclosure provides compositions comprising the particles of this invention together with one or more of the following: a pharmaceutically acceptable302413766 - 41 -Attorney Docket No. UTSDP4503WO- 1001386307diluent; a carrier; a solubilizer; an emulsifier; a preservative; and / or an adjuvant. Such compositions may contain an effective amount of the particles. Thus, the use of the particles as provided herein in the preparation of a pharmaceutical composition or medicament is also included. Such compositions can be used in the treatment of a variety of diseases as described herein.

[0210] Acceptable formulation components for pharmaceutical preparations are nontoxic to recipients at the dosages and concentrations employed. In addition to the particles provided herein, compositions according to the disclosure may contain components for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as acetate, borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); and / or tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0211] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. Suitable vehicles or carriers for such compositions include water (e.g., sterile water) for injection, physiological saline solution, and / or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.302413766 - 42 -Attorney Docket No. UTSDP4503WO- 1001386307

[0212] Compositions comprising particles of this disclosure may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the particles may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0213] In some embodiments, formulation components are present in concentrations that are acceptable to the site of administration. In some embodiments, buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 4.0 to about 8.5, or any number therebetween or any range thereof, or alternatively, between about 5.0 to 8.0. Pharmaceutical compositions can comprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.

[0214] In some embodiments, pharmaceutical compositions of the disclosure are in the form of sustained- or controlled-delivery formulations. In some embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections can be utilized. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules, polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2-hydroxyethyl-methacrylate), ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0215] In some embodiments, pharmaceutical compositions provided herein are to be used for in vivo administration, wherein the in vivo administration is sterile. Sterilization may be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle, or a sterile pre-filled syringe ready to use for injection.

[0216] Compositions provided herein may be formulated for transdermal delivery, optionally with the inclusion of microneedles, microprojectiles, patches, electrodes, adhesives, backings, and / or packaging, or formulations for jet delivery, in accordance with known techniques. See, e.g., U.S. Pat. Nos. 8,043,250; 8,041,421; 8,036,738; 8,025,898; 8,017,146.302413766 - 43 -Attorney Docket No. UTSDP4503WO- 1001386307

[0217] In some embodiments, components used to formulate pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0218] The present disclosure also provides kits for producing multi-dose or single-dose administration units. For example, kits according to the invention may each contain both a first container having a dried composition and a second container having an aqueous diluent, including for example single and multi-chambered pre-filled syringes (e.g., liquid syringes, lyosyringes, or needle-free syringes). Kits may also include, in one or more suitable containers, the materials necessary to make and / or use any of the particles and / or compositions disclosed herein.

[0219] Kits that include particles of this invention and / or a pharmaceutical composition as described herein are also provided herein. Some kits include particles and / or compositions in a container (e.g., vial or ampule), and may also include instructions for use of the particles and / or composition in the various methods disclosed above. The particles and / or composition can be in various forms, including, for instance, as part of a solution or as a solid (e.g., lyophilized powder). The instructions may include a description of how to prepare (e.g., dissolve or resuspend) the particles in an appropriate fluid and / or how to administer the particles for the treatment of the diseases and disorders described herein.

[0220] The kits may also include various other components, such as buffers, salts, complexing metal ions and other agents described above in the section on pharmaceutical compositions. These components may be included with the chimeric protein or may be in separate containers. The kits may also include other therapeutic agents for administration with the particles. Examples of such agents include, but are not limited to, agents to treat the disorders or conditions described above.

[0221] The following examples are provided solely to illustrate certain aspects of the particles and compositions that are provided herein and thus should not be construed to limit the scope of the claimed invention.302413766 - 44 -Attorney Docket No. UTSDP4503WO- 1001386307EXAMPLES

[0222] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0223] As discussed herein, the inventors surprisingly found that conjugation of three agonists (e.g., agonists of CD3, agonists of 4-1 BB, and agonists of IL-2R) to a particle resulted in greater T cell activation and anti-tumor efficacy in vitro and / or in vivo relative to use of the three agonists alone. The data demonstrated that spatial-temporal delivery of multiple agonists improved T cell activation and cancer immunotherapy by promoting simultaneous immunomodulation.Example 1 : Preparation and Characterization of Exemplary Tumor Infiltrating Lymphocyte (TIL) Activating Particles (TIL-NPs)

[0224] See FIG. 1A for a simplified graphical description. A 1:2 wt / wt mixture of N3-PEG(5K)-PLGA (30K) (polyethylene glycol and poly(lactic-co-glycolic) acid) and m ethoxy- PEG (2 K)-PLGA (20K) were used to prepare azide-functionalized PEG-PLGA NP cores, which were purified via Amicon Ultra-15 Centrifugal Filter Units (50 kDa cutoff). DBCO-functionalized anti-CD3, anti-4-1 BB, and IL-2 protein were prepared via amine-NHS ester reaction with DBCO-PEGs-NHS ester. The target degree of functionalization was 15 for anti-4-1 BB, 9foranti-CD3, and 10 for IL-2. Functionalized antibodies and cytokine were purified via Zeba Spin Desalting Columns. Each antibody / protein contained an average of four conjugated DBCO. Then DBCO-functionalized anti-CD3, anti-4-1 BB, and IL-2 were conjugated to the azide-functionalized PEG-PLGA NPs through stain-promoted azide-alkyne cycloaddition at 4 °C for 15-18 h (in the dark) to form co-stimulatory molecules functionalized NPs (exemplary “TIL-NPs”). The target degree of bioconjugation was 50 pg of immune-activating molecules per milligram of NPs.

[0225] The sizes of different NPs were characterized by dynamic light scattering method and the results thereof are shown in FIG. 2A. The successful conjugation of NP cores with anti-CD3, anti-4-1BB, and IL-2 were further verified by FACS binding assay as shown in FIG. 2B.The results showed that inventors successfully engineered exemplary TIL-NPs containing agonists of CD3, agonists of 4-1BB, and agonists of IL-2, in the form of anti-CD3, anti-4-1 BB,302413766 - 45 -Attorney Docket No. UTSDP4503WO- 1001386307and IL-2. These TIL-NPs averaged 126.2 nm in diameter and had a polydispersity index of 0.036 (FIG. 2A), and each milligram of TIL-NPs contained approximately 50 pg immune activating molecules. In vitro FACS binding assay confirmed the successful conjugation of anti-CD3, anti-4-1 BB, and IL-2 to the azide-functionalized NPs (FIG. 2B).Example 2: TIL-NPs Improved TIL Anti-Cancer Efficacy In Vitro

[0226] To test the ability of TIL-NPs to amplify and activate TILs in vitro, a series of coculturing experiments were performed. These experiments included culturing of murine colon adenocarcinoma cells (MC38; 5000 cells per well) alone (“MC38”), or coculturing of MC38 cells with TILs isolated from tumors of MC38- bearing mice. In coculturing experiments TILs were added either alone, or in combination with non-conjugated (“free”) anti-CD3 (6 ng / ml), free anti-4-1 BB (40 ng / ml), and free IL-2 protein (20 ng / ml) (“MC38+TILs+free antibodies”), or in combination with TIL-NPs comprising anti-CD3, anti-4-1 BB, and IL-2 (MC38+TILS+TIL-NPs) prepared as described in Example 1. The number of TILs per well depended on the various Target / effector ratios. 48 h after co-culture, the viabilities of MC38 cells were evaluated by MTS assay (FIG. 3A) and IFN-y concentrations in the supernatant were analyzed by ELISA assay (FIG. 3B) (t-test was used to compare the groups).

[0227] At all T:E ratios tested, the results showed that TILs were stimulated by TIL-NPs and that these TILs displayed significantly improved anticancer activity against MC38 cells when compared to that of unstimulated TILs or TILs administered with free antibodies and IL-2 (FIG.3A). The results showed that IFN-y levels in all coculture groups were elevated, with a significant increase in the experimental TIL group treated with the TIL-NPs relative to control TILs left unstimulated or administered with free antibodies and IL-2 (FIG. 3B).Example 3: TIL-NPs Displayed Improved Anti-Cancer Efficacy In Vivo Relative to Free Agonists Alone

[0228] See FIG. 1B for a schematic representation. The therapeutic efficiency of intratumoral injectable TIL-NPs was evaluated in C57BL / 6J mice in vivo, which had MC38 subcutaneous tumors implanted in the right (treatment side) and left (non-treatment side) flanks (MC38 bilateral tumor model). Tumors were left untreated, or treated with intra tumoral (i.t.) injection of free antibodies (anti-CD3 (1.5 pg) and anti-4-1 BB (10 pg)) and IL-2 (5 pg) (per injection), or i.t. injection of TIL-NPs. Both treatments were provided 3 times, 1 injection every 3-days. The tumor growth of the treated primary and untreated secondary tumor were monitored following treatment to assess systemic treatment response.

[0229] The results showed that intratumorally injected TIL-NPs were highly effective in eradicating the treated primary tumor when compared to that of control and free antibodies groups (FIGS. 4A-4B). Surprisingly, all of the primary tumors were completely regressed302413766 - 46 -Attorney Docket No. UTSDP4503WO- 1001386307(complete response (CR) rate of 100%). Importantly, the TIL-NPs also provided a robust systemic response, with 60% of the untreated secondary tumors also showing a complete response. Additionally, administration of TIL-NPs significantly prolonged mice's survival (FIG.4C). Together, these results highlighted the surprising and promising therapeutic potential, for use either in vitro and / or in vivo, of particles comprising a CD3 agonist (e.g., anti-CD3), a 4-1BB agonist (e.g., anti-4-1 BB), and an IL-2R agonist (e.g., IL-2).Example 4: Additional Characterization of TIL-NPs

[0230] To test the ability of TIL-NPs to amplify and activate TILs in vitro, a series of coculturing experiments will be performed. These experiments include culturing of cancer cells alone (as control), or coculturing of cancer cells with TILs isolated from tumor-bearing mice that were previously inoculated with the same / cancer cell type. In coculturing experiments TILs will be added 1) alone, or in combination (or pre-activated) with 2) non-conjugated (“free”) CD3 agonist, free 4-1 BB agonist, and free IL-2R agonist, 3) free CD3 agonist and free 4-1 BB agonist, 4) free CD3 agonist and free IL-2R agonist, 5) free 4-1 BB agonist and free IL-2R agonist, 6) any one of free 4-1 BB agonist, free CD3 agonist, or free IL-2 agonist protein, 7) particles conjugated to 4-1 BB agonist and IL-2R agonist, 8) particles conjugated to CD3 agonist and IL-2R agonist, 9) particles conjugated to CD3 agonist and 4-1 BB agonists, 10) particles conjugated to any of one of 4-1BB agonist, CD3 agonists, and IL-2R agonists, and / or 11) TIL-NP comprising agonists of CD3 (e.g., anti-CD3), agonists of 4-1BB (e.g., anti-4-1 BB), and agonists of IL-2R (e.g., IL-2). The viability of cancer cells will be evaluated and IFN-y concentrations in the supernatant will be analyzed. The results are expected to show that TILs stimulated by TIL-NPs expand well and to a significantly greater extent that the other treatments and controls. The results are also expected to show that TILs co-administered with TIL-NP and / or pre-activated with TIL-NP display significantly improved anticancer activity against cancer cells when compared to that of unstimulated TILs or any of the other treatments. The results are expected to show that IFN-y levels in all coculture groups are elevated, with a significant increase in the experimental TIL group treated with the TIL-NPs relative to one or more control TILs left unstimulated or TILs administered (or preactivated) with other test / control combinations.

[0231] To test the in vivo therapeutic efficiency of intratumoral injectable TIL-NP, tumor bearing mice are implanted in the right (treatment side) and left (non-treatment side) flanks (e.g., MC38 bilateral tumor model). Tumors are left untreated, or treated with intra tumoral (i.t.) injection of 1) non-conjugated (“free”) CD3 agonist, free 4-1 BB agonist, and free IL-2R agonist, 3) free CD3 agonist and free 4-1 BB agonist, 4) free CD3 agonist and free IL-2R agonist, 5) free 4-1 BB agonist and free IL-2R agonist, 6) any one of free 4-1 BB agonist, free CD3 agonist, or free IL-2 agonist protein, 7) particles conjugated to 4-1 BB agonist and IL-2R agonist, 8)302413766 - 47 -Attorney Docket No. UTSDP4503WO- 1001386307particles conjugated to CD3 agonist and IL-2R agonist, 9) particles conjugated to CD3 agonist and 4-1 BB agonists, 10) particles conjugated to any of one of 4-1 BB agonist, CD3 agonists, and IL-2R agonists, and / or 11) TIL-NP particles comprising agonists of CD3 (e.g., anti-CD3), agonists of 4-1 BB (e.g., anti-4-1 BB), and agonists of IL-2R (e.g., IL-2). Treatments include injections 3 times with a 3-day interval between each injection. The tumor growth of the treated primary and untreated secondary tumor will be monitored following treatment to assess systemic treatment response. The results are expected to show that intratumorally injected TIL-NPs are highly effective in eradicating the treated primary tumor when compared to that of control groups or any of the other treatments. The TIL-NPs are also expected to provide a robust systemic response, with significant portions of the untreated secondary tumors also showing a complete response and to a greater extent than the other treatments. Administration of TIL-NPs are expected to significantly prolong mice survival relative to controls. These results will further highlight the surprising and promising therapeutic potential, for use either in vitro and / or in vivo, of particles comprising a CD3 agonist (e.g., anti-CD3), a 4-1 BB agonist (e.g., anti-4-1 BB), and an IL-2R agonist (e.g., IL-2), relative to the combination of agonists delivered as soluble agents and / or one or more other control groups or other treatments. Example 5: In Vitro Functional Activation and Cytotoxic Activity of Human TILs Mediated by TIL-NPs

[0232] To evaluate the functional activation and cytotoxic capability of human tumor-infiltrating lymphocytes (TILs) mediated by the disclosed TIL-NPs, TILs were isolated from renal cell carcinoma (RCC) patient tumor tissue. Tumor tissue was mechanically dissociated and enzymatically digested to obtain a single-cell suspension, followed by lymphocyte enrichment using density gradient centrifugation.

[0233] Isolated human TILs were cultured in complete RPMI-1640 medium and divided into the following treatment groups: (i) TILs alone (untreated), (ii) TILs treated with free antibodies, comprising anti-CD3 antibody and anti-41 BB antibody, and interleukin-2 (IL-2), and (iii) TILs treated with TIL-NPs comprising a nanoengager (e.g., nanoparticle of the disclosure) formulation integrating anti-CD3, anti-41 BB, and IL-2.

[0234] After a defined activation period, treated or untreated TILs were co-cultured with human renal carcinoma target cells to assess TIL-mediated cytotoxicity. Human RCC cell lines 786-0 and ACHN were used as tumor targets. Tumor cells cultured in the absence of TILs served as target-only controls. Co-culture experiments were conducted at defined target-to-effector (T) ratios, including 1:5 and 1:10. Following incubation, tumor cell viability was quantified using a standard cell viability assay.302413766 - 48 -Attorney Docket No. UTSDP4503WO- 1001386307

[0235] The results showed that TILs treated with TIL-NPs exhibited markedly enhanced cytotoxic activity against renal carcinoma cells compared with all control groups. When cocultured with 786-0 or ACHN cells, TIL-NP-treated TILs significantly reduced tumor cell viability at both tested target-to-effector ratios (FIG. 5).

[0236] By contrast, untreated TILs demonstrated limited cytotoxic activity, while TILs treated with free antibodies induced only moderate tumor cell killing. Tumor cells cultured in the absence of TILs maintained high viability, confirming that tumor cell death was mediated by activated TILs rather than nonspecific effects.

[0237] These results demonstrated that nanoengager-mediated stimulation effectively induced functional activation of human TILs, resulting in potent cytotoxic responses against cancer cells, such as renal carcinoma cells.Example 6: In Vivo Antitumor Efficacy of TIL-NPs in a Humanized Renal Cell Carcinoma Mouse Model

[0238] To evaluate the in vivo antitumor efficacy of the disclosed TIL-NPs in a human immune context, a humanized renal cell carcinoma mouse model was established. Briefly, NOD-Prkdcscid / Il2r9’nul1(NSG) mice were engrafted with human peripheral blood mononuclear cells (PBMCs) to generate a functional human immune system (FIG. 6A).

[0239] After human immune reconstitution, human renal carcinoma cells were subcutaneously inoculated into both the right and left flanks of each mouse to establish bilateral tumors. When tumors reached a predefined volume, mice were randomized into treatment groups, including (i) PBS control, (ii) free antibodies comprising anti-CD3 antibody, anti-41 BB antibody, and interleukin-2 (IL-2), and (iii) TIL-NPs comprising a nanoengager (e.g., nanoparticle of the disclosure) formulation including anti-CD3, anti-41 BB, and IL-2.

[0240] Therapeutic agents were administered locally to the right flank tumor at defined time points. Tumor volumes were measured at regular intervals using calipers, and body weight was monitored throughout the study to assess systemic tolerability. At the experimental endpoint, tumor tissues were collected for histological and immunohistochemical analyses.

[0241] The results showed that administration of TIL-NPs resulted in potent antitumor activity in humanized mice bearing bilateral renal carcinoma tumors. Tumors treated with TIL-NPs exhibited pronounced growth inhibition and near-complete regression compared with tumors in the PBS control and free antibody groups (FIG. 6B). Notably, although treatment was administered only to the right flank tumor, significant suppression of tumor growth was also observed in the untreated contralateral tumor, indicating induction of a systemic antitumor302413766 - 49 -Attorney Docket No. UTSDP4503WO- 1001386307immune response (FIG.60). In contrast, mice treated with free antibodies showed only limited tumor growth inhibition, and control mice exhibited progressive tumor growth on both flanks.

[0242] Body weight remained relatively stable in TIL-NP-treated mice throughout the study, suggesting acceptable systemic tolerability. Immunohistochemical analysis further revealed increased infiltration of CD8+T cells in tumors from the TIL-NP-treated group (FIG. 7), consistent with enhanced cytotoxic T-cell-mediated antitumor immunity.302413766 - 50 -

Claims

Attorney Docket No. UTSDP4503WO- 1001386307CLAIMSWhat is claimed is:

1. A particle comprising:an agonist of cluster of differentiation 3 (CD3);an agonist of 4-1BB (CD137); andan agonist of interleukin 2 (IL-2) receptor (IL-2R);wherein the agonist of CD3, the agonist of 4-1BB, and the agonist of IL-2R are bound to a core of the particle.

2. The particle of claim 1 , wherein the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprises a ligand, an antibody, an active fragment of a ligand, and / or an active fragment of an antibody.

3. The particle of claim 2, wherein the agonist of CD3, the agonist of 4-1 BB, and / or the agonist of IL-2R comprises the antibody or the active fragment of an antibody.

4. The particle of claim 1 , wherein the hydrodynamic diameter of the particle is from 50 to 500 nm.

5. The particle of claim 1 , comprising at least a 5% greater concentration of agonist of 4-1 BB relative to the agonist of CD3, the agonist of IL-2R, or both the agonist of CD3 and the agonist of IL-2R.

6. The particle of claim 1, wherein there is or is about 30 to 100 pg of total combined agonist of CD3, agonist of 4-1 BB, and agonist of IL-2R per 1000 pg of particle.

7. The particle of claim 1 , wherein at least one of the agonists of CD3, agonist of 4-1 BB, and agonist of IL-2R are conjugated to the particle by click chemistry.

8. The particle of claim 1, wherein the particle comprises PEG-PLGA particle cores at least 50 kDa in mass.

9. The particle of claim 8, wherein the PEG-PLGA particle cores comprise a 1:10 to 10:1 wt / wt mixture of N3-PEG(5K)-PLGA (30K) and methoxy-PEG(2K)-PLGA (20K).

10. The particle of claim 1, wherein the particle is not a lipid nanoparticle.

11. A composition comprising a population of particles of claim 1.

12. A kit comprising the composition of claim 11.302413766 - 51 -Attorney Docket No. UTSDP4503WO- 100138630713. Use of the composition of claim 11 in the production of a medicament for activating and / or inducing a tumor infiltrating lymphocyte (TIL) cell response.

14. Use of the composition of claim 11 in the production of a medicament for treatment of cancer.

15. A method of activating a TIL cell, comprising contacting the TIL cell with the particle of claim 1.

16. A method of inducing a TIL cell immune response, comprising contacting the TIL with the particle of claim 1.

17. A method of inducing a TIL cell immune response in a subject in need thereof, comprising administering to the subject an effective amount of the particle of claim 1.

18. A method of activating TIL cells in a subject in need thereof, comprising administering to the subject an effective amount of the particle of claim 1.

19. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the particle of claim 1.

20. A method of making the particle of claim 1, the method comprising combining purified functionalized PEG-PLGA particle cores at least 50 kDa in mass with functionalized agonist of CD3, functionalized agonist of 4-1 BB, and functionalized agonist of IL-2R.302413766 - 52 -

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