Particles with tunable elasticity for cellular engineering and serum profiling

Abstract

Disclosed are polymeric nanoparticles and microparticles having a tunable elastic modulus and having one or more biological proteins conjugated to a surface thereof and their use in immunotherapies for treating diseases, such as cancer or infectious diseases.

Description

PARTICLES WITH TUNABLE ELASTICITY FOR CELLULAR ENGINEERING AND SERUM PROFILING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 695,524 filed September 17, 2024, which is incorporated herein by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant EB028239 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Adoptive cell therapy (ACT) is an immunotherapy strategy for cancer that has seen widespread clinical success. During ACT, patient-derived lymphocytes are stimulated with the antigen of interest ex vivo, proliferated, then returned to the patient to initiate an antigen-specific antitumor response. While effective, this process is resource-intensive and logistically impossible for many patients. Particulate artificial antigen presenting cells (aAPCs) offer a potential “off-the- shelf” alternative to ex vivo ACT. While particulate aAPCs perform well in vitro, they have had limited success in vivo due to poor bioavailability after injection. Barriers to bioavailability of aAPCs include rapid clearance, unfavorable biodistribution, and inadequate interactions with CD8+ T cells at sites of interest. Thus, there is a need for aAPCs having improved bioavailability and other improved characteristics for use in immunotherapies for treating diseases, such as cancer or infectious diseases. SUMMARY

[0004] In some aspects, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) having an immune targeting Signal 1 protein and a costimulatory Signal 2 protein conjugated to a surface thereof, wherein the aAPC comprises a hydrogel having a tunable elasticity.

[0005] In certain aspects, the hydrogel has a stiffness ranging from about 50 kPa to about 5,000 kPa. In certain aspects, the aAPC has a particle size having a range selected from about 100 nm to 4000 nm, 500 nm to 3000 nm, and 1000 nm to 2000 nm.1 43105.601_P18311-02

[0006] In certain aspects, the hydrogel comprises one or more of poly(ethylene) glycol (PEG), a PEG-derivate, alginate, chitosan, collagen, gelatin, hyaluronic acid, polyacrylamide, polycaprolactone, polyester, or polyvinyl alcohol to form the hydrogel. In particular aspects, the hydrogel comprises a PEG hydrogel. In more particular aspects, the hydrogel has a volume fraction of PEG ranging from about 0.15 to about 0.5.

[0007] In particular aspects, the Signal 1 protein is selected from a major histocompatibility complex (MHC) + antigen peptide and an anti-CD3 antibody. In certain aspects, the costimulatory Signal 2 protein is selected from an antibody or antigen-binding fragment thereof that specifically binds to CD28, CD80 (B7-1), CD86 (B7-2), B7- H3, 4-1BBL, 4-1BB, CD27, CD30, CD134 (OX- 40L), B7h (B7RP-1), CD40, LIGHT, an antibody or antigen-binding fragment thereof that specifically binds to HVEM, an antibody or antigen-binding fragment thereof that specifically binds to CD40L, an antibody or antigen binding fragment thereof that specifically binds to OX40, and an antibody or antigen-binding fragment thereof that specifically binds to 4-lBB. In particular aspects, the costimulatory Signal 2 protein comprises an anti-CD28 (αCD28) antibody.

[0008] In certain aspects, the aAPC further comprises one or more proteins or small molecules having a binding affinity for one or more blood serum proteins. In certain aspects, the aAPC further comprises an immunomodulatory material. In particular aspects, the immunomodulatory material comprises a genetic material.

[0009] In certain aspects, the aAPC further comprises a paramagnetic particle. In particular aspects, the paramagnetic particle comprises an iron-dextran particle.

[0010] In certain aspects, the aAPC further comprises a shape selected from a sphere, a prolate ellipsoid, a tri-axial ellipsoid, and an oblate ellipsoid.

[0011] In other aspects, the presently disclosed subject matter provides an in vitro method for identifying, isolating, or detecting one or more antigen-specific T cells, the method comprising:

[0012] (a) contacting a plurality of unpurified immune cells comprising one or more antigen- specific T cells with a plurality of aAPCs disclosed herein;

[0013] (b) separating antigen-specific T cells associated with the plurality of aAPCs from cells not associated with the plurality of aAPCs;

[0014] (c) recovering antigen-specific T cells associated with the plurality of aAPCs; and

[0015] (d) expanding the recovered antigen-specific T cells in culture for a period of time to provide a composition comprising antigen-specific T cells.2 43105.601_P18311-02

[0016] In certain aspects, the plurality of unpurified immune cells comprising one or more antigen- specific T cells are obtained from a sample comprising one or more of a peripheral blood mononuclear cell (PBMC) sample, memory T cells, naive T cells, previously activated T cells, and tumor infiltrating lymphocytes.

[0017] In certain aspects, the plurality of unpurified immune cells comprising one or more antigen-specific T cells are obtained from a sample comprising one or more of bone marrow, lymph node tissue, spleen tissue, and a tumor.

[0018] In certain aspects, the plurality of unpurified immune cells are obtained from a patient or a donor. In particular aspects, the donor comprises a donor who is HLA-matched to an adoptive transfer recipient. In particular aspects, the plurality of unpurified immune cells are obtained from a patient and the patient has one or more diseases, disorders, or conditions selected from the group consisting of a cancer, an infectious disease, and an autoimmune disease.

[0019] In certain aspects, the one or more antigen-specific T cells are selected from the group consisting of cytotoxic CD4+T cells, CD4+helper T cells, CD8+cytotoxic T lymphocytes, T- helper 17 (Th17) cells, regulatory T cells (Tregs), and combinations thereof.

[0020] In certain aspects, the aAPC comprises a paramagnetic particle and the separating of the antigen-specific T cells associated with the plurality of aAPCs from the cells not associated with the plurality of aAPCs is by magnetic separation.

[0021] In other aspects, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition, the method comprising administering to a subject in need of treatment thereof a composition comprising one or more aAPCs as disclosed herein in vivo or one or more antigen-specific T cells prepared by the methods disclosed herein in vitro.

[0022] In certain aspects, the disease, disorder, or condition is selected from a cancer, an infectious disease, and an autoimmune disease.

[0023] In certain aspects, the disease, disorder, or condition is a cancer and the one or more antigen-specific T cells comprise cytotoxic T cells specific for one or more tumor-associated peptide antigens to the subject in need of treatment thereof. In particular aspects, the cancer comprises a solid tumor or a hematological malignancy. In more particular aspects, the cancer is selected from the group consisting of a melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and3 43105.601_P18311-02neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, a neuroblastoma, and a glioma.

[0024] In certain aspects, the infectious disease comprises an infectious disease caused by one or more pathogens selected from bacteria, viruses, prions, fungi, parasites, helminths, and combinations thereof. In particular aspects, the infectious disease is selected from acquired immunodeficiency syndrome (AIDS), hepatitis, a cytomegalovirus (CMV) infection, and post- transplant lymphoproliferative disorder (PTLD).

[0025] In certain aspects, the disease, disorder, or condition is an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture's syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, and Hashimoto's thyroiditis.

[0026] In other aspects, the presently disclosed subject matter provides a method for diagnosing a disease or a disease state in a subject, the method comprising:

[0027] (a) obtaining a blood serum sample from the subject;

[0028] (b) contacting the blood serum sample with a plurality of aAPCs disclosed herein, wherein the plurality of aAPCs includes one or more aAPCs which can be the same or different and / or a plurality of non-APC particles alone or in combination with the plurality of aAPCs;

[0029] (c) separating one or more blood serum proteins associated with the plurality of aAPCs from blood serum proteins not associated with the plurality of aAPCs; and

[0030] (d) determining an identity and / or an amount of the one or more blood serum proteins associated with the plurality of aAPCs to diagnose a disease or disease state in the subject.

[0031] In certain aspects, the plurality of non-APC particles includes one or more non-APC particles having a same or different elastic modulus. In certain aspects, the plurality of non-APC particles includes one or more non-APC particles that bind differentially to blood serum protein.

[0032] In other aspects, the presently disclosed subject matter provides a non-viral method for delivering an immunomodulatory material to a T cell, the method comprising administering an aAPC having an immunomodulatory material conjugated thereto to a cell.

[0033] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects4 43105.601_P18311-02will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES

[0034] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0035] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0036] FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e, FIG. 1f, and FIG. 1g show the synthesis and characterization of PEG aAPCs with tunable elasticity. FIG. 1a) Synthesis schematic. FIG. 1b) transmission electron microscopy (TEM) images of particles. FIG. 1c) Bulk polyethylene glycol diacrylate (PEGDA) gel elasticity measured by rheometry. FIG. 1d) Conjugation results for total protein conjugation for each particle group. FIG. 1e) Particle size stability measured via dynamic light scattering (DLS) before and after conjugation, where T0 = before conjugation and T1 = after conjugation. ns = no significance determined by t-test with α = 0.05. FIG. 1f) Conjugation schematic. FIG. 1g) Physicochemical characterization of particles. PDI = polydispersity index.

[0037] FIG. 2a, FIG. 2b, FIG. 2c, and FIG. 2d show antigen specific in vitro T-cell stimulation with soft and hard aAPCs. FIG. 2a) conjugation results. FIG. 2b) CD8+ T-cell proliferation after 7 days measured by number of cells. “+” indicates a positive control of commercial beads containing anti-CD3 and anti-CD28 and “-” indicates a negative control of no stimulation. FIG. 2c) CD8+ T-cell proliferation by day 3 measured by CellTrace™ Violet (CTV) dilution. FIG. 2d) generation breakdown data from CTV plots after 3 days of incubation. Significance in FIG. 2a) and FIG. 2b) determined by One Way Anova and Two Way Anova, respectively, with Tukey’s multiple corrections. Asterisks are used to denote various levels of significance: ns = no significance; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; and **** indicates p<0.0001.

[0038] FIG.3a, FIG.3b, FIG.3c, FIG.3d, FIG.3e, FIG.3f, FIG.3g, and FIG.3h demonstrate the effects of particle aAPC elasticity on T-cell phenotype and functionality. D7 phenotyping of FIG. 3a) naïve T cells, FIG. 3b) memory T cells, and FIG. 3c) effector T cells. Mean fluorescence intensity of intracellular cytokine staining of FIG. 3d) CD107, FIG. 3e) IFN-γ, FIG. 3f) TNF-α,5 43105.601_P18311-02and FIG. 3g) IL-2 on D7. FIG. 3h) Cytokine polyfunctionality of cells after 7 days of aAPC stimulation. Significance in FIG. 3a-FIG. 3c determined by Two Way Anova with Tukey’s multiple corrections. Significance in FIG. 3d-FIG. 3g determined by One Way Anova with Tukey’s multiple corrections. Asterisks are used to denote various levels of significance: ns = no significance; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; and **** indicates p<0.0001.

[0039] FIG.4a, FIG.4b, FIG.4c, FIG.4d, FIG.4e, FIG.4f, and FIG.4g show that aAPC elasticity drives in vivo clearance, biodistribution, and in vitro cellular uptake and binding. FIG.4a) Specific T cell binding. FIG.4b) Nonspecific T cell binding. FIG.4c) In vivo clearance profiles of particles injected intravenous (IV). FIG. 4d) Half-life of IV-administered particles. FIG. 4e) Organ biodistribution 24 hours after IV administration of particles. FIG. 4f) Uptake of particles by macrophages. FIG. 4g) Nonspecific macrophage binding by particles. Significance in FIG. 4d) determined by One Way Anova with Tukey’s multiple corrections. Significance in FIG. 4a, FIG. 4b, FIG. 4e, FIG. 4f, and FIG. 4g) determined by Two Way Anova with Tukey’s multiple corrections. Asterisks are used to denote various levels of significance: ns = no significance; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; and **** indicates p<0.0001.

[0040] FIG.5a, FIG.5b, FIG.5c, FIG.5d, and FIG.5e demonstrate that aAPC elasticity modulates magnitude and composition of the protein corona. FIG. 5a) Clustered heatmap of missing (white) and valid (black) values of all proteins detected on the corona of each sample. FIG. 5b) Relative abundance of the six most significantly differently expressed proteins in a comparison of 50 kPa to 5000 kPa as measured by FragPipe Analyst software (p < 0.01). FIG.5c) Volcano plot showing the significantly upregulated proteins (toward 5000 kPa) or downregulated (toward 50 kPa) in the protein corona. Statistically significant protein (adjusted p < 0.1). Nonsignificant proteins are unlabeled and shown in grey. Pathway enrichment analysis showing pathways significantly FIG. 5d) upregulated (toward 5000 kPa) or FIG.5e) downregulated (toward 50 kPa) using the Hallmark database. Size is proportional to the number of genes included in the pathway.

[0041] FIG. 6a, FIG. 6b, FIG. 6c, FIG. 6d, FIG. 6e, FIG. 6f, FIG. 6g, FIG. 6h, FIG. 6i, and FIG. 6j demonstrate that aAPC elasticity influences in vivo T cell stimulation in an antigen-specific model of adoptive T cell transfer. FIG. 6a) Experimental schematic. FIG. 6b) % CD8+ T cells, FIG. 6c) % Thy1.1 CD8+ T cells, and FIG. 6d) CTV MFI in blood on day 3. FIG. 6e) CTV intensity, FIG. 6f) % CD8+ T cells, and FIG. 6g) % Thy1.1+ T cells in spleen on Day 7. FIG. 6h)6 43105.601_P18311-02CTV histogram, FIG. 6i) Thy1.1+ T cells, and FIG. 6j) CTV intensity in the lymph nodes on Day 7. Significance determined by One Way Anova with Tukey’s multiple corrections. Asterisks are used to denote various levels of significance: ns = no significance; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; and **** indicates p<0.0001.

[0042] FIG. 7A, FIG. 7B, and FIG. 7C show in vitro nonspecific T cell stimulation with soft and stiff aAPCs: FIG. 7A) Signal 1 (anti-CD3) and signal 2 (anti-CD28) conjugation to PEG aAPCs. FIG. 7B) Day 7 / Day 1 Fold proliferation of antigen nonspecific CD8+ T cells. FIG. 7C) CTV proliferation peaks for 0.1x dose PEG aAPCs.

[0043] FIG. 8 is a representative gating schematic for phenotype analysis.

[0044] FIG. 9 is a representative gating schematic for intracellular cytokine analysis.

[0045] FIG. 10 shows principal component analysis of protein corona groups and replicates.

[0046] FIG. 11 shows proteomics volcano plots of 50 kPa vs. 850 kPa and 850 kPa vs. 5000 kPa comparisons.

[0047] FIG. 12A, FIG. 12B, and FIG. 12C show a pathway enrichment analysis for FIG. 12A- FIG.12B) 5000 kPa vs.850 kPa and FIG.12C-FIG.12D) 50 kPa vs.850 kPa. FIG.12A) and FIG. 12C) represent upregulated pathways, and FIG. 12B) and FIG. 12D) represent downregulated pathways.

[0048] FIG. 13 is a heatmap of genes involved in the coagulation pathway enrichment analysis from the Hallmark database.

[0049] FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, and FIG. 14F show gene ontology analysis for (FIG. 14A-FIG. 14B) 5000 kPa vs. 50 kPa, (FIG. 14C-FIG. 14D) 5000 kPa vs. 850 kPa and (FIG. 14E-FIG. 14F) 50 kPa vs. 850 kPa. FIG. 14A, FIG. 14C, and FIG. 14E represent upregulated gene groups, and FIG. 14B, FIG. 14D, and FIG. 14F represent downregulated genes groups.

[0050] FIG. 15 is a representative gating schematic for in vivo T cell proliferation.

[0051] FIG. 16 shows representative organ images for biodistribution studies.

[0052] FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, and FIG. 17E show the synthesis and characterization of PEG nano particle aAPCs with tunable stiffness, size, and shape. FIG. 17A) Schematic describing the PEG ellipsoidal nanoparticle synthesis process. FIG. 17B) TEM images of spherical PEG nanoparticles. FIG. 17C) TEM images of ellipsoidal PEG nanoparticles. FIG.7 43105.601_P18311-0217D) Size and polydispersity (PDI) profiles of spherical PEG nanoparticles as measured by DLS. FIG. 17E) Shape descriptors of ellipsoidal PEG nanoparticles as measured by NIH ImageJ.

[0053] FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D demonstrate that spherical aAPC particle size and elasticity affect T cell proliferation. FIG. 18A) Spherical PEG microparticle T cell proliferation on Day 7 of stimulation. FIG. 18B) Spherical PEG nanoparticle T cell proliferation on Day 7 of stimulation. FIG. 18C) Protein conjugation efficiency on spherical microparticles. FIG. 18D) Protein conjugation efficiency on spherical nanoparticles.

[0054] FIG. 19A, FIG. 19B, FIG. 19C, and FIG. 19D demonstrate that spherical aAPC particle size and elasticity affect particle clearance in vivo. Spherical PEG nanoparticle blood FIG. 19A) clearance curves and FIG. 19C) Half-life as a function of elasticity. Spherical PEG microparticle blood FIG. 19B) Clearance curves and FIG. 19D) Half-life.

[0055] FIG.20A, FIG.20B, FIG.20C, FIG.20D, FIG.20E, and FIG.20F show nanoparticle PEG aAPC shape and elasticity influence proliferation of Ag-specific T cells. FIG. 20A) Spherical and FIG. 20B) ellipsoidal PEG nanoparticle aAPC day 3 CTV proliferation results as a function of dose and elasticity in an antigen-specific T cell stimulation assay. FIG. 20C) Spherical and FIG. 20D) ellipsoidal conjugation efficiencies of signal 1 (Dbgp100) and signal 2 (anti-CD28). FIG. 20E) Spherical and FIG. 20F) ellipsoidal PEG nanoparticle Day 7 fold proliferation of CD8+ T cells in an antigen-specific T cell stimulation assay.

[0056] FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D demonstrate that nanoparticle PEG aAPC shape and elasticity influence T cell phenotype and functionality. FIG. 21A) Spherical and FIG. 21B) ellipsoidal PEG nanoparticle aAPC day 7 T cell phenotype as a function of elasticity in an antigen-specific T cell stimulation assay. FIG. 21C) Spherical and FIG. 21D) ellipsoidal PEG nanoparticle aAPC day 7 T cell functionality as a function of elasticity in an antigen-specific T cell stimulation assay. (** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001).

[0057] FIG. 22A, FIG. 22B, FIG. 22C, and FIG. 22D demonstrate that nanoparticle PEG aAPC shape and elasticity influence macrophage uptake. Comparison of macrophage uptake FIG. 22A) and binding FIG. 22B) of 850 kPa PEG spherical and ellipsoidal microparticles. Macrophage uptake FIG. 22C) and binding FIG. 22D) of ellipsoidal PEG nanoparticles as a function of dose and elasticity.8 43105.601_P18311-02DETAILED DESCRIPTION

[0058] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0059] A. Artificial Antigen Presenting Cells Having a Tunable Elasticity

[0060] In some embodiments, the presently disclosed subject matter provides an artificial antigen presenting cell (aAPC) having an immune targeting Signal 1 protein and a costimulatory Signal 2 protein conjugated to a surface thereof, wherein the aAPC comprises a hydrogel having a tunable elasticity.

[0061] As used herein, an “artificial antigen presenting cell” (aAPC) is an artificial biomimetic particle-based platform that has been made in vitro and has not been made naturally by a body. An “antigen presenting cell” is a cell that comprises a molecule that is capable of binding to the T cell receptor (TCR) on a T cell and has other factors which direct the T cell response. As used herein, the term “capable of” refers to having the capacity or ability, for example, a molecule that is “capable of” binding to a receptor is a molecule that has a three-dimensional structure having the capacity or ability to interact and / or bind with a receptor.

[0062] In certain embodiments, the hydrogel has a stiffness ranging from about 50 kPa to about 5,000 kPa, including about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, and 5000 kPa.

[0063] Further, the presently disclosed aAPCs have at least one dimension in the range of about 1 nm to about 2,500 nm, including any integer value between 1 nm and 2,500 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, and 2500 nm and all integers and fractional integers in between). In more particular embodiments, the aAPC has a9 43105.601_P18311-02particle size or at least one dimension ranging from about 100 nm to 4000 nm, 500 nm to 3000 nm, and 1000 nm to 2000 nm, including 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, and 4000 nm. In such embodiments, the aAPC can be referred to as a “microparticle”. Thus, the term “microparticle” includes particles having at least one dimension in the range of about one micrometer (µm), i.e., 1 × 10-6meters, to about 1000 µm.

[0064] In certain embodiments, the hydrogel comprises or is composed of one or more of poly(ethylene) glycol (PEG), a PEG-derivate, alginate, chitosan, collagen, gelatin, hyaluronic acid, polyacrylamide, polycaprolactone, polyester, or polyvinyl alcohol to form the hydrogel. In particular embodiments, the hydrogel comprises a poly(ethylene) glycol (PEG) hydrogel. In more particular embodiments, the hydrogel has a volume fraction of PEG ranging from about 0.15 to about 0.5, including a 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 volume fraction of PEG.

[0065] In certain embodiments, the aAPCs further comprise at least two ligands, an antigen presenting complex (e.g., a major histocompatibility complex (MHC), including a peptide-MHC), referred to herein as “Signal 1,” and a costimulatory ligand, e.g., a lymphocyte activating ligand, referred to herein as “Signal 2,” bound to a surface thereof.

[0066] Antigen presenting complexes comprise an antigen binding cleft, which harbors an antigen for presentation to a T cell or T cell precursor. Antigen presenting complexes can be, for example, MHC class I or class II molecules, and can be linked or tethered to provide dimeric or multimeric MHC. In some embodiments, the MHC are monomeric, but their close association on the paramagnetic particle is sufficient for avidity and activation. In some embodiments, the MHC are dimeric. Dimeric MHC class I constructs can be constructed by fusion to immunoglobulin heavy chain sequences, which are then associated through one or more disulfide bonds (and with associated light chains). In some embodiments, the Signal 1 complex is a non-classical MHC-like molecule, such as member of the CD1 family (e.g., CD1a, CD1b, CD1c, CD1d, and CD1e). MHC multimers can be created by direct tethering through peptide or chemical linkers, or can be multimeric via association with streptavidin through biotin moieties. In some embodiments, the antigen presenting complexes are MHC class I or MHC class II molecular complexes involving fusions with immunoglobulin sequences, which are extremely stable and easy to produce, based on the stability and secretion efficiency provided by the immunoglobulin backbone.10 43105.601_P18311-02

[0067] MHC class I molecular complexes having immunoglobulin sequences are described in U.S. Pat. No. 6,268,411, which is hereby incorporated by reference in its entirety. These MIIC class I molecular complexes may be formed in a conformationally intact fashion at the ends of immunoglobulin heavy chains. MHC class I molecular complexes to which antigenic peptides are bound can stably bind to antigen-specific lymphocyte receptors (e.g., T cell receptors). In various embodiments, the immunoglobulin heavy chain sequence is not full length, but comprises an Ig hinge region, and one or more of CH1, CH2, and / or CH3 domains. The Ig sequence may or may not comprise a variable region, but where variable region sequences are present, the variable region may be full or partial. The complex may further comprise immunoglobulin light chains.

[0068] Exemplary MHC class I molecular complexes comprise at least two fusion proteins. A first fusion protein comprises a first MHC class I α chain and a first immunoglobulin heavy chain (or portion thereof comprising the hinge region), and a second fusion protein comprises a second MHC class I α chain and a second immunoglobulin heavy chain (or portion thereof comprising the hinge region). The first and second immunoglobulin heavy chains associate to form the MHC class I molecular complex, which comprises two MHC class I peptide-binding clefts. The immunoglobulin heavy chain can be the heavy chain of an IgM, IgD, IgG1, IgG3, IgG2β, IgG2α, IgG4, IgE, or IgA. In some embodiments, an IgG heavy chain is used to form MHC class I molecular complexes. If multivalent MHC class I molecular complexes are desired, IgM or IgA heavy chains can be used to provide pentavalent or tetravalent molecules, respectively.

[0069] Exemplary class I molecules include HLA-A, HLA-B, HLA-C, HLA-E, and these may be employed individually or in any combination. In some embodiments, the antigen presenting complex is an HLA-A2 ligand.

[0070] Exemplary MHC class II molecular complexes are described in U.S. Pat. No. 6,458,354, U.S. Pat. No. 6,015,884, U.S. Pat. No. 6,140,113, and U.S. Pat. No. 6,448,071, which are hereby incorporated by reference in their entireties. MHC class II molecular complexes comprise at least four fusion proteins. Two first fusion proteins comprise (i) an immunoglobulin heavy chain (or portion thereof comprising the hinge region) and (ii) an extracellular domain of an MHC class IIβ chain. Two second fusion proteins comprise (i) an immunoglobulin κ or λ light chain (or portion thereof) and (ii) an extracellular domain of an MHC class IIα chain. The two first and the two second fusion proteins associate to form the MHC class II molecular complex. The extracellular11 43105.601_P18311-02domain of the MHC class IIβ chain of each first fusion protein and the extracellular domain of the MHC class IIα chain of each second fusion protein form an MHC class II peptide binding cleft.

[0071] The immunoglobulin heavy chain can be the heavy chain of an IgM, IgD, IgG3, IgG1, IgG2β, IgG2α, IgG4, IgE, or IgA. In some embodiments, an IgG1 heavy chain is used to form divalent molecular complexes comprising two antigen binding clefts. Optionally, a variable region of the heavy chain can be included. IgM or IgA heavy chains can be used to provide pentavalent or tetravalent molecular complexes, respectively.

[0072] Fusion proteins of an MHC class II molecular complex can comprise a peptide linker inserted between an immunoglobulin chain and an extracellular domain of an MHC class II polypeptide. The length of the linker sequence can vary, depending upon the flexibility required to regulate the degree of antigen binding and receptor cross linking. Immunoglobulin sequences in some embodiments are humanized monoclonal antibody sequences.

[0073] The presently disclosed aAPCs also can have a costimulatory molecule bound thereto. Such costimulatory molecules can be referred to herein as a “Signal 2” and are generally a T cell affecting molecule, that is, a molecule that has a biological effect on a precursor T cell or on an antigen-specific T cell. Such biological effects include, for example, differentiation of a precursor T cell into a CTL, helper T cell (e.g., Th1, Th2), or regulatory T cell; and / or proliferation of T cells. Thus, T cell affecting molecules include T cell costimulatory molecules, adhesion molecules, T cell growth factors, and regulatory T cell inducer molecules. In some embodiments, an aAPC comprises at least one such ligand; optionally, an aAPC comprises at least two, three, or four such ligands.

[0074] In certain embodiments, Signal 2 is a T cell costimulatory molecule. T cell costimulatory molecules contribute to the activation of antigen-specific T cells. Such molecules include, but are not limited to, molecules that specifically bind to CD28 (including antibodies), CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, antibodies that specifically bind to OX40, and antibodies that specifically bind to 4-1BB. In some embodiments, the costimulatory molecule (signal 2) is an antibody (e.g., a monoclonal antibody) or portion thereof, such as F(ab')2, Fab, scFv, or single chain antibody, or other antigen binding fragment. In some embodiments, the antibody is a humanized monoclonal antibody or portion12 43105.601_P18311-02thereof having antigen-binding activity, or is a fully human antibody or portion thereof having antigen-binding activity.

[0075] Adhesion molecules can be used to mediate adhesion of the aAPC to a T cell or to a T cell precursor. Representative adhesion molecules include, for example, ICAM-1 and LFA-3.

[0076] In some embodiments, Signal 1 is provided by peptide-HLA-A2 complexes, and Signal 2 is provided by B7.1-Ig or anti-CD28. An exemplary anti-CD28 monoclonal antibody is 9.3 mAb (Tan et al., J. Exp. Med. 1993177:165), which may be humanized in certain embodiments and / or conjugated to the bead as a fully intact antibody or an antigen-binding fragment thereof.

[0077] Some embodiments employ T cell growth factors, which affect proliferation and / or differentiation of T cells. Examples of T cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens. If desired, cytokines can be present in molecular complexes comprising fusion proteins, or can be encapsulated by the aAPC. Particularly useful cytokines include IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21 gamma interferon, and CXCL10. Optionally, cytokines are provided solely by media components during expansion steps.

[0078] In certain embodiments, the aAPC further comprises one or more proteins or small molecules having a binding affinity for one or more blood serum proteins. As used herein, the term “molecule” generally refers to two or more atoms held together by covalent bonds. Therefore, a molecule can be relatively small, such as the size of a peptide, or it can be relatively big, such as the size of a protein comprising several polypeptides. As used herein, a molecule is not restricted by size.

[0079] Molecules can be directly attached to particles by adsorption or by direct chemical bonding, including covalent bonding. See, Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, New York, 1996. A molecule itself can be directly activated with a variety of chemical functionalities, including nucleophilic groups, leaving groups, or electrophilic groups. Activating functional groups include alkyl and acyl halides, amines, sulfhydryls, aldehydes, unsaturated bonds, hydrazides, isocyanates, isothiocyanates, ketones, and other groups known to activate for chemical bonding. Alternatively, a molecule can be bound to a particle through the use of a small molecule-coupling reagent. Non-limiting examples of coupling reagents include carbodiimides, maleimides, n-hydroxysuccinimide esters, bischloroethylamines, bifunctional aldehydes such as glutaraldehyde, anhydrides and the like. In other embodiments, a molecule can be coupled to a particle through affinity binding such as a biotin-streptavidin linkage or coupling, as is known in13 43105.601_P18311-02the art. For example, streptavidin can be bound to a particle by covalent or non-covalent attachment, and a biotinylated molecule can be synthesized using methods that are well known in the art.

[0080] If covalent binding to a particle is contemplated, the support can be coated with a polymer that contains one or more chemical moieties or functional groups that are available for covalent attachment to a suitable reactant, typically through a linker. For example, amino acid polymers can have groups, such as the s-amino group of lysine, available to couple a molecule covalently via appropriate linkers. This disclosure also contemplates placing a second coating on a particle to provide for these functional groups.

[0081] Activation chemistries can be used to allow the specific, stable attachment of molecules to the surface of particles. There are numerous methods that can be used to attach proteins to functional groups. For example, the common cross-linker glutaraldehyde can be used to attach protein amine groups to an aminated particle surface in a two-step process. The resultant linkage is hydrolytically stable. Other methods include use of cross-linkers containing n-hydrosuccinimido (NHS) esters which react with amines on proteins, cross-linkers containing active halogens that react with amine-, sulfhydryl-, or histidine-containing proteins, cross-linkers containing epoxides that react with amines or sulfhydryl groups, conjugation between maleimide groups and sulfhydryl groups, and the formation of protein aldehyde groups by periodate oxidation of pendant sugar moieties followed by reductive amination.

[0082] In some embodiments, the aAPCs have magnetic properties. Representative methods for preparing aAPCs, including magnetic aAPCs, are provided in International PCT Patent Application Publication No. WO2021262846 for Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T Cells, to Schneck et al., published December 3, 2021, which is incorporated herein in its entirety. In certain embodiments, the aAPC further comprises a paramagnetic particle. Paramagnetic materials have a small, positive susceptibility to magnetic fields. These materials are attracted by a magnetic field and the material does not retain the magnetic properties when the external field is removed. Exemplary paramagnetic materials include, without limitation, magnesium, molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beads suitable for magnetic enrichment are commercially available (e.g., DYNABEADS®, MACS MICROBEADS™, Miltenyi Biotec, and the like). In some14 43105.601_P18311-02embodiments, the aAPC particle comprises an iron dextran bead (e.g., a dextran-coated iron-oxide bead).

[0083] In certain embodiments, the aAPC has a spherical shape. In other embodiments, the aAPC has a non-spherical or an asymmetrical shape. Representative asymmetric artificial antigen presented cells are disclosed in International PCT Patent Application Publication No. WO2013086500 for Artificial Antigen Presenting Cells Having a Defined and Dynamic Shape, to Green et al., published June 13, 2013, which is incorporated herein by reference in its entirety.

[0084] For example, in some embodiments, the presently disclosed non-spherical or asymmetric aAPCs include: a three-dimensional microparticle or nanoparticle having an asymmetrical shape, wherein the asymmetrical shape has at least one surface having a radius of curvature along at least one axis which is in one of the following ranges: (a) 1 nm – 10 nm; (b) 11 nm - 100 nm; (b) 101 nm - 400 nm; (c) 401 nm – 1µm; (d) 10µm - 20µm; (e) 20 µm – 100 µm (f) 101 µm - 1mm. In some embodiments, the non-spherical or asymmetric aAPCs include: a three-dimensional microparticle or nanoparticle having an asymmetrical shape defined by a dimension (a) along an x-axis, a dimension (b) along a y-axis, and a dimension (c) along a z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other dimension (a), (b), or (c).

[0085] As the particle becomes flatter, the radius of curvature becomes larger. Conversely, as a surface on the particle becomes more curved, the radius of curvature becomes smaller. In some embodiments, the particle has at least one surface that has a radius of curvature that does not include the range from about 1 micron to about 10 microns.

[0086] In some embodiments, the non-spherical or asymmetric shape comprises a prolate ellipsoid, which is defined by the equation a > b = c. In other embodiments, the non-spherical shape comprises a tri-axial ellipsoid, which can be described by the equation a > b > c. In yet other embodiments, the non-spherical shape comprises an oblate ellipsoid, which can be described by the equation a = b > c. In other embodiments, the non-spherical shape has a dimension (a) along the x axis is equal to the dimension (b) along the y axis, both of which are much less than dimension (c) along the z-axis, such that a = b <<c and the three-dimensional particle comprises a rod.

[0087] In certain embodiments, the aAPC has an aspect ratio ranging from about 1.1 to about 5. In other embodiments, the aspect ratio has a range from about 5 to about 10. In some embodiments, the aspect ratio has a range from about 1.5 to about 3.5, including 1.5, 2, 2.5, 3, and 3.5.15 43105.601_P18311-02

[0088] B. In Vitro Method for Identifying, Isolating, or Detecting One or More Antigen- Specific T Cells

[0089] In other embodiments, the presently disclosed subject matter provides an in vitro method for identifying, isolating, or detecting one or more antigen-specific T cells, the method comprising:

[0090] (a) contacting a plurality of unpurified immune cells comprising one or more antigen- specific T cells with a plurality of aAPCs disclosed therein;

[0091] (b) separating antigen-specific T cells associated with the plurality of aAPCs from cells not associated with the plurality of aAPCs;

[0092] (c) recovering antigen-specific T cells associated with the plurality of aAPCs; and

[0093] (d) expanding the recovered antigen-specific T cells in culture for a period of time to provide a composition comprising antigen-specific T cells.

[0094] Accordingly, in some embodiments, the presently disclosed methods involve enrichment and expansion of antigen-specific T cells, including, but not limited to, cytotoxic T lymphocytes (CTLs), helper T cells, and regulatory T cells. In some embodiments, the presently disclosed methods involve enrichment and expansion of antigen-specific CTLs.

[0095] Precursor T cells can be obtained from a patient or from a suitable HLA-matched donor. Precursor T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, tumors, and combinations thereof. In some embodiments, the T cells are obtained from a PBMC sample from a patient. In some embodiments, the PBMC sample is used to isolate the T cell population of interest, such as CD8+, CD4+ or regulatory T cells. In some embodiments, precursor T cells are obtained from a unit of blood collected from a patient or a donor using any number of techniques known to the skilled artisan, such as Ficoll separation. For example, precursor T cells from the circulating blood of a patient or a donor can be obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells and precursor T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Leukapheresis is a laboratory procedure in which white blood cells are separated from a sample of blood.

[0096] Cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. Washing steps can be accomplished by methods known to those in the art, such as by using a semi-automated “flow-16 43105.601_P18311-02through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells directly re-suspended in a culture medium.

[0097] If desired, precursor T cells can be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. In certain embodiments, the sample from which the T cells are obtained can be used without any isolation or preparatory steps.

[0098] If desired, subpopulations of T cells can be separated from other cells that may be present. For example, specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. Other enrichment techniques include cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry, e.g., using a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.

[0099] In certain embodiments, leukocytes are collected by leukapheresis, and are subsequently enriched for CD8+ T cells using known processes, such as magnetic enrichment columns that are commercially available. The CD8-enriched cells are then enriched for antigen-specific T cells using magnetic enrichment with the aAPC reagent. In various embodiments, at least about 105, or at least about 106, or at least about 107CD8-enriched cells are isolated for antigen-specific T cell enrichment.

[0100] More particularly, in certain embodiments, the plurality of unpurified immune cells comprising one or more antigen-specific T cells are obtained from a sample comprising one or more of a peripheral blood mononuclear cell (PBMC) sample, memory T cells, naive T cells, previously activated T cells, and tumor infiltrating lymphocytes.

[0101] In certain embodiments, the plurality of unpurified immune cells comprising one or more antigen-specific T cells are obtained from a sample comprising one or more of bone marrow, lymph node tissue, spleen tissue, and a tumor.

[0102] In certain embodiments, the plurality of unpurified immune cells are obtained from a patient or a donor. In particular embodiments, the donor comprises a donor who is HLA-matched to an adoptive transfer recipient. In particular embodiments, the plurality of unpurified immune cells are obtained from a patient and the patient has one or more diseases, disorders, or17 43105.601_P18311-02conditions selected from the group consisting of a cancer, an infectious disease, and an autoimmune disease.

[0103] In certain embodiments, the one or more antigen-specific T cells are selected from the group consisting of cytotoxic CD4+ T cells, CD4+ helper T cells, CD8+ cytotoxic T lymphocytes, T-helper 17 (Th17) cells, regulatory T cells (Tregs), and combinations thereof.

[0104] In certain embodiments, the aAPC comprises a paramagnetic particle and the separating of the antigen-specific T cells associated with the plurality of aAPCs from the cells not associated with the plurality of aAPCs is by magnetic separation.

[0105] The ratio of particular ligands on the same particle can be varied to increase the effectiveness of the particle in antigen or costimulatory ligand presentation. For example, aAPCs can be coupled with Signal 1 and Signal 2 at a variety of ratios, such as about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 5:1, about 3:1, about 2:1, about 1:1, about 0.5:1, about 0.3:1; about 0.2:1, about 0.1:1, or about 0.03:1. The total amount of protein coupled to the supports may be, for example, about 250 mg / mL, about 200 mg / mL, about 150 mg / mL, about 100 mg / mL, or about 50 mg / mL of particles. Because effector functions such as cytokine release and growth may have differing requirements for Signal 1 versus Signal 2 than T cell activation and differentiation, these functions can be determined separately.

[0106] The aAPCs present antigen to T cells and thus can be used to both enrich for and expand antigen-specific T cells, including from naive T cells. The peptide antigens will be selected based on the desired therapy, for example, cancer, type of cancer, infectious disease, and the like. In some embodiments, the method is conducted to treat a cancer patient, and neoantigens specific to the patient are identified, and synthesized for loading aAPCs. In some embodiments, between three and ten neoantigens are identified through genetic analysis of the tumor (e.g., nucleic acid sequencing), followed by predictive bioinformatics. As shown herein, several antigens can be employed together (on separate aAPCs), with no loss of functionality in the method. In some embodiments, the antigens are natural, non-mutated, cancer antigens, of which many are known. This process for identifying antigens on a personalized basis is described in greater detail below.

[0107] A variety of antigens can be bound to antigen presenting complexes. The nature of the antigens depends on the type of antigen presenting complex that is used. For example, peptide antigens can be bound to MHC class I and class II peptide binding clefts. Non-classical MHC-like molecules can be used to present non-peptide antigens such as phospholipids, complex18 43105.601_P18311-02carbohydrates, and the like (e.g., bacterial membrane components such as mycolic acid and lipoarabinomannan). Any peptide capable of inducing an immune response can be bound to an antigen presenting complex. Antigenic peptides include tumor-associated antigens, autoantigens, alloantigens, and antigens of infectious agents.

[0108] The terms “cancer-specific antigen (CSA)” and “tumor-specific antigen (TSA)” are used interchangeably herein and refer to a protein, carbohydrate, or other molecule that is uniquely expressed by and / or displayed on cancer cells and is not expressed by or displayed on other cells in the body (e.g., normal healthy cells). In contrast, the terms “cancer-associated-antigen (CAA)” and “tumor-associated-antigen (TAA)” are used interchangeably herein and refer to a protein, carbohydrate, or other molecule that is not uniquely expressed by or displayed on a tumor cell and instead also is expressed on normal cells under certain conditions. Cancer-specific antigens and cancer-associated antigens are well known in the art. In some embodiments, the CSA or CAA comprises one or more antigenic cancer epitopes associated with a malignant cancer or tumor, a metastatic cancer or tumor, or a leukemia. A cancer “neoantigen” is a novel cancer-specific antigen that arises as a consequence of tumor-specific mutations (T.N. Schumacher and R.D. Schreiber, Science, 348(6230):69-74 (2015); and T.C. Wirth and F. Kühnel, Front Immunol., 8: 1848 (2017)).

[0109] “Tumor-associated antigens” include unique tumor antigens expressed exclusively by the tumor from which they are derived, shared tumor antigens expressed in many tumors but not in normal adult tissues (oncofetal antigens), and tissue-specific antigens expressed also by the normal tissue from which the tumor arose. Tumor associated antigens can be, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressors, fusion proteins, or oncoviral proteins.

[0110] A variety of tumor-associated antigens are known in the art, and many of these are commercially available. Oncofetal and embryonic antigens include carcinoembryonic antigen and alpha-fetoprotein (usually only highly expressed in developing embryos but frequently highly expressed by tumors of the liver and colon, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal, hepatic, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many carcinomas), pancreatic oncofetal antigen, 5T4 (expressed in gastriccarcinoma), alphafetoprotein19 43105.601_P18311-02receptor (expressed in multiple tumor types, particularly mammary tumors), and M2A (expressed in germ cell neoplasia).

[0111] Tumor-associated differentiation antigens include tyrosinase (expressed in melanoma) and particular surface immunoglobulins (expressed in lymphomas).

[0112] Mutated oncogene or tumor-suppressor gene products include Ras and p53, both of which are expressed in many tumor types, Her-2 / neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene product, myc (associated with lung cancer), ras, p53, nonmutant associated with breast tumors, MAGE-1, and MAGE-3 (associated with melanoma, lung, and other cancers). Fusion proteins include BCR-ABL, which is expressed in chromic myeloid leukemia. Oncoviral proteins include HPV type 16, E6, and E7, which are found in cervical carcinoma.

[0113] Tissue-specific antigens include melanotransferrin and MUC1 (expressed in pancreatic and breast cancers); CD10 (previously known as common acute lymphoblastic leukemia antigen, or CALLA) or surface immunoglobulin (expressed in B cell leukemias and lymphomas); the α chain of the IL-2 receptor, T cell receptor, CD45R, CD4+ / CD8+ (expressed in T cell leukemias and lymphomas); prostate specific antigen and prostatic acid-phosphatase (expressed in prostate carcinoma); GP 100, MelanA / Mart-1, tyrosinase, gp75 / brown, BAGE, and S-100 (expressed in melanoma); cytokeratins (expressed in various carcinomas); and CD19, CD20, and CD37 (expressed in lymphoma).

[0114] Tumor-associated antigens also include altered glycolipid and glycoprotein antigens, such as neuraminic acid-containing glycosphingolipids (e.g., GM2 and GD2, expressed in melanomas and some brain tumors); blood group antigens, particularly T and sialylated Tn antigens, which can be aberrantly expressed in carcinomas; and mucins, such as CA-125 and CA-19-9 (expressed on ovarian carcinomas) or the underglycosylated MUC-1 (expressed on breast and pancreatic carcinomas).

[0115] “Antigens of infectious agents” include components of protozoa, bacteria, fungi (both unicellular and multicellular), viruses, prions, intracellular parasites, helminths, and other infectious agents that can induce an immune response.

[0116] Bacterial antigens include antigens of gram-positive cocci, gram positive bacilli, gram- negative bacteria, anaerobic bacteria, such as organisms of the families Actinomycetaceae, Bacillaceae, Bartonellaceae, Bordetellae, Captophagaceae, Corynebacteriaceae,20 43105.601_P18311-02Enterobacteriaceae, Legionellaceae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pasteurellaccae, Pseudomonadaceae, Spirochaetaceae, Vibrionaceae and organisms of the genera Acinetobacter, Brucella, Campylobacter, Erysipelothrix, Ewingella, Francisella, Gardnerella, Helicobacter, Levinea, Listeria, Streptobacillus and Tropheryma.

[0117] Antigens of protozoan infectious agents include antigens of malarial plasmodia, Leishmania species, Trypanosoma species and Schistosoma species.

[0118] Fungal antigens include antigens of Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccicioides, Sporothrix, organisms of the order Mucorales, organisms inducing choromycosis and mycetoma and organisms of the genera Trichophyton, Microsporum, Epidermophyton, and Malassezia.

[0119] Viral peptide antigens include, but are not limited to, those of adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus, poxviruses, HIV, influenza viruses, and CMV. Particularly useful viral peptide antigens include HIV proteins such as HIV gag proteins (including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (M) protein and influenza virus nucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis B DNA polymerase, hepatitis C antigens, and the like.

[0120] Antigens, including antigenic peptides, can be bound to an antigen binding cleft of an antigen presenting complex either actively or passively, as described in U.S. Pat. No. 6,268,411, which is hereby incorporated by reference in its entirety. Optionally, an antigenic peptide can be covalently bound to a peptide binding cleft.

[0121] If desired, a peptide tether can be used to link an antigenic peptide to a peptide binding cleft. For example, crystallographic analyses of multiple class I MHC molecules indicate that the amino terminus of β2M is very close, approximately 20.5 Angstroms away, from the carboxyl terminus of an antigenic peptide resident in the MHC peptide binding cleft. Thus, using a relatively short linker sequence, approximately 13 amino acids in length, one can tether a peptide to the amino terminus of β2M. If the sequence is appropriate, that peptide will bind to the MHC binding groove (see U.S. Pat. No. 6,268,411).

[0122] Antigen-specific T cells which are bound to the aAPCs can be separated from cells which are not bound using magnetic enrichment, or other cell sorting or capture technique. Other21 43105.601_P18311-02processes that can be used for this purpose include flow cytometry and other chromatographic means (e.g., involving immobilization of the antigen-presenting complex or other ligand described herein). In one embodiment antigen-specific T cells are isolated (or enriched) by incubation with beads, for example, antigen-presenting complex / anti-CD28-conjugated paramagnetic beads (such as DYNABEADS®), for a time period sufficient for positive selection of the desired antigen- specific T cells.

[0123] In some embodiments, a population of T cells can be substantially depleted of previously active T cells using, e.g., an antibody to CD44, leaving a population enriched for naive T cells. Binding aAPCs to this population would not substantially activate the naive T cells, but would permit their purification.

[0124] In still other embodiments, ligands that target NK cells, NKT cells, or B cells (or other immune effector cells), can be incorporated into a paramagnetic particle, and used to magnetically enrich for these cell populations, optionally with expansion in culture as described below. Additional immune effector cell ligands are described in PCT / US2014 / 25889, which is hereby incorporated by reference in its entirety.

[0125] Without wishing to be bound by theory, removal of unwanted cells may reduce competition for cytokines and growth signals, remove suppressive cells, or may simply provide more physical space for expansion of the cells of interest.

[0126] Enriched T cells are then expanded in culture within the proximity of a magnet to produce a magnetic field, which enhances T cell receptor clustering of aAPC bound cells. Cultures can be stimulated for variable amounts of time (e.g., about 0.5, 2, 6, 12, 36, 48, or 72 hours, as well as continuous stimulation) with aAPC. The effect of stimulation time in highly enriched antigen- specific T cell cultures can be assessed. Antigen-specific T cell can be placed back in culture and analyzed for cell growth, proliferation rates, various effector functions, and the like, as is known in the art. Such conditions may vary depending on the antigen-specific T cell response desired. In some embodiments, T cells are expanded in culture from about 2 days to about 3 weeks, or in some embodiments, about 5 days to about 2 weeks, or about 5 days to about 10 days. In some embodiments, the T cells are expanded in culture for about 1 week, after which time a second enrichment and expansion step is optionally performed. In some embodiments, 2, 3, 4, or 5 enrichment and expansion rounds are performed.22 43105.601_P18311-02

[0127] After the one or more rounds of enrichment and expansion, the antigen-specific T cell component of the sample will be at least about 1% of the cells, or in some embodiments, at least about 5%, at least about 10%, at least about 15%, or at least about 20%, or at least about 25% of the cells in the sample. Further, these T cells generally display an activated state. From the original sample isolated from the patient, the antigen-specific T cells in various embodiments are expanded from about 100-fold to about 10,000 fold, such as at least about 1000-fold, at least about 2000- fold, at least about 3,000 fold, at least about 4,000-fold, or at least about 5,000-fold in various embodiments. After the one or more rounds of enrichment and expansion, at least about 106, or at least about 107, or at least about 108, or at least about 109antigen-specific T cells are obtained.

[0128] The effect of aAPC on expansion, activation and differentiation of T cell precursors can be assayed in any number of ways known to those of skill in the art. A rapid determination of function can be achieved using a proliferation assay, by determining the increase of CTL, helper T cells, or regulatory T cells in a culture by detecting markers specific to each type of T cell. Such markers are known in the art. CTL can be detected by assaying for cytokine production or for cytolytic activity using chromium release assays.

[0129] In addition to generating antigen-specific T cells with appropriate effector functions, another parameter for antigen-specific T cell efficacy is expression of homing receptors that allow the T cells to traffic to sites of pathology (Sallusto et al., Nature 401, 708-12, 1999; Lanzavecchia & Sallusto, Science 290, 92-97, 2000).

[0130] For example, effector CTL efficacy has been linked to the following phenotype of homing receptors, CD62L+, CD45RO+, and CCR7-. Thus, an aAPC-induced and / or expanded CTL population can be characterized for expression of these homing receptors. Homing receptor expression is a complex trait linked to initial stimulation conditions. Presumably, this is controlled both by the costimulatory complexes as well as cytokine milieu. One important cytokine that has been implicated is IL-12 (Salio et al., 2001). As discussed below, aAPC offer the potential to vary individually separate components (e.g., T cell effector molecules and antigen presenting complexes) to optimize biological outcome parameters. Optionally, cytokines such as IL-12 can be included in the initial induction cultures to affect honing receptor profiles in an antigen-specific T cell population.

[0131] Optionally, a cell population comprising antigen-specific T cells can continue to be incubated with either the same aAPC or a second aAPC for a period of time sufficient to form a23 43105.601_P18311-02second cell population comprising an increased number of antigen-specific T cells relative to the number of antigen-specific T cells in the first cell population. Typically, such incubations are carried out for 3-21 days, preferably 7-10 days.

[0132] Suitable incubation conditions (culture medium, temperature, etc.) include those used to culture T cells or T cell precursors, as well as those known in the art for inducing formation of antigen-specific T cells using DC or artificial antigen presenting cells. See, e.g., Latouche & Sadelain, Nature Biotechno. 18, 405-09, April 2000; Levine et al., J. Immunol. 159, 5921-30, 1997; Maus et al., Nature Biotechnol. 20, 143-48, February 2002. See also the specific examples, below.

[0133] To assess the magnitude of a proliferative signal, antigen-specific T cell populations can be labeled with CFSE and analyzed for the rate and number of cell divisions. T cells can be labeled with CFSE after one-two rounds of stimulation with aAPC to which an antigen is bound. At that point, antigen-specific T cells should represent 2-10% of the total cell population. The antigen- specific T cells can be detected using antigen-specific staining so that the rate and number of divisions of antigen-specific T cells can be followed by CFSE loss. At varying times (for example, 12, 24, 36, 48, and 72 hours) after stimulation, the cells can be analyzed for both antigen presenting complex staining and CFSE. Stimulation with aAPC to which an antigen has not been bound can be used to determine baseline levels of proliferation. Optionally, proliferation can be detected by monitoring incorporation of 3H-thymidine, as is known in the art.

[0134] C. In Vivo Method for Treating a Disease, Disorder, or Condition

[0135] In other embodiments, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition, the method comprising administering to a subject in need of treatment thereof a composition comprising one or more aAPCs as disclosed herein in vivo or one or more antigen-specific T cells prepared by the methods disclosed herein in vitro.

[0136] In certain embodiments, the disease, disorder, or condition is selected from a cancer, an infectious disease, and an autoimmune disease.

[0137] C.1 Methods for Treating a Cancer

[0138] Cancers that can be treated or evaluated according to the presently disclosed methods include cancers that historically illicit poor immune responses or have a high rate of recurrence. Exemplary cancers include various types of solid tumors, including carcinomas, sarcomas, and lymphomas. In various embodiments the cancer is melanoma (including metastatic melanoma),24 43105.601_P18311-02colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma. In some embodiments, the cancer is a hematological malignancy, such as chronic myelogenous leukemia, childhood acute leukemia, non-Hodgkin's lymphomas, chronic lymphocytic leukemia, malignant cutaneous T-cells, mycosis fungoids, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, and discoid lupus erythematosus.

[0139] In various embodiments, the cancer is stage I, stage II, stage III, or stage IV. In some embodiments, the cancer is metastatic and / or recurrent. In some embodiments, the cancer is preclinical, and is detected in the screening system described herein (e.g., colon cancer, pancreatic cancer, or other cancer that is difficult to detect early).

[0140] C.2 Methods for Treating an Infectious Disease

[0141] In other embodiments, the presently disclosed subject matter includes a method for treating an infectious disease. Infectious diseases that can be treated include those caused by bacteria, viruses, prions, fungi, parasites, helminths, and the like. Such diseases include AIDS, hepatitis, CMV infection, and post-transplant lymphoproliferative disorder (PTLD).

[0142] CMV, for example, is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplants. This is due to the immunocompromised status of these patients, which permits reactivation of latent virus in seropositive patients or opportunistic infection in seronegative individuals. A useful alternative to these treatments is a prophylactic immunotherapeutic regimen involving the generation of vims-specific CTL derived from the patient or from an appropriate donor before initiation of the transplant procedure. PTLD occurs in a significant fraction of transplant patients and results from Epstein-Barr virus (EBV) infection. EBV infection is believed to be present in approximately 90% of the adult population in the United States. Active viral replication and infection is kept in check by the immune system, but, as in cases of CMV, individuals immunocompromised by transplantation therapies lose the controlling T cell populations, which permits viral reactivation. This represents a serious impediment to25 43105.601_P18311-02transplant protocols. EBV may also be involved in tumor promotion in a variety of hematological and non-hematological cancers.

[0143] Other viral pathogens potentially treated by the presently disclosed methods include, but are not limited to adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus, poxviruses, HIV, influenza viruses, and COVID-19.

[0144] C.3 Methods for Treating an Autoimmune Disease

[0145] Autoimmune diseases that can be treated include systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, Goodpasture's syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, and Hashimoto's thyroiditis. In some embodiments, the patient is suspected of having an autoimmune disease or immune condition (such as those described in the preceding sentence), and the evaluation of T cell responses against a library of paramagnetic aAPCs as described herein, is useful for identifying or confirming the immune condition.

[0146] The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein.

[0147] In general, the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of26 43105.601_P18311-02ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like.

[0148] D. Methods for Diagnosing a Disease or a Disease State

[0149] In other embodiments, the presently disclosed subject matter provides a method for diagnosing a disease or a disease state in a subject, the method comprising:

[0150] (a) obtaining a blood serum sample from the subject;

[0151] (b) contacting the blood serum sample with a plurality of aAPCs disclosed herein, wherein the plurality of aAPCs includes one or more aAPCs which can be the same or different and / or a plurality of non-APC particles alone or in combination with the plurality of aAPCs;

[0152] (c) separating one or more blood serum proteins associated with the plurality of aAPCs from blood serum proteins not associated with the plurality of aAPCs; and

[0153] (d) determining an identity and / or an amount of the one or more blood serum proteins associated with the plurality of aAPCs to diagnose a disease or disease state in the subject.

[0154] In certain embodiments, the plurality of non-APC particles includes one or more non-APC particles having a same or different elastic modulus. In certain embodiments, the plurality of non- APC particles includes one or more non-APC particles that bind differentially to blood serum protein.

[0155] In such embodiments, the presently disclosed aAPCs can be mixed with blood serum samples and “pull down” serum proteins that bind to them. Particles with different elastic modulus can bind to a different selection of proteins from the same serum sample. Using a pool of particles that span elastic modulus can best profile proteins present in the serum to get a “finger print” of proteins that can then be correlated to disease state in a diagnostic manner. In some embodiments, the particles can be multiplexed.

[0156] E. Non-viral Method for Delivering an Immunomodulatory Material to a T cell

[0157] In other embodiments, the presently disclosed subject matter provides a non-viral method for delivering an immunomodulatory material to a T cell, the method comprising administering an aAPC having an immunomodulatory material conjugated thereto to a cell. See, for example, International PCT Patent Application Publication No. WO2024077071 for Nanoparticles for Delivery of Immunoregulatory Materials to T Cells, to Schneck, published April 11, 2024, which is incorporated herein by reference in its entirety.27 43105.601_P18311-02

[0158] F. Reagents / Kits

[0159] In other embodiments, the presently disclosed subject matter provides a kit comprising the presently disclosed aAPCs together and, in some embodiments, with components for performing an enrichment and expansion process. Suitable containers for the presently disclosed aAPCs include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Optionally, one or more different antigens can be bound to the paramagnetic particles or can be supplied separately. Kits may comprise, alternatively or in addition, one or more multi-well plates or culture plates for T cells. In some embodiments, kits comprise a sealed container comprising paramagnetic particles, a magnet, and optionally test tubes and / or solution or buffers for performing magnetic enrichment.

[0160] A kit can further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to an end user, including other buffers, diluents, filters, needles, and syringes.

[0161] Kits also may contain reagents for assessing the extent and efficacy of antigen-specific T cell activation or expansion, such as antibodies against specific marker proteins, MHC class I or class II molecular complexes, TCR molecular complexes, anticlonotypic antibodies, and the like.

[0162] A kit can also comprise a package insert containing written instructions for methods of inducing antigen-specific T cells, expanding antigen-specific T cells, using paramagnetic particles in the kit in various protocols. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.

[0163] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed aAPCs can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.28 43105.601_P18311-02

[0164] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0165] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0166] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly an aAPC disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As29 43105.601_P18311-02used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0167] Further, the aAPCs disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the aAPCs, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0168] The timing of administration of an aAPC disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of an aAPC described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of an aAPC described herein and at least one additional therapeutic agent can receive an aAPC and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0169] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the aAPC described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either an aAPC or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.30 43105.601_P18311-02

[0170] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0171] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of an aAPC described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0172] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0173] Qa / QA + Qb / QB = Synergy Index (SI)

[0174] wherein:

[0175] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0176] Qais the concentration of component A, in a mixture, which produced an end point;

[0177] QBis the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0178] Qb is the concentration of component B, in a mixture, which produced an end point.

[0179] Generally, when the sum of Qa / QAand Qb / QBis greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.31 43105.601_P18311-02

[0180] Depending on the specific conditions being treated, the “agent(s)” may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intradermal, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, intralymphatic administration, and intratumoral administration, or intraocular injections or other modes of delivery.

[0181] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0182] Use of pharmaceutically acceptable inert carriers to formulate the aAPCs herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The aAPCs can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the aAPCs of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0183] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.

[0184] In particular embodiments, the aAPC disclosed herein is administered intranasally in a form selected from the group consisting of a nasal spray, a nasal drop, a powder, a granule, a32 43105.601_P18311-02cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures.

[0185] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the aAPCs according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the aAPC is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the aAPC(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the aAPC(s), and the preference and experience of the attending physician.

[0186] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active aAPCs into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0187] Pharmaceutical preparations for oral use can be obtained by combining the active aAPCs with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl- cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0188] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone,33 43105.601_P18311-02carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active aAPC doses.

[0189] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active aAPCs may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

[0190] Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0191] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%, + / -10%, + / -5%, + / -4%, + / -3%, + / - 2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0192] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0193] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The34 43105.601_P18311-02present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. EXAMPLES

[0194] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make aAPCs of the disclosure by other methods. EXAMPLE 1

[0195] Particle Elasticity Influences Polymeric Artificial Antigen Presenting Cell Effectiveness In Vivo via CD8+ T cell Activation, Macrophage Uptake, and the Protein Corona

[0196] Overview

[0197] In this Example, a particulate artificial antigen presenting cell (aAPC) platform with tunable elasticity was prepared to study, in part, the effects of particle elasticity on aAPC effectiveness in vivo. We found that elasticity significantly impacted T cell activation, particle clearance, and organ biodistribution. We further investigated possible mechanisms to explain these findings.

[0198] Biomaterial properties, such as elasticity, have been shown to vastly impact the bioavailability and particle-cell interactions, but this impact has yet to be investigated in the context of aAPCs for in vivo T-cell stimulation. Previous literature likewise indicates that biomaterial properties, especially elasticity, can modulate T-cell activation in vitro.

[0199] With the goal of creating a more biomimetic, next-generation particulate aAPC, we developed a poly(ethylene) glycol hydrogel particle platform with tunable elasticity to investigate the impact of elasticity on antigen-specific T cell activation for in vivo adoptive transfer. Using35 43105.601_P18311-02this knowledge, we were able to gain more precise control over in vivo T cell activation and investigate possible mechanisms including the effects of aAPC elasticity on T cell binding, macrophage uptake, and the protein corona.

[0200] Introduction

[0201] One of the major challenges in the field of cancer immunotherapy is mounting an effective attack against solid tumors while minimizing off-target effects. In recent years, cytotoxic CD8+ T cells have emerged as an attractive candidate for cancer immunotherapy interventions due to their function as powerful effector arms of the adaptive immune system and their ability to precisely target cells expressing antigens of interest. Raskov et al., 2021.

[0202] Substantial progress in the treatment of solid cancers has been made using T cells as demonstrated by the FDA approval of adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T cells. While ACT and CART therapies have been successful in the clinic, there remain significant drawbacks to their widespread use, such as high cost, the resource intensive process of patient T-cell extraction and ex vivo expansion, and manufacturing complexity. Bailey et al., 2023. Because of this, particulate artificial antigen presenting cells (aAPCs) have become a promising next generation approach for cellular engineering and T-cell immunotherapy of solid cancers. Wang et al., 2017; Est-Witte et al., 2021.

[0203] aAPCs mimic the function of natural antigen presenting cells (APCs) and provide a platform for the activation and expansion of antigen-specific T cells. aAPCs can be designed to express co-stimulatory molecules and cytokines, enabling the generation of T cells with specific targets and tunable effector functions. This characteristic is particularly useful in the context of cancer and immune disorders where natural APC function is dysregulated and nonspecific expansion of T cells may have deleterious effects. Bandola-Simon and Roche, 2019; Li et al., 2020.

[0204] The use of particulate aAPCs offers several advantages over traditional methods of two- dimensional (2D) T-cell expansion, such as the ability to control the timing and magnitude of T- cell activation, the potential to reduce cost and resources by generating a T-cell response in vivo, and the capacity for large-scale, cell-free manufacturing. Est-Witte et al., 2021; Ben-Akiva et al., 2019. While aAPCs have shown effective therapeutic T cell expansion-ex vivo, in vivo therapeutic T-cell expansion remains challenging due to the potential for off-target effects and poor biodistribution.36 43105.601_P18311-02

[0205] To address these issues, researchers have incorporated design principles from the field of particulate drug delivery. aAPCs are typically designed to be in the range of 100 nm to 10 µm and have been made from a variety of materials, including polymers, lipids, and inorganic materials. Wang et al., 2017.

[0206] Biomaterial properties, such as particle size, shape, surface chemistry, and material, have been investigated and found to modulate in vivo aAPC effectiveness significantly. Meyer and Sunshine, 2016; Meyer et al., 2015; Ben-Akiva et al., 2017; Rhodes et al., 2021. While many biomaterial properties of particulate aAPCs and how they function in vivo have been studied, the impact of particle elasticity remains poorly understood. To date, aAPC stiffness has only been well-studied ex vivo using 2D surfaces.

[0207] T cells are known to be sensitive to changes in mechanical stiffness from both the extracellular matrix and APCs, with stiffness cues playing a major role in T-cell activation, differentiation, and function. De et al., 2019; Du et al., 2023; Meng et al., 2020. In the context of artificial antigen presenting cells, stiffness has been shown to modulate the effectiveness of 2D aAPCs ex vivo at T-cell stimulation, skew T-cell differentiation toward effector or memory phenotypes, and influence cytokine production, depending on the range of stiffness investigated. Platzman et al., 2015; Hickey et al., 2019; Saitakis et al., 2017.

[0208] While the role of elasticity in particulate aAPCs has not yet been well-investigated, the role of elasticity of micro- and nanoparticles for drug delivery has been studied extensively. Nano- and micro-particle elasticity has been shown to drive particle clearance, biodistribution, cell uptake, targeting, and resulting efficacy in several preclinical particle-based therapeutics for cancer drug delivery. Guo et al., 2018; Anselmo and Mitragotri, 2017; Anselmo et al., 2015; Hui et al., 2019.

[0209] In this Example, we aim to elucidate the role of particle elasticity in polymeric aAPCs for in vivo T cell expansion. Without wishing to be bound to any one particular theory, it is thought that optimizing particle stiffness will improve in vivo particle aAPC effectiveness by increasing aAPC bioavailability while maximizing effectiveness of T-cell activation in vivo.

[0210] Results and discussion

[0211] Particle Synthesis and Characterization

[0212] To create a particulate aAPC platform with tunable elasticity, we chose poly(ethylene glycol) (PEG) as the particle bulk material due to its low cost, wide availability, established ease37 43105.601_P18311-02of elasticity modulation, and significance as an anti-fouling material in the field of particulate drug delivery. Anselmo et al., 2015; Cui et al., 2015; Van Thienen et al., 2008

[0213]

[0214] Previously, we have shown that larger microparticle polymeric aAPCs are more efficient at T-cell activation but exhibit poor biodistribution, Sunshine et al., 2014, while polymeric nanoparticle aAPCs have better biodistribution profiles but have less efficient T-cell activation. Meyer and Sunshine, 2016. To balance these effects, a particle size between approximately 1000 nm to 2000 nm was chosen. PEG particle synthesis was accomplished via a water-in-oil emulsion and UV photocrosslinking (FIG.1a). Anselmo et al., 2015. The elasticity of the PEG particles was altered by changing the volume fraction (Vf) of PEG in solution from 0.15-0.5. The stiffness of each resulting gel was measured by bulk rheometry (FIG. 1c), and three groups were chosen to represent the largest possible stiffness range of the material. The groups chosen were approximately 50 kPa, 850 kPa, and 5000 kPa, which corresponded to 0.15, 0.30, and 0.50 PEG Vf, respectively. These stiffnesses are within the range of biological tissue and are an order of magnitude softer than current existing polymeric aAPCs. Akhtar et al., 2011; Doshi et al., 2009.

[0215] Transmission electron microscopy (TEM) revealed consistent spherical morphology across stiffnesses (FIG. 1c), and dynamic light scattering (DLS) showed a size profile for each group of 1-3 µm, polydispersity index (PDI) around 0.1-0.4, and zeta potentials between -25 and -38 mV (FIG.1g). To create aAPCs, immune targeting signal 1 and costimulatory signal 2 were conjugated directly to PEG particles via NHS chemistry (FIG. 1d-FIG. 1e). The size of the resulting aAPCs did not change significantly after protein conjugation, indicating that the particles remained stable during this process (FIG. 1f).

[0216] In Vitro T-Cell Stimulation

[0217] After thorough characterization, PEG particles from each elasticity group were conjugated with either antigen-specific MHC class I Db dimer loaded with gp100 peptide (Dbgp100) or nonspecific anti-CD3 as signal 1 and costimulatory anti-CD28 as signal 2. The amount of protein added to each particle group was adjusted to result in no significant differences in the amount of signal 1 and signal 2 per particle across stiffness groups (FIG. 2a). Particles were then added to primary murine CD8+ T cells isolated from a transgenic mouse strain engineered to express a T cell receptor specific to mouse premelanosome protein (PMEL) and the T cell Thy1.1 allele, Overwijk et al., 1998, or C57 / BL6 mice for antigen-specific or -nonspecific stimulations,38 43105.601_P18311-02respectively, and incubated at 37 ºC for 7 days. Results of nonspecific stimulations are shown in FIG. 1 (see also FIG. 7). On day 3, cells were analyzed for qualitative proliferation using a cell proliferation tracking dye and compared to cells with no aAPCs as a negative control and Dynabeads (Thermo Fisher Scientific; Waltham, MA) containing anti-CD3 and anti-CD28 at one bead / cell as a positive control (FIG. 2c). All particle groups and the positive control beads displayed proliferation peaks, and all particle groups for the 1x dose showed significant proliferation compared to the no stimulation control (p<0.05) (FIG. 2d). On day 7, cells were collected and counted to quantify proliferation via the number of cells compared with the number of cells on day 1 of incubation. Fold proliferation of antigen-specific CD8+ T cells increased significantly with increasing stiffness from 50 kPa-5000 kPa and exhibited a dose response (FIG. 2b).

[0218] After activation of naïve T cells, the resulting cell phenotype can play a major role in the efficacy of anti-cancer immune responses. Naïve T cells differentiate into either effector or memory T cell phenotypes, with effector T cells responsible for the immediate anti-cancer response and memory T cells responsible for long-term anti-cancer responses and antigen recognition. Waldman et al., 2020.

[0219] Thus, we were interested in characterizing the phenotype and functionality of CD8+ T cells generated after exposure to PEG aAPCs to determine if there were any differences across particle elasticity groups. To investigate T-cell phenotype, cells were collected at day 7, filtered, and stained for T-cell phenotype markers CD44 and CD62L. Cells were analyzed via flow cytometry and sorted into 3 groups: naïve (CD62L+, CD44-), effector (CD62L-, CD44+), and memory (CD62L+, CD44+) CD8+ T cells. Both aAPC dose and aAPC elasticity were found to be significant factors in determining T-cell phenotype (FIG. 3a-FIG. 3c). The proportion of naïve cells increased with decreasing dose for all elasticity groups, with a slight increase in the proportion of naïve cells in the stiffest group (FIG.3a), corresponding to the proliferation dose response seen in FIG. 2. For memory T cells, differences in elasticity were most apparent in the lowest dose (0.01×), with significantly increasing presence of memory T cells from 50 kPa-5000 kPa (FIG. 3b). Effector T cells also varied with both dose and elasticity (FIG. 3c). At the highest dose, there were no significant differences among elasticity groups. At the 0.1× dose, there was a significantly higher proportion of effector cells at the intermediate elasticity (850 kPa), and at the lowest dose, effector cells significantly decreased from soft to hard particles. This dose-dependent response39 43105.601_P18311-02may suggest that elasticity-driven phenotype changes could be more prevalent when stimulation and availability of signals 1 and 2 are limited. Other groups studying biomaterial properties in aAPCs have observed phenotype, as well as proliferation, changes are compounded by ligand density, with the effects of ligand density overpowering stiffness effects in some cases. Hickey et al., 2019; Wauters et al., 2022; Majedi et al., 2019.

[0220] In addition to T-cell phenotype, the resulting functionality of activated T cells is essential for successful aAPC therapy. While generation of effector T cells is necessary for a robust anti- cancer response, it is possible to unintentionally shift these populations into nonfunctional “exhausted” T cell phenotypes through repeated low dose stimulation, which is often seen in chronic infections or cancer. Waldman et al., 2020. Likewise, changes in T-cell function have previously been associated with changes in 2D substrate stiffness ex vivo. De et al., 2019; Saitakis et al., 2017.

[0221] To determine T-cell functionality after stimulation, cells at the intermediate 0.1x dose were collected, filtered, and restimulated at day 7 of incubation for 6 hours. Cells were then stained for relevant markers of anti-cancer functionality including lytic function surface marker CD107a and cytokines IFN-γ, TNF-α, and IL-2, and analyzed via flow cytometry. In each stiffness group, cells showed multifunctionality by having a significantly higher number of cells expressing three or more cytokines simultaneously than the no stimulation control (FIG. 3h; p < 0.01, p < 0.05, p < 0.01, and p < 0.05 for the 50 kPa, 850 kPa, 5000 kPa, and positive control beads, respectively). There were no significant differences in multifunctionality across stiffness groups. When comparing the amounts of each individual cytokine expressed, only TNF-α showed a significant relationship between expression and aAPC stiffness, with TNF-α expression decreasing with increasing stiffness (FIG. 3d-FIG. 3g). Only the 50-kPa group showed a significant increase in TNF-α expression when compared to the no aAPC control (p < 0.0001). TNF-α is a pleiotropic cytokine and can mediate a variety of physiological processes. In CD8+ T cells, expression of TNF-α can either be pro-inflammatory or anti-inflammatory, so it is difficult to make any conclusions as to the anti-cancer contribution of the stiffness-mediated increase in TNF-α. Chen et al., 2018.

[0222] In Vitro T-cell binding

[0223] To elucidate a possible mechanism behind the increase in T cell proliferation seen as a function of elasticity, we investigated the impact of elasticity on aAPC specific and nonspecific T40 43105.601_P18311-02cell binding (FIG. 4a-FIG. 4b). Binding of APCs to T cells is the first step in initiating the T-cell receptor (TCR) signaling cascade, resulting in T-cell activation. PEG aAPCs were conjugated with anti-CD3 and anti-CD28 and mixed with primary T cells isolated from C57BL / 6 mice. To measure nonspecific binding, soluble anti-CD3 and anti-CD28 were incubated with T cells to block binding sites prior to aAPC incubation. At the highest dose, there were only slight differences in binding, while at the intermediate and lower doses, nonspecific binding significantly decreased from 50 kPa to 5000 kPa (FIG. 4b). While it is clear elasticity impacts aAPC binding to T cells, the trends seen in T-cell proliferation cannot be fully explained by T-cell binding alone. It is likely there are a number of factors influencing T cell proliferation in response to changes in particle elasticity. For example, Meng et al. recently reported T cell activation and metabolism are regulated through mechanosensing via the YAP pathway, with increasing proliferation in hard (40 kPa) compared to soft (4 kPa) 3D bulk substrates. Meng et al., 2020. In this same system, they found that immunological synapse size was also changed by modulating mechanical stiffness, both of which would significantly impact T cell activation. Majedi et al., 2020.

[0224] In Vivo Clearance and Biodistribution

[0225] After establishing the impact of elasticity on PEG aAPCs in vitro, we sought to determine the impact of elasticity on PEG aAPC clearance and biodistribution in vivo. Both particle clearance and biodistribution contribute to aAPC bioavailability, which is one of the main challenges in developing effective particulate aAPCs. To investigate the impact of aAPC elasticity on clearance and biodistribution, fluorescent particles were injected intravenously into mice, and blood samples were collected at 10, 20, 30, 40, 60, and 240 minutes after injection to assess any changes in clearance of unconjugated PEG particles in the blood or organ biodistribution as a result of varying particle elasticity. The clearance of PEG particles in the blood was found to be significantly affected by particle elasticity (FIG. 4c), with softer particles circulating for longer in the blood. The half-life of the 50-kPa particles was approximately 5-fold higher than that of the 5000 kPa particles (FIG.4d), which is consistent with trends seen in PEG nanoparticles of similar elasticities. Anselmo and Mitragotri, 2017; Anselmo et al., 2015.

[0226] After 8 hours, mouse organs were harvested, and the amount of particle signal from each organ was quantified to determine the biodistribution of particles as a function of particle elasticity (FIG. 4e). Significant differences were found in the lungs and the liver, with increasing stiffness corresponding with more signal in the liver and less signal in the lungs. No significant changes in41 43105.601_P18311-02biodistribution as a result of elasticity were found in the heart, spleen, or kidneys. These results along with historical data in the field further suggest that elasticity is an important physiological parameter when designing micro- and nanoparticles for drug delivery, as it modulated not only circulation times, but also eventual organ biodistribution. Depending on the application, particle elasticity could be utilized to direct cargoes to specific organs to potentially reduce off target effects.

[0227] In Vitro Macrophage Uptake

[0228] Previous studies have indicated that the higher blood half-life seen in soft particles than in hard particles may be due to elasticity-directed differences in macrophage uptake. Anselmo and Mitragotri, 2017. Macrophages are responsible for first-pass clearance of foreign bodies and danger signals in blood and tissue as part of the reticuloendothelial system. As such, they are one of the main drivers of clearance of micro- and nanoparticles in vivo. To understand macrophage uptake as a possible mechanism driving the differences seen in clearance and biodistribution as a function of aAPC elasticity, particles were incubated with RAW 264.7 macrophages to measure macrophage particle binding and uptake. After incubation at 37 ºC, macrophages internalized and bound approximately 4-fold more of the 5000-kPa particles than the 50-kPa particles (FIG. 4f), indicating an elasticity-driven effect on macrophage uptake. Interestingly, when macrophages were prevented from internalization by decreasing the incubation temperature to 4 ºC, overall particle signal per cell decreased, but the trends remained the same, suggesting that particle elasticity also drives nonspecific particle binding to macrophages (FIG. 4g).

[0229] Protein Corona Analysis

[0230] Once particles are in a biofluid-like serum, their surfaces become coated with biological molecules forming a protein corona, and this occurrence has recently been shown to have a significant impact on micro- and nanoparticle clearance and biodistribution in vivo. Mishra et al., 2021; Li et al., 2021; Tomak et al., 2021.

[0231] The protein corona has been shown to greatly impact pharmacological behavior of injected particles in the blood, as well as cell-particle interactions in vivo. Mishra et al., 2021; Panico et al., 2022.

[0232] It is known that biomaterial properties such as size, shape, degree of PEGylation, material, curvature, elasticity, and surface chemistry can significantly modulate the magnitude and content of the corona. Panico et al., 2022; Tengjisi et al., 2022; Partikel et al., 2019. Thus, thorough study42 43105.601_P18311-02of the impact of PEG aAPC elasticity on protein corona formation would give vital information as to possible mechanisms behind the differences we observed in particle biodistribution, as well as in T-cell activation and may elucidate new avenues of investigation into biomaterial-based optimization of aAPCs.

[0233] To study the protein corona on our PEG aAPC platform, we incubated particles with varying elasticities in whole mouse serum for 1 hour at 37 ºC. Particles were then washed, and the corona was separated from the particles and analyzed via liquid chromatography mass spectrometry (LC-MS). LC-MS data was then processed and analyzed using a combination of the proteomics analysis pipeline FragPipe, Yu et al., 2020; Polasky et al., 2020; Teo et al., 2021; Yu et al., 2020; Chang et al., 2020; Geiszler et al., 2021; Tsou et al., 2015, and proteomics visualization software Quickomics. Gao et al., 2021.

[0234] Analysis revealed the magnitude of protein corona formation, the composition of the protein corona, and the pathway enrichment by the protein corona were all significantly impacted by PEG aAPC elasticity. FIG. 5a shows a clustering map of every protein detected on the PEG aAPC coronas, with the x axis displaying each stiffness and replicate (N = 3), and the y axis displaying the clustering of proteins. As shown in the x axis, the algorithm clustered replicates within each group together, which, along with the principal component analysis (PCA) of the LC- MS data (FIG. 10), indicates that replicates are not significantly different within each group; however, there are significant differences across groups, with the 850 kPa and 5000 kPa groups clustered more closely to each other than the 50 kPa group. In FIG. 5a, the color of each bar indicates whether the protein was detected on each replicate, with a black bar indicating detection and a white bar indicating no detection, or a missing value. The groups are listed in order of highest total amount of protein to least amount of protein, and it is clear than the softest group (50 kPa) is adsorbing fewer proteins than the 850 kPa and 5000 kPa groups, with the 850 kPa group adsorbing the highest number of proteins. The differences in composition of the protein corona as a function of stiffness can be visualized via a volcano plot (FIG. 5c). Genes encoding for each protein that are in black and labeled passed the threshold for statistical significance. Additional protein corona analysis for comparisons between each stiffness group can be found in FIG. 11 and FIG. 12.

[0235] FIG.5b shows the top six most significantly changed protein-encoding genes in the protein corona as determined by absolute fold change as a function of elasticity. Gene P01648 is not well studied but is known to be involved in the complement pathway. Parhami-Seren et al., 2001; Capra,43 43105.601_P18311-021981. The complement pathway is a primary protein corona regulator of particle clearance, and the upregulation of this protein suggests the complement pathway could be involved in elasticity- mediated differences in particle clearance in vivo. Panico et al., 2022. In particular, other groups studying particulate protein corona effects found that increases in complement-associated proteins in the corona corresponded with increased macrophage uptake as well as increased accumulation in the liver, which is consistent with our findings. Chinen et al., 2017.

[0236] Lipocalin 2 (Lcn2) is a protein expressed in response to toll-like receptor (TLR) activation in multiple cells and has been implicated as a modulator of inflammation and macrophage polarization. Santiago-Sánchez et al., 2020. While Lcn2 is involved in many cell functions, it is interesting to note that high expression of Lcn2 has been associated with deactivated macrophages, which would support our findings that macrophage uptake increased from 50 kPa to 5000 kPa. Santiago-Sánchez et al., 2020; Warszawska et al., 2013.

[0237] Intracellular adhesion molecule 1 (ICAM-1) is perhaps the most significant of these proteins in regulating both T-cell activation and macrophage activity. ICAM-1 is responsible for initiating T-cell activation via APCs by binding APCs to T cells and facilitating translation of TCR signals to T-cell proliferation and differentiation. Zumwalde et al., 2013. Specifically, upregulation of ICAM-1 is known to skew T-cell differentiation towards a memory phenotype. Cox et al., 2013.

[0238] In macrophages, ICAM-1 was found to regulate macrophage activation, and increases in ICAM-1 expression have been implicated in higher clearance of particles via phagocytosis. Panico et al., 2022; Zhong et al., 2021. While very little is known about how the protein corona interacts with T cells, our findings indicate that harder particles correlate to higher T-cell activation and skewing toward a memory phenotype. Likewise, harder particles were associated with higher macrophage uptake and particle clearance. Taken together, it is possible that the ICAM-1 pathway may mediate aAPC effectiveness via elasticity-driven protein corona effects. Triose phosphate isomerase (TPI1) is a part of the glycolysis pathway, and higher TPI1 expression is implicated in T-cell differentiation toward effector cells via glycolytic metabolism, which is consistent with our findings that softer (50 kPa) particles skewed differentiation towards an effector phenotype. Crompton et al., 2016.

[0239] ASlc25a5 has been rigorously studied and has a variety of functions in immune cells, including driving macrophage inflammation and activation and impairing B-cell activity. It has44 43105.601_P18311-02been demonstrated, however, to have no impact on T-cell activation, so its effects on this study are difficult to interpret. Cho et al., 2015; Saragovi et al., 2020; Moon et al., 2021.

[0240] Finally, matrix metalloproteinase 19 (Mmp19) is involved in modification of the extracellular matrix and has been demonstrated to be essential for T-cell development and T-cell responses in mice. In studies examining the impacts of particles on macrophage function, higher expression of Mmp19 was linked to higher production of TNF-α across cell types and was found to be abundant in unstimulated macrophages as well as macrophages destined for apoptosis. Beck et al., 2008; Matysiak-Kucharek et al., 2018; Huang et al., 2012; Park et al., 2010. Interestingly, we found that T cells stimulated by soft particles (50 kPa) secreted significantly more TNF-α than the harder groups, and the trends in corona expression of Mmp19 are consistent with our findings concerning macrophage uptake as a function of particle elasticity.

[0241] While individual protein changes in the corona are meaningful, the current understanding of the protein corona is that these biomolecules function in networks rather than in isolation, and as such, it is important to consider upregulation of pathway and gene categories in addition to individual genes. Tomak et al., 2021. Pathway enrichment analysis using the hallmark database revealed significant changes as a function of elasticity (FIG. 5d-FIG. 5e). Corresponding gene ontology results can be found in FIG. 14. FIG. 5e shows that pathways upregulated on 5000 kPa particles include coagulation, complement, and UV response, while FIG. 5e shows that downregulated pathways include coagulation, androgen response, mTORC1 signaling, and xenobiotic metabolism. Of particular relevance to the in vivo clearance and biodistribution findings of this study are the changes in coagulation and complement. Both coagulation and complement pathways have been identified as some of the main drivers of opsonization and macrophage uptake by phagocytosis. Panico et al., 2022.

[0242] The finding that the complement pathway is upregulated in 5000 kPa compared to 50 kPa particles would offer a potential explanation for the increase in macrophage uptake and decrease in clearance times observed in the stiffer particles. Interestingly, the coagulation pathway is both significantly upregulated and downregulated when comparing the corona of 50 kPa to 5000 kPa particles. A breakdown of the clusters of genes that are upregulated or downregulated can be found in FIG. 13. More research is needed to determine how individual and clusters of genes in the coagulation pathway influence particle clearance and biodistribution.

[0243] In Vivo T-cell Stimulation45 43105.601_P18311-02

[0244] Finally, we tested our PEG aAPC particle platform in a murine model of adoptive cell transfer to determine the effect of aAPC elasticity on antigen-specific T-cell activation in vivo (FIG. 6a). aAPCs of varying elasticities were conjugated with Dbgp100 and anti-CD28. Particles were then mixed with primary CD8+ T cells from a PMEL, Overwijk et al., 1998, mouse stained with a proliferation dye (CTV) and injected into a lymphodepleted C57 / BL6 mouse. Blood samples were collected 3 days after injection, and secondary lymphoid organs were harvested 7 days after injection. Samples were then stained for PMEL surface marker Thy1.1 and analyzed via flow cytometry to determine the frequency of proliferated PMEL (Thy1.1+) cells. Figs. 6b-c represent results from blood samples collected on day 3 after injection. On day 3, all particle groups showed a significant decrease in CTV intensity, indicating unstimulated PMEL T cells remained circulating only in the no treatment group (FIG. 6d). There was a significantly higher frequency of CD8+ T cells only in the intermediate elasticity group (850 kPa), and while trends in frequency of Thy1.1+ cells were consistent with the results of CD8+ cells in the blood, there was no statistical significance between groups (FIG. 6c). On day 7, both splenic CD8+ T-cell frequency (FIG. 6f) and the frequency of splenic Thy1.1+ T cells (FIG. 6g) were significantly increased only in the 850-kPa group compared to the no treatment control. In the lymph nodes, all groups showed significantly lower CTV signal than in the no treatment control (FIG. 6j), while only the softest group (50 kPa) showed significant frequency of Thy1.1+ cells, with a decreasing trend from 50 kPa-5000 kPa (FIG. 6i). Overall, proliferation was significantly highest in the intermediate elasticity group (850 kPa) in both the blood and spleen. This biphasic response in vivo may be due to the competing functions of elasticity that we have observed, with harder aAPCs generating higher proliferation of T cells in vitro, but also having reduced circulation half-life in vivo. Interestingly, the softest group (50 kPa) showed the most significant proliferation in the lymph nodes.

[0245] Summary

[0246] Using the presently disclosed PEG aAPC platform, we were able to thoroughly investigate the impact of particulate aAPC elasticity on in vitro and in vivo antigen-specific T-cell activation across a range of particle elasticities (50 kPa–5000 kPa). In vitro, elasticity drove T-cell proliferation, differentiation, and, to a lesser extent, functionality. Harder particles (5000 kPa) enabled higher T-cell proliferation and skewed T-cell differentiation toward memory phenotypes at low particle doses. Softer particles (50 kPa) induced poor proliferation but skewed T cells toward46 43105.601_P18311-02an effector phenotype and significantly increased TNF-α production. In vivo, softer particles circulated approximately 5x longer than harder particles and preferentially accumulated in the lungs while harder particles were cleared quickly and preferentially accumulated in the liver. In an in vivo model of antigen-specific adoptive T-cell transfer, the intermediate elasticity particle group (850 kPa) outperformed the other groups and had the highest proliferation of antigen-specific T cells in the blood and the spleen. The softest group (50 kPa) had the highest T cell proliferation in the lymph node.

[0247] We investigated three possible mechanisms as potential explanations for the trends seen in our results: T cell binding, macrophage uptake, and protein corona modulation. aAPC elasticity significantly impacted all three mechanisms, with harder particles binding more efficiently to T cells and macrophages, thus offering a potential explanation for the higher T cell activation and macrophage uptake seen in harder particles. We also found significant changes in the magnitude and composition of the protein corona formed around aAPCs depending on their elasticity, and trends in protein expression indicate complement proteins and ICAM-1 may be important mechanistic pathways modulated by aAPC elasticity in vivo through differential protein corona formation. Taken together, these results underscore the importance of considering elasticity as an essential design criterion the in vivo interactions between particulate biomaterials and cellular surfaces, and in particular, for the engineering of particulate aAPC therapeutics. Likewise, we have uncovered several possible mechanisms that may be influenced by particle elasticity which require further exploration and may be useful for a range of biomaterial applications.

[0248] Experimental

[0249] Particle Synthesis

[0250] Particles were synthesized via a water-in-oil emulsion method. To prepare the water phase of the emulsion, 0.15, 0.3, or 0.5 volume fraction (Vf) of poly(ethylene glycol) diacrylate (PEG) (Mn 700 Da, Sigma Aldrich; St. Louis, MO) was combined with 45 mg of acrylic acid N- hydroxysuccinimide ester (Sigma Aldrich; St. Louis, MO), 30 µL of 2-carboxyethyl acrylate (Sigma Aldrich; St. Louis, MO), and volume was brought up to 3 mL with Millipore water. For uptake and binding studies, a crystal of methacryloxyethyl thiocarbamoyl rhodamine B (Polysciences, Inc.; Washington PA) was added to the mixture to make the particles fluorescent. The oil phase consisted of a mixture of 900 mg Span80 (Sigma Aldrich; St. Louis, MO), 300 mg Tween80 (Sigma Aldrich; St. Louis, MO) and 45 mL of cyclohexane (Sigma Aldrich; St. Louis,47 43105.601_P18311-02MO). The water phase was added to the cyclohexane mixture and emulsified by a T-25 digital ULTRA-TURRAX IKA tissue homogenizer (IKA Works; Wilmington, NC) at a speed of 8,000, 14,000, or 15,000 rpm for 0.15, 0.3, or 0.5 Vf PEG particles, respectively. 300 µL of 2-hydroxy- 2-methylpropriophenone (Sigma Aldrich; St. Louis, MO) was immediately added dropwise to the emulsion. The entire solution was then poured into a 35-mm glass petri dish and placed directly under a UV BLAK-RAY B100APR lamp (Analytik Jena US; Upland, CA) for 10-15 minutes to crosslink emulsified PEG droplets into gel particles. After crosslinking, the solution was transferred to a 50 mL conical tube and centrifuged at 3200 × g for 5 min. The cyclohexane supernatant was discarded, and particles were resuspended in ethanol to remove any leftover surfactant. Particles were then centrifuged again and washed twice with water. After washing with water, particles were passed through a 40-µm filter to remove any large solid PEG debris. The particles were then centrifuged once more and resuspended in 3 mL of water. To determine particle concentrations, 200 µL of solution was transferred to a pre-weighed microcentrifuge tube and lyophilized overnight. The tube was then weighed, and concentration of particles was determined via the dry weight of PEG material / 200 µL of solution. The PEG stock suspension was diluted to 5 mg / mL, aliquoted, and stored at -20 ºC for up to 3 months.

[0251] Particle Characterization

[0252] To confirm formation of spherical particles, samples of the PEG particle suspension were imaged under a Hitachi 7600 transmission electron microscope (TEM) (Hitachi High-Tech; Tokyo, Japan). Samples were transferred to a carbon film 400-mesh copper grid (Electron Microscopy Sciences; Hatfield, PA), dried overnight, then imaged via TEM. To determine particle diameter, polydispersity index (PDI), and zeta potential, particles were analyzed via dynamic light scattering (DLS) using a ZetaSizer Pro (Malvern Panalytical Ltd; Malvern, UK).

[0253] Bulk hydrogel modulus was measured via an Ares G2 oscillatory shear rheometer. Bulk gels 8 mm in diameter were formed by mixing the PEG solutions described above at 0.1-0.75 Vf with 1% 2-methyl-propiphenone, pouring into 8-mm PDMS molds, and incubating under UV light for 5 minutes. Gels were then removed from the PDMS molds and placed on the lower plate of the rheometer. Gels were subjected to 0.1-50% oscillatory strain sweeps at 1-Hz oscillation frequency at room temperature to obtain the equilibrium elastic storage modulus (Ge) at tan(δ) = 1. Zuidema et al., 2014.

[0254] Protein Conjugation48 43105.601_P18311-02

[0255] Particles were conjugated with signal 1 (rat anti-mouse CD3 or mouse Dbgp100) and costimulatory signal 2 (hamster anti-mouse CD28) (Bio X Cell; Lebanon, NH) to create PEG aAPCs. 5 µg of each signal was mixed together and added to 1 mg of particles at 2 mg / mL of particles in PBS and placed on a rotator at 4 ºC for 24 hours. After 24 hours, particles were removed from the rotator and washed 3x with PBS. Particles were then stored at -20 ºC until use. To measure the amount of each antibody conjugated to the PEG particles, fluorescent secondary antibodies for each primary antibody were mixed at 5-µg secondary / mg aAPC. Secondary antibodies were added to aAPCs at 2 mg aAPC / mL PBS and incubated for one hour on a rotator at room temperature. aAPCs were then washed 3x with PBS to remove excess secondary antibodies. Sample fluorescence was then measured via a Biotek Synergy 2 Plate Reader (Agilent Technologies; Santa Clara, CA) and compared to a standard secondary antibody curve to determine the amount of each signal per 1-mg particles.

[0256] In Vitro T-Cell Stimulation

[0257] All animal experiments were conducted following the guidelines of the Johns Hopkins University Animal Care and Use Committee (ACUC). Primary mouse T cells were freshly isolated from PMEL or C57 / BL6 mice between 8-14 weeks of age for Dbgp100 antigen-specific or nonspecific stimulations, respectively (Jackson Labs; Bar Harbor, ME). Mice were humanely euthanized, and spleens and lymph nodes were collected. Spleens and lymph nodes were passed through a 100 µm cell filter then treated with 4mL of ACK lysis buffer for 1 minute to lyse red blood cells. The ACK lysis was promptly quenched with 46mL of PBS, and cells were centrifuged at 500 x g. Cells were then resuspended in 50 mL of PBS and passed through a second 100-µm cell filter to separate any remaining debris from cell lysis. CD8+ T cells were isolated from the resulting lymphocyte suspension using a Miltenyi CD8a+ Cell Isolation Kit following the manufacturer’s instructions (Miltenyi Biotec; Auburn, CA). For proliferation studies, a portion of the CD8+ T cells were labeled with CellTrace Violet (CTV) proliferation dye (ThermoFisher Scientific; Waltham, MA).100,000 cells were then mixed with either 1 mg, 0.1 mg, or 0.01 mg of aAPCs and cultured in B’ media (RMPI supplemented with L-glutamine, 1% non-essential amino acids, 1% MEM vitamin solution, 1% sodium pyruvate, 0.1% β-mercaptoethanol, 1% 10 mg / mL penicillin / streptomycin, 10% fetal bovine serum (FBS), and 1% T cell growth factor cocktail). A small portion of the cells were stained with calcein AM viability dye (Thermo Fisher Scientific; Waltham, MA), plated, and analyzed to get baseline object counts using a CellInsight CX7 Pro49 43105.601_P18311-02HCS Content Analysis Platform (Thermo Fisher Scientific; Waltham, MA). After three days of incubation, the labeled cells were collected and analyzed via flow cytometry. After seven days of incubation, the unlabeled cells were stained with calcein and analyzed via the CX7. Fold proliferation was calculated as # objects per field 7 days after plating / # objects per field immediately after plating.

[0258] For phenotyping studies, unlabeled cells after 7 days of incubation with PEG aAPCs were passed through a 30-µm filter and stained at 4 ºC for 30 minutes for viability, CD62L, and CD44 (Biolegend; San Diego, CA). Cells were washed then analyzed via flow cytometry to determine the amount of naïve (CD62L+, CD44-), effector (CD62L-, CD44+), and memory (CD62L+, CD44+) CD8+ T cells.

[0259] For the T-cell functionality assay, unlabeled cells after 6 days of incubation with PEG aAPCs were passed through a 30-µm filter and split into two groups: restimulation and no stimulation. Two solutions of 1:100 FITC anti-CD107a (Biolegend; San Diego, CA), 0.2:100 GolgiStop and GolgiPlug Protein Transport Inhibitors (BD Biosciences; San Jose, California) were prepared in B’ media.0.2:100 of 1X cell stimulation cocktail (Thermo Fisher Scientific; Waltham, MA) was added to the solution intended for the restimulation group.100 µL of solution was added to each well and incubated at 37 ºC for 6 hours. After 6 hours, cells were washed and stained with 1:100 BV CD8a (Biolegend; San Diego, CA) and 1:1000 LIVE / DEAD Fixable Yellow (Thermo Fisher Scientific; Waltham, MA) at 4 ºC for 30 minutes. Cells were then washed and fixed overnight with 100 µL of Cytofix / Cytoperm Fixation and permeabilization solution (BD Biosciences; San Jose, California). Cells were then washed with 1X BD PERM / Wash buffer (BD Biosciences; San Jose, California) and stained with 1:100 APC-Cy7 anti-IFN-γ, APC anti-IL-2, and PE anti-TNFα (Biolegend; San Diego, CA) for 1 hour at 4 ºC. Cells were then washed and analyzed via flow cytometry. Cells were gated using the unstimulated group as a negative control (FIG. 9).

[0260] Particle Cell Uptake and Binding

[0261] RAW 264.7 murine macrophages were plated at 35,000 cells / well in a 96-well plate and cultured in DMEM + L-glutamine supplemented with 10% FBS and 1% penicillin / streptomycin. 1 mg, 0.1 mg, 0.01 mg, and 0.001 mg of fluorescent particles were split across 6 wells each. One half of the samples were immediately placed at 4 ºC to prevent cellular uptake and one half of samples were incubated at 37 ºC for uptake conditions. After 1 hour of incubation, cells were50 43105.601_P18311-02washed 3x with PBS and trypsinized. Cells were then resuspended in FACS buffer (PBS + 2% FBS + 0.08% sodium azide), stained for viability, and analyzed via flow cytometry for binding (4 ºC) and uptake (37 ºC).

[0262] To measure specific and nonspecific binding of particles to primary murine T cells, primary T cells were isolated from C57BL / 6 mouse spleens and lymph nodes using a Pan T cell isolation kit following the manufacturer’s instructions (Miltenyi; Auburn, CA). Cells were split into two groups: specific and nonspecific binding. The nonspecific binding group was then treated with 1 µg of soluble anti-CD3 and 1µg of soluble anti-CD28 per well to block aAPC binding sites. Cells were then plated at 50,000 cells / well and incubated with 1 mg, 0.1 mg, 0.01 mg, or 0.001 mg of anti-CD3 / anti-CD28 fluorescent aAPCs at 37 ºC for 1 hour. After incubation, cells were washed 3x, stained with a viability dye, and analyzed via flow cytometry.

[0263] In Vivo Clearance and Biodistribution

[0264] PEG particles were fabricated as described above with an added 100 µL of 5-mg / mL LI- COR 800CW infrared carboxylate dye (LI-COR Biosciences; Lincoln, NE) to the liquid PEG solution before crosslinking to label the particles for in vivo imaging. 1 mg of labeled particles in 100 µL of PBS was injected retro-orbitally into a C57 / BL6 mouse under anesthesia (n = 3-5 per group). After injection, blood was collected from the retro-orbital sinus at 10 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, and 4 hours using Miro-Hematocrit Capillary Tubers (Thermo Fisher Scientific; Waltham, MA). Tubes were imaged under a LI-COR Pearl Trilogy imaging system (LI-COR Biosciences; Lincoln, NE). After 8 hours, mice were euthanized, and liver, spleen, lungs, heart, and kidneys were excised and imaged using the LI-COR Pearl Trilogy system to determine particle biodistribution. Images of the blood in the hematocrit tubes were normalized across time points using the LI-COR system software and exported. NIH ImageJ, Abramoff et al., 2004, was used to measure the mean fluorescence intensity of each tube, and each tube was normalized to the 10 minute sample to account for variation in initial particle fluorescence across groups. Clearance plots were made in GraphPad Prism (GraphPad Software, Inc.; San Diego, CA) and fitted with a one-phase decay curve to generate half-life data. To quantify biodistribution, each organ was placed together for each mouse and imaged using the LI-COR Pearl Trilogy system. NIH ImageJ was used to measure the mean fluorescence of each organ, and the organs were normalized to total fluorescence in each animal.

[0265] Protein Corona Analysis51 43105.601_P18311-02

[0266] 50-kPa, 850-kPa, and 5000-kPa particles were treated with 0.1-M TRIS buffer at pH 8 for 10 minutes at room temperature to quench surface NHS via NHS hydrolysis. Quenched particles were then washed 3 times and incubated with 50 µL of normal mouse serum (Bio-Rad Laboratories; Hercules, CA) for 1 hour at 37 ºC. Particles were then washed 3 times to remove any unbound protein. 50 µL of PreOmics iST prep kit lysis buffer (PreOmics GmbH; Planegg, Germany) was added to the particles and samples were heated to 95 ºC for 5 min to remove proteins from particles. Particles were then centrifuged and the supernatant containing the protein corona components was stored at -80 ºC for further processing.

[0267] After the proteins were separated from the particles, samples were processed for LC-MS analysis using a PreOmics iST prep kit following the manufacturer’s instructions (PreOmics GmbH; Planegg, Germany). Peptides were separated using a 60-minute gradient from 4-30% buffer B (buffer A: 0.1% formic acid, buffer B: 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 nL / minute. The mass spectrometer was set to acquire spectra in a data-dependent acquisition (DDA) mode. Briefly, the full MS scan was set to 300-1,200 m / z in the Oribitrap Exploris 480 with a resolution of 120,000 (at 200 m / z) and an AGC target of 5x105. MS / MS was performed in the ion trap using the top speed mode (2 seconds), an AGC target of 1x104, and an HCD collision energy of 35.

[0268] Raw data was searched using the FragPipe computational platform, Yu et al., 2020; Polasky et al., 2020; Teo et al., 2021; Yu et al., 2020; Chang et al., 2020; Geiszler et al., 2021; Tsou et al., 2015, (version 19.1). The final protein.tsv report was analyzed using the FragPipe- Analyst tool and visualized using Quickomics. Gao et al., 2021. The protein identifications and corresponding label-free quantification values were obtained by analyzing the raw data files using FragPipe with standard in-house parameters. Statistical analysis was performed based on the combined protein.tsv file, which involved filtering out contaminant proteins and removing proteins that were not consistently identified or quantified under the same conditions. The MaxLFQ intensity values were transformed to log2 scale, and missing values were imputed using the Missing not At Random (MNAR) method, which utilizes random draws from a left-shifted Gaussian distribution of 1.8 standard deviations apart with a width of 0.3. Differential expression analyses were conducted using protein-wise linear models and empirical Bayes statistics. The Limma package from R Bioconductor was utilized to generate a list of differentially expressed proteins for each pairwise comparison, with a significance cutoff of an adjusted p-value of 0.152 43105.601_P18311-02(Benjamini-Hochberg method) and a [log2 fold change] of 1 applied to determine significantly regulated proteins in each comparison.

[0269] In Vivo T cell Stimulation

[0270] 50-kPa, 850-kPa, and 5000-kPa aAPCs were evaluated for efficacy in vivo by intravenous injection and adoptive transfer of CD8+ PMEL T cells into C57 / BL6 mice. One day prior to injection, sublethal irradiation (500cGy) was applied to C57 / BL6 mice using an MSD Nordion Gammacell irradiator (Johns Hopkins Molecular Imaging Center) to induce transient lymphopenia. The next day, 1 × 106Thy1.1+ PMEL CD8+ T cells were isolated from PMEL spleens and lymph nodes, labeled with CellTrace Violet, and mixed with 2-mg aAPCs for one hour on ice. After incubation, the aAPC + cell mixture was injected retro-orbitally into Thy1.2+ C57BL / 6 mice (n = 3-4 per group). Three days after injection, blood samples were collected, treated with ACK lysis buffer for 1 minute on ice, and stained with PE anti-Thy1.1, APC anti- CD8, and LiveDead near IR for 30 minutes at 4 ºC. After staining, samples were washed 3 times with FACS buffer and analyzed via flow cytometry to determine the percentage of CD8+ T cells / live cells, the percentage of Thy1.1+ cells / live cells, and the CTV fluorescence intensity of live cells in the sample. Seven days after injection, mice were euthanized, and spleens and lymph nodes were harvested from each mouse. Spleens and lymph nodes were mashed and passed through a 100-µm cell filter. Spleens were then treated with 4 mL of ACK lysis buffer for 1 minute to lyse red blood cells. The ACK lysis was promptly quenched with 46 mL of PBS and cells were centrifuged at 500 xg. Cells were then resuspended in 50 mL of PBS and passed through a second 100-µm cell filter. All lymphocytes were then stained and analyzed via flow cytometry using the same method described above for blood cells on day 3. All animal studies were performed using a protocol approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.

[0271] Statistical Analysis

[0272] For all experiments in this study excluding protein corona analysis, GraphPad Prism 9 was used for statistical analysis with p<0.05 considered significant. All in vitro cell experiments were performed with n = 3-4 biological replicates. For in vivo experiments, n = 3-5 mice were used per group. T-tests, One-way ANOVA, Two-way ANOVA were performed as appropriate with Tukey’s post-test to determine significance between groups. In Figures asterisks are used to denote53 43105.601_P18311-02various levels of significance: * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; and **** indicates p<0.0001. EXAMPLE 2

[0273] Nanoparticle Elasticity and Shape Influence Polymeric Artificial Antigen Presenting Cell Effectiveness

[0274] Introduction

[0275] With recent clinical successes in the field of immunotherapy, Hickey et al., 2018, there has become a need to develop new biotechnologies that can better modulate the behavior immune cells. Of specific interest to the field of immunotherapy due to their role in generation of adaptive immunity is the interaction between antigen presenting cells (APCs) and T cells. Ben-Akiva et al., 2017.

[0276] A particle can be constructed to function as an artificial cell, in this case an artificial antigen presenting cell (aAPC), by mimicking features of natural biological APCs. While certain key aAPC parameters, such as ligand surface density, have been investigated and found to be important for aAPC / T-cell stimulation, the physical parameters of the particle have not been well investigated, yet are critical features of acellular encounters. Hickey et al., 2019; Meyer et al., 2015; Ben-Akiva et al., 2018.

[0277] Previous literature indicates that shape and size play a major role in the effectiveness of particulate aAPC systems. Ben-Akiva et al., 2017; Meyer et al., 2015; Meyer et al., 2018. There is evidence that elasticity exerts influence over the proliferation and functionality of T cells in 2D aAPC systems, De La Zerda et al., 2018; Hickey et al., 2019, and particle pharmokinetics, Guo et al., 2018; Anselmo and Mitragotri, 2017; Anselmo et al., 2015, but the impact on 3D particulate aAPC effectiveness in relation to the combined parameters of particle size, shape, and stiffness has not been previously investigated.

[0278] To investigate the impact of particulate aAPC elasticity and its influence on other key parameters, such as size and shape, the poly(ethylene glycol) (PEG)-based platform described in Example 1 was expanded to have tunable size and shape in addition to tunable elasticity. After determining the impact of elasticity on spherical microparticle PEG aAPCs, we sought to elucidate the relationships between elasticity, size, and shape on aAPC particle effectiveness. We used our54 43105.601_P18311-02PEG aAPC platform to compare the effects of nanoparticle elasticity and anisotropy across the same elasticity range as in Example 1 (50 kPa–5000 kPa).

[0279] Materials and Methods

[0280] Spherical PEG nanoparticle synthesis: PEG particles were synthesized using a single water-in-oil emulsion approach with homogenization micro scale spherical particles (described in Example 1) and sonication (60 % amplitude on ice for 2 minutes) resulting in nano scale spherical droplets. Droplets were then exposed to UV light for 5-15 minutes to crosslink droplets into solid particles. Elasticity was altered by modulating the volume fraction of PEG in each particle. The resulting elasticity range (50-5000kPa) was determined via bulk hydrogel rheometry described in Example 1.

[0281] Ellipsoidal PEG nanoparticle synthesis: To create ellipsoidal particles, PEG particle synthesis was adapted to a 1D thin film stretching procedure. Meyer et al., 2015. In brief, particles were emulsified via homogenizer or sonicator as described in Example 1, then frozen under continuous stirring at 4 °C, as Mn= 700 PEG has a freezing temperature of 15 °C (FIG. 17). Solid nanodroplets were then spun down at 20,000 × g at 4 °C using a floor ultracentrifuge and resuspended in chilled poly(vinyl alcohol) (PVA) film casting solution (8.5 mL glycerol + 50 g PVA + 500mL MilliQ water). The resulting solution was then poured into rectangular cassettes and allowed to dry at 4 °C to create a thin film. Films were then mechanically stretched at 70 °C in one direction to create anisotropic PEG nano droplets. Stretched films were then exposed to UV light for 10 minutes to crosslink droplets into solid, ellipsoidal particles. Films were then dissolved in MilliQ water and particles were washed 2× with water.

[0282] Transmission electron microscopy: For transmission electron microscopy (TEM), NPs were formulated as above and then diluted 1:10 serially in Millipore water. 5 µL of each sample was added to a carbon film 400 mesh copper grid (Electron Microscopy Sciences; Hatfield, PA), dried overnight, then imaged via a Hitachi 7600 transmission electron microscope (Hitachi High- Tech; Tokyo, Japan). NIH ImageJ was used to analyze size and shape descriptors of ellipsoidal particles (area, max length, and aspect ratio.

[0283] Protein Conjugation: Particles were conjugated with signal 1 (rat anti-mouse CD3 or mouse Dbgp100) and costimulatory signal 2 (hamster anti-mouse CD28) (Bio X Cell; Lebanon, NH) to create PEG aAPCs. 5 µg of each signal was mixed together and added to 1 mg of particles at 2 mg / mL of particles in PBS and placed on a rotator at 4 °C for 24 hours. After 24 hours, particles55 43105.601_P18311-02were removed from the rotator and washed 3x with PBS. Particles were then stored at -20 ºC until use. To measure the amount of each antibody conjugated to the PEG particles, fluorescent secondary antibodies for each primary antibody were mixed at 5-µg secondary / mg aAPC. Secondary antibodies were added to aAPCs at 2-mg aAPC / mL PBS and incubated for one hour on a rotator at room temperature. aAPCs were then washed 3x with PBS to remove excess secondary antibodies. Sample fluorescence was then measured via a Biotek Synergy 2 Plate Reader (Agilent Technologies; Santa Clara, CA) and compared to a standard secondary antibody curve to determine the amount of each signal per 1 mg particles as well as the conjugation efficiency (100 × µg antibody conjugated / µg antibody added).

[0284] In Vitro T Cell Stimulation: Primary mouse T cells were isolated from fresh PMEL or C57 / BL6 mice between 8-14 weeks of age for Dbgp100 antigen-specific or nonspecific stimulations, respectively (Jackson Labs; Bar Harbor, ME). Spleens and lymph nodes were passed through a 100-µm cell filter then treated with 4 mL of ACK lysis buffer for 1 minute to lyse red blood cells. The ACK lysis was promptly quenched with 46 mL of PBS and cells were centrifuged at 500 × g. Cells were then resuspended in 50 mL of PBS and passed through a second 100-µm cell filter to separate any remaining debris from cell lysis. CD8+ T cells were isolated from the resulting lymphocyte suspension using a Miltenyi CD8a+ Cell Isolation Kit following the manufacturer’s instructions (Mitlenyi; Auburn, CA). For proliferation studies, a portion of the CD8+ T cells were labeled with CellTrace violet (CTV) proliferation dye (ThermoFisher Scientific; Waltham, MA).100,000 cells were then mixed with either 1 mg, 0.1 mg, or 0.01 mg of aAPCs and cultured in B’ media (RMPI supplemented with L-glutamine, 1% non-essential amino acids, 1% MEM vitamin solution, 1% sodium pyruvate, 0.1% β-mercaptoethanol, 1% 10 mg / mL penicillin / streptomycin, 10% fetal bovine serum (FBS), and 1% T cell growth factor cocktail). A small portion of the cells were stained with calcein AM viability dye (Thermo Fisher Scientific; Waltham, MA), plated, and analyzed to get baseline object counts using a via a CellInsight CX7 Pro HCS Content Analysis Platform (Thermo Fisher Scientific; Waltham, MA). After three days of incubation, the labeled cells were collected and analyzed via flow cytometry. After seven days of incubation, the unlabeled cells were stained with calcein and analyzed via the CX7. Fold Proliferation was calculated as # objects per field on day 7 / # objects per field on day 0.

[0285] For phenotyping studies, unlabeled cells after 7 days of incubation with PEG aAPCs were passed through a 30-µm filter and stained for viability, CD62L, and CD44. Cells were washed then56 43105.601_P18311-02analyzed via flow cytometry to determine the amount of naïve (CD62L+, CD44-), effector (CD62L-, CD44+), and memory (CD62L+, CD44+) CD8+ T cells.

[0286] For the cytokine functionality assay, unlabeled cells after 6 days of incubation with PEG aAPCs were passed through a 30-µm filter and split into two groups: restimulation and no stimulation. Two solutions of 1:100 FITC anti-CD107a (Biolegend; San Diego, CA), 0.2:100 GolgiStop and GolgiPlug Protein Transport Inhibitors (BD Biosciences; San Jose, California) were prepared in B’ media. 0.2:1001X cell stimulation cocktail (Thermo Fisher Scientific; Waltham, MA) was added to the solution intended for the restimulation group.100 µL of solution was added to each well and incubated at 37 ºC for 6 hours. After 6 hours, cells were washed and stained with 1:100 BV CD8a (Biolegend; San Diego, CA) and 1:1000 LIVE / DEAD Fixable Yellow (Thermo Fisher Scientific; Waltham, MA) at 4 ºC for 30 minutes. Cells were then washed and fixed overnight with 100 µL of Cytofix / Cytoperm Fixation and permeabilization solution (BD Biosciences; San Jose, California). Cells were then washed with 1X BD PERM / Wash buffer (BD Biosciences; San Jose, California) and stained with 1:100 APC-Cy7 anti-IFN-γ, APC anti-IL-2, and PE anti-TNFα (Biolegend; San Diego, CA) for 1 hour at 4 ºC. Cells were then washed and analyzed via flow cytometry. Cells were gated using the unstimulated group as a negative control.

[0287] Nanoparticle Cell Uptake and Binding: (as described in Example 1) RAW 264.7 murine macrophages were plated at 35,000 cells / well in a 96-well plate and cultured in DMEM + L- glutamine supplemented with 10% FBS and 1% penicillin / streptomycin. 1 mg, 0.1mg, 0.01mg, and 0.001 mg of fluorescent particles were split across 6 wells each. One half of the samples were immediately placed at 4 ºC to prevent cellular uptake and one half of samples were incubated at 37 ºC for uptake conditions. After 1 hour of incubation, cells were washed 3× with PBS and trypsinized. Cells were then resuspended in FACS buffer (PBS + 2% FBS + 0.08% sodium azide), stained for viability, and analyzed via flow cytometry for binding (4 ºC) and uptake (37 ºC).

[0288] To measure specific and nonspecific binding of particles to primary murine T cells, primary T cells were isolated from C57 / BL6 mouse spleens and lymph nodes using a Pan T cell isolation kit following the manufacturer’s instructions (Miltenyi; Auburn, CA). Cells were split into two groups: specific and nonspecific binding. The nonspecific binding group was then treated with 1 µg of soluble anti-CD3 and 1 µg of soluble anti-CD28 per well to block aAPC binding sites. Cells were then plated at 50,000 cells / well and incubated with 1 mg, 0.1mg 0.01mg, or 0.001 mg of anti-57 43105.601_P18311-02CD3 / anti-CD28 fluorescent aAPCs at 37 ºC for 1 hour. After incubation, cells were washed 3×, stained with a viability dye, and analyzed via flow cytometry.

[0289] In Vivo Clearance and Biodistribution: (as described in Example 1) PEG particles were fabricated as described above with an added 100 µL of 5-mg / mL LI-COR 800CW infrared carboxylate dye (LI-COR Biosciences; Lincoln, NE) to the liquid PEG solution before crosslinking to label the particles for in vivo imaging. 1 mg of labeled particles in 100 µL of PBS was injected retro-orbitally into a C57 / BL6 mouse under anesthesia (n = 3-5 per group). After injection, blood was collected retro-orbitally at 10 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, and 4 hours using Miro-Hematocrit Capillary Tubers (Thermo Fisher Scientific; Waltham, MA).

[0290] Tubes were imaged under a LI-COR Pearl Trilogy imaging system (LI-COR Biosciences; Lincoln, NE). After 8 hours, mice were euthanized and liver, spleen, lungs, heart, and kidneys were excised and imaged using the LI-COR Pearl Trilogy system to determine particle biodistribution. Images of the blood in the hematocrit tubes were normalized across time points using the LI-COR system software and exported. NIH ImageJ, Abramoff et al., 2004, was used to measure the mean fluorescence intensity of each tube, and each tube was normalized to the 10- minute sample to account for variation in initial particle fluorescence across groups. Clearance plots were made in GraphPad Prism (GraphPad Software, Inc.; San Diego, CA) and fitted with a one-phase decay curve to generate half-life data. To quantify biodistribution, each organ was placed together for each mouse and imaged using the LI-COR Pearl Trilogy system. NIH ImageJ was used to measure the mean fluorescence of each organ, and the organs were normalized to total fluorescence in each animal.

[0291] Results and Discussion

[0292] As shown in FIG. 17, the adapted particle synthesis methods from Example 1 resulted in spherical nanoparticles with a size approximately 200 nm as measured by DLS. The ellipsoidal synthesis procedure resulted in anisotropic particles with no significant differences in size and shape distribution across elasticities (FIG. 17E).

[0293] Results from spherical nanoparticle studies reveal that particles conjugate similar amounts of signals 1 and 2 but smaller size corresponds to decreased proliferation (FIG. 18A-FIG. 18B). Elasticity trends, however, remain consistent across different sizes (FIG. 18A-FIG. 18B). In vivo, nanoparticles circulate for much longer than microparticles but exhibit the same trends in58 43105.601_P18311-02elasticity, albeit less dramatic, with softer particles (50 kPa) circulating longer than stiffer particles (5000kPa) (FIG. 19A-FIG. 19B).

[0294] In addition to comparing the effects of spherical PEG particle size and elasticity, we were interested in determining the interplay of the effect of shape and elasticity at the nanoscale. Previously, we have shown that more anisotropic particles are more efficient aAPCs due to their improved ability to circulate longer in the blood and improve T cell stimulation via increase surface area contact. Meyer et al., 2015; Song et al., 2019.

[0295] Thus, we sought to determine if this phenomenon is adjusted in any way by particle elasticity, which we determined in Example 1 plays a major role in aAPC effectiveness. Using the same PMEL / Dbgp100 antigen-specific CD8+ T cell stimulation assay described previously in Example 1, we compared nanospherical PEG aAPCs to nanoellipsoidal PEG aAPCs at 50, 850, and 5000 kPa. The first round of results for this comparison are shown in FIG. 20. The spherical nanoparticles conjugated about 2 times more signal 1 than their ellipsoidal counterparts but had similar conjugation efficiencies of signal 2 (FIG.20C-FIG.20D). On Day 3, (FIG.20A-FIG.20B) there were few differences in CTV proliferation profiles between elasticities and shape. On Day 7, (FIG. 20E-FIG. 20F) Spherical particles appeared to follow similar trends as seen in spherical microparticles, with increasing stiffness corresponding to increasing T cell activation, while ellipsoidal particles seemed to have the opposite trend.

[0296] While it was difficult to determine the effects of shape combined with elasticity on T cell proliferation, the effects of shape combined with elasticity on T cell phenotype and functionality were more clearly defined. FIG. 21A-FIG. 21B shows elasticity influences spherical, but not ellipsoidal T cell phenotype. In spherical particles, higher stiffness correlated with higher percentages of effector cells, while in ellipsoidal particles, there was little difference across elasticity. This is an interesting finding, and may be explained by the surface area contact hypothesis we put forward in our previous work. Meyer et al., 2015; Meyer et al., 2018.

[0297] We have established that surface area contact is a main driver of signal and T cell receptor binding, initiating the T cell activation cascade. In the case of the ellipsoidal particles, there is maximized T cell surface area contact, whereas in the spherical particles, T cell surface area contact is limited. Because of this, it is possible that elasticity plays a secondary role to surface area contact. We found in our previous work with microparticle elasticity that density of signals 159 43105.601_P18311-02and 2 may overshadow effects of elasticity, indicating that elasticity effects on T cell phenotype may be weaker than other biomaterial effects.

[0298] FIG. 21B-FIG. 21C show elasticity, but not shape, influences antigen-specific T cell functionality. Similar to findings in PEG microparticle aAPCs (Example 1), we found that both spherical and ellipsoidal particles showed elasticity-driven differences in expression of various cytokines. The most prominent trend was in the expression of TNF-α, which is difficult to interpret due to the pleiotropic effects of TNF-α. It is not clear why elasticity overshadows the role of shape in T cell functionality, but it may indicate that our surface area contact hypothesis does not fully explain the interplay between aAPC elasticity and shape on T cell activation for adoptive transfer. More research is needed to fully describe this relationship.

[0299] Finally, we wanted to investigate the effects of shape and elasticity on particle clearance. The first step to doing so is investigating the effects of shape and elasticity on macrophage uptake, as macrophage uptake is one of the main drivers of particle clearance by the reticuloendothelial system. Sanchez et al., 2017; Garapaty and Champion, 2017.

[0300] We incubated spherical and ellipsoidal particles at 37 ºC and 4 ºC to simulate conditions for uptake and binding, respectively. In agreement with our previous work with PGLA particles, Meyer et al., 2018, we found that spherical particles were internalized and bound by macrophages at an order of magnitude higher than ellipsoidal particle (FIG. 22A-FIG. 22B). When nanoscale ellipsoidal particles were compared across elasticity, interestingly, the softer nanoparticles were taken up more significantly than the stiffer nanoparticles (FIG.22C-FIG.22D). This is the opposite trend of what we have seen with spherical microparticles in Example 1, and more research is needed both in vitro and in vivo to fully elucidate the interplay of shape, elasticity, and size on PEG aAPC clearance and biodistribution.

[0301] Summary

[0302] aAPC particle size, shape, and elasticity have complex relationships to both each other as well as aAPC effectiveness. Researchers have studied particle size, shape, and elasticity separately, but it is clear from this work that these biomaterial variables have interdependent effects on T cell activation and function, and it would be useful for the field to continue to study these phenomena. While the field has extensively studied the role of chemical properties of aAPCs (proteins on the surface), the finding of this current work suggest that the physical properties of aAPCs (including size, shape and stiffness) are also key design parameters.60 43105.601_P18311-02REFERENCES

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[0376] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.67 43105.601_P18311-02

Claims

THAT WHICH IS CLAIMED:

1. An artificial antigen presenting cell (aAPC) having an immune targeting Signal 1 protein and a costimulatory Signal 2 protein conjugated to a surface thereof, wherein the aAPC comprises a hydrogel having a tunable elasticity.

2. The aAPC of claim 1, wherein the hydrogel has a stiffness ranging from about 50 kPa to about 5,000 kPa.

3. The aAPC of claim 1 or claim 2, comprising a hydrogel particle having a particle size having a range selected from about 100 nm to 4000 nm, 500 nm to 3000 nm, and 1000 nm to 2000 nm.

4. The aAPC of any one of claims 1 to 3, wherein the hydrogel comprises one or more of poly(ethylene) glycol (PEG), a PEG-derivate, alginate, chitosan, collagen, gelatin, hyaluronic acid, polyacrylamide, polycaprolactone, polyester, or polyvinyl alcohol to form the hydrogel.

5. The aAPC of claim 4, wherein the hydrogel has a volume fraction of PEG ranging from about 0.15 to about 0.

5.

6. The aAPC of claim 1, wherein the Signal 1 protein is selected from a major histocompatibility complex (MHC) + antigen peptide and an anti-CD3 antibody.

7. The aAPC of claim 1, wherein the costimulatory Signal 2 protein is selected from an antibody or antigen-binding fragment thereof that specifically binds to CD28, CD80 (B7-1), CD86 (B7-2), B7- H3, 4-1BBL, 4-1BB, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, an antibody or antigen-binding fragment thereof that specifically binds to HVEM, an antibody or antigen-binding fragment thereof that specifically binds to CD40L, an antibody or antigen binding fragment thereof that specifically binds to OX40, and an antibody or antigen- binding fragment thereof that specifically binds to 4-lBB.68 43105.601_P18311-028. The aAPC of claim 7, wherein the costimulatory Signal 2 protein comprises an anti-CD28 (αCD28) antibody.

9. The aAPC of any one of claims 1 to 8, further comprising one or more proteins or small molecules having a binding affinity for one or more blood serum proteins.

10. The aAPC of any one of claims 1 to 9, further comprising an immunomodulatory material.

11. The aAPC of claim 10, wherein the immunomodulatory material comprises a genetic material.

12. The aAPC of any one of claims 1 to 11 further comprising a paramagnetic particle.

13. The aAPC of claim 12, wherein the magnetic particle comprises an iron-dextran particle.

14. The aAPC of any one of claims 1 to 13, further comprising a shape selected from a sphere, a prolate ellipsoid, a tri-axial ellipsoid, and an oblate ellipsoid.

15. An in vitro method for identifying, isolating, or detecting one or more antigen- specific T cells, the method comprising: (a) contacting a plurality of unpurified immune cells comprising one or more antigen- specific T cells with a plurality of aAPCs of any one of claims 1 to 14; (b) separating antigen-specific T cells associated with the plurality of aAPCs from cells not associated with the plurality of aAPCs; (c) recovering antigen-specific T cells associated with the plurality of aAPCs; and (d) expanding the recovered antigen-specific T cells in culture for a period of time to provide a composition comprising antigen-specific T cells.69 43105.601_P18311-0216. The method of claim 15, wherein the plurality of unpurified immune cells comprising one or more antigen-specific T cells are obtained from a sample comprising one or more of a peripheral blood mononuclear cell (PBMC) sample, memory T cells, naive T cells, previously activated T cells, and tumor infiltrating lymphocytes.

17. The method of claim 15 or claim 16, wherein the plurality of unpurified immune cells comprising one or more antigen-specific T cells are obtained from a sample comprising one or more of bone marrow, lymph node tissue, spleen tissue, and a tumor.

18. The method of any one of claims 15 to 17, wherein the plurality of unpurified immune cells are obtained from a patient or a donor.

19. The method of claim 18, wherein the donor comprises a donor who is HLA- matched to an adoptive transfer recipient.

20. The method of claim 18, wherein the plurality of unpurified immune cells are obtained from a patient and the patient has one or more diseases, disorders, or conditions selected from the group consisting of a cancer, an infectious disease, and an autoimmune disease.

21. The method of any one of claims 16 to 20, wherein the one or more antigen- specific T cells are selected from the group consisting of cytotoxic CD4+T cells, CD4+helper T cells, CD8+cytotoxic T lymphocytes, T-helper 17 (Th17) cells, regulatory T cells (Tregs), and combinations thereof.

22. The method of claim 15, wherein the aAPC comprises a paramagnetic particle and the separating of the antigen-specific T cells associated with the plurality of aAPCs from the cells not associated with the plurality of aAPCs is by magnetic separation.

23. A method for treating a disease, disorder, or condition, the method comprising administering to a subject in need of treatment thereof a composition comprising one or more70 43105.601_P18311-02aAPCs of any one of claims 1 to 14 in vivo or one or more antigen-specific T cells prepared by the method of any one of claims 15-22 in vitro.

24. The method of claim 23, wherein the disease, disorder, or condition is selected from a cancer, an infectious disease, and an autoimmune disease.

25. The method of claim 24, wherein the disease, disorder, or condition is a cancer and the one or more antigen-specific T cells comprise cytotoxic T cells specific for one or more tumor-associated peptide antigens to the subject in need of treatment thereof.

26. The method of claim 24, wherein the cancer comprises a solid tumor or a hematological malignancy.

27. The method of claim 26, wherein the cancer is selected from the group consisting of a melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, a neuroblastoma, and a glioma.

28. The method of claim 24, wherein the infectious disease comprises an infectious disease caused by one or more pathogens selected from bacteria, viruses, prions, fungi, parasites, helminths, and combinations thereof.

29. The method of claim 28, wherein the infectious disease is selected from acquired immunodeficiency syndrome (AIDS), hepatitis, a cytomegalovirus (CMV) infection, and post- transplant lymphoproliferative disorder (PTLD).

30. The method of claim 24, wherein the disease, disorder, or condition is an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis,71 43105.601_P18311-02Goodpasture's syndrome, Graves' disease, pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiac disease, and Hashimoto's thyroiditis.

31. A method for diagnosing a disease or a disease state in a subject, the method comprising: (a) obtaining a blood serum sample from the subject; (b) contacting the blood serum sample with a plurality of aAPCs of any one of claims 1 to 14, wherein the plurality of aAPCs includes one or more aAPCs which can be the same or different and / or a plurality of non-APC particles alone or in combination with the plurality of aAPCs; (c) separating one or more blood serum proteins associated with the plurality of aAPCs from blood serum proteins not associated with the plurality of aAPCs; and (d) determining an identity and / or an amount of the one or more blood serum proteins associated with the plurality of aAPCs to diagnose a disease or disease state in the subject.

32. The method of claim 31, wherein the plurality of non-APC particles includes one or more non-APC particles having a same or different elastic modulus.

33. The method of claim 31, wherein the plurality of non-APC particles includes one or more non-APC particles that bind differentially to blood serum protein.

34. A non-viral method for delivering an immunomodulatory material to a T cell, the method comprising administering an aAPC of claim 10 or claim 11 to a cell.72 43105.601_P18311-02

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