Compositions of an artificial lymph node matrix and methods of preparing the same

Abstract

Disclosed are an artificial lymph node (aLN) comprising an extracellular matrix (ECM) hydrogel conjugated with an antigen presenting complex (Signal 1), a co-stimulatory ligand (Signal 2), and T cell-stimulating cytokine (Signal 3) and methods of their use for stimulating one or more T cells and treating a disease, disorder, or condition selected from a cancer, an infectious disease, and an autoimmune disease.

Description

COMPOSITIONS OF AN ARTIFICIAL LYMPH NODE MATRIX AND METHODS OF PREPARING THE SAMERELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Application No. 63 / 553,953 filed on February 15, 2024, the contents of which are herein incorporated by reference.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grants R21CA185819, EB029341, and EB028239 awarded by NIH. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The text of the computer readable sequence listing filed herewith, titled “JHU_42820_601_SequenceListing.xml”, created February 11, 2025, having a file size of 4,534 bytes, is hereby incorporated by reference in its entirety.BACKGROUND

[0004] T cells are a critical mediator of antigen- specific immune responses and are common targets for engineering anti-tumor immunotherapy. To this end, engineering strategies have boosted, directed, and potentiated T cell immune responses for therapeutic applications.Biomaterials scaffolds have previously been used to stimulate antigen presenting cells to elicit antigen- specific immune responses. The structural and molecular features that directly stimulate and expand naive, endogenous, tumor-specific T cells in vivo, however, have not been defined. Accordingly, although such biomaterial scaffolds have been used for the delivery of antigen and boosting of pre-existing T cell responses, they have not been used to directly stimulate and expand naive, endogenous, antigen- specific T cells in vivo as they exhibit either low efficacy or require extensive ex vivo preparation.SUMMARY

[0005] In some aspects, the presently disclosed subject matter provides an artificial lymph node(aLN) comprising an extracellular matrix (ECM) hydrogel conjugated with an antigen presentingcomplex (Signal 1 ), a co-stimulatory ligand (Signal 2), and T cell-stimulating cytokine (Signal 3). In certain aspects, Signal 3 comprises a T cell- stimulating cytokinc / antibody complex.

[0006] In certain aspects, the antigen presenting complex (Signal 1) is selected from a major histocompatibility complex (MHC) molecule, a human leukocyte antigen (HLA) molecule, and an anti-CD3 antibody. In certain aspects, the MHC molecule is a monomer or a dimer. In certain aspects, the antigen presenting complex (Signal 1) is an MHC class I molecule, an MHC class II molecule, or a combination of an MHC class I molecule and an MHC class II molecule. In particular aspects, the antigen presenting complex (Signal 1) is selected from an MHC-Ig dimer or an HLA-Ig dimer. In more particular aspects, the antigen presenting complex (Signal 1) comprises an MHC molecule loaded with a peptide or an HLA molecule loaded with a peptide. In certain aspects, the peptide is selected from GP100: KVPRNQDWL SEQ ID NO: 1; SIY: SIYRYYGL SEQ ID NO: 2; OVA: SIINFEKL SEQ ID NO: 3, and MART-1: ELAGIGILTV SEQ ID NO: 4.

[0007] In certain aspects, the co-stimulatory ligand (Signal 2) comprises an antibody that specifically binds to a protein expressed on a T cell, wherein the protein expressed on a T cell is selected from CD28, CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, HVEM, CD40L, 0X40, and combinations thereof. In particular aspects, the co-stimulatory ligand (Signal 2) is selected from anti-CD28 (a-CD28) antibody, anti-CD27 (a-CD27) antibody, and anti-41BB (ot-41BB) antibody.

[0008] In certain aspects, the co-stimulatory ligand (Signal 2) comprises at least a first antibody that specifically binds to a protein expressed on a T cell and at least a second antibody that specifically binds to a protein expressed on a T cell. In certain aspects, the at least first antibody that specifically binds to a protein expressed on a T cell and the at least second antibody that specifically binds to a protein expressed on a T cell are present in a ratio of first antibody: second antibody selected from about 99:1, 98:2, 97:3, 96:4, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 4:96, 3:97, 2:98, and 1:99. In particular’ aspects, the at least first antibody that specifically binds to a protein expressed on a T cell and the at least second antibody that specifically binds to a protein expressed on a T cell are selected from a-CD28 and a-CD27 or a-CD28 and a-41BB.

[0009] In certain aspects, the T cell-stimulating cytokine is selected from IL-2, IL-4, IL-7, IL- 10, IL-12, IL-12p70, IL-15, IL-21, CXCL10, and gamma interferon (IFN-y). In particular, aspects,the T cell-stimulating cytokine / antibody complex (Signal 3) comprises an anti-IL-2 antibody complexed with IL-2 (IL-2+Ab). In more particular aspects, the anti-IL-2 antibody comprises M AB 602.

[0010] In certain aspects, the hydrogel comprises a hyaluronic acid (HA) hydrogel. In particular aspects, the HA hydrogel comprises a thiol-modified HA hydrogel. In certain aspects, the aLN further comprises one or more adhesion molecules. In particular aspects, the one or more adhesion molecules are selected from arginylglycylaspartic acid (RGD), collagen, and intercellular adhesion molecule 1 (ICAM-1).

[0011] In other aspects, the presently disclosed subject matter provides a microgel comprising the aLN as describe herein.

[0012] In other aspects, the presently disclosed subject matter provides a method for stimulating one or more T cells, the method comprising contacting the one or more T cells with one or more aLNs described herein, or a microgel thereof. In certain aspects, the one or more T cells comprise antigen- specific CD8+ T cells. In particular aspects, the contacting of the one or more T cells with the one or more aLNs occurs in vivo. In more particular aspects, the method comprises administering the one or more aLNs in combination with one or more naive T cells. In even more particular aspects, administering the one or more aLNs in combination with one or more naive T cells creates a T cell-activating niche. In certain aspects, administering the one or more aLNs results in one or more of recruitment of host immune cells, coordination of host immune cells, and providing an immuno- stimulatory microenvironment for antigen-specific T cell activation and expansion. In more certain aspects, the contacting of the one or more T cells with the one or more aLNs induces in vivo antigen- specific CD8+ T cell stimulation without ex vivo priming or expansion. In certain aspects, the contacting of the one or more T cells with the one or more aLNs enables in situ manipulation of antigen- specific responses for an immunotherapy.

[0013] In other aspects, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition, or providing prophylactic protection to immunosuppressed patients, the method comprising administering a therapeutically effective amount of an artificial lymph node (aLN) as described herein or a microgel thereof, to a subject in need of treatment thereof. In certain aspects, the method further comprises administering naive, wild type CD8+ T cells in combination with one or more aLNs. In particular aspects, the disease, disorder, orcondition is selected from a cancer, an infectious disease, and an autoimmune disease. In more particular aspects, the cancer comprises a solid tumor or a hematological malignancy. In even 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 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. In certain aspects, administering the naive, wild type CD8+ T cells in combination with the one or more aLNs results in activation and expansion of tumor-targeted T cells that kill target cancer cells, slows tumor growth, and increases survival.

[0014] 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 aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0015] 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.

[0016] 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:

[0017] FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, FIG. IF, and FIG. 1G demonstrate that conjugation of an IL2+Ab complex allows for sustained cytokine signaling and improved T cell proliferation and poly functionality. FIG. 1A) Schematic diagram showing the components of the HA hydrogel-based aLN (created with BioRender). FIG. IB) Graph of the Day 3 CFSE dilution and FIG. 1C) Day 7 fold expansion of CD8+ T cells stimulated on hydrogel surfaces with different cytokine formats. The graph displays the comparison between HA hydrogel conjugated to anti-IL-2 antibody complexed with IL-2 (IL-2+Ab), with soluble IL-2 and without IL-2 (analyzed by one-way ANOVA; error bars show s.e.m.; n = 5-8, CFSE plot is a representativestaining, and FIG. 8D shows statistical replicates). FIG. ID) Day 3 CFSE and FIG. IE) Day 7 proliferation comparison of HA hydrogel conjugated with IL- 2+ Ab and HA hydrogel with soluble IL-2 at 0.1, 1, and 10 pg / mL (analyzed by two-way ANOVA; error bars show s.e.m.; n = 5-8, CFSE plot is a representative staining where FIG. 8D, FIG. 8E show statistical replicates). FIG. IF) Percentage of SIY antigen- specific (SIY+) CD8+ T cells of the total CD8+ T cells on day 7, which were stimulated on the HA hydrogel conjugated to IL-2+Ab or the HA hydrogel fed continuously by a cocktail of cytokines (error bars show s.e.m.; n = 6-9). FIG. 1G) Polyfunctionality as determined by SIY+ CD8+ T cells that are co-positive for 1, 2, or 3 of IFNy, TNFa, CD 107a markers on day 7 by intracellular staining (averaged pie chart from 3 replicates, *p < 0.05 between conditions expressing all 3 markers, paired student’s t-test, two-tailed, n = 6 9).

[0018] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F, and FIG. 2G illustrate various aspects of generating injectable aLNs for CD8+ T cell activation and stimulation efficacy ex vivo. FIG. 2A) Schematic illustrating the process of generating injectable aLN (created with BioRender). FIG. 2B) Immunofluorescent image of an aLN particle. FIG. 2C) 7-Day fold expansion of T cells stimulated by aLN with varying storage modulus (G’) (n = 3). FIG. 2D) 7- Day fold expansion of T cells stimulated by aLN with varying signal densities (n = 3). FIG. 2E) Fold expansion over a seven-day culture comparing stimulation with Dynabeads, aLN, and no stimulation (n = 3). FIG. 2F) Representative flow plot and theoretical fold expansion of B6 CD8+ T cells after being plated with OVA-specific aLN (n = 4). FIG. 2G) Representative flow plot and theoretical fold expansion of B6 CD8+ T cells after being plated with SIY-specific aLN (n = 4).

[0019] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, and FIG. 3H demonstrate that in vivo CD8+ T cell activation by aLN leads to functional antigen- specific killing. FIG. 3A) Schematic diagram showing the in vivo killing assay of adoptive T cell therapy via subcutaneous (s.c.) or intraperitoneal (i.p.) injection. FIG. 3B) Percent killing of target cell population within the LN on day 7 by different treatment groups (error bars show s.e.m.; *p < 0.05, ***p < 0.001, n = 4, one-way ANOVA with Tukey’s post-test). FIG. 3C) Thyl.l percentage of CD8+ T cells from the LN from mice in treatment groups indicated on day 7 after treatment (error bars show s.e.m.; *p < 0.05, n = 4, one-way ANOVA with Tukey’s post-test). FIG. 3D) Mean fluorescent intensity (MFI) of CellTrace Violet (CTV) labeled adoptivelytransferred Thyl .1 +, CD8+ T cells within the LN by different treatment groups on day 7 (error bars show s.c.m.; ***p < 0.001, n = 4, one-way ANOVA with Tukcy’s post-test). FIG. 3E) Percent killing of target cell population within the LN on day 21 by different treatment groups (error bars show s.e.m.; *p < 0.05, one-way ANOVA with Tukey’s post-test, n = 3). FIG. 3F) CD45.2+ percentage of CD8+ T cells in the LN from mice in treatment groups indicated on day 7 after treatment. FIG. 3G) Percent killing of target cell population (OVA-pulsed splenocytes) within the lymph node (LN) on day 7 by different treatment groups (F, G *p < 0.05, **p < 0.01; n = 4; student’s T test). FIG. 3H) Granzyme B expression of naive OT I cells, ex vivo-activated OT I cells, and in vi vo-activated OT I cells after 7 days of culture (*p < 0.05; one-way ANOVA with Tukey’s post-test; n = 3). (FIG. 3A- FIG. 3D created with BioRender).

[0020] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, and FIG. 4G demonstrate that the aLN can expand functional antigen- specific cells from endogenous populations in vivo. FIG. 4A) Timeline of in vivo cell killing with and without irradiation. The group receiving irradiation was given a central dose of 500 cGy to lymphodeplete the mice on day 1. On day 0, 5 xlO6naive B6 CD8+ T cells were injected s.c. with aLN, and target cells were injected on day 8. Mice were harvested the next day and cells were run on flow cytometry. FIG. 4B) Percent target cell killing in the LN on day 9 (n = 4). FIG. 4C) Enrichment of OVA-specific T cells in the aLN injection site after B6 CD8+ T cells were co-injected with either aLN or hydrogel with no signals (“blank aLN”) (n = 4). FIG. 4D) Percent killing of target cell population within the lymph node on day 9 by different treatment groups (n = 4). FIG. 4E) CD 127 expression of cells expanded in vitro or in vivo with aLN for 9 days (n = 4-6). FIG. 4F, FIG. 4G) Representative flow plots and enrichment of OVA-specific T cells from B6 CD8+ T cells in vitro and in vivo, compared to naive freshly- isolated CD8+ T cells (n = 4-5). (FIG. 4B- FIG. 4E, FIG. 4G **p < 0.01, ***p < 0.001; student’s T-test).

[0021] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H, FIG. 51, and FIG. 5J demonstrate that host immune cells infiltrate the aLN matrix and promote antigenspecific T cell expansion and functionality. FIG. 5A) Schematic of aLN explant timeline. 5 x 106naive B6 CD8+ T cells were injected in healthy mice and were harvested on days 3, 6, and 9 and analyzed by flow cytometry to detect helper cell infiltration. FIG. 5B- FIG. 5G) Percent (left) and total number (right) of host and injected cells staining: FIG. 5B) CD3+, FIG. 5C) CD3+CD8+, FIG. 5D) CD3+CD4+, FIG. 5E) CD3-, FIG. 5F) CD3-CDl lb+, and FIG. 5G)CD3-CD1 lb- (n = 6). FIG. 5H) Representative images from the CODEX fluorescence imaging analysis on aLN samples collected on days 3 and 9. FIG. 51, FIG. 5 J) Cell type quantification using single-cell segmentation of CODEX images taken at 5 hours (marked as day 0), 3 days, and 9 days.

[0022] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 61, FIG. 6J, FIG. 6K, FIG. 6E, FIG. 6M, FIG. 6N, FIG. 60, FIG. 6P, and FIG. 6Q demonstrate that aLN reduces tumor burden and extends survival in prophylactic and treatment cancer models. FIG. 6A) Timeline of prophylactic tumor treatment performed with MC38-OVA and B16-OVA tumor lines. FIG. 6B- FIG. 6E) The aLN was co-injected s.c. with 1 x 106naive OT-I CD8+ T cells on day -7 and 1 x 106MC-38 OVA cells were injected s.c. on the opposite flank on day 0. Tumor measurements and survival were monitored (n = 6). FIG. 6F- FIG. 61) The aLN was co-injected s.c. with 1 x 106naive OT-I CD8+ T cells on day -7 and 2 x 105B16 OVA cells were injected s.c. on the opposite flank on day 0. Tumor measurements and survival were monitored (n = 5). FIG. 6 J) Timeline of tumor treatment model, performed with MC-38 OVA tumor line. 1 x 106MC-38 OVA cells were injected s.c. on day 0. On day 6, when tumors were palpable (schematic not to scale), 1 x 106naive OT-I CD8+ T cells were co-injected with aLN on the opposite flank. FIG. 6K- FIG. 6N) Tumor measurements and survival were monitored (n = 4 - 5). FIG. 60) Timeline of tumor treatment model in conjunction with anti-PD-1. On day 0, 2 x 105B16 OVA cells were injected s.c.. On day 6, aLN was co-injected s.c. with 1 x 106naive B6 CD8+ T cells on the opposite flank. In the group receiving anti-PD-1, the 200 pg was delivered i.p. on days 6, 9, 12, and 15. FIG. 6P- FIG. 6Q) Tumor measurements and survival were monitored (n = 7). Results in FIG. 6D, FIG. 6H, and FIG. 6M were analyzed by 2-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, whereas results in FIG. 6E, FIG. 61, FIG. 6N, and FIG. 6Q were analyzed by log-rank test; *p < 0.05. (FIG. 6A, FIG. 6J, FIG. 60 created with BioRender).

[0023] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E demonstrate that aLN expands MART- 1 -specific CD8+ T cells from human PBMCs of healthy donors. FIG. 7A) Diagram of workflow for expanding MART- 1- specific CD8+ T cells from human PBMCs (created with BioRender). FIG. 7B) Representative flow plots generated from 4 donors on days 0 and 11 showing the CD8+MART-1+ T cell population. FIG. 7C) Number of MART- 1 -specific CD8+ T cells over the course of an 11-day culture as measured by tetramer staining (tet+). FIG. 7D) Fold expansion of CD8+ MART-1+ T cells from each donor as determined by manual counting andflow cytometry. FIG. 7E) Percentage of CD8+MART-1+ T cells expressing IFNy, TNFa, and IL-2 after rcstimulation for 6 hours with aCD3 / aCD28 Dynabcads.

[0024] FIG. 8A and FIG. 8B demonstrate that adding Signal 3 to the HA hydrogel leads to IL-2 protection and enhanced long-term CD8+ T cell activation. FIG. 8A) Comparison of IL-2 encapsulated into the aLN and the aLN with soluble IL-2+Ab in the culture media (error bars show s.e.m.; n = 5-8, CFSE plot is a representative staining). FIG. 8B) Fold CD8+ T cell activation at different levels of equivalent IL-2 in culture (error bars show s.e.m.; n = 5-6).

[0025] FIG. 9 and FIG. 9B demonstrate production of MAB602 antibody. MAB602 was expressed recombinantly through transient transfection of human embryonic kidney (HEK) 293F cells and purified via protein G affinity chromatography followed by size-exclusion chromatography (SEC). FIG. 9A) MAB602 eluted as a monodisperse peak and FIG. 9B) migrated at the predicted molecular weight under both reducing and non-reducing SDS-PAGE conditions.

[0026] FIG. 10A, FIG. 10B, and FIG. 10C demonstrate that adding Signal 3 to the HA hydrogel enhances CD8+ T cell proliferation. FIG. 10A-FIG. 10C) Replicates of CFSE and generational analysis (error bars show s.e.m.; n = 6). FIG. 10A) Comparison between aLN and various cytokine support: IL-2+Ab conjugated, IL-2 soluble in media, no IL-2, or no stimulation (no aLN). FIG. 10B) aLN with IL-2 soluble in media and FIG. 10C) aLN with IL-2+Ab conjugated at 0.1, 1, and 10 pg / mL of equivalent IL-2 in culture.

[0027] FIG. 11 A and FIG. 1 IB illustrate ex vivo activation of SIY-specific CD8+ T cells on aLN. FIG. HA) Representative flow cytometry plots of CD8+ T cells stimulated on aLN with and without IL-2+Ab. FIG. 1 IB) Number of SIY+ CD8+ T cells of total CD8+ T cells on day 7 stimulated on the aLN with either a soluble cocktail of cytokines for signal 3 or IL-2+Ab as signal 3 (error bars show s.e.m.; n = 6-9).

[0028] FIG. 12A, FIG. 12B, and FIG. 12C show cytokine release analysis of ex vivo activation of SIY-specific CD8+ T cells on aLN. FIG. 12A) Representative flow cytometry plots of CD8+ T cells stimulated on aLN with and without IL-2+Ab. FIG. 12B, FIG. 12C) Functionality by FIG. 12B) percent and FIG. 12C) MFI of SIY+ CD8+ T cells positive for IFNy, TNFa, and CD107a markers at day 7 by intracellular staining (error bars show s.e.m.; n = 3, *p < 0.05, paired student’s t-test, two-tailed).

[0029] FIG. 13 shows results from an in vitro killing assay using aLN-activated cells. B6 CD8+ T cells were activated on SIY-spccific aLN with IL-2+Ab conjugated for 7 days. Cells were then collected and incubated with B16-SIY tumor cells, and killing was analyzed after 18 hours.

[0030] FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, and FIG. 14E are schematics of how a microgel particle form of aLN was prepared. FIG. 14A) SEM imaging of bulk hydrogel. FIG. 14B) Picture of housing device for 150-pm diameter stainless steel filter used to particulate microgel aLN from HA hydrogel formed within a syringe. A picture taken by epifluorescence microscopy where hydrogels were labeled with a maleimide-fluorescent probe (scale bar = 500 pm). FIG. 14C) Picture of inner components of the filter device taken from Millipore Sigma’s website: http: / / www.emdmillipore.com / US / en / product / Swinny-Filter-Holder-13mm-stainless- steel,MM_NF-XX3001200#anchor_UG. FIG. 14D) Structure of aLN particles evaluated by scanning electron microscopy (SEM). Scale bars indicated in images. FIG. 14E) Immunofluorescent image of multiple aLN microgel particles.

[0031] FIG. 15A and FIG. 15B show aspects of an in vitro migration assay. FIG. 15A) Schematic of migration assay setup. 1 x 106PMEL CD8+ T cells were added above the compacted aLN. At the indicated time points, the number of cells in lower chamber were counted. FIG. 15B) Quantification of the release of cells into the lower chamber over 24 hours.

[0032] FIG. 16 illustrates aspects of generating injectable aLN for CD8+ T cell activation and stimulation efficacy ex vivo. aLN made at day 0 were plated with B6 CD8+ at 2 weeks, 1 month, and 3 months post fabrication. Fold proliferation was measured after a 7-day culture (n = 3).

[0033] FIG. 17A, FIG. 17B, and FIG. 17C demonstrate that in vivo CD8+ T cell activation by aLN leads to functional antigen- specific killing as analyzed within the spleen. FIG. 17A) Percent killing of target cell population within the spleen by different treatment groups as analyzed by day 7 (error bars show s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001, n = 4, one-way ANOVA with Tukey’s post-test). FIG. 17B) Thyl.l+ percentage of CD8+ T cells from the spleen from mice in treatment groups indicated on day 7 after treatment (error bars show s.e.m.; n = 4). FIG. 17C) CTV mean fluorescent intensity (MFI) of adoptively transferred Thyl.l+, CD8+ T cells within the spleen by different treatment groups as analyzed by day 7 (error bars show s.e.m.; ***p < 0.001, n = 4, one-way ANOVA with Tukey’s post-test).

[0034] FIG. 18A and FIG. 18B show toxicity analysis of aLN injections. FIG. 18 A) Complete blood count (CBC) analysis of mice receiving various no injections (control), aLN alone, aLNwith 1 x 106PMEL CD8+ T cells harvested after 24h, and aLN with 1 x 106PMEL CD8+ T cells harvested after 9 days (n = 3; ** p < 0.01, Student’s T test against control group). FIG. 18B) Representative histology images of the heart, kidney, liver, lungs, and spleen of the same testing groups (n = 3).

[0035] FIG. 19A and FIG. 19B demonstrate that an optimized aLN improves in vitro culture and can expand antigen specific cells from endogenous populations in vivo. FIG. 19A) Percent killing of target cell population (SIY -pulsed splenocytes) within the draining lymph node by different treatment groups. (**p < 0.01, n = 4, student’s T test). FIG. 19B) Percent killing of target population (OVA-pulsed splenocytes) within the lymph node by different treatment groups. < 0.0001, n = 4, one-way ANOVA).

[0036] FIG. 20A and FIG. 20B show that host immune cells infiltrate the aLN matrix and promote antigen-specific T cell expansion and functionality. Percent (left) and total number (right) of host and injected cells staining: FIG. 20A) total cells, and FIG. 20B) CD3+CD8+KbOVA+.

[0037] FIG. 21 demonstrates that H & E staining on explanted aLN samples show cell flux over 9 days. 5 x 106naive B6 CD8+ T cells were injected in healthy mice and were explanted 5 hours, 3 days, or 9 days late. Explants were sectioned and stained with hematoxylin and eosin (H & E). aLN particles are shown in grey / light purple.

[0038] FIG. 22A, FIG. 22B, and FIG. 22C show single-cell segmentation of CODEX images. A deep learning algorithm was applied to CODEX images at hour 5, day 3, and day 9 to perform single-cell segmentation. FIG. 22A) Representative images of single-cell segmentation at each time point. FIG. 22B, FIG. 22C) Quantification of specific cell types in the microenvironment.

[0039] FIG. 23 A, FIG. 23B, FIG. 23C, FIG. 23D, and FIG. 23E demonstrate that cells stimulated on the aLN stalling from splenocytes rather than purified CD8+ T cells show enhanced enrichment for SIY-specific T cells and increased cytokine secretion. FIG. 23A) Number of SIY-specific CD8+ T cells after stimulation from total splenocytes compared to purified CD8+ T cells (n = 9-11). FIG. 23B, FIG. 23C) ICS results of antigen- specific cells expanded from either splenocytes or purified CD8+ T cells for 7 days (analyzed by Student’s t test). Cell expressing all 4 of CD107a, IFNy, IL-2, and TNFa are shown in (FIG. 23B); expression of individual cytokines is shown in (FIG. 23C). FIG. 23D) Number of SIY-specific CD8+ T cells after stimulation from total splenocytes compared to CD4+ T cell-depletedsplenocytes (n = 3-4). FIG. 23E) Correlation between CD4+ T cell infiltration into the aLN and enrichment for antigen- specific CD8+ T cells within the aLN (n = 6). (A, B: *p < 0.05, ***p < 0.001; student’s t test).

[0040] FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D show expansion of CM V- specific CD8+ T cells from human PBMCs or isolated CD8+ T cells. FIG. 24A) Either PBMCs or isolated C8+ T cells were incubated with CMV-specific aLN for 7 days and then analyzed by flow cytometry to determine enrichment for antigen- specific T cells within the CD8+ T cell population. FIG. 24B) Percent of CD4+ T cells in culture on day 0 plotted against the percentage of MARTI specific CD8+ T cells on day 11. FIG. 24C) Percent of CD4+ T cells in culture on day 11 plotted against the percentage of MARTI specific CD8+ T cells on day 11. FIG. 24D) Percent of CD8+MART1+ T cells expressing one or more of IFNy, TNFa, and IL-2 for each donor.

[0041] FIG. 25 is a schematic of how host helper cells may interact with donor cells in the aLN.

[0042] FIG. 26 is a schematic diagram showing the aLN microenvironment and its advantages in T cell activation and expansion.

[0043] FIG. 27 A, FIG. 27B, and FIG. 27C show: (FIG. 27 A) Phenotype of CD8+ T cells is impacted by Signal 2 composition. (FIG. 27B, FIG. 27C) CD8+ T cells are sensitive not only to Signal 2 composition, but also Signal 2 ratio.

[0044] FIG. 28A and FIG. 28B demonstrate that Signal 2 composition affects persistence of CD8+ T cells in vivo.DETAILED DESCRIPTION

[0045] 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.

[0046] In some embodiments, the presently disclosed subject matter provides a T cellstimulating scaffold referred to herein as an “artificial lymph node (aLN),” which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the costimulatory signal anti-CD28 (Signal 2), and a tethered T cell-stimulating cytokine (Signal 3).

[0047] As described herein, the aLN is a dynamic immune-stimulating scaffold that enables the direct, in vivo antigen-specific CD8+ T cell activation and expansion. The aLN mimics key functions of both antigen presenting cells and secondary lymphoid organs; along with direct T cell stimulation, the aLN recruits host immune cells and coordinates them, providing a stimulatory microenvironment for T cell expansion. Co-injecting the aLN with naive, wild type CD8+ T cells, results in robust activation and expansion of tumor-targeted T cells that kill target cells in an antigen- specific manner and slows tumor growth in both prophylactic and therapeutic cancer models. The presently disclosed subject matter demonstrates that the aLN induces potent in vivo antigen- specific CD8+ T cell stimulation without need for ex vivo priming and enables in situ manipulation of antigen-specific responses for immunotherapies.

[0048] Artificial Lymph Nodes (aLNs)

[0049] In some embodiments, the presently disclosed subject matter provides an artificial lymph node (aLN) comprising an extracellular matrix (ECM) hydrogel conjugated with an antigen presenting complex (Signal 1), a co-stimulatory ligand (Signal 2), and T cell- stimulating cytokine (Signal 3). In certain embodiments, Signal 3 comprises a T cell- stimulating cytokine / antibody complex.

[0050] 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. In other 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., CD la, CD lb, CDlc, CD Id, and CDle). 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 molecularcomplexes 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.

[0051] 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 CHI, 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.

[0052] Exemplary MHC class I molecular complexes comprise at least two fusion proteins. A first fusion protein comprises a first MHC class I a 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 a 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, IgGl, IgG3, IgG2p, IgG2a, 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.

[0053] 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. 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 lip chain. Two second fusion proteins comprise (i) animmunoglobulin K or light chain (or portion thereof) and (ii) an extracellular domain of an MHC class Ila chain. The two first and the two second fusion proteins associate to form the MHC class II molecular complex. The extracellular domain of the MHC class lip chain of each first fusion protein and the extracellular domain of the MHC class Ila chain of each second fusion protein form an MHC class II peptide binding cleft.

[0054] The immunoglobulin heavy chain can be the heavy chain of an IgM, IgD, IgG3, IgGl, IgG2p, IgG2a, IgG4, IgE, or IgA. In some embodiments, an IgGl 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.

[0055] 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.

[0056] In certain embodiments, the antigen presenting complex (Signal 1) is selected from a major histocompatibility complex (MHC) molecule, a human leukocyte antigen (HLA) molecule, and an anti-CD3 antibody. In certain embodiments, the MHC molecule is a monomer or a dimer. In certain embodiments, the antigen presenting complex (Signal 1) is an MHC class I molecule, an MHC class II molecule, or a combination of an MHC class I molecule and an MHC class II molecule. In particular embodiments, the antigen presenting complex (Signal 1) is selected from an MHC-Ig dimer or an HLA-Ig dimer. In more particular embodiments, the antigen presenting complex (Signal 1) comprises an MHC molecule loaded with a peptide or an HLA molecule loaded with a peptide. In certain embodiments, the peptide is selected from GP100: KVPRNQDWL SEQ ID NO: 1; SIY: SIYRYYGL SEQ ID NO: 2; OVA: SIINFEKL SEQ ID NO: 3, and MART-1: ELAGIGILTV SEQ ID NO: 4.

[0057] The co-stimulatory ligand is referred to herein as a “Signal 2.” Such co-stimulatory ligands 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., Thl, Th2), or regulatory T cell; and / or proliferation of T cells. Thus, T cell affecting molecules include T cell co-stimulatory molecules, adhesion molecules, T cell growth factors, and regulatory T cellinducer molecules. In some embodiments, an aAPC comprises at least one such ligand; optionally, an aAPC comprises at least two, three, or four such ligands.

[0058] In certain embodiments, signal 2 is a T cell co- stimulatory 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 0X40, and antibodies that specifically bind to 4- IBB. In some embodiments, the co- stimulatory 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 portion thereof having antigen-binding activity, or is a fully human antibody or portion thereof having antigen-binding activity.

[0059] In particular embodiments, the co-stimulatory ligand (Signal 2) is selected from anti- CD28 (ot-CD28) antibody, anti-CD27 (0C-CD27) antibody, and anti-41BB (a-41BB) antibody. In more particular embodiments, the co-stimulatory ligand comprises an anti-CD28 (a-CD28) antibody.

[0060] In certain embodiments, the co-stimulatory ligand (Signal 2) comprises at least a first antibody that specifically binds to a protein expressed on a T cell and at least a second antibody that specifically binds to a protein expressed on a T cell. In certain embodiments, the at least first antibody that specifically binds to a protein expressed on a T cell and the at least second antibody that specifically binds to a protein expressed on a T cell are present in a ratio of first antibody: second antibody selected from about 99:1, 98:2, 97:3, 96:4, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 4:96, 3:97, 2:98, and 1:99. In particular embodiments, the at least first antibody that specifically binds to a protein expressed on a T cell and the at least second antibody that specifically binds to a protein expressed on a T cell are selected from a-CD28 and CX-CD27 or a- CD28 and a-41BB.

[0061] In certain embodiments, the T cell- stimulating cytokine is selected from IL-2, IL-4, IL-7, IL-10, IL-12, IL-12p70, IL-15, IL-21, CXCL10, and gamma interferon (IFN-y). In particular, embodiments, the T cell- stimulating cytokine / antibody complex (Signal 3) comprises an anti-IL-2 antibody complexed with IL-2 (IL-2+Ab). In more particular embodiments, the anti-IL-2 antibody comprises MAB602.

[0062] In certain embodiments, the hydrogel comprises a hyaluronic acid (HA) hydrogel. In particular embodiments, the HA hydrogel comprises a thiol-modified HA hydrogel.

[0063] The term “hydrogel,” as used herein, refers to a three-dimensional network composed of hydrophilic polymers crosslinked either through covalent bonds or via physical intramolecular or intermolecular interactions. Hydrogels can absorb large amounts of water or biological fluids (up to several thousand percent), and swell readily without dissolving. The high hydrophilicity of hydrogels is primarily due to the presence of hydrophilic moieties such as carboxyl, amide, amino, and hydroxyl groups distributed along the backbone of polymeric chains. In the swollen state, hydrogels are soft and rubbery, closely resembling living tissues. Many hydrogels, such as chitosan and alginate -based hydrogels, exhibit desirable biocompatibility (see, e.g., El-Sherbiny, I.M., and Yacoub, M.H. Global Cardiology Science & Practice, 2013(3): 316-342 (2013); and Kyung et al, J. Appl. Polym. Sci., 83: 128-136 (2002)). Since their discovery more than 50 years ago, hydrogels have been employed in a variety of applications including, for example, drug delivery, wound healing, ophthalmic materials, and tissue engineering (see, e.g., El-Serbiny and Yacoub, supra; Hoffman, A.S., Ann. NY Acad. Sci., 944: 62-73 (2001); and Peppas et al., Eur. J. Pharm. Biopharm., 50: 27-46 (2000)).

[0064] Hydrogels typically reach their equilibrium swelling when a balance occurs between osmotic driving forces, which encourage the entrance of water or biological fluids into the hydrophilic hydrogel matrix, and the cohesive forces exerted by the polymer strands within the hydrogel. These cohesive forces resist the hydrogel expansion and the extent of these forces depends particularly on the hydrogel crosslinking density. Generally, the more hydrophilic the polymer forming the hydrogel, the higher the total water amount absorbed by the hydrogel. Likewise, the higher the crosslinking extent of a particular hydrogel, the lower the extent of the gel swelling. Hydrogels in their dried forms are referred to in the art as “xerogels,” while dry porous hydrogels resulting from the use of drying techniques (e.g., freeze-drying or solvent extraction) are referred to in the art as “aerogels” (see, e.g., Guenet, J.M., Thermoreversible gelation of polymers and biopolymers; Academic Press, New York (1992), p. 89).

[0065] Hydrogels can be classified based on a variety of characteristics, such as, for example origin, durability, response to stimuli, charge, structure, and composition. With respect to origin,hydrogels can be classified as natural, synthetic or semi-synthetic. Most synthetic hydrogels are synthesized by traditional polymerization of vinyl or vinyl-activated monomers. The equilibrium swelling values of these synthetic hydrogels vary widely according to the hydrophilicity of the monomers and the crosslinking density. Natural hydrogels typically are made of natural polymers including, for example, polynucleotides, polypeptides, and polysaccharides that can be obtained from a variety of sources (e.g., collagen from mammals and chitosan from shellfish exoskeletons). With respect to durability, hydrogels can be classified as durable (such as most polyacrylate -based hydrogels) or biodegradable (such as polysaccharide -based hydrogels), depending on their stability characteristics in a physiological environment. Biodegradable hydrogels have recently been developed in which degradable polymers inside the hydrogel matrices undergo chain scission to form oligomers of low molecular weight. The resulting oligomers are either eliminated by the organism or undergo further degradation. Such biodegradable hydrogels can be used in both biomedical and non-biomedical applications (see e.g., Zhu, W. and Ding, J., J. Appl Polym Sci., 99: 2375 (2006)). With respect to response to environmental stimuli, “smart” hydrogels have been developed that exhibit changes in swelling behavior, network structure, and / or mechanical characteristics in response to various environmental stimuli such as pH, temperature, light, ionic strength or electric field (see, e.g., Gutowska et al., J Control Release, 22: 95-104 (1992); Ferreira et al, Int J Pharm., 794: 169-180 (2000); and D’Emanuele, A. and Staniforth, J.N., Pharm Res., 8: 913-918 (1991)). These changes typically disappear upon removal of the stimulus and the hydrogels are restored to their original state in a reversible manner.

[0066] In certain embodiments, the hydrogel comprises an extracellular matrix. The term “extracellular matrix (ECM)” is well known in the art as the non-cellular component present within all tissues and organs that provides structural support to cells and performs other important functions. ECM is composed of an interlocking meshwork of fibrous proteins, including collagen, elastin, fibronectin, and laminin as well as polysaccharides such as glycosaminoglycans (GAGs), which typically form proteoglycans upon covalent linkage to proteins (see, e.g., Alberts et al, Molecular Biology of the Cell, Garland Science, London (2007)).

[0067] The hydrogel described herein may be generated using natural polymers, such as polynucleotides, polypeptides, and polysaccharides. Such natural polymers may be obtained orderived from any natural source, including, for example, a living organism (a mammal, a fish, an insect, or a plant). For example, chitosan is a natural polymer obtained from shellfish exoskeletons, while collagen is a natural polymer obtained from mammals. Other natural polymers that may be used in hydrogels include, but are not limited to, hyaluronic acid (HA), an amphiphilic peptide, alginate, collagen, fibrin, gelatin, chondroitin sulfate, carboxymethylcellulose, dextran, agarose carbomer, and derivatives thereof. It will be appreciated that hydrogels based on natural polymers are particularly suited for tissue engineering applications due to their intrinsic characteristics of biological recognition (e.g., presentation of receptor-binding ligands and susceptibility to cell-triggered proteolytic remodeling and degradation).

[0068] In some embodiments, the hydrogel of the present disclosure may be generated using a synthetic polymer. Examples of suitable synthetic polymers include, but are not limited to, poly(ethylene glycol) (PEG), poly(ethylene glycol) diacrylate (PEGDA), poly(lactic acid) (PLA), poly(ethylene oxide) (PEG), polyvinyl alcohol) (PVA), poly(hydroxyl-ethyl methacrylate) (PHEMA), methacrylated dextran-graft-lysine (Dex-MA- LA), methacrylamide-modified gelatin (Gel-MA), and derivatives thereof. Hydrogels based on synthetic polymers may exhibit less immunogenicity then natural polymer-based hydrogels, and may provide greater control over material characteristics and tissue responses.

[0069] In particular embodiments, the hydrogel comprises hyaluronic acid (HA). HA is a linear’ polysaccharide and is the only non-sulfated glycosaminoglycan found distributed throughout the ECM, including lymphoid tissues (Jackson, D.G., Immunol. Rev., 230: 216-231 (2009)). Hyaluronic acid impacts cell motility and adhesion, differentiation, gene expression, and proliferation (Toole, B. P., Nat. Rev. Cancer 4, 528 (2004); Entwistle et al., J. Cell. Biochem., 61, 569-577 (1996); and Ponta et al., Nat. Rev. Mol. Cell Biol., 4: 33 (2003)). HA also can be easily modified through tunable chemistry, enabling the addition of adhesive ligands (Lei et al., Biomaterials, 32: 39-47 (2011); Shu et al., J. Biomed. Mater. Res., Part A, 79: 902-912 (2006)) conjugation of drugs or growth factors (Peattie et al., Biomaterials, 25: 2789-2798 (2004)), and control of the elastic modulus and porosity of the hydrogel containing HA (Li et al., Faseb J., 27: 1127-1136 (2013); and Tan et al., Biomaterials, 30, 6844-6853 (2009)). HA and its derivatives have been clinically used as medical products for over three decades (Kuo, J.W., Practical Aspects of Hyaluronan Based Medical Products, Boca Raton: CRC / Taylor & Francis; (2006)).More recently, HA has become recognized as an important building block for the creation of new biomatcrials with utility in tissue engineering and regenerative medicine. HA-containing hydrogels and methods of producing HA-containing hydrogels are described in, e.g., Burdick, J. A. and G.D. Prestwich, Adv Mater., 23(12): H41-H56 (2011); and Xu et al., Soft Matter, 8(12): 3280-3294 (2012). In some embodiments, the hydrogel comprises hyaluronic acid crosslinked with polyethylene glycol diacrylate (PEGDA). Any suitable amount of PEGDA may be incorporated into the HA hydrogel.

[0070] HA hydrogels described herein can be prepared using any suitable method known in the art. Such methods may include, for example, emulsification, lyophilization, emulsificationlyophilization, spray drying, solvent casting-leaching, gas foaming-leaching, photolithography, photocrosslinking, electrospinning, microfluidics, micromolding, and 3D-organ / tissue printing (see, e.g., El-Serbiny and Yacoub, supra, and Xu et al., supra). The hydrogel may be conjugated with one or more molecules or substances that provide stimulatory signals required for T cell activation.

[0071] In certain embodiments, the hydrogel has a shear modulus of about 20 Pa to about 1600 Pa, including about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, and 1600 Pa. Hydrogels suitable for use with the presently disclosed aLNs and methods are disclosed in International PCT Patent Application Publication No. W02020206128 for Artificial T Cell Stimulating Matrix For Immunotherapy to Schneck et al., published October 8, 2020, which is incorporated herein by reference in its entirety.

[0072] In some embodiments, one or more adhesion proteins are included in the presently disclosed aLN. Without wishing to be bound to any one particular theory, it is thought that surface molecules besides those directly involved in the immunological synapse can be incorporated into the aLN to alter how the T cells interact with the hydrogel. For example, it has been previously shown that incorporation of the cell adhesion peptide RGD into the aLN results in an increase in proliferation after 7 days; however, it did not rescue proliferation in all conditions, for example, on stiffer gels. Hickey, 2019. Beyond improving the interaction between T cells and aLN, it also has been shown that including cell adhesion motifs on the substrate improves the ability of T cells to migrate across the substrate. Hailemichael et al., 2013; Stephan et al., 2015. The ability for T cells to migrate in and out of the matrix in vivo can be vital to ensuring that activated cells can leave the aLN and for naive cells to enter the aLN.Accordingly, in some embodiments, the role of various adhesion molecules (including, but not limited to RGD, collagen, and ICAM-1) in improving activation, proliferation, and migration of T cells, as well as other host immune cells that may be beneficial to the microenvironment, can be investigated. To this end, in certain embodiments, RGD, collagen, and ICAM-1 can be incorporated separately into aLN expressing MHC Class I and II, and the optimized Signals 2 and 3.

[0073] In certain embodiments, the aLN further comprises one or more adhesion molecules. In particular embodiments, the one or more adhesion molecules are selected from RGD, collagen, and ICAM- 1. In more particular embodiments, the one or more adhesion molecules is RGD.

[0074] In other embodiments, the presently disclosed subject matter provides a microgel comprising the aLN as describe herein.

[0075] Methods for Stimulating T Cells

[0076] In other embodiments, the presently disclosed subject matter provides a method for stimulating one or more T cells, the method comprising contacting the one or more T cells with one or more aLNs described herein, or a microgel thereof. In certain embodiments, the one or more T cells comprise antigen- specific CD8+ T cells. In particular embodiments, the contacting of the one or more T cells with the one or more aLNs occurs in vivo. In more particular embodiments, the method comprises administering the one or more aLNs in combination with one or more naive T cells. In even more particular embodiments, administering the one or more aLNs in combination with one or more naive T cells creates a T cell-activating niche. In certain embodiments, administering the one or more aLNs results in one or more of recruitment of host immune cells, coordination of host immune cells, and providing an immuno-stimulatory microenvironment for antigen- specific T cell activation and expansion. In more certain embodiments, the contacting of the one or more T cells with the one or more aLNs induces in vivo antigen- specific CD8+ T cell stimulation without ex vivo priming or expansion. In certain embodiments, the contacting of the one or more T cells with the one or more aLNs enables in situ manipulation of antigen-specific responses for an immunotherapy.

[0077] In other embodiments, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition, or providing prophylactic protection to immuno suppressed patients, the method comprising administering a therapeutically effective amount of an artificial lymph node (aLN) as described herein or a microgel thereof, to a subjectin need of treatment thereof. In certain embodiments, the method further comprises administering naive, wild type CD8+ T cells in combination with one or more aLNs. In particular embodiments, the disease, disorder, or condition is selected from a cancer, an infectious disease, and an autoimmune disease. In more particular embodiments, the cancer comprises a solid tumor or a hematological malignancy. In even more particular embodiments, 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. In certain embodiments, administering the naive, wild type CD8+ T cells in combination with the one or more aLNs results in activation and expansion of tumor- targeted T cells that kill target cancer cells, slows tumor growth, and increases survival.

[0078] Methods for Treating a Disease, Disorder, or Condition

[0079] In some embodiments, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition, the method comprising administering a therapeutically effective amount of a presently disclosed artificial lymph node (aLN) to a subject in need of treatment thereof.

[0080] In certain embodiments, the method further comprises administering naive, wild type CD8+ T cells in combination with one or more aLNs.

[0081] In certain embodiments, the disease, disorder, or condition is selected from a cancer, an infectious disease, and an autoimmune disease, or to provide prophylactic protection to an immuno suppressed patient.

[0082] Methods for Treating Cancer

[0083] In some embodiments, the disease, disorder, or condition is a cancer. In particular embodiments, the cancer is a solid tumor or a hematological malignancy. 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), 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, 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.

[0084] In certain embodiments, the cancer is a stage I, stage II, stage III, or stage IV cancer. 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).

[0085] Methods for Treating an Infectious Disease

[0086] In other embodiments, the presently disclosed subject matter includes a method for treating an infectious disease. The infectious disease may be one in which enrichment and expansion of antigen-specific immune cells (such as CD8+ or CD4+ T cells) ex vivo for adoptive transfer to the patient could enhance or provide for a productive immune response. 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 posttransplant lymphoproliferative disorder (PTLD).

[0087] 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 therapieslose the controlling T cell populations, which permits viral reactivation. This represents a serious impediment to transplant protocols. EBV may also be involved in tumor promotion in a variety of hematological and non-hematological cancers.

[0088] 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.

[0089] Methods for Treating an Autoimmune Disease

[0090] In some embodiments, the presently disclosed subject matter provides a method for treating an autoimmune disease. Representative autoimmune diseases include, but are not limited to, 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.

[0091] 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.

[0092] 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 ofordinary 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.

[0093] Reagents / Kits

[0094] In other embodiments, the presently disclosed subject matter provides a kit comprising the presently disclosed aLNs or the components for preparing the presently disclosed aLNs. Suitable containers for the presently disclosed aLNs or components thereof 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). Kits may comprise, alternatively or in addition, one or more multi-well plates or culture plates for T cells.

[0095] 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.

[0096] 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, ant clonotypic antibodies, and the like.

[0097] A kit can also comprise a package insert containing written instructions for methods of preparing the presently disclosed aLNs and uses thereof. 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.

[0098] 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%, 4-7-5%, + / -4%, + / -3%, + / -2%, and + / -!%. 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.

[0099] 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.

[0100] 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. The present 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 nonlimiting, 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

[0101] 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 compounds of the disclosure by other methods.EXAMPLE 1In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix

[0102] Overview

[0103] This Example describes an artificial lymph node (aLN) matrix, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), a co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ, such as vaccines. This dynamic immune- stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naive, wild type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen- specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen- specific responses for immunotherapies .

[0104] Background

[0105] The therapeutic use of T cells is a burgeoning field with applications in cancer immunotherapy, infectious diseases, and autoimmunity. Interest, in part, is due to the exquisite specificity of antigen-specific T cells, the ability to induce immunological memory, and the remarkable clinical responses that have been observed thus far. Current adoptive T cell therapies (ACTs), including CAR-T, TCR, and endogenous T cell therapy (ETC), however, routinely require extensive ex vivo manufacturing and have relatively restricted clinical applications. Thus, the generation of de novo cancer-specific T cell stimulation in vivo is an area of high interest.

[0106] Cancer vaccines have been investigated for this effort but have historically failed due to the challenge of eliciting a cellular response from vaccines, as well as the fact that many cancer patients have dysfunctional dendritic cells. Gajewski and Cron, 2020. Other studies have investigated nanoparticle- and microparticle-based artificial antigen presenting cells (aAPCs) for in vivo activation of antigen-specific T cells; Est-Witte et al., 2021; Rhodes et al., 2021; Meyer et al., 2014; however, the difficulty of achieving sufficient accumulation of aAPCs in the T cell zones of secondary lymphoid organs limits the efficacy of this approach. Meyer et al., 2014; Kosmides et al., 2017; Sun et al., 2020.

[0107] Alternatively, synthetic scaffolds have been developed to support in vivo growth of T cells. Such scaffolds have the advantage of concentrating T cells and stimulating them. These early iterations, however, relied on non-specific T cell stimulation, limiting their use to either extracted tumor-infiltrating lymphocytes (TILs), ex vzvo-enriched antigen- specific cells, or engineered cells. Weiden et al., 2018; Stephan et al., 2015; Agarwalla et al., 2022. Moreover,this reliance could result in non-specific expansion of endogenous off-target T cells that may have unintended autoimmunity implications. Additionally, these systems lack other environmental cues found in the lymph node, such as ECM binding and interactions with other immune cells, which significantly influence cell signaling through mechanical and biochemical stimuli. Engler et al., 2006.

[0108] Scope of Example

[0109] This Example provides a new approach to generating T cells in vivo for cancer immunotherapy by constructing a T cell-stimulating scaffold that integrates structural support with immunological cues to induce the formation of an immuno-stimulatory microenvironment that mimics T cell activation within the lymph node. This platform, termed herein as the “artificial lymph node (aLN),” is comprised of a hyaluronic acid (HA) hydrogel that incorporates Signal 1 (peptide-loaded-MHC complex), Signal 2 (anti-CD28), and Signal 3 (immune effector cell-biased IL-2 cytokine / anti-IL-2 antibody complex), concurrently facilitating ECM signaling through HA binding while also triggering antigen- specific T cell activation through a mechanism distinct from vaccination. The bulk gel can be formed into microparticles and then co-injected subcutaneously with naive T cells to create a T cell-activating niche.

[0110] In vivo, the aLN forms a dynamic microenvironment that mimics natural T cell stimulation by (1) presenting T cell-stimulating signals, (2) providing ECM signaling, Hickey et al., 2019, and (3) facilitating interaction between T cells and supporting cells recruited from the host immune system. In this Example, we investigate the aLN matrix properties, signaling cues, and the interaction with the host immune cells to enhance antigen- specific T cell activation from naive, wild type populations of CD8+ T cells. The aLN is shown to expand rare, antigen-specific T cells in vivo without the need for ex vivo manipulation. Furthermore, aLN-activated T cells have the ability to kill target cells and extend survival in multiple tumor models.

[0111] Results

[0112] Engineering local presentation of Signal 3 on a T cell-stimulating HA hydrogel

[0113] The lymph node not only acts as a space to locally concentrate T cells with antigen presenting cells, but also provides an environment for T cells to proliferate and differentiate after their initial activation for a sustained period. A critical component required is cytokine support, commonly referred to as Signal 3. Engineering control of Signal 3 directly into stimulation platforms has largely been limited to the release of encapsulated IL-2. Pires and Hammond,2021 . This approach mimics cell-secretion of IL-2, but the cytokine is quickly depleted by renal filtration (half-life <5 minutes), Ac ct al., especially in vivo, and thus needs to be consistently replenished or supplemented by soluble cytokines. While the aLN can sustainably release IL-2, as shown by the proliferation of cells in culture with IL-2-encapsulating aLNs (FIG. 8A, FIG. 8B), without wishing to be bound to any one particular theory it is thought that incorporating a tethered anti-IL-2 antibody into the platform would allow better control of the dose of cytokine delivered and reduce the risk of cytokine being rapidly depleted in vivo (FIG. 1A). Further, the antibody may anchor, protect, potentiate, and bias IL-2 activity in a fashion previously unexplored. We were specifically interested in the use of anti-cytokine antibodies known to direct the activity of IL-2 toward immune effector cells (i.e., T effector cells and natural killer [NK] cells). Krieg et al., 2010; Letourneau, et al., 2010.

[0114] To realize this tethered antibody approach, a thiol-modified hyaluronic acid (HA) hydrogel that incorporated an anti-CD3 antibody (Signal 1), an anti-CD28 antibody (Signal 2), and an anti-IL-2 antibody complexed with IL-2 (Signal 3) was created. To bias IL-2 delivery toward immune effector cells, the anti-IL-2 antibody MAB602 was employed (FIG. 9), which has been demonstrated to extend the half-life of IL-2 while selectively potentiating its activity on naive CD8+T cells to elicit targeted, durable immuno- stimulatory responses. Krieg et al., 2010; Letourneau et al., 2010; Silver et al., 2021; Boyman et al., 2006; Spangler et al., 2015. The tethered and biased anti-IL-2 antibody (IL- 2+ Ab) approach was compared to the standard use of soluble IL-2 to support T cell proliferation.

[0115] Conjugating the anti-IL-2 antibody complexed with IL-2 (IL-2+Ab) to the aLN led to robust CD8+ T cell expansion as measured by day 3 CFSE dilution (FIG. IB, FIG. 10A).Soluble IL-2 also promoted expansion by day 3, whereas the aLN without IL-2 did not support T cell growth (FIG. IB, FIG. 10A). The importance of persistent cytokine stimulation was modeled in vitro by not replenishing the IL-2 in the media after day 0 for any group during the day 7 proliferation. By day 7, the aLN with IL-2+Ab conjugated promoted greater CD8+ T cell proliferation than equivalently dosed soluble IL-2 conditions (FIG. 1C). These data indicate that conjugated IL- 2+ Ab complexes extend the proliferative effect of IL-2 in culture in the context of the aLN.

[0116] The IL-2+Ab complex was compared to equimolar soluble IL-2 by titrating the dose from 10 pg / mL to 0.1 pg / mL with equivalent concentrations of Signals 1 and 2. All conditions led torobust day 3 expansion, as indicated by CFSE dilution (FIG. 1D, FIG. 10B, C). The aLN group with conjugated IL-2+Ab complexes, however, promoted greater CD8+ T cell activation on day 7 at all doses (FIG. IE). The consistently higher extent of proliferation for IL-2+Ab complexes indicates that concentrating IL-2 on the aLN could potentiate CD8+ T cell activation.

[0117] The native lymph node can expand rare antigen- specific CD8+ T cells from an endogenous population of T cells. To mimic this function in the aLN platform, an antibody presenting the murine Kb MHC allele was conjugated to the aLN and loaded with model antigen SIY (KbSIY) as Signal 1 and anti-CD28 as Signal 2. CD8+ T cells from B6 mice were stimulated on the aLN (with conjugated IL-2+Ab complex) and were compared to aLN stimulation with continuous feeding of soluble cytokines. At day 7, cells were stained with Kb- presenting antibody loaded with SIY or non-cognate peptide to evaluate the percentage and number of SIY+ CD8+ T cells that were induced. Their functionality was then evaluated by intracellular cytokine staining (ICS). Ahmadzadeh et al., 2009; Pollack et al., 2014; Wherry, 2011; Wherry et al., 2003.

[0118] SIY-specific CD8+ T cells stimulated on the aLN with conjugated IL- 2+ Ab complex, compared to aLN with soluble cytokines, resulted in similar numbers of SIY-antigen- specific T cells (FIG. IF, FIG. 11), but almost three times the number of polyfunctional SIY+ T cells that were positive for IFNy, TNFa, and CD107a (FIG. 1G, FIG. 12). This increase in functionality derived largely from an increase in the percentage of T cells that were positive for IFNy (FIG. 12B, C). Additionally, SIY-specific T cells activated with the aLN conjugated with IL-2+Ab are effective at killing B16-SIY tumor cells in vitro (FIG. 13). These results demonstrate that the aLN hydrogel conjugated with the IL-2+Ab complexes can activate and expand highly functional antigen-specific T cells from a diverse TCR repertoire while providing local, sustained cytokine support.100119} Generating Injectable aLN for CD8+ T Cell Activation

[0120] In addition to local cytokine support, we sought to provide the structural support necessary to create a T cell-stimulating immune niche. The hyaluronic acid gel formulation used here has been described in detail in a previous publication Hickey et al., 2019. Briefly, the hyaluronic acid is thiol-modified and gelled via a PEGDA crosslinker to form a dense matrix (FIG. 14). By modifying the crosslinker density, we are able to achieve a range of stiffness from 0.5 - 10 kPa. Hickey et al., 2019. The hydrogel is biocompatible and degrades slowly viahyaluronidases. Advanced BioMatrix Glycosil® Version 2.0,” can be found under https: / / advanccdbiomatrix.com / glycosil.html, n.d.

[0121] Microgel particles were generated from this formulation to provide several advantages compared to a bulk hydrogel. In vitro, the particles sink to the bottom of the well and can interact with T cells in a pseudo-3D manner. Once injected, the microparticles compact to form a granular- hydrogel,’ Daly et al., 2019, effectively creating a dual -porous hydrogel scaffold. This method allows us to maintain a controlled signal density and matrix stiffness while permitting cell infiltration between particles for cell-cell interactions, T cell expansion, and cellular migration.

[0122] To generate microgel versions of the aLN, the aLN hydrogel was passed through a stainless- steel mesh filter with a pore size of 150 pm (FIG. 2A, FIG. 14B, C). Yao et al., 2022. The resulting microgel particles were irregularly shaped, with sizes ranging from 50 pm to 300 pm (FIG. 2B, FIG. 14D, FIG. 14E). When these particles are compacted via centrifugation over a 70-pm mesh, cells migrate through the matrix within 24 hours (FIG. 15). To optimize the aLN, both stiffness (FIG. 2C) and signal density (FIG. 2D) were varied, as T cells are quite sensitive to both parameters. Hickey et al., 2019. On day 7, CD8+ T cell fold proliferation peaked around 2 kPa and drastically declined at higher stiffnesses (FIG. 2C). This observation was consistent with our previous work, which showed that hydrogels with a storage modulus (G’) in the range of 0.5 to 1 kPa resulted in a higher degree of TCR complex clustering and fold proliferation as compared to more stiff gels. Hickey et al., 2019. As expected, CD8+ T cell expansion also showed dependence on signal density (FIG. 2D). Fold proliferation peaked at 4 pg / mL and dropped off at higher signal densities, possibly due to overstimulation. aLN microparticles optimized for signal density and stiffness resulted in 4-fold greater proliferation compared to a standard T cell-activating system (anti-CD3 / anti-CD28-conjugated Dynabeads) in a 7-day culture (FIG. 2E). Additionally, aLN microparticles were stable at 4°C storage for at least three months (FIG. 16).

[0123] Optimized microgel particles with loaded Kb MHC (with either SIY or another model antigen OVA), were used to expand antigen- specific cells from naive B6 CD8+ T cell populations. On day 7, cells were harvested and analyzed by flow cytometry to determine the expansion of OVA+ or SIY+ CD8+ T cells. OVA- and SIY-specific T cell populations grew from less than 0.01%, Murphy and Weaver, 2017; Jenkins and Moon, 2012, to 5.7% and 39.2%of CD8+ T cells, respectively, in 7 days of culture. Thus, the aLN can expand the populations of OVA- and SIY-spccific T cells by approximately 2,400- and 11,000-fold, respectively (FIG. 2F, FIG. 2G).

[0124] In Vivo Antigen-specific CD8+ T Cell Activation by aLN Leads to Functional Killing

[0125] With an injectable aLN capable of providing prolonged stimulatory signaling, the efficacy of the aLN to stimulate T cells in vivo was tested using an in vivo killing assay (FIG. 3A). Schutz et al., 2014. The effect of the injection route and the importance of ex vivo priming of T cells before co-injection in vivo with aLN were investigated.

[0126] B6 mice were used as the host and were treated with 1 x 106pmel transgenic, Thyl.l+, CD8+ effector T cells on day 0, with varying injection routes, activation statuses, and with the presence or absence of aLN (FIG. 3A). Transgenic pmel CD8+ T cells are specific for Dbspl0° and express the congenic marker Thy 1.1. Labeling them with CellTrace Violet (CTV), allowed us to assess their in vivo activation and proliferation. On day 6, Thy 1.2+ B6 splenocytes were CFSE labeled, pulsed with cognate peptide, and injected i.v. to serve as target cells. The next day, host lymph nodes and spleens were harvested for evaluation of adoptively transferred effector cell proliferation (pmel, CTV, Thy 1.1) and target cell killing.

[0127] Interestingly, DO treatment with naive pmel CD8+ T cells co-injected subcutaneously (s.c.) with the aLN provided the most effective killing of all the groups (approximately 65%) (FIG. 3B, FIG. 17A). Intraperitoneal (z.p.) injections of the same set of cells along with aLN resulted in significantly reduced killing (approximately 5%), indicating that s.c. injection of aLN and CD8+ T cells provides a better microenvironment for aLN-mediated CD8+ T cell stimulation in vivo. This observation is possibly due to the ability of aLN microparticles to compact and form a stable scaffold upon s.c. injection. As expected, 3-day activated CD8+ T cells effectively killed cognate target cells (approximately 45%), but 3-day activated CD8+ T cells co-injected with aLN were less effective at killing target cells (approximately 25%) than either preactivated cells injected alone or naive cells injected with aLN.

[0128] To detect the presence and activation status of pmel cells in the periphery, the lymph nodes and spleens were harvested and stained for Thy 1.1+ CD8+ T cells and CTV dilution. For 3-day activated CD8+ T cells co-injected with aLN, there was a higher frequency of adoptively transferred CD8+ T cells in the lymph node (approximately 0.9%) compared to pre-activated CD8+ T cells without aLN (approximately 0.43%) (FIG. 3C, FIG. 17B). Additionally, pre-activated CD8+ T cells co-injected with aLN had a much lower CTV MFI, indicating robust in vivo expansion, compared to pre-activated CD8+ T cells without aLN (FIG. 3D, FIG. 17C). While pre-activated cells co-injected with aLN showed significant expansion via CTV dilution, that group showed slightly lower percentage of Thy 1.1+ cells in the spleen and LN than naive cells with aLN (though not statistically significant). This observation may be due to the preactivated cells dying off gradually following injection due to exhaustion, or due to their migration to tissue(s) that we did not analyze for Thyl.l presence. In contrast, naive CD8+ T cells co-injected with the aLN consistently showed the largest presence of Thy 1.1+ cells (approximately 6.51%) and the highest dilution of CTV, indicating significant in vivo expansion of functional CD8+ T cells. No toxicity was seen in host mice 24 hours and 9 days after treatment injections, as determined by complete blood count (CBC) testing and histology analysis of the heart, lungs, liver, kidneys, and spleen, other than a slight increase in platelet count on day 9 (FIG. 18).

[0129] The persistence of aLN-stimulated T cells was analyzed via in vivo killing on day 21. B6 mice were injected s.c. with 1 x 106naive pmel CD8+ T cells in the presence of either aLN or HA microgels with no signals conjugated (“blank aLN”). On day 21, cells co-injected with aLN still demonstrated an average of 16% target cell killing, while cells injected with no stimulation demonstrated only 1% killing (FIG. 3E).

[0130] To test the generality of our approach, we studied the in vivo activity of the aLN using OT-I transgenic CD8+ T cells, which are specific for the ovalbumin antigen. Naive OT-I cells (CD45.2+) were injected s.c. into B6 mice (CD45.1+) either with blank aLN (no signals conjugated) or KbOVA-specific aLN. In the group that received KbOVA aLN, the persistence of injected cells (FIG. 3F) and significant target-cell killing of OVA-pulsed splenocytes (FIG. 3G) were observed.

[0131] In vitro versus in vivo OT-I T cell activation with aLN also was compared. Interestingly, OT-I CD8+ T cells that were co-injected with aLN and then isolated 7 days later showed about a 3.5-fold higher Granzyme B expression after re-stimulation compared to OT-I cells that were plated with aLN ex vivo for 9 days (FIG. 3H). This finding suggests that in vivo activation of T cells may be superior to ex vivo activation. These results demonstrate the potent capacity of the aLN to elicit in vivo antigen- specific CD8+ T cell stimulation without additional ex vivo priming.

[0132] Activating Rare Antigen-specific CD8+ T cells in vivo from a Diverse TCR Repertoire

[0133] The use of transgenic mice represents T cell therapies, such as CAR or TCR T cell therapy, where the T cells share one TCR. There arc advantages, however, to expanding antigenspecific cells from broad repertoires, including being able to expand polyclonal populations that all recognize one antigen. Isser et al., 2021. We tested the ability of the aLN to activate antigenspecific cells from wild type B6 mice, which have a broad, diverse TCR repertoire capable of recognizing many different antigens.

[0134] The ability of the aLN to activate adoptively transferred CD8+ T cells from wild type B6 mice was initially studied by analyzing in vivo killing in irradiated and non-irradiated host mice. Irradiated mice received lymphodepleting doses of radiation, 500 cGy, Wrzesinski et al., 2010, on day -1. On day 0, aLN specific for model antigen SIY were co-injected with 6 x 106naive B6 CD8+ T cells. Target cells pulsed with SIY peptide were injected on day 8, and mice were harvested on day 9 (FIG. 4A). The group that was not lymphodepleted, and thus had a fully intact host immune system, showed 13 times more target cell killing than the group that was lymphodepleted (FIG. 4B). This effect was antigen specific as control aLN, aLN with no signals conjugated (“blank aLN”), had no activity (FIG. 19A).

[0135] Since lymphodepletion negatively affected functional activation, it was important to determine if host cells directly mediate in vivo killing. We analyzed killing through cognate aLN with or without transferred cells. Injection of cognate aLN without adoptively transferred cells did not result in detectable target cell killing (FIG. 19B). Additionally, we investigated the system for its specificity to ensure that the immune reaction is not non-specific. Expansion of T cells using non-cognate aLN also did not result in target cell killing (FIG. 19B). Therefore, killing was mediated by the adoptively transferred cells in an antigen specific manner.

[0136] The in vivo activity of the aLN also was studied using another antigen, OVA. KbOVA- specific aLN were able to enrich for OVA-specific T cells up to 12% within the aLN injection site (FIG. 4C). These cells were also highly functional and had activity outside the aLN site, as shown by effective target cell killing in the LN (FIG. 4D). Again, we directly compared cells activated in vivo with aLN against cells activated ex vivo with aLN. On day 9, both groups were harvested and analyzed by flow cytometry. Interestingly, compared to ex vi vo-activated cells, in if v -activatcd cells showed higher expression of CD 127, a marker for T cell memory, indicating that these cells may persist longer in the body (FIG. 4E). Ldpez-Cantillo et al., 2022.Additionally, when directly compared to the ex vivo expansion of antigen- specific cells, in vivoexpanded cells showed a 6-fold increase in the percentage of antigen-specific cells (FIG. 4F, FIG. 4G). These data indicate that the aLN can activate and expand different rare antigenspecific T cells from an endogenous repertoire in vivo, and possibly that the in vivo activation of T cells may be superior to ex vivo activation.

[0137] Probing aLN interactions with the host immune system

[0138] While in vivo killing was entirely dependent on the adoptively transferred cells (FIG. 19B), the aLN showed enhanced efficacy in mice with an intact immune system (FIG. 4B). To understand the role the host immune cells played in the aLN expansions of the injected cells, recruitment of host immune cells to the aLN was studied by co-injecting CD45.1+ B6 CD8+ T cells and KbOVA-specific aLN into CD45.2+ mice. aLNs were explanted on days 3, 6, and 9 to analyze infiltrating cells (FIG. 5A-FIG. 5G, FIG. 20). The total number of cells in the aLN remains approximately constant throughout the 9-day period; however, the number and percentage of host cells in the injection site increase over time (FIG. 20A). There is an initial decrease of the number and percent of total CD3+ cells and CD3+CD8+ by day 3, but these populations gradually increase again by day 9 (FIG. 5B, C). These populations not only include the adoptively transferred cells, but also infiltration of host CD4+ and CD8+ T cells (FIG. 5C, FIG. 5D). OVA-specific CD8+ T cells began to be detectable on day 3 and steadily increased until day 9 (FIG. 20B).

[0139] The majority of the infiltrating cells in the aLN on day 3 are CD3- (FIG. 5E). CD3- CDllb- cells, which may include granulocytes, platelets, and erythrocytes, rapidly infiltrate the aLN but then decrease in number and percent by day 9 (FIG. 5F). CD3-CD1 lb-1- cells, which may include macrophages, monocytes, dendritic cells, and NK cells, gradually increase by day 9 (FIG. 5G). The ebb and flow of these populations generally follow what has been shown to occur both during wound healing and in response to biomaterial injection. Davenport Huyer et al., 2020.

[0140] With a majority of the cells as CD3-, this observation suggests that the aLN also causes secondary immunostimulatory functions either directly or indirectly through CD8+ stimulation. Consequently, we wanted to examine the local environment with greater granularity in the cell types represented and organization induced by the aLN. To do so we employed CODEX multiplexed fluorescence microscopy, a technique using iterative imaging and DNA-barcoded antibodies that enables multiple antibodies to be imaged simultaneously. Kennedy-Darling et al.,2021 ; Black et al., 2021 ; Hickey et al., 2022; Hickey et al., 2023. We designed a panel that would be able to identify adoptively transferred T cells and major adaptive and innate immune cell types (Table 1, Table 2).

[0141] Table 1. CODEX staining conditions and cycle information.

[0142] Table 2. CODEX marker staining evaluation.

[0143] CODEX fluorescence microscopy images (FIG. 5H, Table 1, Table 2) of aLN explants 3 days and 9 days post-injection align with our flow cytometry data. By day 3, there was a large number of CD1 Ib-t- host leukocytes that infiltrated the aLN, along with a small increase in host CD4+ T cells (FIG. 5H). This trend of increasing immune infiltration continued 9 days after the implantation (FIG. 5H), also supported by traditional histology (FIG. 21). More specifically, CODEX images show that by day 9 the aLN microenvironment contains a high diversity ofimmune cells, including CD8+ T cells (a mixture of both transferred and host cells), dendritic cells (CDl lc+), macrophages (F4 / 80+), NK cells (NKp46), monocytes and neutrophils (Ly6G+), and a small number of B cells. These populations were quantified using a deep-learning algorithm to perform single-cell segmentation and processing. Hickey et al., 2023.

[0144] We quantified the fluorescence of each cell marker for every cell profiled across each of the days and plotted the major cell categories (FIG. 51, FIG. 5J, FIG. 22). The diversity of cells in the microenvironment is indicative of higher order immune responses occurring and could provide support for the observed more robust response with the endogenous immune system intact. Not only was there a greater diversity, but also we observed T cells interspersed and in direct contact with a variety of antigen-presenting cells including dendritic cells, different macrophage phenotypes, and myeloid cells (CDl lb+). Hickey et al., 2023. These results suggest that the aLN microparticle injection is serving as a direct T cell-activator and may be a catalyst to create a microenvironment that supports T cell expansion beyond the typical artificial antigen- presenting cell technologies.

[0145] To model the in vivo effects of host immune cells in the aLN, we further probed the importance of specific helper cells in T cell expansion ex vivo. Antigen-specific cells were expanded from either splenocytes or purified CD8+ T cells and the number of CD8+ T cells per well was held constant. Cells expanded from splenocytes showed an increased number of SIY+ CD8+ T cells present by day 7 (FIG. 23A). These cells were more functional, showing modest increases in stimulatory cytokine release, most notably IFNy (FIG. 23B, FIG. 23C). When CD4+ T cells were depleted from total splenocytes, there is a significant decrease in the total number of SIY-specific T cells after a 7-day culture, indicating the importance of CD4+ T cells in CD8+ T cell expansion (FIG. 23D). Additionally, amongst the host helper cells that migrated in and out of the aLN, CD4+ T cells were most directly correlated with the enrichment of antigen- specific cells within the aLN (FIG. 23E). These results are consistent with the idea that CD4+ helper T cells, along with other immune cells, have a critical role in the success of CD8+ T cell activation and expansion. Zajac and Cox, 2010; Hickey et al., 2020.

[0146] In Vivo aLN stimulated T Cells Inhibit Tumor Growth. As shown hereinabove, aLN injected in vivo can expand and functionally activate adoptively transferred T cells in an antigenspecific specific manner. We tested the ability of this treatment to induce T cells that recognize and kill tumor cells in the distal flank using several tumor models. The effect of aLN injectionswas tested in two prophylactic tumor prevention models, MC38-OVA, a murine colon cancer model, and B16-OVA, a murine melanoma model (FIG. 6A). CD8+ T cells were isolated from OT-I mice, and 1 x 106CD8+ T cells were co-injected with OVA-specific aLN on day -7. On day 0, MC-38 OVA tumor cells were injected on the opposite flank. aLN-treated mice had slower tumor growth (FIG. 6B-D) and statistically significant prolonged survival (FIG. 6E). Similar results were obtained using B16-OVA tumors, which are known to be more aggressive and less immunogenic (FIG. 6 F-FIG. 61).

[0147] The aLN also was tested in a tumor treatment model. MC38-OVA tumor cells were injected on day 0, and once the tumors were palpable, OT-I CD8+ T cells with the aLN were injected on the opposite flank on Day 6 (FIG. 6J). Significant reductions in tumor growth (FIG. 6K-FIG. 6M) and prolonged survival (FIG. 6N) were seen in treated animals.

[0148] We tested the ability for the aLN to stimulate tumor- specific T cells from fully naive B6 CD8+ T cells in a tumor treatment model, as well as tested the effects of combining the aLN platform with immune checkpoint blockade (ICB). On day 0, B16-OVA tumor cells were injected on one flank, and aLN with B6 CD8+ T cells was injected on the opposite flank on day 6. In the groups also receiving ICB, mice were given 200 pg anti-PDl i.p. on days 6, 9, 12, and 15 (FIG. 60). Tumor growth (FIG. 6P) and survival (FIG. 6Q) were monitored. The group receiving a sham hydrogel injection and anti-PD-1 saw the fastest tumor growth. The aLN treatment alone resulted in extended survival, while the group receiving both aLN and anti-PD-1 treatment had the slowest growth and significantly extended survival. These results confirm the function of aLN-stimulated T cells in stimulating tumor- specific T cells from a broad CD8+ T cell repertoire, as well as its potential to be used in conjunction with other immunotherapies.

[0149] Expansion of human antigen-specific T cells using aLN. To test the translatability of the aLN platform, the aLN was used to expand antigen-specific CD8+ T cells from human PBMCs. As with murine models, a greater enrichment / expansion was seen when bulk PBMCs were stimulated compared to purified CD8+ T cells. Within the CD8+ population, there was more than a 9-fold greater enrichment of antigen-specific CD8+ T cells when the starting cell population was PBMCs (FIG. 24A). PBMCs harvested from four healthy donors were plated with aLN with HLA-A*02-MART-l as Signal 1 and anti-CD28 as Signal 2 (FIG. 7A). Cells were harvested on day 11 and analyzed by flow cytometry using MART-1 tetramers to detect antigen-specific cells. Robust antigen- specific expansion was observed and measured by thepercentage (FIG. 7B), total cell number (FIG. 7C), and fold expansion (FIG. 7D) of MART-1 + CD8+ T cells. Similar to our observation in the murine antigen- specific expansions, the percentages of CD4+ T cells in aLN culture both on day 0 and day 11 positively correlated to the percentages of MART-1+ CD8+ T cells on day 11 (R2= 0.77 and 0.98, respectively) (FIG. 24B, FIG. 24C).

[0150] The functionality of MART- 1- specific cell expanded cells was tested by intracellular cytokine staining. MART-1 specific cells expressed one or more of IFNy, TNFa, and IL-2, indicating polyfunctional cells (FIG. 7E, FIG. 24D). Collectively, these studies with human immune cells help demonstrate the potential translatability of the aLN system, motivating the continued study of artificial T cell niches.[Q0151]Discussion

[0152] The aLN platform incorporates several essential T cell-activating components, providing T cells with Signals 1, 2, and 3, ECM cues, and helper cells from the host, which work in concert to improve T cell activation over traditional stimulation / expansion methods, such as nano / microparticle-based artificial antigen presenting cells (aAPCs). Hickey et al., 2019. The matrix material, hyaluronic acid, is also a biocompatible material that has been FDA-approved for other applications in humans, https: / / www.fda.gov / medical-devices / aesthetic-cosmetic- devices / fda-approved-dermal-fillers#materials. As the first aLN matrix for direct, in vivo antigen- specific activation of CD8+ T cells, this Example addresses the current technical gap in generating T cell responses in situ from fully naive T cell populations.

[0153] The aLN offers compositional versatility, including control over the stiffness, concentrations / densities of Signals 1, 2, and 3, presentation configuration of Signal 3, and the ability to conjugate additional supporting cues. In this Example, the aLN uniquely presents and protects an immune effector cell-biased Signal 3 in a localized fashion and provides HA as a cell adhesion signaling. The matrix-bound IL-2- Ab complex extended the stability and potency of IL-2 and provided a local presentation of IL-2 at the interface of the matrix and T cells, thus amplifying cytokine signaling efficiency and reducing the overall amount of IL-2 needed to support aLN function. Additionally, the IL-2 antibody clone employed reduces the inherent bias of IL-2 towards T regulatory cells by blocking cytokine interaction with the IL-2 receptor alpha chain’ Krieg et al., 2010; Letourneau et al., 2010, enhancing the in vivo performance of the system in the context of cancer therapy. Spangler et al., 2015. We also showed that the aLN canencapsulate cytokine molecules for extended release, demonstrating the system’s modularity. The aLN platform can easily incorporate various cytokines, chcmokincs, and metabolismaltering drugs to further enhance or tailor the T cell-stimulating microenvironment.

[0154] To showcase the function of the in vivo aLN- stimulated T cells and their potential applicability for cancer therapeutics, we tested the ability of the aLN to activate both transgenic CD8+ T cells and endogenous CD8+ T cells in vivo. The use of transgenic cells is analogous to the deployment of engineered TCR and chimeric antigen receptor (CAR) T cell therapies, wherein all CD8+ T cells have identical antigen specificities. Fesnak et al., 2016.

[0155] We demonstrated effective antigen- specific in vivo activation comparable to direct ex vivo T cell activation with functional killing activity. In vzvo-activated cells were able to inhibit tumor growth and extend survival in two murine models using distal treatment injections, which has not been previously shown using T cell stimulating scaffolds. We then established that the aLN can expand functional rare, tumor- specific cells from a naive, wild type population, demonstrating its potential in endogenous T cell therapy. Additionally, we show the platform’s potential to be combined with other immunotherapies such as ICB . An important potential limitation, however, should be discussed. It is known that cancer patients often have a detectable amount of tumor- specific T cells in circulation, Fehlings et al., 2019, which may be analogous to the group tested in FIG. 3, which received a 3-day pre-activation prior to being co-injected with hydrogel. Further testing of whether the presence of previously activated tumor-specific T cells in patients will be of importance in future translational studies to determine which patients may be responders or non-responders to this therapy.

[0156] Mechanistically we found that the influx of host immune cells was highly beneficial, highlighting the importance of host cells in supporting the activation of CD8+ T cells, which is consistent with our understanding of the natural LN microenvironment: Endogenously, many cells interact with and influence T cell activation. Hickey et al., 2020; Laidlaw et al., 2016; Lauvau and Goriely, 2016. Macrophages and dendritic cells, licensed by CD4+ T cells, provide co-stimulation and cytokine support. Activated CD4+ T cells provide soluble cytokine support, including IL-2, for T cell expansion. Macrophages and monocytes can also release stimulating cytokines and chemokines that attract other helper immune cells. Finally, CD8+ T cells provide themselves and neighboring T cells with a cocktail of cytokines, including IL-2, that stimulate growth (FIG. 25). The aLN may facilitate the concentration of all these cell types and create amore natural T-cell-stimulating microenvironment. Moreover, because the aLN microenvironment develops similarly to tertiary lymphoid organs (TLOs), Aoyama ct al., 2021, it may also be of use in foundational and therapeutic studies of TLOs. Additional studies will further analyze the phenotype of the recruited cells, including transcription factor staining of CD4+ T cells, and determine potential inhibitory or inflammatory signals generated by these cells.

[0157] The aLN provides several advantages over other T cell stimulation platforms. Firstly, the aLN can expand antigen- specific T cells directly from splenocytes that have an endogenous repertoire of T cells. Secondly, the aLN hydrogel microparticles are fully separable from the enriched cells using cell strainers, allowing for a wide range of assays to be performed after enrichment. Finally, the aLN is modular and can independently tune Signals 1, 2, and 3, and the number of T cell binding sites to modulate contact between the aLN and T cells.

[0158] In vivo, the aLN also provides unique advantages. The aLN creates a dynamic space that develops into a lymph-node-mimicking microenvironment, not unlike tertiary lymphoid structures. Aoyama et al., 2021. First, upon injection, the aLN microgels are compacted to provide a higher degree of cell-cell interaction in addition to cell-matrix interactions. T cells may be in closer proximity, allowing them to take advantage of autocrine and paracrine signaling provided by nearby cells. Additionally, the compacted aLN allows T cells to interact with aLN microgel particles in a three-dimensional space, which has been shown to improve cell phenotype and proliferation compared to 2D culture systems. Daly et al., 2019; Jensen and Teng, 2020; Perez Del Rio, 2018. Finally, in vivo T cell culture can allow host immune cells to infiltrate the aLN and support T cell expansion as they do in the natural lymph node (FIG. 26).

[0159] Along with other recent translational advances, the aLN platform for de novo, in vivo antigen- specific T cell responses has the potential to make immunotherapy more widely accessible to patients. aLN platforms can inspire new studies to enrich our understanding of the immunological functions of T cells, the mechanism of immune niche formation, and the interactions between immune cells and biomaterials. These studies will also provide insights into new strategies to combat a broader range of medical conditions, such as autoimmune disorders, infectious diseases, and tissue remodeling.

[0160] Experimental

[0161] Anz7zzaZs. B6, 2C, OT-I, and PMEL transgenic mice were maintained per guidelines approved by the Johns Hopkins University Institutional Animal Care and Use Committee. C57BL / 6J CD45.1+ and CD45.2+ mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All mice were between 8 and 12 weeks old, and both male and female mice were used.

[0162] MHC-Ig, HIA-Ig, and Peptides. Soluble MHC-Ig and HLA-Ig dimers loaded with peptides were produced in-house as described. Kosmides et al., 2018; Hickey and Schneck, 2018; Chiu et al., 2011. Peptides used for murine experiments: GP100: KVPRNQDWL, SIY: SIYRYYGL, and OVA: SIINFEKL. Peptides used for human experiments: MART-1: ELAGIGILTV. Peptides were purchased from GenScript (New Jersey, USA).

[0163] IL-2 + 11-2 Antibody Immunocomplex Preparation. The mouse immunoglobulin (IgG) 2a heavy chain (HC) and kappa light chain (LC) of MAB602 were separately cloned into the gWiz mammalian expression vector (Genlantis) using Gibson Assembly (New England Biolabs). Plasmids were purified using ZymoPURETM II Plasmid Midiprep Kit (Zymo Research), and constructs were verified by Sanger sequencing.

[0164] MAB602 was expressed recombinantly in human embryonic kidney (HEK) 293-F cells via transient co-transfection of plasmids encoding the heavy and light chains. Heavy and light chain plasmids were titrated in small-scale co-transfection tests to determine optimal DNA ratios for large-scale expression. HEK 293F cells were grown to 1.2 x 106cells / mL and diluted to 1.0 x 106cells / mL on the day of transfection. Plasmid DNA (filter sterilized through a 0.22-pm PES filter, Corning) and polyethyleneimine (PEI, Polysciences) were independently diluted to 0.05 and 0.1 mg / mL, respectively, in OptiPro medium (Thermo Invitrogen), and incubated at 20°C for 15 min. Equal volumes of diluted DNA and PEI were mixed and incubated at 20°C for an additional 15 min. Subsequently, the DNA / PEI mixture was added to a flask containing the diluted HEK cells, which was then incubated at 37°C with shaking for 3-5 days. Secreted protein was harvested from HEK 293F cell supernatants via Protein G (Thermo Scientific) affinity chromatography, followed by size-exclusion chromatography on an AKTA fast protein liquid chromatography (FPLC) instrument using a Superdex 200 column (Cytiva). All proteins were stored in HEPES -buffered saline (HBS, 150 mM NaCl in 10 mM HEPES pH 7.3). Purity was verified by SDS-PAGE analysis.

[0165] The MAB602 anti-IL2 antibody was thiolated with the above procedures and stored with 0.5 wt / vol% PEGDA. IL-2 was added at a 1:1 molar ratio to minimize unbound IL-2 in solution and stored at 4 °C until use.

[0166] Preparation of aLN. Anti-CD3 and anti-CD28 antibodies were purchased respectively from BioXCell (145-2C11; West Lebanon, NH, USA) and BioLegend (37.51; San Diego, CA, USA). Antibodies and MHC-Ig dimers were partially reduced with 100-mM dithiothreitol (DTT) for 30 min at room temperature to expose free thiol groups. After conjugation, antibodies / MHC- Ig were thoroughly washed through a centrifugation filtration with a 50-kDa MWCO filter. PEGDA crosslinker was added to reduced MHC-Ig dimers, anti-CD3, and anti-CD28 antibody solutions to a final concentration of 0.5% PEGDA in preparation for crosslinking HA and conjugation of the signaling cues.

[0167] Hydrogels were prepared as previously described. Hickey et al., 2019. Briefly, thiol- modified hyaluronic acid (HA) (ESI BIO, Alameda, CA, USA) was resuspended with 1 mL sterile dH2O and incubated at 37°C for 30 min until completely dissolved to form 1% HA solution in lx PBS. To form hydrogels, the appropriate antibodies were added to the HA, followed by a crosslinker (polyethylene glycol diacrylate (PEGDA) with a molecular weight of 3400 (Laysan Bio, Arab, AL)) at a 4:1 volume ratio. The mixture was aspirated into a syringe and allowed to gel for 1 h.

[0168] To produce microgel aLN, the bulk gel was particulated using the stainless steel Swinny Filter Holder 13 mm (Millipore Sigma, Burlington, MA) with a stainless-steel mesh with an intermesh diameter of 150 pm. This device was autoclaved to be sterilized each time prior to use. For ex vivo T cell activation, a solution of complete media was used to prime the device. Then a syringe with 500 pL of complete media was placed on the side furthest from the mesh. Then the preformed aLN hydrogel was attached to the device. Then pressure was exerted on the syringe with the hydrogel to push it through the mesh. Then the solution was pushed back and forth through the mesh with both syringes for a total of 5 times before eluting into culture dishes with cells. For in vivo injections, the procedure was the same, except that the solution used was PBS.

[0169] aLN staining and imaging. aLN microparticles were first activated by 50-mM EDC- HC1 (ProteoChem) and 20 mM NHS (Sigma Aldrich) in 0.1 M MES buffer (Sigma Aldrich) for 30 min. After washing the microparticles with PBS 3 times, microgels were added to 0.1 mg / mL FITC-labeled bovine albumin (Thermo Fisher) for 2 h for conjugation. aLN microparticles werewashed with PBS another 3 times to remove the excessive bovine albumin. 100 pL of the solution containing microparticles was added to a glass slide, covered by a cover slip, and dried at 4°C for 2 h. Microparticle slides were then imaged by ZEISS Apotome 3 Microscope to determine the shape and morphology of aLN microparticles.

[0170] CD8+ T Lymphocyte Isolation. Murine cells were obtained from adult mouse lymph nodes and spleens. Obtained cells were treated with ACK lysing buffer to lyse red blood cells and were filtered through cell strainers to isolate splenocytes. CD8+ T lymphocytes were then isolated from splenocytes by negative selection using CD8+ isolation kits and magnetic columns from Miltenyi Biotech (Auburn, CA, USA) according to the manufacturer’s protocol.

[0171] E Vivo T Cell Culture and Activation. For ex vivo T cell expansion, isolated CD8+ T cells were cultured in the T cell culture media (RPMI supplemented with L-glutamine, non- essential amino acids, vitamin solution, sodium pyruvate, P-mercaptoethanol, 10% fetal bovine serum, ciprofloxacin, and a cocktail of T cell growth factors as described previously). Oelke et al., 2000. On day 3 or 4 of the culture, cells were fed half the volume of the initial T cell culture media with twice the concentration of the T cell growth factor cocktail. When IL-2 or IL-2+Ab complexes were used at indicated doses, the cocktail of T cell growth factors was not added. For stimulation on the hydrogel, cells were plated on the surface of the hydrogel with concentrations of the stimulatory antibody (either anti-CD3 and anti-CD28 or pMHC-Ig and anti-CD28) conjugated to the HA hydrogel.

[0172] T Cell Proliferation Assay. CD8+ T cells were isolated as previously described and resuspended in 1 mL of T cell culture media. Cells were mixed with 1 pL of CellTrace™ carboxyfluorescein succinimidyl ester (CFSE) dye (ThermoFisher) in 1 mL of T cell culture media per 3 million cells and incubated at 37°C for 20 min. CFSE-stained cells were washed with 50-mL T cell culture media to remove any unstained dye and were plated. On day 3 of culture, cells were harvested and stained with a 1: 100 PBS solution of APC-conjugated rat antimouse CD8a, clone 53-6.7 (BD Pharmingen) for 15 min at 4°C. The CFSE fluorescence intensity was measured using a BD FACSCalibur flow cytometer. Cell proliferation was analyzed using FlowJo with diluted CFSE fluorescence peaks, signifying the population after each round of cell division. A subset of the cells was allowed to expand for 7 days, and viable cells were counted with a hemocytometer to determine fold expansion.

[0173] Expansion of Rare Antigen-Specific T cells. B6 CD8+ T cells were stimulated on HA hydrogel surfaces as described previously for 7 days. To detect antigen- specific CD8+ T cells, cells were stained with 1 pg of either cognate or non-cognate biotinylated pMHC-Ig dimer, with a 1:100 ratio of APC-conjugated rat anti-mouse CD8a, clone 53-6.7 (BD Pharmingen) in FACS wash buffer for 1 h at 4°C. Samples were washed and then stained with a 1:350 ratio of PE- labeled streptavidin (BD Pharmingen) and a 1:1000 ratio of LIVE / DEAD™ Fixable Green Dead Cell Stain (ThermoFisher) in PBS for 15 min at 4°C. Cells were washed and read on a BD FACSCalibur. To determine the percent of antigen-specific cells, the following gates were used in the respective order: live+, lymphocyte-i- (forward scatter by side scatter), CD8+, and Dimer+. The Dimer-i- gate was determined by comparing the non-cognate to the cognate stain. To determine the percentage of antigen-specific cells, the percentage of Dimer-i- of the cognate MHC-Ig stain was subtracted from the non-cognate MHC-Ig stain. This number was multiplied by the percentage of CD8+ T cells and the number of cells counted to obtain the number of antigen-specific cells.

[0174] Antigen-specific T Cell Phenotype Assays. For analysis of cellular memory phenotype, on day 7 of culture, the number of cells was counted using a hemocytometer. After counting, less than 500,000 cells were stained with 1 g of either cognate or non-cognate biotinylated pMHC- Ig dimer, with a 1:100 ratio of PerCP-conjugated rat anti-mouse CD8a, clone 53-6.7 (BD Pharmingen) in FACS wash buffer for 1 h at 4°C. Samples were washed and stained with a 1:350 ratio of PE-labeled streptavidin (BD Pharmingen), a 1:100 PBS solution of APC- conjugated rat anti-mouse CD8a, clone 53-6.7 (BD Pharmingen), FITC-conjugated rat antimouse CD62L, clone MEL- 14 (BD Pharmingen), APC-conjugated rat anti-mouse CD44, clone IM7 (BioLegend), and 1:1000 of LIVE / DEAD Fixable Aqua Dead Cell Stain (ThermoFisher) for 15 min at 4°C. Cells were then washed with FACS wash buffer to be read on BD LSRII flow cytometer and analyzed using FlowJo to measure the population of naive T cells (CD62L+CD44-), effector T cells (CD62L-CD44+), and memory T cells (CD62L+CD44+).

[0175] Antigen-specific T Cell Cytokine Functionality Assay. On day 7 of culture, approximately 500,000 CD8+ T cells were isolated from each condition and separated into cognate or non-cognate groups. Cells were stained with 1 pg of either cognate or non-cognate biotinylated pMHC-Ig dimer for 1 h at 4°C. After washing, samples were stained with a 1:350 ratio of PE-labeled streptavidin (BD Pharmingen, San Diego, CA, USA). Then 10 pL solution of1 :50 FTTC anti-CD107a, 1 :350 BD GolgiStop Protein Transport Inhibitor (BD Biosciences), and 1:350 BD GolgiPlug Protein Transport Inhibitor (BD Bioscicnccs) in PBS was added to the samples and incubated with 100 pL of complete media for 37 °C for 6 h. Cells were then washed and stained with 1:100 PBS solution of PerCP-conjugated anti-mouse CD8a, clone 53-6.7 (BioLegend), and 1:1000 of LIVE / DEAD AmCyan Fixable Aqua Dead Cell Stain (ThermoFisher) at 4°C for 30 min. Cells were then fixed and permeabilized overnight with 100 pL BD Cytofix / Cytoperm Fixation and Permeabilization Solution (BD Biosciences). Cells were washed with lx BD PERM / Wash buffer with 2% BSA and stained with 1:100 solution of APC- conjugated rat anti-mouse IFNy, clone XMG1.2 (BD Pharmingen) and PE-Cy7-conjugated rat anti-mouse TNFa, clone MP6-XT22 (Biolegend) in PERM / Wash buffer with 2% BSA at 4°C for 1 h. Stained cells were read on a BD LSR II flow cytometer.

[0176] In Vitro Killing Assay. Cultured tumor cells were harvested and stained with CFSE. 5 x 103tumor cells were added to each well of a 96 well plate, with each testing condition in triplicate. CD8+ T cells that had been cultured with aLN for 7 days were harvested the same day, and added to the wells with tumor cells at the indicated effector : tumor (E:T) ratios. The cells were incubated together at 37C for 18 hours, then harvested and run on flow cytometry to determine % killing of the tumor cells. The percent killing was calculated as follows: % of in vitro killing = (1 - (% live tumor cells) / (% live tumor cells in tumor only condition)) x 100. Vitro Migration Assay. Hydrogel microparticles were added to a culture plate with inwell inserts of pore size 70 pm. The hydrogel microparticles were added above the inserts and the plate was centrifuged to compact the particles to mimic the pressure in a subcutaneous injection. CD8+ T cells (l x 106cells) were added above the hydrogel and cells were counted from the bottom chamber at the indicated time points.

[0178] In Vivo Killing Assay using Transgenic Mouse Model. On day 0, Thy 1.2 B6 mice were injected s.c. with indicated treatment groups, including the adoptive transfer of 1 x 106transgenic pmel CD8+ T cells that were either naive or pre-activated and labeled with CellTrace™ Violet (ThermoFisher) per manufacturer’s instructions. CD8+ T cells that were preactivated for 3 days prior on anti-CD3, anti-CD28 HA hydrogels as previously described. Selected treatment groups received aLN hydrogels containing DbgplOO, anti-CD28, and IL- 2+Ab at a concentration of 10 pg / mL of aLN gel. For each group of four mice, 250 pL of microgel aLN were formed in a syringe and eluted in a total of 500 pL of PBS as previouslydescribed and then pre-mixed with isolated naive or activated pmel CD8+ T cells, where each mouse received 100 pL of total injection material cither with subcutaneous or intraperitoneal injections.

[0179] On day 6, two populations of splenocytes were differentially labeled with high and low levels of CFSE, where the high group was incubated with cognate gplOO peptide for pmel CD8+ T cells. Specifically, target cells were harvested from splenocytes of Thyl.2 B6 mice. 4 x 107splenocytes were labeled with a high concentration of CFSE (0.25 pM), and another was labeled with a low concentration of CFSE (0.025 pM) in 1 mL of PBS at 37°C for 20 min (Invitrogen, Eugene, OR). Five- volume of excess fetal bovine serum (FBS) was added to quench the reaction, incubated at 37°C for another 5 min, and then washed with serum-free media twice. CFSE-high cells were then incubated at 37 °C for 1 h with 1 pg of GP100 peptide for every 1 x 107splenocytes in media without serum. The cells were washed twice with PBS, counted, and mixed at a 1:1 ratio with control nontarget, CFSE-low splenocytes. 1 x 107mixed splenocytes were injected into each recipient mice intravenously in 100 pL volume, including Thyl.2 B6 mice that received no treatment.

[0180] On day 7 post-treatment, the lymph nodes and spleen were harvested for evaluation of treated cell proliferation and target killing of the gplOO-i- population. Cells were counted and 1 x 107lymphocytes were stained with a 1:100 ratio of APC -conjugated rat anti-mouse CD8a, clone 53-6.7 (BioLegend), and a 1:100 ratio of PerCP-conjugated mouse anti-mouse CD90.1 (Thyl.l), clone OX -7 (BioLegend) in PBS for 30 min at 4°C. Cells were washed by centrifugation and read on a BD LSRII flow cytometer. The percent killing was calculated as follows: % of in vivo killing = 100 - ([(% specific peptide-pulsed cells in treatment / % unspecific B6 cells in treatment) / (% specific peptide-pulsed in no treatment controls / % unspecific B6 cells in no treatment controls)] x 100). The experiment was repeated in the same manner using OT I transgenic CD8+ T cells and SIINEK-pulsed splenocytes. For experiments using B6 CD8+ T cells rather than transgenic cells, 3-6 x 106CD8+ T cells were injected on day 0. Peptide-pulsed target cells were injected i.v. on day 8, and mice were sacrificed on day 9.

[0181] Scanning Electron Microscopy. Injection sites were explanted and fixed with 4% paraformaldehyde, followed by 4% osmium tetroxide, and dehydrated with serial solutions of ethanol and hexamethyldisilazane. Samples were coated with 10 nm of AuPd (EM ACE600, Leica Microsystems, Deerfield IL), and secondary electron micrographs were captured with ascanning electron microscope (MIRA, TESCAN, Bmo-Kohoutovice, Czech Republic) at an acceleration voltage of 5 kV.

[0182] Prophylactic Tumor Inhibition Assays. On day 7, B6 mice were injected s.c. with 1 x 106naive OT-I CD8+ T cells and KbOV A- specific aLN. On day 0, either 1 x 106MC38-OVA or 2 x 105B16-OVA tumor cells were injected s.c. on the opposite flank. Tumor sizes were measured using calipers and reported by multiplying the longest measured length by the perpendicular direction of the tumor. For all mice, routine behavior, grooming, and feeding observations were be made daily. Animals showing obvious signs of severe distress and / or pain, including inability to feed or drink or excessive weight loss (>20%), were humanely sacrificed by CO2 inhalation. Tumors were measured every 2-3 days and mice with tumors over 2000 mm3in size were euthanized. In addition, if ulceration of subcutaneous tumors occurred, those animals were euthanized.

[0183] Tumor Treatment Assays. On day 0, B6 mice were injected with 1 x 106MC38-OVA tumor cells. On day 6, once tumors were palpable, 1 x 106naive OT-I CD8+ T cells were coinjected s.c. with KbOVA specific aLN on the opposite flank from the tumors. Tumor sizes were measured using calipers and reported by multiplying the longest measured length by the perpendicular direction of the tumor. Mice were monitored as described above.

[0184] Culture and Analysis of Human Antigen-Specific CD8+ T cells. The of The Johns Hopkins Institutional Review Board approved this study protocol using human cells, and all healthy volunteers gave written informed consent (Human IRB protocol number: NA_00027947). Frozen PBMCs from healthy donors were thawed in a 37°C water bath for 1 min in a 0.02% benzonase solution. Cells were washed three times in warm T cell culture media and transferred to a T-25 flask to be kept standing upright at 37 °C overnight. For ex vivo T cell culture, PBMCs were plated at 500,000 cells / 100 p L in a 96-well U-bottom plate. For each well, 30 pL of peptide-loaded and washed microparticles and 60 pL of 2x human cytokine media were added (T cell culture media with 10% human AB serum supplemented with IL-2, IL-4, IL-6, IL- ip, and IFNy). On days 3 and 5 of culture, cells were fed 100 pL of warm 2x human cytokine media. Cells were harvested on day 7 and washed through a 70-pm filter tube in warm media to remove the microparticles. After counting, the PBMCs were rcplatcd at 30,000 cclls / 100 pL with 30 pL of newly made microparticles and 60 pL of human cytokine media. Cells were harvested, counted, and analyzed by flow on day 11.

[0185] To analyze cells for antigen-specific expansion and cytokine release, approximately 500,000 CD8+ T cells were isolated from each condition and separated into cognate or noncognate groups. Cells were incubated at 37°C for 6 h in 100 pL of media containing 1:350 BD GolgiPlug Protein Transport Inhibitor (BD Biosciences). The media for the group to be stimulated also contained 250,000 anti-CD3 / anti-CD28 DynaBeads. Cells were washed and resuspended in PBS containing Human BD Fc Block (BD Biosciences) and 1 pg of MART-1 tetramer (MBL) and allowed to incubate for 30 min at room temperature. After washing, cells were stained with APC Cy7 CD3 and APC CD8 (BioLegend) at 4°C for 30 min. Cells were washed, then fixed and permeabilized with 100 pL BD Cytofix / Cy toperm Fixation and Permeabilization Solution (BD Biosciences). After incubation at 4°C for 20 min, cells were washed and stained with FITC IFNy, PeCy7 TNFa, and BV421 IL-2 (BioLegend) stain diluted 1:50 in perm / wash buffer for 1 h at 4°C. Cells were washed and resuspend in 200 pL / well of PBS to run on flow cytometry.

[0186] CODEX Multiplexed Imaging. aLN were explanted on the given day and were embedded in OCT and immediately frozen. After freezing, explants were sectioned to a thickness of 7 pm using a cryostat and arranged on slides. The tissue arrays were stained with the validated panels of CODEX antibodies and imaged. Black et al., 2021.

[0187] Briefly, this entailed cyclic stripping, annealing, and imaging of fluorescently labeled oligonucleotides complementary to the oligonucleotide conjugated to the antibody. Each array underwent CODEX multiplexed imaging; metadata from each CODEX run can be found in Table 1. Raw imaging data were processed using the Akoya Phenocylcer Fusion 1.6.0 software for image stitching, drift compensation, deconvolution, and cycle concatenation. After the upload, images were evaluated for specific signals. Any markers that produced an untenable pattern or a low signal-to-noise ratio were excluded from the ensuing analysis. Uploaded images were visualized in Image J (https : / / imagej .nih.gov / ij / ) .

[0188] CODEX single-cell segmentation. To obtain quantitative single cell information, we segmented individual cells and extracted single-cell protein expression. Processed data were segmented using the CellVisionSegmenter, a neural network R-CNN-based single-cell segmentation algorithm68. The CellVisionSegmenter software can be downloaded at github.com / michacllccl / CcllScg.

[0189] Cell-type analysis. Cells were identified and classified into 16 cell types and states based on marker expression for murine studies. Cell type identification was done following the strategies we have developed. Hickey et al., 2023; Hickey et al., 2021. Briefly, nucleated cells were selected by gating DRAQ5, Hoechst double -positive cells, followed by z-normalization of protein markers used for clustering (some phenotypic markers were not used in the unsupervised clustering). The data were overclustered with Leiden-based clustering with the scanpy Python package. Clusters were assigned a cell type based on average cluster protein expression and location within image. Impure clusters were split or reclustered following mapping back to original fluorescent images.EXAMPLE 2Signal 2 molecules for producing memory-like effector CD8+ T cells, Thl helper CD4+ T cells, and cytotoxic CD4+ T cells

[0190] The activation of naive, antigen- specific T cells requires multiple signals to sufficiently induce proliferation and differentiation. Traditional aAPCs have presented Signal 1 (peptide- MCH / HLA complex) and Signal 2 (co- stimulation). aAPC technologies have almost exclusively presented a-CD28 as Signal 2; however, further research has pointed to the importance of additional co- stimulatory molecules in controlling T cell differentiation. Chacon et al., 2013; Croft et al., 2003; Hernandez-Chacon et al., 2011; Oh et al., 2015; Rudolf et al., 2008; Zheng et al., 2014.

[0191] These research efforts have suggested that Signal 2 presentation affects the resulting T cell phenotype, including their memory status, which has notable effects in cancer immunotherapy. Ross et al., 2018. Without wishing to be bound to any one particular theory, it is thought that, in CD8+ T cell activation, alternative Signal 2 molecules on aAPCs may result in memory precursor effector cells (MPECs) rather than short-lived effector cells (SLECs). Joshi et al., 2007. For CD4+ T cell activation, in which more lineages exist, Signal 2 variation is an even more important determinate of cell fate. Chen et al., 2013.

[0192] In this Example, we will determine which Signal 2 molecules most effectively create memory CD8+ T cells and helper Thl CD4+ T cells. Additionally, we will investigate how different Signal 2 compositions affect interactions between the cell types, so as to optimize CD4+ T cell support to CD8+ T cells in co-culture. Recent work, has revealed that CD4+ T cells can have lytic activity and when studied in model systems can be important in tumorimmunotherapy and also seen in clinical responses including ACT' Isser et al., 2022; Melenhorst ct al., 2022; Fu ct al., 2013; Oh ct al., 2020. We therefore also propose to study the impact of Signal 2 on CD4+ lytic activity.

[0193] Effect of Signal 2 molecules on the generation of memory-like CD8+ T cells.

[0194] Preliminary Data. Several studies have shown that effector CD8+ T cells may lead to strong initial anti-tumor effects. If these cells do not develop into memory cells, however, those effects are short-lived and can lead to cancer recurrance. Dolina et al., 2021; Liu et al., 2020.

[0195] As such, our focus will be on choice of Signal 2 molecules that facilitate development of a memory phenotype, which can be skewed through the activation milieu. For example, receptors in the TNF-family, such as CD27 and 4-1BB, have been shown to increase expansion and survival of CD8 T cells. Chacon et al., 2013; Croft, 2003; Hernandez-Chacon et al., 2011; Oh et al., 2015. Stimulation through two or more of these receptors increases expansion further.Rudolf et al., 2008; Zeng et al., 2014.

[0196] These molecules, however, have not been directly compared or simultaneously expressed on in vivo scaffolds. Determining the combination of signals that results in enhanced expansion of antigen- specific T cells with a memory phenotype will improve uses of aLN for both ex vivo analysis and therapeutic applications of the aLN platform. In a preliminary experiment, we tested the activation of antigen specific CD8+ T cells from naive B6 T cells with aLN of various Signal compositions, including the canonical Signal 2 a-CD28 ex vivo (FIG. 27 A). We saw that the addition of a-41BB led to about a two-fold higher ratio of central memory to effector cells.Additionally, the ratio of specific Signal 2 molecules influences the proliferation and phenotype of ex vivo cultures T cells (FIG. 27B, FIG. 27C). We will continue to build on these results by investigating alternative Signals 2’s and the ratio of Signal 2 molecules and performing extensive analysis of the resulting phenotype, functional memory, and anti-cancer function.

[0197] Effects of top aLN compositions on in vivo stimulated T cells

[0198] Preliminary Data. To test in vivo use of aLN, we co-injected naive CD8+ T cells with aLN presenting a cognate antigen and three different co-stimulation combinations: 0C-CD28, a- CD28+0C-CD27, and ot-CD28+a-41BB. The function of these cells was tested in vivo on days 7 and 21 by analyzing target cell killing of i.v. injected peptide-pulsed splenocytes. While each group of aLN-stimulated cells showed similar function on day 7, by day 21 the groups that received co-stimulation with either a-CD27 or 0C-41BB showed significantly higher killingfunction than those that received stimulation with only 0C-CD28 (FIG. 28). These results show strong evidence that including additional co-stimulatory molecules on the aLN produce CD8+ T cells that persist longer or function better in vivo.

[0099] Experimental Design. To test the synergy between CD4+ and CD8+ co-expansion in vivo, we will inject naive B6 CD4+ and CD8+ T cells with Class I and Class II aLNs, subcutaneously, as optimized in the previous sub-aims. After 8 days, we will i.v. inject target cells, splenocytes pulsed with OVA. On day 9, we will harvest spleens, lymph nodes, and aLN to detect T cell proliferation and migration, and analyze target cell killing via flow cytometry. Since it is important to create both an immediate therapeutic response as well as a long-lasting response, we will repeat this experiment with a longer timeline (2, 3, and 4 weeks) to determine functional memory response generation. We will also repeat these experiments with another affinity high antigen complex, KbSIY, and a low-affinity DbGPlOO antigen. Throughout these experiments, we will compare the inclusion of CD4+ specific aLN to the injection of only CD8+ specific aLN. We hypothesize that stimulating Thl CD4+ T cells in conjunction with CD8+ T cells will increase the expansion of antigen- specific cells and improve their function and memory phenotype.EXAMPLE 3Investigate Inclusion of Adhesion Proteins in aLN on Cell Infiltration and Egress

[0200] In some embodiments, adhesion proteins are included in the presently disclosed aLN. Without wishing to be bound to any one particular theory, it is thought that surface molecules besides those directly involved in the immunological synapse can be incorporated into the aLN to alter how the T cells interact with the hydrogel. For example, it has been previously shown that incorporation of the cell adhesion peptide arginylglycylaspartic acid (RGD) into the aLN results in an increase in proliferation after 7 days; however, it did not rescue proliferation in all conditions, for example, on stiffer gels. Hickey, 2019. Beyond improving the interaction between T cells and aLN, it also has been shown that including cell adhesion motifs on the substrate improves the ability of T cells to migrate across the substrate. Hailemichael et al., 2013; Stephan et al., 2015. The ability for T cells to migrate in and out of the matrix in vivo can be vital to ensuring that activated cells can leave the aLN and for naive cells to enter the aLN. Accordingly, in some embodiments, the role of various adhesion molecules (including, but not limited to RGD, collagen, and ICAM-1) in improving activation, proliferation, and migration ofT cells, as well as other host immune cells that may be beneficial to the microenvironment, can be investigated. To this end, in certain embodiments, RGD, collagen, and intercellular adhesion molecule 1 (ICAM-1) can be incorporated separately into aLN expressing MHC Class I and II, and the optimized Signals 2 and 3.We will incorporate RGD, collagen, and ICAM-1 separately into aLN expressing MHC Class I and II, and the optimized Signals 2 and 3 from Aims 1 and 2. Naive CD4+ and CD8+ T cells will be co-cultured in each aLN, and we will record antigen- specific T cell activation and proliferation after 7 days. This will be repeated with peptides of low, medium, and high affinity, as the benefit of adhesion proteins may be more prominent in T cell clones that have low affinity for their target. To test the effect of adhesion proteins on the migratory capacity of naive and activated T cells, we will use a Nikon Eclipse Ti inverted time-lapse microscope to monitor T cell migration at distinct stages of a 9-day culture: days 0, 3, 7, and 9 to represent naive, activated, and proliferating T cells. CD4+ and CD8+ T cells will be differentially labelled to determine how the adhesion molecules influence each cell individually, as well as how they interact with each other. To track single-cell migration, we will take time-lapse images at 30 sec. intervals for 30 min. Movements of individual T cells will be quantified using Volocity software (Improvision). To track how addition of these adhesion molecules affects T cell migration in the matrix in vivo, we will inject aLN with or without each adhesion molecule into the flank on day 0, and measure cell migration in two ways: (1) by explanting the aLN days 3, 6, and 9 and quantify each cell type in the matrix, and (2) fluorescently labeling the T cells prior to injection and using IVIS imaging to track cell migration daily after injection until day 9.REFERENCES

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[0272] 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.

Claims

THAT WHICH IS CLAIMED IS:

1. An artificial lymph node (aLN) comprising an extracellular matrix (ECM) hydrogel conjugated with an antigen presenting complex (Signal 1), a co-stimulatory ligand (Signal 2), and T cell-stimulating cytokine.

2. The aLN of claim 1, wherein the antigen presenting complex (Signal 1) is selected from a major histocompatibility complex (MHC) molecule, a human leukocyte antigen (HLA) molecule, and an anti-CD3 antibody.

3. The aLN of claim 2, wherein the MHC molecule is a monomer or a dimer.

4. The aLN of claim 2, wherein the antigen presenting complex (Signal 1) is an MHC class I molecule, an MHC class II molecule, or a combination of an MHC class I molecule and an MHC class II molecule.

5. The aLN of claim 2, wherein the antigen presenting complex (Signal 1) is an MHC-Ig dimer or an HLA-Ig dimer.

6. The aLN of any one of claim 1 to 5, wherein the antigen presenting complex (Signal 1) comprises an MHC molecule loaded with a peptide or an HLA molecule loaded with a peptide.

7. The aLN of claim 6, wherein the peptide is selected from GP100: KVPRNQDWL SEQ ID NO: 1; SIY: SIYRYYGL SEQ ID NO: 2; OVA: SIINFEKL SEQ ID NO: 3, and MART-1: ELAGIGILTV SEQ ID NO: 4.

8. The aLN of claim 1, wherein the co-stimulatory ligand (Signal 2) comprises an antibody that specifically binds to a protein expressed on a T cell, wherein the protein expressed on a T cell is selected from CD28, CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27,CD30, CD134 (OX-40L), B7h (B7RP-1 ), CD40, LIGHT, HVEM, CD40L, 0X40, and combinations thereof.

9. The aLN of claim 8, wherein the co-stimulatory ligand (Signal 2) is selected from anti-CD28 (a-CD28) antibody, anti-CD27 (a-CD27) antibody, and anti-41BB (a-41BB) antibody.

10. The aLN of claim 8, wherein the co-stimulatory ligand (Signal 2) comprises at least a first antibody that specifically binds to a protein expressed on a T cell and at least a second antibody that specifically binds to a protein expressed on a T cell.

11. The aLN of claim 10, wherein the at least first antibody that specifically binds to a protein expressed on a T cell and the at least second antibody that specifically binds to a protein expressed on a T cell are present in a ratio of first antibody: second antibody selected from about 99:1, 98:2, 97:3, 96:4, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 4:96, 3:97, 2:98, and 1:99.

12. The aLN of claim 10, wherein the at least first antibody that specifically binds to a protein expressed on a T cell and the at least second antibody that specifically binds to a protein expressed on a T cell are selected from a-CD28 and a-CD27 or a-CD28 and a-41BB.

13. The aLN of claim 1, wherein the T cell- stimulating cytokine is selected from IL- 2, IL-4, IL-7, IL- 10, IL- 12, IL-12p70, IL- 15, IL-21, CXCL10, and gamma interferon (IFN-y).

14. The aLN of claim 1, wherein Signal 3 comprises a T cell- stimulating cytokine / antibody complex.

15. The aLN of claim 14, wherein the T cell- stimulating cytokine / antibody complex (Signal 3) comprises an anti-IL-2 antibody complexed with IL-2 (IL-2+Ab).

16. The aLN of claim 15, wherein the anti-IL-2 antibody comprises MAB602.

17. The aLN of any one of claims 1 to 16, wherein the hydrogel comprises a hyaluronic acid (HA) hydrogel.

18. The aLN of claim 17, wherein the HA hydrogel comprises a thiol-modified HA hydrogel.

19. The aLN of claim 1, further comprising one or more adhesion molecules.

20. The aLN of claim 19, wherein the one or more adhesion molecules are selected from arginylglycylaspartic acid (RGD), collagen, and intercellular adhesion molecule 1 (ICAM- 1).

21. A microgel comprising the aLN of any one of claims 1 to 20.

22. A method for stimulating one or more T cells, the method comprising contacting the one or more T cells with one or more aLNs of any one of claims 1-20, or a microgel thereof.

23. The method of claim 22, wherein the one or more T cells comprise antigenspecific CD8+ T cells.

24. The method of claim 22 or claim 23, wherein the contacting of the one or more T cells with the one or more aLNs occurs in vivo.

25. The method of any one of claims 22 to 24, comprising administering the one or more aLNs in combination with one or more naive T cells.

26. The method of claim 23, wherein administering the one or more aLNs in combination with one or more naive T cells creates a T cell-activating niche.

27. The method of any one of claims 22 to 26, wherein administering the one or more aLNs results in one or more of recruitment of host immune cells, coordination of host immune cells, and providing an immuno-stimulatory microenvironment for antigen- specific T cell activation and expansion.

28. The method of any one of claims 22 to 27, wherein the contacting of the one or more T cells with the one or more aLNs induces in vivo antigen-specific CD8+ T cell stimulation without ex vivo priming or expansion.

29. The method of any one of claims 22 to 28, wherein the contacting of the one or more T cells with the one or more aLNs enables in situ manipulation of antigen-specific responses for an immunotherapy.

30. A method for treating a disease, disorder, or condition, or providing prophylactic protection to immunosuppressed patients, the method comprising administering a therapeutically effective amount of an artificial lymph node (aLN) of any one of claims 1 to 20 to a subject in need of treatment thereof.

31. The method of claim 30, further comprising administering naive, wild type CD8+ T cells in combination with one or more aLNs.

32. The method of claim 30 or claim 31, wherein the disease, disorder, or condition is selected from a cancer, an infectious disease, and an autoimmune disease.

33. The method of claim 32, wherein the cancer comprises a solid tumor or a hematological malignancy.

34. The method of claim 32, 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 oralcancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, a neuroblastoma, and a glioma.

35. The method of any one of claim 32 to claim 34, wherein administering the naive, wild type CD8+ T cells in combination with the one or more aLNs results in activation and expansion of tumor-targeted T cells that kill target cancer cells, slows tumor growth, and increases survival.

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