Anti-CD3 granular hydrogel for immune protection of cell therapies

Abstract

The present invention provides biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 antibody presenting engineered biomaterials, as well as methods of making the biomaterials. The invention provides anti-CD3 antibody presenting engineered biomaterials, that are hydrogels bound to anti-CD3 antibody. The invention provides methods of using biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 antibody presenting engineered biomaterials, for immunomodulation, inducing immune suppression, and specific immune tolerance to prevent or reduce the risk of rejection of cellular or tissue grafts and / or the treatment of autoimmune disorders such as type 1 diabetes.

Description

Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 ANTI-CD3 GRANULAR HYDROGEL FOR IMMUNE PROTECTION OF CELL THERAPIES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 63 / 695,761, filed September 17, 2024, and U.S. Provisional Application No. 63 / 786,553, filed April 10, 2025, each of which is incorporated by reference in its entirety for all purposes. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant No. R01 DK132387, awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING

[0003] The application includes sequences in an electronic sequence listing named 637910SEQLST.XML of size 5.5 KB, created September 16, , 2025, which is incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0004] Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin producing cells, leaving patients dependent on exogenous insulin therapies. Despite advances in continuous glucose monitoring and insulin pumps, patients remain at risk for severe hypoglycemic episodes. Clinical islet transplant therapy, through transplantation of cadaveric donor islets, achieves insulin independence in most patients and offers the potential for a durable, physiological cure. However, clinical application is severely limited due to limited donor tissue availability and graft longevity. One of the key hurdles thought to contribute to the ultimate failure of transplanted grafts is immune-mediated rejection. After islet allotransplantation into patients with T1D, the grafts undergo destruction from both allogeneic adaptive responses and autoreactive responses against islet antigens. Current drug regimens are targeted against allogeneic recognition of the transplanted tissue, but can compromise graft function as well as 1 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 increase the susceptibility for life-threatening pathogenic infections. These limitations have created a significant need for the protection of transplanted cells from immune attack.

[0005] To address this, biomaterials have been explored to encapsulate transplanted islets and shield them from the host immune system. While this strategy has shown some promise, traditional bulk hydrogels are hindered by their size and nanoporous structure, which restricts cell infiltration and revascularization. Additionally, the need to maintain oxygen and nutrient diffusion limits the scaffold’s volume, often resulting in local hypoxia and nutrient deprivation for the encapsulated islets. [1-3] Efforts to overcome these limitations and enhance encapsulated islet survival include oxygen-generating beads, co-encapsulation with mesenchymal stem cells (MSCs) or endothelial cells, tethered angiogenic growth factors, macroporous scaffolds, and the incorporation of ECM protein to recapitulate the islet basement membrane and niche. [4-9]

[0006] More recently, microporous annealed particles (MAPs), a form of granular hydrogel composed of interlinked packed microgels, have emerged as a promising platform for tissue engineering and cell delivery.[10-13] Unlike bulk hydrogels, MAP hydrogels feature interconnected porous networks that promotes nutrient diffusion, cell migration, and integration with host tissue. [14, 15] These materials are tunable, injectable due to their shear-thinning properties, and compatible with the same chemistries used in traditional hydrogel systems.

[0010] Islets can be embedded in the pores of MAP hydrogels and injected with minimal shear forces.

[0016]

[0007] Granular hydrogels are also being explored for localized immunomodulatory therapy.

[0017]

[0008] There is a need in the art for improved granular hydrogels and immunomodulatory biomaterials for treatment of autoimmune disorders such as type 1 diabetes and for treatment of allograft rejection. BRIEF SUMMARY OF THE CLAIMED INVENTION

[0009] In one aspect, the invention provides an anti-CD3 hydrogel biomaterial comprising: a) a hydrogel comprising maleimide-terminated four-arm polyethylene glycol (PEG-4MAL) and a thiol-terminated four-arm polyethylene glycol (PEG-SH); and b) an anti-CD3 antibody conjugated to the hydrogel. 2 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0010] In some anti-CD3 hydrogel biomaterials, the hydrogel is a microgel. In some anti-CD3 hydrogel biomaterials, the microgel has a 10 μm diameter.

[0011] In some anti-CD3 hydrogel biomaterials, the microgel is formed by reacting an N- hydroxysuccinimde (NHS)-PEG-thiol with PEG-4MAL via batch emulsion polymerization. In some anti-CD3 hydrogel biomaterials, the anti-CD3 antibody is conjugated to the hydrogel via an amide linkage to at least one arm of the PEG-SH.

[0012] In some anti-CD3 hydrogel biomaterials, the hydrogel is functionalized with at least one guest molecule and at least one host molecule. In some anti-CD3 hydrogel biomaterials, the host molecule is a cyclodextrin or a cucurbit[n]uril. In some anti-CD3 hydrogel biomaterials, the cyclodextrin is β-cyclodextrin. In some anti-CD3 hydrogel biomaterials, the guest molecule is adamantane, phenylalanine, ferrocene, or p-xylylenediamine. In some anti-CD3 hydrogel biomaterials, the host molecule is β-cyclodextrin and the guest molecule is adamantane.

[0013] In another aspect, the invention provides a method of making a biomaterial displaying an anti-CD3 antibody, the method comprising reacting a N-hydroxysuccinimde (NHS)-polyethylene glycol (PEG)-thiol with a maleimide-terminated four-arm polyethylene glycol (PEG-4MAL) macromer to generate an NHS-activated microgel via batch emulsion polymerization; and capturing an anti-CD3 antibody via the NHS moiety to form the biomaterial displaying the anti- CD3 antibody.

[0014] In some methods, the microgel has a 10 μm diameter.

[0015] Some methods further comprise the step of functionalizing the PEG-4MAL with at least one guest molecule and at least one host molecule prior to reacting the N-hydroxysuccinimde (NHS)-polyethylene glycol (PEG)-thiol with the PEG-4MAL.

[0016] In another aspect, the invention provides a biomaterial produced by any of the above- mentioned methods.

[0017] In another aspect, the invention provides a method of making an anti-CD3 microgel, comprising: solubilizing four-arm polyethylene glycol (PEG-4MAL) in aqueous buffer at pH 5.4; solubilizing four-arm polyethylene glycol (PEG)-thiol (PEG-SH) in aqueous buffer at pH 3 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 5.4; solubilizing sulfo-SMCC; reacting the solubilized sulfo-SMCC with the solubilized PEG-SH to form a solubilized NHS-coupled PEG-SH; combining the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL at a 1:1 stoichiometric ratio to form a hydrogel; emulsifying the hydrogel with mineral oil to form an NHS-reactive microgel; and conjugating the NHS-reactive microgel with an anti-CD3 monoclonal antibody to form the anti-CD3 microgel.

[0018] Some methods further comprise the step of functionalizing the solubilized PEG-4MAL with at least one guest molecule and at least one host molecule prior to combining the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL. In some methods, the host molecule is a cyclodextrin or a cucurbit[n]uril. In some methods, the cyclodextrin is β-cyclodextrin. In some methods, the guest molecule is adamantane, phenylalanine, ferrocene, or p-xylylenediamine. In some methods, the host molecule is β-cyclodextrin and the guest molecule is adamantane.

[0019] In another aspect, the invention provides an anti-CD3 microgel produced by any of the above-mentioned methods.

[0020] In another aspect, the invention provides a method of inducing immune tolerance to a graft cell or a graft tissue in a subject in need thereof, comprising transplanting into the subject any of the above-mentioned anti-CD3 hydrogel biomaterials and the graft cell or the graft tissue.

[0021] In some methods, the graft cell is a pancreatic islet cell. In some methods, the graft cell is a stem cell derived beta cell cluster (sBC). In some methods, the stem cell derived beta cell cluster (sBC) is a pancreatic beta cell cluster.

[0022] In some methods, the graft tissue comprises a pancreatic islet cell. In some methods, the graft tissue is a stem cell derived beta cell cluster (sBC). In some methods, the stem cell derived beta cell cluster (sBC) is a pancreatic beta cell cluster.

[0023] In another aspect, the invention provides a method of treating type 1 diabetes in a subject in need thereof, comprising transplanting into the subject any of the above-mentioned anti-CD3 hydrogel biomaterials and pancreatic islet cells or stem cell derived beta cell clusters (sBC).

[0024] In some methods, pancreatic islet cells are transplanted into the subject. In some methods, stem cell derived beta cell clusters (sBC) are transplanted into the subject. 4 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0025] Some methods further comprise administering to the subject an immunosuppressive drug. In some methods, the immunosuppressive drug is rapamycin, cyclophosamide busulfan, fludarabine, methotrexate, sulfasalazine, hydroxychloroquine, azathioprine, tocilizumab, etanercept, adalimumab, anakinra, abatacept, rituximab, certolizumab, golimumab, cyclosporine, dexamethasone, methylprednisolone, prednisone, tacrolimus, or triamcinolone. In some methods, the immunosuppressive drug is rapamycin.

[0026] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A-E depict the synthesis of anti-CD3 microgels. (A) Bioconjugate chemistry reactions for generating anti-CD3 microgels. Reaction scheme for maleimide-thiol Michael-type addition reaction, which is the basis for microgel generation. (B) 4-arm PEG-SH precursor is made amine-reactive by reacting one of the four arms with a Sulfo-SMCC which added an NHS ester group. (C) Depicts the approach for generating microgels from PEG-MAL and PEG-SH. 4- arm PEG-MAL is reacted with NHS-conjugated 4-arm PEG-SH as a water-in-oil emulsion to form microgels ready for functionalization with amine-containing biomolecules. (D) Depicts the NHS-ester to amine conjugation reaction used for functionalizing NHS-conjugated microgels with biomolecules. (E) Depicts the reaction of biomolecules (e.g. anti-CD3 antibody) with NHS- conjugated microgels to form biomolecule-functionalized microgels.

[0028] Figure 2 a schematic showing a general approach to making microgels (without biomolecule functionalization). 4-arm PEG-MAL and 4-arm PEG-SH are solubilized in aqueous solution, mixed, then vortexed as a water-in-oil emulsion to generate microgel droplets. Microgels are then collected by centrifugation and wash steps.

[0029] Figure 3 depicts a schematic showing a general approach to making amine-reactive microgels ready for biomolecule functionalization. 4-arm PEG-MAL and 4-arm PEG-SH-NHS are solubilized in aqueous solution, mixed, then vortexed as a water-in-oil emulsion to generate microgel droplets. Microgels are then collected by centrifugation and wash steps.. Figures 4A-C depict Synthesis of anti-CD3 microgels. (A) Reaction scheme for anti-CD3 microgels. PEG-SH 5 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 is reacted with a bifunctional crosslinker sulfo-SMCC. The functionalized macromer is then mixed with PEG-MAL and made into microgels through a water in oil emulsion. Washed microgels are then surface labeled with anti-CD3 or an isotype control. (Left) Isotype control microgels will be unable to interact and prevent the migration of activated autoimmune T cells towards transplanted islets. (Right) Depicts that unfunctionalized microgels allow for penetration of the material by T cells migrating through the interstitial spaces, while anti-CD3 microgels trap and eliminate T cells such that they cannot reach the transplanted cells in the interior of the construct. ( (B) Microscopy images were collected showing surface-coating of PEG-MAL microgels with anti-CD3 antibody, with results quantified in plot in panel B. Quantified mean fluorescence intensity of anti-CD3 over microgel area for sulfo-smcc titration.(C) Percent release of Armenian hamster IgG from sulfo-smcc microgels over 3 days. (D) Activated and expanded diabetogenic NOD.A2 T cells were co-cultured with titrated anti-CD3 microgels overnight before being analyzed for flow cytometry. (E) Percent CD4+T Cells of live total for titrated amount of anti-CD3. (F) Percent CD8+T cells of Live total.

[0030] Figures 5A-F depict Transwell migration assay for T cells through anti-CD3 microgels. (A) Scheme for transwell migration assay. Activated diabetogenic NOD.A2 CD3+ T Cells were placed on top of anti-CD3 microgels or isotype microgels in a transwell insert. In the bottom well a chemokine gradient was created by placing CXCL9 and CXCL10 in islet media. After 18 hours wells were analyzed for migration. (B) Microscopy images were collected showing T cells in the transwell stained for viability, with results quantified in panel B. Viability in the gel analyzed as the number of dead cells over the number of live cells in the gel. (C) Number of cells that migrated through the microgels towards the bottom well for a no gel control, isotype microgels and anti-CD3 microgels. (D) Viability of cells that migrated through to the bottom well as determined by Trypan Blue. (E) Percent release of Armenian hamster IgG from sulfo- SMCC microgels over 3 days.

[0031] Figures 6A-6K depict Transwell Invasion Assay of NOD.A2 T Cells towards sBCs. (A) Schematic of transwell invasion assay. 7.5e5 Activated, Expanded NOD.A2 T cells were placed on top of microgels with or without aCD3 in a transwell insert. 50 sBCs were placed in the bottom of the 24 well dish. Samples were collected after 24 and 48 hours. Analysis of (B) CD4+ T cell, (C) Live percentage of CD4+ T cells, and (D) CD8+ T cell populations within the 6 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 transwell after 24 and 48 hours. Analysis of (E) CD4+, (F) live percentage of CD4+, (G) CD8+, and (H) live percentage of CD8+ in the bottom well after 24 and 48 hours. Analysis of the % chromogranin A (CHGA+) and live percentage of CHGA+ after 24 and 48 hours. (K) Analysis of the percent C-peptide+ of the CHGA+ population in the bottom well after 24 and 48 hours.

[0032] Figures 7A-G depict Autoimmune model of type 1 diabetes in a humanized mouse for assessing immunomodulatory biomaterials. (A) Timeline of the study. On day -4 iPSCs were transplanted with anti-CD3 microgels or isotype microgels under the kidney capsule of an NSG.A2 mouse (anti-CD3 n = 6, isotype n = 4). On day 0 all mice received an adoptive cell transfer of activated diabetogenic NOD.A2 T Cells. Then monitored via IVIS over the next 12 days. (B) Macroscopic image of the sBC clusters surrounded by anti-CD3 microgel. Image shows sBCs + anti-CD3 microgels implanted under the mouse kidney capsule. (C) Images were collected showing intravital bioluminescence imaging of stem cell derived beta cells (sBCs) transplanted under the kidney capsule in NSG.A2 mice adoptively transferred with diabetogenic T cells from NOD.A2 mice, with results quantified in panel C. Quantification of the total flux measured from the mice. Two-way ANOVA.(D) Explanted kidneys for anti-CD3 and isotype microgel treatment groups bioluminescence detection via IVIS. Boxes indicate the grafts. (E) Quantification of the total flux in explanted kidneys. Chi Square analysis, Chi Square = 1.66. P- values displayed. All data displayed as mean ± s.e.m

[0033] Figures 8A-E depicts Insulin and HNA expression by sBCs 12 days after transplantation. Microscopy images were collected showing explanted kidney capsule grafts, immunostained for insulin and human nuclear antigen (HNA), counterstained for DNA < and PEG microgels labeled with a fluorescent tracer dye, with results quantified in panels B and C. (A) Implant area including the capsule and gel. (B) Insulin MFI normalized by the implant area for isotype and anti-CD3 after 12 days. (C) HNA MFI normalized by implant area for isotype and anti-CD3 after 12 days. (D) Representative relationship of sBC distance from kidney surface and insulin MFI. Slope for anti-CD3 microgels, m = 0.04, slope for isotype microgels, m = 0.01. Data represented by mean ± s.e.m., analyzed by nested T-test (A, B, C) or student’s t-test (D).

[0034] Figures 9A-E depict T Cell infiltration in anti-CD3 microgels. Microscopy images were collected of CD3 and CD4+ T Cells into gel and sBC graft for isotype anti-CD3 microgels, 7 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 stained for insulin, PEG, CD4, and CD3. Microscopy images were collected showing CD4+ and CD3+ T cell infiltration around sBC for isotype and anti-CD3 microgels. Microscopy images were collected of CD3+ and CD8+ T Cells into gel and sBC graft for isotype and anti-CD3 microgels, stained for insulin, PEG, CD8, and CD3. Results are quantified in panels A-F. (A-B) Expression and quantification of CD3+ T Cells in implant area (A) and gel area (B). (C-D) Expression and quantification of CD4+ T Cells in implant area (C) and gel area (D). (E-F) Expression and quantification of CD8+ T Cells in implant area (E) and gel area (F). Data represented by mean and s.e.m., analyzed by nested t-test, p values displayed.

[0035] Figures 10A-G depict autoimmune model of type 1 diabetes in a humanized HLA mouse for assessing immunomodulatory biomaterials. Subcutaneous transplant of sBCs in humanized autoimmune model of type 1 diabetes. (A) Timeline of study. On day -16800-1200 sBCs were injected with 200-400 ml of microgels + / - aCD3 in the dorsal subcutaneous space of an NSG.A2 mouse. On day 0 all mice received an adoptive cell transfer of 1e6 activated diabetogenic NOD.A2 T cells. Grafts were then monitored via IVIS over the next 42 days. Images were collected showing intravital bioluminescence imaging of stem cell derived beta cells (sBCs) transplanted subcutaneously in NSG.A2 mice adoptively transferred with diabetogenic T cells from NOD.A2 mice, with results quantified in panel B Microscopy images were collected showing intravital fluorescence imaging of PEG-microgels transplanted subcutaneously in NSG.A2mice. (B Quantification of the graft survival as measured by IVIS from the mice. (C) Blood glucose measurements over time of mice transplanted with the hydrogel and sBCs showing conversion to diabetes from the transferred T cells. (D) Intraperitoneal glucose tolerance test (IPGTT) on day 22 in mice transplanted with the hydrogel and sBCs. (E) Area under the curve (AUC) quantification of IPGTT. P-value displayed on the graph. Data displayed as mean ± s.e.m. (F) Percent diabetes incidence days post-adoptive cell transfer. (G) Human and (H) mouse c-peptide measured pre-act (Day -7) and post-ACT (day 33).

[0036] Figures 11A-H depicts Insulin and HNA expression by sBCs in subcutaneous space. (A) macroscopic image of the explanted sBC and microgel graft on day 42. (B) Tissue section of microgels and sBC graft immunostained for Insulin, CD3+, and CD8+ T cells. Scale bar = 50 m. (C-D) Flow cytometry analysis of draining lymph nodes of ACT controls and microgels + / - aCD3 of (C )%CD4+ and (D) %CD8+. Analysis of cell populations in subcutaneous grafts by 8 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 flow cytometry of (E) %CHGA+ (human endocrine cell marker), (F) %Cpep+ of CHGA+, (G) %CD4+ and (H) %CD8+. Data analyzed by one-way anova with tukey’s multiple comparisons (C, D), or student’s t-test (E, F, G, H).

[0037] Figures 12A-H depict results using guest-host microgels and PEG microgels.

[0038] Figures 13A-H depict results using guest-host microgels and PEG microgels.

[0039] Figures 14A-D depict results of studies on islets transplanted in guest-host MAP hydrogels in hyperglycemic conditions.

[0040] Figures 15A-G depict results of studies on effect of CD3 conjugated to guest-host MAP for the protection of transplanted sBCs.

[0041] Figures 16A-K depict results of studies on the immunoprotective effects of aCD3+ microgels.

[0042] Figures 17A-G depict results of studies on protection of sBCs by CD3+ MAP from an autoimmune attack.

[0043] Figures 18A- L depict results of studies on guest-host MAP grafts.

[0044] Figures 19A-E depict results of studies on CD3+ MAP activity using a transwell migration assay toward a CXCL9 / 10 gradient.

[0045] Figures 20A-E depict results of studies modeling autoimmune rejection of transplanted human sBCs. DEFINITIONS

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic 9 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4thed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0047] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term “an antibody” or “at least one antibody” can include a plurality of antibodies, including mixtures thereof. The conjunction "or" is to be interpreted in the inclusive sense, i.e., as equivalent to "and / or," unless the inclusive sense would be unreasonable in the context.

[0048] In general, the term "about" indicates insubstantial variation in a quantity of a component of a composition not having any significant effect on the activity or stability of the composition. When the specification discloses a specific value for a parameter, the specification should be understood as alternatively disclosing the parameter at "about" that value. Also, the use of "comprise," "comprises, " "comprising,” "contain," "contains," "containing," "include," "includes," and "including" are not intended to be limiting.

[0049] Unless specifically noted, embodiments in the specification that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components. Embodiments in the specification that recite "consisting essentially of" various components are also contemplated as "consisting of". "Consisting essentially of" means that additional component(s), composition(s), or method step(s) that do not materially change the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. 10 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0050] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions, such as "not including the endpoints"; thus, for example, "within 10-15" includes the values 10 and 15. One skilled in the art will understand that the recited ranges include the end values, as whole numbers in between the end values, and where practical, rational numbers within the range (e.g., the range 5-10 includes 5, 6, 7, 8, 9, and 10, and where practical, values such as 6.8, 9.35, etc.). When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0051] Unless otherwise apparent from the context, the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM or standard deviation) of a stated value. Unless otherwise apparent from the context, the term “about” encompasses values within ±5% or ±10% of a stated value.

[0052] Statistical significance means p 0.05.

[0053] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an anti-CD3 granular hydrogel ” or “at least one anti-CD3 granular hydrogel” can include a plurality of anti-CD3 granular hydrogels including mixtures thereof.

[0054] The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0055] The term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.

[0056] The term “symptom” refers to a subjective evidence of a disease, as perceived by the subject. A "sign" refers to objective evidence of a disease as observed by a physician. 11 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0057] The term "individual" or “subject” refers to a human or a non-human animal. The non- human subject may be, for example, a non-human animal, and may be, for example, a non- human mammal, bird, reptile, amphibian, or fish.. The “mammal” may include any animal classified as such, including humans, non-human primates, primates, baboons, chimpanzees, monkeys, cynomolgus, marmoset, rhesus, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, ferrets, guinea pigs, hamsters, gerbils etc.

[0058] Examples of an autoimmune disorder include type 1 diabetes, multiple sclerosis (MS), lupus (systemic lupus erythematosus, SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, psoriasis, psoriatic arthritis, autoimmune hepatitis, myasthenia gravis, Graves’ Disease, Hashimoto’s thyroiditis, vitiligo, and Sjogren’s syndrome.

[0059] Examples of an allograft rejection include islet cells (e.g., pancreatic islet cells), splenocytes, PBMCs, bone marrow cells, mesenchymal stem cells, hematopoietic stem cells, stem cells, induced pluripotent stem cells, human beta cell products, stem cell derived beta cell clusters (sBCs), hepatocytes, dendritic cells, macrophages, endothelial cells, cardiac myocytes, thymic epithelial cells and vascular cells, and immune cells, including T cells, etc., depending on the condition being treated.

[0060] The terms "therapeutically effective dose," or "therapeutically effective amount," refer to that amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of an autoimmune disorder. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, or reduce the risk of occurrence of one or more symptoms of an autoimmune disorder. The effective amount can be determined by methods well known in the art and as described in subsequent sections of this description.

[0061] The terms "treatment," "therapeutic method," and their cognates refer to treatment and prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely 12 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 receptiveness to treatment of a subject having the disorder. Treatment may include slowing or reversing the progression of a disorder.

[0062] The term “immune cell” refers to a white blood cell, for example a granulocyte, lymphocyte, or monocyte. The term “phagocyte” refers to a cell that can ingest a foreign particle, bacterium, or dead or dying cell. Exemplary phagocytes are white blood cells (such as neutrophils, monocytes, macrophages, mast cells, and dendritic cells). The term “monocyte” refers to a type of leukocyte or white blood cell that can differentiate into a macrophage or a monocyte-derived dendritic cell. The term “macrophage” refers to a white blood cell of the innate immune system that engulfs and digests pathogens, such as cancer cells, microbes, cellular debris, and foreign substances, which do not have proteins that are specific to healthy body cells on their surface, by phagocytosis. This process is called phagocytosis, which acts to defend the host against infection and injury. The term “neutrophil” refers to a type of white blood cell. A neutrophil is a type of phagocyte. The term “dendritic cell” refers to an antigen- presenting cell of the mammalian immune system and whose main function is to process antigen material and present it on the cell surface to the T cells of the immune system.

[0063] The term “adsorption” refers to the electrostatic association of an entity to the microgel without the formation of a chemical bond. The phrase “conjugated to” refers to the formation of a chemical bond via a chemical reaction of two functional groups. The term “functionalized” refers to the addition of a functional reactive group to facilitate downstream conjugation of two entities.

[0064] The term “hydrogel” refers to a water swollen polymer material, e.g., water-swollen polymer networks, with dimensions much larger than a cell (such as >500 μm). The term “microgel”(also referred to as a “hydrogel microparticle”) refers to a hydrogel with smaller dimensions (such as on the order of 10s or 100s of μm). In some embodiments the term “hydrogel microparticle” refers to small microgels formed by emulsion, microfluidic device, or fracturing of macroscopic hydrogels. The term “granular hydrogel” refers to a type of hydrogel composed of compacted microgels, often referred to as granules or hydrogel microparticles, that come together to form a cohesive, porous network. 13 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0065] The term “macromer” refers to a large, reactive polymeric molecule that contains functional groups capable of participating in chemical reactions, such as polymerization or crosslinking. These macromers serve as building blocks for creating more complex polymer networks or structures.

[0066] The term “click’ chemistry” refers to the simple, highly reactive, and highly efficient conjugation of two entities via two reactive groups with a selective affinity towards each other.

[0067] The term “allogeneic” refers to cells, tissues, or organs that are taken from one individual and transplanted into another genetically different individual of the same species. The term “xenogeneic” refers to cells, tissues, or organs that are derived from a different species and used in another species. The term "autologous” refers to cells, tissues, or organs that are obtained from and used in the same individual. The term “autogeneic” refers to processes, tissues, or responses that arise from or are derived from the individual's own body. The term “syngeneic” refers to cells, tissues, or organs obtained from one individual and used in a different, genetically identical individual.

[0068] The term “allograft” refers to a graft of cells, tissues, or organs that are taken from one individual and transplanted into another genetically different individual of the same species.

[0069] Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that an antibody or other biologically entity is typically at least 50% w / w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w / w pure of interfering proteins and contaminants from production or purification. Often an isolated monoclonal antibody or other biological entity is the predominant macromolecular species remaining after its purification.

[0070] Specific binding of an antibody to its target antigen means an affinity and / or avidity of at least 106, 107, 108, 109, 1010, 1011, or 1012M-1. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated 14 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody binds one and only one target.

[0071] The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0072] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).

[0073] An immunoglobulin light or heavy chain variable region (also referred to herein as a “light chain variable domain” (“VL domain”) or “heavy chain variable domain” (“VH domain”), respectively) consists of a “framework” region interrupted by three “complementarity determining regions” or “CDRs.” The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR-H2, and CDR-H3. When the 15 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 application discloses a VL sequence with R as the C-terminal residue, the R can alternatively be considered as being the N-terminal residue of the light chain constant region. Thus, the application should also be understood as disclosing the VL sequence without the C-terminal R.

[0074] The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional definitions include, the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia Kabat CDR in which CDR- H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular’s antibody modelling software; and, the contact definition of Martin et al (bioinfo.org.uk / abs), and IMGT definition (imgt.org / IMGTScientificChart / Numbering / IMGTnumberingCDR_VK.html; also see Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010) and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010)) (see Table 1). Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. When an antibody is said to comprise CDRs by a certain definition of CDRs (e.g., Kabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Kabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present. For example, an antibody comprising CDRs defined by Kabat includes among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions. 16 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0075] Table 1: Conventional Definitions of CDRs Using Kabat Numbering Composite IMGT*CDR-H1 by Chothia can end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places insertions of extra residues at 35A and 35B, whereas Chothia numbering places them at 31A and 31B. If neither H35A nor H35B (Kabat 17 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 numbering) is present, the Chothia CDR-H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0076] The term “antibody” includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv. Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.

[0077] The term “antibody” also includes a bispecific antibody and / or a humanized antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). Some bispecific or bifunctional antibodies have two different heavy / light chain pairs and two different binding sites.

[0078] Bispecific antibodies can also be: (1) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so- called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; or (5) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fc-region. Examples of platforms useful for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab). 18 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0079] The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0080] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography or cryogenic electron microscopy (cryo-EM) of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0081] Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, 10x, 20x or 100x) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the references antibody by at least 75%, 90% or 99%. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. 19 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0082] The term “polypeptide” refers to a polymer of amino acids of any length including natural full length proteins, fragments and synthetic peptides. The term also includes a polymer that has been modified, such as a polypeptide having a modified peptide backbone.

[0083] The term “polynucleotide” refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. A polynucleotide may be single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and a pol comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0084] The term “fragment” when referring to a polypeptide means a polypeptide that is shorter or has fewer amino acids than the full-length polypeptide. The term “fragment” when referring to a polynucleotide means a polynucleotide that is shorter or has fewer nucleotides than the full- length polynucleotide. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment. A fragment can also be, for example, a functional fragment or an immunogenic fragment.

[0085] For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.

[0086] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do 20 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0087] “Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0088] Unless otherwise stated, sequence identity / similarity values include the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10. 21 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0089] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” an anti-CD3 granular hydrogel may contain an anti-CD3 granular hydrogel alone or in combination with other ingredients. When the disclosure refers to a feature comprising specified elements, the disclosure should alternative be understood as referring to the feature consisting essentially of or consisting of the specified elements. Moreover, elements that are shown or described as being combined with other elements, can, in various embodiments, exist as stand-alone elements. DETAILED DESCRIPTION I. General

[0090] The present invention provides biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 hydrogel biomaterials. The invention provides methods of using the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, in inducing immune tolerance to a graft cell or a graft tissue in a patient in need thereof, comprising transplanting into the subject the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, and a graft cell or a graft tissue. The invention provides methods of using the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, in treating type 1 diabetes in a patient in need thereof, comprising transplanting into the subject the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel and pancreatic islets or stem-cell derived beta cell clusters (sBCs).

[0091] The anti-CD3 hydrogel biomaterials comprise: a) a hydrogel comprising maleimide- terminated four-arm polyethylene glycol (PEG-4MAL) and a thiol-terminated four-arm polyethylene glycol (PEG-SH); and b) an anti-CD3 antibody conjugated to the hydrogel. Some of the anti-CD3 hydrogel biomaterials are microgels, for example a microgel of 10 μm diameter.

[0092] The microgel can be formed by reacting an N-hydroxysuccinimde (NHS)-PEG-thiol with PEG-4MAL via batch emulsion polymerization. In some anti-CD3 hydrogel biomaterials the anti-CD3 antibody is conjugated to the hydrogel via an amide linkage to at least one arm of the 22 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 PEG-SH by first functionalizing at least one arm of the PEG-SH with Sulfo-SMCC to make it amine-reactive. The anti-CD3 hydrogel biomaterials can be made by a method comprising reacting N-hydroxysuccinimde (NHS)-PEG-SH macromer (generated by reacting four-arm PEG- SH macromer with Sulfo-SMCC) with maleimide-terminated four-arm polyethylene glycol (PEG-4MAL) macromer to generate an NHS-activated microgel via batch emulsion polymerization and capturing an anti-CD3 antibody via the NHS moiety on the microgel.

[0093] An exemplary method of preparing the anti-CD3 hydrogel biomaterials is: 1. Solubilize four-arm polyethylene glycol (PEG-4MAL) in aqueous buffer at pH 5.4; 2. Solubilize four-arm polyethylene glycol (PEG)-thiol (PEG-SH) in aqueous buffer at pH 5.4; 3. Solubilize sulfo-SMCC; 4. React the solubilized sulfo-SMCC with the solubilized PEG-SH to form a solubilized NHS-coupled PEG-SH; 5. Combine the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL at a 1:1 stoichiometric ratio. This mixture will crosslink into a hydrogel in several minutes; 6. Emulsify the NHS-coupled PEG-SH and the PEG-4MAL mixture with mineral oil and emulsion stabilizer before it completes crosslinking, then allow the emulsion droplets to complete crosslinking to form NHS-reactive microgels; 7. Purify and wash the generated microgels to remove mineral oil; 8. Conjugate the NHS-reactive microgels with an anti-CD3 monoclonal antibody to form the anti-CD3 microgel.

[0094] Several studies have demonstrated that biomolecules tethered to microgels can modulate immune responses and promote graft acceptance in allogeneic transplant models. For example, PD-L1 conjugated onto microgels has been shown to establish immune tolerance in allogeneic islet transplantation.[18, 19] Similarly, FasL, a pro-apoptotic member of the TNG family, has 23 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 been immobilized on PEG-MAL microgels using biotin-streptavidin chemistry to induce apoptosis in effector T cells in both mice and rhesus macaques. This approach increased regulatory T cell (CD4+Foxp3+) populations at the graft site and significantly prolonged survival, through it required systemic immunosuppression with rapamycin. [20, 21] FasL has also been co-delivered with IL-2, IL-2D, to exploit synergistic immunomodulatory pathways, leading to localized immune tolerance.

[0022] However, complete protection was not achieved due to persistent granzyme-B-expressing CD8+T cells at the graft site. While these immunomodulatory biomaterials have shown great potential in allograft settings their efficacy in in autoimmune models of T1D has not been thoroughly investigated. Therefore, there is a critical need for biomaterial that can actively suppress autoreactive T cells and protect transplanted cells from autoimmune attack.

[0095] Clinical islet transplantation is dependent on the availability of cadaveric donor islets, limiting the scalability of this therapy. To overcome this, protocols have been established to induce the differentiation of human pluripotent stem cells (hPSCs) into pancreatic cells as an attractive, unlimited source of functional, insulin-producing cells for cell therapies. sBC clusters have shown control over normal blood sugar levels in immune-compromised animals and have quickly progressed to phase ½ clinical trials (NCT04786262) with very promising results. sBC transplants are safe and result in insulin independence in most trial participants > 90 days post-transplant.

[0023]

[0096] The invention provides an immunomodulatory PEG-based microgel platform conjugated with an anti-CD3 antibody to prevent autoimmune rejection of transplanted sBCs. Anti-CD3, or teplizumab, is a monoclonal antibody therapy that targets the CD3 complex on T cells, leading to their functional inactivation and deletion. Teplizumab, a modified form of anti-CD3 with reduced Fc receptor binding, is the first FDA-approved drug to delay the onset of clinical T1D in high- risk individuals.

[0024] In its clinical trials (TrialNet 10), a single course of teplizumab significantly slowed progression to clinical type 1 diabetes in high risk individuals who had at least two autoantibodies.

[0025] Soluble aCD3 has also shown efficacy in prolonging graft survival in allogeneic transplantation models. For example, repeated administration of Fc nonbinding CD3 antibody F(ab’)2-fragments, enabled long-term acceptance of allogeneic BALB / c islets grafted under the kidney capsule of a diabetic C57BL / 6 mouse.

[0026] 24 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0097] aCD3 was localized on the surfaces of PEG microgels to create a porous immunomodulatory biomaterial capable of attenuating autoreactive T cell responses. Antibody conjugation was optimized to ensure retention of CD3-binding activity and tunability. Next, anti- CD3 microgels were demonstrated to arrest the migration of activated diabetogenic CD3+ T cells from a NOD.A2 mouse. Finally, sBCs were co-transplanted with anti-CD3 microgels both under the kidney capsule and into the subcutaneous space of an NSG.A2 mouse and the grafts challenged with an adoptive transfer of NOD.A2 CD3+ diabetogenic T cells. This approach allowed evaluation of the efficacy of localized anti-CD3 delivery in preventing autoimmune- mediated rejection of sBCs.

[0098] The purpose of this study was to examine the effect of anti-CD3 linked to PEG microgels on protecting transplanted islets and stem-cell-derived beta-like clusters (sBCs) in a humanized mouse model of type 1 diabetes. Anti-CD3 microgels were generated by binding the antibody to NHS-functionalized microgels. To create NHS-functionalized microgels the inventors used a bifunctional crosslinker with NHS and maleimide reactive groups (sulfo-SMCC) to incorporate it into the microgel core by Michael-type addition with PEG-SH (Figure 4A). NHS-microgels were incubated with anti-CD3 and isotype controls to react with lysine residues containing an amine group on the antibody to create surface-coated anti-CD3 microgels (Figure 4B). One concern was the location of the NHS and amine group binding site on the antibody. However, amine-based conjugations are widely practiced and work well without disrupting the function or specificity of antibodies. The inventors wanted to avoid the conjugation of the NHS-microgel to the antigen binding sites, rendering the antibody inert to immune protection. Fc regions on the bottom of the antibodies can bind to resting T cells and Fcγ receptor-bearing cells which can result in cytokine release and toxicity in the surrounding environment

[0029] . Future studies can include the effect of microgel-conjugated anti-CD3 on resting T cells. The inventors can also test the conjugation of Fab fragments or teplizumab onto PEG microgels.

[0099] By titrating the amount of sulfo-SMCC conjugated to the PEG-SH macromer, the amount of anti-CD3 bound to the microgels was controlled. The amount of sulfo-SMCC on the microgels was linearly related to the mean fluorescence intensity (MFI) of anti-CD3 bound to the microgel (Figure 4B). In the unfunctionalized PEG microgel control it was found that binding occurred onto the 3D contours of the microgels. This binding may result from surface adsorption 25 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 of the antibodies onto the microgels. In the titrated sulfo-SMCC samples from 0.25 mM to 1 mM, an even distribution of the antibody surrounding individual microgels was found. This difference in the binding pattern shows that NHS-ester is available on the microgel surfaces to covalently attach. Furthermore, this suggests that the sulfo-SMCC conjugation method can be used to coat microgels with a wide range of molecules for immune modulation or other regenerative medicine applications. Examples include monoclonal antibodies, growth factors, cytokines, and cell signaling molecules.

[0100] In a transwell invasion assay, the inventors investigated whether the covalent attachment of the anti-CD3 onto microgels affected the recognition and attachment of CD3+T cells. In this study, the inventors investigated the migration of activated diabetogenic CD3+primary T cells from a NOD.A2 mouse through anti-CD3 and isotype microgels towards chemokine gradients containing CXCL9 and CXCL10. The results indicated that cells invaded the gel interior after 18 hours and many of the cells in the no-gel control and isotype groups migrated into the lower wells. In contrast, the anti-CD3 microgel group showed drastically reduced migration into the lower wells. This suggests that the anti-CD3 conjugated onto the microgels was able to impede the migration of activated CD3+ diabetogenic T cells. These findings are significant because they demonstrate the potential of anti-CD3 microgels to modulate T cell behavior and prevent the infiltration of autoreactive T cells towards a target. Whereas many previous biomaterial immunomodulatory strategies for islet transplantation have targeted allogeneic rejection, this method targets specifically autoimmune rejection [18, 20].

[0101] The anti-CD3 microgels were able to attenuate an autoimmune rejection through a humanized autoimmune model of type 1 diabetes. The use of mouse models with human immune cell engraftment has emerged as a method to test human sBC pseudo islet tolerance. The model used by the inventors is a severely immunodeficient host (NSG-HLA-A2 / HHD, abbreviated NSG.A2 or NSG-A2) (for example Strain #:014570, The Jackson Laboratory, Bar Harbor, ME) that has been reconstituted with activated diabetogenic T cells from a NOD-cMHCI- -A2, abbreviated NOD.A2 or NOD-A2) (for example, Strain #:031856, The Jackson Laboratory, Bar Harbor, ME) mouse (Figures 9A-F). NSG-HLA-A2 / HHD mice are immunodeficient (NOD scid gamma background) and express human HLA class I heavy and light chains. The NOD-cMHCI- -A2 strain carries knock-out alleles for the classical MHC class I genes, H2-D1 and H2-K1, and 26 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 expresses the human HLA-A2 transgene. This strain is used to develop HLA-humanized NOD models for use in type 1 diabetes (T1D) therapy development. sBCs carry tremendous potential as an abundant and renewable source of functional beta cells for cell replacement therapies. Many current studies rely on cell encapsulation in hydrogels, such as alginate, and systematic immunosuppression for survival. There is a significant need for sustained and localized immune suppression without the encapsulation of transplanted sBCs. The inventors hypothesized that anti-CD3 microgels could act as an immune barrier, while still providing a regenerative microenvironment to sBCs transplanted under the kidney capsule in a humanized mouse model. The data show that anti-CD3 microgels resulted in the extended survival of sBCs from 8 days for isotype microgels to 12 days after adoptive cell transfer (Figure 7C). Furthermore, there were significantly more grafts that had remaining signal in situ for anti-CD3 microgels over isotype microgels (Figure 7D-7E). In histology the anti-CD3 microgels had approximately three times more insulin signal than isotype microgels (Figure 8B). These data suggest that anti-CD3 microgels were able to impede the destruction of transplanted sBCs for four days.

[0102] The infiltration of autoreactive T cells was investigated through immunohistochemistry. There was significant infiltration of T cells into the implant surrounding the sBCs for both isotype and anti-CD3 microgels (Figure 9A-F). In both groups there was minimal infiltration into the microgel interstitial spaces by CD3+, CD4+, and CD8+ T Cells, but there was massive infiltration in the implant and regions surrounding the sBCs (Figure 8A-C, 9A-F). It appears that the initial T cells that migrated through the microgel were arrested but there were some that made it to the sBCs. These were then activated and were able to expand and propagate at the site, moving the microgels out of the way and blocking the area between the sBC and microgel. It appears that the addition of secondary crosslinking mechanisms on the microgels may resist the rapid expansion and occlusion of the sBC from the immunomodulatory microgel. Guest-host interactions, are reversible, non-covalent interactions that dissociated when shear force is applied and reassociate when shear force is removed

[0014] . Previously it has been shown that guest-host interactions conjugated onto the surfaces of microgels demonstrate self-healing and stabilization of microgel scaffolds in vivo and in vitro [16, 27]. The number and strength of guest-host interactions can be tailored to withstand the force generated by cells proliferating and can protect the sBCs from being walled off from the immunomodulatory microgel. 27 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0103] Furthermore, one limitation of this study was the use of the kidney capsule as a transplantation site. The volume under the kidney capsule is limited, fitting approximately 20-40 μL before the capsule ruptures. This limits the amount of microgel that can be transplanted in addition to the volume of sBC that is required for the graft to maintain normoglycemia. Due to this limited amount, and the inability to precisely measure the amount of gel that is being delivered to the kidney capsule, it is challenging to estimate the dosing of the anti-CD3 that is being delivered. This variability and limitation on the amount of microgels that can be delivered prevents the encapsulation and protection of sBCs delivered into the microgels. The location of the sBC in the microgel graft was significantly related to its functionality and survival in vivo. sBCs in anti-CD3 microgels closest to the kidney capsule surface underwent the most loss whereas those further away from the capsule had greater survival. In isotype microgels there was a positive linear relationship between insulin MFI and distance from the kidney surface, but it was significantly lower than the anti-CD3 microgels. It appears that there is a relationship between the infiltration of T cells and the order that they encounter the sBCs, as represented in the isotype microgels, but it is the found that the anti-CD3 microgels were able to slow the infiltration and extend the survival of the sBCs. The heterogeneity of the location of the sBCs in the graft is one limitation of the kidney capsule site. To consistently implant the same number of sBCs in a controlled manner, they must be transplanted in a separate injection following the microgels. This results in the inability to control the location of the sBCs in the graft and leaves some sBCs exposed or closer to the kidney surface than others within the anti-CD3 microgels. Figures 10A-H show that sBCs transplanted with a larger gel volume subcutaneously that performs better than the kidney capsule model of Figures 7A-E, 8A-D, and 9A-F.

[0104] The addition of secondary crosslinking mechanisms on the microgels may resist the rapid expansion and occlusion of the sBC from the immunomodulatory microgel. Guest-host interactions, are reversible, non-covalent interactions that dissociated when shear force is applied and reassociate when shear force is removed.

[0014] Previously it has been shown that guest-host interactions conjugated onto the surfaces of microgels demonstrate self-healing and stabilization of microgel scaffolds in vivo and in vitro. [16, 27] A combinatorial microgel was created that contained both guest-host interactions and aCD3 to prevent the infiltration and expansion of T cells in the graft. The inventors injected the sBCs in the guest-host microgels into the subcutaneous space and monitored its engraftment and viability over 60 days. It was found that 28 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 aCD3 conjugated to microgels resulted in the survival of 75% of injected grafts, whereas without the addition of aCD3 grafts started to fail at day 20 (Figure 10B). 87.5% of mice that received the microgels without aCD3 started to develop diabetes 20 days after adoptive cell transfer whereas only 37.5% of mice who received the aCD3 microgel graft developed diabetes during the study. The inventors hypothesize that this is due to the arrest and subsequent anergy or death of circulating T cells in the subcutaneous graft. By transplanting into the subcutaneous space over the kidney capsule, more volume was afforded to transplant both the biomaterial and sBCs in a more reliable method. sBCs were able to survive in the microgels that were swollen with the sBC maturation media and key small molecules (cysteine and glutamine) before transplant. II. Anti-CD3 microporous annealed particle hydrogel protects stem cell derived beta cells from autoreactive T cells in a humanized model of type 1 diabetes

[0105] Abstract

[0106] Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic β cells, leaving patients dependent on exogenous insulin and at risk of severe hypoglycemia. Stem cell–derived β-like cells (sBCs) offer a promising approach for beta cell replacement therapy, but clinical translation is limited by immune-mediated rejection, recurrent autoimmunity, and inhospitable transplantation sites. Biomaterials have been investigated to provide localized immunoisolation and immunomodulation, but clinical translation has been hindered by foreign body responses and depletion of therapeutic agents. Here, the inventors present a porous, guest-host interlinked PEG- based microporous annealed particle (MAP) hydrogel functionalized with αCD3 antibodies (αCD3⁺ MAP) to provide a localized immunomodulatory microenvironment for subcutaneous sBC transplantation. Guest-host MAP hydrogels supported rapid vascularization, minimal foreign body response, and supported the engraftment of syngeneic islets subcutaneously. Surface-conjugated αCD3 attenuated autoreactive T cell responses in a human-HLA mouse model of T1D, halting T cell migration in vitro and protecting transplanted sBCs from immune- mediated destruction following adoptive transfer of diabetogenic T cells in vivo. Remarkably, subcutaneous αCD3⁺ MAP also conferred protection to endogenous pancreatic islets, demonstrating systemic immune modulation from a localized platform. These findings establish αCD3⁺ MAP hydrogels as a promising strategy for localized immune tolerance, supporting 29 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 vascularized engraftment and long-term survival of β cell grafts without the need for systemic immunosuppression.

[0107] Introduction

[0108] Type 1 diabetes (T1D) is a chronic disease characterized by the immune-mediated destruction of insulin producing pancreatic beta cells, leaving patients dependent on exogenous insulin therapies. Despite advances in continuous glucose monitoring and insulin pumps, patients remain at risk for severe hypoglycemic episodes and secondary complications including neuropathy, kidney failure, and cardiovascular disease. [1A] The generation of stem cell derived beta-like cells (sBCs) holds tremendous promise as a personalized and scalable source of functional, insulin-producing cells for beta cell replacement therapy. Results from initial clinical trials using sBCs have reported normalized blood sugar levels in addition to insulin- independence in 83% of patients 90 days post-transplant. [2A] The immune-mediated allorejection and resurgence of an autoimmune attack of sBC grafts are currently mitigated via systemic administration of broad-spectrum immunosuppressive agents. However, long-term delivery of immunosuppressive agents leads to significant side effects, including opportunistic infections and malignancies. [3A] In addition to the immune-mediated challenges, current transplantation into the intrahepatic milieu is inhospitable to beta cell replacement therapies leading to complications such as thrombosis, immediate blood-mediated inflammatory responses, and amyloidosis, ultimately leading to graft loss. Further, utilization of alternative sites such as the liver, omentum, and subcutaneous delivery have largely struggled due to delayed vascularization resulting in ischemia of the beta cell or islet graft with no fine-tuned control over the transplanted cells microenvironment. [4A]

[0109] Biomaterials have long been used in investigating treatments for T1D as encapsulation devices for immuno-isolation of cellular therapies and for localized immunomodulatory drug delivery. [5A, 6A] However, these approaches often struggle with clinical translation, due to foreign body response to the material and the depletion of loaded immunomodulatory agents. [5A, 7A] In addition, biomaterial design parameters (for example, porosity, stiffness, and charge) have frequently been used as modulators of immune response and have recently garnered increasing interest to reduce foreign body response to promote graft survival in many different 30 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 transplant sites. [8A] Recent advances in microporous annealed particle (MAP) hydrogels have highlighted their potential to improve engraftment and survival of transplanted beta cells. Beyond providing rapid vascularization, efficient nutrient exchange, MAP hydrogels can be engineered to present tethered immunomodulatory proteins on their surfaces design to interact with defined immune cell subsets, driving either apoptosis or differentiation into a regulatory phenotype. [9A] This capability allows for localized and sustained engagement of immune cell subsets, reducing the risk of autoimmune attack and supporting long-term graft survival. Together, these features position MAP hydrogels as a promising next-generation platform for cell replacement therapies in T1D.

[0110] In this study, the inventors developed an immunomodulatory guest-host interlinked PEG- based microgel platform conjugated with an CD3 antibody to prevent autoimmune rejection of transplanted sBCs in a subcutaneous site. Previously, the inventors have established a MAP hydrogel interlinked with guest-host molecules (guest-host MAP) as an injectable microenvironment for the delivery of islets and to enable rapid cell migration. Here the inventors demonstrate that the porosity and addition of guest-host molecules results in minimal foreign body response and the development of vasculature in the subcutaneous space. The inventorsconjugated CD3 to the surfaces of guest-host MAP ( CD3+ MAP) that attenuates autoreactiveT cell responses in a human-HLA mouse model of T1D. CD3, or teplizumab, is a monoclonal antibody therapy that targets the CD3 complex on T cells, leading to their functional inactivation and deletion. Teplizumab, a modified form of CD3 with reduced Fc receptor binding, is the first FDA-approved drug to delay the onset of clinical T1D in high-risk individuals. [10A] In its clinical trials (TrialNet 10A), a single course of teplizumab significantly slowed progression to clinical type 1 diabetes in high risk individuals who had at least two autoantibodies. [11A] Soluble CD3 has also shown efficacy in prolonging graft survival in allogeneic transplantation models. [12A] However, the use of tethered CD3 in as a localized immunosuppressive microenvironment has yet to be studied. To investigate the protection of human sBCs by CD3+ MAP in a reproducible manner, the inventors established an HLA-humanized T1D model using NOD-cMHCI--A2, which lacks endogenous MHC class I genes and expresses a human HLA-A2 transgene, in combination with NSG-HLA-A2 / HHD mice, which carries the same transgene with the addition of a SCID mutation, that renders them immunodeficient. T cells isolated from 31 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 diabetic NOD-cMHCI--A2 mice can directly interact and engage with HLA-A2 expressed on sBCs. These cells are adoptively transferred from the NOD-cMHCI--A2 to NSG-HLA-A2 / HHD mice to create a controlled induction of autoimmune diabetes. CD3+ MAP hydrogel arrested the migration of activated T cells, in vitro. sBCs transplanted with CD3+ MAP in immune deficient mice were resistant to immune challenge by adoptive transfer of diabetogenic T cells. Subcutaneous CD3+ MAP hydrogels conferred protection against immune-mediated destruction of islets in the pancreas. The approach demonstrates a localized immune tolerance approach that has the potential to improve engraftment and survival of beta cell replacement therapies without systemic immune suppression.

[0111] DISCUSSION

[0112] This study, for example as in Example 6, aimed to generate a localized immunomodulatory niche using CD3+MAP for enhancing the engraftment and survival of transplanted sBCs in the context of autoimmune diabetes. The findings demonstrate that guest- host MAP hydrogels not only provide a mechanically resilient and cytocompatible scaffold that supports vascular integration in the subcutaneous space but also serves as a framework for the tethered presentation of immunomodulatory proteins such as αCD3 to attenuate autoreactive T cell responses.

[0113] Compared to PEG microgels, guest-host MAP exhibited enhanced mechanical resilience and recovery following high-strain deformation, supporting their use as injectable materials capable of withstanding the dynamic stresses of transplantation. Guest-host MAP hydrogels did not elicit cytotoxic effects on murine islets in vitro, suggesting that the guest-host interactions themselves do not compromise cell viability. In vivo, guest-host MAP demonstrated a distinct advantage in modulating the host immune response. While both guest-host MAP and PEG microgels exhibited minimal fibrotic encapsulation, guest-host MAP supported extensive cellular infiltration and the formation of vessel-like structures, confirmed by CD31+staining. These findings highlight the importance of microporosity and dynamic guest-host interactions in creating an immune-permissive environment that supports neovascularization. Robust vascular integration is particularly critical in the subcutaneous niche, which is otherwise poorly vascularized and has historically been inhospitable to beta cell grafts. 32 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0114] Functionally, guest-host MAP hydrogels supported the survival and glucose-regulating capacity of transplanted syngeneic islets in a streptozotocin-induced diabetes model. Islets embedded within guest-host MAP restored normoglycemia within four weeks and maintained glucose responsiveness comparable to naïve controls. Histological analysis confirmed that engrafted islets became fully vascularized and integrated into the host tissue, consistent with the improved metabolic outcomes observed in vivo. By contrast, islet-only controls failed to establish stable grafts, underscoring the necessity of a supportive biomaterial scaffold for long- term function in the subcutaneous space. This distinguishes MAP from conventional encapsulation strategies that often suffer from fibrotic encapsulation and limited nutrient exchange. The observed vascularization and integration are essential for long-term β cell graft survival, particularly in subcutaneous sites where delayed perfusion has historically been a barrier to clinical translation.

[0115] By conjugating αCD3 to the hydrogel surface, the inventors created a localized, sustained immunomodulatory interface that engages and functionally impairs autoreactive T cells. In vitro assays confirmed that αCD3+ MAP reduces T cell viability and prevents directed migration toward β cell targets, suggesting that the tethered antibody is both accessible and active when presented within the microgel matrix. Release kinetics studies indicated minimal antibody leaching, supporting the notion that immune modulation occurs locally at the graft site rather than systemically.

[0116] In vivo, the inventors leveraged an HLA-humanized adoptive transfer model of autoimmune diabetes to evaluate the immunoprotective function of CD3+MAP in a controlled manner. This approach allowed the inventors to precisely introduce diabetogenic T cells while avoiding confounding contributions from B cells, NK cells, or other innate immune compartments. Under this stringent challenge, CD3+MAP grafts exhibited markedly improved survival compared to controls, with preserved graft signal, increased HNA+ and insulin+ cell retention, and higher human C-peptide levels. Moreover, αCD3+ MAP protected not only the local graft but also preserved endogenous pancreatic islets, suggesting systemic benefit through deletion or arrest of autoreactive T cells. Flow cytometry analysis supported this interpretation, revealing reduced proportions of CD4+ T cells in draining lymph nodes without clear induction of an anergic phenotype, consistent with αCD3-mediated depletion or functional inactivation. 33 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0117] These findings highlight several mechanistic insights. First, localized presentation of αCD3 within a supportive biomaterial niche can limit autoreactive T cell infiltration and function without broad systemic immunosuppression. Second, the protection of both transplanted sBCs and endogenous pancreatic islets indicates that αCD3+ MAP may act as a sink for autoreactive T cells, promoting their deletion during trafficking through the graft site. Third, the dual role of MAP, serving as both a physical scaffold for vascularization and a biochemical platform for tethered immunomodulation, addresses two critical and often competing challenges in cell replacement therapy of graft survival in poorly vascularized transplant sites versus the resistance to autoimmune destruction.

[0118] While these results are promising, several limitations warrant consideration. The injection of islets in MAP hydrogels with or without guest-host does induce loss in the first couple of days after transplantation. Furthermore, although robust vascularization is seen in the graft, earlier time points have not been assessed, and the delayed vascularization is hypothesized to be contributing to the loss of islets after injection. Notably, the αCD3 antibody used throughout these studies still contains the Fc region, as opposed to the Fab fragment variations that are now being employed in clinical trials. [10A] Previously, the presence of this Fc region has shown to promote extensive T cell proliferation and cytokine production through the engagement of the Fc region with Fc receptors (FcR) on monocytes and macrophages. [21A, 22A] However, this data has only been collected in the soluble form of the antibody, and there is no conclusive evidence that suggests that immobilized antibodies would cause mitogenic activity to the degree that is seen when delivered systemically. To increase translatability in future studies, the attachment of Teplizumab will be assessed in a human T cell challenge context. Furthermore, the adoptive transfer model, while providing precise control over T cell subsets, is a humanized-HLA matched model and does not fully encompass the challenges associated with protection from transplanted human T cells. In the future, long-term studies in fully immune-competent and humanized models will be necessary to evaluate the durability of protection and potential for immune escape.

[0119] In summary, the inventors demonstrate that αCD3+ MAP hydrogels arrest autoreactive T cell infiltration, preserve graft viability, and protect endogenous islets from immune-mediated destruction in a stringent subcutaneous HLA-humanized adoptive transfer model. This work 34 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 introduces a strategy for localized, durable immune modulation in type 1 diabetes, offering an alternative to systemic immunosuppression and advancing the clinical feasibility of subcutaneous β cell replacement therapies. III. Antigens Targeted by Hydrogels

[0120] Antigens targeted by hydrogels can include T cell surface molecules including thymocyte globulin (aTG), CD3, CD4, CD8, CD28, and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT). An exemplary antigen targeted by a hydrogel of the invention is CD3. Human CD3 is a complex including CD3 delta (e.g., Swiss Prot P04234), CD3 gamma (e.g., Swiss Prot PO9693), two molecules of CD3 epsilon (e.g., Swiss Prot P07766) and two molecules of CD3 zeta (e.g., Swiss Prot P20963). Reference to human CD3 and its subunits includes the exemplified human forms and other known allelic variants in humans as indicated in the Swiss Prot database.

[0121] Mouse CD3 is a complex including CD3 delta (e.g., Swiss Prot P04235), CD3 gamma (e.g., Swiss Prot P11942), two molecules of CD3 epsilon (e.g., Swiss Prot P22646) and two molecules of CD3 zeta (e.g., Swiss Prot P24161). Reference to mouse CD3 and its subunits includes the exemplified mouse forms and other known allelic variants in mice as indicated in the Swiss Prot database. IV. Immunomodulatory Molecules Conjugated to Hydrogels

[0122] Biomaterials of the invention comprise an immunomodulatory molecule conjugated to a hydrogel. Some immunomodulatory molecules are antibodies. Some immunomodulatory molecules are biomolecules that modulate immune cell behavior.

[0123] Some antibodies recognize T cell surface molecules including thymocyte globulin (aTG), CD3, CD4, CD8, CD28, and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT). An exemplary antibody is an antibody against CD3. An exemplary antibody against CD3 is anti-mouse CD3ε (clone 145-2C11, available for example at Biolegend). An exemplary hamster anti-mouse CD3ε (clone 145-2C11) heavy chain amino acid sequence is GenBank: AAA86877.1 (SEQ ID NO:1). An exemplary hamster anti-mouse CD3ε (clone 145- 2C11) light chain amino acid sequence is GenBank: AAA86876.1 (SEQ ID NO:2).

[0043] 35 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0124] An exemplary antibody against CD3 is teplizumab, a humanized antibody against CD3ε with the Fc region removed (USP 5,885,573) having a heavy chain amino acid sequence of SEQ ID NO:3 and a light chain amino acid sequence of SEQ ID NO: 4 [World Wide Web at genome.jp / dbget-bin / www_bget?dr:D09013].

[0125] In some embodiments an Fab fragment of an antibody against is conjugated to the hydrogel biomaterial. In some embodiments an Fab fragment of an antibody against CD3 is conjugated to the hydrogel biomaterial.

[0126] Exemplary biomolecules that modulate immune cell behavior are FasL, CTLA4Ig, PD- L1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and CD47.

[0127] In some embodiments, the immunomodulatory molecule is a monoclonal antibody, a polyclonal antibody, a growth factor, a cytokine, or a cell signaling molecule. V. Type 1 Diabetes

[0128] Type 1 diabetes is an autoimmune disease in which immune cells mistakenly attack pancreatic beta cells, causing deficiency of insulin and elevation of blood glucose (19). Type 1 diabetes (T1D) is an autoimmune disease characterized by loss of insulin-producing β-cell mass, and thereby glycemic control, due to a coordinated immune response against β-cell specific antigens requiring CD4+and / or CD8+T cells. Restoration of β-cell mass through allogeneic islet transplantation is currently the preferred clinical intervention to improve glycemic control in patients with severe glycemic instability. However, longevity of allogeneic grafts is limited not only by host immune responses, but also by secondary graft failure due to toxic effects of chronic immunosuppression required to control rejection.

[0129] Treatments for type 1 diabetes include treatment with insulin, amylin analog pramlintide, metformin, GLP-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, SGLT2 inhibitor

[0045] . Treatment for type 1 diabetes includes pancreatic transplant and islet transplantation, and sBC transplantation

[0019] . Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. 36 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 VI. Hydrogels

[0130] The hydrogel may be any pharmaceutically acceptable hydrogel that is suitable for administration into the target subject. A hydrogel typically is formed when an organic polymer (natural or synthetic) is crosslinked via covalent, ionic, or hydrogen bonds to create a three- dimensional open-lattice structure which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include macromer-based materials (including PEG macromers) assembled using different crosslinking methods (such as Michael-type addition, thiol-ene, click reactions, etc.), polysaccharides (such as alginate), polyphosphazines, and polyacrylates, or block copolymers such as PLURONICS or TETRONICS, polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature, free radical polymerization, click reactions or pH, respectively.

[0131] The biomaterial can be a polyethylene glycol (PEG) hydrogel or microgel. In further specific embodiments, the hydrogel is synthesized from maleimide-terminated 4-arm poly(ethylene) glycol (PEG-4MAL) macromers, such as by batch emulsion (e.g., as in Example 5 and Figure 2). See Headen et al.,

[0036] . The PEG-4MAL platform enables stoichiometric, covalent incorporation of thiol-containing molecules, and provides improved crosslinking efficiency for formation of structurally defined hydrogels. See Phelps et al., (2012)

[0044] . PEG- 4MAL exhibits minimal toxicity in vivo, and it is rapidly excreted in the urine, important considerations for clinical applications.

[0132] Sulfo-SMCC can be reacted with PEG-thiols to produce an NHS-activated PEG-thiol (NHS-PEG-thiol). Amine-reactive hydrogels or microgels (for example NHS-reactive hydrogels or microgels) can be produced by reacting NHS-PEG-thiol with PEG-4MAL macromer, and generating 10 μm diameter microgels via batch emulsion. See, e.g., Figures. 2 and 3. The resulting microgels display NHS-group capable of conjugating to a biomolecule, for example to an anti-CD3 antibody. See, e.g., Figures 1E and 3.

[0133] In some embodiments the biomaterial comprises or is formulated with (e.g., admixed with or blended with) an additional therapeutic agent, such as an immunosuppressant. Examples of suitable immunosuppressive drugs include rapamycin, cyclophosamide busulfan, fludarabine, methotrexate, sulfasalazine, hydroxychloroquine, azathioprine, tocilizumab, etanercept, 37 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 adalimumab, anakinra, abatacept, rituximab, certolizumab, golimumab, cyclosporine, dexamethasone, methylprednisolone, prednisone, tacrolimus and triamcinolone. In some embodiments, the immunosuppressive drug is rapamycin.

[0134] Some hydrogels are microgels (hydrogel microparticle). Some hydrogels are granular hydrogels. Some microgels are guest-host microgels. Guest-host microgels are microgels functionalized with guest -host molecules to provide an interlinking reversible interactions between the PEG-MAL microgels [14, 16, 27]. Exemplary host molecules include cyclodextrins and cucurbit[n]urils. An exemplary host molecule is β-cyclodextrin. Exemplary guest molecules include adamantane, phenylalanine, ferrocene, and p-xylylenediamine.

[0035] . In some hydrogels the host molecule is β-cyclodextrin and the guest molecule is adamantane.

[0135] Some biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel are guest-host microgels. Guest-host interactions in combination with the immunomodulatory antibody on the PEG microgels can prevent the spread of microgels away from the sBCs and enforce the proliferating T cells to interact with the immunomodulatory antibody on the surface of the gels. Some anti-CD3 microgels are guest-host microgels. Guest-host interactions in combination with the anti-CD3 on the PEG microgels can prevent the spread of microgels away from the sBCs and enforce the proliferating T cells to interact with anti-CD3 on the surface of the gels.

[0136] Anti-CD3 hydrogels of the invention may be prepared by conjugating an anti-CD3 antibody to an amine-reactive microgel.

[0137] An amine-reactive microgel is prepared by: solubilizing four-arm polyethylene glycol (PEG-4MAL) in aqueous buffer at acidic pH; solubilizing four-arm polyethylene glycol (PEG)- thiol (PEG-SH) in aqueous buffer at acidic pH; solubilizing sulfo-succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC); reacting the solubilized sulfo-SMCC with the solubilized PEG-SH to form a solubilized NHS-coupled PEG-SH; combining the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL to form a hydrogel; and emulsifying the hydrogel with mineral oil to form an NHS-reactive microgel. Any water soluble amine-to-sulfhydryl crosslinker may be used to introduce amine-reactivity. Examples include sulfo-SIAB, sulfo-MBS, sulfo-LC-SPDP, sulfo-KMUS, sulfo-GMBS, sulfo-EMCS, SM(PEG)n. 38 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 An amine-containing biomolecule, for example an anti-CD3 antibody, is conjugated to the amine-reactive microgel (e.g. NHS-reactive microgel). Other exemplary biomolecules that can be conjugated include a monoclonal or a polyclonal antibody, a growth factor, a cytokine, or a cell signaling molecule.

[0138] In some methods, the amine-reactive biomolecule is an immunomodulatory molecule. In some methods, the amine-reactive biomolecule is an antibody. In some methods, the antibody is an anti-CD3 antibody. In some methods, the amine-reactive biomolecule is a biomolecule that modulates immune cell behavior.

[0139] In some methods, the four-arm polyethylene glycol (PEG-4MAL) is solubilized in aqueous buffer at pH 5.4. In some methods, the four-arm polyethylene glycol (PEG)-thiol (PEG- SH) is solubilized in aqueous buffer at pH 5.4. The reaction occurs, is specific, and microgels can be manufactured at neutral pH (7.0) and physiological pH (7.4). Crosslinking proceeds rapidly; and an acidic pH of 5.4 slows down the reaction to provide additional time for mixing and formation of the emulsion to improve usability during manufacturing.

[0140] In some methods, the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL are combined at a 1:1 stoichiometric ratio to form a hydrogel.

[0141] Some microgels have 10 μm diameter. Microgels of larger or smaller diameter can be manufactured by controlling the vortexing speed during emulsion mixing, or using alternative manufacturing approaches such as microfluidic devices.

[0142] Exemplary procedures are shown in Examples 1 and 5 and in Figures 1-3 and 4A.

[0143] An exemplary protocol for preparing an anti-CD3 granular hydrogel is: 1. Separately solubilize two different commercially available macromers: 4-arm polyethylene glycol maleimide (PEG-MAL) and 4-arm polyethylene glycol thiol (PEG-SH) in aqueous buffer at pH 5.4. pH is skewed away from neutral to slow down the reaction. 2. Solubilize commercially available sulfo-SMCC, a heterobifunctional crosslinker with a thiol-reactive functional group on one end (maleimide) and an amine- 39 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 reactive functional group (N-hydroxysuccinimide ester (NHS-ester)) on the other end, separated by a cyclohexane spacer. The sulfo-SMCC is reacted with PEG-SH to substitute an NHS-ester for some of the thiol groups. (Figure 1B) 3. Combine solubilized PEG-4MAL and NHS-functionalized PEG-SH at a 1:1 stoichiometric ratio to form a hydrogel. (Figure 1C) 4. Vortex the mixed precursor solutions in mineral oil to make an emulsion before the cross-linking reaction completes, to make NHS-reactive microgels. (Figure 3) and (Figure 2) 5. NHS-reactive microgels are incubated with an anti-CD3 monoclonal antibody. The NHS-ester available on the surface of the microgels can react with free primary amine groups on the antibody. After incubation, any excess anti-CD3 in solution is removed, and the inventors are left with microgels that have anti-CD3 available to interact on the surface of the microgels.

[0144] Exemplary methods to prepare a PEG-MAL hydrogel are in Figures 1A-E. Exemplary methods to make a microgel are in Figure 2. Exemplary methods to conjugate a biomolecule to a hydrogel are in Figures 1D, 1E,and 4A.

[0145] Some methods to prepare a hydrogel comprise the step of functionalizing the solubilized PEG-4MAL with at least one guest molecule and at least one host molecule prior to combining the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL. VII. Pharmaceutical Compositions and Methods of Use

[0146] Biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 microgels, and methods of the invention are useful in the treatment of a disorder, for example an autoimmune disorder or an allograft rejection disorder. Exemplary autoimmune disorders are type 1 diabetes, multiple sclerosis (MS), lupus (systemic lupus erythematosus, SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, psoriasis, psoriatic arthritis, autoimmune hepatitis, myasthenia gravis, Graves’ Disease, Hashimoto’s thyroiditis, vitiligo, and Sjogren’s syndrome. Patients amenable to treatment include individuals at risk of a disorder, for example an autoimmune disorder or an allograft rejection disorder, but not showing symptoms, as well as 40 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 patients presently showing symptoms. Optionally, presence or absence of symptoms, signs, or risk factors of a disease is determined before beginning treatment.

[0147] In prophylactic applications, a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel or a pharmaceutical composition comprising the same is administered to a patient susceptible to, or otherwise at risk of, an autoimmune disorder or an allograft rejection disorder in regime (dose, frequency, and route of administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the disorder. In therapeutic applications, a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, is administered to a patient suspected of, or already suffering from, an autoimmune disorder or an allograft rejection disorder in a regime (dose, frequency, and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disorder. Exemplary biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 hydrogels, comprise a drug useful for treating an autoimmune disorder, for example rapamycin.

[0148] A regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods of the invention, or if a more favorable outcome is demonstrated in treated patients versus control patients in a controlled clinical trial (e.g., a phase II, phase II / III or phase III trial) at the p < 0.05 or 0.01 or even 0.001 level.

[0149] Effective doses vary depending on many different factors, such as means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.

[0150] The dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

[0151] A biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, can be administered in such doses daily, on alternative 41 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 days, weekly, fortnightly, monthly, quarterly, or according to any other schedule determined by empirical analysis.

[0152] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries. The formulation depends on the route of administration chosen.

[0153] An effective amount of a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, or a pharmaceutical composition comprising the same is an amount that is sufficient to generate a desired response, such as to reduce or eliminate a sign or symptom of a condition or disease. In some embodiments, an "effective amount" is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any autoimmune disorder. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with an autoimmune disorder.

[0154] The pharmaceutical compositions of the invention can be readily employed in a variety of therapeutic or prophylactic applications, e.g., for treating an autoimmune disorder. In various embodiments, the pharmaceutical compositions can be used for treating or preventing an autoimmune disorder. Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, topical, intravenous, oral, subcutaneous, intraarterial, intra-articular, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, parenteral, or intramuscular routes. A subcutaneous or intramuscular injection is most typically performed in the arm or leg muscles as well as the abdomen. The pharmaceutical compositions of the invention can be transplanted beneath the abdominal rectus sheath of a subject. 42 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0155] Some biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 granular hydrogels, are administered with pancreatic islets or sBCs. Biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 microgels of the invention may be subcutaneously transplanted. The subcutaneous space offers more space for biomaterial and sBC engraftment, where the volumes and dosing of the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 microgels, can be better evaluated. In some methods, sBCs are mixed with biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 microgels, and embedded fully within the gel before transplantation. It has been shown that guest-host MAP microgels transplanted in the subcutaneous space demonstrated vascularization over 31 days and could potentially support the revascularization of sBCs in vivo [for example as in Figures 10A-G, Example 4].

[0156] For prophylactic applications, the pharmaceutical composition is provided in advance of any symptom. The pharmaceutical compositions can be administered prophylactically to individuals who have a known genetic risk of an autoimmune disorder. Such individuals include those having relatives who have experienced such an autoimmune disorder, and those whose risk is determined by analysis of genetic or biochemical markers (e.g., mutations associated with an autoimmune disorder, for example mutations associated with type 1 diabetes).

[0157] For therapeutic applications, the pharmaceutical composition is provided at or after the onset of a symptom of disease, for example after development of a symptom of an autoimmune disorder, or after diagnosis of an autoimmune disorder. The pharmaceutical composition of the invention can be combined with other agents known in the art for treating or preventing an autoimmune disorder.

[0158] The subject of application of the present invention may be human or non-human. The non-human subject may be, for example, a non-human animal, and may be, for example, a non- human mammal, bird, reptile, amphibian, or fish. Examples of the non-human mammal include rodents (for example, mice and rats), dogs, cats, horses, pigs, cows, sheep, goats, primates, and the like. In addition, all aforementioned embodiments are applicable to domesticated, agricultural, or zoo-maintained mammals experiencing an autoimmune disorder, as well as to 43 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 humans. For example, biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, may be administered to humans, non- human primates, primates, baboons, chimpanzees, monkeys, cynomolgus, marmoset, rhesus, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, ferrets, guinea pigs, hamsters, gerbils etc. Biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, may be administered to house pets such as dogs, cats, rabbits, ferrets, guinea pigs, hamsters and gerbils, as well as to agricultural animals, such as horses, sheep, cows, and pigs, or to animals such as camel, cynomolgus, marmoset, rhesus and chimpanzee. Biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, may be administered to a human.

[0159] Biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, can be administered to an animal model of disease, for example to an animal model of type 1 diabetes. The therapeutic efficacy of biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, can be evaluated in an animal model of type 1 diabetes, for example a murine model of type 1 diabetes. In some methods the animal model of type 1 diabetes is a severely immunodeficient host (NSG-HLA-A2 / HHD) that has been reconstituted with activated diabetogenic T cells from a NOD-cMHCI- -A2 mouse (for example, Figures 7A-E, Figures 10A-H, Examples 3, 4, and 5).

[0160] An exemplary murine autoimmune model for transplantation of human sBCs is a combination of NOD-cMHCI- / -A2 (NOD.A2) and NSG-HLA-A2 / HHD (NSG.A2) mice. The NOD.A2 mouse is a non-obese diabetic mouse that develops spontaneous autoimmune diabetes that has been engineered to express human HLA Class I instead of mouse MHC Class I. The modification of the MHC Class I allows immune cells from NOD.A2 mice to target and attack human sBCs. The NSG.A2 mouse is from the same background as the NOD.A2 mouse but has a SCID mutation making it immunodeficient. In this model, the inventors adoptively transfer T cells isolated from diabetic NOD.A2 mice into an NSG.A2 mouse that has been transplanted with sBCs. Adoptively transferred T cells from the NOD-A2 mouse will properly invade and destroy pancreatic islets as well as grafted sBCs in the kidney capsule of an NSG-A2 mouse.

[0040] . Other exemplary animal models of type 1 diabetes include destruction of endogenous 44 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 pancreatic beta cells with streptozotocin (STZ), alloxan, or selective depletion using a diphtheria toxin receptor-mediated cell-knockout; and models for transplantation of human sBCs in a combination of NSG mice with or without induction of type 1 diabetes using chemical, toxin, or cell-based methods combined with additional mutations such as double knockout of MHC Class I and MHC Class II combined with reconstitution of a human immune system from an allogeneic donor, a human immune system from a donor with type 1 diabetes, or reconstitution with T cells engineered to express a diabetogenic T cell receptor or other receptor that is specific to the sBC graft. [37, 34, 42, 38, 39].

[0161] A biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, is useful in development of drugs for treatment of disease, for example an autoimmune disorder. A biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, is useful in development of drugs for treatment of allograft rejection. A biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, is useful in treatment of allograft rejection and for allogeneic immune rejection management. A biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 granular hydrogel, has the advantage of localized function.

[0162] The dosage and the frequency of administration of the pharmaceutical composition useful in methods of the present invention may be determined appropriately by a person having ordinary skill in the art (for example, a doctor) according to a target pathological condition. VIII. Methods of Inducing Immune Tolerance

[0163] The biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, can be used to effect immunomodulation, for example in a patient or subject having a cellular or tissue graft and / or to treat type 1 diabetes in a patient or subject. The biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials can be used to prevent or reduce the risks of rejection of cellular or tissue grafts in a patient or subject and / or the treatment of type I diabetes in a patient or subject. 45 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0164] The biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, described herein are useful for inducing immunosuppression, for example in a patient or subject having a cellular or tissue graft and / or to treat type 1 diabetes in a patient or subject. The biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, can be used to induce immunosuppression in a subject in need thereof comprising administering to the subject a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 hydrogel biomaterial, in an amount effective to induce immune tolerance.

[0165] The biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, can be used to induce specific immune tolerance. For example, administering a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 hydrogel biomaterial, along with a graft (e.g., a graft cell or a graft tissue) may induce specific immune tolerance to the graft cell or the graft tissue. The invention provides methods of inducing specific immune tolerance in a subject in need thereof comprising administering to the subject a graft cell or a graft tissue and a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 hydrogel biomaterial, in an amount effective to induce immune tolerance to the graft cell or the graft tissue.

[0166] As used herein a “graft cell” refers to a donor cell (or tissue or organ comprising a cell), that is administered to a subject in need thereof. Types of graft cells include islet cells (e.g., pancreatic islet cells), splenocytes, PBMCs, bone marrow cells, mesenchymal stem cells, hematopoietic stem cells, stem cells, induced pluripotent stem cells, human beta cell products, stem cell derived beta cell clusters (sBCs), hepatocytes, dendritic cells, macrophages, endothelial cells, cardiac myocytes, thymic epithelial cells, neural cells, and vascular cells, and immune cells, including T cells, etc., depending on the condition being treated. In accordance with these methods a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 hydrogel biomaterial, induces specific immune tolerance to the graft cells. As used herein a “graft tissue” refers to a donor tissue that is administered, implanted, or transplanted into a subject in need thereof. The graft tissue may comprise one or more types of cells, extracellular matrix components, and / or structural or functional biological materials. Types 46 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 of graft tissue include, but are not limited to, pancreatic islets, liver tissue, thymic tissue, skin grafts, cardiac tissue, vascular grafts (e.g., arteries or veins), bone marrow tissue, mucosal tissue, lymphoid tissue (e.g., spleen or lymph node tissue), intestinal tissue, lung tissue, kidney tissue, and engineered or biofabricated tissues derived from stem cells or donor sources. The specific type of graft tissue may be selected based on the therapeutic purpose, such as immune reconstitution, metabolic support, organ repair, or tissue regeneration.

[0167] For example, a subject may be administered pancreatic islet cells to treat diabetes. The subject may be administered pancreatic islet cells and a hydrogel engineered to display an immunomodulatory molecule, for example anti-CD3, (an anti-CD3 hydrogel biomaterial) in order to specific induce immune tolerance to the pancreatic islet cells.

[0168] In any embodiments, the graft cell may be administered as a preparation of isolated cells or as part of a tissue or organ.

[0169] In some embodiment, the graft cell or graft tissue is allogeneic. In some embodiments, the graft cell or graft tissue is xenogenic. In some embodiment, the graft cell or graft tissue is from a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.

[0170] In some embodiments, the graft cell or graft tissue is autologous or autogeneic (from the subject being treated). For example, an autologous graft cell may be derived from autologous tissue by induced pluripotency and differentiation of the induced pluripotent cells to the desired autologous graft cell. In some embodiments, cells from the subject are used to induce immune tolerance to self that has been interrupted in autoimmune disease. Exemplary cells suitable for use in these embodiments include mobilized hematopoietic stem cells, PBMCs, dendritic cells, and the like. In some embodiments, the cells are chosen from those that naturally express self antigens that are targeted in the autoimmune disease. For example, type I diabetes is an autoimmune disease wherein the body reacts and rejects pancreatic islet (β) cells. In early stages of diabetes, before all islet cells are rejected, it can be possible to induce tolerance to islet cells and thereby prevent the progression of diabetes. 47 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0171] In some methods, the subject is in need of immune tolerance to a graft cell, and a method of inducing immune tolerance comprises administering a biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 hydrogel biomaterial, as described herein and the graft cell. In these embodiments, the graft cell is selected based on the condition to be treated. For example, when the subject is in need of the treatment or prevention of type 1 diabetes, the graft cell may be pancreatic islet cells, human beta cell products, or sBCs.

[0172] A biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example an anti-CD3 hydrogel biomaterial (such as an anti-CD3 microgel), and graft cell may be administered in the same composition, or may be administered separately. In some embodiments, the graft cell is encapsulated by the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterial. For example, the graft cell may be entrapped within the hydrogel or microgel biomaterial. In some embodiments, the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial (such as an anti-CD3 microgel), and graft cell are administered to the same site in the subject, such as by local injection into approximately the same site. In some embodiments, the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial (such as an anti-CD3 microgel), and graft cell are transplanted into the same site in the subject (e.g., co-transplantation). In accordance with any of these embodiments, the methods may achieve long-term, specific immunosuppression at the site of the graft.

[0173] In some methods, the administering is by transplantation. In some embodiments, allogeneic islet graft acceptance is achieved by simple co-transplantation of unmodified islets, human beta cell products, or sBCs and the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial, without long term immunosuppression.

[0174] In some embodiments the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial (such as an anti-CD3 microgel), is administered with an additional therapeutic agent, such as an immunosuppressive 48 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 drug, such as rapamycin or any of the others mentioned above. In such embodiments, the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial (such as an anti-CD3 microgel), and immunosuppressive drug may be formulated together (e.g., the hydrogel may comprise the immunosuppressive drug), or they may be administered in separate compositions, simultaneously or sequentially in any order. In some embodiments, a shorter course of immunosuppressive drug may be required than when no biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, no anti-CD3 hydrogel biomaterial is administered.

[0175] In some embodiments, the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial (such as anti-CD3 microgels), that comprise an immunosuppressive drug provide controlled release of the drug. In some embodiments, biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 hydrogel biomaterials (such as anti-CD3 microgels), that comprise an immunosuppressive drug provide controlled release of the drug within the graft microenvironment, or contain the graft in the form of a capsule engineered with these immunomodulatory molecules (for example anti-CD3).

[0176] As noted above, the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial (such as an anti-CD3 microgel), may administered in an amount effective to induce immunosuppression or induce specific immune tolerance. Effective amounts of the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial, will vary depending on the subject being treated, the route of administration, and the nature and severity of the condition to be treated. The amounts of the biomaterial comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 hydrogel biomaterial, used in the examples below are illustrative:

[0177] Thus, the localized immunomodulatory biomaterial-enabled approach described herein may provide an alternative to chronic immunosuppression for clinical islet transplantation.

[0178] Thus, in accordance with specific embodiments, described herein are biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example, the anti-CD3 49 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 hydrogel biomaterials wherein the immunomodulatory molecule, for example anti-CD3, is displayed on a biocompatible material, such as a hydrogel, such as a polyethylene glycol (PEG) hydrogel. The biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 hydrogel biomaterials, are useful, for example, for immunomodulation, such as for preventing or reducing the risks of rejection of cellular or tissue grafts, such as for preventing or reducing the risks of foreign graft rejection, for preventing or reducing the risks of rejection of pancreatic islet transplantation, and / or for preventing or reducing the risks of rejection of stem cells, human pancreatic beta cell products (such as may be used for the treatment of type 1 diabetes), and in conjunction with other treatments and / or the treatment of other disorders that may benefit from cellular or tissue grafts. Thus, for example, the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example the anti-CD3 hydrogel biomaterials, described herein are useful in the treatment of autoimmune diseases, such as type I diabetes, the prevention of rejection of cellular and tissue grafts, such stem cells, pancreatic islets, hematopoietic stem cells, hepatocytes, mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, human beta cell products derived from stem cells, stem-cell derived beta cell clusters (sBCs), and in conjunction with the treatment of various hematopoietic and immune deficiency disorders through the use of stem cells.

[0179] Anti-CD3 hydrogels have been shown to be effective in arresting migration of activated diabetogenic CD3+ T cells from a NOD.A2 mouse in transwell invasion assay (Example 2, and Figures 8A-D). Anti-CD3 hydrogels have been shown to result in extended survival of sBCs transplanted into a mouse model of type 1 diabetes (Example 3 and Figures 7A-E). IX. Kits

[0180] The invention further provides kits (e.g., containers) comprising biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 granular hydrogel compositions, and related materials, such as instructions for use (e.g., package insert). The instructions for use may contain, for example, instructions for administration of the biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example anti-CD3 granular hydrogel compositions, and optionally one or more additional agents. The containers of biomaterials comprising an immunomodulatory molecule conjugated to a hydrogel, for example 50 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 anti-CD3 granular hydrogel compositions, may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses.

[0181] Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products

[0182] Kits can also include a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0183] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. EXAMPLES

[0184] Example 1: Preparation of Anti-CD3 microgels

[0185] The inventors investigated the effect of anti-CD3 linked to PEG microgels for the protection of transplanted stem-cell-derived beta-like clusters (sBCs) in a humanized mouse 51 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 model of type 1 diabetes. Polyethylene glycol maleimide (PEG-MAL) and PEG-thiol (PEG-SH) are useful in generating microgels

[0014] . Polyethylene glycol maleimide (PEG-MAL) and PEG- thiol (PEG-SH) are highly reactive macromers that are easily functionalized with proteins, peptides, or molecules of interest

[0027] . To add proteins of interest sulfo-SMCC, a small bifunctional crosslinker with N-hydroxysuccinimide-ester (NHS) and maleimide (MAL), was reacted with PEG-SH for 30 minutes (Figure 4A). The functionalized PEG-SH macromer was added to an equal volume of PEG-MAL, quickly mixed, and immediately transferred to mineral oil to create an emulsion. The NHS-functionalized microgels were washed with 0.3% Tx-100 and 1X PBS before being incubated with Armenian hamster anti-CD3 to react with an amine group on the antibody. Two types of microgels were created, one with the anti-CD3 Armenian hamster antibody and the other with an Armenian hamster isotype control.

[0186] The effect of anti-CD3 (aCD3) conjugated to PEG-MAL microporous annealed particles was evaluated for the protection of transplanted stem cell-derived beta cells (sBCs) in a humanized mouse model of type 1 diabetes. It has been demonstrated that microgels can be generated using a batch emulsion of polyethylene glycol maleimide (PEG-Mal) and PEG-thiol (PEG-SH).

[0014] It has been also shown that these highly reactive macromers are easily functionalized with proteins, peptides, or molecules of interest.

[0027] To functionalize microgels with aCD3, PEG-SH were first reacted with sulfo-SMCC, a hetero-bifunctional crosslinker containing N-hydroxysuccinimide (NHS)-ester and maleimide groups. (Figure 4A) The modified PEG-SH was then mixed in equal volume with PEG-MAL, quickly mixed, and immediately emulsified in mineral oil. The resulting NHS-functionalized microgels were washed with 0.3% Triton X-100 (TX-100) and 1X PBS, then incubated with Armenian hamster anti-CD3 (aCD3) to react with an amine group on the antibody. Two types of microgels were created, one with the anti-CD3 Armenian hamster antibody (aCD3+ microgels) to allow NHS-mediated coupling to primary amines on the antibody.

[0187] To determine whether anti-CD3 can bind to PEG microgels, the molar amount of sulfo- SMCC reacting with PEG-SH was varied. 40 μL of 0.2 mg / ml soluble anti-CD3 was incubated with the approximately 200 μL microgels overnight in 500 μL of 1X PBS, and then washed with 0.3% TX-100 and 1X PBS. Microgels were blocked with 10% bovine serum albumin (BSA) and then labeled them with an anti-Armenian hamster secondary antibody. There was a linear 52 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 relationship between the amount of sulfo-SMCC on the microgels and the MFI of the anti-CD3 (Figure 4B). Some binding of the anti-CD3 to the 3D-contours of the unfunctionalized microgels was observed, which suggests there is a minor amount of adsorption onto the microgels. In the sulfo-SMCC conjugated microgels, there is an even distribution of the binding around individual microgels. This difference in binding pattern is evidence for covalent attachment of the anti-CD3 onto the NHS-conjugated microgels.

[0188] Two types of microgels were generated: one conjugated with aCD3 (aCD3+ microgels) and one with an Armenian hamster isotype control antibody (microgels). To assess aCD3 binding efficiency, the molar amount of sulfo-smcc reacting with PEG-SH was varied. Then, 40 μl of 0.2 mg / ml soluble aCD3 was incubated overnight with approximately 200 μl NHS-functionalized microgels in 500 μl of 1X PBS, followed by washing with 0.3% TX-100 in 1X PBS. The microgels were blocked overnight with 10% bovine serum albumin (BSA) and then labeled them with an anti-Armenian hamster secondary antibody. A linear relationship was observed between the amount of sulfo-SMCC conjugated to the PEG-SH and the mean fluorescence intensity (MFI) of the secondary antibody onto the resulting NHS-functionalized microgels (Figure 4B). Some of the aCD3 binding was also detected on unfunctionalized microgels, suggesting a minor amount of nonspecific adsorption. In contrast, NHS-functionalized microgels showed a uniform antibody distribution, demonstrating evidence of covalent attachment of aCD3 to the NHS- functionalized microgel surface.

[0189] To confirm that this was binding of the anti-CD3 antibody and not binding of the secondary antibody to remaining NHS-sites on the microgel NHS-conjugated microgels were treated with secondary antibodies only. NHS esters are known to hydrolyze easily and have a half-life of 4-5 hours at pH 7.0.

[0028] Three days after the generation of the microgels the secondary antibodies were added to the titrated NHS-conjugated microgels to allow enough time for the NHS-ester groups to hydrolyze. There was no significant binding of the secondary antibody to the microgels. This result confirms that there was binding of the anti-CD3 to the NHS-conjugated microgels, and that the inventors were able to control the amount conjugated onto the microgels.

[0190] Example 2 Bioactivity of the anti-CD3 conjugated microgels 53 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0191] The activity of the antibody was tested after it was covalently attached to PEG microgels using a transwell invasion assay (Figure 5A). To investigate the bioactivity of the anti-CD3 conjugated microgels, the inventors evaluated the migration of activated diabetogenic CD3+ primary T Cells from a NOD.A2 mouse through anti-CD3 microgels and isotype microgels towards a CXCL9 and CXCL10 chemokine gradient. After 18 hours, cells had invaded through the gel interior and cells had migrated into the lower well. The inventors analyzed the viability of cells remaining in the transwell insert that were positioned within the microgels. The cells in the isotype microgels had a significantly higher viability than those within the anti-CD3 microgels. (Figure 5B) In the bottom well, there were significantly fewer cells for anti-CD3 microgels than the isotype and no-gel control. (Figure 5C). Cells that migrated into the bottom well were more viable in the no-gel control and isotype control than in the anti-CD3 microgels (Figure 5D). These results suggest that anti-CD3 microgels can arrest the migration of activated diabetogenic T cells toward a target. Furthermore, these results suggest that anti-CD3 microgels have the potential to be used in vivo to block the infiltration of autoreactive T cells towards transplanted sBCs and islets. The inventors assessed the release kinetics of surface-conjugated Armenian hamster IgG from sulfo-SMCC microgels at 37°C. About 4% of the antibody released from the microgels over 3 days. Based on this data, the inventors confirmed the covalent conjugation was stable and would not result in a burst release of anti-CD3 when implanted in vivo.

[0192] The ability of the microgels to protect sBCs from an autoimmune attack was assessed. The experiment above was repeated but sBCs were included in the bottom well of the transwell insert (Figure 6A). After 48hrs there was a significantly lower percentage of live CD4+ T cells within the transwell in aCD3+ microgels, but no difference in CD8+ T cells (Figure 6B, C, D). In the bottom well, the inventors analyzed the numbers of T cells able to travel through. T cells that did travel through had significantly lower viability when going through aCD3+ microgels for both CD4+ and CD8+ T cells (Figure 6E, F, G, H). No differences were found in the frequencies of ChromagraninA+ or Cpep+ cells between aCD3+ microgels (Figure 6I, J, K).

[0193] Example 3 Immune protective effects of anti-CD3 microgels Transplanted in Mouse Models of Type 1 Diabetes 54 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0194] The inventors studied an autoimmune model for transplantation of human sBCs using a combination of NOD-cMHCI- / -A2 (NOD.A2) and NSG-HLA-A2 / HHD (NSG.A2) mice. The NOD.A2 mouse is non-obese diabetic mouse that develops spontaneous autoimmune diabetes that has been engineered to express human HLA Class I instead of mouse MHC Class I. The modification of the MHC Class I allows immune cells from NOD.A2 mice to target and attack human sBCs. The NSG.A2 mouse is from the same background as the NOD.A2 mouse but has a SCID mutation making it immunodeficient. In this model, the inventors adoptively transfer T cells isolated from diabetic NOD.A2 mice into an NSG.A2 mouse that has been transplanted with sBCs. The inventors have demonstrated that adoptively transferred T cells from the NOD- A2 mouse will properly invade and destroy pancreatic islets as well as grafted sBCs in the kidney capsule of an NSG-A2 mouse [23, 40]. By transplanting sBCs before introducing an immune challenge, the inventors can ensure the engraftment of the sBCs before the destruction of the graft, allowing specific observation of autoimmune rejection of the graft and not rejection due to inflammation or innate responses. The sBCs were engineered to express luciferase as a bioluminescent reporter. Using this model, the inventors can systematically test immunomodulatory biomaterials and other immune evasion strategies in real time in a model of autoimmune diabetes.

[0195] The immune protective effects of anti-CD3 microgels transplanted with sBCs were evaluated in the humanized NSG-A2 / NOD-A2 mouse model (Figure 7A). The inventors transplanted approximately 800 sBCs into the kidney capsule of an NSG-A2 mouse with approximately 40 μL of anti-CD3 or isotype microgels (Figure 7B). After four days the inventors adoptively transferred activated CD3+ T cells isolated from a diabetogenic NOD.A2 mouse to induce diabetes. The viability and location of the implanted luciferase-expressing sBCs and microgel grafts under the kidney capsule were monitored by transdermal bioluminescence for 12 days using IVIS in vivo imaging systems (Figure 7C). Microgels were labeled with Alexa Fluor- 647 to visualize their location using IVIS during the experiment (Figure 7C). The microgels stayed localized within the kidney at day 12 for both the anti-CD3 microgel group and the isotype group. D-Luciferin was administered to the mice subcutaneously to assess sBC survival through transdermal bioluminescence during the experiment. The sBCs in the anti-CD3 microgels survived longer than the sBCs in the isotype control microgels, with a statistical difference in total corrected flux on days 10 and 11 (Figure 7C). The sBCs in the isotype control 55 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 group lost signal on day 10, and the sBCs in the anti-CD3 microgel group lost signal on day 12. To observe the survival of the sBCs in kidney grafts, the bioluminescent signal of the explanted kidneys in situ (Figure 7D-7E) was measured. Significantly more anti-CD3 microgel grafts (4 of 6) showed signal than isotype microgel grafts (1 of 4) after 12 days. The data suggest that anti- CD3 microgels were more resistant to immune invasion and could delay rejection within the first 10 days but were insufficient for full graft survival. Animal weight and blood glucose were monitored throughout the surgery and showed no significant differences between the experimental groups.

[0196] Example 4 Immunohistochemistry of anti-CD3 microgels anti-CD3 microgels Transplanted in Mouse Models of Type 1 Diabetes

[0197] The grafts were then assessed using immunohistochemistry to determine the mechanism of destruction and sBC survival. Cryosections of the graft were stained for human nuclear antigen (HNA) and insulin to determine graft survival. The gels well compacted within the kidney capsule, with lots of cells infiltrating the microgel graft. (Figure 8A). However, instead of migrating through the microgel interstitial spaces, cells appear in large clusters surrounding sBCs and the capsule area. This suggests that infiltrating innate immune cells or adaptive immune cells propagate and expand within the gel to form a barrier between the sBC and the host. Insulin expression and intensity were significantly higher in anti-CD3 microgels than in isotype microgels (Figure 8B). Anti-CD3 microgels had higher HNA expression than isotype microgels (Figure 8C). The data suggest that anti-CD3 microgels extended the survival of the grafted sBCs and that they remained viable and functional after 12 days.

[0198] The inventors were interested in the relationship between the distance of the sBC from the kidney surface and the viability of the sBC. The inventors hypothesized that adoptively transferred T cells migrated from the kidney vasculature and through the gel towards sBC targets. The inventors measured the distance of each sBC and its correlating distance perpendicular to the kidney surface (Figure 8D). There was a positive correlation between the distance from the kidney surface and the insulin MFI in anti-CD3 microgels (m = 0.04) and it was significantly greater than that in isotype microgels (m = 0.01). This suggests that the closer 56 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 sBCs were first to undergo an autoimmune attack and those further within the anti-CD3 microgels were more likely to survive.

[0199] To further elucidate the autoimmune rejection of the transplanted sBCs immunohistochemistry was performed on the invading CD3, CD4, and CD8+ T cells in the kidney capsule graft (Figure 9A-F). The inventors found that there were significantly more CD3+ T cells invading into the isotype microgels than in the anti-CD3 microgel grafts (Figure 9A-B). However, the number of CD4+ T Cells in the gel was not significantly different (Figure 9C-D). For both CD4+ and CD8+ T cells there were no significant differences between the isotype microgels and anti-CD3 microgels in the number of cells in the implant area or in the gel area (Figure 9C-F). Overall, there were more CD4+ T cells in the implant but more CD8+ T cells in the gel (Figure 9E-F). These results suggest that T cells migrated to the graft and were able to infiltrate into the gel however, cells that were able to travel to the sBCs were able to proliferate and expand within the graft.

[0200] Figure 10 depicts autoimmune model of type 1 diabetes in a humanized HLA mouse for assessing immunomodulatory biomaterials, where transplants were performed subcutaneously. One limitation of this study was the use of the kidney capsule as a transplantation site. The volume under the kidney capsule is limited, fitting approximately 20-40 μl before the capsule ruptures. This limits the amount of microgel that can be transplanted in addition to the volume of sBC that is required for the graft to maintain normoglycemia. Due to this limited amount, and the inability to precisely measure the amount of gel that is being delivered to the kidney capsule, it is extremely challenging to estimate the dosing of the anti-CD3 that is being delivered. This variability and limitation on the amount of microgels that can be delivered prevents the encapsulation and protection of sBCs delivered into the microgels. The location of the sBC in the microgel graft was significantly related to its functionality and survival in vivo. sBCs in anti- CD3 microgels closest to the kidney capsule surface underwent the most loss whereas those further away from the capsule had greater survival. In isotype microgels there was a positive linear relationship between insulin MFI and distance from the kidney surface, but it was significantly lower than the anti-CD3 microgels. The inventors speculate that there is a relationship between the infiltration of T cells and the order that they encounter the sBCs, as represented in the isotype microgels, but it was found that the anti-CD3 microgels were able to 57 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 slow the infiltration and extend the survival of the sBCs. The heterogeneity of the location of the sBCs in the graft is one limitation of the kidney capsule site. To consistently implant the same number of sBCs in a controlled manner, they must be transplanted in a separate injection following the microgels. This results in the inability to control the location of the sBCs in the graft and leaves some sBCs exposed or closer to the kidney surface than others within the anti- CD3 microgels.

[0201] To improve on this study, guest-host interactions were used in combination with anti-CD3 conjugated to the PEG microgels injected into the subcutaneous space (Figure 10A). It was hypothesized that these interactions could prevent the spread of microgels away from the sBCs and enforce the proliferating T cells to interact with anti-CD3 on the surface of the gels. The transplant site was moved from the kidney capsule to the subcutaneous space, that offers vastly more space for biomaterial and sBC engraftment, and where the volumes and dosing of the anti- CD3 microgels could be better evaluated. Furthermore, sBCs can be more reliably mixed with anti-CD3 microgels and embedded fully within the gel before transplantation, and the location can be better controlled. 600 sBCs mixed with 200 L of aCD3+GH+ microgels were transplanted into the subcutaneous space and its engraftment and viability were monitored against adoptive cell transfer of NOD.A2 activated T Cells over 45 days (Figure 11A). Microgels were packed into the back of a syringe, and a small pipette containing a small volume of media plus the sBCs were inserted into the back of the syringe in the middle of the microgel volume. The back of the syringe was replaced and injected into the right dorsal side of the mouse. The bioluminescence signal of the sBCs were monitored over 33 days post-adoptive cell transfer (Figure 11B). sBC grafts engrafted over 16 days in the animal and were localized in the area of the transplanted gel. aCD3 microgels maintained a steady signal post adoptive cell transfer, whereas microgels without that the IVIS signal was undetectable 33 days after adoptive cell transfer. sBCs transplanted with isotype microgels had undetectable IVIS 33 days after adoptive cell transfer (Figure 10B). The inventors quantified the graft survival of each group over the days post-ACT by detectable graft signal (Figure 10B). 75% of aCD3 grafts survived and had a detectable signal 45 days post-ACT, whereas only 25% of isotype control grafts survived (Figure 10B). This data suggests that the anti-CD3 has a protective effect on the sBCs against an autoimmune rejection in vivo. Blood glucose was tracked over the time course of the 58 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 study and found that the adoptively transferred T cells trafficked not only to the graft but to the pancreas as well and caused diabetes in all but one isotype control and three anti-CD3 microgel mice (Figure 10F). This data leads the inventors to believe that T cells that traffic to the graft are arrested and anergized or killed in the anti-CD3 grafts leading to a decrease in the total number of T cells in the animal, whereas in the isotype grafts T cells can destroy the graft and able to migrate to the pancreas and cause destruction. The inventors were also able to confirm the transition to diabetes through intraperitoneal glucose tolerance test (IPGTT) at day 22 post- adoptive cell transfer (Figure 10D). Mice with isotype control mice have a higher area-under- curve as compared to the anti-CD3 mice (Figure 10E).

[0202] The subcutaneous grafts in one cohort were explanted, with half of the graft processed for analysis of cell populations and the other half for the histology (Figure 11A). The subcutaneous sections were analyzed for the presence of sBCs by HNA and insulin MFI. The inventors also stained for infiltrating T cells (Figure 11B). Grafts with no anti-CD3 had large numbers of infiltrating CD3+ T Cells and CD8+ T cells within the gel and surrounding the sBC with little to no remaining insulin signal. In the aCD3 microgels it was found that there was remaining insulin signal, and found few numbers of CD3+ T cells surrounding the sBC. The draining lymph nodes were collected and processed for quantification by flow cytometry. It was found that in mice with aCD3+ microgel grafts, there was a smaller frequency of circulating live CD4+ and CD8+ T Cells (Figures 11C-D). In the graft, there was a higher frequency of Chromagranin A+ (a human endocrine cell marker), and higher percentage of Cpep+ cells (Figures 11E-F). However, there was no significant differences in the number of T cells present in + / - aCD3 microgels.

[0203] Example 5 Materials and Methods

[0204] Anti-CD3 microgels process description The base gel is composed of two cross-shaped polymers that react at their end functional groups to form an extended nano meshwork (Figures 1-2). The method includes additional steps to conjugate a biomolecule to the surface of the gel (steps 3-4 below). 6. The inventors separately solubilized two different commercially available macromers: 4-arm polyethylene glycol maleimide (PEG-MAL) and 4-arm polyethylene glycol thiol (PEG-SH) in aqueous buffer at pH 5.4. The inventors 59 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 skewed the pH away from neutral to slow down the reaction. When these macromers were combined at a 1:1 stoichiometric ratio, a hydrogel was formed. (Figures 1A and 1C) 7. The inventors formed microgels by vortexing the mixed precursor solutions in mineral oil to make an emulsion before the cross-linking reaction completes. (Figure 2) 8. To make microgels ready to bind biomolecules, the inventors solubilized commercially available sulfo-SMCC, a heterobifunctional crosslinker with a thiol-reactive functional group on one end (maleimide) and an amine-reactive functional group (N-hydroxysuccinimide ester (NHS-ester)) on the other end, separated by a cyclohexane spacer. The sulfo-SMCC was reacted with PEG-SH to substitute an NHS-ester for some of the thiol groups. (Figure 1B) 9. The NHS-coupled PEG-SH was mixed with PEG-MAL and emulsified to make NHS reactive microgels. (Figure 3) 10. The microgels were then incubated with an anti-CD3 monoclonal antibody. The NHS-ester available on the surface of the microgels can react with free primary amine groups on the antibody. After incubation, any excess anti-CD3 in solution was removed, and the inventors were left with microgels that have anti-CD3 available to interact on the surface of the microgels. The anti-CD3 microgels were designed to resist T cell infiltration to transplanted pancreatic beta cells. (Figure 1E)

[0205] Immunomodulatory Microgel Fabrication: PEG-MAL (4-arm, 20 kDa) and PEG-SH (4- arm, 20kDa) were dissolved in 1X PBS with 1% HEPES at pH 5.4 (70.8 mg / ml, 1.7 mM). The reduced pH helps to slow down the crosslinking reaction between PEG-MAL and PEG-SH which at neutral pH is so quick it hinders mixing and handling

[0030] . At pH 5.4 the gelation of PEG-MAL with PEG-SH takes approximately 34 seconds.

[0014] Sulfo-SMCC was dissolved in 1X PBS with 1% HEPES at pH 5.4 (0.22 mg / ml, 0.5 mM) and reacted with PEG-SH for 30 minutes at 25°C. PEG-MAL was added to the functionalized PEG-SH macromer at a 1:1 volume ratio, quickly pipetted up and down several times to mix thoroughly, then transferred to a 30x volume of mineral oil with 2% vol / vol span 80 surfactant in a 50 ml conical tube. The tube was immediately vortexed for 30 seconds to generate a water in oil emulsion, then allowed to finish 60 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 crosslinking for 30 minutes at 25°C on a rocker to generate microgels of a final polymer concentration of 6 % wt / vol. Microgels were centrifuged at 3,000 x g for 5 minutes and washed three times with 0.3% Triton X-100 in 1X PBS at pH 7.2, and three times with 1X PBS. After washing procedures, microgels were suspended in a 1:2 volume ratio with 1X PBS, and 200 μL of 0.2 mg / ml anti-mouse CD3ε (Biolegend, 145-2C11) or the Armenian Hamster IgG isotype control were added to the microgels and left for 16 hours to react in suspension. Post-incubation, microgels were centrifuged at 3,000 x g and the supernatant was removed and washed with 1X PBS.

[0206] Immunomodulatory Microgels with Guest-Host Molecules Fabrication: PEG-MAL (4- arm, 20kDa) and PEG-SH (4-arm, 20kDa) were dissolved in 1X PBS with 1% HEPES at pH 5.4 (70.8 mg / ml). Sulfo-smcc was dissolved in 1X PBS at pH 5.4 (0.44 mg / ml, 1.0 mM) and reacted with PEG-SH for 30 minutes at 25°C. Adamantane-thiol was dissolved in 1X PBS with 1%HEPES and 10% DMSO at pH 5.4 (0.84 mg / ml, 0.25 mM) and mono-6-mercapto- -cyclodextrinwas dissolved in 1X PBS with 1% HEPES at pH 5.4 (5.6 mg / ml, 0.25 mM). Adamantane-thioland mono-6-mercapto- -cyclodextrin were added dropwise to the PEG-4MAL macromer at 1adamantane: 1 -cyclodextrin molar ratio and reacted for 30 minutes at 25°C. Sulfo-smcc functionalized PEG-SH was added to the functionalized PEG-4MAL macromer at a 1:1 volume ratio, quickly pipetted up and down several times to mix thoroughly, then transferred to a 30x volume of mineral oil with 2 % vol / vol Span 80 surfactant in a 50 ml conical tube. The tube was immediately vortexed for 30 seconds to generate an emulsion, then allowed to finish crosslinking for 30 minutes at 25°C on a rocker to generate microgels of a final polymer concentration of 6 % wt / vol. Microgels were centrifuged at 3,000 x g for 5 minutes and washed three times with 0.3% Triton X-100 in 1X PBS at pH 7.2, and three times with 1X PBS. After washing procedures, microgels were suspended in a 1:2 volume ratio with 1X PBS, and 200 μL of 0.2 mg / ml anti- mouse CD3ε (Biolegend, 145-2C11) or the Armenian Hamster IgG isotype control were added to the microgels and left for 16 hours to react in suspension. Post-incubation, microgels were centrifuged at 3,000 x g and the supernatant was removed and washed with 1X PBS.

[0207] hPSC culture and differentiation of stem cell derived beta-like cells: sBCs were generated using methods consistent with previous publications

[0031] . Undifferentiated hPSC Mel1INS-GFPreporter cells were maintained on hES qualified Matrigel (Corning #354277) in mTeSR+ 61 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 media (STEMCELL Technologies #05826).

[0032] Differentiation to stem cell derived beta like cells (sBC) was carried out in suspension‐based, bioreactor magnetic stirring system (Reprocell #ABBWVS03A-6, #ABBWVDW-1013, #ABBWBP03N0S-6) as follows. Confluent hPSC cultures were dissociated into single‐cell suspension by incubation with TrypLE (Gibco #12-604- 021) for 8 min at 37 C. Detached cells were quenched with mTESR+ media. Cells were then counted using a MoxiGo II cell counter (Orflow), followed by seeding 0.5 × 106cells / ml in mTeSR+ media supplemented with 10 μM ROCK inhibitor (Y-27632, R&D Systems #1254-50). Bioreactors were placed on a magnetic stirring system set at 60 rpm in a cell culture incubator with 5% CO2to induce sphere formation for 48 hr. To induce definitive endoderm differentiation, spheres were collected in a 50 ml Falcon tube, allowed to settle by gravity, washed once with RPMI (Gibco #11-875-093) + 0.2% FBS, and re‐suspended in d1 media [RPMI containing 0.2% FBS, 1:5,000 ITS (Gibco #41400-045), 100ng / ml Activin A (R&D Systems #338-AC-01M), and 3 μM CHIR99021 (STEMCELL Technologies #72054)]. Differentiation media was changed daily by letting spheres settle by gravity for 3-10 min. Most supernatant was removed by aspiration; fresh media was added, and bioreactors were placed back on stirrer system. sBC differentiation was based on published protocol

[0033] with modifications as outlined below. Differentiation medias are as: day 2-3: RPMI containing 0.2% FBS, 1:2,000 ITS, and 100 ng / ml Activin A; d4-5: RPMI containing 2% FBS, 1:1,000 ITS, and 50 ng / ml KGF (Prepotech #100- 19-1MG); d6: DMEM with 4.5g / L D-glucose (Gibco #11960-044) containing 1:100 SM1 (STEMCELL Technologies #5711), 1:100 NEAA (Gibco #11140-050), 1mM Sodium Pyruvate (Gibco #11360-070), 1:100 GlutaMAX (Gibco #35050-061), 3 nM TTNPB, (R&D Systems #0761), 250 nM Sant-1 (R&D Systems #1974), 250 nM LDN (STEMCELL Technologies #72149), 30 nM PMA (Sigma Aldrich #P1585-1MG), 50 μg / ml 2-phospho-L-ascorbic acid trisodium salt (VitC) (Sigma #49752-10G); d7: DMEM containing 1:100 SM1, 1:100 NEAA, 1mM Sodium Pyruvate, 1:100 GlutaMAX, 3nM TTNPB, and 50 μg / ml VitC; d8-9: DMEM containing 1:100 SM1, 1:100 NEAA, 1mM Sodium Pyruvate, 1:100 GlutaMAX, 100 ng / ml EGF (R&D Systems #236-EG-01M), 50 ng / ml KGF, and 50 μg / ml VitC; d10-16: DMEM containing 2% fraction V BSA, 1:100 NEAA, 1mM Sodium Pyruvate, 1:100 GlutaMAX, 1:100 ITS, 10ug / ml Heparin (Sigma #H3149-250KU), 2 mM N-Acetyl-L-cysteine (Cysteine) (Sigma #A9165-25G), 10 μM Zinc sulfate heptahydrate (Zinc) (Sigma #Z0251-100g), 1x BME, 10 μM Alk5i II RepSox (R&D Systems #3742 / 50), 1 μM 3,3’,5-Triiodo-L-thyronine sodium salt 62 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 (T3) (Sigma #T6397), 0.5 μM LDN, 1 μM Gamma Secretase Inhibitor XX (XXi) (AsisChem #ASIS-0149) and 1:2501M NaOH to adjust pH to ~7.4; d17-23: CMRL (Gibco #11530-037) containing 1% BSA, 1:100 NEAA, 1 mM Sodium Pyruvate, 1:100 GlutaMAX, 10ug / ml Heparin, 2mM Cysteine, 10uM Zinc, 1x BME, 10 μM Alk5i II RepSox, 1 μM T3, 50 μg / ml VitC, and 1:250 NaOH to adjust pH to ~7.4. All medias also contained 1x PenStrep.

[0208] Autoimmune diabetes model and Adoptive Cell Transfer: The mouse strain NOD-cMHCI- / -A2 (NOD.A2) (Jax Stock No: 031856) and NSG-HLA-A2 / HHD (NSG.A2) (Jax Stock No: 014570) was provided by Dr. Dave Serreze at The Jackson Laboratory. Mice were maintained in University of Florida Institutional Animal Care Services and genotype was confirmed by PCR using Transnetyx. All experiments involving animals were approved by the University of Florida Animal Care and Use Committee (IACUC no. 202200000503). Four days before adoptive cell transfer NSG.A2 mice received a transplant of about 400 sBCs under the left kidney capsule with about 40 μL of microgels, similarly to transplantation of pancreatic islets as described

[0041] . Mice were anesthetized with 1.5-2% isoflurane, shaved and cleaned with alternate washes of sterile saline and 2% chlorhexidine. A subcutaneous injection of buprenorphine and meloxicam was administered as a post-operative analgesic. Splenocytes from NOD.A2 were collected and frozen after blood glucose read over 250 mg / dl. Splenocytes were thawed and isolated for CD3+ T Cells using the EasySepTMMouse T Cell Isolation Kit (Stemcell Technologies, no.19851).6-well plates were coated with anti-CD28 and anti-CD3 overnight in 1X PBS at 4°C. Plates were then blocked for 4 hours with splenocyte media at 25°C. Isolated T cells were plated and left to activate for 3 days. After 3 days, T cells were removed from coated plates and allowed to expand in a T-75 flask for 3 days. On day 0, five million activated T cells were injected via the tail vein into the mice. Weight and blood glucose measurements were taken every 2-3 days. 12 days after transplant the graft-bearing kidneys, pancreas, and spleen were recovered for immunohistochemistry analysis.

[0209] Dynamic Glucose-Stimulated Insulin-Secretion Assay: Dynamic insulin secretion was measured using a BioRep Technologies perifusion machine (catalog no. PERI4-115-1810-076). sBC clusters were placed on a poly-l-lysine coated coverglass before being placed into the perifusion chamber, and various solutions were perfused through the system at 100 uL / min by aperistaltic pump; cells and solutions were kept at 37 C. the perifusion program consisted of a 3063 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 min preincubation step with Krebs-Ringer bicarbonate buffer containing a low glucose concentration (2.8mmol / L; “low glucose”) followed by the following run programs: 1) 30min low glucose concentration, 20min high glucose concentration (20 mmol / L; “High glucose”), 20 min high glucose with 3-isobutyle-1-methyxanthine (IBMX; 50 mol / L), 20 min low glucose, 10min KCL, and 10 min low glucose. Perfiusion flow-through was collected in 96-well platesand stored at 4 C overnight or -20 C if longer storage was needed for future analysis. Cell pelletswere recovered and lysed with acid / ethanol solution before being frozen overnight for assessment of total insulin content.

[0210] Longitudinal Monitoring of the sBC grafts by transdermal bioluminescence: Following transplantation, the sBC grafts’ viability was longitudinally monitored by transdermal bioluminescence. D-Luciferin was prepared at a concentration of 30 mg / ml and filtered through a 0.22 μm Spin-X® centrifuge tube filter (Costar no. 8160) at each timepoint. Mice were anesthetized with 1.5-2% isoflurane and sterile ocular lubricant was applied. Hair on the left lateral side of the mice was removed with depilatory cream. D-Luciferin was injected subcutaneously at 75 mg / kg five minutes prior to imaging. Transdermal bioluminescence was captured with an IVIS® Spectrum In Vivo Imaging System. For bioluminescence imaging, the system was set to take images without any emission filter (open) and sequential imaging was performed until signal peaked. Transplants were longitudinally monitored at days -4, 0, 4, 8, 10, 11, and 12 after adoptive cell transfer. In vivo bioluminescence imaging data was analyzed using PerkinElmer, Inc Living Image® 4.5.5 software. The total flux (o / s) was estimated with a circular region of interest (ROI) over the left kidney region. A corrected total flux (CTF) was determined by measuring the background signal and applying equation below.3.2% paraformaldehyde (Thermo Fisher no. 047377) at 4°C in 1X PBS overnight followed by three washes with 1X PBS. The tissues were immersed in 15% w / v sucrose in 1X PBS overnight, followed by 30% w / v sucrose overnight then snap frozen in Optimal Cutting Temperature (O.C.T.) compound (Thermo Fisher) using 2-methylbutane (> 99% purity) chilled with liquid nitrogen. Frozen tissue blocks were cut with a cryostat into serial 20 μm sections and mounted on 64 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 Superfrost Plus microscope slides (Thermo Fisher). Cryosections were incubated three times for 5 minutes in 0.3% Triton X-100. Samples were the blocked and permeabilized in 1X PBS with 0.3% Triton X-100 + 10% donkey serum for 1 hour at 25°C. Primary antibodies were incubated overnight in PBS with 0.3% Triton X-100 + 1% donkey serum at 4°C. Secondary antibodies were incubated at 1:200 dilutions in 1X PBS in 0.3% Triton X-100 for 2 hours at 25°C. Coverslips were mounted with ProLongTMGold (Thermo Fisher)

[0212] Example 6. Studies on Anti-CD3 microporous annealed particle hydrogel protects stem cell derived beta cells from autoreactive T cells in a humanized model of type 1 diabetes

[0213] Results

[0214] To enhance the survival and engraftment of transplanted sBCs by promoting vascular integration and mitigating the foreign body response, the inventors explored the use of MAP hydrogels as a supportive biomaterial platform. Previous studies have demonstrated the reliable and consistent generation of guest-host MAP through batch emulsion. In brief, polyethylene glycol maleimide (PEG-MAL) is covalently conjugated to Adamantane (guest) and - cyclodextrin (host) prior to the addition of a PEG-thiol (PEG-SH) crosslinker and formation of microgels in a batch emulsion. Resulting microgels allow islets and sBCs to be embedded within the interstitial spaces. The microgels are linked together through guest-host interactions present on the surface and are composed of both chemical and physical crosslinks (Figure 12A). The inventors found that the batch emulsion method was highly reproducible and consistent for the generation of PEG microgels over several iterations (Figure 12B, C). The average diameter (19.4 m) and PDI (0.29) were not significantly different across all groups.

[0215] To assess the mechanical properties of guest-host MAP the inventors performed a low amplitude (1%) oscillatory frequency sweep from 10 to 0.01 Hz and found that all groups displayed viscoelastic behavior over the entire frequency range (Figure 12D). The guest-host MAP had a significantly higher storage modulus (1500 Pa) than PEG microgels (390 Pa) over the entire frequency range. The inventors found that guest-host MAP demonstrated full recovery of the storage modulus following high-strain deformation while maintaining low yield stresses over PEG microgels that progressively lost structural integrity upon exposure to high strains, 65 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 indicating its suitability for injectable delivery and mechanical resilience in a transplant setting (Figure 12E, F). The inventors investigated the potential cytotoxic effects of the guest-host particles by culturing islets isolated from C57BL / 6 mice with either guest-host MAP or PEG microgels. Islet viability was comparable between groups, with no decline observed over seven days (Figure 12G). In both microgel types, islets maintained a rounded morphology with minimal cell shedding throughout the culture period (Figure 12H, I). These results indicate that guest-host interactions do not induce acute cytotoxicity and that guest-host MAP hydrogels are safe for maintaining islet viability.

[0216] The inventors sought to determine whether the microporous architecture and guest-host interactions of guest-host MAP could mitigate the foreign body response and promote vasculature in the subcutaneous space, establishing a microenvironment that supports the long- term engraftment of transplanted beta cells. To evaluate the immunological response to the material, guest-host MAP and PEG microgels were administered subcutaneously into C57BL / 6 mice. Implants were retrieved for histological analysis at day 31. Both microgel types exhibited minimal fibrotic encapsulation and showed evidence of integration with the surrounding host tissue (Figure 13A,B). The inventors observed a marked difference in cellular infiltration and vascular integration between guest-host MAP and PEG microgels. In guest-host MAP implants, infiltrating cells were organized into elongated, vessel-like structures spanning the scaffold and were positive for basement membrane markers (Figure 13C). Cellular infiltration into the guest- host MAP hydrogel extended approximately 0.9 mm from the surface, whereas cells within the PEG microgels exhibited a more uniform dense distribution, with an average infiltration of 3.9 mm (Figure 13D, E). To confirm vascular integration, the inventors stained for CD31 and found a greater density of vessels and a significant increase in CD31+area within guest-host MAP hydrogels (Figure 13F, G). These results indicate that guest-host MAP facilitates blood vessel formation and tissue integration, which are critical for the survival and function of transplanted beta cells.

[0217] To evaluate the functional capacity of islets transplanted in guest-host MAP hydrogels in hyperglycemic conditions, the inventors utilized a streptozotocin (STZ)-induced model of diabetes in C57BL / 6 mice. Syngeneic mouse islets were encapsulated within guest-host MAP hydrogels and transplanted subcutaneously. Blood glucose was monitored longitudinally to 66 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 assess graft function and restoration of normoglycemia (Figure 14A). Mice receiving islets within guest-host MAP hydrogels achieved normoglycemia within four weeks (Figure 14B), whereas islet-only controls dispersed upon transplantation and died due to rapid hypoglycemia caused by insulin dumping from the graft. Intraperitoneal glucose tolerance tests revealed no significant differences between guest-host MAP and naïve controls (Figure 14C). Grafts were explanted after 70 days for histological analysis, revealing fully vascularized islets within the guest-host MAP demonstrated and extensive intra-islet CD31+area (Figure 14D). These findings demonstrate that guest-host MAP provide a supportive subcutaneous microenvironment that promotes engraftment, vascular integration, and functional recovery to normoglycemic levels.

[0218] In this study, the inventors evaluated the effect of CD3 conjugated to guest-host MAP for the protection of transplanted sBCs. To functionalize guest-host MAP with CD3, the inventors first reacted the crosslinker PEG-SH with sulfo-SMCC, a hetero-bifunctional crosslinker containing N-hydroxysuccinimide (NHS)-ester and maleimide groups. (Figure 15A) The modified PEG-SH was then mixed in equal volume with PEG-MAL and emulsified in mineral oil. The resulting NHS-functionalized microgels were then incubated with Armenianhamster anti-mCD3 ( CD3, clone 145-2C11) that has previously been shown to cure T1D in 64to 80% of NOD mice with 5 days of treatment after disease onset. [13A, 14A] To assess CD3 conjugation efficiency, the inventors varied the molar amount of sulfo-smcc reacting with PEG- SH and incubated with CD3 and labeled with an Armenian-hamster secondary antibody. The inventors observed a significant binding of the antibody on the surfaces of the microgels, displaying a uniform distribution (Figure 15B). The inventors found significant differences between the amount of sulfo-SMCC conjugated to the PEG-SH and the mean fluorescence intensity (MFI) of the secondary antibody onto the resulting NHS-functionalized microgels (Figure 15C).

[0219] Release kinetics of surface-conjugated CD3 from sulfo-smcc microgels at 37°C showed approximately 4% antibody release over three days (Figure 15D). The functional activity of the conjugated antibody was assessed using a co-culture assay with the titrated CD3 microgels and isolated, activated, CD3+ T Cells from a diabetic NOD-cMHCI--A2 mouse (Figure 15E). Increasing surface concentrations of CD3, corresponding to higher sulfo-smcc amounts, 67 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 resulted in a dose-dependent decrease in the percentage of live CD4+ T Cells (Figure 15F) and a lower percentage of live CD8+ T cells at the highest titrations (Figure 15G), demonstrating effective T cell engagement and functional activity of surface-conjugated CD3. The inventors further assessed CD3+ MAP activity using a transwell migration assay toward a CXCL9 / 10 gradient (Figure 19A). Fewer T cells migrated through CD3+ MAP compared with isotype or no-gel controls, and cells retained within the CD3+ MAP showed reduced viability (Figure 19B-E). These results indicate that CD3+ MAP can arrest migration of activated T cells, suggesting its potential to block infiltration of autoreactive T cells towards transplanted sBCs and islets in vivo.

[0220] To investigate the immunoprotective effects of the aCD3+ microgels, the inventors generated sBCs through a direct differentiation process from human Mel1INS-GFPESCs (Figure 16A). sBCs at day 23 were assessed for expression of the pINS.GFP reporter and quantified for key pancreatic cell markers (Figure 16B, C). Consistent with previous reports, ESCs effectively generate into insulin+sBCs. [15A, 16A, 17A, 18A, 19A, 20A] Day 23 sBCs were dissociated into single cells, filtered, and cryopreserved. Upon thawing, clusters were reaggregated and cultured in our optimized beta cell maturation media for 5 days (PRObeta), as previously described. The inventors next evaluated the capacity of CD3+MAP to protect sBCs from autoimmune attack using a transwell migration assay in which activated, diabetogenic NOD- cMHCI--A2 CD3+ T cells were challenged to migrate through CD3+ MAP towards sBCs seeded in the bottom well (Figure 16D). Analysis of sBCs in the bottom well revealed no differences in viability, as measured by the human endocrine marker Chromagranin A (CHGA), nor in the proportion of C-peptide+ (Cpep+) cells among downgated live human cells (Figure 16E-G). In contrast, the T cells that successfully migrated through CD3+ MAP exhibited reduced viability, with CD4+ T cell impaired at 24 hours and CD8+ T cells at both 24 and 48 hours (Figure 16H-K). These findings indicate that CD3+ MAP can attenuate T cell viability during infiltration towards a beta cell target.

[0221] To model autoimmune rejection of transplanted human sBCs, the inventors employed a humanized-HLA model combining NOD-cMHCI⁻ / ⁻A2 and NSG-HLA-A2 / HHD mice. The inventors confirmed this model by isolating T cells from diabetic NOD-cMHCI⁻ / ⁻A2 mice then 68 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 activating and expanding them before adoptively transferring into NSG-HLA-A2 / HHD recipients transplanted with firefly-luciferase expressing sBCs. Adoptively transferred T cells effectively infiltrate and destroy both endogenous islets and grafted sBCs, recapitulating autoimmune pathology (Figure 20A-C). By transplanting sBCs prior to T cell transfer, the inventors ensured initial engraftment and distinguished autoimmune rejection from innate or inflammatory loss (Figure 20D). This system provides a reproducible platform to evaluate immunomodulatory biomaterials and immune evasion strategies for beta cell replacement in autoimmune diabetes in a humanized-HLA model.

[0222] To investigate the protection of sBCs by CD3+ MAP from an autoimmune attack, the inventors transplanted 600 sBCs embedded in CD3+ MAP into the subcutaneous space of an NSG-HLA-A2 / HHD mouse (Figure 6A). This approach allows stable engraftment prior to immune challenge and enables precise control of the number and type of immune cells introduced through adoptive cell transfer (ACT), thereby avoiding confounding contributions from B cells, NK cells, and other innate effectors present in fully immune competent NOD- cMHCI⁻ / ⁻A2. Following ACT of activated NOD-cMHCI⁻ / ⁻A2 T cells, grafts were monitored via bioluminescence imaging for engraftment and viability over 45 days post ACT (Figure 17A, B). Grafts containing CD3+ MAP maintained a stable signal post ACT, whereas control microgels exhibited loss of signal by day 33. At day 45 post ACT 85% of CD3+ MAP grafts remained detectable compared to 50% of the Armenian hamster isotype control, demonstrating a protective effect of CD3+ MAP (Figure 17C). Longitudinal blood glucose monitoring showed that transferred T cells trafficked both to subcutaneous graft and the pancreas, leaving only 25% of guest-host MAP mice and 80% of CD3+ MAP mice diabetes-free by day 45 post ACT (Figure 17D). Analysis of human C-peptide and mouse C-peptide revealed significantly higher human and mouse c-peptide post-ACT in the CD3+ MAP mice compared to the guest-host MAP control. Explanted grafts demonstrated increased numbers of human nuclear antigen+ and insulin+ cells remaining in CD3+ MAP compared to the guest-host MAP control.

[0223] The inventors found that guest-host MAP grafts had large numbers of infiltrating CD3+ T Cells within the gel and surrounding the sBC with little to no remaining insulin signal (Figure 18A, B). Flow cytometric analysis of the collected grafts showed no significant differences in the 69 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 number of T cells within the graft (Figure 18C). To understand the effect of CD3+ MAP systemically the inventors collected the draining lymph nodes and analyzed by flow cytometry. CD3+ MAP grafts had significantly smaller proportions of circulating CD4+ T cells, however there was no difference in the anergy markers CD73+FR4+ between the two groups. There were no differences between proportions of CD8+T Cells or their anergic markers (Figure 18I). In the pancreas, there were significantly more islets per mouse and a higher insulin MFI in CD3+MAP (Figure 18J). However, there were significantly more CD3+T cells in the guest-host MAP (Figure 18K, L). Together, these results support CD3+MAP induction of deletion or arrest of circulating autoreactive T cells, thereby limiting autoimmune destruction and preserving sBC viability.

[0224] Methods:

[0225] Chemicals and Reagents: 4-arm PEG-MAL (PEG-4MAL, 20 kDa) was purchased from Laysan Bio. 4-arm PEG-thiol (PEG-4SH, 20 kDa) was purchased from Jenkem Technology. 1- adamantane-thiol, span 80, mineral oil, and triton-X 100 were obtained from Sigma Aldrich. Mono(6-mercapto-6-deoxy)-β-cyclodextrin was obtained from Zhiyuan Biotechnology.

[0226] Guest-host MAP and PEG Microgel Fabrication: Guest-host MAP microgels were synthesized by dissolving PEG-4MAL (20kDa) in 1X phosphate buffered saline (PBS) with 1% HEPES at pH 5.4 (70.8 mg / ml, 1.7 mM). The reduced pH helps to slow down the crosslinking reaction which at neutral pH is so quick it hinders mixing and handling. [23A] At pH 5.4 the gelation of PEG-4MAL with PEG-4SH takes approximately 34 seconds. [24A] Adamantane- thiol was dissolved in 1X PBS with 1% HEPES and 10% DMSO at pH 5.4 (0.84 mg / ml, 0.5 mM) and mono-6-mercapto-β-cyclodextrin was dissolved in 1X PBS with 1% HEPES at pH 5.4 (5.6 mg / ml, 0.5 mM). Adamantane-thiol and mono-6-mercapto-β-cyclodextrin were added dropwise to the PEG-4MAL macromer at 1 adamantane:1 β-cyclodextrin molar ratio and reacted for 30 minutes at 25°C. PEG-4SH (20 kDa) was dissolved in 1X PBS with 1% HEPES at pH 5.4 (48.4 mg / ml, 1.2 mM) and reacted with a trace amount of Alexa Fluor-MAL for 5 minutes at 25°C to help aid in microgel visualization. PEG-4SH was added to the functionalized PEG- 4MAL macromer at a 1:1 volume ratio, quickly pipetted up and down several times to mix thoroughly, then transferred to a 30x volume of mineral oil with 2% vol / vol span 80 surfactant in 70 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 a 50 ml conical tube. The tube was immediately vortexed for 30 seconds to generate an emulsion, then allowed to finish crosslinking for 30 minutes at 25°C on a rocker plate to generate microgels with a final weight percentage of 6% wt / vol. PEG microgels were synthesized by dissolving PEG-4MAL (20 kDa) macromer in 1X PBS with 1% HEPES at pH 5.4 (70.8 mg / ml, 1.4 mM). PEG-4SH (20 kDa) was dissolved in 1X PBS with 1% HEPES at pH 5.4 (59.14 mg / ml, 1.4 mM) and reacted with a trace amount of Alexa Fluor-MAL for 5 minutes. PEG-4SH was added dropwise to the PEG-4MAL macromer at a 1:1 volume ratio, and microgels were formed by emulsion the same as above. For all groups, microgels were centrifuged at 3,000 x g for 5 minutes and washed three times with 0.3 % Triton X-100 in 1X PBS at pH 7.2, and twice with 1X PBS.

[0227] Microgel Characterization: Microgel diameter was taken by imaging several batches of microgels using confocal microscopy. The images were processed using a gaussian filter, binarizing the image, and then using StarDist in the FIJI distribution of ImageJ to determine the microgel diameter. The Feret’s diameter of each microgel was considered as the diameter. The particle size data were then analyzed by frequency distribution with a bin size of 5, the average diameter and standard deviation were used to determine the PDI (Equation 1), where σ is the standard deviation and <D> is the average diameter. [25A] [1A]200 μl of media was placed on top of the gel and islet mixture, then placed in the incubator. Media was switched every two days, islets were imaged on day one, five, and seven using CalceinAM, Ethidium homodimer-1 and Hoescht to assess viability. 4-5 islets per group were imaged on a confocal microscope with a 20x. Numbers of cells were quantified by the StarDist package in FIJI. [26A, 27A]

[0229] Subcutaneous microgel transplantation: C57Bl / 6 were anesthetized with 1.5-2% isoflurane, shaved, and cleaned with alternate washes of sterile saline and 2% chlorhexidine. Subcutaneous injection of meloxicam was administered as post-operative analgesic. 20 μl of guest-host MAP and PEG microgels were injected into the upper left and lower left quadrant of the dorsum. 71 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

[0230] αCD3+ MAP Fabrication: PEG-MAL (4-arm, 20kDa) and PEG-SH (4-arm, 20kDa) were dissolved in 1X PBS with 1% HEPES at pH 5.4 (70.8 mg / ml). Sulfo-smcc was dissolved in 1X PBS at pH 5.4 (0.44 mg / ml, 1.0 mM) and reacted with PEG-SH for 30 minutes at 25°C. Adamantane-thiol and mono-6-mercapto-β-cyclodextrin were functionalized to PEG-MAL as described above. Sulfo-SMCC functionalized PEG-SH was added to the functionalized PEG- 4MAL macromer at a 1:1 volume ratio, microgel generation and washing steps were completed as described above. Microgels were suspended in a 1:2 volume ratio with 1X PBS, and 200 μL of 0.2 mg / ml anti-mouse CD3ε (Biolegend, 145-2C11) or Armenian Hamster IgG isotype control were added to the microgels reacted for 16 hours. Post-incubation, microgels were centrifuged at 3,000 x g and the supernatant was removed and washed with 1X PBS.

[0231] Human stem cell culture and sBC differentiation: Undifferentiated human pluripotent stem (hPSC) Mel1INS-GFPreporter cells[28A] were maintained on hESC qualified Cultrex (Biotechne #3434-005-002) in mTeSR+ media (STEMCELL Technologies #05826). Differentiation to stem cell-derived beta-like cells (sBC) was carried out in suspension‐based, magnetic stirring platforms (Reprocell #ABBWVS03A-6, #ABBWVDW-1013, #ABBWBP03N0S-6) as described [29A, 30A]. Briefly, 90% confluent hPSC cultures were dissociated into single‐cell suspension by incubation with TrypLE (Gibco #12-604-021). Dissociation was halted with mTeSR+ media, and cells were counted using a Countess 3 cell counter (ThermoFisher Scientific), followed by seeding 0.5 × 106cells / mL in mTeSR+ media supplemented with 10 μM ROCK inhibitor in bioreactors. 3D sphere formation was performed for 48-72 hours. Differentiation media was changed daily by letting spheres settle by gravity for 3-5min. Most supernatant was removed by aspiration; fresh media was added, and bioreactors were placed back on stirrer system. sBC differentiation and cryopreservation was based on our published protocol[31A, 32A] with modifications as outlined below with key experiments using select additions or subtractions from medias as described in the results and figures. Differentiation medias are as follows: induction of definitive endoderm differentiation using d1 media [RPMI containing 0.2% FBS, 1:5,000 ITS (Gibco #41400-045), 200 ng / ml Activin A (R&D Systems #338-AC-01M), and 3 μM CHIR99021 (STEMCELL Technologies #72054)] and day 2-3: RPMI containing 0.2% FBS, 1:2,000 ITS, and 100 ng / ml Activin A; d4-5: RPMI containing 2% FBS, 1:1,000 ITS, and 50 ng / ml KGF (Prepotech #100-19-1MG); d6-7: DMEM with 4.5 g / L D-glucose (Gibco #11960-044) containing 1:50 N-21 MAX (Biotechne #AR008), 72 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 1:100 NEAA (Gibco #11140-050), 1mM Sodium Pyruvate (Gibco #11360-070), 1:100 GlutaMAX (Gibco #35050-061), 3 nM TTNPB, (R&D Systems #0761), 250 nM Sant-1 (R&D Systems #1974), 250 nM LDN (STEMCELL Technologies #72149), 30 nM PMA (Sigma Aldrich #P1585-1MG), 50 μg / ml 2-phospho-L-ascorbic acid trisodium salt (VitC) (Sigma #49752-10G); d8-9: DMEM containing 1:50 N-21 MAX, 1:100 NEAA, 1 mM Sodium Pyruvate, 1:100 GlutaMAX, 100 ng / ml EGF (R&D Systems #236-EG-01M), 50 ng / ml KGF, and 50 μg / ml VitC; d10-15: DMEM containing 1:50 N-21 MAX, 1:100 NEAA, 1 mM Sodium Pyruvate, 1:100 GlutaMAX, 10 μg / ml Heparin (Sigma #H3149-250KU), 2 mM N-Acetyl-L-cysteine (Cysteine) (Sigma #A9165-25G), 10 μM Zinc sulfate heptahydrate (Zinc) (Sigma #Z0251-100g), 1x BME, 10 μM Alk5i II RepSox (R&D Systems #3742 / 50), 1 μM 3,3’,5-Triiodo-L-thyronine sodium salt (T3) (Sigma #T6397), 0.5 μM LDN, 1uM Gamma Secretase Inhibitor XX (XXi) (AsisChem #ASIS-0149) and 1:2501 M NaOH to adjust pH to ~7.4. For experiments presented in Figures 4, 5, 6, 7, 8 and Supplementary Figures 4, 5, 7 and 810μM ROCKi (RnD systems #1254 / 50) was added; d16-30: CMRL (Gibco #11530-037) containing 1:50 N-21 MAX, 1:100 NEAA, 1:100 GlutaMAX, 10ug / ml Heparin, 2mM Cysteine, 10 μM Zinc, 1x BME, 1 μM T3, 50ug / ml VitC, 1:1000 Trace Elements A (Corning # 25-021-CI), 1:1000 Trace Elements B (Corning # 25-022- CI) with or without 10 μM Alk5i II RepSox and 1:250 NaOH to adjust pH to ~7.4. All medias with exception of mTeSR+ also contained 1x PenStrep.

[0232] Cryopreservation and thawing of hPSC-derived beta-like cells: Day 23 sBC were dissociated into single cells and cryopreserved as described[29A, 32A]. Briefly, cells were quenched with 2% FBS in PBS and filtered using a cell strainer into FACS 5 mL tubes. Cells were counted using a Countess 3 cell counter (ThermoFisher Scientific) and resuspended at 3 x 106cells / 100 μL of CryoStor® CS10 (StemCell Technologies). Cells were cryopreserved overnight before transfer to liquid nitrogen for long-term storage. For thawing, 1 mL of warm sBC media (d16-30 media described above) was added to the thawed cryovial dropwise before the entire volume was transferred into 5 mL of sBC media, counted and seeded in Aggerwell 800 plates to generate clusters with 3,000 cells / cluster. After 24 hours, a partial media change was done to remove any debris. Fully formed clusters were generated after 48-72 hours.

[0233] Autoimmune diabetes model and Adoptive Cell Transfer: The mouse strain NOD-cMHCI- / -A2 (Jax Stock No: 031856) and NSG-HLA-A2 / HHD (Jax Stock No: 014570) was provided by 73 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 Dr. Dave Serreze at The Jackson Laboratory. Mice were maintained in University of Florida Institutional Animal Care Services and genotype was confirmed by PCR using Transnetyx. Splenocytes from NOD-cMHCI- / -A2 were collected and frozen after blood glucose read over 250 mg / dl. Splenocytes were thawed and isolated for CD3+ T Cells using the EasySepTMMouse T Cell Isolation Kit (Stemcell Technologies, no.19851). 6-well plates were coated with anti-CD28 and anti-CD3 overnight in 1X PBS at 4°C. Plates were then blocked for 4 hours with splenocyte media at 25°C. Isolated T cells were plated and left to activate for 3 days. After 3 days, T cells were removed from coated plates and allowed to expand in a T-75 flask for 3 days. One million activated T cells were injected via the tail vein into the mice.

[0234] Longitudinal Monitoring of the sBC grafts by transdermal bioluminescence: Following transplantation, the sBC grafts’ viability was longitudinally monitored by transdermal bioluminescence. D-Luciferin was prepared at a concentration of 30 mg / ml and filtered through a 0.22 μm Spin-X® centrifuge tube filter (Costar no. 8160) at each timepoint. Mice were anesthetized with 1.5-2% isoflurane and sterile ocular lubricant was applied. Hair on the left lateral side of the mice was removed with depilatory cream. D-Luciferin was injected subcutaneously at 75 mg / kg five minutes prior to imaging. Transdermal bioluminescence was captured with an IVIS® Spectrum In Vivo Imaging System. For bioluminescence imaging, the system was set to take images without any emission filter (open) and sequential imaging was performed until signal peaked. Transplants were longitudinally monitored at days -4, 0, 4, 8, 10, 11, and 12 after adoptive cell transfer. In vivo bioluminescence imaging data was analyzed using PerkinElmer, Inc Living Image® 4.5.5 software. The total flux (o / s) was estimated with a circular region of interest (ROI) over the left kidney region. A corrected total flux (CTF) was determined by measuring the background signal in the lef and applying equation 6-1.

[0235] Serum C-peptide Analysis: For serum C-peptide, mice were fasted for 6 hours followed by an intraperitoneal injection of 2g / kg bodyweight glucose in a 20% glucose solution. Peripheral blood was collected via tail puncture at 30 minutes post-injection into anti-coagulant serum tubes. Blood tubes were spun down at 10,000 x g for 10 minutes and serum was isolated into new tubes. Human C-peptide was quantified using a human specific C-peptide ELISA 74 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 (ALPCO: 80-CPTHU-CH05). Mouse C-peptide was quantified using a mouse specific C-peptide ELISA.

[0236] Flow Cytometry: For gel, sBC, T cell, single cell suspensions were made by washingclusters with PBS and incubating with TrypLE or Collagenase-D at 37 C for 12-15 min. Cellswere quenched with 2% FBS in PBS and filtered using a cell strainer into FACS 5ml tubes and incubated for 30 min on ice for surface markers / live dead staining then fixed with 4% paraformaldehyde for 5 min at room temperature and stained in CAS buffer with 0.4% Triton X (CAS-T) overnight at 4ºC for intracellular markers. After incubation, the cells were washed and resuspended in FACS buffer for analyses on 5-laser Cytek Aurora. Analysis and graphs were made using FloJo software v10.9 (BD Life Sciences). The following antibodies were used with their respective dilution: PE / Cyanine7 anti-mouse FR4 (Biolegend #125012, 1:100), PerCP / Cyanine5.5 anti-mouse CD73 (Biolegend #127214, 1:100), Brilliant violet 750 anti- mouse CD4 (Biolegend #100467, 1:200), Brilliant violet 510 anti-mouse CD8a (Biolegend #100751, 1:200), PE mouse anti-human Chromagranin A (BD Pharmingen #564563, 1:50), C- peptide mouse monoclonal antibody (OriGene Technologies #BM270, 1:500, self-conjugated using Antibody labeling kit Alexa fluor 488 (Invitrogen #A88062).

[0237] Histology and Immunofluorescence: Dorsal sections containing the gel grafts and graft- bearing kidneys were fixed with 3.2% paraformaldehyde at 4°C in 1X PBS overnight followed by three washes with 1X PBS. The tissues were immersed in 15 % w / v sucrose in PBS overnight, and were followed by 30 % w / v sucrose overnight then snap frozen in Optimal Cutting Temperature (O.C.T) compound using 2-methylbutane (>99% purity) chilled with liquid nitrogen. Frozen tissue blocks were cut with a cryostat into serial 20 μm sections and mounted on superfrost plus microscope slides. Hematoxolin and eosin stains were done by the University of Florida Molecular Pathology core and imaged using an Olympus VS200. Using cell profiler, images were separated into color images and the nuclei identified as objects from the hemotoxolin stain. Using the relate objects and find object neighbors module in cell profiler, the nearest distance of the nuclei to the surface of the graft was measured. Cryosections were incubated three times for 5 minutes in 0.3% Triton X-100. Samples were then blocked and permeabilized in PBS with 0.3 % Triton X-100 + 10% donkey serum for 1 hour at 25°C. Primary antibodies were incubated overnight in PBS with 0.3% Triton X-100 + 1% donkey serum at 4°C. 75 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 Secondary antibodies were incubated at 1:200 dilutions in PBS with 0.3% Triton X-100 for 1 hour at room temperature. Some sections were stained with Hoescht Nuclear Yellow at 1:50 in 1X PBS with 0.3% TritonX-100, then washed with 0.3% Triton X-100 three times before coverslips were mounted with ProLong Gold. The following primary antibodies were used for immunofluorescence staining with their respective dilution: rabbit anti-laminin (Abcam #ab11575, 1:300), goat anti-ColIV (Abcam #ab6586,1:40), goat anti-CD31 (Biotechne # AF3628, 1:600), guinea pig anti-insulin (DAKO, 1:1000), rabbit anti-HNA (1:200), rat anti-CD3 (Biolegend #100202, 1:50), rabbit anti-CD8 (Bioss #bs-0648R-TR, 1:200).

[0238] Statistical analysis: Means among three or more groups were compared by a one-way analysis of variance (ANOVA) in GraphPad Prism 8 software. If deemed significant, Tukey's post hoc pairwise comparisons were performed. Means between two groups were compared by two-tailed Student's t-test. A confidence level of 95% was considered significant. Survival curves were analyzed using a Kaplan-Meier curve and analysis. The statistical test used, error bars, and definition of n are all indicated in the individual figure legends.

[0239] Microscopy: Microgels and tissue sections were imaged on a Leica SP8 confocal laser- scanning microscope using 10× / 0.3 and 20× / 0.8 numerical aperture Plan-Apochromat air objectives at 1024 × 1024-pixel resolution. 76 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 SEQUENCE LISTING

[0240] SEQ ID NO:1 145.2c11 heavy chain, partial [Nothocricetulus migratorius] GenBank: AAA86877.1 >AAA86877.1 145.2c11 heavy chain, partial [Nothocricetulus migratorius] MNSGLQLVFFVLTLKGIQGEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYI TSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSAKTTA P

[0241] SEQ ID NO:2 145.2c11 kappa light chain [Nothocricetulus migratorius] GenBank: AAA86876.1 >AAA86876.1 145.2c11 kappa light chain [Nothocricetulus migratorius] MRAPTVYPVLLFLWFTGAICDIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYY TNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRADAKPTVSIFPP SSEQLGTGSATLVCFVNNFYPKDINVKWKVDGSEKRDGVLQSVTDQDSKDSTYSLSSTLSLTKADYERHN LYTCEVTHKTSTAAIVKTLNRNEC

[0242] SEQ ID NO:3 Teplizumab (Heavy chain) QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTI SRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0243] SEQ ID NO:4 Teplizumab (Light chain) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDY TFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 77 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01

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Santini-González, J., et al., Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I. Front Endocrinol (Lausanne). 2022 Nov 25;13:989815.. 24. Herold, K.C., et al., An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes. New England Journal of Medicine, 2019. 381(7): p. 603-613. 25. AntiCD3 Mab (Teplizumab) For Prevention of Diabetes In Relatives At-Risk for Type 1 Diabetes Mellitus, A. National Institute of, et al., Editors. 2009. 26. You, S., et al., Induction of Allograft Tolerance by Monoclonal CD3 Antibodies: A Matter of Timing. American Journal of Transplantation, 2012. 12(11): p. 2909-2919. 27. Widener, A.E., A. Roberts, and E.A. Phelps, Single versus dual microgel species for forming guest-host microporous annealed particle PEG-MAL hydrogel. Journal of Biomedical Materials Research Part A, 2023. 111(9): p. 1379-1389. 28. Hermanson, G.T., Chapter 20 - Antibody Modification and Conjugation, in Bioconjugate Techniques (Third Edition), G.T. Hermanson, Editor. 2013, Academic Press: Boston. p. 867-920. 29. Carpenter, P.A., et al., Non-Fc Receptor-Binding Humanized Anti-CD3 Antibodies Induce Apoptosis of Activated Human T Cells. The Journal of Immunology, 2000. 165(11): p. 6205-6213. 79 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 30. Darling, N.J., et al., Controlling the kinetics of thiol-maleimide Michael-type addition gelation kinetics for the generation of homogenous poly(ethylene glycol) hydrogels. Biomaterials, 2016. 101: p. 199-206. 31. Castro-Gutierrez, R., et al., Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition. Frontiers in Endocrinology, 2021. 12. 32. Micallef, S.J., et al., INSGFP / whuman embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells. Diabetologia, 2012. 55(3): p. 694-706. 33. Russ, H.A., et al., Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. The EMBO Journal, 2015. 34(13): p. 1759-1772. 34. Barra J.M., et al., Combinatorial genetic engineering strategy for immune protection of stem cell-derived beta cells by chimeric antigen receptor regulatory T cells. Cell Rep. 2024 Nov 26;43(11):114994. 35. Barrow, S.J. et al., Cucurbituril-Based Molecular Recognition. Chem. Rev. 2015, 115, 22, 12320–12406. 36. Headen. D.M. et al., Microfluidic-based generation of size-controlled, biofunctionalized synthetic polymer microgels for cell encapsulation. Advanced Materials, 26:3003-3008 (2014). 37. Hu X., et al., Human hypoimmune primary pancreatic islets avoid rejection and autoimmunity and alleviate diabetes in allogeneic humanized mice. Sci Transl Med. 2023 Apr 12;15(691):eadg5794 38. Khosravi-Maharlooei M., et al.,. Modeling human T1D-associated autoimmune processes. Mol Metab. 2022 Feb;56:101417. 39. Matsuoka K., et al., Generation of mouse models for type 1 diabetes by selective depletion of pancreatic beta cells using toxin receptor-mediated cell knockout. Biochem Biophys Res Commun. 2013 Jul 5;436(3):400-5. 40. Racine J.J., et al., Improved Murine MHC-Deficient HLA Transgenic NOD Mouse Models for Type 1 Diabetes Therapy Development. Diabetes. 2018 May;67(5):923-935. 41. Szot G.L., et al., Transplantation of pancreatic islets into the kidney capsule of diabetic mice. J Vis Exp. 2007;(9):404. 42. Yoshihara E., et al., Immune-evasive human islet-like organoids ameliorate diabetes. Nature. 2020 Oct;586(7830):606-611. 43. Alegre M.L., et al., An anti-murine CD3 monoclonal antibody with a low affinity for Fc gamma receptors suppresses transplantation responses while minimizing acute toxicity and immunogenicity. J Immunol. 1995 Aug 1;155(3):1544-55. 44. Phelps E.A. et al., Advanced Materials, 201224: 64-70, 62. 45. DiMeglio L.A., et al. Type 1 diabetes. Lancet. 2018; 391(10138):2449-2462. 80 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 ADDITIONAL REFERENCES 1A. Atkinson, M.A., G.S. Eisenbarth, and A.W. Michels, Type 1 diabetes. The Lancet, 2014. 383(9911): p. 69-82. 2A. Association, A.D., Stem Cell-derived Islet Therapies Shown to Reduce the Need for Injectable Insulin in Breakthrough studies demonstrate stem cell-based technologies as a potential alternative treatment for type 1 diabetes 2025: Chicago, IL 3A. Shapiro, A.M.J., M. Pokrywczynska, and C. Ricordi, Clinical pancreatic islet transplantation. Nature Reviews Endocrinology, 2017. 13(5): p. 268-277. 4A. Smink, A.M., M.M. Faas, and P. De Vos, Toward Engineering a Novel Transplantation Site for Human Pancreatic Islets. Diabetes, 2013. 62(5): p. 1357-1364. 5A. Stabler, C.L., et al., Engineering immunomodulatory biomaterials for type 1 diabetes. Nature Reviews Materials, 2019. 4(6): p. 429-450. 6A. Brusko, T.M., H.A. Russ, and C.L. Stabler, Strategies for durable β cell replacement in type 1 diabetes. Science, 2021. 373(6554): p. 516-522. 7A. Anderson, J.M., A. Rodriguez, and D.T. Chang, Foreign body reaction to biomaterials. Seminars in Immunology, 2008. 20(2): p. 86-100. 8A. Widener, A.E., A. Roberts, and E.A. Phelps, Granular Hydrogels for Harnessing the Immune Response. Advanced Healthcare Materials, 2024. 9A. Lei, J., et al., FasL microgels induce immune acceptance of islet allografts in nonhuman primates. Science Advances, 2022. 8(19). 10A. Herold, K.C., et al., An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes. New England Journal of Medicine, 2019. 381(7): p. 603-613. 11A. AntiCD3 Mab (Teplizumab) For Prevention of Diabetes In Relatives At-Risk for Type 1 Diabetes Mellitus, A. National Institute of, et al., Editors. 2009. 12A. You, S., et al., Induction of Allograft Tolerance by Monoclonal CD3 Antibodies: A Matter of Timing. American Journal of Transplantation, 2012. 12(11): p. 2909-2919. 13A. Chatenoud, L., et al., [Remission of established disease in diabetic NOD mice induced by anti-CD3 monoclonal antibody]. C R Acad Sci III, 1992. 315(6): p. 225-8. 14A. Chatenoud, L., et al., Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proceedings of the National Academy of Sciences, 1994. 91(1): p. 123-127. 15A. Barra, J.M., et al., Cryopreservation of Stem Cell–Derived β-Like Cells Enriches for Insulin-Producing Cells With Improved Function. Diabetes, 2024. 73(10): p. 1687-1696. 16A. Barra, J.M., et al., Combinatorial genetic engineering strategy for immune protection of stem cell-derived beta cells by chimeric antigen receptor regulatory T cells. Cell Reports, 2024. 43(11): p. 114994. 17A. Docherty, F.M., et al., ENTPD3 Marks Mature Stem Cell–Derived β-Cells Formed by Self-Aggregation In Vitro. Diabetes, 2021. 70(11): p. 2554-2567. 18A. Nair, G.G., et al., Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells. Nature Cell Biology, 2019. 21(2): p. 263-274. 19A. Russ, H.A., et al., Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro. The EMBO Journal, 2015. 34(13): p. 1759- 1772. 20A. Shilleh, A.H., S. Beard, and H.A. Russ, Enrichment of stem cell-derived pancreatic beta- like cells and controlled graft size through pharmacological removal of proliferating cells. Stem Cell Reports, 2023. 18(6): p. 1284-1294. 81 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 21A. Bresson, D. and M. Von Herrath, Humanizing Animal Models: A Key to Autoimmune Diabetes Treatment. Science Translational Medicine, 2011. 3(68): p. 68ps4-68ps4. 22A. Van Wauwe, J.P., J.R. De Mey, and J.G. Goossens, OKT3: a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J Immunol, 1980. 124(6): p. 2708- 13. 23A. Darling, N.J., et al., Controlling the kinetics of thiol-maleimide Michael-type addition gelation kinetics for the generation of homogenous poly(ethylene glycol) hydrogels. Biomaterials, 2016. 101: p. 199-206. 24A. Widener, A.E., et al., Guest–host interlinked PEG-MAL granular hydrogels as an engineered cellular microenvironment. Biomaterials Science, 2021. 25A. Karmakar, S., Particle size distribution and zeta potential based on dynamic light scattering: techniques to characterise stability and surface distribution of charged colloids particle. Recent Trends Materials: Physics Chem. Recent Trends in Materials Physics and Chemistry. 2019. 117-159. 26A. Schmidt, U., et al., Cell Detection with Star-Convex Polygons. 2018, Springer International Publishing. p. 265-273. 27A. Schindelin, J., et al., Fiji: an open-source platform for biological-image analysis. Nature Methods, 2012. 9(7): p. 676-682. 28A. Micallef, S.J., et al., INS(GFP / w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells. Diabetologia, 2012. 55(3): p. 694-706. 29A. Castro-Gutierrez, R., et al., Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition. Front Endocrinol (Lausanne), 2021. 12: p. 707881. 30A. Santini-Gonzalez, J., et al., Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I. Front Endocrinol (Lausanne), 2022. 13: p. 989815. 31A. Russ, H.A., et al., Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro. The EMBO journal, 2015. 34(13): p.1759-1772. 32A. Barra, J.M., et al., Cryopreservation of Stem Cell-Derived beta-Like Cells Enriches for Insulin-Producing Cells With Improved Function. Diabetes, 2024. 73(10): p. 1687-1696. 82 LEGAL02 / 46561991v1

Claims

Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 WHAT IS CLAIMED IS:

1. An anti-CD3 hydrogel biomaterial comprising: a) a hydrogel comprising maleimide-terminated four-arm polyethylene glycol (PEG-4MAL) and a thiol-terminated four- arm polyethylene glycol (PEG-SH); and b) an anti-CD3 antibody conjugated to the hydrogel.

2. The anti-CD3 hydrogel biomaterial of claim 1, wherein the hydrogel is a microgel.

3. The anti-CD3 hydrogel biomaterial of claim 2, wherein the microgel has a 10 μm diameter.

4. The anti-CD3 hydrogel biomaterial of claim 2, wherein the microgel is formed by reacting an N-hydroxysuccinimde (NHS)-PEG-thiol with PEG-4MAL via batch emulsion polymerization.

5. The anti-CD3 hydrogel biomaterial of claim 1, wherein the anti-CD3 antibody is conjugated to the hydrogel via an amide linkage to at least one arm of the PEG-SH.

6. The anti-CD3 hydrogel biomaterial of any one of claims 1-5, wherein the hydrogel is functionalized with at least one guest molecule and at least one host molecule.

7. The anti-CD3 hydrogel biomaterial of claim 6, wherein the host molecule is a cyclodextrin or a cucurbit[n]uril.

8. The anti-CD3 hydrogel biomaterial of claim 7, wherein the cyclodextrin is β-cyclodextrin.

9. The anti-CD3 hydrogel biomaterial of claim 6, wherein the guest molecule is adamantane, phenylalanine, ferrocene, or p-xylylenediamine.

10. The anti-CD3 hydrogel biomaterial of claim 6, wherein the host molecule is β-cyclodextrin and the guest molecule is adamantane.

11. A method of making a biomaterial displaying an anti-CD3 antibody, the method comprising 83 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 reacting a N-hydroxysuccinimde (NHS)-polyethylene glycol (PEG)-thiol with a maleimide-terminated four-arm polyethylene glycol (PEG-4MAL) macromer to generate an NHS-activated microgel via batch emulsion polymerization; and capturing an anti-CD3 antibody via the NHS moiety to form the biomaterial displaying the anti-CD3 antibody.

12. The method of claim 11, wherein the microgel has a 10 μm diameter.

13. The method of claim 11 or claim 12, further comprising the step of functionalizing the PEG-4MAL with at least one guest molecule and at least one host molecule prior to reacting the N-hydroxysuccinimde (NHS)-polyethylene glycol (PEG)-thiol with the PEG-4MAL.

14. A biomaterial produced by the method of any one of claims 11-13.

15. A method of making an anti-CD3 microgel, comprising: solubilizing four-arm polyethylene glycol (PEG-4MAL) in aqueous buffer at pH 5.4; solubilizing four-arm polyethylene glycol (PEG)-thiol (PEG-SH) in aqueous buffer at pH 5.4; solubilizing sulfo-SMCC; reacting the solubilized sulfo-SMCC with the solubilized PEG-SH to form a solubilized NHS-coupled PEG-SH; combining the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL at a 1:1 stoichiometric ratio to form a hydrogel; emulsifying the hydrogel with mineral oil to form an NHS-reactive microgel; and conjugating the NHS-reactive microgel with an anti-CD3 monoclonal antibody to form the anti-CD3 microgel. 84 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 16. The method of claim 15, further comprising the step of functionalizing the solubilized PEG-4MAL with at least one guest molecule and at least one host molecule prior to combining the solubilized NHS-coupled PEG-SH and the solubilized PEG-4MAL.

17. The method of claim 16, wherein the host molecule is a cyclodextrin or a cucurbit[n]uril.

18. The method of claim 17, wherein the cyclodextrin is β-cyclodextrin.

19. The method of claim 16, wherein the guest molecule is adamantane, phenylalanine, ferrocene, or p-xylylenediamine.

20. The method of claim 16, wherein the host molecule is β-cyclodextrin and the guest molecule is adamantane.

21. An anti-CD3 microgel produced by the method of any one of claims 15- 20.

22. A method of inducing immune tolerance to a graft cell or a graft tissue in a subject in need thereof, comprising transplanting into the subject the anti-CD3 hydrogel biomaterial of any one of claims 1-10 and the graft cell or the graft tissue.

23. The method of claim 22, wherein the graft cell is a pancreatic islet cell.

24. The method of claim 22, wherein the graft cell is a stem cell derived beta cell cluster (sBC).

25. The method of claim 24, wherein the stem cell derived beta cell cluster (sBC) is a pancreatic beta cell cluster.

26. The method of claim 22, wherein the graft tissue comprises a pancreatic islet cell.

27. The method of claim 22, wherein the graft tissue is a stem cell derived beta cell cluster (sBC). 85 LEGAL02 / 46561991v1Atty Docket No.: 049648 / 637910 UF Ref. No.T19515WO01 28. The method of claim 27, wherein the stem cell derived beta cell cluster (sBC) is a pancreatic beta cell cluster.

29. A method of treating type 1 diabetes in a subject in need thereof, comprising transplanting into the subject the anti-CD3 hydrogel biomaterial of any one of claims 1-10 and pancreatic islet cells or stem cell derived beta cell clusters (sBC).

30. The method of claim 29, wherein pancreatic islet cells are transplanted into the subject.

31. The method of claim 29, wherein stem cell derived beta cell clusters (sBC) are transplanted into the subject.

32. The method of any one of claims 22-31, further comprising administering to the subject an immunosuppressive drug.

33. The method of claim 32, wherein the immunosuppressive drug is rapamycin, cyclophosamide busulfan, fludarabine, methotrexate, sulfasalazine, hydroxychloroquine, azathioprine, tocilizumab, etanercept, adalimumab, anakinra, abatacept, rituximab, certolizumab, golimumab, cyclosporine, dexamethasone, methylprednisolone, prednisone, tacrolimus, or triamcinolone.

34. The method of claim 33, wherein the immunosuppressive drug is rapamycin. 86 LEGAL02 / 46561991v1

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