Synthetic viscoelastic activating cells for t cell engineering

Abstract

Conventional CAR-T cell therapies have shown remarkable success in treating blood cancers and lymphomas. However, several issues with this technology persist, including cancer recurrence. Here we describe a scalable technology platform to produce synthetic viscoelastic activating cells (SynVACs) with programmable mechanical and chemical activities as artificial antigen presenting cells (aAPCs). The disclosure presented herein shows that the viscoelastic properties of the described SynVACs have a profoundly beneficial effect on expanding T cells. For example, in comparison to conventional rigid or elastic microspheres, SynVACs exhibit robust improvements on T cell expansion, T memory stem cell (TMSC) formation, chimeric antigen receptor (CAR) transduction efficiency, tumor killing efficiency, and in vivo long-term persistence of CAR-T cells.

Description

[0001] SYNTHETIC VISCOELASTIC ACTIVATING CELLS FOR T CELL ENGINEERING

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. Section 1 19(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63 / 515,228, filed July 24, 2023, entitled "SYNTHETIC VISCOELASTIC ACTIVATING CELLS FOR T CELL ENGINEERING”, the contents of which is incorporated by reference herein.

[0004] TECHNICAL FIELD

[0005] Embodiments of the disclosure concern at least the fields of immunology and medicine.

[0006] BACKGROUND OF THE INVENTION

[0007] Cancer remains a leading cause of death in the United States and worldwide, with a significant impact on public health. Although early diagnosis and traditional therapies such as surgery', radiation therapy and chemotherapy have significantly improved the treatment of various cancers, there are still limitations such as side effects, resistance to therapies, uncurable genetic mutations, and recurrence. To address these limitations, researchers are exploring immunotherapies such as chimeric antigen receptor (CAR)-T cell therapy1-4. Current CAR-T cell therapies have shown remarkable success in treating blood cancers and lymphomas5. However, several issues persist, including cancer recurrence and the lack of long-term immunity against cancer. In the context of solid tumors, the immunosuppressive tumor environment and the incompetency of existing CAR-T cell expansion methods present additional challenges that need to be addressed6-8. Therefore, there is an urgent need for the development of more effective CAR-T cell expansion techniques, which can enhance the cancer-killing capability of these cells and promote long-lasting immunity against cancer. Addressing these challenges will significantly impact cancer treatment, including solid tumors, ultimately improving patient outcomes and quality of life.

[0008] T cell sternness, characterized by the capacity to self-renew and differentiate into multiple T cell subsets, is a vital feature for sustaining a long-lasting and effective immune response against cancer9. Particularly, T memory stem cells (TMSCs), a subset of memory T cells with stem-like properties, are becoming increasingly recognized for their critical role in sustaining a durable and effective immune response after adoptive transfer10 12. TMSCs possess both a self-renewal ability and multipotent capacity to differentiate into various antigen-specific T cell subsets, rendering them exceptionally potent in generating a robust and persistent immune response13. Although cytokines and small molecules have been explored to enhance the generation and maintenance of TMSCs14, 15, the results may vary due to the lack of control of other niche factors, such as the mechanical properties of synthetic antigen-presenting cells (APCs) or matrices.

[0009] T cell activation is a critical first step in the adaptive immune response, as it defends the body following the initial interaction between T lymphocytes and APCs16. Recently, various approaches of synthetic APCs have been developed to induce ex vivo and in vivo expansion of T cells17’19, using advanced biomaterials that replicate specific bioactive signals of the APC surface in both two-dimensional (2D)20, 21and three-dimensional (3D)22’24microenvironment. While current ex vivo T cell stimulation platforms are useful for efficiently enriching and activating antigenspecific T cells, the conditions remain to be optimized. For example, the dominant method for the activation of the T cells is the use of anti-CD3 / CD28 antibody-coated paramagnetic beads such as Dynabeads (Gibco)25. However, T cells activated by Dynabeads generally result in suboptimal cell expansion rates, fewer CD8+cy totoxic T cells, and loss of sternness due to the intrinsic differences between Dynabeads and APCs26. Particularly, Dynabeads made of polystyrene are stiff (20-40 MPa)27and have very distinct mechanical properties compared to APCs that are much softer and display viscoelastic behavior28, potentially compromising the engagement and activation level of T cell receptors (TCRs)29 30.

[0010] Viscoelasticity of the extracellular matrix (ECM) and surrounding cells plays a significant role in shaping cellular behavior and function31. Viscoelasticity reflects the ability of a substance to resist deformation and return to its original shape after being subjected to stress. Many cell types have been shown to respond to alterations in the viscoelastic properties of their environment32. However, whether the viscoelastic property of synthetic APCs regulates T cell activation and expansion remains unexplored33.

[0011] There is a need in the art for methods and materials useful to facilitate TCR activation and T cell differentiation, for example in CAR-T cell expansion methodologies.

[0012] SUMMARY OF THE INVENTION

[0013] In order to determine and characterize the viscoelastic material properties of synthetic antigen presenting cells and the effects of these properties on T cell activation, we developed a methodology that can generate novel synthetic activating cells having various viscoelastic properties (“SynVACs”). As shown by the data presented below, we discovered that the SynVACs disclosed herein have surprisingly robust effects on T cell expansion, and are therefore useful to augment therapeutic regimens such as those that utilize activated T cells to target lymphomas and solid tumors.

[0014] As disclosed herein, we further developed a scalable technology platform that can fabricate viscoelastic alginate microspheres having well-defined viscoelasticity7, and then observed the effects of synthetic activating cells having different viscoelastic properties on T cell activation and expansion. As shown in illustrative working embodiments of the invention, the SynVACs disclosed herein are observed produce robust effects on T cell activation, T memory stem cell formation, and chimeric antigen receptor (CAR) introduction into T cells. As discussed below, the material properties of the SynVACs disclosed herein allow them to generate CAR-T cells having superior functional properties as compared to CAR-T cells produced with similar conventional materials that are used to stimulate T cells such as Dynabeads (e.g., the SynVACs disclosed herein exhibit significantly higher efficiencies for use with T cells in CAR-T therapies than conventional materials).

[0015] Embodiments of the invention disclosed herein include compositions comprising synthetic viscoelastic activating cells (SynVACs) that are useful for T cell engineering, including T cell activation, expansion, and differentiation. As noted above, T cell-based therapies hold promise as a treatment that utilizes a patient's T cells to fight diseases, particularly cancer. In this context, embodiments of the invention include using the synthetic viscoelastic activating cells disclosed herein in methods designed to modify T cells to better target cancer cells and / or genetically modify them to express a chimeric antigen receptor (CAR) that recognizes and attacks cancer cells.

[0016] As discussed in detail below, in certain methods of the invention, SynVACs are prepared using a droplet formation microfluidic device with two phases, an aqueous solution phase, and an oil phase. In illustrative working embodiments of the invention designed to create structurally homogeneous SynVACs in the size range of 5-10 pm by acidic dissociation of calcium-EDTA complex, we use a premixed solution of alginate and calcium-EDTA as the aqueous solution phase. In these methods, we utilized alginate polymers with different molecular weights (LVLG. 70 KD MW, and MVG, 180 KD MW) and controlled the viscoelasticity of the polymer matrix by modulating the composition ratios of the LVLG and MVG alginate. We used different concentrations of calcium-EDTA to independently alter the stiffness of the SynVACs, enabling us to obtain SynVACs with different viscoelasticity and stiffness that possess mechanical properties similar to cellular APCs such as dendritic cells. Furthermore, we precisely control the ligand density on SynVACs based on advanced bioorthogonal chemistry. The SynVACs made by these methods more closely mimic cellular APCs as compared to Dynabeads, in terms of both mechanical properties and density of activation signals.

[0017] Embodiments of the invention also include methods of using the SynVACs disclosed herein, for example in methods designed to stimulate T cells, particularly peripheral blood mononuclear cell (PBMC)-derived T cells. Typically such methods employ steps that use SynVACs similarly to how conventional anti-CD3 / CD28 Dynabeads are utilized. Illustrative methods of the invention include those that that involve collecting PBMCs from a healthy donor and stimulating the collected T cells with SynVACs (or Dynabeads). In these methods, the T cells can be further supplemented with growth factors such as human IL-2 to support T cell expansion. Flow cytometry analysis can be used to compare the T cell activation, exhaustion, and memory’ phenotype between different artificial antigen presenting cells such as those disclosed herein and conventional materials such as Dynabeads.

[0018] As discussed below; the SynVACs disclosed herein are found to be superior to Dynabeads in promoting T cell activation, as evidenced by a higher CD8 / CD4 ratio, higher T memory' stem cell level, lower exhaustion phenotype, and greater expansion fold. The stimulation effect of SynVACs in comparison to commercial anti- CD3 / CD28 Dynabeads on primary mouse T cells shows consistent results. These characteristics indicate that the SynVACs can generate a more robust T cell response with longer therapeutic effects. Therefore, the present invention provides novel methods of stimulating T cells that have a variety of therapeutic applications, particularly in the field of immunotherapy for cancer or other immune-related diseases.

[0019] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1. Development of SynVACs to mimic the viscoelastic properties of native APCs using a microfluidic device, (a) SynVACs employ a biomaterial-based approach, utilizing ionically crosslinked alginate networks to create a tunable viscoelastic system that mimics the mechanical properties of native APCs. Scale bar, 15 pm. (b) SEM images of the microfluidic device at the crosslinking area. Scale bar, 100 pm. (c) SynVACs are fabricated based on a pH-induced internal gelation method. Scale bar, 50 pm. (d) Representative images of antibody-coated elastic beads, and SynVACs compared to Dynabeads. Scale bar, 15 pm. (e) The size of microbeads is controlled by using a microfluidic device with various channel widths (n = 5). (I) The size distribution of elastic beads and SynVACs. (g) The size of microbeads is controlled by using a microfluidic device with various flow rates (n = 5). (h) Quantification of cell viability (determined by a LIVE / DEAD staining kit) at day 14 by co-culturing elastic beads and SynVACs with Jurkat T cells (n = 3). (i) Production and harvest rate of SynVACs per microfluidic device (n = 3). (j) Stability of GFP- labeled SynVACs over time in RPMI media with 10% FBS (n = 3). (k) The strategy for removing SynVACs from T cells after co-culture involved a single centrifugation step (600 g, 5 minutes) (left). Quantitative of removal efficiency (right) (n = 5). In h- k. data represent mean ± standard deviation (s.d.). In e. g, i. k, significance was determined by a two-tailed, unpaired t-test (ns: not significant). In h, significance was determined by a one-way ANOVA and Tukey’s multiple comparison test (ns: not significant).

[0022] Figure 2. Characterization of SynVACs in terms of stiffness, viscoelasticity, and ligand density, (a) An alginate gel can be ionically or covalently crosslinked to provide viscoelastic or elastic properties, respectively. The molecular weight (MW) of the alginate polymer can control the viscoelasticity of crosslinked alginate networks. As the MW increases, a dense network with high physical entanglement and overlap of the polymer chain forms, resulting in an alginate gel with high stiffness and low viscoelasticity, and vice versa. The covalently crosslinked network is elastic since the covalent crosslinks preserve the memory of the initial state. The SynVACs can be transformed into elastic beads by covalent crosslinking and subsequent Ca2+ removal, (b) Quantitative assessment of the compressive modulus of SynVACs, elastic beads, and human APCs conducted using AFM indentation technique, (n = 12). (c) Quantification of the time scale at which an initially applied stress is relaxed to half its original value (n = 7). The stress relaxation time reflects the viscoelastic property of the gels, (d) Shear storage and shear loss modulus as a function of frequency for ionically and covalently crosslinked gels, (e) Schematic of antibody conjugation on SynVACs and elastic beads using the TCO- tetrazine ligation method, (f) Representative flow cytometry histograms showing the fluorescence intensity of SynVACs and elastic beads expressing various levels of anti-CD3 densities (n = 3). (g) Effect of antibody density on human T cell activation by the analysis of an early T cell activation marker CD69. SynVACs coated with different amounts of antibodies (anti-CD3 / anti-CD28 with a ratio of 1: 1) were cocultured with Jurkat cells (human T lymphocyte cell line) for 18 hours, and the percentage of CD69+ cells was analyzed by flow cytometry (n = 5). (h) Confocal microscopy images of individually labeled SynVAC, displaying a distinct fluorescent signal approximately 100 nanometers thick around the equatorial section, indicative of surface-conjugated antibodies. Scale bar. 2 pm. In b and c, data represent mean ± s.d. In b and c, significance was determined by one-way ANOVA with Tukey’s multiple comparison test (ns: not significant). In g, significance was determined by a two-tailed, unpaired t-test (ns: not significant).

[0023] Figure 3. Polyclonal expansion of primary human T cells by SynVACs results in a higher T memory stem cell population, (a) Experimental timeline of human primary T cells activated by Dynabeads compared to SynVACs. (b) SEM images of interactions between T cells and Dynabeads at day 1 (left). SEM images of interactions between T cells and SynVACs at day 1 (right). Scale bar, 2 gm. (c) Immunofluorescence staining of CD3E, beta-actin, and nuclei (DAPI) in primary7human T cells after 3 days of culture with Dynabeads. elastic beads, and SynVACs. Scale bar, 5 pm. (d) Quantification of CD3s cluster number in primary human T cells after 3 days of culture with Dynabeads, elastic beads, and SynVACs. Scale bar: 5 pm. (e) Quantification of CD3s cluster size in primary human T cells, (n = 50). (f) Fold expansion of human T cells, (n = 3). (g) PD-1+and TIM3+double-positive T cells of expanded PBMCs being cultured with Dynabeads, elastic beads (El, E2), and SynVACs (VI, V2) respectively, at day 10, and day 14, as determined by flow cytometry analysis, (n = 3). (h-k) Flow cytometry analysis of the total number of CD8+T cells (h), CD8+CCR7+CD45RO’ CD95hiTMSCs (i), CD4+T cells (j), and CD4+CD25+FOXP3+Treg cells (k) in expanded T cells being cultured with Dynabeads, elastic beads, and SynVACs, respectively, at day 10 and day 14. (n = 3). The 'Bare beads' group was treated with viscoelastic microbeads lacking antibody conjugation, all within an IL-2 enriched medium. In contrast, the other control group was comprised solely of T cells, which were similarly supplemented with IL-2, but without the inclusion of any beads. In d-k, data represent mean ± s.d. Significance was determined by one-way ANOVA and Tukey’s multiple comparison test (ns: not significant).

[0024] Figure 4. SynVACs enhance the transduction efficiency and tumor killing capacity of CAR-T cells, (a) CAR expression levels in CAR19-T cells on day 6 postactivation by Dynabeads. elastic beads, and SynVACs. (b) Comparison of CAR transduction efficiency between Dynabeads and SynVACs (n = 5). (c) Evaluation of in vitro tumor-killing capability of MCAR-T cells against OVCAR3 and OVCAR8 tumor cells, and CAR19-T cells against Nalm6 and Raji tumor cells. CAR-T cells were expanded for 13 days using Dynabeads or SynVACs and then co-cultured at various effector-to-target cell ratios (n = 3). (d) IFN-y secretion was measured via an ELISA assay 24 hours post-culture with tumor cells (n = 3). (e) Phenotypic analysis of tumor-killing efficiency in CAR-T cells 24 hours post-co-culture with tumor cells. (f) Quantitative analysis of flow cytometry results (n = 3). The percentage is determined by the isoty pe antibody staining. In b, d, and f, data represent mean ± s.d. In b and f, Significance was determined by a two-tailed, unpaired t-test (ns: not significant). In d, Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test (ns: not significant).

[0025] Figure 5. Single-cell RNA sequencing analysis reveals distinct activation patterns and gene expression profiles in CAR-T cells activated by SynVACs and Dynabeads. (a) Experimental workflow showing how and when CAR-T cells were collected for single-cell RNA sequencing, (b) UMAP plot showing the distribution of CAR-T cells activated by Dynabeads or SynVACs with 11 distinct clusters identified at day 14. (c) Pie Chart of the 11 distinct clusters in SynVACs group demonstrating more CD8+TMSCs (1), less CD4+Th 17 cells (9), less CD8+terminally differentiated effector memory T cells (4), and less Treg cells (10) compared with the Dynabeads group, (d) Gene expression signatures and specific markers used for identifying 11 distinct clusters, (e) Individual cells in the UMAP embedding colored by expression of CD8A, CI)4. CCR7. and IFNG. (f) Violin plots of overall expressed genes reveal distinct expressions of CCR7 and IFNG genes demonstrating enhanced sternness and tumor killing efficiency of SynV AC-activated CAR-T cells. In f, significance was determined by a z-test.

[0026] Figure 6. In vivo efficacy of SynVAC-activated CAR19 T cells in a human lymphoma Raji xenograft mouse model, (a) Experimental yvorkflow showing SynVAC-activated CAR19-T cell generation and therapy on a human lymphoma Raji xenograft mouse model, where 1 x 106Raji cells were intravenously (i.v.) injected into NSG mice on day 0 and 3 x 106CAR19-T cells were i.v. injected on day 4. Bioluminescence imaging (BLI) was used to monitor tumor growth, (b) Bioluminescent imaging of NSG mice inoculated with luciferase Raji cells, and then either mock treated (Vehicle) or treated with CAR19-T cells activated by SynVACs or Dynabeads at various time points (n = 5). (c) Quantification of bioluminescent signal (n = 5). (d) CD3+, CD45+double positive cells in different organs at day 40 post-injection of CAR-T cells activated by Dynabeads / SynVACs, as determined by FACs analysis, (e) Characterization of CD4 / 8 ratio, CAR expression, and CD62L expression in blood, (f) Quantification of the flow cytometry results in e (n = 5). In f, data represent mean ± s.d. Significance was determined by a two-tailed, unpaired t- test.

[0027] Figure 7. In vivo efficacy of SynVAC-activated MCAR-T cells in a human ovarian solid tumor xenograft mouse model, (a) Experimental workflow illustrating SynVAC-activated MCAR-T cell generation and therapy in a human ovarian solid tumor xenograft mouse model, where 1 x 1060VCAR8 cells were i.p. (intraperitoneal) injected into NSG mice on day 0 and 3 x 106MCAR-T cells were i.v. injected on day 4. Bioluminescence imaging (BLI) is utilized to monitor tumor growth, (b) Bioluminescent imaging of NSG mice inoculated with luciferized 0VCAR8 cells, and then either mock treated (Vehicle) or treated with MCAR-T cells activated by SynVACs or Dynabeads at various time points after treatment, (c) Quantification of bioluminescent signal, (d) Bioluminescent imaging of ovarian cancer metastases in various organs at day 40. (e) Quantification of bioluminescent signal in different organs (n = 8). (f) Phenotype and CAR expression level of MCAR-T cells collected from peritoneal ascites of the experimental mice, as determined by flow cytometry, (g) Quantification of the flow cytometry data (n = 8). In e and g, data represent mean ± s.d. In e, significance was determined by a one-way ANOVA and Tukey’s multiple comparison test (ns: not significant). In g. significance was determined by a two- tailed, unpaired t-test.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0030] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation or by an Arabic numeral, the full citation of which is found preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.

[0031] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology', cell biology and recombinant DNA, which are within the skill of the art. See. e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory' Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology' (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane. eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)).

[0032] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0033] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. ■‘Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0034] All numerical designations, e.g., pH. temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0035] T cell activation is a critical first step in the adaptive immune response, as it defends the body following the initial interaction between T lymphocytes and APCs16. Recently, various approaches of synthetic APCs have been developed to induce ex vivo and in vivo expansion of T cells17 19, using advanced biomaterials that replicate specific bioactive signals of the APC surface in both two-dimensional (2D)20’21and three-dimensional (3D)22'24microenvironment. While current ex vivo T cell stimulation platforms are useful for efficiently enriching and activating antigenspecific T cells, the conditions remain to be optimized. For example, the dominant method for the activation of the T cells is the use of anti-CD3 / CD28 antibody-coated paramagnetic beads such as Dynabeads (Gibco)25. However, T cells activated by Dynabeads generally result in suboptimal cell expansion rates, fewer CD8+cytotoxic T cells, and loss of sternness due to the intrinsic differences between Dynabeads and APCs26. Particularly, Dynabeads made of polystyrene are stiff (20-40 MPa)27and have very distinct mechanical properties compared to APCs that are much softer and display viscoelastic behavior28, potentially compromising the engagement and activation level of T cell receptors (TCRs)29, 30. T cell sternness, characterized by the capacity to self-renew and differentiate into multiple T cell subsets, is a vital feature for sustaining a long-lasting and effective immune response against cancer9. Particularly, T memory stem cells (TMSCs), a subset of memory T cells with stem-like properties, are becoming increasingly recognized for their critical role in sustaining a durable and effective immune response after adoptive transfer10'12. TMSCs possess both a self-renewal ability and multipotent capacity to differentiate into various antigen-specific T cell subsets, rendering them exceptionally potent in generating a robust and persistent immune response13. Although cytokines and small molecules have been explored to enhance the generation and maintenance of TMSCs14,the results may vary due to the lack of control of other niche factors, such as the mechanical properties of synthetic antigen-presenting cells (APCs) or matrices.

[0036] Viscoelasticity of the extracellular matrix (ECM) and surrounding cells plays a significant role in shaping cellular behavior and function31. Viscoelasticity reflects the ability of a substance to resist deformation and return to its original shape after being subjected to stress. Many cell types have been shown to respond to alterations in the viscoelastic properties of their environment32. However, whether the viscoelastic property of synthetic APCs regulates T cell activation and expansion remains unexplored33. We postulated that the stiffness and viscoelasticity of APCs play a crucial role in regulating TCR activation and T cell differentiation. To demonstrate this, we then developed a scalable technology platform to produce synthetic viscoelastic activating cells (SynVACs) that mimic the stiffness (kPa) and viscoelasticity of native APCs, and further demonstrated the robust effects of SynVACs on T cell expansion and as a potential therapy of lymphoma and solid tumors, in comparison with purely elastic beads and the Dynabeads - a clinically used product as a benchmark.

[0037] The invention disclosed herein has a number of embodiments. For example, embodiments of the invention include compositions of matter comprising alginate microparticles designed to form synthetic activating cells having viscoelastic properties ('‘SynVACs’’). Typically in these compositions, the alginate microparticles comprise alginate polymers having molecular weights from 35 KD to 600 KD. In certain of these embodiments, the alginate polymers have molecular weights less than 500 KD, less than 400 KD, less than 300 KD. less than 200 KD, or less than 100 KD. Typically these compositions further comprise an agent that ionically crosslinks the alginate polymers such as a calcium-EDTA complex. Optionally, the agent that ionically crosslinks the alginate polymers is present in concentration from lOmM to 100 mM (e.g., 15 nM-50mM as shown in Table 1). In certain embodiments of the invention, the alginate microparticles are designed to exhibit a diameter from 5-20 micrometers (e.g., from 8-10 micrometers or 7-9 micrometers).

[0038] In typical embodiments of the invention, the alginate microparticles further exhibit certain material properties that facilitate their ability to stimulate T lymphocytes For example, in certain embodiments, the alginate microparticles exhibit a stiffness from 1 Kpa to 30 Kpa (e.g., from 2 Kpa to 25 Kpa as shown in Table 1) under physiological conditions. In some embodiments of the invention, the alginate microparticles exhibit a stress relaxation time (tl / 2 (s)) from 5 seconds to 1000 seconds (e.g., approximately 8-12 tl / 2 (s)) under physiological conditions. In some embodiments of the invention, the alginate microparticles exhibit a loss modulus of 200 Pa - 6000 Pa at 1-10% strain (e.g., a loss modulus of 300-800 Pa at 1-10% strain as shown in Figure 2diii) under physiological conditions. Typically, the alginate microparticles are coupled to one or more polypeptide ligands such as antibodies. In illustrative embodiments of the invention, the one or more polypeptide ligands comprise an antibody that binds CD28 and / or an antibody that binds CD3. In some embodiments of the invention, the alginate microparticles do not comprise a polypeptide ligand that functions in cell-cell or cell-extracellular matrix (ECM) adhesion (e.g., an integrity). In illustrative working embodiments of the invention, the alginate microparticles were shown to comprise from IO45to IO63polypeptide ligands / bead. Embodiments of the invention include methods of making alginate microparticles having a selected viscoelasticity and / or stiffness profiles. Typically these methods comprise the steps of: selecting amounts of alginate polymers having a selected molecular weights; disposing the alginate polymers in an aqueous solution; disposing the aqueous solution in a microfluidic device selected to utilize aqueous solution phases and oil phases, and then forming the alginate microparticles via droplet formation using pH-induced internal gelation. Typically in these methods, the aqueous solution comprises an ionic crosslinking agent (e.g., a calcium-EDTA complex) that ionically crosslinks the alginate polymers. As discussed below, typically these methods are selected to form alginate microparticles having selected material properties such as alginate microparticles that: exhibit a diameter from 5-20 micrometers; exhibit a stiffness from 1 Kpa to 30 Kpa under physiological conditions; exhibit a stress relaxation time (tl / 2 (s)) from 5 seconds to 1000 seconds under physiological conditions; and / or exhibit a loss modulus of 200 Pa - 6000 Pa at 1-10% strain under physiological conditions. Embodiments of the invention also comprise coupling one or more polypeptide ligands to a surface of the microparticles, for example methods where polypeptide ligands are coupled to a surface of the microparticles using a Tetrazine-TCO click reaction.

[0039] Embodiments of the invention include methods of using the SynVACs disclosed herein to activate or stimulate T cells. Embodiments of the invention include methods of modulating an activity of T cells (e.g., stimulating T cell growth or differentiation, inducing T cell expansion and the like), the method comprising combining the T cells with an alginate microparticle SynVACs disclosed herein such that an activity of the T cell is modulated. Typically in these methods, the one or more polypeptide ligands bound to the surface the microparticles comprise an antibody that binds CD28 and / or an antibody that binds CD3; and do not comprise a polypeptide ligand that functions in cell-cell or cell-extracellular matrix (ECM) adhesion (e.g., an integrin). In certain embodiments, T cell is selected to be one expressing CD8. In certain embodiments of the invention, the T cell is selected to be one that comprises a chimeric antigen receptor (CAR). In some embodiments of the invention, the T cell is obtained from a patient diagnosed wi th a malignancy.

[0040] Further aspects and embodiments of the invention are disclosed in the Examples below.

[0041] EXAMPLES

[0042] EXAMPLE 1: SYNTHETIC VISCOELASTIC ACTIVATING CELLS (SYNVACS) FOR T CELL ENGINEERING AND CANCER THERAPY

[0043] Fabrication of viscoelastic microbeads by using a high-throughput microfluidic device

[0044] Based on the current limitations of existing platforms, our goal was to develop cutting-edge technology to enhance the sternness, CAR transduction rate, tumor-killing efficiency, and in vivo persistence of CAR-T cells for cancer immunotherapy. Therefore, we designed SynVACs as engineering APCs with finetuned mechanical properties (viscoelasticity and stiffness) and surface ligands / antibodies to achieve the desired T cell activation (Figure la). Alginate is an inert material with excellent biocompatibility, tunable mechanical properties, and well-controlled surface chemistry for molecular conjugation, which makes it an ideal candidate for the material base of the platform34-35. We fabricated alginate-based SynVACs using a high-throughput microfluidic device via a pH-induced internal gelation method. The SEM image reveals the channel design of the microfluidic device, specifically the crosslinking area for microbead formation (Figure lb). At the T-junction, an alginate solution with pH-responsive calcium meets an acidic oil phase to induce particle formation and ionic crosslinking. Data in Figure 1c demonstrates the formation of alginate beads.

[0045] In developing the invention, we optimized device and fabrication parameters to achieve monodispersed elastic or viscoelastic microparticles with defined sizes. We made viscoelastic alginate beads using low molecular weight (MW) of alginate (75 kDa) and calcium-based ionic crosslinking. In this work, we utilized clinical-grade sodium alginate. Alginate is widely acknowledged as a non-antigenic material, showing excellent biocompatibility, particularly in vitro, where it finds extensive utilization. Additionally, our previous studies have demonstrated no obvious antigenicity7when alginate is used in form of microparticles or macroscopic hydrogels in vivo™’19In its unmodified state, alginate demonstrates little to none cell binding, offering an inert background conducive to the incorporation of precisely defined biological signals. The maximum stiffness achievable by alginate gels may be below 300 kPa36, which is sufficient to cover the dynamic range of native APCs. As disclosed herein, we engineered alginate beads with the mechanical properties mimicking APCs (kPa level). Dynabeads made of polystyrene have a stiffness of up to 20-40 MPa27, 37Since Dynabeads have been optimized with CD3 / CD28 antibody coating for T cell activation and expansion in clinical settings, we use Dynabeads as a benchmark for functional comparison of expanded T cells. To fabricate elastic beads, we utilized higher MW (120 kDa) alginate to first generate ionically crosslinked microbeads, then convert them into covalently crosslinked microbeads via carbodiimide-based chemistry. Figure Id displays the resultant viscoelastic beads and elastic beads, showing transparency compared to rigid polystyrene Dynabeads. The diameter of the microbeads can be adjusted by modifying the channel width of the microfluidic device (Figure le). Since the use of alginate with varying molecular weights could lead to changes in the solution's viscosity, subsequently causing differences in the bead size produced, we used cellulose to counterbalance potential viscosity changes. Carboxy methyl cellulose (CMC) can be easily washed out of microbeads aft er wards'3 39without interferences with mechanical property and antibody modification process. As disclosed herein, we made viscoelastic and elastic beads with diameters ranging from 7-9 micrometers (Figure If), mirroring the size of dendritic cells. Furthermore, the bead size can be fine-tuned within a narrow range by adjusting the oil phase flow' rate within the channel (Figure 1g). Our experiments indicate that neither viscoelastic nor elastic beads affect cell viability when cocultured with human Jurkat T cells, suggesting that the fabricated beads are biocompatible with immune cells (Figure Ih). A single microfluidic chip can produce approximately 13 million microbeads within an hour, and around 11 million beads can be collected, with losses potentially due to multiple centrifugation steps during the collection process (Figure li). This process can be easily scaled up with a parallel microfluidic system for the high-throughput production of viscoelastic beads, which is critical for their prospective clinical application.

[0046] To assess the long-term stability of beads in culture, we conjugated green fluorescence protein (GFP) onto SynVACs during fabrication and maintained the microbeads in RPMI culture media (with 10% fetal bovine serum) or HEPES buffer at 4 "Cfor 15 days, the typical period for human CAR-T cell expansion ex vivo (Figure Ij). The evaluation of the mean fluorescence intensity (MFI) revealed no significant changes during this period, indicating that SynVACs were stable in solution. To remove these alginate microbeads from the culture, the beads can be quickly dissolved within a few minutes by introducing chelating agents like EDTA or citrate (Figure Ij). As an alternative approach, SynVACs can be readily separated from cells by using a physical centrifugation method, resulting in a removal rate of alginate beads (lower density than cells) greater than 97% (Figure Ik). A higher removal rate can be achieved through multiple centrifugation processes. Consistent with the stability data, the amount of residual alginate in the supernatant of SynVAC and T cell co-culture was less than 50 ng / ml. This low level of alginate had no detrimental effects on T cells in our experiments, similar to previous in vitro40and vivo19studies.

[0047] Modulation and characterization of the mechanical properties of SynVACs and elastic beads

[0048] In this work, four types of alginate microbeads with defined viscoelasticity and stiffness were fabricated, including: (1) VI: low stiffness viscoelastic beads (2) V2: high stiffness viscoelastic beads (3) El : low stiffness elastic beads and (4) E2: high stiffness elastic beads. The formulation and parameters for each type of microbead are shown in Table 1. For viscoelastic beads in the SynVAC group (VI and V2), we utilized lower molecular weight alginate polymer (75 kDa) to achieve fast stress relaxation (high viscoelasticity), and varied calcium concentration (15 mM and 50 mM) to ionically crosslink alginate with low (15 kPa) and high (25 kPa) stiffness, while maintaining a half-stress relaxation time around 10 seconds (Figure 2a-c). A calcium concentration between 10 mM and 50 mM induced a significant increase in alginate gel stiffness but had a negligible effect on the relaxation time. To fabricate beads with elastic properties (El and E2), we generated microbeads with higher molecular weight (120 kDa) alginate polymer using the same method, crosslinked them further with varying concentrations of adipic acid dihydrazide (AAD), and subsequently removed calcium-based crosslinking with sodium citrate. This process of converting viscoelastic crosslinks to elastic covalent bonds enables microbeads with tunable stiffness and slow stress relaxation (Figure 2a-c). With this slow stress relaxation and covalent crosslinking, stiffness is the dominant mechanical property7, and modulation of the stiffness with AAD (betw een 5 and 25 mM) had no significant effect on the viscoelastic property (half-stress relaxation time of around 1000 seconds). The stiffness of SynVACs, elastic beads, and native APCs was also directly measured using the atomic force microscope (AFM) indentation method, showing consistent results as 2D gels of the same formulation (Figure 2b). Additionally, the variation of the stiffness of the fabricated microbeads was similar to 2D gel, demonstrating the stable fabrication process. Moreover, the SynVACs and elastic beads utilized in the AFM assessments were pre-conjugated with antibodies. This ensures that the measurement results reflect the properties of the actual microbeads employed in T cell activation experiments. Among all the groups fabricated, the low stiffness, high viscoelastic properties of VI (15 kPa, T1 / 2 10 seconds) possess properties most similar to that of mouse dendritic cells with a stiffness of 11 kPa and stress relaxation time of 15 seconds41. In addition, our side-by-side characterization of stress relaxation of SynVAC beads and the human native APCs (monocytes) by using AFM showed similar results, while elastic beads did not show any stress relaxation.

[0049] Further rheological tests were performed to evaluate the viscoelasticity of VI and El beads. Frequency sweep analysis demonstrated that the storage modulus of viscoelastic gels and elastic gels were similar; however, the loss modulus of viscoelastic gels was 50 times higher than that of the elastic gel. Shear-thinning of viscoelastic gels was 10 times more than the elastic gels. Strain sweep tests also revealed that the viscoelastic gels exhibited a >20-fold higher loss modulus compared to the elastic gels. Consistently, the frequency-dependent and strain-dependent loss factor (tan 5), which represents the ratio of the energy lost to the energy stored in a material during the deformation, revealed higher viscoelasticity for the viscoelastic gels than elastic gels (Figure 2d).

[0050] Surface modification of SynVACs and elastic beads with T cell activation signals

[0051] To provide T cell activation signals, SynVACs and elastic beads were modified by conjugating signaling molecules onto these alginate microbeads based on bioorthogonal chemistry for (1) polyclonal T cell activation using activating antibodies (anti-CD3 and anti-CD28), and (2) antigen-specific CAR-T cell enrichment using mesothelin protein and anti-CD28 (Figure 2e). Tetrazine- Trans-Cyclooctene (TCO) clicks reaction occurs rapidly and selectively, resulting in a high conjugation rate and stable covalent linkage between the antibodies and SynVACs42. Additionally, employing Tetrazine-TCO click chemistry with a short PEG linker for antibody conjugation to SynVACs ensures that the ligand's functionality' is directly' influenced by the underlying material properties.

[0052] To evaluate the specific ligand density in different formulations quantitatively, we constructed a series of stable SynVACs and elastic beads that differ only in their amount of FITC-labeled anti-CD3 conjugation as we varied the amount of the tetrazine-antibodies utilized in the formulation. The different level of conjugated antibody density was first visualized by immunofluorescent images. To quantify the number of antibodies on each bead, we used flow cytometry analysis based on the calibration beads with known antibody densities (Figure 2f). The antibody density or in other means the ligand spacing has been proven to be critical for T cell activation as it directly affects the interactions between T cell receptors (TCRs) and their corresponding ligands on APCs43, 44Our data demonstrated that an antibody density higher than IO5 9on SynVACs or elastic beads ensures efficient activation of mouse primary T cells and human Jurkat cells (Figure 2g), as evidenced by the flow cytometry analysis of CD69. an early activation marker. This result aligns with previously published data indicating that a ligand spacing of less than 50 nm greatly enhances T cell activation on 2D surface45. We also included Dynabeads as a direct benchmark in our experiments. We estimated the antibody density on Dynabeads using the same method. Our findings indicate the presence of two distinct antibody types on the Dynabeads' surface, with a molar ratio close to 3: 1, and the total antibody count on the Dynabeads' surface is approximately 278,958 ± 2,790. With a diameter of 4.5 micrometers for the Dynabeads, the antibody spacing is calculated to be around 15.9 ± 0.1 nm.. similar to our beads (Table 1). To further substantiate our results, we assessed early T cell activation using SynVACs (VI). elastic beads (El), and Dynabeads in parallel experiments with Jurkat NFAT-zsGreen reporter cells. The activation levels recorded at the 18-hour interval are in agreement with our flow cytometry analysis, reinforcing the validity of our approach and reflecting consistency with previously published data24. Therefore, we chose a ligand density of 105 9for all bead formulations in the subsequent studies. It is noteworthy that our initial data showed the 2D gel system led to significantly lower T cell activation rates. As a result, we focused on the bead-based system for suspension culture of T cells, a decision that is consistent with previous studies indicating less than optimal T cell expansion with 2D gel systems46.

[0053] To further characterize the spatial distribution of antibody conjugation, we performed fluorescence staining and confocal microscopy to examine the conjugated antibodies in individual microbeads. This analysis revealed a distinct ~100-nanometer fluorescent layer on the bead surface. Additionally, we utilized X-ray Photoelectron Spectroscopy (XPS) to analyze the bead surface composition. XPS results showed nitrogen presence, confirming antibody surface modification. Quantitatively, an average of 5.3% of alginate on the elastic bead surface and 6.4% on the SynVACs surface were modified, as determined by the amino acid to alginate monomer ratio.

[0054] Polyclonal expansion of primary human and mouse T cells

[0055] We then employed SynVACs, elastic beads, and Dynabeads (as a commercial benchmark) for the polyclonal expansion of primary human T cells in peripheral blood mononuclear cells (PBMCs) (Figure 3a). Four types of SynVACs and elastic beads with distinct mechanical properties were fabricated by conjugating anti-CD3 and anti-CD28 in a 1: 1 molar ratio, as detailed in Table 1. Dynabeads were used as a gold standard control. Physical interactions between the beads and T cells were examined by using scanning electron microscopy (SEM) (Figure 3b). After coculturing SynVACs and human T cells for 24 hours, we observed the formation of the immunologic synapse (IS)-like structure (Figure 3b), which was confirmed by immunofluorescent staining for CD3 Epsilon at day 3 (Figure 3c) and as early as 1 hour. Through quantitative analysis, it was found that SynVACs significantly increased both the number and size of CD3 clusters in human T cells (Figure 3d-e). The relative size variations of T cells in Figures 3b and c are primarily due to the inherent growth of T cells upon activation, typically expanding to 12-15 micrometers by day 3, and potential dehydration-induced cell shrinkage during SEM sample preparation.

[0056] Following these examinations of T cell-beads interactions at early time points, we performed long-term culture for up to 2 weeks, which is in line with standard clinical protocols for CAR-T cell activation and proliferation, and investigated the impacts of SynVAC, elastic beads and Dynabeads on T cell expansion. Interestingly, SynVACs resulted in a T cell expansion rate that was more than 4-fold higher than that of the elastic beads at day 14, and 1.5-fold higher than that of Dynabeads (Figure 3f). The total T cell counts on Days 10 and 14 relative to the initial T cell seeding number (100,000) were observed. There was a statistically significant increase in the T cell expansion rate when using lower-stiffness SynVACs (VI) compared to higher- stiffness SynVACs (V2). We also noted that the proliferation activated by the elastic bead group was less pronounced compared to that by Dynabeads as Dynabeads have been optimized for antibody density and the bead size. When compared to Dynabeads, there are reports on significant or modest improvement by various artificial APC (aAPC) systems22, 4 / , 4S. It is possible that, in addition to aAPC. differences in T cell sources, activation states, and culture conditions, may account for the different effects. In addition, while rapid proliferation is often seen as a positive outcome, it is essential to consider the biological relevance and the quality of the T cell response. Regarding exhaustion markers PD-1 and LAG-3, no substantial differences were detected among the various conditions after 14 days of culture (Figure 3g). Remarkably, while Dynabeads prominently promoted CD4-biased skewing, consistent with a previous report47, all SynVACs formulations induced rapid and substantial CD8-biased skewing, with VI promoting the highest number of CD8+T cells by day 14 (Figure 3h). which may directly target and eliminate tumor cells, making them more desirable for cancer immunotherapy49, 50. On the other hand, we recognize the importance of CD4+T cells in supporting this response and sustaining immunological memory, vital for a comprehensive antitumor immune strategy51. Future studies may explore the synergistic effects of a balanced CD8+ / CD4+T cell response to enhance the efficacy and durability of antitumor immunity. Furthermore, the SynVAC formulations (VI) resulted in a 6-fold higher proportion of CD8+CCR7+CD45RO" CD95hlTMSCs compared to Dy nabeads and elastic beads (Figure 3i), based on the analysis by fluorescence-activated cell sorting (FACS). These TMSCs were also positive for other markers such as CXCR3, CD58, and CD1 la. This finding is significant as TMSCs are known to possess a remarkable capacity for self-renewal, differentiation into effector cells, and long-term persistence, w here the persistence and functional capacity of the infused T cells are crucial for therapeutic success. Conversely, SynVACs (VI) produced significantly fewer CD4+T cells by day 14 compared to Dynabeads (Figure 3j). Consequently, SynVACs (VI) yielded a significantly lower number of CD4+CD25+FOXP3+regulatory T (Treg) cells compared to Dynabeads (Figure 3k). A reduced proportion of Treg cells in the activated T cell population may result in enhanced anti-tumor immune responses, as Treg cells are known to suppress the activity of other immune cells52. In addition to Day 10 and 14, early assessments at day 7 revealed phenotypic alterations in T cells and superior initial efficacy of SynVACs over elastic beads, with an increase in TMSCs and CD8+cells, alongside a reduction in Tregs, compared to Dynabeads. These trends are consistent with the observations at days 10 and 14, although the effects are less pronounced.

[0057] Furthermore, our analysis shows that T cells retain CD25 expression when activated by SynVACs, elastic beads, and Dynabeads, affirming sustained activation over a two-week period of cell expansion. In addition, our data demonstrate no significant differences in T cell expansion, CD4 / CD8 ratio and CD3+CCR7+CD45RO" CD95hlTMSCs when using human PBMCs or PBMC-derived T cells (isolated by CD3+) as the starting cell source for T cell expansion. These results justify our choice of PBMCs as a viable starting material for CAR T cell production, aligning with established practices in both preclinical and clinical research.

[0058] Similarly, for the polyclonal activation of primary mouse T cells, we employed SynVACs (VI), elastic beads (El), and Dynabeads. We recorded timelapse movies to observe the physical interactions between T cells and SynVACs / Dynabeads. Interestingly, on day 7, cells in the Dynabeads group formed large aggregates, while SynVACs led to relatively loose, sheet-like structures. This observation suggests that SynVACs may reduce the risk of overstimulation and promote better cell-cell interactions and nutrient exchange, potentially facilitating sustained T cell activation and expansion. Additionally, clusters in the Dynabead group dissipated after day 7, whereas the majority of clusters in the SynVACs groups persisted until day 10. This phenomenon further highlights the potential advantages of SynVACs in T cell activation and expansion, as they appear to create a more conducive environment for T cell interactions and persistence. FACS data analysis showed consistent effects of SynVACs on human primary T cells with respect to higher fold expansion, no significant exhaustion. CD8-biased skewing, more CD44’ CD62L+CD95+TMSCs formation, lower Treg formation, and in SynVAC co-culture.

[0059] In addressing concerns regarding the role of calcium ions (Ca2+) from alginate gels in T cell activation and differentiation, we conducted a thorough evaluation and show that the calcium released from the alginate beads has a negligible effect on T cell activation and differentiation. First, SynVACs undergo rigorous washing and dialysis processes, and contain minimal free calcium ions. Secondly, we typically use 500,000 beads in a 24-well plate with 500 ml media to activate an equivalent number of PBMCs, and the calculations suggest a less than 0.5% change of calcium concentration in the culture media. Thirdly, we measured calcium concentration and did not find a significant increase in calcium ion concentration in the culture media. Consequently, we believe that the viscoelastic properties of the alginate, rather than calcium release, are the predominant factors influencing the observed T cell phenotypes.

[0060] In vitro CAR transduction into T cells and tumor cell killing activity

[0061] Next, we assessed the performance of SynVACs in CAR-T cell generation and in vitro tumor cell killing assays. These evaluations are critical for determining the efficiency and effectiveness of SynVACs in generating CAR-T cells with high transduction efficiency and potent tumor-killing capabilities. FACS data analysis revealed that SynV AC-activated T cells exhibited a remarkably higher CAR transduction efficiency of up to -90% at day 6, whereas Dynabeads and elastic beads achieved only 42% and 17%, respectively (Figure 4a-b), consistent with the average level of CAR transduction in previous reports (2O-32%)10’53. Our further studies with multiple donors confirm the reproducibility of the improved CAR transduction efficiency. Staining with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by flow cytometry analysis, showed that the highest proliferation rate was in T cells activated by SynVACs on day 3 post-CAR transduction, where might contribute to significantly enhanced transduction efficiency.

[0062] In vitro tumor cell killing assays demonstrated that SynV AC-activated CAR-T cells exhibited potent cytotoxic effects against various cancer cell lines, including 0VCAR3, 0VCAR8, Nalm6, and Raji cells, suggesting that SynVACs can enhance the functionality and specificity of CAR-T cells (Figure 4c). Specifically, the Sy nV AC -expanded CAR-T cells demonstrate a more pronounced reduction in live tumor cells across all effector-to-target (E:T) ratios compared to the CAR-T cells expanded by Dynabeads or elastic beads. This suggests that the SynV AC -expanded CAR-T cells possess superior cytotoxic capabilities in vitro. ELISA assays revealed that SynV AC-activated CAR-T cells exhibited the highest levels of interferon-gamma (IFN-y) secretion, while no significant differences were observed between Dynabeads and elastic bead-activated CAR-T cells (Figure 4d). This finding suggests that SynVACs enhance the functional capacity of CAR-T cells by promoting the secretion of IFN-y, a critical cytokine involved in antitumor immune responses. Consistent results were observed when employing primary mouse T cells where SynVACs VI promoted the formation of IFN-g+TNF-a+CD8+T cells at day 7. FACS analysis further demonstrated that post tumor stimulation, SynV AC -activated CAR-T cells exhibited significantly higher expression of proteins such as CD69, Perforin, and Granzyme B. compared to Dynabead-activated CAR-T cells (Figure 4e-f). CD69 is an early activation marker of T cells, while Perforin and Granzyme B are critical components of the cytotoxic granules responsible for inducing target cell apoptosis. The enhanced production of these proteins in SynV AC-activated CAR-T cells indicate a more potent cytotoxic potential and a stronger activation status compared to their Dynabead-activated counterparts. Taken together, these results reinforce the idea that modulating the mechanical properties, especially the viscoelasticity' of synthetic APCs, like SynVACs, can influence the phenoty pic and functional properties of the generated CAR-T cells, which could be strategically utilized for optimizing the CAR- T cell manufacturing process.

[0063] Antigen-specific enrichment of MCAR-T cells using mesothelin / anti-CD28 conjugated SynVACs

[0064] We also evaluated the performance of SynVACs in antigen-specific activation of T cells. This assessment is crucial for understanding how efficiently SynVACs can specifically activate T cells in response to the presence of target antigens, which is a key factor in determining the overall effectiveness of immunotherapies. We used mesothelin and anti-CD28 conjugated SynVACs to activate mesothelin-specific CAR- T (MCAR-T) cells. Mesothelin is a cell surface glycoprotein overexpressed in various solid tumors, making it an attractive target for CAR-T cell therapy54. The anti-CD28 molecule serves as a co-stimulatory signal, enhancing T cell activation and expansion. At day 5, we found that SynVACs and aAPCs (a cell line overexpressing human CD83 / CD86 / 4-1BBL co-stimulatory receptors and human mesothelin) significantly increased the proportion of MCAR-T cells from 74.3% to 95.2% and 94.4% respectively, whereas Dynabeads surprisingly led to a reduced proportion (i.e.. 74.3% to 63.0%). Further investigation revealed that, although aAPCs demonstrated equal capacity as SynVACs in enriching MCAR-T cells, they were unable to promote CD8- biased skewing like SynVACs, as evidenced by the CD4-CD8 ratio. These findings emphasize the unique advantages of SynVACs in promoting not only the expansion of MCAR-T cells but also their preferential CD8-biased skewing, which is crucial for enhancing the cytotoxic potential of CAR-T cells in cancer immunotherapy. Moreover, antigen-specific activation using SynVACs further highlights the versatility and adaptability of the SynVAC platform for personalized immunotherapies of various cancer types.

[0065] Single cell RNA sequencing (scRNAseq) analysis of expanded T cells To further elucidate the differences in gene expression and subpopulations of the CAR-T cells being activated by SynVACs (leading formulation) and Dynabeads (as a gold standard control), respectively we conducted single cell RNA sequencing (scRNAseq) at day 14 before the expanded cells were used for in vivo studies (Figure 5a). These included 9,400 T cells activated by Dynabeads and 7,874 T cells activated by SynVACs. Bioinformatic analysis of gene expression mapped in two dimensions via uniform manifold approximation and projection (UMAP) identified 11 clusters (Figure 5b). Primarily, 7 clusters were comprised of CD8+T cells, 3 clusters were CD4+T cells, and 1 cluster consisted of double-negative T cells. Figure 5c visualizes the changes in the percentages of the 11 distinct clusters in pie charts. Each cluster’s subtype was then identified based on protein markers and gene signatures (Figure 5d)

[0066] To explore the CCR7+memory T cell subpopulations, we focused on clusters 1 and 8, which displayed a gene-expression profile of TMSCs, featuring high levels of LEF1, TCF7, CCR7, SELL and IL7R. Consistent with our findings in Figures 3-4, SynVACs increased the percentage of CD8+TMSCs from 6.8% to 14.4% (Cluster 1) and CD8+effector memory T cells from 26.2% to 45.1% (Clusters 3 and 5), but decreased the percentage of CD8+terminally differentiated effector memory T cells from 14.9% to 4.2% (Table 2). It was noted that the expression levels of CD95 and CD28 were relatively low in cluster 1 (CD8+TMSCs); if we only consider CD95+cells in this cluster, SynVAC also significantly increased this population (4.9%) in comparison to Dynabeads (2.2%). On the other hand, SynVACs decreased CD4+T helper 17 cells from 18.0% to 4.3% (cluster 9) and CD4+regulatory T cells from 9.5% to 1.0% (cluster 10), although SynVACs increased CD4+TMSCs from 2.0% to 3.5% (Table 2) Cluster 4 is characterized as terminally differentiated due to its higher expression of exhaustion markers such as TIGIT and effector genes like NK.G7, GNLY, and GZMB, coupled with lower levels of LEF1 and sternness-associated markers, aligning with the TEMRA profile. Cluster 7 is labeled as exhausted because it presents high CXCR6 expression, indicative of tissue residency, and elevated TIGIT levels, alongside a reduced expression of sternness and effector genes, consistent with an exhausted phenotype. Taken together, scRNAseq analysis provides more comprehensive information on the subpopulations of expanded T cells and confirms the beneficial effects of SynVACs on the increase of T cell sternness and CD8+subpopulations.

[0067] Another noteworthy observation was that the SynV AC-activated CAR-T cells exhibited higher expression levels of CD8A, CCR7, and IFNG, compared to Dynabead-activated cells (Figure 5e-f). CD8A gene is associated with cytotoxic T cells, which are instrumental in eradicating cancer cells. The CCR7 gene is an important marker for stem-like T cells, and its high expression is associated with improved therapeutic potential in CAR-T cell therapy. The elevated gene expression of IFNG in SynV AC-activated CAR-T cells indicate a potentially more potent antitumor immune response, as IFNG enhances the ability of the immune system to detect and eradicate cancer cells. The enhanced gene expression of CD8A, CCR7, and IFNG in the SynVACs group suggests amplified anti-tumor responses and sustained immune protection.

[0068] Exploring the heterogeneity between SynVACs and Dynabeads populations, we conducted gene enrichment analysis using the EnrichR package, which further revealed that SynV AC-activated CAR-T cells displayed an upregulation of several critical biological processes and pathways, such as DNA metabolic processes, DNA replication, and transcription regulation. These findings suggest that SynVACs- mediated activation may promote CAR-T cell proliferation and expansion, which is essential for robust and lasting anti-tumor responses. The upregulation of DNA metabolic processes and DNA replication implies that the SynV AC-activated CAR-T cells undergo more active cell division and growth compared to those activated by Dynabeads. This increased proliferation capacity may enhance CAR transduction rates and tumor-killing efficacy observed in the SynVACs group. The RNA sequencing data provided is indeed insightful, yet it is understood that the true value lies in perturbing the identified signals to ascertain their functional roles. Moving forward, we plan to conduct targeted perturbation studies to unravel the functional implications of these genetic variations, with the goal of deepening our grasp of T cell programming and advancing the efficacy of CAR-T cell therapies.

[0069] In vivo antitumor efficacy and long-term persistence of CAR-T cells in a xenograft B cell lymphoma model

[0070] Subsequently, we assessed the in vivo antitumor efficacy of SynV AC-activated CAR19 T cells in a human lymphoma Raji xenograft mouse model (Figure 6a). This evaluation is essential to determine the therapeutic potential of SynV AC-activated CAR-T cells in a physiologically relevant setting and to provide valuable insights into their ability to target and eliminate cancer cells in a living organism. We observed that in the untreated control group, all mice succumbed to the disease by day 28 (Figure 6b). In contrast, SynV AC-activated CAR19-T cells effectively eliminated cancer cells in all of the treated mice by 28 days post-injection. The Dynabeads group, on the other hand, displayed significant cancer recurrence (Figure 6c). Additionally, survival curves were included to demonstrate the long-term outcomes following treatment. Statistical analysis revealed a significant difference in tumor burden between the Dynabeads and SynVACs treatment groups. Furthermore, on day 40, we examined the persistence of CAR19-T cells in various tissues of the mice and found a marked increase in the SynVACs group across multiple organs and tissues, such as blood, spleen, and liver (Figure 6d). Upon further investigation of the blood, we observed that the phenotype of T cells in the SynVACs group displayed significantly elevated levels of CD8-biased skewing, CAR expression, and CD62L expression (Figure 6e-f). The elevated levels of CD62L observed in CAR19-T cells activated by SynVACs indicate a greater presence of T cell subpopulations such as naive T cells, TMSCs, and central memory T cells. This observation has significant implications for the potential effectiveness and durability of CAR-T cell therapy. Additional experiments were conducted to evaluate the performance of CAR-T cells activated by elastic beads, as this group is pivotal when asserting the effects of altered viscoelasticity. The CAR- T cells activated by elastic beads demonstrated a pattern of cancer recurrence comparable to that of the Dynabeads group. These findings underscore the superior efficacy of SynV AC-activated CAR19-T cells in not only eliminating cancer cells but also maintaining long-term persistence in vivo. The enhanced persistence and phenotype characteristics of CAR19-T cells in the SynVACs group suggest robust T cell function, which may contribute to better cancer control and reduced chances of recurrence.

[0071] In vivo antitumor efficacy and long-term persistence of MCAR-T cells in a xenograft ovarian cancer model

[0072] Finally, we assessed the in vivo antitumor efficacy of SynV AC-activated MCAR-T cells in a human ovarian cancer xenograft mouse model (Figure 7a). While we have utilized lymphoma animal models, their limitations in fully representing solid tumors necessitate a further investigation of SynVACs' effectiveness in solid tumor contexts for a more comprehensive understanding of their therapeutic potential. By day 33, untreated control mice had all developed ovarian solid tumors, while CAR-T cells activated by Dynabeads only slightly decreased the tumor growth trend. In contrast, we observed that although SynVACs were unable to completely eradicate cancer cells, they significantly reduced tumor cell recurrence and even had a probability of completely preventing tumor cell recurrence in one of the eight mice (Figure 7b-c). A significant difference in tumor burden between the Dynabeads and SynVACs treatment groups was evident upon statistical analysis. Additionally, on day 40, we examined the metastasis of ovarian tumor cells in different tissues and organs of mice. Compared to the vehicle control group and the Dynabead-activated CAR-T treatment group, the SynV AC group effectively prevented tumor cell proliferation and metastasis, particularly in the lungs, bone tissue, and brain tissue (Figure 7d-e). Specifically, we have focused on the infiltration and phenotypic characterization of CAR-T cells in the peritoneal ascites of experimental mice, which also reflects their presence at the ovarian cancer tumor sites. Our findings reveal a notable increase in T cell retention in the SynVACs group, rising from 1.75% to 7.06%, in comparison to the Dynabeads group. Moreover, the proportion of CAR-T cells within the tumor notably increased from 27.3% to 61.6%. Additionally, we observed a significant rise in the percentage of CD62L+CAR-T cells within the tumor, escalating from 23.4% to 43.3% (Figure 7f-g). These findings reveal that CAR-T cells activated by SynVACs not only inhibit tumor grow th and metastasis but also promote the in vivo longevity of CAR-T cells, increase the proportion of CAR-expressing cells, and elevate the population of CD62L+T cells such as naive T cells. TMSCs and central memory T cells, especially in solid tumor environments such as ovarian cancer. In summary, SynVACs show great potential for significantly enhancing the effectiveness of CAR- T cell therapy against solid tumors, leading to better cancer control and reduced chances of recurrence.

[0073] Methods

[0074] High-throughput microfluidic platform for fabrication of alginate viscoelastic micro beads

[0075] The microfluidic device was designed utilizing AutoCAD, followed by fabrication via conventional photolithography technique. Initially, photosensitive epoxy (SU-8 2015, MicroChem) was spun onto a 4-inch silicon wafer to a thickness of 13 pm. This wafer underwent a soft bake at 95 °C for 6 minutes, and UV exposure through a chrome mask bearing the desired channel patterns. Following a 10-minute development phase in SU-8 developer (MicroChem) and a rinse with isopropyl alcohol, the master mold took shape on the wafer. Poly dimethylsiloxane (PDMS) prepolymer and curing agent were mixed, poured onto the silicon substrate, and cured at 65 °C for 3 hours to produce a PDMS slab. This slab as then subjected to punch- outs to create inlet and outlet points, a rinse with 50% ethanol to clean the channel, and an oxygen plasma treatment using Plasma Prep II (SPI Supplies) to prepare for bonding. The assembled devices were placed in a 65 °C oven for roughly 30 minutes to strengthen bonding and left at room temperature for 24 hours to ensure optimal hydrophobicity before experiments. A custom "torch-like" component was designed and 3D-printed using an Elegoo 4K printer (ELEGOO Inc) and sterilized with 70% ethanol before being fitted to the microfluidic device to act as a reservoir. This reservoir, possessing a 5 ml capacity, served as a storage area for the SynVACs produced in the oil phase, facilitating manual collection later on. During the production process, a microscope was used for real-time monitoring of the size and production rate of the alginate beads to promptly address any unexpected issues within the chip.

[0076] Fabrication of the SynVACs and elastic beads with well-defined mechanical properties

[0077] To minimize batch-to-batch variation, we utilized Novamatrix's PRONOVA sodium alginate, manufactured under Good Manufacturing Practice (GMP) guidelines and adhering to ISO standards. This particular grade has been regulated by the FDA as Generally Regarded as Safe (GRAS) for clinical use, ensuring its safety' and reliability. Alginate viscoelastic microbeads were fabricated within the microfluidic device via a pH-induced internal gelation method58, 59The core stream was loaded with a fluid comprised of 3% alginate (VLVG, MW 70 kD, NovaMatrix), 1% carboxymethyl cellulose (used to stabilize laminar flow), and Ca-EDTA in deionized (DI) water. The concentration of Ca-EDTA was adjusted to modulate the stiffness of the resultant viscoelastic beads. The sheath stream contained 1% surfactant (157 FSH, Krytox), and 0.5% acetic acid, all dissolved in fluorocarbon oil (Novec 7500, 3M). Both fluids, sterilized with a 0.22-micron filter prior to use, were fed into the microfluidic device via separate inlets, facilitated by syringe pumps (Harvard Apparatus). After the formation and temporary storage of the microbeads in the reservoir, they were collected into a new tube containing 20% perfluoro- 1 -octanol (PFO) (Sigma) and 0.2% acetic acid in fluorocarbon oil, which promoted crosslinking. Subsequently, HEPES-C buffer (20mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid, 140 rnM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, pH=7.2) was added to the collection tube. The tube was centrifuged at 1000g for 1 minute to transition the microbeads from the oil phase to the HEPES buffer. Finally, the buffer containing the microbeads was collected, rinsed twice (6000 g, 5 min) for a thorough cleaning, and stored at 4 ''C for further experimentation.

[0078] Elastic beads were fabricated by converting ionically crosslinked viscoelastic microbeads into permanently crosslinked covalent microbeads. To begin with, we first fabricated viscoelastic microbeads using fluid containing 3% alginate (MVG: VLVG = 1 :2, average MW = 120 kD) and 25 mM Ca-EDTA. The resulting microbeads were resuspended in MES-Ca buffer (100 mM 2-(N-morpholino) ethane sulfonic acid, 300 mM sodium chloride, 2 mM calcium chloride, pH=6) for 1 hour to adjust the pH to 6. Subsequently, the microbeads were gathered by centrifugation (6000 g, 5 min) and resuspended in MES-Ca buffer containing 320 mM l-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), hydroxy benzotriazole (HoBt), and adipic acid dihydrazide (AAD) for overnight incubation. The concentration of AAD and HoBt was adjusted to modulate the stiffness of the resultant elastic beads. Finally, a sodium citrate buffer (77 mM NaCl, 55 mM sodium citrate) was introduced to the elastic beads for 2 hours to chelate calcium ions and completely remove the ionic crosslink bonds. The covalent beads were then rinsed with HEPES-T buffer (HEPES buffer with 0.5% Tween20) and stored at 4 for future use.

[0079] Preparation of SynVACs and elastic beads for T cell stimulation

[0080] SynVACs with well-defined spatial organization of ligands may enhance CAR-T cell activation, resulting in improved expansion, persistence, and therapeutic efficacy60. To prepare SynVACs and elastic beads across different scenarios, trans- CycloOctyne (TCO) - Tetrazine ligation was employed to covalently conjugate antibodies / proteins onto the microbeads. Briefly, 4 million microbeads were resuspended in 800 pl MES buffer containing 200 mM EDC hydrochloride and 200 mM sulfo-NHS for an overnight reaction to activate carboxylic groups. Microbeads were then washed with HEPES-CT buffer (HEPES buffer containing 2 mM calcium chloride and 0.5% Tween20) to adjust the pH to 7. TCO-PEG6-amine (2 pmol / million beads, Click Chemistry Tools) was added to the solution and allowed to conjugate onto the microbeads overnight at room temperature. The reaction mixture was placed on a roller during this process to ensure thorough mixing. The microbeads were dialyzed against HEPES-CT buffer using a 1000 KD dialysis bag (Spectrum laboratories) to remove excess TCO (HEPES-CT buffer was replaced 2-3 times per day). Antibody-Tetrazine conjugation was performed according to the manufacturer's protocol. Anti-CD3 (Biolegend 317302) and anti-CD28 (Biolegend,302902) antibodies were mixed at a 1 : 1 ratio for polyclonal T cell activation. Mesothelin protein (Aero Biosystems, MSN-H526x) and anti-CD28 antibodies were mixed at a 1 : 1 ratio for antigen-specific CAR-T enrichment. The mixture was concentrated in 100 pl PBS buffer using an Amicon Ultra-2 spin column (Sigma Aldrich, USA). The concentrated mixture was mixed with Tetrazine-PEG5-NHS (Sigma Aldrich, USA) at a molar ratio of 1 :5 and reacted for 30 min. The antibody / protein-tetrazine complex was then desalted using a spin column and rinsed with PBS 5 times to remove unreacted Tetrazine-PEG5-NHS. The purified antibody / protein-tetrazine complex was mixed with glycerol at a 1 : 1 ratio and preserved at -20 °C. To refine the ligand density of SynVACs and elastic beads, we adjusted the dose of the antibody / protein-tetrazine for the tetrazine-TCO ligation with TCO-labeled microbeads.

[0081] Mechanical property and ligand density characterization of SynVACs

[0082] Rheological measurements were performed using an Anton Parr Rheometer. A 3% alginate solution (70 kD, VLVG) was crosslinked with various concentrations of calcium crosslinker to generate an 8 mm disk of gel, supplemented with 1% carboxymethyl cellulose. The disk had an average thickness of about 2 mm. For the formation of the elastic hydrogel, a previously established protocol for elastic beads was followed. Specifically, a 3% alginate solution (120 kD, MVG: VLVG = 1:2) was crosslinked with a certain concentration of calcium crosslinker to create an 8 mm gel disk. Gel samples were equilibrated with MES buffer (pH=6) for 1 hour, followed by immersion in an MES solution containing different concentrations of adipic acid dihydrazide (AAD) and corresponding hydroxybenzotriazole (HoBt) under constant stirring. This ensured an even distribution of AAD within the alginate matrix. The mixture was left to react at room temperature overnight, allowing the formation of an alginate gel with both ionic and covalent bonds. Subsequently, the gel samples were immersed in a sodium citrate buffer for 2 hours to chelate and remove the calcium ions from the gel. Lastly, the resulting covalent alginate gel was thoroughly rinsed with deionized water and equilibrated in HEPES buffer for 24 hours to ensure the complete removal of unreacted components and stabilization of the gel's mechanical properties.

[0083] For the rheological test, an 8-mm PP025 measuring plate was used. The viscoelastic gel sample was carefully placed onto the center of the rheometer plate with a spatula. The cantilever was lowered to the preferred gap height (1 mm was used in this study). Oscillatory strain sweeps (0.1-500%, 1 Hz), oscillatory frequency sweeps (0.1-100 Hz, 1% strain), and time sweeps (0.5% strain, 1 Hz, 2 min) were all conducted at room temperature to measure the storage (G', Pa) and loss (G", Pa) moduli. A shear ramp (0.01-100 s ') was used to examine the relationship between viscosity and shear rate. All experiments were repeated at least thrice. Rheological measurements of elastic hydrogels were performed using the same procedure.

[0084] The compressive moduli and stress relaxation properties of the viscoelastic gel were evaluated via compression tests on gel disks (8 mm in diameter, 2 mm thick, equilibrated in RPMI for 24 h) using a method from a previously published study61. The gel disks w ere compressed with a deformation rate of 1 / 120 mm / s for 30 seconds using a Chatillon TCD225 series force measurement system. The slope of the stressstrain curves (first 5-10% of strain) was used as the initial compressive modulus. Thereafter, the strain was held constant while the load was recorded over time. Stress relaxation w as calculated by measuring the time it took for the stress to decrease to half from the maximum stress. No prestress was applied to the gels for these measurements. Compression and stress relaxation measurements of elastic hydrogels were performed using the same procedure.

[0085] The average antibody density per Sy nV AC was determined by quantitative flow cytometry. Briefly, IxlO5SynVACs from each experimental group were conjugated with the appropriate antibody: Anti-CD3 FITC (Biolegend 300305). SynVACs were then washed and resuspended in HEPES-CT buffer for analysis in a Flow Cytometer (BD LSRFortessa Cell Analyzer). A standard curve was constructed using Quantum Simply Cellular anti-Mouse IgG beads (#815. Bang Laboratories) stained with Anti-CD3 FITC. For elastic beads, the number of antibodies per bead was determined using the same procedure. The average mesothelin protein density per SynVAC was determined based on a standard curve constructed using Quantum Simply Cellular anti-Mouse IgG beads (#815, Bang Laboratories) stained with Anti- mesothelin FITC.

[0086] AFM for SynVACs mechanical property and cell mechanics methods

[0087] Antigen-presenting cells (primary' monocytes) are extracted from human PBMCs using the CD 14 MicroBeads kit (Miltenyi Biotec) on the same day as the test. SynVACs, elastic beads, and APCs were placed on a JPK NanoWizard 4a BioScience AFM and indented by a Bruker SAA-SPH-1UM probe with a spring constant k = -0.25 N / m (the exact k value of each probe was determined by LDV calibration and utilized for the specific test). After the force spectroscopies were obtained, Young’s modulus was determined by fitting the data to a Hertz / Sneddon model using JPK Data Processing62. To measure viscoelasticity, the height after reaching 10 nN was maintained constant on the surface of SynVACs, elastic beads, and APCs, and the stress relaxation profile was obtained by recording the vertical deflection force over the relaxation time.

[0088] X-ray Photoelectron Spectroscopy (XPS) Analysis for SynVACs ligand density The elastic beads and SynVACs were carefully prepared and mounted for XPS measurements using an Axis Ultra DLD spectrometer (Kratos Analytical Inc.; Chestnut Ridge, NY). The analysis was conducted under ultrahigh vacuum conditions to prevent contamination and interference from atmospheric gases. We used a monochromatic Al Kot X-ray source ( = 1486.6 eV) for excitation and scanned a wide range of binding energies to capture the complete elemental profile of the bead surfaces. The core level spectra were acquired for Carbon (C Is) and Nitrogen (N Is), and the peak areas were integrated to calculate the atomic ratios. The presence of nitrogen on the surfaces was particularly indicative of antibody conjugation, given that antibodies contain nitrogen-rich amino acids. The data were analyzed and quantified to determine the percentage of antibody conjugation on the alginate monomer at bead surfaces.

[0089] Biosafety evaluation of SynVACs for CAR-T expansion

[0090] A biosafety test was conducted to ensure that alginate-based SynVACs could be effectively separated from T cells through physical centrifugation, thereby ensuring no residual SynVACs in the activated T cells prior to therapeutic applications. In detail, pre-labeled SynVACs were co-cultured with primary mouse T cells at a 1: 1 ratio for 24 hours. Post-culture, the cells were stained with Hoechst 33342 (1 : 1000, Thermo Fisher) at room temperature for 10 minutes. The co-culture mixture was then subjected to centrifugation (600g, 5 minutes) and resuspended in buffer solution. The bead-to-cell ratio before and after the washing procedure was confirmed via FACS analysis.

[0091] To ensure the absence of residual alginate monomers, which could potentially trigger innate immune responses if co-injected with CAR-T cells, the alginate residue in the supernatant was quantified via high-performance liquid chromatography (HPLC). For HPLC analysis, a carbon stationary phase (Kromasil 300-5-E18, 4.6 x 250 mm) was chosen. Elution was performed with a mobile phase consisting of 40% acetonitrile and 60% DI H2O, at a flow rate of 1 mL / min, and ultraviolet (UV) detection was set at 254 nm. The system was equilibrated with the mobile phase for 20 minutes before the first injection. A sodium alginate standard solution was prepared by dissolving 250 mg of accurately weighed VLVG in 5 ml distilled water to create a 5% stock solution. Calibration standards were prepared by diluting varying amounts of the VLVG stock solution to yield a concentration range of 0.05 to 20 pg / mL using distilled water.

[0092] T cell culture

[0093] The Human Jurkat T cell line and Jurkat NFAT-zsGreen reporter cell line were gifts from the Christopher Seet Lab at UCLA. Primary mouse T cells were isolated from the spleen of C57BL / 6 mice using a pan T cell isolation kit, with CD3+T cells procured for polyclonal activation studies. Healthy donors human PBMCs were sourced from the UCLA / CFAR Virology Core Laboratory, in compliance with federal and state regulations, with no identifying information provided. The human Burkitt's lymphoma cell line Raji, acute lymphoblastic leukemia cell line NALM6, ovarian cancer cell lines OVCAR3 and OVCAR8, chronic myelogenous leukemia cell line K.562, and embryonic kidney (HEK) 293T cells were obtained from ATCC.

[0094] Stable tumor cell lines expressing firefly luciferase and enhanced green fluorescence protein dual -reporters (FG) were generated by transducing parental tumor cell lines with lentiviral vectors encoding the target gene(s). Post lentivector transduction (72 hours), cells were flow cytometry sorted to isolate gene-engineered cells, thereby establishing stable cell lines. Four stable tumor cell lines were created for this study, including Raji-FG, NALM6-FG, OVCAR3-FG, and OVCAR8-FG cell lines. An aAPC was generated by engineering the K562 human chronic myelogenous leukemia cell line (ATCC) to overexpress human CD83 / CD86 / 4-1BBL costimulatory receptors. The aAPC-MSLN cell lines were further engineered from the parental aAPC line to overexpress human MSLN.

[0095] Jurkat cells and primary mouse T cells were cultured in ATCC modified

[0096] Roswell Park Memorial Institute (RPMI) 1640 medium enriched with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 50pM 2-mercaptoethanol, and 1 OOmg / ml normocin. The reporter Jurkat media was supplemented with an additional Ing / ml puromycin. Human PBMCs from healthy donors were cultured in complete lymphocyte culture medium composed of RPMI 1640 supplemented with 10% FBS, 1 % Penicillin-Streptomycin-Glutamine (P / S / G), 1 % MEM non-essential amino acids (NEAA), 10 mM HEPES, ImM sodium pyruvate, 2-mercaptoethanol (50 mM), and 100 mg / ml normocin. Raji, NALM6, OVCAR3, and OVCAR8 cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% P / S / G.

[0097] Cell viability assays

[0098] After Jurket T cells were mixed with SynVACs or elastic beads at a 1:1 ratio, the mixture was incubated for 3 hours in a 96- well plate with C I O4cells per well. To assess the cell viability, the L1VE / DEAD Cell Imaging Kit (Invitrogen, R37601) was used according to the manufacturer's protocol. Fluorescence images were collected using a Zeiss Axio Observer Z1 inverted fluorescence microscope and analyzed using ImageJ software.

[0099] In vitro polyclonal T-cell expansion studies

[0100] Isolated mouse primary' T cells or human PBMCs were activated by SynVACs, elastic beads, or Dynabeads (T Cell- Activator, Gibco), respectively. An initial number of 5 x 105PBMCs (roughly 20% of the total number were T cells) were seeded in the starting culture media supplied with 30 lU / ml recombinant IL-2 (Biolegend) and activated by SynVACs, elastic beads or Dynabeads at a bead-to-cell ratio of 1: 1. For the negative controls, one group called “Bare beads” was treated with viscoelastic microbeads without antibody conjugation in IL-2 enriched media, while the other control group consisted of T cells alone, also supplemented with IL-2 but without any beads. Fresh media containing 30 lU / ml IL-2 was added to the cells to keep the cell density below 2 x 106cells / ml through the whole culture process. Cell numbers were counted on day 7, day 10, and day 14. Fold expansion was calculated by dividing the number of cells at the respective time point by the number of cells seeded at the start of the culture.

[0101] Lentiviral vectors for CAR introduction

[0102] Lentiviral vectors used in this study were all constructed from a parental lentivector pMNDW63. The Lenti / CAR19 vector was constructed by inserting into the pMNDW a synthetic gene encoding CD19-targeting CAR. The Lenti / MCAR vector was constructed by inserting into the pMNDW a synthetic gene encoding mesothelin- targeting CAR. The synthetic gene fragments were obtained from GenScript and IDT. Lentiviruses were produced using Human Embryonic Kidney (HEK) 293T cells (ATCC), following a standard transfection protocol using the Trans-IT-Lenti Transfection Reagent (Minis Bio) and a centrifugation concentration protocol using the Amicon™ Ultra Centrifugal Filter Units, according to the manufacturer's instructions (MilliporeSigma)64

[0103] In vitro CAR-T cell generation

[0104] On day 0, human PBMCs from healthy donors were activated by SynVACs, elastic beads, or Dynabeads (T cell-Activator, Gibco), respectively. An initial number of 5 x 105PBMCs (roughly 20% of the total number were T cells) were seeded in the starting culture media supplied with 30 lU / ml recombinant IL-2 and activated by SynVACs, elastic beads, or Dynabeads at a bead-to-cell ratio of 1: 1. On day 2. cells were transduced with Lenti / CAR19 or Lenti / MCAR virus for another 24 hours. The resulting CAR-T cells were expanded for another 2-3 weeks in CIO medium, fresh media supplied with 30 lU / ml recombinant IL-2 was supplemented if needed, and then the generated CAR-T cells were cryopreserved for future use.

[0105] In vitro antigen-specific CAR-T cell enrichment studies

[0106] Human MCAR-T cells were re-stimulated using MSLN-conjugated SynVACs, MSLN-expressing artificial artificial-presenting cells, or Dynabeads, respectively. An initial number of 1 x 106MCAR-T cells (roughly 70% of the total number were CAR+cells) were seeded in the starting culture media supplied with 30 lU / ml recombinant IL-2 (Biolegend) and activated by SynVACs at a bead-to-cell ratio of 1 : 1. CAR expression and T cell phenotype was analyzed using flow cytometry at day 5.

[0107] Flow cytometry analysis of T cell phenotypes

[0108] Cells were harvested at certain time points and the T cell phenotypes were evaluated using flow cytometry’. Four hours prior to the flow analysis, a protein transport inhibitor (BD Biosciences) was added to the aliquoted cells to increase the signal of intracellular markers. Fold expansion was calculated at the same time, by dividing the number of cells at a specific time point by the number of seeded cells at the beginning of the culture. To evaluate the CAR transduction rate, we analyzed CAR expression using a fluorophore-conjugated antibody specific to the extracellular domain of the CAR construct (e.g., anti-mesothelin, anti-CD19) on the T cell surface via flow cytometry on days 7 and 10.

[0109] All flow cytometry' stains were performed in PBS for 30 min on ice. The samples were stained with Mouse Fc Block (anti-mouse CD16 / 32) or Human Fc Receptor Blocking Solution (TrueStain FcX) before antibody staining. Antibody staining was performed at a certain dilution according to the manufacturer’s instructions. Fluorochrome-conjugated antibodies specific for human CD4 (Clone OKT4), CD8 (Clone SKI), CD45 (Clone H130), TCRaP (Clone 126), CD3 (Clone HIT3a), CD4 (Clone OKT4), CD8 (Clone SKI), CD45RO (Clone UCHL1), CD58 (clone TS2 / 9), CDl la (clone TS2 / 4), CXCR3 (clone G025H7), CD19 (Clone HIB19), Granzyme B (Clone QA16A02), Perforin (Clone dG9), CD69 (Clone FN50), CD45RA (Clone HI100), CD62L (Clone DREG-56), CD95 (Clone G043H7), CD25 (Clone BC96). PD-1 (Clone A17188A). Tim-3 (F38-2E2), FOXP3 (clone 206D), IFN-y (Clone B27), TNF-a (Clone Mabl l), mouse CD4 (Clone GK1.5), CD8 (Clone 53-6.7), IFN-y (Clone XMG1.2), TNF-a (Clone MP6-XT22), PD-1 (Clone 29F.1A12), Tim-3 (Clone B8.2E12), CCR7 (Clone 4B12), CD25 (Clone PC61), CD 44 (Clone IM7), CD95 (Clone SA367H8), CD62L (Clone MEL-14), CD95 (Clone SA367H8), Sca-1 (Clone D7), and streptavidin were purchased from Biolegend. Fluorochrome-conjugated antibodies specific for human MSLN (Clone 420411) were purchased from R&D Systems. Goat anti-mouse IgG F(ab’)2 secondary antibody was purchased from Thermo Fisher. Human Fc Receptor Blocking Solution (TrueStain FcX) was purchased from Biolegend, and Mouse Fc Block (anti-mouse CD16 / 32) was purchased from BD Biosciences. Intracellular cytokines were stained using a Cell Fixation / Permeabilization Kit (BD Biosciences). Stained cells were analyzed using LSRII (BD Biosciences). FlowJo vlO software was utilized to analyze the data.

[0110] SEM imaging of the interactions between microbeads and T cells

[0111] Human CD3+T cells were isolated from PBMCs using a human pan T isolation kit (Miltenyi Biotec) and stimulated with SynVACs or Dynabeads for 24 hours. The activated human T cells, along with SynVACs or Dynabeads, were then fixed using 4% glutaraldehyde, refrigerated for 2 hours, and subsequently post-fixed with 1% osmium tetroxide for another 2 hours. After fixation, the T cells were rinsed with HEPES-C buffer and then dehydrated through a graduated series of ethanol concentrations (75%, 85%, and 95%), with each step lasting 30 minutes. The samples were then dried using a critical point dryer. The specimens were subsequently mounted on specimen stubs and sputter-coated with a gold-palladium layer to prepare them for SEM imaging.

[0112] Immunofluorescent visualization of T Cell activation

[0113] Human CD3+T cells were isolated from PBMCs using a human pan T isolation kit (Miltenyi Biotec) and stimulated with SynVACs or Dynabeads for 72 hours. On day 3, activated human T cells were harvested and adhered to a 12 mm cover glass (Citoglas) that had been pre-treated with Poly-L-Lysine (Sigma). The cells were then fixed with 100% methanol at -20 °C for 5 minutes, followed by blocking with HEPES-C buffer containing 5% donkey serum. The samples were subsequently incubated with primary antibodies against CD3E (1 :400, Abeam, ab52959), P-actin (1:800, Cell Signaling, #4970), and NFAT1 (1 :50, Cell Signaling, #4389) at 4 °C overnight. After triple washing with HEPES-CT buffer, the cells were incubated with an appropriate secondary antibody for 1 hour. Nuclei were visualized by staining with 4,6-diamidino-2-phenylindole (DAPI; D3571 , Thermo Fisher) at a 1 : 1000 dilution for 10 minutes. Confocal images were acquired using a Leica SP8-STED confocal microscope and further analyzed using ImageJ software.

[0114] Single cell RNA sequencing

[0115] CAR-T cells activated by SynVACs and Dynabeads were cultured and harvested at day 14, followed by sorting with a FACSAria II flow' cytometer. The sorted cells were immediately dispatched to the UCLA Technology’ Center for Genomics and Bioinformatics (TCGB) Core for single cell TCR sequencing. The sequencing was executed with a 10X Genomics Chromium™ Controller Single Cell Sequencing System, as per the manufacturer's guidelines and the TCGB Core's standard protocol. Library preparation was accomplished using the Illumina TruSeq RNA Sample Prep Kit (Cat#FC- 122- 1001). and the sequencing was performed with 150 bp paired-end reads (5,000 reads / cell) on an Illumina NovaSeq system. Lastly, the reads were mapped to the human T cell receptor reference genome (hg38) using Cell Ranger VDJ. This allowed for the visualization of the frequencies of alpha or beta chain recombination events in CAR-T cells activated by SynVACs and Dynabeads. The processed cell matrix, data tables (e.g., expression values), and metadata have been made available in the public repository Gene Expression Omnibus (GEO) database (GSE242531).

[0116] Enzyme-linked immunosorbent cytokine assays (ELISA)

[0117] The ELISAs for detecting human cytokines were performed following a standard protocol from BD Biosciences. Supernatants from cell culture assays were collected and assayed to quantify human IFN-y. The capture and biotinylated pairs for detecting cytokines were purchased from BD Biosciences. The streptavidin-HRP conjugate was purchased from Invitrogen. Human cytokine standards were purchased from eBioscience. The samples were analyzed for absorbance at 450 nm using an Infinite M1000 microplate reader (Tecan).

[0118] In vitro tumor killing assay

[0119] Tumour cells (1 x 104cells per well) were co-cultured with effector cells (at ratios indicated in figure legends) in T cell culture medium in Coming 96-well clear bottom black plates for 24 hours. At the end of the culture, live tumor cells were quantified by adding D-luciferin (150 pg / ml; Caliper Life Science) to cell cultures and reading out luciferase activities using an Infinite Ml 000 microplate reader (Tecan).

[0120] In vivo bioluminescence live animal imaging (BLI)

[0121] BLI was performed using a Spectral Advanced Molecular Imaging (AMI) HTX imaging system (Spectral Instrument Imaging). Live animal imaging was acquired 5 minutes after intraperitoneal (i.p.) injection of D-Luciferin (1 mg / mouse) for total body bioluminescence, and 15 minutes after i.p. injection of D-luciferin (3 mg / mouse) for tissue bioluminescence. Imaging results were analyzed using AURA imaging software (Spectral Instrument Imaging).

[0122] In vivo anti-tumor efficacy study of CAR19-T cells in human Raji xenograft NSG mouse model

[0123] The experimental design is shown in Figure 6a. Briefly, on day 0, NSG mice received intravenously (i.v.) inoculation of Raji-FG cells (1 x 106cells per mouse). On day 4, the experimental mice received i.v. injection of vehicle (100 pl PBS per mouse), or CAR19-T cells (3 x 106CAR-T cells in 100 pl PBS per mouse). During the experiment, mice were monitored for their tumor loads to be measured using BLI. On day 40, the experimental mice w ere euthanized and their tissues w ere collected for further analysis. In vivo anti-tumor efficacy study of MCAR-T cells in human OVCAR8 xenograft

[0124] NSG mouse model

[0125] The experimental design is shown in Figure 7a. Briefly, on day 0. NSG mice received Intraperitoneally (i.p.) inoculation of OVCAR8-FG cells (1 x 106cells per mouse). On day 4, the experimental mice received i.v. injection of vehicle (100 pl PBS per mouse), or MCAR-T cells (3 x 106CAR-T cells in 100 pl PBS per mouse). During the experiment, mice were monitored for their tumor loads to be measured using BLI. On day 40, the experimental mice were euthanized and their tissues were collected for further analysis.

[0126] Statistical analysis

[0127] Data is represented as the mean ± standard deviation (SD). When necessary, a two-tailed Student's t-test was employed to identify statistically significant differences between the two groups, utilizing GraphPad Prism 8 for computations. In instances of comparison among more than two groups, a one-way analysis of variance (ANOVA) was conducted, followed by Tukey's multiple comparison test. Levels of statistical significance were denoted as follows: not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001.

[0128] As discussed above, we have developed a microfluidic platform for generating SynVACs that mimic the mechanical properties of APCs and present activation signals for T cell engineering. Our synthetic APCs represent an innovative approach that integrates both chemical and mechanical programmability to accurately replicate the dynamic behavior of natural antigen-presenting cells with high fidelity. This endeavor not only exemplifies the forefront of biomimetic engineering but also offers potential advancements in immunotherapy by closely mirroring the complex functionalities of natural APCs. First, microfluidic technology enables precise control over the size and shape of the microspheres, resulting in highly uniform particle populations. This uniformity is crucial for ensuring consistent and reproducible interactions with T cells. Secondly, microfluidic techniques facilitate the rapid and efficient production of microspheres, potentially enabling large-scale manufacturing of SynVACs for widespread application. This scalability is essential for translating our research findings into practical, real-world solutions. Thirdly, thanks to their mechanical properties, microbead-based activation systems more closely replicate the physiological interactions between T cells and antigen-presenting cells (APCs), thereby facilitating a more natural engagement of T cells. Finally, SynVACs that mimic the mechanical properties of APCs can facilitate proper T cell-SynVAC interactions, leading to more effective T cell activation, CD8+T cell-biased skewing, higher CAR transduction efficiency, enhanced CAR-T cell sternness, and more robust and durable immune responses against cancer cells, ultimately potentially improving remission durations and reducing the risk of cancer recurrence.

[0129] We found that SynVACs significantly enhance CAR transduction efficiency compared to Dynabeads, which has been further supported by in vivo experiments. The phenotypic analysis and CAR expression levels of CAR19-T cells collected from the blood reveal a notably high proportion of CAR-expressing cells. A higher transduction efficiency ensures that a larger proportion of T cells express the desired CAR or transgene, which can lead to more potent and effective therapeutic responses against target cells, such as tumor cells55. Additionally, with higher transduction efficiencies, a smaller number of starting T cells may be required to generate a therapeutic dose of CAR-T cells, potentially preserving more of the patient's healthy T cell population and minimizing unwanted cytokine storms.

[0130] Moreover, we have discovered that T cells activated by SynVACs exhibit a significantly higher tumor-killing efficiency compared to those activated by Dynabeads, evidenced by elevated expression levels of CD69. perforin, granzyme B, and IFN-y. Elevated CD69 expression suggests that SynVACs may promote a more efficient and rapid activation of T cells compared to conventional methods. Increased expression of perforin and granzyme B molecules in SynV AC -activated T cells highlights their heightened cytotoxic potential and their ability to effectively eliminate target cells, such as cancer cells. Elevated IFN-y expression in SynV AC-activated T cells underscores their enhanced functionality and capacity to mount robust antitumor immune responses. Taken together, these findings demonstrate that SynVACs not only significantly enhance T cell activation and expansion but also promote the generation of highly functional and cytotoxic T cells endowed with potent antitumor capabilities. This outcome is consistent with the observed CD8+cell-biased skewing induced by SynVACs

[0131] Interestingly, a recent study demonstrates that culturing CD8+T cells in slow-relaxing (1,000-10,000 seconds at 60% stress relaxation) viscoelastic collagen gel for 3 days before or after co-culture with Dynabeads enhances the tumor killing activity of T cells while fast-relaxing gel increases long-term memory genes56. However, it is essential to note that this study explores the effects of a viscoelastic matrix within a significantly different experimental system compared to ours, leading to some different findings. Firstly, their primary objective is to investigate the mechanical properties of a 3D collagen matrix on T cells, which is more relevant to in vivo conditions when T cells reside in collagen-rich tissues. T cells were in the collagen matrix for a three-day period and then Dynabeads were used for T cell expansion. In contrast, our research aims to develop a microfluidic system for the fabrication of viscoelastic artificial cells, enabling the activation and expansion of T cells over the entire two-week duration crucial for CAR-T cell production. We provide a comprehensive solution for clinical translation, aiming to enhance CAR-T therapy. Secondly, we utilize non-adherent alginate microbeads for suspension culture, exclusively presenting T cell activation signals. In contrast, the 3D collagen matrix involves potentially cell-matrix interactions. Thirdly, the stiffness of the collagen gel was below 1 kPa. in contrast to our 10 kPa condition. Fourthly, the previous study indicates that a slow-relaxing gel is more effective in inducing tumor cell killing, while a fast-relaxing gel increases the expression of long-term memory genes. Conversely, our approach demonstrates that a fast-relaxing gel is more effective in activating T cells for both tumor-killing activity and the formation of T memory' stem cells. Importantly, we focus on investigating the activation and differentiation of naive T cells, rather than CD8+cells as in their study. Another recent article presents the development of the MASTER scaffold, an innovative alginate-based platform that accelerates in vivo CAR-T cell manufacturing to a single day, enhancing their persistence and efficacy against distal tumors in mice24. This method focuses primarily on accelerating the efficiency of CAR-T cell manufacturing. Although alginate is used, the study does not explore the impact of viscoelasticity on T cell activation, and there is no experimental data indicating attempts to modulate the viscoelastic properties of alginate. We believe that the conclusions of our paper could be greatly beneficial to their proposed system, which could enhance the activation of CAR-T cells within their system by further regulating the viscoelastic properties of the alginate scaffold. Therefore, our objectives, experimental systems, and findings are distinct from those presented in the recent publication, offering novel insights into the mechanochemical effects of viscoelastic artificial cells.

[0132] Stem-like T cells are a unique subset of T cells that possess properties of both stem cells and immune cells, including TMSCs, naive T cells, and central memory T (TCM) cells. These cells can self-renew, and differentiate into various effector and memory' T cell subsets12-57As disclosed herein, we demonstrate that CAR-T cells activated by SynVACs exhibited a higher proportion of cells expressing high levels of CCR7 across the entire RNA transcriptome. This observation is particularly significant because cells expressing high levels of CCR7 are associated with TCM cells and TMSCs, yvhich are known to exhibit increased self-renewal capacity, longterm persistence, and improved anti -tumor efficacy. The expansion of these CCR7- expressing subpopulations suggests that SynVACs might promote the generation of more potent CAR-T cells with a greater ability to control tumor growth and achieve durable responses in cancer immunotherapy. The observation that SynVACs greatly enhance the long-term persistence of CAR-T cells in vivo in a B lymphoma xenograft model has significant implications for adoptive cell therapies. Persistence is an essential factor influencing the therapeutic efficacy of CAR-T cells, as it determines the duration of their antitumor activity within the host. The long-term persistence of CAR-T cells allows for the continuous recognition and elimination of tumor cells, leading to a more effective and durable treatment response. This finding underscores the potential advantages of using SynVACs for CAR-T cell activation and expansion, as it may ultimately lead to significantly enhanced clinical outcomes in patients receiving CAR-T cell therapy.

[0133] In the ovarian solid tumor xenograft models, we observed a remarkable reduction in tumor size when treated with Sy nV AC -activated CAR-T cells compared to both Dynabead-activated CAR-T cells and the vehicle control group. Furthermore, we found a significant decrease in metastatic cancer cells within vital organs such as the lung, pancreas, and uterus in the SynV AC-treated group. These findings emphasize the superior antitumor efficacy of SynV AC-activated CAR-T cells in combating not only primary tumors but also metastatic cancer cells that have spread to distant organs. This enhanced therapeutic performance may be attributed to the significantly enhanced expansion, persistence, and function of the SynV AC-activated T cells. If these cells can effectively infiltrate and target tumor cells, this may ultimately lead to better tumor control and potentially prolong patient survival.

[0134] In conclusion, our disclosure data demonstrates the remarkable potential of SynVACs as a powerful tool for T cell activation and expansion. Our promising findings highlight the advantages of SynVACs over traditional Dynabeads, paving the way for more effective and durable CAR-T cell therapies, and have significant implications for the development of next-generation adoptive cell therapies, ultimately enhancing treatment outcomes and overall survival for cancer patients.

[0135] TABLES Mechanical and chemical property Mechanical manipulation conditions Chemical crosslinking conditions

[0136] Table 1 : The formulation of SynVACs and elastic beads.

[0137] Table 2: Average percentage of the 14 subpopulations based on scRNAseq data.

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[0198] CONCLUSION

[0199] This concludes the description of embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. All publications mentioned are incorporated herein by reference to disclose and describe aspects, methods and / or materials in connection with the cited publications.

Claims

CLAIMS:

1. A composition of matter comprising alginate microparticles, wherein the alginate microparticles: comprise alginate polymers having molecular weights from 35 kDa to 600 kDa; comprise an agent that ionically crosslinks the alginate polymers; are coupled to one or more polypeptide ligands; and exhibit a diameter from 5-20 micrometers.

2. The composition of claim 1, wherein the alginate microparticles: exhibit a stiffness from 1 Kpa to 30 Kpa under physiological conditions; exhibit a stress relaxation time (tl / 2 (s)) from 5 seconds to 1000 seconds under physiological conditions; exhibit a loss modulus of 200 Pa - 6000 Pa at 1-10% strain under physiological conditions; comprise from IO45to IO6 3polypeptide ligands / bead; and / or do not comprise a polypeptide ligand that functions in cell-cell or cell- extracellular matrix (ECM) adhesion.

3. The composition of claim 1, wherein the agent that ionically crosslinks the alginate polymers is present in concentration from 10 mM to 100 mM.

4. The composition of claim 3, wherein the agent that ionically crosslinks the alginate polymers comprises a calcium-EDTA complex.

5. The composition of claim 1, wherein the one or more polypeptide ligands comprise an antibody that binds CD28 and / or an antibody that binds CD3.

6. A method of making alginate microparticles having a selected viscoelasticity and / or stiffness comprising the steps of: selecting amounts of alginate polymers having a selected molecular weights; disposing the alginate polymers in an aqueous solution; disposing the aqueous solution in a microfluidic device selected to utilize aqueous solution phases and oil phases, forming the alginate microparticles via droplet formation using pH-induced internal gelation.

7. The method of claim 6, wherein the aqueous solution comprises a calcium- EDTA complex that ionically crosslinks the alginate polymers,8. The method of claim 6. wherein the method is selected to form alginate microparticles that: exhibit a diameter from 5-20 micrometers; exhibit a stiffness from 1 Kpa to 30 Kpa under physiological conditions; exhibit a stress relaxation time (tl / 2 (s)) from 5 seconds to 1000 seconds under physiological conditions; and / or exhibit a loss modulus of 200 Pa - 6000 Pa at 1-10% strain under physiological conditions.

9. The method of claim 6. further comprising coupling one or more polypeptide ligands to a surface of the microparticles.

10. The method of claim 9. wherein the polypeptide ligands are coupled to a surface of the microparticles using a Tetrazine-TCO click reaction.

11. A method of modulating a physiological activity of a T cell, the method comprising combining the T cell with alginate microparticles of claim 1 such that a physiological activity of the T cell is modulated.

12. The method of claim 11 , wherein the T cell is selected to express CD8.

13. The method of claim 12, wherein the T cell comprises a chimeric antigen receptor (CAR).

14. The method of claim 13, wherein the T cell is obtained from a patient diagnosed with a malignancy.

15. The method of claim 11, wherein one or more polypeptide ligands bound to the surface the microparticles comprise an antibody that binds CD28 and / or an antibody that binds CD3; and do not comprise a polypeptide ligand that functions in cell-cell or cell-extracellular matrix (ECM) adhesion (e.g., an integrin).

16. The method of claim 11, wherein the physiological activity is the growth of the T cell.

17. The method of claim 11, wherein the physiological activity is active T cell division.

18. The method of claim 11 , wherein the physiological activity is the differentiation of the T cell.

19. The method of claim 11 , wherein the alginate microparticles : exhibit a stiffness from 1 Kpa to 30 Kpa under physiological conditions;exhibit a stress relaxation time (t 1 / 2 (s)) from 5 seconds to 1000 seconds under physiological conditions; exhibit a loss modulus of 200 Pa - 6000 Pa at 1-10% strain under physiological conditions; comprise from 1045to 106 3polypeptide ligands / bead; and do not comprise a polypeptide ligand that functions in cell-cell or cell-extracellular matrix (ECM) adhesion.

20. The method of claim 11, wherein the T cell is combined with alginate microparticles in vitro.

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