Synthetic viscoelastic activated cells for t cell engineering

Abstract

Conventional CAR-T cell therapies have shown a significant success in the treatment of hematological cancers and lymphoma. However, this technique still has several problems, including cancer recurrence. Herein, a scalable technical platform is described to produce synthetic viscoelastic activated cells (SynVAC) with programmable mechanical and chemical activity as artificial antigen presenting cells (aAPC). The disclosure presented herein shows that the viscoelastic properties of the described SynVAC have great beneficial effects on expansion of T cells. For example, SynVAC exhibits robust improvements in T cell expansion, T memory stem cell (TMSC) formation, chimeric antigen receptor (CAR) transduction efficiency, tumor killing efficiency, and long-term in vivo persistence of CAR-T cells compared to conventional rigid or elastic microspheres.

Description

Cross-reference to related applications

[0001] This application claims the benefit of commonly-assigned US Provisional Patent Application No. 63 / 515,228, filed July 24, 2023, entitled “Synthetic Viscoelastic Activated Cells for T Cell Engineering,” pursuant to 35 USC Section 119(e), the contents of which are incorporated herein by reference. Technical Field

[0002] The implementation schemes disclosed herein involve at least the fields of immunology and medicine. Background Technology

[0003] Cancer remains a leading cause of death in the United States and worldwide, with a significant impact on public health. While early diagnosis and traditional therapies such as surgery, radiation therapy, and chemotherapy have significantly improved the treatment of various cancers, limitations remain, including side effects, resistance to treatments, incurable genetic mutations, and relapses. To address these limitations, researchers are exploring immunotherapies, such as chimeric antigen receptor (CAR)-T cell therapy. 1-4 Current CAR-T cell therapy has shown remarkable success in treating hematological malignancies and lymphomas. 5 However, several problems remain, including cancer recurrence and the lack of long-term immunity against cancer. In the context of solid tumors, the immunosuppressive tumor environment and the inadequacy of existing CAR-T cell expansion methods present additional challenges that need to be addressed. 6-8 Therefore, there is an urgent need to develop more effective CAR-T cell expansion technologies that can enhance the cancer-killing ability of these cells and promote durable immunity against cancer. Addressing these challenges will significantly impact cancer treatment (including solid tumors) and ultimately improve patient outcomes and quality of life.

[0004] T cell stemness (characterized by the ability to self-renew and differentiate into multiple T cell subsets) is an important feature for maintaining a long-term and effective immune response against cancer. 9 In particular, T memory stem cells (TMSCs), a subset of memory T cells with stem cell-like properties, are increasingly recognized for their crucial role in maintaining a durable and effective immune response after adoptive transfer. 10-12 TMSCs possess self-renewal capacity and the multipotent ability to differentiate into various antigen-specific T cell subsets, making them particularly potent in generating robust and durable immune responses. 13 Although cytokines and small molecules have been explored to enhance the generation and maintenance of TMSCs. 14, 15However, the results may vary due to the lack of control over other niche factors, such as the mechanical properties of synthetic antigen-presenting cells (APCs) or the matrix.

[0005] T cell activation is a crucial first step in the adaptive immune response because it protects the body following the initial interaction between T lymphocytes and APCs. 16 Recently, it has been used in two-dimensional (2D) applications. 20, 21 And three-dimensional (3D) 22-24 Advanced biomaterials that replicate specific bioactive signals on the surface of APCs in microenvironments have led to the development of various methods for synthesizing APCs to induce T cell expansion in vitro and in vivo. 17-19 While current ex vivo T cell stimulation platforms can be used for efficient enrichment and activation of antigen-specific T cells, conditions still need optimization. For example, a primary method for activating T cells is using paramagnetic beads coated with anti-CD3 / CD28 antibodies, such as Dynabeads (Gibco). 25 However, due to the inherent differences between Dynabeads and APCs, T cells activated by Dynabeads typically result in suboptimal cell proliferation rates and fewer CD8+ cells. + Cytotoxic T cells and stem cell loss 26 In particular, Dynabeads made of polystyrene are rigid (20-40 MPa). 27 Furthermore, it possesses very different mechanical properties compared to APC, which is softer and exhibits viscoelastic behavior. 28 This potentially impairs the binding and activation levels of T cell receptors (TCRs). 29, 30 .

[0006] The viscoelasticity of the extracellular matrix (ECM) and surrounding cells plays a crucial role in shaping cell behavior and function. 31 Viscoelasticity reflects a material's ability to resist deformation and return to its original shape after being subjected to stress. Many cell types have been shown to respond to changes in viscoelasticity in response to their environment. 32 However, whether the viscoelastic properties of synthetic APCs regulate T cell activation and expansion remains to be explored. 33 .

[0007] There is a need in the art for methods and materials that can be used to promote TCR activation and T cell differentiation (e.g., in CAR-T cell expansion methods). Summary of the Invention

[0008] To determine and characterize the viscoelastic material properties of synthetic antigen-presenting cells and their impact on T cell activation, a method for generating novel synthetic activating cells (“SynVAC”) with a variety of viscoelastic properties was developed. As shown in the data presented below, the SynVAC disclosed herein exhibits a surprisingly robust effect on T cell expansion and can therefore be used to enhance therapeutic regimens, such as those targeting lymphomas and solid tumors with activated T cells.

[0009] As disclosed herein, a scalable technology platform has been further developed to fabricate viscoelastic alginate microspheres with defined viscoelasticity, and the effects of synthetically activated cells with varying viscoelasticity on T cell activation and expansion were then observed. As shown in the illustrative embodiments of the invention, the SynVAC disclosed herein exhibits robust effects on T cell activation, T memory stem cell formation, and chimeric antigen receptor (CAR) introduction into T cells. As described below, the material properties of the SynVAC disclosed herein enable it to generate CAR-T cells with superior functional properties compared to CAR-T cells generated using similar conventional materials for stimulating T cells, such as Dynabeads (e.g., the SynVAC disclosed herein demonstrates significantly higher efficiency than conventional materials when used with T cells in CAR-T therapy).

[0010] Embodiments of the invention disclosed herein include compositions comprising synthetic viscoelastic activated cells (SynVAC), which can be used in T cell engineering, including T cell activation, expansion, and differentiation. As described above, T cell-based therapies are promising treatments that utilize a patient's T cells to combat disease, particularly cancer. In this context, embodiments of the invention include the use of the synthetic viscoelastic activated cells disclosed herein in methods of designing T cells to better target cancer cells and / or genetically modifying them to express chimeric antigen receptors (CARs) that recognize and attack cancer cells.

[0011] As discussed in detail below, in some methods of the present invention, a droplet-forming microfluidic device having two phases (an aqueous phase and an oil phase) is used to prepare SynVACs. In an illustrative working embodiment of the present invention, which aims to produce structurally homogeneous SynVACs with a size range of 5-10 μm through the acidic dissociation of a calcium-EDTA complex, a premixed solution of alginate and calcium-EDTA is used as the aqueous phase. In these methods, alginate polymers with different molecular weights (LVLG, 70 kD MW and MVG, 180 kD MW) are utilized, and the viscoelasticity of the polymer matrix is ​​controlled by adjusting the composition ratio of LVLG and MVG alginate. The stiffness of the SynVAC is independently varied using different concentrations of calcium-EDTA, making it possible to obtain SynVACs with different viscoelasticities and stiffnesses that have mechanical properties similar to those of cellular APCs such as dendritic cells. Furthermore, the ligand density on the SynVAC is precisely controlled based on advanced bioorthogonal chemistry. In terms of mechanical properties and activation signal density, SynVACs prepared by these methods more closely mimic cellular APCs than Dynabeads.

[0012] Embodiments of the invention also include methods using the SynVAC disclosed herein, such as in methods designed to stimulate T cells, particularly peripheral blood mononuclear cell (PBMC)-derived T cells. Typically, such methods employ steps similar to those using conventional anti-CD3 / CD28 Dynabeads. Exemplary methods of the invention include those involving collecting PBMCs from a healthy donor and stimulating the collected T cells with SynVAC (or Dynabeads). In these methods, the T cells may be further supplemented with growth factors such as human IL-2 to support T cell expansion. Flow cytometry analysis can be used to compare T cell activation, exhaustion, and memory phenotypes between different artificial antigen-presenting cells (such as those disclosed herein) and conventional materials (such as Dynabeads).

[0013] As described below, SynVAC, disclosed herein, is found to be superior to Dynabeads in promoting T cell activation, as demonstrated by a higher CD8 / CD4 ratio, higher levels of T memory stem cells, a lower exhaustion phenotype, and a greater expansion fold. SynVAC showed consistent results in stimulating primary mouse T cells compared to commercial anti-CD3 / CD28 Dynabeads. These characteristics suggest that SynVAC can generate more robust T cell responses with longer therapeutic effects. Therefore, this invention provides a novel method for stimulating T cells with diverse therapeutic applications, particularly in the field of immunotherapy for cancer or other immune-related diseases.

[0014] Other objects, features, and advantages of the invention will become apparent to those skilled in the art from the following detailed description. However, it should be understood that while the detailed description and specific examples indicate some embodiments of the invention, they are given by way of illustration rather than limitation. Many changes and modifications can be made within the scope of the invention without departing from its spirit, and the invention includes all such modifications. Attached Figure Description

[0015] Figure 1 SynVAC, a microfluidic device used to develop viscoelasticity mimicking that of natural APC, is shown in the following figures: (a) SynVAC employs a biomaterial-based approach that utilizes an ionically cross-linked alginate network to create a tunable viscoelastic system mimicking the mechanical properties of natural APC. Scale bar, 15 μm. (b) SEM image of the microfluidic device in the cross-linked region. Scale bar, 100 μm. (c) SynVAC fabricated using a pH-induced internal gelation method. Scale bar, 50 μm. (d) Representative image of antibody-coated elastic beads and SynVAC compared to Dynabeads. Scale bar, 15 μm. (e) Control of bead size (n = 5) using microfluidic devices with different channel widths. (f) Size distribution of elastic beads and SynVAC. (g) Control of bead size (n = 5) using microfluidic devices with different flow rates. (h) Quantitative cell viability on day 14 by co-culturing elastic beads and SynVAC with Jurkat T cells (determined by a LIVE / DEAD staining kit) (n = 3). (i) Yield and yield of SynVAC per microfluidic device (n = 3). (j) Stability of GFP-labeled SynVAC over time in RPMI medium containing 10% FBS (n = 3). (k) Strategy for removing SynVAC from T cells after co-culture involving a single centrifugation step (600 g, 5 min) (left). Quantification of removal efficiency (right) (n = 5). In hk, data represent mean ± standard deviation (sd). In e, g, i, k, significance was determined by a two-tailed unpaired t-test (ns: not significant). In h, significance was determined by one-way ANOVA and Tukey's multiple comparison test (ns: not significant).

[0016] Figure 2Characterization of SynVAC in terms of stiffness, viscoelasticity, and ligand density. (a) Alginate gels can be ionicly or covalently crosslinked to provide viscoelastic or elastic properties, respectively. The molecular weight (MW) of the alginate polymer controls the viscoelasticity of the crosslinked alginate network. As MW increases, a dense network with high physical entanglement and overlap of polymer chains is formed, resulting in alginate gels with high stiffness and low viscoelasticity, and vice versa. Covalently crosslinked networks are elastic because covalent crosslinking retains the memory of the initial state. SynVAC can be converted into elastic beads through covalent crosslinking and subsequent Ca2+ removal. (b) Quantitative evaluation of the compressive modulus of SynVAC, elastic beads, and human APC using AFM indentation technique (n = 12). (c) Quantification of the timescale of initial stress relaxation to half its original value (n = 7). Stress relaxation time reflects the viscoelastic properties of the gel. (d) Shear storage modulus and shear loss modulus of ionicly crosslinked and covalently crosslinked gels as a function of frequency. (e) Schematic diagram of antibody conjugation on SynVAC and elastic beads using the TCO-tetraazine conjugation method. (f) Representative flow cytometry histograms showing fluorescence intensity of SynVAC and elastic beads expressing different levels of anti-CD3 density (n = 3). (g) Effect of antibody density on human T cell activation by analyzing CD69, an early T cell activation marker. SynVACs coated with different amounts of antibody (1:1 anti-CD3 / anti-CD28 ratio) were co-cultured with Jurkat cells (human T lymphocyte line) for 18 hours, and the percentage of CD69+ cells was analyzed by flow cytometry (n = 5). (h) Confocal microscopy image of individually labeled SynVACs showing a prominent fluorescence signal approximately 100 nm thick around the equatorial section, indicating surface-conjugated antibodies. Scale bar, 2 μm. In b and c, data represent mean ± sd. In b and c, significance was determined by one-way ANOVA and Tukey's multiple comparison test (ns: not significant). In g, significance was determined by a two-tailed unpaired t-test (ns: not significant).

[0017] Figure 3Polyclonal expansion of primary human T cells via SynVAC resulted in a higher T memory stem cell population. (a) Experimental timeline of human primary T cells activated by Dynabeads compared to SynVAC. (b) SEM image of the interaction between T cells and Dynabeads on day 1 (left). SEM image of the interaction between T cells and SynVAC on day 1 (right). Scale bar, 2 μm. (c) Immunofluorescence staining of CD3ε, β-actin, and nucleus (DAPI) in primary human T cells after 3 days of culture with Dynabeads, elastic beads, and SynVAC. Scale bar, 5 μm. (d) Quantification of the number of CD3ε clusters in primary human T cells after 3 days of culture with Dynabeads, elastic beads, and SynVAC. Scale bar: 5 µm. (e) Quantification of the size of CD3ε clusters in primary human T cells. (n = 50). (f) Fold expansion of human T cells. (n = 3). (g) PD-1 inhibitors of amplified PBMCs cultured with Dynabeads, elastic beads (E1, E2), and SynVAC (V1, V2) on days 10 and 14, respectively. + and TIM3 + Double-positive T cells, as determined by flow cytometry analysis (n = 3). (hk) CD8+ in expanded T cells cultured with Dynabeads, elastic beads, and SynVAC on days 10 and 14, respectively. + T cells (h), CD8 + CCR7 + CD45RO - CD95 hi TMSC (i), CD4 + T cells (j) and CD4 + CD25 + FOXP3 + Flow cytometry analysis of the total number of Treg cells (k). (n = 3). The “naked bead” group was treated with viscoelastic microbeads lacking antibody conjugation, all in IL-2-rich medium. In contrast, another control group contained only T cells, similarly supplemented with IL-2, but without any beads. In dk, data represent mean ± sd. Significance was determined by one-way ANOVA and Tukey’s multiple comparison test (ns: not significant).

[0018] Figure 4SynVAC enhances the transduction efficiency and tumor-killing ability of CAR-T cells. (a) CAR expression levels in CAR19-T cells on day 6 after activation by Dynabeads, elastic beads, and SynVAC. (b) Comparison of CAR transduction efficiency between Dynabeads and SynVAC (n = 5). (c) Evaluation of in vitro tumor-killing ability 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 SynVAC and then co-cultured at various effector cell to target cell ratios (n = 3). (d) IFN-γ secretion was measured by ELISA 24 hours after culturing with tumor cells (n = 3). (e) Phenotypic analysis of tumor-killing efficiency in CAR-T cells 24 hours after co-culturing with tumor cells. (f) Quantitative analysis of flow cytometry results (n = 3). Percentages were determined by staining with allotype antibodies. In b, d, and f, the data represent mean ± sd. In b and f, significance was determined by a two-tailed unpaired t-test (ns: not significant). In d, significance was determined by a one-way ANOVA followed by a Tukey multiple comparison test (ns: not significant).

[0019] Figure 5 Single-cell RNA sequencing analysis revealed distinct activation patterns and gene expression profiles in CAR-T cells activated via SynVAC and Dynabeads. (a) An experimental workflow showing how and when to collect CAR-T cells for single-cell RNA sequencing. (b) A UMAP plot showing the distribution of CAR-T cells activated via Dynabeads or SynVAC on day 14, in which 11 distinct clusters were identified. (c) A pie chart of the 11 distinct clusters in the SynVAC group, showing a higher CD8 count compared to the Dynabeads group. + TMSC (1), fewer CD4 + Th17 cells (9), fewer CD8 cells + Terminal differentiation effector memory T cells (4) and fewer Treg cells (10). (d) Gene expression signatures and specific markers used to identify 11 distinct clusters. (e) Individual cells in UMAP embeddings stained by expression of CD8A, CD4, CCR7, and IFNG. (f) Violin plots of overall gene expression reveal the distinct expression of CCR7 and IFNG genes, illustrating the enhanced stemness and tumor-killing efficiency of SynVAC-activated CAR-T cells. In f, significance was determined by z-test.

[0020] Figure 6 In vivo efficacy of SynVAC-activated CAR19 T cells in a human lymphoma Raji xenograft mouse model. (a) Experimental workflow demonstrating SynVAC-activated CAR19-T cell generation and therapy in a human lymphoma Raji xenograft mouse model, wherein 1 x 10⁻⁶ cells were administered on day 0. 6 Raji cells were intravenously (iv) injected into NSG mice, and on day 4, 3 x 10 cells were injected intravenously. 6 (a) CAR19-T cells. Tumor growth was monitored using bioluminescence imaging (BLI). (b) Bioluminescence imaging of NSG mice at different time points inoculated with luciferase Raji cells and then treated with a mimic (middle) or with CAR19-T cells activated by SynVAC or Dynabeads (n = 5). (c) Quantification of bioluminescence signals (n = 5). (d) CD3+ levels in different organs on day 40 after injection of CAR-T cells activated by Dynabeads / SynVAC. + CD45 + Double-positive cells, as determined by FAC analysis. (e) Characterization of CD4 / 8 ratio, CAR expression, and CD62L expression in blood. (f) Quantification of flow cytometry results from e (n = 5). In f, data represent mean ± SD. Significance was determined by a two-tailed unpaired t-test.

[0021] Figure 7 In vivo efficacy of SynVAC-activated MCAR-T cells in a mouse model of human ovarian solid tumor xenograft. (a) Experimental workflow illustrating the generation and treatment of SynVAC-activated MCAR-T cells in a mouse model of human ovarian solid tumor xenograft, wherein 1 x 10⁻⁶ cells were administered on day 0. 6 One OVCAR8 cell was injected intraperitoneally (ip) into NSG mice, and 3 x 10 cells were administered intravenously on day 4. 6(a) Bioluminescence imaging (BLI) of MCAR-T cells. Tumor growth was monitored using bioluminescence imaging. (b) Bioluminescence imaging of NSG mice seeded with luciferinated OVCAR8 cells and then treated with a mimic (middle) or with MCAR-T cells activated by SynVAC or Dynabeads at different time points after treatment. (c) Quantification of bioluminescence signals. (d) Bioluminescence imaging of ovarian cancer metastases in various organs at day 40. (e) Quantification of bioluminescence signals in different organs (n ​​= 8). (f) Phenotypic and CAR expression levels of MCAR-T cells collected from peritoneal fluid of experimental mice, as determined by flow cytometry. (g) Quantification of flow cytometry data (n = 8). In e and g, data represent mean ± sd. In e, significance was determined by one-way ANOVA and Tukey's multiple comparison test (ns: not significant). In g, significance was determined by a two-tailed unpaired t-test. Detailed Implementation

[0022] In the description of the embodiments, reference may be made to the accompanying drawings, which form a part of this document, and the ear embodiments in which the invention can be practiced are illustrated by way of illustration. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. Many techniques and procedures described or referenced herein are fully understood and commonly used by those skilled in the art. Unless otherwise defined, all technical terms, symbols, and other scientific terms or terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this invention pertains. In some instances, terms having their commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from what is commonly understood in the art.

[0023] Throughout this disclosure, various publications, patents, and published patent specifications are referenced either by identifying references or by Arabic numerals, with their full references preceding the claims. The disclosures of these publications, patents, and published patent specifications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention pertains.

[0024] Unless otherwise stated, the practice of this technique will employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols in Molecular Biology (FM Ausubel et al., eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., (1995)); Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual; and Animal Cell Culture (RI Freshney, ed., (1987)).

[0025] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0026] As used herein, the term "comprising" is intended to mean that a compound, composition, or method includes the described elements but does not exclude other elements. When used to define compounds, compositions, and methods, "consisting substantially of" should mean excluding other elements that are of any significant importance to the composition. Thus, a composition consisting substantially of elements as defined herein will not exclude trace contaminants, such as contaminants from separation and purification methods, as well as pharmaceutically acceptable carriers, preservatives, etc. "Containing" should mean excluding other components beyond trace elements. Embodiments defined by each of these transitional terms are within the scope of this art.

[0027] All numerical designations, such as pH, temperature, time, concentration, and molecular weight (including range), are approximate values ​​with increments of 1%, 5%, or 10% (+) or (-). It should be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about." It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary, and their equivalents are known in the art.

[0028] T cell activation is a crucial first step in the adaptive immune response because it protects the body following the initial interaction between T lymphocytes and APCs. 16 Recently, it has been used in two-dimensional (2D) applications. 20, 21 And three-dimensional (3D) 22-24 Advanced biomaterials that replicate specific bioactive signals on the surface of APCs in microenvironments have led to the development of various methods for synthesizing APCs to induce T cell expansion in vitro and in vivo. 17-19 While current ex vivo T cell stimulation platforms can be used for efficient enrichment and activation of antigen-specific T cells, conditions still need optimization. For example, a primary method for activating T cells is using paramagnetic beads coated with anti-CD3 / CD28 antibodies, such as Dynabeads (Gibco). 25 However, due to the inherent differences between Dynabeads and APCs, T cells activated by Dynabeads typically result in suboptimal cell proliferation rates and fewer CD8+ cells. + Cytotoxic T cells and stem cell loss 26 In particular, Dynabeads made of polystyrene are rigid (20-40 MPa). 27 Furthermore, it possesses very different mechanical properties compared to APC, which is softer and exhibits viscoelastic behavior. 28 This potentially impairs the binding and activation levels of T cell receptors (TCRs). 29, 30 .

[0029] T cell stemness (characterized by the ability to self-renew and differentiate into multiple T cell subsets) is an important feature for maintaining a long-term and effective immune response against cancer. 9 In particular, T memory stem cells (TMSCs), a subset of memory T cells with stem cell-like properties, are increasingly recognized for their crucial role in maintaining a durable and effective immune response after adoptive transfer. 10-12 TMSCs possess self-renewal capacity and the multipotent ability to differentiate into various antigen-specific T cell subsets, making them particularly potent in generating robust and durable immune responses. 13 Although cytokines and small molecules have been explored to enhance the generation and maintenance of TMSCs. 14, 15 However, the results may vary due to the lack of control over other niche factors, such as the mechanical properties of synthetic antigen-presenting cells (APCs) or the matrix.

[0030] The viscoelasticity of the extracellular matrix (ECM) and surrounding cells plays a crucial role in shaping cell behavior and function. 31Viscoelasticity reflects a material's ability to resist deformation and return to its original shape after being subjected to stress. Many cell types have been shown to respond to changes in viscoelasticity in response to their environment. 32 However, whether the viscoelastic properties of synthetic APCs regulate T cell activation and expansion remains unexplored. 33 It is hypothesized that the stiffness and viscoelasticity of APCs play a crucial role in regulating TCR activation and T cell differentiation. To demonstrate this, a scalable technology platform was subsequently developed to generate synthetic viscoelastic activated cells (SynVAC) that mimic the stiffness (kPa) and viscoelasticity of natural APCs. The robust effect of SynVAC on T cell expansion and its potential as a therapy for lymphoma and solid tumors were further demonstrated in comparisons with pure elastic beads and the clinically used product Dynabeads (as benchmarks).

[0031] The present invention disclosed herein has several embodiments. For example, embodiments of the invention include material compositions comprising alginate microparticles designed to form synthetically activated cells (“SynVAC”) with viscoelastic properties. Typically, in these compositions, the alginate microparticles comprise an alginate polymer having a molecular weight of 35 kD to 600 kD. In some of these embodiments, the alginate polymer has a molecular weight of 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 for ionically crosslinking the alginate polymer, such as a calcium-EDTA complex. Optionally, the agent for ionically crosslinking the alginate polymer is present at a concentration of 10 mM to 100 mM (e.g., 15 nM-50 mM, as shown in Table 1). In some embodiments of the invention, the alginate microparticles are designed to exhibit a diameter of 5-20 micrometers (e.g., 8-10 micrometers or 7-9 micrometers).

[0032] In typical embodiments of the invention, the alginate microparticles further exhibit certain material properties that enhance their ability to stimulate T lymphocytes. For example, in some embodiments, the alginate microparticles exhibit stiffness of 1 kPa to 30 kPa (e.g., 2 kPa to 25 kPa, as shown in Table 1) under physiological conditions. In some embodiments of the invention, the alginate microparticles exhibit stress relaxation times (t1 / 2 (s)) of 5 to 1000 seconds (e.g., approximately 8-12 t1 / 2 (s)) under physiological conditions. In some embodiments of the invention, the alginate microparticles exhibit a loss modulus of 200 Pa to 6000 Pa at 1%-10% strain under physiological conditions (e.g., a loss modulus of 300-800 Pa at 1%-10% strain, as shown in Table 1). Figure 2(as shown in diii). Typically, alginate microparticles are conjugated to one or more polypeptide ligands, such as antibodies. In illustrative embodiments of the invention, one or more polypeptide ligands include antibodies binding to CD28 and / or antibodies binding to CD3. In some embodiments of the invention, the alginate microparticles do not contain polypeptide ligands (e.g., integrins) that function in cell-cell or cell-extracellular matrix (ECM) adhesion. In illustrative working embodiments of the invention, the alginate microparticles are shown to contain 10 4.5 Up to 10 6.3 One polypeptide ligand / bead.

[0033] Embodiments of the present invention include methods for preparing alginate microparticles having selected viscoelastic and / or stiffness spectra. Typically, these methods include the following steps: selecting an amount of alginate polymer having a selected molecular weight; placing the alginate polymer in an aqueous solution; placing the aqueous solution in a microfluidic device selected to utilize both an aqueous and an oil phase; and then forming alginate microparticles via droplet formation using pH-induced internal gelation. Typically in these methods, the aqueous solution contains an ionic crosslinking agent (e.g., a calcium-EDTA complex) that ionicly crosslinks the alginate polymer. As described below, these methods are typically chosen to form alginate microparticles having selected material properties, such as exhibiting a diameter of 5-20 micrometers; exhibiting a stiffness of 1 kPa to 30 kPa under physiological conditions; exhibiting a stress relaxation time (t1 / 2 (s)) of 5 to 1000 seconds under physiological conditions; and / or exhibiting a loss modulus of 200 Pa to 6000 Pa at 1%-10% strain under physiological conditions. Embodiments of the present invention also include coupling one or more polypeptide ligands to the surface of a microparticle, for example, a method of coupling polypeptide ligands to the surface of a microparticle using a tetrazine-TCO click reaction.

[0034] Embodiments of the present invention include methods for activating or stimulating T cells using the SynVAC disclosed herein. Embodiments of the present invention include methods for modulating T cell activity (e.g., stimulating T cell growth or differentiation, inducing T cell expansion, etc.), comprising combining T cells with the alginate microparticles SynVAC disclosed herein, thereby modulating T cell activity. Typically in these methods, one or more polypeptide ligands bound to the surface of the microparticles comprise antibodies binding to CD28 and / or binding to CD3; and do not comprise polypeptide ligands (e.g., integrins) that function in cell-cell or cell-extracellular matrix (ECM) adhesion. In some embodiments, the T cells are selected as T cells expressing CD8. In some embodiments of the present invention, the T cells are selected as T cells comprising a chimeric antigen receptor (CAR). In some embodiments of the present invention, the T cells are obtained from patients diagnosed with malignant tumors.

[0035] Further aspects and embodiments of the present invention are disclosed in the following examples. Example

[0036] Example 1: Synthetic viscoelastic activating cells (SynVAC) for T-cell engineering and cancer therapy

[0037] Preparation of viscoelastic microspheres using high-throughput microfluidic devices

[0038] Given the current limitations of existing platforms, our goal is to develop cutting-edge technologies to enhance the stemness, CAR transduction rate, tumor-killing efficiency, and in vivo persistence of CAR-T cells for cancer immunotherapy. Therefore, SynVAC was designed as an engineered APC with finely tuned mechanical properties (viscoelasticity and stiffness) and surface ligands / antibodies to achieve the desired T cell activation. Figure 1 a) Alginate is an inert material with excellent biocompatibility, tunable mechanical properties, and well-controlled surface chemistry for molecular conjugation, making it an ideal candidate for the material basis of a platform. 34, 35 SynVAC based on alginate was fabricated using a high-throughput microfluidic device via a pH-induced internal gelation method. SEM images reveal the channel design of the microfluidic device, particularly the cross-linking regions for microbead formation. Figure 1 b). At the T-junction, an alginate solution containing pH-responsive calcium encounters the acidic oil phase to induce particle formation and ionic cross-linking. Figure 1 The data in c proves the formation of alginate beads.

[0039] In developing this invention, apparatus and manufacturing parameters were optimized to obtain monodisperse elastic or viscoelastic microparticles with defined sizes. Viscoelastic alginate beads were prepared using low molecular weight (MW) alginate (75 kDa) and calcium-based ionic crosslinking. In this work, clinical-grade sodium alginate was utilized. Alginate is widely recognized as a non-antigenic material, exhibiting excellent biocompatibility, particularly in vitro, where it is widely used. Furthermore, our previous studies have shown that alginate does not exhibit significant antigenicity when used in vivo in the form of microparticles or macro-hydrogels. 18, 19 In its unmodified state, alginate exhibits almost no cell binding, providing an inert background conducive to the incorporation of precisely defined biological signals. The maximum achievable stiffness of alginate gels is likely below 300 kPa. 36 This is sufficient to cover the dynamic range of natural APC. As disclosed in this paper, alginate beads with mechanical properties (kPa level) simulating APC have been engineered. Dynabeads made of polystyrene exhibit stiffness up to 20-40 MPa.27, 37 Since Dynabeads have been optimized with CD3 / CD28 antibody coating for T cell activation and expansion in a clinical setting, Dynabeads were used as a benchmark for functional comparison of expanded T cells. To fabricate the elastic beads, alginate with a high MW (120 kDa) was utilized to first generate ionically cross-linked microbeads, which were then converted into covalently cross-linked microbeads via carbodiimide-based chemistry. Figure 1 Figure d shows the resulting viscoelastic and elastic beads, demonstrating transparency compared to rigid polystyrene Dynabeads. The diameter of the microbeads can be adjusted by modifying the channel width of the microfluidic device. Figure 1 e). Because using alginates of different molecular weights can lead to variations in solution viscosity, subsequently resulting in differences in the size of the generated beads, cellulose is used to balance the potential viscosity variations. Carboxymethyl cellulose (CMC) can be easily eluted from the microbeads subsequently. 38, 39 This process does not interfere with mechanical properties or antibody modification. As disclosed herein, viscoelastic beads and elastic beads with diameters ranging from 7 to 9 micrometers were prepared. Figure 1 f), which reflects the size of the dendritic cells. Furthermore, the bead size can be finely adjusted within a narrow range by regulating the oil phase flow rate within the channel. Figure 1 g). Our experiments showed that neither viscoelastic beads nor elastic beads affected cell viability when co-cultured with human Jurkat T cells, indicating that the manufactured beads are biocompatible with immune cells. Figure 1 h). A single microfluidic chip can generate approximately 13 million microbeads per hour and collect approximately 11 million beads, with losses likely due to multiple centrifugation steps during the collection process. Figure 1 i). This process can be easily scaled up using parallel microfluidic systems for high-throughput production of viscoelastic beads, which is crucial for their future clinical applications.

[0040] To assess the long-term stability of the beads in culture, green fluorescent protein (GFP) was conjugated to SynVAC during manufacturing, and the microbeads were maintained at 4°C for 15 days in RPMI medium (containing 10% fetal bovine serum) or HEPES buffer, which is a typical timeframe for in vitro expansion of human CAR-T cells. Figure 1 Evaluation of mean fluorescence intensity (MFI) revealed no significant change during this period, indicating that SynVAC is stable in solution. To remove these alginate microbeads from the culture, the beads can be rapidly dissolved within minutes by introducing a chelating agent such as EDTA or citrate. Figure 1As an alternative method, SynVAC can be easily separated from cells using physical centrifugation, resulting in a removal rate of over 97% for alginate beads (lower density than cells). Figure 1 (k). Higher removal rates can be achieved through multiple centrifugation processes. Consistent with stability data, the residual alginate level in the SynVAC and T cell co-culture supernatant was less than 50 ng / ml. In our experiments, this low level of alginate did not have a detrimental effect on T cells, which is consistent with previous in vitro studies. 40 and in the body 19 The studies are similar.

[0041] Adjustment and characterization of the mechanical properties of SynVAC and elastic beads

[0042] In this work, four types of alginate microbeads with defined viscoelasticity and stiffness were fabricated, including: (1) V1: low-stiffness viscoelastic beads; (2) V2: high-stiffness viscoelastic beads; (3) E1: low-stiffness viscoelastic beads; and (4) E2: high-stiffness viscoelastic beads. The formulation and parameters of each type of microbead are shown in Table 1. For the viscoelastic beads (V1 and V2) in the SynVAC group, a lower molecular weight alginate polymer (75 kDa) was used to achieve rapid stress relaxation (high viscoelasticity), and the calcium concentrations (15 mM and 50 mM) were varied to ionically crosslink alginate with low (15 kPa) and high (25 kPa) stiffness, while maintaining a half-stress relaxation time of approximately 10 seconds. Figure 2 ac). Calcium concentrations between 10 mM and 50 mM induced a significant increase in the stiffness of the alginate gel, but had a negligible effect on relaxation time. To fabricate beads with elastic properties (E1 and E2), microspheres were generated using the same method with higher molecular weight (120 kDa) alginate polymers, which were further crosslinked with different concentrations of adipic acid dihydrazide (AAD), followed by removal of the calcium-based crosslinks with sodium citrate. This process of converting viscoelastic crosslinks into elastic covalent bonds enabled the microspheres to have tunable stiffness and slow stress relaxation (ac). Figure 2 In this case of slow stress relaxation and covalent crosslinking, stiffness is the dominant mechanical property, and adjusting the stiffness with AAD (between 5 and 25 mM) has no significant effect on viscoelastic properties (approximately 1000 seconds of half-stress relaxation time). The stiffness of SynVAC, elastic beads, and natural APC was also directly measured using atomic force microscopy (AFM) indentation, showing results consistent with those of the same formulation of 2D gels. Figure 2b). Furthermore, the stiffness variation of the fabricated microbeads was similar to that of 2D gels, demonstrating a stable fabrication process. Additionally, SynVAC and elastic beads used in AFM evaluation were pre-conjugated with antibodies. This ensured that the measurements reflected the properties of the actual microbeads used in the T cell activation assay. Among all fabricated groups, V1 exhibited the lowest stiffness and highest viscoelasticity (15 kPa, T...). 1 / 2 The 10-second stress relaxation time (10-second stress relaxation time) is most similar to that of mouse dendritic cells with a stiffness of 11 kPa and a stress relaxation time of 15 seconds. 41 Furthermore, side-by-side characterization of stress relaxation of SynVAC beads and human natural APCs (monocytes) using AFM showed similar results, while the elastic beads showed no stress relaxation.

[0043] Further rheological tests were conducted to evaluate the viscoelasticity of the V1 and E1 beads. Frequency sweep analysis showed that the storage moduli of the viscoelastic and elastomeric gels were similar; however, the loss modulus of the viscoelastic gel was 50 times higher than that of the elastomeric gel. The shear thinning of the viscoelastic gel was more than 10 times that of the elastomeric gel. Strain sweep tests also revealed that the viscoelastic gel exhibited a loss modulus >20 times higher than that of the elastomeric gel. Consistently, frequency-dependent and strain-dependent loss factors (tanδ), representing the ratio of energy lost during deformation to energy stored in the material, revealed the higher viscoelasticity of the viscoelastic gel compared to the elastomeric gel. Figure 2 d).

[0044] Surface modification of SynVAC and elastic beads using T cell activation signals

[0045] To provide T cell activation signals, SynVAC and elastic beads were modified by conjugating signal transduction molecules to these alginate microbeads based on bioorthogonal chemistry for (1) activation of polyclonal T cells using activating antibodies (anti-CD3 and anti-CD28), and (2) enrichment of antigen-specific CAR-T cells using mesothelin and anti-CD28. Figure 2 e). The tetrazine-trans-cyclooctene (TCO) click reaction occurs rapidly and selectively, resulting in a high conjugation rate and stable covalent bonding between the antibody and SynVAC. 42 Furthermore, the use of tetrazine-TCO click chemistry with short PEG links for antibody conjugation with SynVAC ensures that the function of the ligand is directly influenced by the properties of the underlying material.

[0046] To quantitatively evaluate the specific ligand density in different formulations, a series of stable SynVAC and elastic beads were constructed, differing only in the amount of their FITC-labeled anti-CD3 conjugation, due to variations in the amount of tetrazine-antibody used in the formulations. The different levels of conjugated antibody density were first visualized using immunofluorescence imaging. To quantify the number of antibodies on each bead, flow cytometry analysis based on calibration beads with known antibody densities was used. Figure 2 f). Antibody density, or in other words, ligand spacing, has been shown to be crucial for T cell activation because it directly affects the interaction between the T cell receptor (TCR) and its corresponding ligand on the APC. 43, 44 Our data indicates that SynVAC or elastic beads have a value higher than 10. 5.9 The antibody density ensured efficient activation of mouse primary T cells and human Jurkat cells. Figure 2 g) as demonstrated by flow cytometry analysis of CD69 (an early activation marker). This result is consistent with previously published data, which indicate that ligand spacing of less than 50 nm significantly enhances T cell activation on 2D surfaces. 45 Dynabeads were also included as a direct benchmark in our experiments. Antibody density on the Dynabeads was estimated using the same method. Our findings indicate the presence of two distinct antibody types on the Dynabead surface with a molar ratio close to 3:1, and the total antibody count on the Dynabead surface was approximately 278,958 ± 2,790. With Dynabeads having a diameter of 4.5 μm, the antibody spacing was calculated to be approximately 15.9 ± 0.1 nm, similar to our beads (Table 1). To further confirm our results, early T cell activation was assessed using SynVAC (V1), elastic beads (E1), and Dynabeads in parallel experiments with Jurkat NFAT-zsGreen reporter cells. Activation levels recorded at 18-hour intervals were consistent with our flow cytometry analysis, reinforcing the validity of our method and reflecting consistency with previously published data. 24 Therefore, in subsequent studies, 10 types of beads were selected for all bead formulations. 5.9 The ligand density was significant. Notably, our initial data showed that the 2D gel system resulted in a significantly lower T cell activation rate. Therefore, we focused on bead-based systems for T cell suspension culture, a decision consistent with previous studies demonstrating the poor T cell expansion efficiency of 2D gel systems. 46 .

[0047] To further characterize the spatial distribution of antibody conjugates, fluorescence staining and confocal microscopy were performed to examine the conjugated antibodies within individual microbeads. This analysis revealed a distinct fluorescent layer of approximately 100 nm on the bead surface. Furthermore, the bead surface composition was analyzed using X-ray photoelectron spectroscopy (XPS). XPS results showed the presence of nitrogen, confirming antibody surface modification. Quantitatively, an average of 5.3% alginate on the surface of the elastic beads and an average of 6.4% alginate on the SynVAC surface were modified, as determined by the ratio of amino acids to alginate monomers.

[0048] Polyclonal expansion of primary human and mouse T cells

[0049] Then, primary human T cells from peripheral blood mononuclear cells (PBMCs) were polyclonally expanded using SynVAC, elastic beads, and Dynabeads (as a commercial baseline). Figure 3 a). Four types of SynVAC and elastic beads with different mechanical properties were manufactured by conjugating anti-CD3 and anti-CD28 in a 1:1 molar ratio, as detailed in Table 1. Dynabeads were used as the gold standard control. The physical interaction between the beads and T cells was examined using scanning electron microscopy (SEM). Figure 3 b). After co-culturing SynVAC and human T cells for 24 hours, the formation of immune synapse (IS)-like structures was observed. Figure 3 b), this on the 3rd day ( Figure 3 c) This was confirmed as early as 1 hour by immunofluorescence staining targeting CD3 ε. Quantitative analysis revealed that SynVAC significantly increased the number and size of CD3 clusters in human T cells. Figure 3 de). Figure 3 The relative size changes of T cells in b and c are mainly 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.

[0050] Following these examinations of T-cell-bead interactions at early time points, a long-term culture of up to 2 weeks was performed, consistent with standard clinical protocols for CAR-T cell activation and proliferation, and the effects of SynVAC, elastic beads, and Dynabeads on T-cell expansion were investigated. Interestingly, at day 14, SynVAC resulted in a T-cell expansion rate more than 4 times higher than that of elastic beads and 1.5 times higher than that of Dynabeads. Figure 3f). Total T cell counts were observed on days 10 and 14 relative to the initial T cell seeding number (100,000). A statistically significant increase in T cell expansion was observed when using the lower-stiffness SynVAC (V1) compared to the higher-stiffness SynVAC (V2). We also noted that proliferation activated by the elastic bead group was less pronounced compared to proliferation activated by Dynabeads, as Dynabeads have been optimized for antibody density and bead size. Significant or modest improvements have been reported for various artificial APC (aAPC) systems compared to Dynabeads. 22, 47, 48 It is possible that, in addition to aAPC, differences in T cell origin, activation state, and culture conditions could also explain the different effects. Furthermore, while rapid proliferation is generally considered a positive result, biological relevance and the quality of the T cell response must be considered. Regarding the exhaustion markers PD-1 and LAG-3, no substantial differences were detected between various conditions after 14 days of culture. Figure 3 g). Notably, while Dynabeads significantly promoted CD4 bias skewness, this is consistent with previous reports. 47 Consistent, but all SynVAC formulations induced rapid and significant CD8 bias, with V1 through day 14 promoting the highest amount of CD8. + T cells ( Figure 3 h) This can directly target and eliminate tumor cells, making them more suitable for cancer immunotherapy. 49, 50 On the other hand, we recognize CD4 + The importance of T cells in supporting this response and maintaining immune memory is crucial for a comprehensive anti-tumor immunotherapy strategy. 51 Future research may explore the balance of CD8. + / CD4 + The synergistic effect of T cell responses enhances the efficacy and durability of antitumor immunity. Furthermore, based on analysis using fluorescence activated cell sorting (FACS), the SynVAC formulation (V1) resulted in a 6-fold higher CD8 count compared to Dynabeads and elastic beads. + CCR7 + CD45RO - CD95 hi The proportion of TMSC ( Figure 3i). These TMSCs were also positive for other markers such as CXCR3, CD58, and CD11a. This finding is significant because TMSCs are known to have remarkable self-renewal, differentiation into effector cells, and long-term persistence, in which the persistence and functional capacity of infused T cells are crucial for therapeutic success. Conversely, compared to Dynabeads, SynVAC (V1) produced significantly fewer CD4+ cells by day 14. + T cells ( Figure 3 j). Therefore, compared to Dynabeads, SynVAC (V1) produces a significantly lower number of CD4+. + CD25 + FOXP3 + Regulatory T (Treg) cells ( Figure 3 (k) A reduced proportion of Treg cells in the activated T cell population may lead to an enhanced anti-tumor immune response, since Treg cells are known to suppress the activity of other immune cells. 52 In addition to days 10 and 14, early assessments on day 7 also revealed phenotypic changes in T cells and the initial efficacy of SynVAC superior to elastic beads, compared to Dynabeads, in TMSCs and CD8. + Cell counts increased, while Treg counts decreased. These trends were consistent with observations on days 10 and 14, although the effects were less pronounced.

[0051] Furthermore, our analysis showed that T cells retained CD25 expression when activated by SynVAC, elastic beads, and Dynabeads, confirming sustained activation during the two-week cell expansion period. Additionally, our data indicate that when using human PBMCs or PBMC-derived T cells (via CD3... + When isolated cells are used as the starting cell source for T cell expansion, T cell expansion, CD4 / CD8 ratio, and CD3+ are observed. + CCR7 + CD45RO - CD95 hi There was no significant difference in TMSCs. These results justify our choice of PBMCs as a viable starting material for CAR T cell generation, consistent with established practices in both preclinical and clinical studies.

[0052] Similarly, SynVAC (V1), elastic beads (E1), and Dynabeads were used for polyclonal activation of primary mouse T cells. Time-lapse movies were recorded to observe the physical interactions between T cells and SynVAC / Dynabeads. Interestingly, on day 7, cells in the Dynabeads group formed large aggregates, while SynVAC resulted in relatively loose, sheet-like structures. This observation suggests that SynVAC may reduce the risk of overstimulation and promote better cell-cell interactions and nutrient exchange, potentially contributing to sustained T cell activation and expansion. Furthermore, the clusters in the Dynabead group dissipated after day 7, while most clusters in the SynVAC group persisted until day 10. This phenomenon further highlights the potential advantage of SynVAC in T cell activation and expansion, as they appear to create a more favorable environment for T cell interactions and persistence. FACS data analysis showed that in SynVAC co-cultures, higher expansion folds, no significant exhaustion, CD8 bias, and more CD44 were observed. - CD62L + CD95 + SynVAC has a consistent effect on human primary T cells in terms of TMSC formation and lower Treg formation.

[0053] To address concerns regarding the role of calcium ions (Ca²⁺) from alginate gel in T cell activation and differentiation, we conducted a comprehensive evaluation and demonstrated that the effect of calcium released from alginate beads on T cell activation and differentiation is negligible. First, SynVAC undergoes a rigorous washing and dialysis process and contains very little free calcium ions. Second, we typically used 500,000 beads in 24-well plates with 500 ml of culture medium to activate the same number of PBMCs, and calculations showed that the change in calcium concentration in the medium was less than 0.5%. Third, we measured calcium concentration and found no significant increase in calcium ion concentration in the culture medium. Therefore, we conclude that the viscoelasticity of alginate, rather than calcium release, is the dominant factor influencing the observed T cell phenotype.

[0054] In vitro transduction of CAR into T cells and its tumor cell killing activity

[0055] Next, the performance of SynVAC in CAR-T cell generation and in vitro tumor cell killing assays was evaluated. These evaluations were crucial for determining the efficiency and effectiveness of SynVAC in generating CAR-T cells with high transduction efficiency and potent tumor-killing capabilities. FACS data analysis revealed that SynVAC-activated T cells exhibited a significantly higher CAR transduction efficiency of up to approximately 90% on day 6, while Dynabeads and elastic beads achieved only 42% and 17%, respectively. Figure 4 ab), which is consistent with the average level of CAR transduction (20%-32%) in previous reports. 10, 53 Further studies using multiple donors confirmed the reproducibility of the improved CAR transduction efficiency. Staining with carboxyfluorescein diacetate succinimide (CFSE) followed by flow cytometry analysis showed that the highest proliferation rate was observed in SynVAC-activated T cells on day 3 post-CAR transduction, which may contribute to the significantly enhanced transduction efficiency.

[0056] In vitro tumor cell killing assays showed that SynVAC-activated CAR-T cells exhibited potent cytotoxic effects against various cancer cell lines, including OVCAR3, OVCAR8, Nalm6, and Raji cells. This indicates that SynVAC can enhance the function and specificity of CAR-T cells. Figure 4 c). Specifically, compared with CAR-T cells expanded via Dynabeads or elastic beads, SynVAC-expanded CAR-T cells exhibited a more significant reduction in viable tumor cells across all effector-to-target (E:T) ratios. This indicates that SynVAC-expanded CAR-T cells possess superior cytotoxicity in vitro. ELISA assays revealed that SynVAC-activated CAR-T cells exhibited the highest levels of interferon-γ (IFN-γ) secretion, while no significant difference was observed between Dynabeads- and elastic bead-activated CAR-T cells. Figure 4 d). This finding demonstrates that SynVAC enhances the functional capacity of CAR-T cells by promoting the secretion of IFN-γ, a key cytokine involved in anti-tumor immune responses. Consistent results were observed when primary mouse T cells were used, where SynVAC V1 promoted IFN-γ secretion on day 7. + TNF-α + CD8 + T cell formation. FACS analysis further demonstrated that, after tumor stimulation, SynVAC-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 4CD69 is an early marker of T cell activation, while perforin and granzyme B are key components of cytotoxic granules responsible for inducing apoptosis in target cells. Enhanced production of these proteins in SynVAC-activated CAR-T cells indicates a more potent cytotoxic potential and a stronger activation state compared to their Dynabead-activated counterparts. In summary, these results reinforce the idea that modulating the mechanical properties, particularly viscoelasticity, of synthetic APCs like SynVAC can influence the phenotypic and functional properties of generated CAR-T cells, which can be strategically used to optimize the CAR-T cell preparation process.

[0057] Antigen-specific enrichment of MCAR-T cells using mesothelin / anti-CD28 conjugated SynVAC.

[0058] The performance of SynVAC in antigen-specific activation of T cells was also evaluated. This assessment is crucial for understanding how SynVAC can efficiently and specifically activate T cells in response to the presence of target antigens, a key factor in determining the overall effectiveness of immunotherapy. Mesothelin-specific CAR-T (MCAR-T) cells were activated using SynVAC conjugated with anti-CD28. Mesothelin is a cell surface glycoprotein overexpressed in various solid tumors, making it an attractive target for CAR-T cell therapy. 54 Anti-CD28 molecules act as co-stimulatory signals, enhancing T cell activation and expansion. On day 5, SynVAC and aAPC (a cell line overexpressing the human CD83 / CD86 / 4-1BBL co-stimulatory receptor and human cortisol) significantly increased the proportion of MCAR-T cells, from 74.3% to 95.2% and 94.4%, respectively, while Dynabeads surprisingly resulted in a decreased proportion (i.e., 74.3% to 63.0%). Further investigation revealed that although aAPCs exhibited the same capacity as SynVAC in enriched MCAR-T cells, they did not promote CD8 bias skewness as SynVAC, as demonstrated by the CD4-CD8 ratio. These findings highlight the unique advantage of SynVAC in not only promoting MCAR-T cell expansion but also promoting its preferential CD8 bias skewness, which is crucial for enhancing the cytotoxic potential of CAR-T cells in cancer immunotherapy. Furthermore, antigen-specific activation using SynVAC further underscores the versatility and adaptability of the SynVAC platform for personalized immunotherapy across various cancer types.

[0059] Single-cell RNA sequencing (scRNAseq) analysis of expanded T cells

[0060] To further elucidate the differences in gene expression and subsets of CAR-T cells activated by SynVAC (the main formulation) and Dynabeads (as the gold standard control), single-cell RNA sequencing (scRNAseq) was performed on day 14 before the expanded cells were used for in vivo studies. Figure 5 a). This included 9,400 T cells activated via Dynabeads and 7,874 T cells activated via SynVAC. Bioinformatics analysis of gene expression mapped in two dimensions via uniform manifold approximation and projection (UMAP) identified 11 clusters ( Figure 5 b). Initially, the 7 clusters contained CD8. + T cells, 3 clusters are CD4 + T cells, and one cluster consists of double-negative T cells. Figure 5 c visualized the percentage changes for 11 different clusters in a pie chart. Then, subtypes of each cluster were identified based on protein biomarkers and gene signatures. Figure 5 d).

[0061] To explore CCR7 + Memory T cell subsets, we focused on clusters 1 and 8, which exhibit the gene expression profile of TMSCs, characterized by high levels of LEF1, TCF7, CCR7, SELL, and IL7R. (This is in contrast to our previous work.) Figure 3-4 Consistent with the findings in [the original text], SynVAC will [do something related to CD8]. + The percentage of TMSC increased from 6.8% to 14.4% (cluster 1), and CD8... + The percentage of effector memory T cells increased from 26.2% to 45.1% (clusters 3 and 5), but the percentage of CD8+ terminally differentiated effector memory T cells decreased from 14.9% to 4.2% (Table 2). Note that cluster 1 (CD8+) + The expression levels of CD95 and CD28 in TMSCs are relatively low; if we only consider CD95 in this cluster... + SynVAC also significantly increased the percentage of cells in this population (4.9%) compared to Dynabeads (2.2%). On the other hand, SynVAC reduced CD4+ cell count. + T helper 17 cells decreased from 18.0% to 4.3% (cluster 9), CD4 + Regulatory T cells decreased from 9.5% to 1.0% (cluster 10), although SynVAC reduced CD4... +TMSCs increased from 2.0% to 3.5% (Table 2). Cluster 4 was characterized by terminal differentiation due to higher expression of exhaustion markers such as TIGIT and effector genes such as NKG7, GNLY, and GZMB, and lower levels of LEF1 and stem cell-related markers, consistent with the TEMRA profile. Cluster 7 was labeled as exhausted because it exhibited high CXCR6 expression (indicating tissue residency), elevated TIGIT levels, and reduced expression of stem and effector genes, consistent with the exhaustion phenotype. In summary, scRNA-seq analysis provided more comprehensive information on the subsets of expanded T cells and confirmed the effect of SynVAC on increasing T cell stemness and CD8+. + Beneficial effects of subgroups.

[0062] Another noteworthy observation was that, compared to Dynabead-activated cells, SynVAC-activated CAR-T cells exhibited higher levels of CD8A, CCR7, and IFNG expression. Figure 5 The CD8A gene is associated with cytotoxic T cells, which play a role in eradicating cancer cells. The CCR7 gene is an important marker of stem cell-like T cells, and its high expression is associated with improved therapeutic potential in CAR-T cell therapy. Elevated gene expression of IFNG in SynVAC-activated CAR-T cells suggests a potentially more potent anti-tumor immune response, as IFNG enhances the immune system's ability to detect and eradicate cancer cells. Enhanced gene expression of CD8A, CCR7, and IFNG in the SynVAC group indicates an enhanced anti-tumor response and sustained immune protection.

[0063] To explore the heterogeneity between the SynVAC and Dynabead populations, gene enrichment analysis was performed using the EnrichR package, which further revealed that SynVAC-activated CAR-T cells exhibited upregulation of several key biological processes and pathways, such as DNA metabolism, DNA replication, and transcriptional regulation. These findings suggest that SynVAC-mediated activation may promote CAR-T cell proliferation and expansion, which is crucial for robust and durable anti-tumor responses. The upregulation of DNA metabolism and DNA replication implies that SynVAC-activated CAR-T cells undergo more active cell division and growth compared to CAR-T cells activated via Dynabeads. This increased proliferative capacity may enhance the CAR transduction rate and tumor-killing efficacy observed in the SynVAC group. The RNA sequencing data provided are indeed insightful, but it should be understood that the real value lies in perturbing the identified signals to determine their functional roles. Looking ahead, we plan to conduct targeted perturbation studies to reveal the functional implications of these genetic variations, with the goal of deepening our understanding of T cell programming and advancing the efficacy of CAR-T cell therapy.

[0064] In vivo antitumor efficacy and long-term durability of CAR-T cells in xenograft B-cell lymphoma model

[0065] Subsequently, the in vivo antitumor efficacy of SynVAC-activated CAR19 T cells was evaluated in a human lymphoma Raji xenograft mouse model. Figure 6 a). This evaluation is crucial for determining the therapeutic potential of SynVAC-activated CAR-T cells in a physiologically relevant context and for providing valuable insights into their ability to target and eliminate cancer cells in living organisms. We observed that in the untreated control group, all mice died of the disease by day 28. Figure 6 b). In contrast, SynVAC-activated CAR19-T cells effectively eliminated cancer cells in all treated mice by 28 days post-injection. On the other hand, the Dynabeads group showed significant cancer recurrence ( Figure 6 c). Furthermore, survival curves were included to demonstrate long-term outcomes after treatment. Statistical analysis revealed a significant difference in tumor burden between the Dynabeads and SynVAC treatment groups. Additionally, on day 40, the persistence of CAR19-T cells in various tissues of mice was examined, and a significant increase in CAR19-T cells was found in multiple organs and tissues (such as blood, spleen, and liver) in the SynVAC group. Figure 6 d). Further blood investigation revealed that the SynVAC group exhibited significantly elevated levels of CD8-biased skewness, CAR expression, and CD62L expression in its T cells. Figure 6 The elevated levels of CD62L observed in CAR19-T cells activated via SynVAC indicate a greater presence of T cell subsets such as naïve T cells, TMSCs, and central memory T cells. This observation is significant for the potential efficacy and durability of CAR-T cell therapy. Additional experiments were performed to evaluate the performance of CAR-T cells activated via elastic beads, as this group is crucial in asserting the effects of altered viscoelasticity. CAR-T cells activated via elastic beads exhibited a cancer recurrence pattern comparable to the Dynabeads group. These findings highlight the superior efficacy of SynVAC-activated CAR19-T cells not only in eliminating cancer cells but also in maintaining long-term persistence in vivo. The enhanced persistence and phenotypic characteristics of CAR19-T cells in the SynVAC group indicate robust T cell function, which may contribute to better cancer control and a reduced chance of recurrence.

[0066] In vivo antitumor efficacy and long-term persistence of mCAR-T cells in xenograft ovarian cancer model

[0067] Finally, the in vivo antitumor efficacy of SynVAC-activated MCAR-T cells was evaluated in a human ovarian cancer xenograft mouse model. Figure 7 a). Although animal models of lymphoma have been used, their limitations in fully representing solid tumors necessitate further investigation into the effectiveness of SynVAC in a solid tumor context to gain a more comprehensive understanding of its therapeutic potential. By day 33, all untreated control mice had developed ovarian solid tumors, and CAR-T cells activated by Dynabeads only slightly reduced the tumor growth trend. In contrast, we observed that while SynVAC was not able to completely eradicate cancer cells, it significantly reduced tumor cell recurrence and even potentially completely prevented tumor cell recurrence in one of eight mice. Figure 7 (bc). After statistical analysis, a significant difference in tumor burden was observed between the Dynabeads and SynVAC treatment groups. Furthermore, on day 40, metastasis of ovarian tumor cells in different tissues and organs of mice was examined. Compared to the vector control group and the Dynabead-activated CAR-T treatment group, the SynVAC group effectively inhibited tumor cell proliferation and metastasis, particularly in the lung, bone, and brain tissues. Figure 7 Specifically, we focused on the infiltration and phenotypic characteristics of CAR-T cells in the peritoneal fluid of mice, reflecting their presence in ovarian cancer tumor sites. Our findings revealed a significant increase in T cell retention in the SynVAC group compared to the Dynabeads group, rising from 1.75% to 7.06%. Furthermore, the proportion of CAR-T cells within the tumor significantly increased from 27.3% to 61.6%. Additionally, intratumoral CD62L was observed. + The percentage of CAR-T cells increased significantly, from 23.4% to 43.3%. Figure 7 These findings reveal that CAR-T cells activated by SynVAC not only inhibit tumor growth and metastasis, but also promote the in vivo lifespan of CAR-T cells, increase the proportion of CAR-expressing cells, and enhance CD62L. + The populations of T cells, such as naive T cells, TMSCs, and central memory T cells, are particularly prominent in solid tumor settings such as ovarian cancer. In summary, SynVAC shows great potential to significantly enhance the efficacy of CAR-T cell therapy against solid tumors, leading to better cancer control and a reduced chance of recurrence.

[0068] method

[0069] High-throughput microfluidic platform for manufacturing alginate viscoelastic microspheres

[0070] The microfluidic device was designed using AutoCAD and subsequently fabricated using conventional photolithography. Initially, photosensitive epoxy resin (SU-8 2015, MicroChem) was spin-coated onto a 4-inch silicon wafer to a thickness of 13 μm. The wafer underwent a soft bake at 95°C for 6 minutes and was then UV exposed through a chromium mask with the desired channel pattern. After a 10-minute development stage in SU-8 developer (MicroChem) and rinsing with isopropanol, a master mold was formed on the wafer. A polydimethylsiloxane (PDMS) prepolymer and curing agent were mixed, poured onto the silicon substrate, and cured at 65°C for 3 hours to produce a PDMS plate. This plate was then stamped to create inlet and outlet points, rinsed with 50% ethanol to clean the channels, and treated with oxygen plasma using Plasma Prep II (SPISupplies) to prepare for bonding. The assembled device was placed in a 65°C oven for approximately 30 minutes to strengthen the bond and then left at room temperature for 24 hours to ensure optimal hydrophobicity before experimentation. A custom "torch-shaped" component was designed and 3D printed using an Elegoo 4K printer (ELEGOO Inc.), sterilized with 70% ethanol, and then installed into a microfluidic device to serve as a reservoir. This reservoir, with a 5 ml capacity, acts as a storage area for SynVAC generated in the oil phase, facilitating subsequent manual collection. During production, microscopy was used to monitor the size and productivity of the alginate beads in real time to quickly address any unexpected issues within the chip.

[0071] Manufacturing of SynVAC and elastic beads with defined mechanical properties

[0072] To minimize batch-to-batch variability, Novamatrix's PRONOVA sodium alginate, manufactured according to Good Manufacturing Practice (GMP) guidelines and in accordance with ISO standards, was used. This particular grade is regulated by the FDA as Generally Regarded as Safe (GRAS), ensuring its safety and reliability. Internal gelation was achieved via a pH-induced method. 58, 59Alginate viscoelastic microbeads were fabricated within a microfluidic device. The core flow was loaded with a fluid containing 3% alginate (VLVG, MW 70kD, NovaMatrix), 1% carboxymethyl cellulose (for stabilizing laminar flow), and Ca-EDTA in deionized (DI) water. The Ca-EDTA concentration was adjusted to regulate the stiffness of the resulting viscoelastic beads. The sheath flow contained 1% surfactant (157 FSH, Krytox) and 0.5% acetic acid, both dissolved in fluorocarbon oil (Novec 7500, 3M). Both fluids, sterilized with a 0.22-micron filter prior to use, were facilitated into the microfluidic device via a separate inlet using a syringe pump (Harvard Apparatus). After the microbeads formed and were temporarily stored in a reservoir, they were collected into new test tubes containing 20% ​​perfluoro-1-octanol (PFO) (Sigma) and 0.2% acetic acid in fluorocarbon oil, which promoted cross-linking. Subsequently, HEPES-C buffer (20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 140 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, pH 7.2) was added to the collection tube. The tube was centrifuged at 1000 g for 1 minute to transfer 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 thorough cleaning, and stored at 4°C for further experiments.

[0073] Elastic beads were fabricated by converting ionically cross-linked viscoelastic microbeads into permanently cross-linked covalent microbeads. Initially, viscoelastic microbeads were fabricated using a 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)ethanesulfonic acid, 300 mM sodium chloride, 2 mM calcium chloride, pH=6) for 1 hour to adjust the pH to 6. Subsequently, the microbeads were collected by centrifugation (6000 g, 5 min) and resuspended overnight in MES-Ca buffer containing 320 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HoBt), and adipic acid dihydrazide (AAD). The concentrations of AAD and HoBt were adjusted to regulate the stiffness of the resulting elastic beads. Finally, sodium citrate buffer (77 mM NaCl, 55 mM sodium citrate) was introduced into the elastic beads for 2 hours to chelate calcium ions and completely remove ionic cross-links. The covalent beads were then rinsed with HEPES-T buffer (HEPES buffer containing 0.5% Tween 20) and stored at 4°C for future use.

[0074] Preparation of SynVAC and elastic beads for T cell stimulation

[0075] SynVAC, with its well-defined ligand spatial organization, can enhance CAR-T cell activation, leading to improved expansion, persistence, and therapeutic efficacy. 60 To prepare SynVAC and elastic beads for different scenarios, antibody / protein covalently conjugated to the beads using trans-cyclooctyne (TCO)-tetraazine linkage. Briefly, 4 million beads were resuspended in 800 μl MES buffer containing 200 mM EDC hydrochloride and 200 mM sulfo-NHS for an overnight reaction to activate the carboxyl groups. The beads were then washed with HEPES-CT buffer (HEPES buffer containing 2 mM calcium chloride and 0.5% Tween 20) to adjust the pH to 7. TCO-PEG6-amine (2 μmol / million beads, Click Chemistry Tools) was added to the solution and allowed to conjugate to the beads overnight at room temperature. During this process, the reaction mixture was placed on a roller to ensure thorough mixing. The beads were dialyzed against the HEPES-CT buffer using 1000 kDa dialysis bags (Spectrum laboratories) to remove excess TCO (HEPES-CT buffer was changed 2-3 times daily). Antibody-tetraazine 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 (Acro 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 μl of PBS buffer using an Amicon Ultra-2 spin column (Sigma Aldrich, USA). The concentrated mixture was then mixed with tetraazine-PEG5-NHS (Sigma Aldrich, USA) at a 1:5 molar ratio and reacted for 30 min. The antibody / protein-tetraazine complex was then desalted using a spin column and washed five times with PBS to remove unreacted tetraazine-PEG5-NHS. The purified antibody / protein-tetraazine complex was mixed with glycerol at a 1:1 ratio and stored at -20°C. To improve the ligand density of SynVAC and elastic beads, the dosage of antibody / protein-tetrazine was adjusted for tetrazine-TCO linkage with TCO-labeled microbeads.

[0076] Mechanical properties and ligand density characterization of SynVAC

[0077] Rheological measurements were performed using an Anton Parr rheometer. A 3% alginate solution (70 kD, VLVG) was crosslinked with various concentrations of calcium crosslinking agents to generate 8 mm gel discs supplemented with 1% carboxymethyl cellulose. The discs had an average thickness of approximately 2 mm. To form an 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 crosslinking agent to produce 8 mm gel discs. The gel samples were equilibrated with MES buffer (pH=6) for 1 hour and then immersed in MES solutions containing different concentrations of adipic dihydrazide (AAD) and the corresponding hydroxybenzotriazole (HoBt) under continuous stirring. This ensured a uniform distribution of AAD within the alginate matrix. The mixture was allowed to react overnight at room temperature, which allowed the formation of alginate gels with both ionic and covalent bonds. Subsequently, the gel samples were immersed in sodium citrate buffer for 2 hours to chelate and remove calcium ions from the gel. Finally, the resulting covalent alginate gel was thoroughly rinsed with deionized water and equilibrated in HEPES buffer for 24 hours to ensure complete removal of unreacted components and to stabilize the mechanical properties of the gel.

[0078] For rheological testing, an 8-mm PP025 measuring plate was used. The viscoelastic gel sample was carefully placed in the center of the rheometer plate using a spatula. The cantilever was lowered to the preferred gap height (1 mm was used in this study). Oscillatory strain scans (0.1%–500%, 1 Hz), oscillation frequency scans (0.1–100 Hz, 1% strain), and time scans (0.5% strain, 1 Hz, 2 min) were performed at room temperature to measure the storage modulus (G', Pa) and loss modulus (G'', Pa). Shear ramps (0.01–100 s⁻¹) were also performed. -1 This is used to examine the relationship between viscosity and shear rate. All experiments were repeated at least three times. Rheological measurements of elastic hydrogels were performed using the same procedure.

[0079] Using the methods described in a previously published study, the compressive modulus and stress relaxation properties of viscoelastic gels were evaluated via compression tests on gel discs (8 mm in diameter, 2 mm thick, equilibrated in RPMI for 24 h). 61The gel disc was compressed for 30 seconds at a deformation rate of 1 / 120 mm / s using a Chatilon TCD225 series force measurement system. The slope of the stress-strain curve (the first 5%-10% of the strain) was used as the initial compressive modulus. Thereafter, the strain was kept constant while the load was recorded over time. Stress relaxation was calculated by measuring the time required for the stress to decrease from its maximum to half. No prestress was applied to the gel for these measurements. Compression and stress relaxation measurements of the elastic hydrogel were performed using the same procedure.

[0080] The average antibody density of each SynVAC was determined by quantitative flow cytometry. In short, 1x10⁻¹⁰ antibodies from each experimental group were used. 5 Each SynVAC was conjugated with an appropriate antibody: anti-CD3 FITC (Biolegend 300305). The SynVACs were then washed and resuspended in HEPES-CT buffer for analysis in a flow cytometer (BD LSR Fortessa Cell Analyzer). A standard curve was constructed using Quantum Simply Cellular anti-mouse IgG beads stained with anti-CD3 FITC (#815, Bang Laboratories). For elastic beads, the same procedure was used to determine the antibody concentration for each bead. The mean mesothelin protein density per SynVAC was determined based on the standard curve constructed using Quantum Simply Cellular anti-mouse IgG beads stained with anti-mesothelin FITC (#815, Bang Laboratories).

[0081] AFM for SynVAC mechanical properties and cellular mechanical methods

[0082] Antigen-presenting cells (primary monocytes) were extracted from human PBMCs on the day of testing using a CD14 MicroBeads kit (Miltenyi Biotec). SynVAC, elastic beads, and APCs were placed on a JPK NanoWizard 4a BioScience AFM and indented using Bruker SAA-SPH-1UM probes with a spring constant k = approximately 0.25 N / m (the precise k value for each probe was determined by LDV calibration and used for specific tests). After obtaining the force spectrum, Young's modulus was determined by fitting the data to a Hertz / Sneddon model using JPK data processing. 62 To measure viscoelasticity, the height of the surface of SynVAC, elastic beads, and APC was kept constant after reaching 10 nN, and stress relaxation profiles were obtained by recording the vertical deflection force during the relaxation time.

[0083] X-ray photoelectron spectroscopy (XPS) analysis of SynVAC ligand density

[0084] Elastic beads and SynVAC were carefully prepared, and XPS measurements were performed using an Axis Ultra DLD spectrometer (Kratos Analytical Inc.; Chestnut Ridge, NY). Analysis was conducted under ultra-high vacuum conditions to prevent contamination and interference from atmospheric gases. Excitation was performed using a monochromatic Al Kα X-ray source (λ = 1486.6 eV), and a wide range of binding energies was scanned to capture the complete elemental spectrum of the bead surface. Nuclear energy level spectra of carbon (C 1S) and nitrogen (N 1S) were obtained, and peak areas were integrated to calculate atomic ratios. Given that the antibody contains nitrogen-rich amino acids, the presence of nitrogen on the surface specifically indicates antibody conjugation. The data were analyzed and quantified to determine the percentage of antibody conjugation on the alginate monomers on the bead surface.

[0085] Biosafety evaluation of SynVAC for CAR-T amplification

[0086] Biosafety testing was conducted to ensure that the alginate-based SynVAC could be effectively separated from T cells via physical centrifugation, thus ensuring that no residual SynVAC remained in activated T cells prior to therapeutic application. Specifically, pre-labeled SynVAC was co-cultured with primary mouse T cells at a 1:1 ratio for 24 hours. After culture, cells were stained with Hoechst 33342 (1:1000, Thermo Fisher) at room temperature for 10 minutes. The co-culture mixture was then centrifuged (600g, 5 minutes) and resuspended in buffer. The bead-to-cell ratio before and after the washing procedure was confirmed by FACS analysis.

[0087] To ensure the absence of residual alginate monomers (which could potentially trigger an innate immune response if co-injected with CAR-T cells), residual alginate in the supernatant was quantified by high-performance liquid chromatography (HPLC). For HPLC analysis, a carbon stationary phase (Kromasil 300-5-E18, 4.6 x 250 mm) was selected. Elution was performed with a mobile phase consisting of 40% acetonitrile and 60% DI H₂O at a flow rate of 1 mL / min, and UV detection was set at 254 nm. The system was equilibrated with the mobile phase for 20 min prior to the first injection. A sodium alginate standard solution was prepared by dissolving 250 mg of accurately weighed VLVG in 5 mL of distilled water to produce a 5% stock solution. Calibration standards were prepared by diluting different volumes of the VLVG stock solution with distilled water to produce concentrations ranging from 0.05 to 20 µg / mL.

[0088] T cell culture

[0089] The human Jurkat T cell line and Jurkat NFAT-zsGreen reporter cell line were gifts from Christopher Seet's laboratory at UCLA. Primary mouse T cells were isolated from the spleen of C57BL / 6 mice using a pan-T cell isolation kit to obtain CD3+ cells. + T cells were used for polyclonal activation studies. Healthy human PBMCs were obtained from the UCLA / CFAR Virology Core Laboratory, complying with federal and state regulations; identification information was not provided. Human Burkitt lymphoma cell line Raji, acute lymphoblastic leukemia cell line NALM6, ovarian cancer cell lines OVCAR3 and OVCAR8, chronic myelogenous leukemia cell line K562, and embryonic kidney (HEK) 293 T cells were obtained from ATCC.

[0090] Stable tumor cell lines expressing firefly luciferase and enhanced green fluorescent protein (FG) dual reporter genes were generated by transducing parental tumor cell lines with lentiviral vectors encoding target genes. After lentiviral vector transduction (72 hours), cells were sorted by flow cytometry to isolate genetically engineered cells, thus establishing stable cell lines. Four stable tumor cell lines were created for this study: Raji-FG, NALM6-FG, OVCAR3-FG, and OVCAR8-FG. aAPCs were generated by engineering the K562 human chronic myeloid leukemia cell line (ATCC) to overexpress the human CD83 / CD86 / 4-1BBL co-stimulatory receptor. The aAPC-MSLN cell line was further engineered from the parental aAPC line to overexpress human MSLN.

[0091] Jurkat cells and primary mouse T cells were cultured in ATCC-modified Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 50 μM 2-mercaptoethanol, and 100 mg / ml normocin. Jurkat medium was supplemented with an additional 1 ng / ml puromycin reporter. Human PBMCs from healthy donors were cultured in a complete lymphocyte medium supplemented with 10% FBS, 1% penicillin-streptomycin-glutamine (P / S / G), 1% MEM non-essential amino acids (NEAA), 10 mM HEPES, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 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.

[0092] Cell viability assay

[0093] After mixing Jurket T cells with SynVAC or elastic beads at a 1:1 ratio, the mixture was plated in 96-well plates at 1 x 10⁻⁶ cells per well. 4 Cells were incubated for 3 hours. To assess cell viability, the LIVE / 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.

[0094] In vitro polyclonal T cell expansion research

[0095] Isolated mouse primary T cells or human PBMCs were activated using SynVAC, elastic beads, or Dynabeads (T Cell-Activator, Gibco), respectively. An initial number of 5 x 10⁻⁶ cells was used. 5 PBMCs (approximately 20% of the total cells were T cells) were seeded in starting medium supplied with 30 IU / ml recombinant IL-2 (Biolegend) and activated at a 1:1 bead-to-cell ratio using SynVAC, viscoelastic beads, or Dynabeads. For negative controls, one group, referred to as "naked beads," was treated with unconjugated viscoelastic microbeads in IL-2-rich medium, while another control group, consisting only of T cells, was also supplemented with IL-2 but without any beads. Fresh medium containing 30 IU / ml IL-2 was added to the cells to maintain a cell density below 2 x 10⁻⁶ throughout the culture. 6Cells / ml. Cell counts were performed on days 7, 10, and 14. Fold expansion was calculated by dividing the cell count at each time point by the initial cell count at the start of culture.

[0096] Lentiviral vectors for CAR introduction

[0097] The lentiviral vectors used in this study were all derived from the parental lentiviral vector pMNDW. 63 Construction. The Lenti / CAR19 vector was constructed by inserting a synthetic gene encoding a CD19-targeting CAR into pMNDW. The Lenti / MCAR vector was constructed by inserting a synthetic gene encoding a mesothelin-targeting CAR into pMNDW. Synthetic gene fragments were obtained from GenScript and IDT. (Following manufacturer's instructions (MilliporeSigma)). 64 Following the standard transfection protocol using Trans-IT-Lenti transfection reagent (Mirus Bio) and using Amicon... TM The centrifugal concentration protocol of Ultra Centrifugal Filter Units uses human embryonic kidney (HEK) 293T cells (ATCC) to produce lentiviruses.

[0098] In vitro CAR-T cell generation

[0099] On day 0, human PBMCs from healthy donors were activated using SynVAC, elastic beads, or Dynabeads (T-cell activators, Gibco), respectively. An initial quantity of 5 x 10⁻⁶ PBMCs was used. 5 PBMCs (approximately 20% of the total cells are T cells) were seeded in starting medium supplied with 30 IU / ml recombinant IL-2 and activated at a 1:1 bead-to-cell ratio using SynVAC, elastic beads, or Dynabeads. On day 2, the cells were re-transduced with Lenti / CAR19 or Lenti / MCAR virus for 24 hours. The resulting CAR-T cells were then expanded in C10 medium for 2–3 weeks, supplemented with fresh medium supplied with 30 IU / ml recombinant IL-2 if necessary, and then cryopreserved for future use.

[0100] In vitro antigen-specific CAR-T cell enrichment study

[0101] Human MCAR-T cells were restimulated using MSLN-conjugated SynVACs, MSLN-expressing artificial presenting cells, or Dynabeads, respectively. An initial number of 1 x 10⁻⁶ cells was used. 6 70% of the total number of MCAR-T cells (approximately 70% of the total are CAR cells) +Cells were seeded in starting medium supplied with 30 IU / ml recombinant IL-2 (Biolegend) and activated by SynVAC at a bead-to-cell ratio of 1:1. CAR expression and T cell phenotype were analyzed by flow cytometry on day 5.

[0102] Flow cytometry analysis of T cell phenotypes

[0103] Cells were harvested at specific time points, and T cell phenotypes were evaluated using flow cytometry. Four hours prior to flow cytometry analysis, a protein transport inhibitor (BD Biosciences) was added to aliquots of cells to enhance the signal of intracellular markers. Fold expansion was calculated by dividing the cell count at specific time points by the initial seeding number. To evaluate CAR transduction rates, CAR expression was analyzed on the surface of T cells by flow cytometry on days 7 and 10 using antibodies specifically conjugated to the extracellular domains of CAR constructs (e.g., anti-mesothelin, anti-CD19).

[0104] All flow cytometry staining was performed on ice in PBS for 30 min. Samples were stained with mouse Fc blocking agent (anti-mouse CD16 / 32) or human Fc receptor blocking solution (TrueStain FcX) prior to antibody staining. Antibody staining was performed at the appropriate dilution according to the manufacturer's instructions. For human CD4 (clone OKT4), CD8 (clone SK1), CD45 (clone H130), TCRαβ (clone I26), CD3 (clone HIT3a), CD4 (clone OKT4), CD8 (clone SK1), CD45RO (clone UCHL1), CD58 (clone TS2 / 9), CD11a (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-γ (clone B27), TNF-α The following antibodies were purchased from Biolegend: clone Mab11, mouse CD4 (clone GK1.5), CD8 (clone 53-6.7), IFN-γ (clone XMG1.2), TNF-α (clone MP6-XT22), PD-1 (clone 29F.1A12), Tim-3 (clone B8.2E12), CCR7 (clone 4B12), CD25 (clone PC61), CD44 (clone IM7), CD95 (clone SA367H8), CD62L (clone MEL-14), CD95 (clone SA367H8), Sca-1 (clone D7), and antibodies conjugated with streptavidin-specific fluorescent dyes. A fluorescent dye-conjugated antibody specific to human MSLN (clone 420411) was 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 blocker (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). Data were analyzed using FlowJo v10 software.

[0105] SEM imaging of the interaction between microbeads and T cells

[0106] Human CD3 was isolated from PBMCs using the Human Pan-T Isolation Kit (Miltenyi Biotec). + T cells were stimulated with SynVAC or Dynabeads for 24 hours. Activated human T cells were then fixed with 4% glutaraldehyde and SynVAC or Dynabeads, chilled for 2 hours, and subsequently post-fixed with 1% osmium tetroxide for another 2 hours. After fixation, the T cells were washed with HEPES-C buffer and then dehydrated by a series of fractions of ethanol (75%, 85%, and 95%), each step lasting 30 minutes. The samples were then dried using a critical point desiccator. The samples were then mounted on a stub and sputter-coated with gold-palladium layers to prepare them for SEM imaging.

[0107] Immunofluorescence visualization of T cell activation

[0108] Human CD3 was isolated from PBMCs using the Human Pan-T Isolation Kit (Miltenyi Biotec). + T cells were stimulated with SynVAC or Dynabeads for 72 hours. On day 3, activated human T cells were harvested and adhered to 12 mm coverslips (Citoglas) pretreated with poly-L-lysine (Sigma). Cells were then fixed with 100% methanol at -20°C for 5 minutes and blocked with HEPES-C buffer containing 5% donkey serum. Samples were then incubated overnight at 4°C with primary antibodies against the following: CD3ε (1:400, Abcam, ab52959), β-actin (1:800, Cell Signaling, #4970), and NFAT1 (1:50, Cell Signaling, #4389). After washing three times with HEPES-C buffer, cells were incubated with appropriate secondary antibodies for 1 hour. Cell nuclei were visualized by staining with 1:1000 diluted 4,6-diamidino-2-phenylindole (DAPI; D3571, ThermoFisher) for 10 minutes. Confocal images were acquired using a Leica SP8-STED confocal microscope and further analyzed using ImageJ software.

[0109] Single-cell RNA sequencing

[0110] CAR-T cells activated by SynVAC and Dynabeads were cultured and harvested on day 14, and then sorted using a FACSAriaII flow cytometer. The sorted cells were immediately sent to the UCLA Technology Center for Genomics and Bioinformatics (TCGB) Core for single-cell TCR sequencing. Following the manufacturer's guidelines and TCGB Core's standard protocol, 10X Genomics Chromium was used for sequencing. TM Sequencing was performed using the Controller single-cell sequencing system. Library preparation was completed using the Illumina TruSeq RNA Sample Preparation Kit (Cat#FC-122-1001), and sequencing was performed on the Illumina NovaSeq system using 150 bp paired-end reads (5,000 reads / cell). Finally, the reads were mapped to the human T-cell receptor reference genome (hg38) using Cell Ranger VDJ. This allowed visualization of the frequency of α- or β-chain recombination events in CAR-T cells activated by SynVAC and Dynabeads. Processed cell matrix, data tables (e.g., expression values), and metadata were available in the Gene Expression Integration (GEO) database (GSE242531), a public repository.

[0111] Enzyme-linked immunosorbent assay (ELISA) for cytokines

[0112] ELISA for detecting human cytokines was performed according to BD Biosciences' standard protocol. Supernatants from cell culture assays were collected and measured to quantify human IFN-γ. Capture and biotinylation pairs for cytokine detection were purchased from BD Biosciences. Streptavidin-HRP conjugates were purchased from Invitrogen. Human cytokine standards were purchased from eBioscience. The absorbance of the samples at 450 nm was analyzed using an Infinite M1000 microplate reader (Tecan).

[0113] In vitro tumor killing assay

[0114] Tumor cells (1 x 10) 4 Cells per well and effector cells (at the ratio shown in the illustration) were co-cultured for 24 hours in T-cell medium in Corning 96-well clear-bottom black plates. At the end of the culture, viable tumor cells were quantified by adding D-luciferin (150 μg / ml; Caliper Life Science) to the cell culture and reading the luciferase activity using an Infinite M1000 microplate reader (Tecan).

[0115] In vivo bioluminescent imaging (BLI) of living animals

[0116] BLI was performed using the Spectral Advanced Molecular Imaging (AMI) HTX imaging system (Spectral Instrument Imaging). In vivo animal imaging was obtained 5 minutes after intraperitoneal (ip) injection of D-luciferin (1 mg / mouse) for whole-body bioluminescence and 15 minutes after intraperitoneal (ip) injection of D-luciferin (3 mg / mouse) for tissue bioluminescence. Imaging results were analyzed using AURA imaging software (Spectral Instrument Imaging).

[0117] In vivo antitumor efficacy of CAR19-T cells in human Raji xenograft NSG mouse model

[0118] Experimental design such as Figure 6 As shown in figure a. In short, on day 0, NSG mice received Raji-FG cells (1 x 10⁶ cells per mouse). 6 CAR19-T cells were intravenously (iv) injected. On day 4, the mice received either a medium (100 μl PBS per mouse) or CAR19-T cells (3 x 10 cells in 100 μl PBS per mouse). 6 The mice were administered CAR-T cells via intravenous injection. Tumor burden was monitored in mice using BLI during the experiment. On day 40, the mice were euthanized and their tissues were collected for further analysis.

[0119] In vivo antitumor efficacy of mCAR-T cells in a human OVCAR8 xenograft NSG mouse model

[0120] Experimental design such as Figure 7 As shown in figure a. In short, on day 0, NSG mice received intraperitoneal (ip) inoculation with OVCAR8-FG cells (1 x 10⁻⁶ cells per mouse). 6 On day 4, the experimental mice received either a medium (100 μl PBS per mouse) or mCAR-T cells (3 x 10 cells in 100 μl PBS per mouse). 6 The mice were administered CAR-T cells via intravenous injection. Tumor burden was monitored in mice using BLI during the experiment. On day 40, the mice were euthanized and their tissues were collected for further analysis.

[0121] Statistical analysis

[0122] Data are expressed as mean ± standard deviation (SD). GraphPad Prism 8 was used for calculations when necessary, and a two-tailed Student's t-test was employed to determine statistical significance between groups. One-way ANOVA was performed for comparisons between more than two groups, followed by Tukey's multiple comparison test. Statistical significance levels are expressed as follows: not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001.

[0123] As described 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 chemical and mechanical programmability to accurately replicate the dynamic behavior of native antigen-presenting cells with high fidelity. This effort not only exemplifies the forefront of biomimetic engineering but also offers potential advancements for immunotherapy by closely reflecting the complex functions of native APCs. First, microfluidic technology enables precise control over the size and shape of microspheres, resulting in highly uniform particle clusters. This uniformity is crucial for ensuring consistent and reproducible interactions with T cells. Second, microfluidic technology facilitates the rapid and efficient production of microspheres, potentially enabling the large-scale production of SynVACs for widespread applications. This scalability is essential for translating our findings into practical, real-world solutions. Third, due to their mechanical properties, microbead-based activation systems more closely replicate the physiological interactions between T cells and antigen-presenting cells (APCs), thereby promoting more natural T cell conjugation. Finally, SynVAC, which mimics the mechanical properties of APCs, can promote appropriate T cell-SynVAC interactions, leading to more effective T cell activation and CD8 activation. + T-cell bias, higher CAR transduction efficiency, enhanced CAR-T cell stemness, and a more robust and durable immune response against cancer cells could ultimately improve remission duration and reduce the risk of cancer recurrence.

[0124] We found that SynVAC significantly enhanced CAR transduction efficiency compared to Dynabeads, a finding further supported by in vivo experiments. Phenotypic analysis and CAR expression levels of CAR19-T cells collected from blood revealed a significantly higher proportion of CAR-expressing cells. Higher transduction efficiency ensures a larger proportion of T cells expressing the desired CAR or transgene, which could lead to a more potent and effective therapeutic response against target cells such as tumor cells. 55Additionally, with higher transduction efficiency, a smaller number of initiating T cells may be needed to generate a therapeutic dose of CAR-T cells, potentially preserving a larger population of healthy T cells in the patient and minimizing unwanted cytokine storms.

[0125] Furthermore, we found that T cells activated by SynVAC exhibited significantly higher tumor-killing efficiency compared to T cells activated by Dynabeads, as evidenced by elevated expression levels of CD69, perforin, granzyme B, and IFN-γ. Elevated CD69 expression indicates that SynVAC promotes more efficient and rapid T cell activation compared to conventional methods. Increased expression of perforin and granzyme B molecules in SynVAC-activated T cells highlights their enhanced cytotoxic potential and their ability to effectively eliminate target cells such as cancer cells. Elevated IFN-γ expression in SynVAC-activated T cells underscores their enhanced function and ability to generate robust anti-tumor immune responses. In summary, these findings demonstrate that SynVAC not only significantly enhances T cell activation and expansion but also promotes the generation of highly functional and cytotoxic T cells with potent anti-tumor capabilities. This result is consistent with the observed SynVAC-induced CD8+ activation. + Cellular bias is consistent.

[0126] Interestingly, recent studies have shown that culturing CD8 in slowly relaxed (60% stress relaxation for 1,000–10,000 seconds) viscoelastic collagen gels before or after co-culturing with Dynabeads can improve the efficacy of culturing CD8 in these gels. + T cells, after 3 days, enhance the tumor-killing activity of T cells, while the rapidly relaxing gel increases long-term memory genes. 56However, it is noteworthy that this study explored the effects of a viscoelastic matrix within an experimental system significantly different from ours, leading to several different findings. First, their primary objective was 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 placed in the collagen matrix for three days and then expanded using Dynabeads. In contrast, our study aimed to develop a microfluidic system for manufacturing viscoelastic artificial cells, enabling the activation and expansion of T cells throughout the entire two-week duration crucial for CAR-T cell generation. We offer a comprehensive clinical translational solution designed to enhance CAR-T therapy. Second, we utilized non-adherent alginate microbeads for suspension culture, which specifically presents T cell activation signals. In contrast, the 3D collagen matrix involves potential cell-matrix interactions. Third, the stiffness of the collagen gel was less than 1 kPa, contrasting with our 10 kPa condition. Fourth, previous studies have shown that slowly relaxing gels are more effective in inducing tumor cell killing, while rapidly relaxing gels increase the expression of long-term memory genes. Conversely, our approach demonstrates that rapidly relaxing gels are more effective in activating T cells for both tumor-killing activity and the formation of T memory stem cells. Importantly, we focused on investigating the activation and differentiation of naïve T cells, rather than CD8 in their studies. + Cells. Another recent article showcases the development of the MASTER scaffold, an innovative alginate-based platform that accelerates in vivo CAR-T cell preparation to a single day, enhancing their durability and efficacy against distant tumors in mice. 24 This method primarily focuses on accelerating the efficiency of CAR-T cell preparation. Although alginate was used, this study did not explore the effect of viscoelasticity on T cell activation, nor were there experimental data indicating an attempt to modulate the viscoelastic properties of alginate. We believe that the conclusions of our paper may be highly beneficial to their proposed system, which could potentially enhance CAR-T cell activation within their system by further modulating the viscoelastic properties of the alginate scaffold. Therefore, our objectives, experimental system, and findings differ from those presented in recent publications, providing new insights into the mechanochemical effects of viscoelastic artificial cells.

[0127] Stem cell-like T cells are a unique subset of T cells that possess characteristics of both stem cells and immune cells. They include TMSCs, naive T cells, and central memory T (TCM) cells. These cells can self-renew and differentiate into various effector T cell subsets and memory T cell subsets. 12, 57As disclosed in this paper, we demonstrate that CAR-T cells activated by SynVAC exhibit a higher proportion of cells expressing high levels of CCR7 across the entire RNA transcriptome. This observation is particularly important because cells expressing high levels of CCR7 are associated with TCM cells and TMSCs, which are known to exhibit increased self-renewal capacity, long-term persistence, and improved antitumor efficacy. The expansion of these CCR7-expressing subsets suggests that SynVAC may promote the generation of more potent CAR-T cells with a stronger ability to control tumor growth and achieve durable responses in cancer immunotherapy.

[0128] The observation that SynVAC significantly enhances the long-term persistence of CAR-T cells in a B-cell xenograft model has important implications for adoptive cell therapy. Persistence is a crucial factor influencing the therapeutic efficacy of CAR-T cells, as it determines the duration of their anti-tumor activity within the host. Long-term persistence of CAR-T cells allows for the sustained recognition and elimination of tumor cells, leading to a more effective and durable therapeutic response. This finding highlights the potential advantages of using SynVAC for CAR-T cell activation and expansion, as it may ultimately result in significantly enhanced clinical outcomes in patients receiving CAR-T cell therapy.

[0129] In an ovarian solid tumor xenograft model, we observed a remarkable reduction in tumor size when treated with SynVAC-activated CAR-T cells compared to the Dynabead-activated CAR-T cell and mediator control groups. Furthermore, we found a significant reduction in metastatic cancer cells in vital organs such as the lung, pancreas, and uterus in the SynVAC treatment group. These findings highlight the superior antitumor efficacy of SynVAC-activated CAR-T cells not only against primary tumors but also against metastatic cancer cells that have spread to distant organs. This enhanced therapeutic performance can be attributed to the significantly enhanced proliferation, persistence, and function of SynVAC-activated T cells. If these cells can effectively infiltrate and target tumor cells, this could ultimately lead to better tumor control and potentially prolong patient survival.

[0130] In summary, our publicly available data demonstrate the compelling potential of SynVAC as a powerful tool for T-cell activation and expansion. Our promising findings highlight the advantages of SynVAC over conventional Dynabeads, paving the way for more effective and durable CAR-T cell therapies and holding significant implications for the development of next-generation adoptive cell therapies, ultimately improving treatment outcomes and overall survival for cancer patients.

[0131] surface

[0132]

[0133] Table 1: Formulation of SynVAC and elastic beads.

[0134]

[0135] Table 2: Average percentage of 14 subpopulations based on scRNAseq data.

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[0201] Conclusion

[0202] The description of embodiments of the present invention concludes here. For purposes of illustration and description, the foregoing description of one or more embodiments of the invention has been presented. The invention is not intended to be exhaustive or to limit it to the precise forms disclosed. Many modifications and variations are possible in light of the foregoing teachings. All referenced publications are incorporated herein by reference to disclose and describe aspects, methods, and / or materials relating to the cited publications.

Claims

1. A composition comprising alginate microparticles, wherein the alginate microparticles: Contains alginate polymers with molecular weights ranging from 35 kDa to 600 kDa; A reagent containing ion-crosslinking agents for the alginate polymer; Coupled to one or more polypeptide ligands; and It shows a diameter of 5-20 micrometers.

2. The composition according to claim 1, wherein the alginate microparticles: It exhibits stiffness ranging from 1 kPa to 30 kPa under physiological conditions; Under physiological conditions, it exhibits stress relaxation times ranging from 5 seconds to 1000 seconds (t1 / 2 (s)); Under physiological conditions, it exhibits a loss modulus of 200 Pa to 6000 Pa at 1%–10% strain; Contains 10 4.5 -10 6.3 One polypeptide ligand / bead; and / or Peptide ligands that do not function in cell-cell or cell-extracellular matrix (ECM) adhesion.

3. The composition according to claim 1, wherein the reagent for ion-crosslinking the alginate polymer is present at a concentration of 10 mM to 100 mM.

4. The composition according to claim 3, wherein the reagent for ion-crosslinking the alginate polymer comprises a calcium-EDTA complex.

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

6. A method for preparing alginate microparticles having selected viscoelasticity and / or stiffness, comprising the following steps: Select the amount of alginate polymer with a selected molecular weight; The alginate polymer was placed in an aqueous solution; The aqueous solution is placed in a microfluidic device selected to utilize both the aqueous and oil phases. The alginate microparticles are formed by using pH-induced internal gelation of droplets.

7. The method of claim 6, wherein the aqueous solution comprises a calcium-EDTA complex of the alginate polymer ionically crosslinked.

8. The method of claim 6, wherein the method is selected to form alginate microparticles, the alginate microparticles being: It exhibits a diameter of 5-20 micrometers; It exhibits stiffness ranging from 1 kPa to 30 kPa under physiological conditions; Under physiological conditions, it exhibits stress relaxation times ranging from 5 to 1000 seconds (t1 / 2 (s)); and / or Under physiological conditions, it exhibits a loss modulus of 200 Pa to 6000 Pa at 1%–10% strain.

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

10. The method of claim 9, wherein the polypeptide ligand is coupled to the surface of the microparticle using a tetrazine-TCO click reaction.

11. A method for regulating the physiological activity of T cells, the method comprising combining the T cells with the alginate microparticles of claim 1, thereby regulating the physiological activity of the T cells.

12. The method of claim 11, wherein the T cells are 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 cells are obtained from a patient diagnosed with a malignant tumor.

15. The method of claim 11, wherein one or more polypeptide ligands bound to the surface of the microparticles comprise antibodies binding to CD28 and / or binding to CD3; and do not comprise polypeptide ligands (e.g., integrins) that function in cell-cell or cell-extracellular matrix (ECM) adhesion.

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

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 cells.

19. The method of claim 11, wherein the alginate microparticles: It exhibits stiffness ranging from 1 kPa to 30 kPa under physiological conditions; Under physiological conditions, it exhibits stress relaxation times ranging from 5 seconds to 1000 seconds (t1 / 2 (s)); Under physiological conditions, it exhibits a loss modulus of 200 Pa to 6000 Pa at 1%–10% strain; Contains 10 4.5 -10 6.3 One polypeptide ligand / bead; and Peptide ligands that do not function in cell-cell or cell-extracellular matrix (ECM) adhesion.

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

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