Biomimetics of immune synapses by graphene oxides

Abstract

A hierarchical 2-D artificial antigen-presenting structure (AAPS) was developed which comprises anti-CD3 (αCD3) and anti-CD28 (αCD28) anchored on graphene oxide (GO). Under rational design, the in-vitro interactions between the αCD3 / αCD28 / GO-AAPSs and T cells delicately mimics the immune synapses between the antigen-presenting cells (APC) and the T cells in vivo, featuring large contact areas as well as high accessibilities to the receptors. αCD3 / αCD28 / GO-AAPS as disclosed herein can stimulate human T cell proliferation and activation without adding external interleukin-2 (IL-2), and the efficiency was several magnitudes higher than the state-of-art commercial αCD3 / αCD28 / Dynabeads-AAPS. Moreover, the antibodies on GO can be programed in variety, density, hierarchy etc. Therefore, such 2-D AAPS provide a novel systematic platform for T cell stimulations.

Description

[0001] BIOMIMETICS OF IMMUNE SYNAPSES BY GRAPHENE OXIDES

[0002] Cross-Reference To Related Applications

[0003] This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application Serial No 63 / 582,680, filed on September 14, 2023, and entitled “BIOMIMETICS OF IMMUNE SYNAPSES BY GRAPHENE OXIDES” which application is incorporated by reference herein.

[0004] TECHNICAL FIELD

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

[0006] BACKGROUND OF THE INVENTION

[0007] T cell-based immunotherapy has gained significant attention due to its promising potential in cancer treatments, particularly following the approval of the first chimeric antigen receptor (CAR) T therapy by the Food and Drug Administration (FDA) in 2017.1 3The success of CAR-T cell therapy depends on the in vitro expansion of functional T cells and efficient transfer of CAR engineering genes facilitated by T cell activation.4-5Consequently, a robust and efficient protocol for activating and expanding T cells is needed in both academia and the medical industry.

[0008] A typical in vitro T cell activation and expansion protocol requires T-cell receptor (TCR) stimulation, ligand costimulation, and prosurvival cytokine supplementation [usually interleukin-2 (IL-2)|.6 9Current conventional in vitro T cell expansion methods use immobilized monoclonal aCD3 and aCD28 on plate substrates10or magnetic microbeads (Beads3728. Dynabeads, ThermoFisher)11’12, along with externally supplemented IL-2. Advanced antigen presentation platforms (APPs) have shown potential for improving T cell expansion efficiency by better mimicking the natural structure and function of antigen-presenting cells (APCs).12 1Examples include fluid lipid bilayers on mesoporous silica microrods12’14and carbon nanotube-polymer composites13. However, these state-of-the-art APPs, including Beads3128, have rigid structures that limit large surface contact formation as in the immunological synapse,1720hindering the complete mimicry of in vivo T cell activation. For example, while in vivo T cell activation can induce substantial IL-2 secretion from T cells for autonomous proliferation,21 23contemporary7in vitro APPs still require external IL-2 supplementation,12 16highlighting the gap between current methods and natural T cell activation achieved by forming immunological synapses with APCs.24 31

[0009] As noted above, human T cell-based therapy shows significant promise in the treatment of cancers and other diseases. In particular, chimeric antigen receptor (CAR)- engineered T (CAR-T) cells have been utilized as front-line therapy for cancers, especially hematopoietic malignancies. However, the conventional materials used to generate these CAR-T cells, such as monoclonal antibodies or Dynabeads, do not provide an architectural platform that optimally reproduces biological immunological synapse formation, a feature which limits their proliferative potential. In view7of this, there is a need in the art for the development of new materials and associated methods that more adequately reproduce biological immunological synapse formation and overcome limitations with conventional materials used to stimulate T cells.

[0010] SUMMARY OF THE INVENTION

[0011] As discussed below, we have developed a flexible graphene oxide antigen presenting platform (GO-APP) that anchors antibodies onto two-dimensional graphene oxides (GOs). By decorating anti-CD3 (aCD3) and anti-CD28 (aCD28) on GO (GO- APP328), we achieved remarkable T cell proliferation. In vitro interactions between GO-APP28and T cells closely mimic in vivo immunological synapses observed between antigen-presenting cells (APCs) and T cells, characterized by extensive contact areas, elevated antigen accessibility, and consequently, robust and sustained activation signals. This immunological synapse mimicry7exhibits a high capacity7for stimulating T cell proliferation while preserving the multifunctionality and high potency of T cells. Meanwhile, it enhances CAR gene engineering efficiency, yielding more than a 5-fold increase in CAR-T cell production compared to the standard protocol utilizing Dynabeads. Notably, GO-APP3 28stimulated appropriate autocrine interleukin-2 (IL-2) in T cells and overcame the in vitro reliance on external IL-2 supplementation, offering an opportunity to culture T cell-based products independent of IL-2 supplementation.

[0012] The invention disclosed herein has a number of embodiments that are based upon the above-noted studies on antibody decorated graphene oxides. For example, embodiments of the invention include compositions of matter comprising a graphene oxide material; a first antibody that selectively binds mammalian CD3 coupled to the graphene oxide material; and a second antibody that selectively binds mammalian CD28 also coupled to the graphene oxide material. As discussed below, there are a number of variations of this material. In illustrative embodiments, the graphene oxide material comprises graphene oxide sheets and / or flakes. Typically, the first antibody and / or the second antibody is coupled to the graphene oxide material via a crosslinker such as one comprising polyethylene glycol (PEG) linking moieties. In embodiments of the invention, the antibody density on the graphene oxide material can be designed to be at least 250 or 500 or 1000 molecules / pm2. In certain embodiments of the invention, the graphene oxide material is also coupled to a cytokine such as human SCF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), TPO (thrombopoietin), or IL-3 (interleukin 3).

[0013] In certain embodiments of the invention, the compositions comprise the graphene oxide materials discussed above combined together with one or more T cells. In embodiments of the invention, the T cell can be, for example, a CD4+ and / or CD8+ T cell or a combination of both (e.g.,ones obtained from a patient diagnosed with a disease or disorder). In some embodiments, the T cell is a CAR-T cell. In preferred graphene oxide compositions, the first and second antibodies are coupled to the graphene oxide material such that a contact area between a T-cell combined with the composition and the composition is at least 5, 10, 25, 50 or 75 pm2. In certain embodiments of the invention, when the graphene oxide composition coupled to the first and second antibody is combined with T cells in culture, the graphene oxide composition stimulates T at least a 50 fold increase in T cell numbers (optionally in the absence of exogenous interleukin-2) within 10, 11 or 12 days in a T cell tissue culture medium (e.g. one disclosed herein) as compared to a control T cell culture lacking the graphene oxide composition.

[0014] Embodiments of the invention also include methods of making the graphene oxide materials disclosed herein, for example by coupling a graphene oxide material to a first antibody that selectively binds mammalian CD3; and also coupling this graphene oxide material to a second antibody that selectively binds mammalian CD28; such that the composition is made. Certain embodiments of these methods include functionalizing the graphene oxide material with carboxyl groups and / or the use of selected linkers (e.g., PEG linkers selected to enhance flexibility) that couple the antibodies to the graphene oxide material. Optionally, the first or the second antibody is coupled to the graphene oxide material via an strepavidin biotin binding interaction. Typically in these methods, the first and / or second antibody is coupled to the graphene oxide material so that antibody density on the graphene oxide material is at least 250 or 500 or 1000 molecules / pm2In certain embodiments of the invention, the first and second antibodies are selectively disposed on the architecture of the graphene oxide material such that a contact area between a T-cell combined with the composition and the composition is at least 5, 10, 25, 50 or 75 pm2.

[0015] Embodiments of the invention include methods of stimulating T cell activation and / or proliferation comprising combining a T cell with an antibody coupled graphene oxide composition disclosed herein such that T cell activation and / or proliferation is stimulated. In certain methods of the invention, the graphene oxide material is selected to stimulate an al least a 50 fold increase in T cell numbers (optionally in the absence of exogenous interleukin-2). In embodiments the T cell can be one obtained from a patient diagnosed with a disease or disorder. In some embodiments of the invention, the disease or disorder is cancer and the T cell is a CAR-T cell. In summary, we have developed a composition designed so that the in-vitro interactions between antibody coupled graphene oxide materials and T cells delicately mimics the immune synapses between the antigen-presenting cells (APC) and the T cells in vivo. The antibody coupled graphene oxide materials disclosed herein can stimulate human T cell proliferation and activation without adding external interleukin- 2 (IL-2), and their efficiencies were surprisingly several magnitudes higher than the state-of-art commercially available material that is used to stimulate T cells (aCD3 / aCD28 / Dynabeads-AAPS). Moreover, the antibodies coupled to graphene oxide materials can be programed in variety, density, hierarchy etc. Therefore, such compositions and associated methods provide a novel systematic platform for a wide variety7of T cell stimulation methodologies.

[0016] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Fig. 1: The design and characterization of a graphene oxide antigen presenting platform (GO-APP) for displaying «CD3 and «CD28 (GO-APP3 28). a, Schematics showing that a T cell interacts with an APC expressing TCR cognate 3 / 28 peptide / MHC complex and CD80 / CD86 (CD28 ligand), with a GO-APP , with an 3 / 28 aCD3 / aCD28 antibody-coated Dynabead (Beads ). and with an aCD3 / aCD28 antibody-coated plate, b, Schematics showing the design and preparation of GO- APP3 28c, An SEM image showing the as-prepared graphene oxide (GO), d, A TEM image negatively stained with uranyl acetate showing the GO-linked protein on the flat region of the GOs. e-g, False colored low vacuum SEM images showing a fixed T cell 3 / 28

[0019] (blue) interacting with a piece of GO-APP (yellow) (e), a dendritic cell (green) (f), 3 / 28 and a Bead (red) (g).

[0020] 3 / 28

[0021] Fig. 2: The applications of GO-APP in T cell culture and CAR-T cell 3 / 28 manufacture, a-d, Study of GO-APP -activated T cell culture from healthy donor PBMCs. a, Experimental design of the T cell proliferation assay, b, T cell proliferation + +

[0022] (n = 4). c, Yield and CD4 and CD8 composition of cultured T cells on day 12 (n = 4).

[0023] Data from 4 different donors are presented, d, ELISA measurements of IL-2 production 3 / 28 in the culture on day 7 (n = 4). e-h, Study of GO-APP -activated T cell culture from

[0024] MACS-sorted CD4 or CD8 T cells, e, Experimental design, f. Sorted CD4 T cell + proliferation with the indicated activation methods (n = 4). g, Sorted CD8 T cell proliferation with the indicated activation methods (n = 4). h, ELISA measurements of IL-2 production in the indicated cultures on day 7 (n = 4). i-L Study of the use of GO- APP3 28in CAR-T cell manufacture, i, Experimental design of CAR-T cell manufacture 3 / 28 with GO-APP . j, Flow cytometry analysis of CAR expression on human T cells stimulated with the indicated reagents and transduced with a retroviral vector or lentivector encoding a BCMA CAR. Data were collected on day 7 in culture and day 5 post transduction, k, Flow cytometry quantification showing CAR (CD 19 CAR) transduction on cultured T cells post lentiviral vector transduction at the indicated 3 / 28 multiplicity of infection (MOI). 1, Plot of CD19 CAR-T cell production in GO-APP

[0025] T cells versus Beads3 28T cells. The seeding cell number was normalized to 1 x It)6cells per condition.

[0026] 3 / 28

[0027] Fig. 3: The applications of GO-APP in CAR-T cell therapy for cancer, a- 3 / 28 d. Study of in vitro GO-APP CAR-T cell tumor-killing efficacy, a, Experimental design of the short-term tumor-killing assay. Human MM. Is multiple myeloma tumor cells and BCMA-targeting CAR-T cells transduced with retroviral vector were used, b, Luciferase activity analysis of short-term in vitro tumor-killing of MM.ls-FG. Tumorkilling data were acquired after 24 hours. E:T = effector to tumor target cell number ratio. Effector cells were counted as total T cells (n = 3). c, Experimental design of the serial tumor-killing assay. Human Raji B cell lymphoma cells and CD19 CAR-targeting CAR-T cells transduced with lentiviral vector were used, d, Luciferase activity analysis of Raji-FG tumor-killing in the serial tumor cell-killing assay. Effector cells were i 3 / 28 counted as CAR cells (n=3 ). e-h, Study of the in vivo antitumor efficacy of GO-APP CAR-T cells, e, Experimental design, a human Raji B cell lymphoma xenograft NSG 3 / 28 mouse model was used here. (Vehicle / PBS: n = 5: GO-APP CAR-T cells and Beads3 28CAR-T cells: n = 6). f, Measurement of Raji-FG tumor load through bioluminescence imaging (BLI) over time, g, Survival analysis, h, Representative images of BLI over time.

[0028] 3 / 28

[0029] Fig. 4: Mechanism study of GO-APP -stimulated T cell activation, a-b, Gene profiling of PBMC-derived activated T cells using deep RNA sequencing. PBMCs from 3 random healthy donors were used, a, Principal component analysis of global gene expression using the first two principal components PCI and PC2. b, Heatmap showing the expression of genes related to T cell activation, T cell signalling, and the IL-2 signalling pathway, c-n, Gene profiling of PBMC-derived activated T cells using single-cell RNA sequencing (scRNA-seq). PBMCs from 2 random healthy donors were combined for this analysis. A total of 30185 cells were recorded in the analysis, c- g, scRNA-seq analysis of total T cells, c, UMAP plot of activated T cells showing the formation of 2 major cell clusters. Each dot represents a single cell and is colored 3 / 28 3z28 according to its activation method (blue: GO-APP , red: Beads ). Note that GO- 3'28 3 / 28

[0030] APP T cells mainly fall in cluster 2. and Beads T cells mainly fall in cluster 1. d, Ingenuity pathway analysis (IPA) showing pathways under common putative upstream regulators enriched in GO-APP3 28T cells compared to Beads3 28T cells. Color saturation indicates the p value of the expression difference, whereas dot size reflects the number of genes regulated under each upstream regulator, e-g, UMAP plots showing the expression of TCR (e), CD28 (f), and IL-2 (g) regulated pathways in 3 / 28 individual T cells. Note that all three major pathways were enriched in GO-APP T cells compared to Beads3 28T cells, h-k, scRNA-seq analysis of CD8 T cells, h-j, 3 / 28 +

[0031] UMAP plots showing 4 major cell clusters. Note that GO-APP CD8 T cells are 3 / 28 + mainly located in clusters 3 and 4; Beads CD8 T cells are mainly located in clusters 1 and 2. h, UMAP plot with annotations showing corresponding activation methods, i, +

[0032] UMAP plot showing the CD8 T cell activation module score. Note that T cells in clusters 3 and 4 exhibited high module scores, j, UMAP plot showing the cytokine production module score. Note that T cells in clusters 3 and 4 exhibit high module scores, k. Violin plots showing the expression distribution of the indicated genes in + +

[0033] CD8 T cells. Note that the indicated genes are related to CD8 T cell function and are highly expressed in GO-APP28CD8 T cells. 1-n, scRNA-seq analysis of CD4 T cells. 1-m, UMAP plot showing the formation of two major cell clusters. Note that GO- 3 / 28 + 3 / 28 +

[0034] APP CD4 T cells are mainly located in cluster 1 ; Beads CD4 T cells are mainly located in cluster 2. 1, UMAP plot with annotations showing corresponding activation + methods, m, UMAP plot showing the cytokine module score. Note that CD4 T cells in cluster 1 exhibit high scores, n, Violin plots showing the expression distribution of the + 4 indicated genes in CD4 T cells. Note that these genes are related to CD4 T cell

[0035] 3Z28 i 3 / 28 function and were highly expressed in GO-APP CD4 T cells compared to Beads +

[0036] CD4 T cells, o, Western blot showing the TCR and IL-2 signaling pathway molecules 3 / 28 3 / 28 in GO-APP T cells and Beads T cells at the protein level, p, Schematics showing 3 / 28

[0037] GO-APP -stimulated T cell activation through a TCR / CD3, CD28, and IL-2 signaling network.

[0038] Fig. 5: The design and characterization of GO-APP3 / 28. (Related to Fig. 1) a, An AFM image showing a representative as-prepared graphene oxide (GO) piece without any decoration, b, A representative AFM image showing a piece of modified GO decorated with antibodies (GO-APP3 28). c, UV-vis spectra analysis of the intermediate structures in the GO-APP3728preparation, d. Schematic of a spherical cap model for calculating the GO-APP3 28contact area on T-cells; see Methods for details.

[0039] Fig. 6: Method development of GO-APP3728in T cell culture. (Related to Fig. 1) a-d, Comparison of T-cell activations using GO-APP3 28and the mixture of GO- APP3and GO-APP28. GO-APP3 28showed superior performance, indicating the significance of concurrent CD3 and CD28 stimulation in close proximity for effective T cell activation, a, Experimental design, b. T cell proliferation (n = 4) c, ELISA measurements of IL-2 production in the culture on day 7 (n = 4). d, ELISA measurements of IFNy production in the culture on day 7 (n = 4). e-g, SEM images showing the as-prepared GO with different sizes: < 5 pm (e), 11.8 ± 2.6 pm (1), and > 80 pm (g). Diameter of pillar reference: 5 pm. (Related to Fig. 1c) h, A false-colored low vacuum SEM image showing oversized (>80 pm) GO-APP3 28sheets (yellow) enveloping multiple T cells (blue). (Related to Fig. le) i, Comparison of T-cell proliferation using GO-APP3728with different sizes, j, Gradient test of GO-APP3728concentration in stimulating T cell expansion. Seeding cell number was normalized to 1 x 10 cells per condition.

[0040] Fig. 7: The applications of GO-APP3728in T cell culture. (Related to Fig. 2) a-f, Study of GO-APP3 28-activated T cell culture from healthy donor PBMCs. a, ELISA measurements of IFN-y production on day 7 (n = 4). (Related to Fig. 2d) b, Flow cytometry analysis of surface CD25 (IL-2 receptor) expression on T cells stimulated with the indicated methods on day 7 (n = 4). (Related to Fig. 2d) c, Flow cytometry analysis of CD4+and CD8+populations in cultured T cells on day 12. (Related to Fig. 2c) d-f, Flow cytometry analysis showing the IL-2 intracellular expression levels in T cells stimulated by the indicated methods on day 7 with gating on CD4+(d) and CD8+(e) populations, f, Quantification of (d) and (e). g-h, Study of T cell culture from PBMCs wi th a titration of external IL-2, g, Experimental design, h, T cell proliferation on day 12 (n = 4). Fig. 8: Single-cell gene profiling of GO-APP3 / 28-stimulated T cell activation. (Related to Fig. 3) a, Gene set enrichment analysis (GSEA) of CD8+T cells for CD8+T cell activation. (Related to Fig. 3i) b. GSEA of CD8+T cells for cytokine production. (Related to Fig. 3j) c, GSEA of CD4+T cells for cytokine production. (Related to Fig. 3m) d, Violin plot showing the expression distribution of the indicated genes encoding the master transcription factors of different Th subtypes in CD4+T cells. (TBX21: Tnl; GATA3: TH2; Foxp3: Treg; RORC: TH 17).

[0041] Fig. 9: Mechanism study of GO-APP3 / 28-stimulated T cell activation at the protein expression level. (Related to Fig. 3) a-b, Flow cytometry analysis of GO- APP3 28T cells and Beads3 28T cells, a, Flow plots showing the indicated intracellular protein expression, b, Mean fluorescence intensity (MFI) measurement from flow cytometry showing the TCR and IL-2 signaling pathway molecules in GO-APP3 28T cells and Beads3 28T cells at the protein level. (Related to Fig. 4o).

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention.

[0044] Two-dimensional (2D) materials, such as graphene oxides, possess high flexibility and great surface modification potential, making them ideal candidates for flexible biomaterials. '’2 54As disclosed herein, we have developed a highly flexible graphene oxide antigen presentation platform (GO-APP) that effectively mimics in vivo immunological synapses (Fig. la). In embodiments of the invention, we conjugated both anti-CD3 (aCD3) and anti-CD28 (aCD28) to pliable GO sheets using supple polyethylene glycol (PEG) linkers, establishing hierarchical flexibility in the GO- APP3 28construct. This design provides extensive interface areas and enhanced accessibility for simultaneous engagement of aCD3 and aCD28, leading to superior T cell activation (Fig. lb).

[0045] As discussed below, the invention disclosed herein has a number of embodiments. For example, embodiments of the invention include compositions of matter comprising a graphene oxide material; a first antibody that selectively binds mammalian CD3 coupled to the graphene oxide material; and a second antibody that selectively binds mammalian CD28 also coupled to the graphene oxide material. As discussed below, there are a number of variations / embodiments of the graphene oxide material. In illustrative embodiments, the graphene oxide material comprises graphene oxide sheets and / or flakes. Typically, the first antibody and / or the second antibody is coupled to the graphene oxide material via a crosslinker such as one comprising polyethylene glycol (PEG) linking moieties. In embodiments of the invention, the antibody density on the graphene oxide material can be designed to be at least 250 or 500 or 1000 molecules / pm2In certain embodiments of the invention, the graphene oxide material coupled to another polypeptide such as a cytokine, for example human SCF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), TPO (thrombopoietin), or IL-3 (interleukin 3).

[0046] In certain embodiments of the invention, the compositions comprise the graphene oxide materials discussed above combined together with a T cell. In embodiments of the invention, the T cell can be, for example, a CD8+T cell (e.g., T cell obtained from a patient diagnosed with a disease or disorder). In some embodiments, the T cell has been engineered to comprise an exogenous polynucleotide sequence, for example a CAR-T cell. In preferred graphene oxide compositions, the first and second antibodies are coupled to the graphene oxide material such that a contact area between a T-cell combined with the composition and the composition is at least 5. 10. 25, 50 or 75 pm2. In certain embodiments of the invention, when the graphene oxide composition coupled to the first and second antibody is combined with T cells in culture, the graphene oxide composition stimulates T at least a 50-fold increase in T cell numbers in an in vitro culture, (optionally in the absence of exogenous interleukin-2).

[0047] Embodiments of the invention also include methods of making the graphene oxide materials disclosed herein, for example by coupling a graphene oxide material to a first antibody that selectively binds mammalian CD3; and also coupling this graphene oxide material to a second antibody that selectively binds mammalian CD28; such that the composition is made. Certain embodiments of these methods include functionalizing the graphene oxide material with carboxyl groups and / or the use of selected linkers (e.g., PEG linkers selected to enhance flexibility) that couple the antibodies to the graphene oxide material. Optionally, the first or the second antibody is coupled to the graphene oxide material via an avidin biotin binding interaction. Typically in these methods, the first and / or second antibody is coupled to the graphene oxide material so that antibody density on the graphene oxide material is at least 250 or 500 or 750 or 1000 molecules / pm2. In certain embodiments of the invention, the first and second antibodies are selectively disposed on the architecture of the graphene oxide material such that a contact area between a T-cell combined with the composition and the composition is at least 5, 10, 25, 50 or 75 pm2.

[0048] Embodiments of the invention include methods of stimulating T cell activation and / or proliferation using the materials disclosed herein. Typically such methods comprise combining a T cell with an antibody coupled graphene oxide composition disclosed herein such that T cell activation and / or proliferation is stimulated (for example in the in vitro culture conditions discussed below). In certain methods of the invention, the graphene oxide material is selected to stimulate an at least a 50 fold increase in T cell numbers (optionally in the absence of exogenous interleukin-2). In embodiments the T cell can be one obtained from a patient diagnosed with a disease or disorder. In some embodiments of the invention, the disease or disorder is cancer and the T cell is a CAR-T cell.

[0049] In one illustrative embodiment of the invention, we synthesized GO sheets using an enhanced Hammer's method,34achieving a mean size of 11.8 ± 2.6 pm (averaging the longest and shortest diameters of 100 GO sheets, Fig. 1c) and a thickness of 1.0 ± 0.2 nm (Fig. 5a) for the predominantly single-layered sheets, which exhibited exceptional flexibility.35The GO surface was functionalized with carboxyl groups via chloroacetic acid treatment, and the successful functionalization was substantiated by consistent UV-visible spectra with reported data (Fig. 5c).33Subsequently, maleimide- (polyethylene glycol)-amine (NH2-PEG-MAL) linkers were conjugated through EDC / NHS coupling. Thiolated streptavidin was then attached, yielding a density of -1216 molecules / pm2Go (determined by BCA protein assay; all densities on GO refer to a single side). Equal amounts of biotinylated aCD3 and aCD28 antibodies (Abs) were conjugated to streptavidin, resulting in an antibody density of -1119 molecules / pm2Go on GO-APP3 / 28(determined by BCA protein assay). This value was consistent with the density estimated from transmission electron microscopy (TEM) imaging (-1043 molecules / pm2Go) after uranyl acetate negative staining (Fig. Id). The final GO- APP3'28thickness increased to 13.2 ± 4.2 nm (Fig. 5b).

[0050] GO-APP3 / 28has been optimized to closely resemble the bona fide immunological synapse. Natural immunological synapses require concurrent CD3 and CD28 stimulation in close proximity for effective T cell activation / 1 9which was corroborated in GO-APP3 / 28with regards to T cell expansion and cytokine secretion (Fig. 6a-d). A high density of bound receptors at the immunological synapse benefits T cell activation.36’37In our design, the density of aCD3 and aCD28 (-559.32 molecules / pm2Go each) accommodates human physiological TCR density (150-570 molecules / pm2),38 41required human pMHC surface density for TCR activation (90- 140 molecules / pm2minimum,42-500 molecules / pm2saturated43), and human physiological CD28 density (100-610 molecules / pm2).38,40’44We further optimized the size of GO-APP3 28, as the contact area plays a crucial role.37GO-APP~28offers extensive conformal contact with T-cells, closely mirrors in vivo APC-T-cell interactions and results in highly effective T-cell activation. (Fig. le). The contact area between the T-cell and GO-APP3 / 28is -80.86 pm2(Fig. 5d), closely resembling the -70 pm2immunological synapse between APC and T-cell (Fig. If) ,14-45-4and considerably greater than the -2.54 gm2contact area between the T-cell and the rigid, spherical commercial Beads3728(Fig. 1g). We observed that both overly small (< 5 pm) and large (>80 pm) GO-APP3728sheets resulted in decreased T cell expansion (Fig. 6e-i). with oversized (>80 pm) GO-APP3 28sheets enveloping multiple T cells. Finally, gradient levels of GO-APP3 28concentration were tested for human T cell expansion, and 0.1 pg / ml was optimal (Fig. 6j). Through the design of GO-APP3728to better mimic the immunological synapse, we have developed a GO-APP3 / 28-based protocol for effective T cell activation and expansion.

[0051] We assessed the capacity of GO-APP3 28for human T-cell activation and expansion in vitro, comparing it to Beads3728and other control methods, with and without external IL-2 supplementation (Fig. 2a). GO-APP3728and Beads3 28displayed superior T cell activation to the controls, with GO-APP3728consistently outperforming Beads3 28(Fig. 2b-c). In a 12-day human peripheral blood mononuclear cell (PBMC) culture with IL- 2 supplementation (30 U / ml, per Beads3728vendor protocol)12,14, GO-APP3728facilitated a more than 100-fold increase in T cell expansion. Comparatively, Beads3'28induced an -40-fold increase (Fig. 2b). Notably, GO-APP3 28was the only method to induce substantial T-cell expansion without IL-2 supplementation (Fig. 2b). Proper in vivo T cell activation through tumor recognition and infection elicits endogenous IL-2 secretion, especially from the CD4+population.21 23Such natural IL-2 production is essential for promoting proliferation, effector function and memory formation.21 23Elevated IL-2 expression is associated with superior T cell stimulation, and the extent of IL-2 production is frequently utilized to assess T cell activation.47The natural machinery of IL-2 production through activation can be achieved with GO-APP3728, likely due to its biomimicry of immunological synapses. Consequently, GO-APP3728effectively eliminated the in vitro dependence on external IL-2 supplementation for T cell activation and expansion. T cells activated by GO-APP3 28(denoted as GO-APP3 28T cells) showed higher levels of IL-2 and interferon-gamma (IFN-y) production, as well as elevated CD25 expression (Fig. 2d, Fig. 7a-b), regardless of IL-2 supplementation. GO-APP3728T cells exhibited a higher proportion of CD4+cells (-64.4% after 12 days) than Beads3728T cells (Fig. 2c, Fig. 7c). Robust and sustained T cell stimuli have been reported to promote a higher ratio of CD4+T cells,1248which can offer benefits in tumor suppression under certain severe cancer scenarios.49’50GO-APP3 28CD4+T cells also exhibited over twofold elevated intracellular IL-2 production compared to Beads3 28CD4+T cells. (Fig. 7d-f).

[0052] We utilized magnetic-activated cell sorting (MACS) to isolate CD4+and CD8+T cells (Fig. 2e) for further studies. Stimulation with GO-APP3 28resulted in enhanced T cell expansion compared to Beads3 28for both sorted CD4+T cells (Fig. 21) and sorted CD8+T cells (Fig. 2g). The overall high expansion of GO-APP3 28CD4+T cells, especially in the absence of IL-2 supplementation, could be attributed to high-level IL- 2 secretion (Fig. 2h) enabled by GO-APP3 28. In contrast, IL-2 production by GO- APP3 28CD8+T cells was comparatively lower, and the expansion of these cells could still benefit from IL-2 supplementation (Fig. 2g-h). To further understand the pivotal role of IL-2 in the GO-APP3 28system, we performed T cell culture studies with a titration of externally supplemented IL-2 (Fig. 7g). In this study, Beads3 28T cell expansion plateaued at 300-3000 U / ml, approaching but not fully achieving the performance of the GO-APP3 28system, which required no IL-2 supplementation (Fig. 7h). Notably, such high levels of IL-2 supplementation to boost Beads3728performance far exceeded the level (30 U / ml) observed in serum during clinical IL-2 therapy, potentially causing toxicity.51 53Moreover, in vitro T cell cultures exposed to excessive IL-2 levels exhibited reduced cytotoxic activity’.54The Beads3728protocol recommended a modest 30 U / ml IL-2 for in vitro cultures.12 14Presuming the IL-2 retains wild-type specific activity of 15 x 106U / mg,55,56IL-2 secretion in GO-APP3728T cells was determined to be ~36 U / ml (Fig. 2d), which aligned with the commonly recommended supplementing concentration (30 U / ml) and was much lower than the 300-3000 U / ml dosage required for Beads3728to approach comparable performance. Therefore, robust natural IL-2 production enabled by GO-APP3728was a pivotal but not the sole explanation for the dramatic activation performance differences between GO-APP3728and Beads3 28. We assessed the therapeutic potential of GO-APP3 28for improving CAR-T cell cancer therapy. GO-APP3 28T cells and Beads3728T cells were subjected to ^-retroviral and lentiviral CAR transduction for tumor-killing assays (Fig. 2i). (Hereafter, all GO- APP3 28T cells were expanded without IL-2 supplementation, while all Beads3728T cells were expanded with 30 U / ml IL-2 supplementation) Efficient transduction for both y- retroviruses57and lentiviruses58,59requires actively dividing T cells, particularly for retroviruses57. Enhanced CD3 and CD28 stimulation promote cell cycle entry for resting T cells.60,61Our results showed that GO-APP3728T cells exhibited superior transduction efficiency for both retroviral and lentiviral transduction compared to Beads3728T cells (Fig. 2j), implying that GO-APP3 28can provide stronger CD3 and CD28 stimulation. Using varying multiplicities of infection (MOIs), we observed that GO-APP3728T cells showed even better CAR transduction (47.2%) at MOI=1 than Beads3 28T cells (33.9%) at MO1=12 (Fig. 2k). This impressive efficiency at low MOIs could reduce viral vector usage and lower CAR-T manufacturing costs. Ultimately, the enhanced T cell expansion and CAR transduction from GO-APP3 28activation resulted in a more than 5-fold increase in CAR-T cell yield compared to the Beads3728method (Fig. 21).

[0053] Next, GO-APP3728CAR-T cells were subjected to tumor-killing assays to evaluate their functionality both in vitro and in vivo. We first used a short-term tumor-killing assay in which the GO-APP3 28CAR-T cells outperformed Beads3728CAR-T cells (Fig. 3a-b). This enhanced short-term killing performance was likely attributed to the improved CAR transduction in the GO-APP3 28CAR-T product relative to the Beads3 28product (Fig. 2j -1). In a more challenging serial tumor-killing assay, lentiviral vector- transduced GO-APP3728CAR-T cells demonstrated superior tumor suppression on days 3 and 5 post-tumor rechallenge when compared to Beads3728CAR-T cells (Fig 3c-d). To further validate the in vivo tumor-killing capacity of GO-APP3728CAR-T cells, we used a Raji human B cell lymphoma xenografted NSG mouse model (Fig. 3e). Raji cells exhibited aggressive in vivo grow th, killing vehicle-treated mice within 3 weeks, during which both GO-APP3728CAR-T cells and Beads3728CAR-T cells suppressed tumor growth well. The superior performance of GO-APP3 28CAR-T cells over Beads3728CAR-T cells became apparent beyond three weeks, with a significantly lower tumor load observed in the GO-APP'728CAR-T-treated group (Fig. 3f and h). Moreover, the survival time of mice was considerably extended in the GO-APP3 28CAR-T-treated group compared to both the Beads3 28CAR-T-treated group and the vehicle-treated group (Fig. 3g). These tumor-killing results highlighted the potential application of GO- APP3 28in CAR-T cell production and the performance of GO-APP3 28CAR-T cells for cell therapy against cancer.

[0054] Several mechanism studies were undertaken to explore the T cell activation driven by GO-APP3 28stimulation in close comparison to Beads3 28. Initially, deep RNA sequencing was carried out with GO-APP3 28T cells and Beads3 28T cells. T cells from three healthy donors were sequenced, and principal component analysis (PCA) revealed that the overall gene profiles of GO-APP3 28T cells significantly differed from those of Beads3728T cells (Fig. 4a). Heatmaps of representative gene expression demonstrated that, in comparison to Beads3728stimulation, GO-APP3728stimulation significantly upregulated genes related to phenotypes of activated T cell transcription factors crucial for T cell signaling and IL-2 signaling pathways (Fig. 4b).

[0055] The activation mechanisms of GO-APP3728and Beads3 28on T cells were further examined through single-cell RNA sequencing (scRNA-seq). Uniform Manifold Approximation and Projection (UMAP) analysis of the T cells exposed two major cell clusters: one activated by GO-APP3728and the other by Beads3728(Fig. 4c). Ingenuity pathway analysis (IPA) comparing GO-APP3 28T cells with Beads3728T cells revealed the top 10 differentially regulated pathways ranked by significance (p value), in which TCR, CD3, IL-2 and CD28 were highlighted among others (Fig. 4d). The upregulation of these pathways in GO-APP3728T cells aligned with our in vitro findings that GO- APP3 28provided more robust activation and expansion of T cells, along with a two- magnitude increase in endogenous IL-2 secretion compared to Beads3'28T cells (Fig. 2d). In addition, Myc-regulated pathways were found to be enriched in GO-APP3728compared to Beads3728. Myc expression closely correlates with cell proliferation and regulates cell cycle progression.62This pathway enrichment corroborated our in vitro results, further highlighting the proliferation advantage of GO- APP '28T cells. We then computed signature gene sets under the TCR, CD28, and IL-2 pathways into three module scores and plotted their levels onto UMAP. The results revealed that genes within these three pathways were significantly enriched in GO-APP3 28T cells compared to Beads3728T cells (Fig. 4e-g). To better comprehend how GO-APP3728- mediated activation impacted CD8+and CD4+T cell components, we extracted CD8+and CD4 T cell populations and analyzed them separately. The UMAP results again showed distinct clustering of GO-APP3 28T cells away from Beads3728T cells within both the CD8+and CD4+populations (Fig. 4h and 1). Superior cytokine production along with a significantly higher CD8+T cell activation signature was present in the gene profile of GO-APP3728T cells in contrast to that of Beads3728T cells (Fig. 4h-j, Fig. 8a-b).63Gene expression analysis pertaining to CD8+T cell function further revealed the enhanced multifunctional effector-like phenotype of GO-APP3'28CD8+T cells simultaneously producing IFN-y, TNFa and cytotoxic molecules (Fig. 4k). For CD4+T cells, the most notable signature unique to GO-APP3728stimulation was enhanced cytokine production,64underscored by IL-2 production, which was barely detectable in Beads3728CD4+T cells (Fig. 41-m, Data Fig. 8c). The overall T helper (Tn) functionality was also improved by GO-APP3 28, as evidenced by the upregulation of genes such as CD40LG. CLL3, and CLL4 (Fig. 4n).65,66It has been reported that IL-2 and IL-2 receptors are associated with the development of Tregcells, and the anti-inflammatory nature of Treg cells is unfavorable in cell therapy since tumor eradication ty pically requires a proinflammatory environment.31,67The expression of signature transcription factors pertained to TH subsets was analyzed, in which GO-APP3 28was found to primarily promote TH I and TH2 functions in CD4+T cells, without any polarization toward THI 7 or Tregcells (Fig. 8d). Sequencing data offered insights into the mechanism of GO-APP3728-mediated T cell activation at the mRNA level. Western blot (Fig. 4o) and flow cytometry (Fig. 9) results further corroborated the sequencing findings by revealing elevated protein expression and phosphorylation in GO-APP3728for representative genes, including many signature transcription factors involved in the TCR, CD28, and IL-2 signaling pathways. The mechanism of action for GO-APP3 28- enabled T cell activation is illustrated in Figure 4p.

[0056] In conclusion, we have successfully developed GO-APP. an antigen-presenting platform based on GO, that was flexible in design, robust in manufacture, and versatile in application. A potential application involves docking aCD3 and aCD28 Abs on GO- APP (GO-APP3 28), thereby achieving biomimicry of immunological synapses for T cell product manufacture. GO-APP3 28shows a high capacity for stimulating T cell expansion while preserving potent effector function. Furthermore, it also enables high efficacy in CAR gene engineering for T cells. Notably, GO-APP3728stimulates strong autocrine IL-2 production, overcoming the dependency of the current state-of-the-art Beads3 28on IL-2 supplementation. This results in exceptional T cell expansion and a multifunctional effector-like phenotype, which translates into better tumor-killing efficacy in vitro and in vivo. One potential constraint of the current GO-APP3728is the separation from the final cell product. The biocompatibility of GO mitigates this concern,68and we observed no toxicity' induced by GO-APP3 28in vitro and in vivo. A viable solution for separation could involve incorporating magnetic nanoparticles onto GO-APP3 28.69GO-APP represents a new approach to explore and modulate the interactions of signaling proteins on the cytomembrane. The ability to successfully design and optimize antigen presentation on GO-APP paves the way for an array of opportunities to develop novel structures for immunological research and therapeutic applications.

[0057] Illustrative Methods

[0058] Graphene oxide (GO) synthesis

[0059] GO was synthesized via an improved Hammer's method.34A mixture of 360 ml H2SO4 (18.4 M), 40 ml H3PO4 (14.8 M), and 3 g graphite flakes was combined, and 18 g KMnO4 was gradually added in six equal portions. The temperature was maintained below 35 °C to mitigate exothermic effects. The reaction was heated to 50 °C and stirred for 12 hours, then cooled to 20 °C and mixed with ~400 ml ice and 3 ml 30% H2O2. The solution was filtered through an 8.0 gm Cyclopore Polycarbonate Membrane and washed using centrifugation and redispersion. The filtrate was washed three times with 10% HC1 and subsequently with water until a neutral pH was achieved. Finally, dialysis was performed for one week using a cellulose membrane (Sigma, D9527), with water changes occurring at least twice daily. Reduplicative rate-zonal centrifugation and selection were precisely implemented until a uniform and narrowly distributed GO size range was achieved.

[0060] GO activation

[0061] Following the preparation of GO, its epoxide and hydroxyl groups were transformed into carboxyl groups via chloroacetic acid under alkaline conditions.33A 0. 16 M NaOH solution was prepared by dissolving 4.8 g NaOH in 10 ml water, which was subsequently combined with 30 ml of 0.5 mg / ml GO. Next, 0.75 g chloroacetic acid was dissolved in 5 ml water and incrementally introduced to the GO mixture. The reaction proceeded under gentle agitation overnight, and the product was purified via multiple centrifugation steps until the pH of the supernatant achieved neutrality.

[0062] NHS / EDC coupling of the MAL-PEG-NH2 linker

[0063] Five milligrams (mass referred to as-dialyzed GO before other treatments) of activated GO was dispersed in 8 ml of 10 mM MES buffer (pH=6). EDC (0. 1 mg) was dissolved in 1 ml of 10 mM MES buffer (pH=6). Sulfo-NHS (0.3 mg) was dissolved in 1 ml of 10 mM MES buffer (pH=6). The as-prepared EDC and Sulfo-NHS solutions were added to the GO mixture and reacted for 20 min under shaking. Then, the mixture was centrifuged and washed with 10 mM PB buffer (pH=7.2). After washing, the mixture was dispersed in 9 ml of 10 mM PB buffer (pH=7.2). MAL-PEG-NH2 (0.3 mg. Mw = 2 K, Advanced Biochemicals, HEP0201) was dissolved in 1 ml of 10 mM PB buffer (pH=7.2). The as-prepared MAL-PEG-NH2 solution was added to the GO mixture and shaken for 2 hours. The mixture was then centrifuged and quenched by 0. 1 mg hydroxylamine in 5 ml PB buffer (pH=7.2) under shaking for 15 min. Next, the mixture was centrifuged and dispersed in PBS buffer (pH=7.4).

[0064] MAL / SH coupling of the streptavidin-thiol

[0065] Five milligrams (mass referred to as-dialyzed GO before other treatments) of GO coupled with the MAL-PEG-NH2 linker (Mw = 2 K) was dispersed in 10 ml of PBS, and then 1 ml of 1 mg / ml streptavidin-thiol (Mw = ~56 kDa) was added. The mixture was gently wobbled for 2 hours at 4 °C and then centrifuged. The amount of streptavidin-thiol remaining in the supernatant was examined by Thermo Fisher Micro BCA Protein Assay. The specific surface area of GO is -1760 m2 / g.70-71The number of streptavidin-thiol linked on 5 mg GO was 1.07 * 1016, and therefore, the average density was 1215.91 molecules / pm2Go. The precipitate was washed by repeated centrifugation in PBS buffer (pH=7.4).

[0066] Linking anti-CD3 (aCD3) and anti-CD28 (aCD28) to streptavidin

[0067] Five milligrams (mass referred to as-dialyzed GO before other treatments) of GO coupled with streptavidin-thiol was precipitated by centrifugation. Ten milliliters of 0.5 mg / ml biotin anti-human aCD3 (OKT3, 3-6 biotin molecules per antibody, Biolegend) and 10 ml of 0.5 mg / ml biotin anti-human aCD28 (CD28.2, 3-6 biotin molecules per antibody, Biolegend) were added. The mixture was dispersed through gentle shaking and pipette mixing. Then, it was gently wobbled at 4 °C for 1 hour. After that, the mixture was centrifuged, and the amount of aCD3 and aCD28 remaining in the supernatant was examined by Thermo Fisher Micro BCA Protein Assay. The antibody density was determined to be ~1118.64 / pm2Go on GO-APP3 28. Therefore, the aCD3 density on GO-APP3 28and the aCD28 density on GO-APP3 28were each ~559.32 / pm2Go. The precipitate was washed by repeated centrifugation in PBS buffer (pH=7.4) and finally dispersed in 50 ml CI O media (complete lymphocyte culture media; see below for the recipe). Negative staining of aCD3 and aCD28 on GO-APP3728

[0068] The TEM grid was treated with H2 / O2 plasma using a Gatan SOLARUS Plasma System. Water-dispersed GO-APP3 28was drop loaded on the TEM grid, and the droplet on the grid was absorbed away from the side by filter paper after ~1 min. The loading of GO-APP was repeated several times. Then, the grid was washed with water and 2% uranyl acetate 1 time (dropped on the grid and then immediately absorbed away from the side by filter paper). After that, 2% uranyl acetate was dropped on the TEM grid. After 1 min, most uranyl acetates were absorbed away from the side by filter paper, leaving the grid wet but without apparent droplets. The grid was dried in air and then ready for TEM imaging under a Titan S / TEM (FEI).

[0069] Low-vacuum Scanning Electron Microscopy (LV-SEM) imaging

[0070] The SEM buffer with pH 7.4 was made with 0.1 M Na-phosphate buffer containing 0.1 M sucrose. The cells were rinsed with warm HBSS. Then, they were fixed with warm 3% glutaraldehyde in SEM buffer, moved to 4 degrees, and stored overnight. The next day, the cells were washed with SEM buffer 2 times for 5 minutes each time. Next, they were fixed with 2% osmium tetroxide in SEM buffer on ice for 1 hour and then washed with SEM buffer 2 times for 5 minutes each time. The cells w ere dehydrated with 50%, 70%, 95%, 100%, and 100% ethanol successively for 15 minutes each time. The final 100% ethanol was replaced with hexamethyldisilazane and then evaporated in the hood. The processed cells were then ready for LV-SEM imaging, which was conducted on an FEI Nova Nano 230 SEM.

[0071] Mice

[0072] NOD.Cg-PrkdcSCIDI12rgtmlwjl / SzJ (NOD / SCID / IL-2Ry7, NSG) mice were maintained in the animal facilities of the University of California, Los Angeles (UCLA). Six- to ten-week-old mice were used for all experiments unless otherwise indicated. All animal experiments were approved by the Institutional Animal Care and Use Committee of UCLA. All mice w ere bred and maintained under specific pathogen-free conditions, and all experiments were conducted in accordance with the animal care and use regulations of the Division of Laboratory7Animal Medicine (DLAM) at UCLA.

[0073] Cell Lines

[0074] The human multiple myeloma cancer cell line MM. I S and B lymphoma Raji were purchased from the American Type Culture Collection (ATCC). Both MM. IS and Raji cells were cultured in RIO media. To make stable tumor cell lines overexpressing firefly luciferase and enhanced green fluorescence protein (Fluc-EGFP) dual reporters, the parental tumor cell lines were transduced with lentiviral vectors encoding the intended gene(s)28. At 72 h post lentivector transduction, cells were subjected to flow cy tometry7sorting to isolate gene-engineered cells for making stable cell lines. Two stable tumor cell lines were generated for this study: MM.1S-FG and Raji-FG.

[0075] Human T cell culture from PBMCs

[0076] Human PBMCs were obtained from the CFAR Gene and Cellular Therapy Core Laboratory at UCLA, without identification information under federal and state regulations. To activate and expand T cells, PBMCs were cultured with Dynabeads™ Human T-Activator CD3 / CD28 (Gibco Thermo Fisher, referred to as Beads3 28in this manuscript) according to the manufacturer’s instructions or with GO-APP'28at the indicated concentrations. aCD3 Ab-coated wells, aCD3 and aCD28 Ab-coated wells, and uncoated wells were included as controls. Complete lymphocyte culture media (denoted as CIO media) was used here for all PBMC -related cultures. CIO was made of RPMI 1640 supplemented with FBS (10% vol / vol), P / S / G (1% vol / vol), MEM NEAA (1% vol / vol), HEPES (10 mM), sodium pyruvate (1 mM), b-ME (50 mM), and Normocin (100 mg / ml).

[0077] Flow cytometry

[0078] Fluorochrome-conjugated antibodies specific for CD3 (OKT3), CD4 (Clone OKT4). CD8 (Clone SKI), CD25 (BC96), CD69 (Clone FN50), CD62L (Clone DREG- 56), PD-1 (clone EH12.2H7), IFN-y (Clone B27), granzyme B (Clone QA16A02), perforin (Clone dG9), TNF-a (Clone Mabl l), IL-2 (Clone MQ1-17H12), and antirabbit IgG (Clone Poly4064) were purchased from BioLegend. Primary antibodies specific for Jak3 (EP909Y), STAT3 (EPR787Y). p-STAT3 (EP2147Y), p-c-Jun (Y172). c-Jun (EP693Y), p-c-Fos (cFosS32-BA9), and c-Fos (EPR20769) were purchased from Abeam. The Human Fc Receptor-blocking Solution (TrueStain FcX) was purchased from BioLegend. Fixable Viability Dye eFluor506 (e506) was purchased from Affymetrix eBioscience. Samples were stained with Fixable Viability Dye eFluor506 (e506) mixed with Human Fc Receptor-blocking Solution (TrueStain FcX), followed by PBS washing to remove e506 and blocking antibodies. Antibody staining was added to all samples at specified dilutions according to the manufacturer’s instructions. Intracellular cytokines, cell signaling components, and transcription factors were stained using a Cell Fixation / Permeabilization Kit (BD Biosciences). Flow cytometry was performed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech), and FlowJo (versions 9 and 10) was used for data analysis.

[0079] Enzyme-linked immunosorbent cytokine assays (ELISA)

[0080] ELIS As for detecting human cytokines were performed following the standard protocol from BD Biosciences. The capture and biotinylated antibody pairs for detecting human IFN-y and IL-2 were purchased from BD Biosciences. The streptavidin-HRP conjugate was purchased from Invitrogen. Human IFN-y and IL-2 standards were purchased from eBioscience. The tetramethylbenzidine (TMB) substrate was purchased from KPL. Culture supernatants were collected and assayed to measure the presence of IFN-y and IL-2 cytokines. Samples were analyzed for absorbance at 450 nm using an Infinite M1000 microplate reader (Tecan).

[0081] Lentiviral vector construction and transduction

[0082] The lentiviral vectors used in this study were constructed from the parental lentivector pMNDW containing the MND retroviral LTR U2 region as an internal promoter and an additional truncated Woodchuck Responsive Element (WPRE) to stabilize viral mRNA.72The pMNDW lentivector mediates high and stable expression of transgenes both in humans and in progeny human immune cells.73To construct the Lenti / BCMACAR-EGFR vector, a synthetic bicistronic DNA fragment encoding BCMACAR-F2A-EGFR was inserted into pMNDW. To construct the Lenti / CD19CAR vector, a synthetic DNA fragment encoding CD19CAR was inserted into pMNDW. Lentiviruses were produced using HEK 293T virus packaging cells according to a standard calcium precipitation protocol and an ultracentrifugation concentration protocol or a tandem tangential flow filtration concentration protocol as previously described.74

[0083] Retroviral vector construction and transduction

[0084] The retroviral vector used in this study was constructed from the parental retro vector MP71, which achieves high-level transgene expression in mouse and human T lymphocytes.75The Retro-BCMACAR-EGFR vector was constructed by inserting a synthetic bicistronic gene encoding BCMACAR-EGFR into MP71. Retroviruses were produced using 293T cells following a standard calcium precipitation protocol and an ultracentrifugation concentration protocol.74Retroviruses were then used to transduce PG-13 packaging cells expressing gibbon ape leukemia virus envelope and the Moloney murine leukemia virus gag-pol proteins76to generate stable cell lines producing Retro- BCMA-CAR virus. For retrovirus production, stable cells were seeded in DIO media for 2 days before harvesting virus supernatants for spin-infection. T cells were transduced with retrovirus in the presence of 10 pg / ml polybrene (Sigma- Aldrich). Retronectin (Takara) could be used to coat one day prior to transduction if more efficient transduction is desired. Three days after seeding, the transduction rate was determined as a percentage of EGFR+cells by flow cytometry.

[0085] In vitro tumor-killing assay A short-term in vitro tumor-killing assay was performed by coculturing MM. 1 s- FG (IxlO4cells per well) and BCMA CAR-T cells at defined ratios in Coming 96-well clear bottom black plates. Cells were cultured in CIO media for 24 hours. After culturing. D-luciferin (150 pg / ml) (Caliper Life Science) was added to the cell cultures to detect live tumor cells, and an Infinite Ml 000 microplate reader (Tecan) was used to measure luciferase activity in accordance with the manufacturer’s instructions. An in vitro high tumor load repetitive challenge assay was performed by coculturing Raji-FG (5xl04cells per well) and CD19 CAR-T cells at a 5: 1 ratio in Coming 96-well clear bottom black plates. Tumor cells (5xl04cells per well) were repetitively added on days 2 and 4 after the coculture setup. Tumor luciferase activity was measured on days 1, 3, and 5 post-coculture following the protocol mentioned above.

[0086] In vivo antitumor efficacy study

[0087] A human Raji xenograft NSG mouse model was used here. The experimental design is shown in Fig. 3e. Briefly, on day 0, NSG mice received intravenous (i.v.) inoculation of Raji-FG cells (1 x 105cells per mouse). On day 4. the experimental mice received i.v. injection of vehicle (100 pl PBS per mouse), GO-APP3 28-activated CD19 CAR-T cells (5 x 106cells in 100 pl PBS per mouse), or Beads3 / 28-activated CAR-T cells (5 x 106cells in 100 pl PBS per mouse). Over the course of the experiment, mice were monitored for survival, and their tumor loads were measured three times per week using BLI imaging.

[0088] Deep RNA sequencing (RNA-Seq)

[0089] T cells were activated and expanded from PBMCs with either Beads3 28with external IL-2 (30 U / ml) or GO-APP3 28in the absence of external IL-2. T cells were collected 7 days after activation. RNA was subsequently extracted with the miRNeasy Micro Kit (Qiagen). RNA libraries were prepared by the Technology Center for Genomics & Bioinformatics at UCLA using the TruSeq Stranded Total RNA Sample Prep kit (Illumina) and sequenced with 50 bp single-end reads on an Illumina HiSeq3000.

[0090] The reads were mapped with STAR 2.5.3a to the human genome (hg38).77The counts for each gene were obtained by using -quantMode GeneCounts in STAR commands, and the other parameters during alignment were set to default. Differential expression analyses were carried out using DESeq2.78Counts normalized by sequencing depth were obtained using the DESeq2 estimateSizeF actors function with default parameters. Principal component analysis (PCA) was performed on the log 10 normalized counts using the R function prcomp. Genes with an adjusted p value < 0.05 were considered significantly differentially expressed.

[0091] Single-cell RNA sequencing (scRNA-seq)

[0092] T cells were activated and expanded from PBMCs with either Beads3 28with external IL-2 (30 U / ml) or GO-APP5 28in the absence of external IL-2. T cells were collected 3 days after activation. scRNA-seq was utilized to study the genome-wide transcriptome profiling of GO-APP3 28and Beads3 28activated T cells from PBMCs. Library construction and scRNA-seq were performed by the UCLA TCGB Core. Cells were quantified using a Cell Countess II automated cell counter (Invitrogen / Thermo Fisher Scientific). A total of 10,000 cells from each experimental group were loaded on the Chromium platform (10X Genomics), and libraries were constructed using the Chromium Next GEM Single Cell 3' Kit v3.1 and the Chromium Next GEM Chip G Single Cell Kit (1 OX Genomics) according to the manufacturer's instructions. Library quality was assessed using the DI 000 ScreenTape on a 4200 TapeStation System (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq using the NovaSeq S4 Reagent Kit (100 cycles; Illumina).

[0093] Data analysis was performed using Cell Ranger Software Suite (1 Ox Genomics). Binary base call (BCL) files were extracted from the sequencer and used as inputs for the Cell Ranger pipeline to generate the digital expression matrix for each sample. Then, the cellranger aggr command was used to aggregate all samples into one digital expression matrix. The matrix was analyzed using Seurat, an R package designed for scRNA-seq. Marker genes were found for each cluster from Human Protein Atlas (proteinatlas.org) and used to define the cell types. Subsequently, seven clusters of cells were analyzed and compared between the different samples.

[0094] Western blot (WB)

[0095] Human T cells were purified from PBMCs and then activated with GO-APP3 28in the absence of IL-2 supplementation or with Beads3 28and 30 U / ml IL-2. Activated T cells from two time points post the setup of the coculture with GO-APP3 28or Beads3728were collected for WB. One at 10 min and the other at 7 days. Total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific) supplemented with a protease / phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentrations were quantified using a BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein were loaded and separated by 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to an Immunobilon-P PVDF membrane (Millipore). The membranes were blocked with SuperBlock™ T20 (TBS) Blocking Buffer (Thermo Fisher Scientific). Antibodies w ere diluted in 5% nonfat milk dissolved in washing buffer TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20). The following antibodies were purchased from Abeam: anti-p-STAT5 (p-Y694), anti-STAT5, anti-p-Akt (p-T308), anti-Akt, anti- ZAP70 (p-Y292), anti-ZAP70, anti-p-c-Jun (p-S63), anti-c-Jun, anti-GAPDH and HRP-labelled goat anti-rabbit IgG secondary antibodies. GAPDH was used as an internal control for total protein extracts. Signals were visualized using a ChemiDoc Image System (Bio-Rad). Data were analyzed using ImageJ software.

[0096] Statistics

[0097] GraphPad Prism 8 software (GraphPad) was used for statistical analysis. Pairwise comparisons w ere made using a 2-tailed Student’s t test. Multiple comparisons were performed using ordinary' 1-way ANOVA, followed by Tukey’s multiple comparisons test. Kaplan-Meier survival curves were analyzed by the log rank (Mantel-Cox) test adjusted for multiple comparisons. Data are presented as the mean ± SEM unless otherwise indicated. In all figures and figure legends, “n” represents the number of samples or animals utilized in the indicated experiments, p values less than 0.05 were considered significant, “ns” denotes not significant; * denotes p value < 0.05; ** denotes p value < 0.01; *** denotes p value < 0.001; **** denotes p value < 0.0001.

[0098] Calculation of the contact areas of Beads3 28and GO-APP3 28on T cells

[0099] The contact area of Beads3 28on T cells was calculated based on a circle model: where r is the radius of the contact area, and r = ~ 0.9 pm according to Fig. 1g. Therefore, Beads ~2.54 pm

[0100] The contact area of GO-APP3 28on T cells was calculated based on a spherical cap model (Fig. 5d): where R is the radius of the sphere and h is the height of the cap. According to Fig. le, R = ~3.9 pm and h = ~3.3 pm. Therefore,

[0101] ^GO-APP=~80.86 pm2

[0102] Data availability

[0103] All deep RNA sequencing data and single-cell RNA sequencing data are available in GEO with accession number GSE233291. Further details regarding the data are available from the corresponding author upon reasonable request.

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[0179] All publications mentioned herein (e.g. those listed above and Liao et al., Int J Mol Sci. 2018 Nov 12:19(11):3564. doi: 10.3390 / ijmsl9113564; Seifi et al., Colloids Surf B Biointerfaces. 2021 Mar;199: 111509; Kong et al., Nat Nanotechnol. 2019 Oct;14(10):927-938. doi: 10.1038 / s41565-019-0555-2. Epub 2019; Zhou et al., Annu Rev Chem Biomol Eng. 2018 Jun 7;9: 17-39. doi: 10.1146; Bellucci et al., Molecules. 2020 Jan 14;25(2):339. doi: 10.3390 / molecules25020339; and Bramini et al., Front Syst Neurosci. 2018 Apr 11;12: 12. doi: 10.3389 / fnsys.2018.00012. eCollection 2018) are incorporated by reference to disclose and describe aspects, methods and / or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

Claims

CLAIMS:

1. A composition of matter comprising: a graphene oxide material; a first antibody that selectively binds mammalian CD3 coupled to the graphene oxide material; and a second antibody that selectively binds mammalian CD28 coupled to the graphene oxide material.

2. The composition of claim 1, wherein: the graphene oxide material comprises graphene oxide sheets or flakes; the first antibody and / or the second antibody is coupled to the graphene oxide material via polyethylene glycol (PEG) linking moieties; and / or the graphene oxide material further comprises a cytokine coupled to the graphene oxide material.

3. The composition of claim 2, wherein the cytokine comprises at least one of: human SCF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), TPO (thrombopoietin), and IL-3 (interleukin 3).

4. The composition of claim 1, wherein the antibody density on the graphene oxide material is at least 250 or 500 or 1000 molecules / pm25. The composition of claim 1, wherein the first and second antibodies are coupled to the graphene oxide material such that a contact area between a T-cell combined with the composition and the composition is at least 5, 10, 25, 50 or 75 |inr.

6. The composition of claim 1, further comprising one or more T cells.

7. The composition of claim 4, wherein the one or more T cells comprise a CD8+ T cell, a CD4+ T cell or a combination of CD4+ and CD8+ T cells.

8. The composition of claim 4, wherein the T cell is a CAR-T cell.

9. The composition of claim 1, wherein when the graphene oxide composition coupled to the first and second antibody is combined with T cells in culture, the graphene oxide composition stimulates T at least a 50 fold increase in T cell numbers in the absence of exogenous interleukin-2.

10. A method of making the composition of claim 1 comprising coupling a graphene oxide material to a first antibody that selectively binds mammalian CD3; and coupling the graphene oxide material to a second antibody that selectively binds mammalian CD28; such that the composition of claim 1 is made.

11. The method of claim 10, wherein the first and / or second antibody is coupled to the graphene oxide material so that antibody densify on the graphene oxide material is at least 250 or 500 or 1000 molecules / pm212. The method of claim 10, wherein the first and second antibodies are coupled to the graphene oxide material such that a contact area between a T-cell combined with the composition and the composition is at least 5, 10, 25, 50 or 75 pm2.

13. The method of claim 10, wherein the method includes functionalizing the graphene oxide material with carboxyl groups.

14. The method of claim 10, wherein the first or the second antibody is coupled to the graphene oxide material via a avidin biotin binding interaction.

15. A method of stimulating T cell activation and / or proliferation comprising combining a T cell with a composition of claim 1-6 such that T cell activation and / or proliferation is stimulated.

16. The method of claim 15, wherein the graphene oxide material stimulates at least a 50 fold increase in T cell numbers in the absence of exogenous interleukin-2.

17. The method of claim 15, wherein the T cell is T cell obtained from a patient diagnosed with a disease or disorder.

18. The method of claim 17, wherein the disease or disorder is cancer.

19. The method of claim 15, wherein the T cell is a CAR-T cell.

20. The method of claim 15, wherein the graphene oxide material comprises sheets of graphene oxide that are from 5 pm to 80 pm in a length and / or a width.

Images