Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, a low-cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19⁺ lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.

Keywords: CAR-T cells; alginate scaffold; cell therapy; immunotherapy; viral transduction.

Figure 1.. Dry macroporous scaffolds mediate retroviral transduction of human activated T cells.

A) Schematic showing the preparation of macroporous alginate scaffolds. B,C) SEM images of dry macroporous scaffolds. D) GFP expression in activated T cells transduced with equal amounts of retrovirus seeded on dry macroporous scaffolds (red) and retronectin-coated plates with spinoculation (blue). Non-transduced control cells are represented in black. E) SEM image of dry nanoporous scaffold. F) GFP expression in T cells transduced with retrovirus seeded on dry macroporous scaffolds (red), hydrated macroporous scaffolds (purple), and dry nanoporous scaffolds (green). Control non-transduced cells are in black. G) Quantification of GFP⁺ cells (***p<0.0001 with respect to dry macroporous scaffolds, Student’s t-test)

Figure 2.. Scaffold-generated CD19.CAR-T cells show similar functional activity to retronectin/spinoculation-derived CAR-T cells in vitro.

A) CD19.CAR expression in T cells transduced on retronectin-coated plates or on macroporous scaffolds in comparison to non-transduced control T cells (NT cells). B) Ex vivo expansion of T cells transduced on retronectin-coated plates or on macroporous scaffolds or NT cells. C, D) Immunophenotypic composition of CAR-T cells obtained via scaffold-mediated or retronectin/spinoculation-mediated transduction and NT cells at day 12 of culture. Analysis was performed gating on CAR-expressing T cells except for NT cells. E) Percentage of CD19⁺ Daudi cells remaining (tumor cells) when co-cultured with scaffold-generated, retronectin/spinoculation-generated CAR-T cells or control NT cells. Tumor cells and T cells were plated at 1:5 effector to target ratio. T cells and tumor cells were quantified by flow cytometry on day 5 of co-culture. ***P < 0.0001 unpaired Student’s t-test. F) IFN-γ and IL-2 release into co-culture supernatant by scaffold-generated, retronectin/spinoculation-generated CAR-T cells and control NT cells after 24 h of coculture with tumor cells as assessed by ELISA. ***P < 0.0001; two-way ANOVA with Tukey correction. Data are represented as the mean ± SD from three experiments, each derived from a different PBMC donor.

Figure 3.. Scaffold-generated CD19.CAR-T cells eradicate tumors in a mouse xenograft model of lymphoma

A) Experimental timeline of the lymphoma xenograft model in NSG mice using the FFLuc-labeled CD19⁺ human Daudi tumor cells. B) Representative tumor bioluminescent images (BLI) of NSG mice inoculated with Daudi cells and treated with control NT cells or treated with CD.19.CAR-T cells generated by scaffold-mediated transduction or retronectin/spinoculation-assisted transduction. C) Kinetics of tumor growth measured by quantification of BLI. **p<0.01 when scaffold or CAR-T cells were compared to control NT cells, one way ANOVA. D) Body weight change and E) survival of tumor-bearing mice treated with control NT cells or CD19.CAR-T cells generated by scaffold-mediated or retronectin spinoculation assisted transduction. *p<0.05, log-rank test.