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 cellsmediated 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.Adoptive T cellular therapy harnesses and redirects a patient's own immune system and has emerged as a promising personalized treatment modality to treat cancer [1][2][3] and various other diseases. [4,5] The most successful example has been the adoptive transfer of chimeric antigen receptor redirected T cells (CAR-T cells) targeting the CD19 expressed by B-cell malginancies, that received FDA approvals in 2017. [6,7] With the compelling success of CD19-specific CAR T-cell therapies, efforts are now being directed toward broadening the application of

Delivery Techniques for Enhancing CAR T Cell Therapy against Solid Tumors

“…Biomaterial-based scaffolds were also developed to transduce T cells. [85] Calcium-crosslinked alginate scaffolds were lyophilized to form 100-200 µm pores, allowing T cell migration, and retroviral particles were seeded in the macroporous scaffolds where they efficiently transduced the T cells that retained full functionality.…”

Absorption rate governs cell transduction in dry macroporous scaffolds

“…Scaffolds were prepared as reported previously. 11,26,33 A solution of ultrapure alginate (Pronova, MVG) in deionized (DI) water was vigorously mixed with an equal volume of calcium-Dgluconate solution in DI water for 15 min. Final mixture alginate concentrations ranged from 0.5% to 2% and calcium-Dgluconate concentrations ranged from 0.1% to 0.3% The resulting mixture was cast 1 mL per well in a 24-well plate and frozen overnight.…”

“…Scaffolds were again synthesized using cryogelation with varying alginate concentrations (0.5%, 1.0%, 1.5%, 2.0%), but with a constant calcium concentration of 0.2%, in line with previous reports. 11,26 Scaffolds were then frozen at −20 °C, −40 °C, −60 °C, or −80 °C and lyophilized. The scaffolds had a cross-sectional area of ∼1.78 cm 2 and a height of ∼4.86 mm [Fig.…”

“…[17][18][19][20][21] CAR T cells have an antigen recognition domain that specifically targets a tumor-associated antigen, enabling CAR T cells to recognize and kill tumor cells in the body. [18][19][20] In the space of CAR T cell therapy, biomaterials have demonstrated utility in improving T cell activation, [22][23][24][25] mediating viral T cell transduction, 11,22,26,27 promoting CAR T cell expansion, 11,25,26,[28][29][30] and releasing CAR T cells in a controlled manner. 11,25 Recently, our lab reported that dry, macroporous alginate scaffolds, known as "Drydux" scaffolds, mediate retroviral transduction of difficult to transduce primary T cells as efficiently as the gold-standard "spinoculation" for CAR T cell generation: centrifugation of activated T cells and virus on Retronectin-coated plates.…”

“…In preliminary efforts to understand the mechanism underpinning Drydux transduction, we demonstrated that cell transduction fails in both dry nanoporous scaffolds and wet macroporous scaffolds, suggesting that scaffold pore size and hygroscopy play central roles in the transduction mechanism. 26 In this paper, we further define the mechanism behind Drydux transduction by delineating the impact of scaffold pore size, stiffness, viral concentration, seeding volume, and absorption rate on transduction of human primary T cells. We synthesized macroporous alginate scaffolds with varying physical properties by changing the alginate concentration, calcium concentration, and freezing temperature.…”