A Rapid Cell Expansion Process for Production of Engineered Autologous CAR-T Cell Therapies

Abstract

The treatment of B-cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials. The anti-CD19 CAR-T cell production method used to support initial trials relied on numerous manual, open process steps, human serum, and 10 days of cell culture to achieve a clinical dose. This approach limited the ability to support large multicenter clinical trials, as well as scale up for commercial cell production. Therefore, studies were completed to streamline and optimize the original National Cancer Institute production process by removing human serum from the process in order to minimize the risk of viral contamination, moving process steps from an open system to functionally closed system operations in order to minimize the risk of microbial contamination, and standardizing additional process steps in order to maximize process consistency. This study reports a procedure for generating CD19 CAR-T cells in 6 days, using a functionally closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process.

Toward precision manufacturing of immunogene T-cell therapies

“…Using this method, we demonstrated that potent CAR T cells can be produced, as characterized by their robust proliferative and cytolytic activities [96]. Following these feasibility studies, a rapid CAR T-cell expansion process has been recently reported that is based on a closedmanufacturing system and serum-free media [97].Thus, limiting manufacturing time could be a practical approach for both process and product improvement.…”

“…Thus, HS substitutes of nonanimal origin may be alternatives in the setting of cell manufacturing and are currently being explored. In this regard, recent studies showed that an immune cell serum replacement (ThermoFisher Scientific) promotes at least equivalently efficient T-cell proliferation as compared with conventional expansion media containing HS [97,114]. It is expected that serum substitution will be integrated into current T-cell manufacturing for economical purposes with the goal of maintaining or improving the quality of T-cell products.…”

Considerations in T Cell Therapy Product Development for B Cell Leukemia and Lymphoma Immunotherapy

“…Cell-based artificial antigen presenting cells can be tuned to express various combinations of stimulatory ligands that preferentially expand a selectable array of T cell subsets (55, 56). Serum free media and shorter culture times are additional enhancements to cell processing that have recently been reported (57). Ultimately, regardless of the technique, ex vivo expansion aims to increase the number of total cells for infusion.…”

Effects of starting cellular material composition on chimeric antigen receptor T‐cell expansion and characteristics

“…When autologous CAR T cells are manufactured, peripheral blood mononuclear cells (PBMNCs) collected by apheresis are typically used as the source of cells to begin manufacturing. The PBMNC concentrates are generally enriched for lymphocytes using density gradient separation, elutriation, or T cell–specific antibodies . The lymphocyte‐enriched PBMNCs are stimulated to proliferate with cytokines and antibodies, transduced with retroviral or lentiviral vectors, and allowed to expand for 7 to 12 days.…”

“…Other methods can be used to manufacture CAR T cells . We hypothesized that the success of CAR T‐cell manufacturing methods that did not use anti‐CD3/anti‐CD28 beads for lymphocyte enrichment and T‐cell stimulation would be less dependent on the composition of the starting material.…”