AW-02. Development of a method for production of a third-generation lentiviral vector for the manufacturing of an anti-CD19 CAR-T cell product

Summary

Cell therapy with genetically modified T cells expressing chimeric antigen receptor (CAR) has achieved significant success in the treatment of B-cell malignancies, such as B-lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL). Antigen-targeted CAR is a chimeric molecule consisting of signaling domains from T cell receptor complex and extracellular antigen-recognizing domain, which can be a single-chain fragment (scFv) of a monoclonal antibody. CAR-T cells are able to effectively recognize and eliminate target antigen-expressing cells. Unlike TCR mediated recognition, antigen recognition by CAR is independent of the major histocompatibility complex (MHC). In comparison to other delivery vectors for CAR gene, lentiviral vectors (LV) have a number of advantages. This includes their capacity for integration into the host genome, driving sustained expression of therapeutic genes. Thus, they are an effective tool for the development of gene and cell therapies. The aim of this work is to optimize the process of developing and obtaining a CAR-T cellular product specific for CD19 antigen, with the CD28 and CD3ζ sequences as costimulatory and signaling domains, respectively.

Materials and methods

The adhesive cell line of human embryonic kidney HEK293T and suspension cell line Expi293F were used for the lentiviral vector (CAR LV) production. The expression plasmid was co-transfected into cells with three packaging plasmids (pLP1(gag/pol), pLP2 (rev) and pLP/VSV-G (VSV G shell) by using polyethylenimine (PEI) or lipofectamine (GenJect-39, ExpiFectamine Transfection Reagent). To assess the functional titers of CAR LV, a human T lymphoblastic leukemia cell line was transduced (Jurkat E6.1). The level of transduction was assessed using flow cytometry by cell staining with recombinant CD19-Fc protein conjugated with Alexa Fluor 647 dye. The expansion level of primary T cells, that were activated and transduced with CAR LV, was measured in culture plates and G-Rex bioreactors after 7-14 days. To assess functional activity of CAR-T cells, cell lines expressing CD19 antigen on their surface (RS4;11, Raji, NALM6, K562-CD19⁺) were used as targets and K562-CD19^– cell line was used as control.

Results

The developed protocol for obtaining lentiviral vector was shown to be effective for the production of lentiviral particles carrying CAR-transgene, with an infectious titer of (6-32)×10⁶ transducing units (TU) per mL (average of 19×10⁶ TU/mL). Such titers enable effective transduction of T-cell line Jurkat E6.1 and primary T cells from healthy donors and patients, achieving a high level of CAR-gene integration in primary T cells (20-70% with MOI=10). The resulting CAR T cellular product demonstrates a high level of expansion (up to 18 times in the culture plates, up to 28 times in the G Rex platform over the course of 8 10 days). In addition, CAR-T cells demonstrated cytotoxic and proliferative activities though functional in vitro tests using CD19-expressing target cell lines (Raji, NALM6, K562-CD19⁺).

Conclusion

As a result of the optimization of the production methodology, an anti-CD19 CAR-T cellular product was obtained with a high level of functional activity in tests. This allowed for the preclinical studies of the efficacy and safety of anti-CD19 CAR-T cell therapy to commence.

Keywords

Immunotherapy, chimeric antigen receptor, lentiviral vector, CAR-T cells, anti-CD19 CAR-T, functional activity, cellular product.