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bioRxiv · 10.1101/2021.03.18.435976

Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

Ukrainskaya, V. M.·Rubtsov, Y. P.·Pershin, D. S.·Podoplelova, N. A.·Terekhov, S. S.·Yaroshevich, I. A.·Sokolova, A. I.·Bagrov, D. V.·Kulakovskaya, E. A.·Shipunova, V. O.·Deyev, S. M.·Maksimov, E. G.·Markov, O. V.·Oshchepkova, A. L.·Zenkova, M. A.·Xie, J.·Gabibov, A. G.·Maschan, M. A.·Stepanov, A. V.·Lerner, R. A.
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Abstract

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia and lymphoma. It also raised an opportunity to design new protocols to target solid tumors. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are based on incubation with high doses of recombinant cytokines to support proliferation, non-specific stimulation with surface-bound antibodies to induce TCR cross-linking, or co-cultivation with antigen-expressing feeder cell lines. These approaches are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. In an effort to develop a specific and improved protocol for CAR-T cell expansion, we took advantage of cell-derived membrane vesicles, and the simple structural demands of the CAR-antigen interaction. Our approach was to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens (antigenic vesicles, AVs), and then use them for stimulation and expansion of CAR-T cells. We developed a rapid, simple, efficient, and inexpensive protocol to generate, stabilize and purify AVs. As proof-of-concept we tested the efficacy of our AV constructs on several CAR-antigen pairs. The data presented in this article clearly demonstrate that our protocol produced AVs with the capacity to induce stronger stimulation, proliferation and functional activity of CAR-T cells than is possible with existing protocols. We predict that this new methodology will significantly improve the ability to obtain improved populations of functional CAR-T cells for therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/435976v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@abbe23org.highwire.dtl.DTLVardef@15007dforg.highwire.dtl.DTLVardef@134e506org.highwire.dtl.DTLVardef@1a7f1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Ukrainskaya, V. M., Rubtsov, Y. P., Pershin, D. S., Podoplelova, N. A., Terekhov, S. S., Yaroshevich, I. A., Sokolova, A. I., Bagrov, D. V., Kulakovskaya, E. A., Shipunova, V. O., Deyev, S. M., Maksimov, E. G., Markov, O. V., Oshchepkova, A. L., Zenkova, M. A., Xie, J., Gabibov, A. G., Maschan, M. A., Stepanov, A. V., Lerner, R. A.. 2021-03-18. Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates. https://doi.org/10.1101/2021.03.18.435976

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