We felt that a promising strategy to obtain high numbers of functional antigen-specific CAR-T cells would be to stimulate CAR-T cells using antigen exposed on cell membrane fragments organized as relatively uniform microcytospheres that lack a nucleus. We reasoned that it would be possible to use the relatively simple structural requirement for functional CAR interaction with its cognate antigen (a mimic of immunoglobulin-antigen interaction) to develop a more manageable methodology for antigen-specific expansion of CAR-T cells. But these advantages are negated by the need to use living cells with their inherent safety considerations, and add the undesired extra steps of feeder cell removal. In the case of feeder/APC the stimulation is more physiological because cells provide additional signals that facilitate stimulation, proliferation, survival, and function of the CAR-T cells. In part, these issues can be solved by using feeder cells expressing specific antigens or designed antigen-presenting cells (APCs). Antigen-specific T cell expansion also presents difficulties, as the antigen should be presented in the context of major histocompatibility complex (MHC) I or II depending on the type of T cell. Another problem is that stimulation of T cells with beads and cytokines results in terminal differentiation of naïve cells into effector T cells with exhausted phenotype. Thus, if CAR-positive cells are originally present in a low percentage they may be significantly diluted.

The main drawback of such non-specific expansion is that CAR-negative ballast cells are also expanding. Cross-linking antibodies are usually immobilized on the surface of polymer beads, magnetic beads, or in some reports, on sophisticated nanotube scaffolds. Non-specific protocols involve the combination of T-cell receptor (TCR) cross-linking by antibodies recognizing constant parts of the TCR and co-stimulatory molecules (anti-CD3 and anti-CD28), with high doses of cytokines, such as IL-2, IL-15, and others. The majority of existing approaches are based on non-specific or antigen-presenting/feeder cell-based protocols. One of the significant challenges in chimeric antigen receptor-modified T cells (CAR-T) therapy is obtaining an adequate number of CAR-T cells with the desired function through donor T cell stimulation and expansion in vitro. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy.

The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. Modern protocols for CAR-T cell expansion 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. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia.

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