Adoptive cell therapy using gene-modified T cells has demonstrated promising clinical outcomes in hematologic malignancies. Production of gene-modified T cells involves the selection of patient T cells, activation via stimulation through the endogenous T cell receptor (TCR) complex and a costimulatory domain, followed by introduction of a tumor antigen-specific TCR or chimeric antigen receptor (CAR) through gene modification. Here we characterize a soluble T cell stimulation reagent, known as an ExpamerTM reagent, in the production of therapeutic CAR T cells.

The Expamer reagent used in these studies is designed to be a late-stage clinical and commercial manufacturing ancillary material with two important attributes that make it highly attractive from a manufacturing and regulatory standpoint; it is a soluble and dissociable reagent. These attributes increase the ease of both introduction and removal from the manufacturing process, giving products manufactured with this reagent consistent product quality and purity. This reagent activates T cells through the simultaneous engagement of the TCR-CD3 complex and the costimulatory receptor CD28 and is compatible with manufacturing of both current and next-generation therapeutics.

Purified healthy donor T cells cultured in the presence of the Expamer reagent rapidly fluxed Ca2+, demonstrating the capacity to induce early TCR signaling. Activation through this reagent additionally promotes upregulation of the cell surface activation marker CD25 and proliferation as measured by CFSE dilution. Following stimulation with this reagent, T cells are readily transduced with a CD19-specific CAR construct. The function of CAR T cells generated with this reagent was measured by effector cytokine production, proliferation, and cytolytic activity in the presence of CD19 expressing and control target cells in vitro. CAR T cells robustly produced IFN-ɣ and IL-2 after activation with a CAR specific antigen. In addition, proliferation in the presence of CD19 expressing target cells was observed as measured by CFSE dilution. Finally, significant cytolytic activity against CD19-expressing target cells was observed.

Collectively, these data provide evidence that functional engineered T cells can be manufactured using the Expamer reagent and support implementation into the production of both current and next-generation therapeutic gene-modified T cells.

The first two authors contributed equally to this work.


Bashour:Juno Therapeutics: Employment. Larson:Juno Therapeutics: Employment. Graef:Juno Therapeutics: Employment. Stemberger:Juno Therapeutics: Employment. Lothar:Juno Therapeutics: Employment. Odegard:Juno Therapeutics: Employment. Ramsborg:Juno Therapeutics: Employment.

Author notes


Asterisk with author names denotes non-ASH members.

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