295 Novel T-cell immunotherapeutics enable the selective generation of more potently cytotoxic CD19 chimeric antigen receptor T-cells (CAR-T cells) from CMV-specific cytotoxic T-cells

Background

CAR-T therapy induces dramatic remissions of relapsed/refractory B-cell malignancies, but remission durability is frequently limited by reduced CAR-T persistence or poor function. We hypothesize CAR-T function and longevity would be increased by selectively deriving CAR-Ts from effector-memory virus-specific CD8+ cytotoxic T-lymphocytes (CTLs). To enable antigen-specific activation of T cells, we developed biologics termed synTacs (synapse for T-cell activation), which deliver TCR and costimulatory signals to selectively activate and expand CMV-specific CTLs. CMV-aCD28-synTacs consist of dimeric Fc domain scaffolds linking HLA-A2 molecules presenting a CMV-derived peptide (NLV/pp65) and an aCD28 binder (figure 1A) that induce selective in vitro and in vivo expansion of CMV-specific CTLs with potent antiviral activity (figure 1B,C).¹ We used CMV-aCD28-synTac to generate CAR-T cells from CMV-specific CTLs and compared their functionality to standard CAR-T cells generated by aCD3/aCD28 activation. We also evaluated an alternative strategy to generate CAR-T cells from CMV-specific T cells in vivo using synTac-based single-chain CMV-MHC-targeted lentiviral vectors.

Methods

We treated purified CD8+ T-cells from a CMV-responsive donor with CMV-aCD28-synTac or aCD3/aCD28 and then transduced them with lentivirus expressing an anti-CD19-CAR and a GFP reporter (figure 2A). CMV-specific CTLs and CAR-T cells were quantified by flow cytometry with tetramer staining or GFP expression, respectively. Functional activity of CD19-CAR-T cells was determined by a B-cell-specific cytotoxic assay. We also assessed the capacity of a pp65-specific lentivirus to selectively transduce CMV-responsive CTLs to potentially generate CAR-T cells by treating pp65+ donor-derived PBMCs with this lentivirus and assessing selective expansion and transduction of CMV-specific CTLs (figure 2B).

Results

After 7 days, CTL treatment with CMV-aCD28-synTac rapidly induced robust activation and >50-fold selective expansion of CMV-specific CTLs (figure 1B). CMV-aCD28-synTac treatment activated CMV-specific T-cells, enabling their selective transduction with an anti-CD19-CAR lentivirus (figure 2C). These CMV-specific anti-CD19-CAR-T cells displayed more potent cytotoxic activity targeting donor B cells than those generated by traditional aCD3/aCD28 activation (figure 3B). We saw similar expansion and selective transduction after 2 weeks using the pp65-targeting lentivirus to generate pp65-responsive CD19 CAR-T cells (figures 2B and 3C,D).

Conclusions

CAR-T cells derived from CMV-specific CTLs after CMV-aCD28-synTac activation exhibit enhanced cytotoxic activity against B cells as compared to standard CAR-Ts. Their co-expression of the CMV-specific T-cell receptor should also enable potent stimulation and activation by in vivo stimulation by endogenous CMV antigen or treatment with CMV-synTacs coupled to aCD28 or other costimulatory ligands or cytokines, enabling more potent and durable treatment of relapsed/refractory B cell malignancies.

Reference

Li M, Garforth SJ, O’Connor KE, et al. T cell receptor-targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice. J Clin Invest. 2021;131(23):e141051.

Ethics Approval

This study was approved by the Albert Einstein College of Medicine’s Ethics Board; approval number 2017–8116.