Adoptive T-cell therapy with gene-modified T-cells expressing a tumor-reactive T-cell receptor (TCR) or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprised of random compositions of CD4+ and CD8+ naïve and memory T-cells, meaning that each patient received a different therapeutic agent. This variation might have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T-cells derived from different subsets (CD4+/CD8+ naïve, central memory, effector memory). T-cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T-cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.

Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

SLAMF7 is under intense investigation as a target for immunotherapy in multiple myeloma. In this study, we redirected the specificity of T cells to SLAMF7 through expression of a chimeric antigen receptor (CAR) derived from the huLuc63 antibody (elotuzumab) and demonstrate that SLAMF7-CAR T cells prepared from patients and healthy donors confer potent antimyeloma reactivity. We confirmed uniform, high-level expression of SLAMF7 on malignant plasma cells in previously untreated and in relapsed/refractory (R/R) myeloma patients who had received previous treatment with proteasome inhibitors and immunomodulatory drugs. Consequently, SLAMF7-CAR T cells conferred rapid cytolysis of previously untreated and R/R primary myeloma cells in vitro. In addition, a single administration of SLAMF7-CAR T cells led to resolution of medullary and extramedullary myeloma manifestations in a murine xenograft model in vivo. SLAMF7 is expressed on a fraction of normal lymphocytes, including subsets of natural killer (NK) cells, T cells, and B cells. After modification with the SLAMF7-CAR, both CD8 and CD4 T cells rapidly acquired and maintained a SLAMF7 phenotype and could be readily expanded to therapeutically relevant cell doses. We analyzed the recognition of normal lymphocytes by SLAMF7-CAR T cells and show that they induce selective fratricide of SLAMF7 NK cells, CD4 and CD8 T cells, and B cells. Importantly, however, the fratricide conferred by SLAMF7-CAR T cells spares the SLAMF7 fraction in each cell subset and preserves functional lymphocytes, including virus-specific T cells. In aggregate, our data illustrate the potential use of SLAMF7-CAR T-cell therapy as an effective treatment against multiple myeloma and provide novel insights into the consequences of targeting SLAMF7 for the normal lymphocyte compartment.

Superagonistic CD28-specific monoclonal antibodies (CD28SA) are highly effective activators of regulatory T-cells (Treg cells) in rats, but a first-in-man trial of the human CD28SA TGN1412 resulted in an unexpected cytokine release syndrome. Using a novel mouse anti-mouse CD28SA, we re-investigate the relationship between Treg activation and systemic cytokine release. Treg activation by CD28SA was highly efficient but depended on paracrine IL-2 from CD28SA-stimulated conventional T-cells. Systemic cytokine levels were innocuous, but depletion of Treg cells prior to CD28SA stimulation led to systemic release of proinflammatory cytokines, indicating that in rodents, Treg cells effectively suppress the inflammatory response. Since the human volunteers of the TGN1412 study were not protected by this mechanism, we also tested whether corticosteroid prophylaxis would be compatible with CD28SA induced Treg activation. We show that neither the expansion nor the functional activation of Treg cells is affected by high-dose dexamethasone sufficient to control systemic cytokine release. Our findings warn that preclinical testing of activating biologicals in rodents may miss cytokine release syndromes due to the rapid and efficacious response of the rodent Treg compartment, and suggest that polyclonal Treg activation is feasible in the presence of antiphlogistic corticosteroid prophylaxis.

Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3′ region of the cAMP response modulator (Crem) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4 + T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4 + T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMPdriven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 (Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/ CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/ CREM and inhibition of NFATc1 and IL-2 induction.adenosin 3′,5′-cyclic monophospate | transcription | lymphocytes