CD7 CAR T Cells for the Therapy of Acute Myeloid Leukemia.

Chimeric antigen receptor (CAR) T cell therapy for the treatment of acute myeloid leukemia (AML) has the risk of toxicity to normal myeloid cells. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients but is absent on normal myeloid and erythroid cells. Since CD7 expression by malignant blasts is also linked with chemoresistance and poor outcomes, targeting this antigen may be beneficial for this subset of AML patients. Here, we show that expression of a CD7-directed CAR in CD7 gene-edited (CD7KO) T cells effectively eliminates CD7+ AML cell lines, primary CD7+ AML, and colony-forming cells but spares myeloid and erythroid progenitor cells and their progeny. In a xenograft model, CD7 CAR T cells protect mice against systemic leukemia, prolonging survival. Our results support the feasibility of using CD7KO CD7 CAR T cells for the non-myeloablative treatment of CD7+ AML.

CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies.

Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7+) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies.

Engineered off-the-shelf therapeutic T cells resist host immune rejection.

Engineered T cells are effective therapies against a range of malignancies, but current approaches rely on autologous T cells, which are difficult and expensive to manufacture. Efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system require abrogating both T- and natural killer (NK)-cell responses, which eliminate foreign cells through various mechanisms. In the present study, we engineered a receptor that mediates deletion of activated host T and NK cells, preventing rejection of allogeneic T cells. Our alloimmune defense receptor (ADR) selectively recognizes 4-1BB, a cell surface receptor temporarily upregulated by activated lymphocytes. ADR-expressing T cells resist cellular rejection by targeting alloreactive lymphocytes in vitro and in vivo, while sparing resting lymphocytes. Cells co-expressing chimeric antigen receptors and ADRs persisted in mice and produced sustained tumor eradication in two mouse models of allogeneic T-cell therapy of hematopoietic and solid cancers. This approach enables generation of rejection-resistant, 'off-the-shelf', allogeneic T-cell products to produce long-term therapeutic benefit in immunocompetent recipients.

Cancer Immunotherapy Using CAR-T Cells: From the Research Bench to the Assembly Line.

The focus of cancer treatment has recently shifted toward targeted therapies, including immunotherapy, which allow better individualization of care and are hoped to increase the probability of success for patients. Specifically, T cells genetically modified to express chimeric antigen receptors (CARs; CAR-T cells) have generated exciting results. Recent clinical successes with this cutting-edge therapy have helped to push CAR-T cells toward approval for wider use. However, several limitations need to be addressed before the widespread use of CAR-T cells as a standard treatment. Here, a succinct background on adoptive T-cell therapy (ATCT)is given. A brief overview of the structure of CARs, how they are introduced into T cells, and how CAR-T cell expansion and selection is achieved in vitro is then presented. Some of the challenges in CAR design are discussed, as well as the difficulties that arise in large-scale CAR-T cell manufacture that will need to be addressed to achieve successful commercialization of this type of cell therapy. Finally, developments already on the horizon are discussed.

Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies.

T cells expressing second-generation chimeric antigen receptors (CARs) specific for CD5, a T-cell surface marker present on normal and malignant T cells, can selectively kill tumor cells. We aimed to improve this killing by substituting the CD28 costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.z CD5 CAR T cells rapidly differentiated into short-lived effector cells. In contrast, 4-1BB costimulation is known to promote development of the central memory subpopulation. Here, we found BB.z CD5 CAR T cells had impaired growth compared with 28.z CD5.CAR T cells, due to increased T-cell-T-cell fratricide. We demonstrate that TRAF signaling from the 4-1BB endodomain upregulated the intercellular adhesion molecule 1, which stabilized the fratricidal immunologic synapse between CD5 CAR T cells. As the surviving BB.z CD5 CAR T cells retained the desired central memory phenotype, we aimed to circumvent the 4-1BB-mediated toxicity using a regulated expression system that reversibly inhibits CAR expression. This system minimized CAR signaling and T-cell fratricide during in vitro expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells in vivo These studies reveal a mechanism by which 4-1BB costimulation impairs expansion of CD5 CAR T cells and offer a solution to mitigate this toxicity. Cancer Immunol Res; 6(1); 47-58. ©2017 AACR.

Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent.

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.