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.

Gomes-Silva D ，Mukherjee M ，Srinivasan M ，Krenciute G ，Dakhova O ，Zheng Y ，Cabral JMS ，Rooney CM ，Orange JS ，Brenner MK ，Mamonkin M ... -  《Cell Reports》

4-1BB enhancement of CAR T function requires NF-κB and TRAFs.

Li G ，Boucher JC ，Kotani H ，Park K ，Zhang Y ，Shrestha B ，Wang X ，Guan L ，Beatty N ，Abate-Daga D ，Davila ML ... -  《JCI Insight》

4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.

Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19+ hematologic malignancies. 4-1BB-costimulated CAR (BBζ) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28ζ) T cells. 4-1BB signaling improves T cell persistence even in the context of 28ζ CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. To specifically study signal transduction by CARs, we developed a cell-free, ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. We observed greater ex vivo survival and subsequent expansion of BBζ CAR T cells when compared to 28ζ CAR T cells. We showed that only BBζ CARs activated noncanonical nuclear factor κB (ncNF-κB) signaling in T cells basally and that the anti-CD19 BBζ CAR further enhanced ncNF-κB signaling after ligand engagement. Reducing ncNF-κB signaling reduced the expansion and survival of anti-CD19 BBζ T cells and was associated with a substantial increase in the abundance of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between BBζ and 28ζ CARs, they demonstrate the necessary and nonredundant role of ncNF-κB signaling in promoting the survival of BBζ CAR T cells, which likely underlies the engraftment persistence observed with this CAR design.

Philipson BI ，O'Connor RS ，May MJ ，June CH ，Albelda SM ，Milone MC ... -  《-》

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.

Mamonkin M ，Mukherjee M ，Srinivasan M ，Sharma S ，Gomes-Silva D ，Mo F ，Krenciute G ，Orange JS ，Brenner MK ... -  《-》

Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia.

We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA-electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti-CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML.

Tasian SK ，Kenderian SS ，Shen F ，Ruella M ，Shestova O ，Kozlowski M ，Li Y ，Schrank-Hacker A ，Morrissette JJD ，Carroll M ，June CH ，Grupp SA ，Gill S ... -  《-》