Preventing Lck Activation in CAR T Cells Confers Treg Resistance but Requires 4-1BB Signaling for Them to Persist and Treat Solid Tumors in Nonlymphodepleted Hosts

Abstract

Purpose: CAR T cells have shown promise against solid tumors, but their efficacy has been limited, due in part, to immunosuppression by CD4+FoxP3+ regulatory T cells (Tregs). Although lymphodepletion is commonly used to deplete Tregs, these regimens are non-specific, toxic, and provide only a narrow window before Tregs repopulate hosts. Importantly, CARs have also been shown to inadvertently potentiate Tregs by providing a source of IL-2 for Treg consumption. We explored whether disruption of the IL-2 axis would confer efficacy against solid tumors without the need for lymphodepletion. Experimental Design: We developed second- (CD28z) and third- (CD28-4-1BBz) generation CARs targeting EGFRvIII. To eliminate secretion of IL-2, two amino acid substitutions were introduced in the PYAP Lck binding-motif of the CD28 domain (ΔCD28). We evaluated CARs against B16 melanomas expressing EGFRvIII. Results: CD28z CARs failed to engraft in vivo. Although 4–1BB addition improved expansion, CD28-4-1BBz CARs required lymphodepletion to treat solid tumors. CARs deficient in Lck signaling, however, significantly retarded tumor growth without a need for lymphodepletion and this was dependent on inclusion of 4–1BB. To evaluate CAR vulnerability to Tregs, we lymphodepleted mice and transferred CARs alone or with purified Tregs. Co-transfer with Tregs abrogated the efficacy of CD28-4-1BBz CARs, whereas the efficacy of ΔCD28-4-1BBz CARs remained unperturbed. Conclusion: In the absence of lymphodepletion, CARs targeting solid tumors are hindered by Treg immunosuppression and poor persistence. Here, CARs were modified to circumvent Treg suppression and to simultaneously improve in vivo engraftment. Modified CARs treated solid tumors without a need for lymphodepletion.

Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells

“…IL: interleukin; MSLN-CAR: fully human anti mesothelin CAR; NECA: 5′-(Nethylcarboxamido) adenosine; ns: Non-significant; SD: standard deviation. (**P < 0.01, ***P < 0.001) express 4-1BB intracellular domains are sensitive to Treg-promoting signals of A2aR is not clearly known [36]. In this study, we did not evaluate Treg markers before or after exposure to NECA and A2aR knockdown/ inhibition.…”

Clinical chimeric antigen receptor‐T cell therapy: a new and promising treatment modality for glioblastoma

“…88 Interestingly, EGFRvIII expression was lost in the patients who had repeat surgery on the control arm of the ACTIV glioblastoma vaccine trial, indicating that antigen expression is inherently unstable. 91 Nonetheless, the finding of intra-tumoral lymphocytic infiltrates comprising mainly endogenous (non-CAR) regulatory T cells after EGFRvIII-CAR-T cell targeting 49 offers the possibility, as shown in an immunocompetent murine model, that protective endogenous immunity could be recruited against antigennegative tumor variants following CAR-T cell therapy, 90 and particularly if changes in CAR design could limit the effect the immunosuppressive effects of regulatory T cells.…”

CAR-T Cells Hit the Tumor Microenvironment: Strategies to Overcome Tumor Escape

“…• Choosing appropriate costimulatory domains (46)(47)(48)(49)(50)(51) • Mutating costimulatory domains (52) • Using alternative cytokines to support engineered CAR-T cells (49,53,54) • Target TAMs with CAR-T cells (55)(56)(57) • Reeducate TAMs toward antitumor phenotype (58- is metabolic rewiring, a process in which the reduced rate of oxidative phosphorylation (OXPHOS) is compensated by enhanced glycolysis. Competition for nutrients, persistent antigenic stimulation and immunosuppressive networks in the TME can lead to T cell exhaustion (112).…”