Preventing Lck activation in CAR T cells confers Treg resistance but requires 4-1BB signaling for them to persist and treat solid tumors in non-lymphodepleted hosts

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 (DCD28). 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 DCD28-4-1BBz CARs remained unperturbed. Conclusions: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.

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