Epitope spreading driven by the joint action of CART cells and pharmacological STING stimulation counteracts tumor escape via antigen-loss variants

Abstract

BackgroundTarget antigen (Ag) loss has emerged as a major cause of relapse after chimeric antigen receptor T (CART)-cell therapy. We reasoned that the combination of CART cells, with the consequent tumor debulking and release of Ags, together with an immunomodulatory agent, such as the stimulator of interferon gene ligand (STING-L) 2′3′-cyclic GMP-AMP (2′3′-cGAMP), may facilitate the activation of an endogenous response to secondary tumor Ags able to counteract this tumor escape mechanism.MethodsMice bearing B16-derived tumors expressing prostate-specific membrane Ag or gp75 were treated systemically with cognate CART cells followed by intratumoral injections of 2′3′-cGAMP. We studied the target Ag inmunoediting by CART cells and the effect of the CART/STING-L combination on the control of STING-L-treated and STING-L-non-treated tumors and on the endogenous antitumor T-cell response. The role of Batf3-dependent dendritic cells (DCs), stimulator of interferon gene (STING) signaling and perforin (Perf)-mediated killing in the efficacy of the combination were analyzed.ResultsUsing an immune-competent solid tumor model, we showed that CART cells led to the emergence of tumor cells that lose the target Ag, recreating the cancer immunoediting effect of CART-cell therapy. In this setting, the CART/STING-L combination, but not the monotherapy with CART cells or STING-L, restrained tumor progression and enhanced overall survival, showing abscopal effects on distal STING-L-non-treated tumors. Interestingly, a secondary immune response against non-chimeric antigen receptor-targeted Ags (epitope spreading), as determined by major histocompatibility complex-I-tetramer staining, was fostered and its intensity correlated with the efficacy of the combination. This was consistent with the oligoclonal expansion of host T cells, as revealed by in-depth T-cell receptor repertoire analysis. Moreover, only in the combination group did the activation of endogenous T cells translate into a systemic antitumor response. Importantly, the epitope spreading and the antitumor effects of the combination were fully dependent on host STING signaling and Batf3-dependent DCs, and were partially dependent on Perf release by CART cells. Interestingly, the efficacy of the CART/STING-L treatment also depended on STING signaling in CART cells.ConclusionsOur data show that 2′3′-cGAMP is a suitable adjuvant to combine with CART-cell therapy, allowing the induction of an endogenous T-cell response that prevents the outgrowth of Ag-loss tumor variants.

mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy

“…CAR T cells recognizing the gp75 mouse melanosomal antigen have been described. 61 The CAR construct carries an EGFP reporter gene to monitor the gene transfer of splenocytes that were preactivated with anti-CD3 and anti-CD28 mAbs. CAR gene transfer attained over 90% transduction efficiency using a retroviral vector ( Figure S9 A).…”

“…Retroviral generation was performed as previously reported. 61 Briefly, isolated CD8 T cells from splenocytes were activated in 24-well plates (Cellstar) coated with anti-CD3 mAb (2 μg/mL, clone 17A2, Biolegend) and supplemented with soluble anti-CD28 mAb (1 μg/mL, clone 37.51, Biolegend) for 48 h in complete medium supplemented with human IL-2 (50U/ml) (Proleukin) at 10 6 /mL density. After 48 h later, lymphocytes were resuspended in retrovirus supernatant containing protamine sulfate (10 μg/mL, SIGMA) and human IL-2 and were spin-inoculated at 2000 x g for 90 min at 32°C.…”

The abscopal effect in patients with cancer receiving immunotherapy

“…Additionally, Woo et al 15 showed that the stimulator of interferon genes mechanism can be upregulated by neoantigen presentation, triggering dendritic cell activation, presentation to CD8 T cells, type I interferon production, and tumor microenvironment infiltration of T cells. Based on this, Conde et al 16 combined chimeric antigen receptor T cell therapy and treatment with the immunomodulatory agent 2′3′-cGAMP in preclinical murine melanoma models to improve tumor microenvironment modulation and enhance epitope spreading with stimulator of interferon genes-L/ligand. This led to tumor growth suppression and improved OS in these murine models, with abscopal effects on distal non-injected lesions.…”

Chimeric Antigen Receptor (CAR) T-Cell Therapy for Patients with Lung Cancer: Current Perspectives

“…In conclusion, lung cancer presents a substantial challenge to the development of effective CAR-based immunotherapies. Nonetheless, advanced genetic engineering technologies that allow precise delivery of cells to the site of disease, recognition of a desired target portfolio, re-education of the immunosuppressive TME and also the stimulation of endogenous immune reactivity and epitope spreading [123][124][125][126][127] together offer hope for future success in this daunting quest.…”