Evaluating TCR mimetic CAR T cell preclinical therapeutics in an immunocompetent MYCN-driven murine neuroblastoma allograft model

Background: Tractable immunocompetent murine models for adoptive cellular therapies could fill a critical gap in preclinical evaluations. Peptide-centric (PC) chimeric antigen receptor (CAR) T cells targeting pMHC of a 9mer peptide derived from the neuroblastoma intracellular oncoprotein PHOX2B presented by HLA-A*24:02 (A24) is now in Phase 1 clinical trial for relapsed neuroblastoma (NCT07007117). To study therapeutic limitations and enhancement strategies, we developed a syngeneic model of PHOX2B/A24-targeting murine (m)PC-CARs. Methods: C57BL/6 (BL/6)-penetrant TH-MYCN-derived allografts and cell lines were engineered with a chimeric human/murine MHC HLA-A*24:02/H-2K b to present conserved PHOX2B 9mer peptide. MSCV retroviral vectors encoding second-generation mPC-CARs containing the clinical scFv conjoined to murine CD28 or 4-1BB with CD3ζ were used to create stable GPE86 producer cell lines. Truncated mCD19 or luciferase was included to monitor transduction efficiency and in vivo trafficking. BL/6 splenocytes were activated and transduced ex vivo with human (h)IL-2, and expanded in hIL-7/15. Multiplex functional assays were performed using flow cytometry, ELISA, real-time cell impedance, and O-link proteomics. Results: Transduction efficiency reproducibly ranged from 40-60%. mPC-CAR-T cells expanded 15 to 20-fold over 14 days while maintaining balanced memory and effector immunophenotypes with minimal exhaustion markers post-manufacture. Against PHOX2B-A24-H-2K b -expressing cell lines, mPC-CAR-T cells with CD28ζ or 4-1BBζ costimulatory domains exhibited robust IFN-gamma, IL-2, and TNFalpha secretion and potent cytotoxicity down to an effector:target ratio of 0.5:1 up to 14 days post-manufacture and after cryopreservation. CD28ζ mPC-CAR T cells maintained 100% cytotoxicity upon serial tumor rechallenge, whereas cytotoxicity of 4-1BBζ was limited to 50% after 3 challenges. In the absence of exogenous cytokine support, tumor exposure induced CD44 + IL7Ralpha - terminal effector phenotype and upregulation of exhaustion markers CD39, CTLA-4, and Tim-3. In vivo studies to assess anti-tumor potency, persistence, and barriers to efficacy are ongoing and will be reported. Conclusion: We created a tractable MHC hybrid TH-MYCN-derived neuroblastoma allograft model in BL/6 mice. This model is currently being deployed to study various PC-CAR enhancement strategies such as mRNA encoded epitope vaccination and cytokine armoring with accompanying spatial transcriptomics that will inform further clinical development of our PHOX2B-directed PC-CAR T cells.