Abstract Background: Fibrolamellar carcinoma (FLC) is a rare and often lethal liver cancer that primarily affects children and young adults. There is no approved systemic therapy for FLC. The FLC transcriptome is defined by an in-frame fusion of exon 1 of DNAJB1 with exons 2-10 of PRKACA, resulting in expression of the chimeric oncoprotein DNAJ-PKAc. Direct pharmacologic inhibition of this oncoprotein has been infeasible due to unacceptable on-target toxicity. However, the shared, tumor-specific expression of DNAJ-PKAc presents an opportunity for neoantigen-targeted immunotherapy. Methods: We are conducting an ongoing clinical trial of a peptide vaccine targeting DNAJ-PKAc in combination with immune checkpoint inhibitors. We used single-cell sequencing and functional assays to identify DNAJ-PKAc-specific T cell receptors (TCRs) from peptide-expanded peripheral blood cells of trial participants. We characterized their HLA restriction, avidity, cytotoxicity, cytokine profiles, and differentiation phenotypes using co-culture systems. Results: We identified seven CD4+ TCRs that recognize DNAJ-PKAc in the context of HLA-DRB1*13:01 (n=2) or HLA-DRB3*01:01 (n=5). When introduced into healthy donor T cells and co-cultured with tumor cells expressing the corresponding HLA allele, these TCRs exhibited variable functional avidity, cytokine production, differentiation, and cytotoxic activity against DNAJ-PKAc-pulsed targets. Among them, JHU12-TCR2 (HLA-DRB3*01:01-restricted) mediated the strongest cytokine production and tumor cell killing despite only moderate avidity. Consistent with this, JHU12-TCR2 showed the greatest in vivo expansion in the original patient, who achieved a near-complete response to immunotherapy. In contrast, a fusion-specific TCR identified from a non-responder, JHU8-TCR2, that shares sequence similarities and clusters with JHU12-TCR2, demonstrated limited cytotoxicity in vitro. Interestingly, although all TCRs recognized the same antigen in matched donor T cells, they drove divergent helper T cell differentiation programs. For example, JHU12-TCR2 favored Th1/Th17 polarization with minimal Treg induction, whereas JHU8-TCR2 promoted a Th2-biased phenotype. Conclusions: We identified and functionally characterized multiple CD4+ TCRs specific for the DNAJ-PKAc oncoprotein, highlighting their potential utility as the basis for TCR-based therapy in FLC. A clinical trial of JHU12-TCR2-based TCR therapy for patients with FLC and HLA-DRB3*01:01 is in development. These findings also suggest that variation in vaccine-induced TCR repertoires may contribute to differences in clinical response, and that intrinsic biophysical or signaling properties of individual TCRs can shape the differentiation fate of CD4+ T cells expressing them. Citation Format: Kayla J. Bendinelli, Allison M. Kirk, Marina Baretti, Abigail M. Gottschall, Heng-Chung Kung, Jeric Peter Hernandez, Waqar Arif, Julie Nauroth, Jennifer Durham, Christopher Thoburn, Amanda Huff, Neeha Zaidi, Alexei Hernandez, Hassan Jamaleddine, Timothy N. West, Justin McCallen, George Coukos, Alexandre Harari, Challice L. Bonifant, Elizabeth Jaffee, Won Jin Ho, Grégoire Altan-Bonnet, Paul G. Thomas, Mark Yarchoan. Development of T cell receptor (TCR) based cellular therapy for fibrolamellar HCC uncovers role of TCR in CD4 T cell differentiation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5620.
Bendinelli et al. (Fri,) studied this question.