Development of T cell receptor (TCR) based cellular therapy for fibrolamellar HCC uncovers role of TCR in CD4 T cell differentiation

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.