Background
Mutations in the RAS family are highly prevalent in human cancers, including up to 35% of non-small cell lung, 45% of colorectal, and 95% of pancreatic cancers. Kirsten rat sarcoma viral oncogene homologue (KRAS) is the most frequently mutated RAS oncogene and patients with KRAS mutations have poor responses to standard treatment regimens. Leveraging Affini-T’s TETHER platform, we present the development and characterization of novel bispecific TCR-based T cell engagers (TCEs) targeting HLA-A*11:01 KRAS-G12D and G12V mutations. These TCEs aim to redirect endogenous T cell cytotoxicity against KRAS mutant cancer cells while limiting off-target responses.
Methods
Bispecific TCR T cell engagers were engineered to engage T cells through stimulation of the CD3 complex and target HLA-A*11:01+ cancer cells presenting either KRAS-G12D or KRAS-G12V peptides via a mutant-specific TCR. Yeast display affinity maturation was performed to enhance the specificity and binding affinity of the TCR for its respective KRAS mutant peptide. In vitro pharmacology assays were conducted to evaluate T cell-mediated activation and killing by the TCEs. In parallel, in vitro tolerability studies were performed to evaluate autoantigen cross-reactivity.
Results
The affinity-matured TCRs for both KRAS-G12D and KRAS-G12V exhibited significantly improved binding specificity and affinity, with a greater than 1000-fold affinity enhancement as compared to the parental TCR. Specificity of these affinity-enhanced TCRs was retained, generating TCRs with up to >7000-fold affinity window for the KRAS mutant peptide compared to KRAS wildtype. In vitro pharmacology assays demonstrated that the TCEs effectively mediated potent T cell cytotoxicity against HLA-A*11:01 human tumor cell lines endogenously harboring the corresponding KRAS mutations. Robust T cell activation, indicated by IFN production, was observed for candidate engagers. Tolerability evaluations reveal minimal off-target effects on normal tissue and favorable off-target profiles in both X-scan mutagenesis scans and an off-target yeast-displayed peptide library. Lastly, all TCRs retain biophysical metrics consistent with favorable developability characteristics.
Conclusions
We report application of the TETHER platform to create highly potent and specific T cell engager molecules against multiple mutated oncogenic driver targets. Our approach provides a promising therapeutic strategy for the treatment of cancers driven by KRAS-G12D and KRAS-G12V mutations. The robust preclinical activity and tolerability profiles of these engagers support their potential for application in managing KRAS-mutant malignancies. Additionally, these data support the potential of the TETHER platform to produce TCR TCEs against additional mutated oncogenic drivers in solid tumors.