Abstract 310: Deciphering the molecular basis for pan-HLA recognition of divarasib-modified pMHCs by newly discovered hit-to-lead antibody AETX-R302

Abstract The practicality of combining targeted therapies using covalent inhibitors of driver oncoproteins with specifically engineered antibodies to selectively kill drug-resistant cancer cells was recently demonstrated (Hattori, Maso et al., Cancer Discov 2023; Maso et al., PNAS 2024). Covalent inhibitors, once bound to their intracellular targets, can survive proteasomal degradation and are subsequently presented by class I MHCs as tumor-specific synthetic neoantigens. Companion antibodies of high specificity and affinity are then generated to selectively recognize these hapten-peptide MHCs (hapten-pMHCs), exploiting the high dissimilarity of hapten-pMHCs from MHCs presenting self-peptides. Therefore, combining the benefits of both targeted and immune therapy, overcomes their weaknesses as monotherapy. The potential of exploiting synthetic neoantigens is well demonstrated by antibody AETX-R114, which recognizes multiple hapten-peptides (sotorasib-conjugated KRASG12C peptides) presented by different HLAs. When reformatted as a T cell engager (TCE), AETX-R114 potently and selectively kills sotorasib-resistant KRASG12C cancer cells expressing different HLAs upon sotorasib treatment in vitro and in vivo. However, its high-affinity hapten-pMHCs cross reactivity, and related cancer cell killing, is restricted to hapten-peptides presented by HLAs belonging to the same HLA superfamily. Here we present the discovery and structural characterization of hit-to-lead antibody AETX-R302, which was raised against hapten-pMHCs presenting divarasib-conjugated KRASG12C peptides (diva-pMHCs). AETX-R302 reveals specific, high affinity (KD ranging between 0.78 and 2.75 nM), and pan-HLA recognition of diva-pMHCs across HLAs A*02 (A2 supertype) and A*03/A*11 (A3 supertype), with minimal free drug competition (IC50 >30 μM), demonstrating that antibodies developed through this platform can effectively break down the supertype barrier while maintaining MHC-restricted recognition. High-resolution cryo-EM structures of AETX-R302 in complex with diva-pMHCs revealed an epitope that involves all three components of the synthetic neoantigen (HLA, hapten, and peptide), exemplifying the three-layer specificity that can be achieved with such antibodies. Its unique binding geometry enables the recognition of HLA residues that are almost completely conserved among HLAs A*02, A*03, and A*11, while allowing a comparable recognition of the hapten when presented by different KRASG12C-derived peptides. Collectively, the cryo-EM structures explain AETX-R302’s unprecedented A2/A3 supertype cross-reactivity and its high specificity and affinity. This information will be exploited to develop TCEs that engage tumor-specific synthetic neoantigen targets with pan-HLA recognition. Citation Format: Lorenzo Maso, Weifeng Liu, Angelina Pizzo, Sarah Mosure, Matthew Southard, Lauren Stopfer, Samantha Sze, Diamond Mensah, Sergio Rodriguez, Anusha Keerthi, Sean Toenjes, Brian Vash, Paul Da Silva Jardine, Rajesh Chopra, Christoph Rader. Deciphering the molecular basis for pan-HLA recognition of divarasib-modified pMHCs by newly discovered hit-to-lead antibody AETX-R302 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 310.