Kraemer, S. D. | Search bioRxiv

bioRxiv2025

EpiTox: A Multi-Modular Framework for Population-Aware Off-Target Prediction Highlighting MAGE-A3 Cross-Reactivity

Targeting intracellular tumor antigens presented by peptide human leukocyte antigen (pHLA) complexes offers a promising immunotherapeutic strategy for patients with limited treatment options. However, development of pHLA-targeted drugs such as T-cell receptor (TCR) mimic antibodies (TCRm) and TCR-based therapeutics remains challenging due to severe offtumor and off-target toxicities arising from incomplete pHLA off-target profiling. The fatal neurological toxicities observed in the MAGEA3 TCR-T cell trial, potentially linked to unanticipated cross-reactivity with EPS8L2- and MAGEA12-derived peptides, underscore the urgent need for more comprehensive safety assessment. While current preclinical de-risking methods utilize sequence similarity searches, they lack the layered integration of genetic context, HLA binding profiles, and structured risk assessment needed to comprehensively evaluate peptide cross-reactivity. To address these limitations, we developed EpiTox, a computational multi-modular platform that systematically identifies and evaluates pHLA off-targets. EpiTox integrates proteome-wide sequence similarity, genetic context, HLA binding profiles, and layered risk assessment to provide a holistic evaluation of potential cross-reactive peptides. Using MAGEA3 as a model, EpiTox identified known toxic off-targets and predicted novel epitopes. Notably, an anti-MAGEA3 therapeutic TCRm antibody bound several newly predicted off-targets, including three SNP-derived peptides. By enabling comprehensive and predictive preclinical safety screening, Epi-Tox supports safer TCRm drug development and may help prevent future clinical failures.

bioinformatics↗

bioRxiv2020

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naive universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.

biochemistry↗