(M1130-01-05) Multi-factored Immunogenicity Risk Assessment Approach for Bispecific Immune Cell Engagers

Purpose: Immunogenicity risk assessment has become an essential component of developability appraisal for biologic drug candidates, including monoclonal (mAb), bispecific (bsAb), and multispecific (msAb) antibody constructs. The incidence of anti-drug antibody (ADA) formation is correlated with CD4+ T cell epitope content, which can be modeled using in silico tools. For antibodies that are used in non-inflammatory contexts, adjusting for Treg epitope content improves the accuracy of the in silico predictions. Here we describe an immunogenicity risk assessment approach for several bsAbs, in which immunoinformatic tools were applied with consideration for clinical and mechanistic factors and subsequently compared to published clinical data.

Methods: We retrieved the amino acid sequences of Teclistamab and Elranatamab, T-cell-redirecting bispecific antibodies that target both CD3 expressed on the surface of T-cell and B-cell maturation antigen expressed on the surface of myeloma cells, Talquetamab, a T-cell redirecting bispecific antibody targeting the novel biomarker GPRC5D on multiple myeloma cells and CD3 on T-cells, Navicixizumab, an anti-DLL4/VEGF bispecific antibody that has demonstrated robust in vivo antitumor efficacy in a number of solid tumor xenografts, Vanucizumab, a bispecific antibody inhibiting vascular endothelial growth factor and angiopoietin-2 simultaneously, designed for the treatment of cancer, and Amivantamab, a fully human bispecific antibody with immune cell-directing activity that targets mesenchymal-epithelial transition (MET) and the Exon 20 mutation of EGFR. Using models for a globally representative set of HLA-DR supertype alleles that cover >95% of the worldwide human population, putative T cell epitopes were mapped for each complete protein sequence and constituent domain using the EpiMatrix algorithm, and each predicted HLA ligand was screened against an internally curated database of human antibody isolates and germline sequences. In addition to evaluating for potential T effector epitopes, the ISPRI platform also considers the contribution of regulatory T cell epitopes (Tregitopes) to immunogenic potential. For each input sequence and domain, ISPRI provided Tregitope-adjusted Immunogenicity Scores, which are correlated with clinical immunogenicity. ISPRI normalizes this score so as to determine how the T cell epitope content of the sequence compares to the T cell epitope content of a random protein of equivalent length. Positive scores indicate greater than expected content, while negative scores indicate less-than-random epitope content. The Tregitope-adjusted Immunogenicity Score can be used to predict the ADA response rate for novel antibody products using a regression model based on a set of clinical benchmark monoclonal antibodies whose observed ADA response rates are known. The JanusMatrix algorithm contained in the ISPRI toolkit was also used to evaluate the conservation of identified T cell epitope clusters (hot spots) with autologous (human) proteins at both the HLA-binding and TCR-interacting face of each epitope. The relative risk of immunogenicity was estimated using the Immunogenicity Scale and calculated using the ISPRI ADA prediction tool. This comprehensive assessment was performed, using ISPRI, for each of the input protein domains and the complete multidomain proteins.

Results: ISPRI-generated Immunogenicity Scores for the bsAbs predicted ADA response rates between 0 and 5%, while observed ADA response rates ranged between 0.5% and 16%. Predicted and observed rates differed by ≤5% for Teclistamab, Vanucizumab, and Amivantamab, and by >5% for Elranatamab, Talquetamab, and Navicixizumab. The ISPRI-generated immunogenicity scores of the complete Vanucizumab, Teclistamab, and Amivantamab bispecific antibodies are low, ranging from -26.23 to -51.76. ISPRI’s regression model to predict ADA resulted in an estimate of minimal ADA response for these antibodies, corroborating the observation of low ADA rates in the clinic. Immunogenicity Scores of the complete Elranatamab, Talquetamab, and Navicixizumab bispecific antibodies are low, ranging from -11.87 to -42.86. However, the ISPRI-generated Immunogenicity Scores for these three bispecific antibodies predicted ADA responses that differed from the observed ADA in the clinic. Notably, clinical sample sizes were small, and differences were greatest for molecules with CD3-targeting domains, where raw (non-Treg-epitope adjusted) scores appeared better correlated with immunogenicity.

Conclusion: In this study, three out of five multi-domain molecules evaluated were shown to have overall immunogenicity risk assessments performed by ISPRI that were consistent with clinical observations. The function or mechanism of the antibody targets may be the cause of the apparent discrepancy between the predicted and observed ADA responses in two of the multidomain molecules. EpiVax’s ISPRI Toolkit not only allows for the rapid in silico analysis and assessment of the immunogenicity risk of complex, multidomain biologics, but also estimates ADA rates. As biologic formats become more complex, it is necessary to consider the immunogenic potential of novel constructs as a whole, and of individual components that might each have distinct T cell epitope characteristics.

References: Zhou Y, Penny HL, Kroenke MA, et al. Immunogenicity assessment of bispecific antibody-based immunotherapy in oncology. Journal for ImmunoTherapy of Cancer 2022;10:e004225. doi:10.1136/jitc-2021-004225