(M1030-01-04) Comprehensive Assessment of Immunogenicity Risk of Host Cell Proteins in Biologics Using In Silico and In Vitro Methods

Purpose: Host cell protein (HCP) impurities are a critical quality attribute (CQA) because they may trigger undesired immune responses with the potential to compromise the safety and efficacy of biologics. The presence of immune responses to Chinese hamster ovary (CHO)-derived HCPs has contributed to the suspension of clinical trials in the past. EpiVax has developed a web-based tool to evaluate the immunogenicity of HCP impurities. This tool, the Interactive Screening and Protein Re-engineering Interface for Host Cell Proteins (ISPRI-HCP) has been in use by industry clients and collaborators since 2003. ISPRI-HCP evaluates the immunogenic potential of proteins based on T cell epitope content and their similarity to the human proteome. We used ISPRI-HCP, to perform an in-silico immunogenicity risk assessment of 140 HCPs identified by a consortium of monoclonal antibody producers. EpiVax will further evaluate and validate the ISPRI-HCP platform by performing in-vitro T cell assays for this set of commonly found HCP impurities.

Methods: In-silico immunogenicity risk assessment of host cell protein impurities was performed using, a web-based platform, ISPRI-HCP. Each input sequence is parsed into overlapping 9-mer frames and evaluated for the potential to bind HLA using the extensively validated EpiMatrix immunoinformatic algorithm. “ Positive hits are considered likely to be T cell epitopes, which can drive anti-HCP immune responses and antibody formation. The density of T cell epitope per unit protein is calculated for each HCP, and then the T cell epitopes in the CHO protein are compared to similar epitopes in the human genome, using JanusMatrix. Each of the 9-mers in the HCP protein are evaluated for humanness by searching for potentially cross-reactive sequences based on the preservation of the TCR-facing residues of HLA ligands. Predicted ligands from an HCP that share the same HLA restriction and TCR-facing contour as epitopes derived from self (human) are presumed to be tolerated. The potential immunogenicity risk was then determined using established thresholds for potential immunogenicity and tolerogenicity. This analysis can identify which HCP have high or low potential for immunogenicity, enabling drug sponsors to focus efforts to remove HCP that have the highest immunogenicity risk.

Results: While detailed reports for each protein will be obtained for the 140 HCP, usually ISPRI-HCP results are depicted on a plot where the Y axis indicates the density of predicted T cell epitopes (EpiMatrix Immunogenicity Score), the X axis indicates the average humanness of those epitopes (JanusMatrix Score), and the size of each marker indicates the relative abundance (e.g. ppm). For this set of proteins, it is clear that HCP immunogenicity varies quite dramatically. For example, for the Jones et al. dataset of monoclonal antibody HCPs, we observe that the results for Phospholipase B-Like 2 Protein show that the protein has a very high T cell epitope density and low cross-conservation with human supports the observation of specific immune response to this impurity. Results for additional HCP will be provided in the presentation. We will be selecting at least 6 HCP for further investigation in vitro, to determine whether the in silico results correlate with human immuine response to overlapping peptide pools representing the putative epitopes and to the whole HCP proteins. PLBL2 and 5 additional CHO HCP impurities will be evaluated in the EpiVax IVIP (In-vitro Immunization Protocol) T cell assay. We expect to publish the results and methods so as to enable drug developers to reduce the risk of immunogencity that can be associated with HCP.

Conclusion: In silico and in vitro analysis of identified HCPs within product preparations for potential immunogenicity is useful for pre-determining the degree of immunogenic risk associated with HCPs. Our process to investigate the in-vitro immune response of HCPs will enhance the development of ISPRI-HCP, providing a better understanding of HCP impurities in biologics, enabling drug developers to improve the safety and efficacy of biologics.

References: Jones M, Palackal N, Wang F, Gaza-Bulseco G, Hurkmans K, Zhao Y, Chitikila C, Clavier S, Liu S, Menesale E, Schonenbach NS, Sharma S, Valax P, Waerner T, Zhang L, Connolly T. "High-risk" host cell proteins (HCPs): A multi-company collaborative view. Biotechnol Bioeng. 2021 Aug;118(8):2870-2885. doi: 10.1002/bit.27808. Epub 2021 May 31. PMID: 33930190.
Fischer SK, Cheu M, Peng K, Lowe J, Araujo J, Murray E, McClintock D, Matthews J, Siguenza P, Song A. Specific Immune Response to Phospholipase B-Like 2 Protein, a Host Cell Impurity in Lebrikizumab Clinical Material. AAPS J. 2017 Jan;19(1):254-263. doi: 10.1208/s12248-016-9998-7. Epub 2016 Oct 13. PMID: 27739010.