(T1530-06-32) Immunogenicity Risk Assessment for Host Cell Proteins in Vaccines and Biologics

Purpose: We have developed a tool that evaluates HCPs that copurify with therapeutic protein, peptide, or vaccine products for immunogenicity. Residual host cell proteins (HCPs), known as “hitchhiker” proteins, can induce detrimental immune responses in patients and compromise the safety and efficacy of a biologic. Indeed, the presence of Chinese hamster ovary (CHO)-derived HCPs contributed to the suspension of two clinical trials in 2012. Our immunogenicity risk assessment tool for therapeutic proteins, the Interactive Screening and Protein Re-engineering Interface, has been in use by industry clients and collaborators since 2003. We have recently developed a web-based tool to evaluate the immunogenic potential of HCPs based on T cell epitopes and their similarity to the human proteome, and to evaluate sets reported HCP impurities.

Methods: We analyzed a set of HCP identified by a consortium of monoclonal antibody producers [Jones et al.], as well as host cell and viral vector proteins identified in a lot of ChAdOx1 nCov-19 vaccine [Krutzke et al.]. Immunogenicity of biologics is driven by activated CD4 + T cells, and anti-drug antibody (ADA) responses may develop in reaction to components of the drug product or contaminants from production host cell lines. The web accessible ISPRI_HCP tool assesses the immunogenicity risk posed by HCPs by evaluating both the potential presence of an HCP of interest and its potential immunogenicity. Each input sequence is parsed into overlapping 9-mer frames and evaluated for potential to bind HLA using the extensively validated EpiMatrix immunoinformatic algorithm, and then evaluated for humanness using the JanusMatrix algorithm. Briefly, JanusMatrix searches for potentially cross-reactive sequences based on the preservation of the TCR-facing residues and established HLA binding preferences of given 9-mer 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.

Results: Our results indicate that the immunogenicity of HCPs can vary dramatically. A careful analysis of identified HCPs within product preparations can help to determine the degree of immunogenic risk of the HCP. Furthermore, visualization of ISPRI HCP results in context with relative abundance in individual product lots can help drug producers to quickly identify the impurities with the highest likelihood of recognition by a patient immune system. Results are depicted on a plot where the Y axis indicates density of predicted T cell epitopes (EpiMatrix Immunogenicity Score), the X axis indicates average humanness of those epitopes (JanusMatrix Score), and the size of each marker indicates the relative abundance (e.g. ppm) measured in the lot. As shown for the Jones dataset of monoclonal antibody HCPs, Phospholipase B-Like 2 Protein is an example of a CHO protein found with high frequency in monoclonal antibody lots, and its position in the upper left quadrant (high epitope density, low cross-conservation with human) supports the observation of specific immune response to this impurity [Fischer et al.]. In the case of the ChAdOx1 nCov-19 vaccine, several viral vector proteins have elevated immunogenicity scores and low human cross-conservation, indicating considerable risk for impurity-driven immune response. Additionally, multiple human host cell proteins persist in the clinical material, with widely ranging immunogenicity scores and human cross-conservation. In a best case scenario, highly cross-conserved human epitopes are well tolerated in this material. In a worst case scenario, however, it is possible that immune responses against the copurifying human host cell proteins or other human epitopes with which they are cross-conserved, might be capable of stimulating auto-immunity, particularly if response against residual viral vector proteins provides an adjuvating effect.

Conclusion: Further development of the ISPRI_HCP tool, in collaboration with biologics industry partners, will enable exploration of immune responses in vitro, enhancing the prediction of immunogenic and tolerated T cell epitopes from HCPs, as well as evaluate the correlations with resultant ADA when patient data are available.

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7. 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.