(W1230-02-11) Comprehensive Assessment of Immunogenicity Risk of Host Cell Proteins in Biologics and Vaccines Using In Silico and In Vitro Methods

Purpose: Host cell protein (HCP) impurities are considered a critical quality attribute because of their potential to compromise the safety and efficacy of biologics by triggering undesired immune responses. In some cases, the presence of immunogenic HCPs has resulted in costly suspensions of late phase clinical trials. To help biologic producers, we have developed a web-based tool ISPRI-HCP (Interactive Screening and Protein Re-engineering Interface for Host Cell Proteins) to evaluate the immunogenicity of HCP impurities. We report the in-silico immunogenicity risk assessment of 140 commonly found Chinese hamster ovary cell (CHO) protein impurities and protein impurities found in the ChAdOx1 nCov-19 vaccine. We are performing in-vitro studies of HCPs for further evaluation as well as validation of the ISPRI-HCP platform.

Methods: We used ISPRI-HCP to classify a set of common HCP impurities according to their immunogenicity risk. These risk assessment thresholds generally correspond with clinical reports when those data are available. In-silico immunogenicity risk assessment of host cell protein impurities was performed using a web-based platform, ISPRI-HCP. ISPRI-HCP estimates the immunogenic potential of protein sequences by evaluating their class II-restricted T cell epitope density, and the relative conservation of these epitopes with other, similar epitopes in the human genome, using the tools EpiMatrix, and JanusMatrix. With EpiMatrix, determines the T cell epitope content by parsing each protein’s sequence into overlapping 9-mer frames and evaluates the potential to bind nine HLA-DR supertypes. Four out of nine positive hits are considered likely to be T cell epitopes and the density of epitopes for each HCP is calculated. JanusMatrix, evaluates the homology of HLA-binding T cell epitope clusters identified in HCPs (e.g., from CHO) to epitopes with similar HLA-DR-restricted epitopes that are present in the human proteome. With JanusMatrix, and then the T cell epitopes in the CHO protein are compared to the human proteome. T cell epitopes with high homology to human proteins are considered to be more tolerated. The combination of EpiMatrix and JanusMatrix Score are used to classify immunogenicity risk based on established thresholds. This analysis can assess which HCPs have high, moderate or low potential for immunogenicity, enabling drug developers to focus efforts to remove unwanted impurities. After in silico analysis, we employ algorithms for computer-assisted-design of peptide arrays to represent each HCP in such a way as to highlight the potential of each putative epitope in that given protein impurity using proper centering and proper flanking, all while saving valuable resources.

Results: ISPRI-HCP results are depicted on a plot such that the Y axis indicates the density of predicted T cell epitopes, providing a measure of immunogenic potential, the X axis indicates the average humanness of those epitopes (indicating potential for tolerance), and the size of each marker indicates relative abundance (e.g. ppm). From the proteins investigated here, it is clear that HCP immunogenicity varies quite dramatically. Analysis of phospholipase B-like 2 Protein, an HCP identified in the Jones et al. dataset of monoclonal antibody HCPs, shows that the protein has a very high T cell epitope density and low cross-conservation with human, agreeing with the clinical observation of specific immune response to this impurity. Hexon, an adenoviral protein impurity present in the ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vaccines, has elevated immunogenicity scores and low human cross-conservation, indicating risk for impurity-driven immune responses and corroborating the findings of Pitkänen et al. which show hexon complexes with platelet factor 4, inducing anti-drug antibodies and triggering spontaneous aggregation of healthy platelets, leading to vaccine-induced thrombotic thrombocytopenia in certain patients. Six HCP have been investigated further in vitro to determine whether the in silico results correlate with human immune response to overlapping peptide pools representing the putative epitopes and to the whole HCP proteins. PLBL2 and 5 additional CHO HCP impurities have so far been evaluated in the In-vitro Immunization Protocol (IVIP) T cell assay, with many more host cell protein impurities scheduled for study. Initial results show a positive correlation between in silico predictions and in vitro immune responses and indicate that further investigation will allow for improvement and fine tuning of in silico platforms used to make these kinds of predictions. We expect to publish the results and methods to enable drug developers to reduce the risk of immunogenicity that can be associated with HCP impurities in biologics and vaccines.

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 and vaccines and enabling developers to improve their safety and efficacy.

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Acknowledgements:

Funding: This work was supported in part by the FDA grant U01FD007660.