In silico methods for immunogenicity risk assessment and human homology screening for therapeutic antibodies

Abstract

In silico immunogenicity risk assessment has been an important step in the development path for many biologic therapeutics, including monoclonal antibodies. Even if the source of a given biologic is 'fully human', T cell epitopes that are contained in the sequences of the biologic may activate the immune system, enabling the development of anti-drug antibodies that can reduce drug efficacy and may contribute to adverse events. Computational tools that identify T cell epitopes from primary amino acid sequences have been used to assess the immunogenic potential of therapeutic candidates for several decades. To facilitate larger scale analyses and accelerate preclinical immunogenicity risk assessment, our group developed an integrated web-based platform called ISPRI, (Immunogenicity Screening and Protein Re-engineering Interface) that provides hands-on access through a secure web-based interface for scientists working in large and mid-sized biotech companies in the US, Europe, and Japan. This toolkit has evolved and now contains an array of algorithms that can be used individually and/or consecutively for immunogenicity assessment and protein engineering. Most analyses start with the advanced epitope mapping tool (EpiMatrix), then proceed to identify epitope clusters using ClustiMer, and then use a tool called JanusMatrix to define whether any of the T cell epitope clusters may generate a regulatory T cell response which may diminish or eliminate anti-drug antibody formation. Candidates can be compared to similar products on a normalized immunogenicity scale. Should modifications to the biologic sequence be an option, a tool for moderating putative immunogenicity by editing T cell epitopes out of the sequence is available (OptiMatrix). Although this perspective discusses the in-silico immunogenicity risk assessment for monoclonal antibodies, bi-specifics, multi-specifics, and antibody-drug conjugates, the analysis of additional therapeutic modalities such as enzyme replacement proteins, blood factor proteins, CAR-T, gene therapy products, and peptide drugs is also made available on the ISPRI platform.

Keywords: Anti-drug antibodies; EpiMatrix; JanusMatrix; T cell epitope; immunogenicity; immunoinformatics; monoclonal antibody; tregitope.

Figure 1.

Screenshot of the ISPRI homepage, with arrows connecting ISPRI silos and tools that enable immunogenicity risk assessment. Analysis usually proceeds by starting with a global and regional assessment of the protein sequence for T cell epitopes, followed by an assessment of regulatory T cell epitope (tregitope) content and then by comparisons to known antigens and the human genome. Once global and regional analysis is performed, additional tools included in the ISPRI platform (OptiMatrix) can be used to support deimmunization.

Figure 2.

Epitope Map. Input sequences are parsed into overlapping 9-mer frames and each frame is assessed for likelihood to bind to the nine HLA-DR supertype alleles. Blue fill indicates putative class II T cell epitopes for the given frame-by-allele assessment; the strength of the score is indicated by the blue shading. Dark blue indicates assessments in the top 1% of the normal distribution, and medium blue fill indicates scores in the top 5% of the normal distribution. The lightest blue shading indicates scores in the top 10% of the normal distribution and are considered ‘near-misses’. Black outlined boxes indicate the relative positions of T cell epitope clusters, or regions of high epitope density, defined by the ClustiMer algorithm. Green fill indicates T cell epitopes for 9-mer frames that are exactly matched to known regulatory T cell epitopes (tregitopes).

Figure 3.

(a)EpiMatrix predicted excess of shortfall in predicted aggregate immunogenicity relative to a Random Protein Standard (per 1,000 9-mer frames analyzed). All scores are adjusted for the presence of tregitopes. *Average of 10 antibodies (VH/VL pairs) known to Induce Anti-Therapeutic Responses in more than 5% of patients, ^†Average of 10 antibodies (VH/VL pairs) known to Induce Anti-Therapeutic Responses in less than 5% of Patients.(b)Tregitope-adjusted EpiMatrix scores for 20,401 proteins derived from the human proteome (UniProt accession UP000005640) were generated.

Figure 4.

Cytoscape figure of human-like epitope.“Non-IgG Tregitope” that was identified in the CDR region of infliximab. This peptide contains several HLA binding motifs (dark grey squares) including one that is extensively conserved with multiple self-peptides that share the same TCR face (dark blue triangles). The JanusMatrix score for this peptide is 3.14. None of the cross-conserved peptides are found in IgG in this instance. This peptide was shown to be associated with IL2 secretion in studies of human T cell responses to infliximab peptides carried out by Vultaggio et al.

Figure 5.

Regional assessment for Immunogenicity. EpiMatrix assessment of an example antibody in the ISPRI toolkit. Each overlapping 9 mer is scored for predicted affinity to one of nine HLA class II alleles. All scores in the top 5% (Z-Score ≥ 1.64) are considered “hits”. Scores in the top 10% are considered elevated, other scores are grayed out for simplicity. Frames containing four or more alleles scoring above 1.64 are referred to as EpiBars and are highlighted in yellow. These frames have an increased likelihood of binding to HLA. Frames conserved in IgG antibodies and believed to be either passively tolerated or actively regulatory are highlighted in green.

Figure 6.

Twenty-two licensed antibodies make up the polynomial regression used for predicting T-dependent ADA responses in ISPRI (orange line). The updated regression model adds 21 new mAb examples with clinical immunogenicity data. Observed immunogenicity indicates the percent of exposed patients with a positive immunogenic response as defined by a positive ADA titer and reported from clinical trials identified in the FDA-approved drug product labels.

Figure 7.

EpiMatrix predicted excess of shortfall in predicted aggregate immunogenicity relative to a Random Protein Standard (per 1,000 9-mer frames analyzed). All scores are adjusted for the presence of tregitopes. Predicted ADA responses are indicated in parentheses. Predicted response = 11.367 + 0.5769 * tregitope-adjusted score + 0.0076 * tregitope-adjusted Score (R²=0.6722). Scores shown represent the combined protein score for the VH/VL sequences. Observed ADA responses for benchmark antibodies are indicated in parentheses on the lefthand side of the scale. *Average of 10 antibodies (VH/VL pairs) known to Induce Anti-Therapeutic responses in more than 5% of patients. †Average of 10 antibodies (VH/VL pairs) known to Induce Anti-Therapeutic responses in less than 5% of patients.