Deimmunisation Project Overview
Immunitrack supports the deimmunisation of biologics by applying highly sensitive affinity and stability assays to assess the binding of drug-derived peptide epitopes to MHC II molecules.
Peptide/MHC interactions have traditionally been assessed by affinity assays but we, and others, have found that stability of the peptide/MHC complex is a better indicator of potential immunogenicity (1-5). Immunitrack’s NeoScreen® immunogenicity prediction platform combines affinity and stability assays to eliminate the false positives that may arise when conducting affinity assays alone.
Typical Deimmunisation Project Workflow
A partner or client typically approaches Immunitrack with the peptide sequence of a biologic they wish to assess for immunogenicity. A meeting takes place to discuss overall project goals, including:
- Which assays to run. We always advise to run stability first and then affinity as this order is most cost-effective.
- How many MHC II alleles should be included in the screening. Immunitrack generally runs a panel of MHC II molecules (see Table 1). Immunitrack MHC Allele Library ▸
- Region to screen. It is possible to screen an entire biologic or a particular region(s) of the protein. Projects concerning humanised antibodies raised against specific targets would generally only require screening of complementarity-determining regions (CDR) for immunogenicity and subsequent deimmunisation steps, while nanobodies or enzymes destined for enzyme replacement therapy are typically screened over their entire protein sequence.
Once the goals have been established, the biologic is assessed in a 3-step process, as illustrated in Figure 1 and outlined below.
Figure 1. Typical Demmunisation Project Workflow.
Step 1: Immunitrack divides the biologic’s peptide sequence into overlapping peptides (13-15 amino acids in length) that will then be analysed for stability and/or affinity, depending on the partner’s wishes, against a representative panel of MHC II molecules (Table 1). For preclinical assessments, we can expand Step 1 as necessary to include MHC alleles from BL6 and BALBc. Stability and affinity assays will determine which of the overlapping peptides constitute potential CD4 T cell epitopes.
Table 1. MHC II Panel Included in Typical Deimmunisation Project. Proposed MHC II alleles and their approximate frequency in a Caucasian population. Data from www.allelefrequencies.net/ or * J Immunol. 2002 Dec 15;169(12):6928-34. DOI: 10.4049/jimmunol.169.12.6928. Note: Additional MHC II alleles can be included to present non-causasian populations as required.
Step 2: Potential CD4 T cell epitopes (identified through Step 1) are alanine-scanned to reveal anchor positions i.e. residues that are critical for MHC II binding. Figure 2 displays the alanine scanning results from a peptide (N3) that had previously been identified through NeoScreen® as a potential CD4 T cell epitope. Initial analysis revealed that N3 bound with reasonable stability to DRB1*0101 and with high stability to DRB3*0101.
Figure 2. Alanine scanning of one 13-mer epitope presented by a subset of MHC II molecules. The numbers in the table represent stability measurements for each epitope (top row) against each MHC II molecule (left most column), expressed as a percentage relative to a reference peptide. The reference peptide is included as a positive control in all stability assays and is set at 100 %. N3 (arrow 1) is the wild-type peptide and the remaining epitopes (N3_1 to N3_15) are N3 derivatives that have a substitution in any one of 15 anchor positions. Arrows 2, 3 and 4 indicate anchor positions 4, 5 and 12, where an alanine substitution decreases binding to MHC II. Arrows 2-4: anchor positions.
Step 3: During this last step, anchor positions identified through Step 2 are substituted with any of the remaining 19 non-alanine amino acids to create a ‘library’ of accepted amino acids at these positions, where biological activity is retained but without binding to relevant MHC II molecules.
The final product following the 3-step workflow described above is typically a list of suggested amino acid changes that may lead to biologics with low or no MHC II binding. Upon request, Immunitrack can use bioinformatics approaches to assist partners in assessing which of these changes are likely to yield a biologic with retained biological activities.