Case Study – Personalised Cancer Vaccine
In 2019, Immunitrack reported its first case of neo-epitope selection that was used in a personalised vaccine to treat a colorectal cancer patient. At the time, this was the first case where in silico neo-epitope prediction methods were combined with in vitro MHC/neo-epitope binding assays in a fully personalised manner.
The project, from analysis of the patient’s tumour sequence data to in vitro identification of immunogenic neo-epitopes took less than 3 weeks and the patient was treated at the National Center for Tumor Diseases (NCT) in Heidelberg.
This case study represents a typical project workflow to identify neo-epitopes for cancer vaccine development.
In Silico Identification of Cancer-Specific Mutations
At the outset of the project, whole exome sequencing (WES) had been performed on two tumour biopsies isolated from the patient. In this case, the patient was suffering from colorectal cancer, but regardless of cancer type WES is typically performed to specifically look for neo-epitopes, which result from mutations in protein-coding regions of the cancer genome.
Figure 1. Whole Exome Sequencing (WES) performed on 2 cancer biopsies and a blood sample.
In collaboration with the University of Copenhagen, Immunitrack analysed the WES data in silico and applied a variant calling protocol to identify cancer-specific mutations. Here, patient DNA was compared between cancer cells and healthy cells from blood and only variants that were unique to the tumour were considered relevant. From these variants, Immunitrack then analysed those that led to missense and frameshift mutations by predicting their likelihood of binding to MHC molecules using net MHC and netMHCpan. 20,000 mutations were identified and analysed.
In total, 100 candidate ‘binders’ were selected for binding analysis on 8 distinct patient MHC alleles, resulting in 800 candidate neo-epitopes for further analysis.
Alleles analysed were MHC I: A*0101, A*2402, B*1301, B*3501, C*0401, C*0702, DRB1*0401, DRB1*1501.
Figure 2. More than 20,000 mutations were identified through variant calling. Around 800 neo-eptiopes selected as high affinity binders to patient MHC I & II.
NeoScreen Is Used to Select Stably-Binding Neo-Epitopes
All 800 candidate neo-epitopes were assessed for their ability to bind stably to MHC molecules using Immunitrack’s unique NeoScreen platform. NeoScreen stability assays work by measuring the decay of MHC/epitope complexes under stress-inducing conditions, such as pH or temperature changes or urea denaturation. From this, a half life can be determined for each MHC/peptide complex. Reference peptides are included in all assays and the data yielded from test epitopes is expressed as a % of the reference peptide.
In this case study, 200 of the 800 tested epitopes were identified as highly stable when compared to the reference peptide.
Figure 3. Perform MHC/epitope stability measurement of 100 peptides with highest predicted affinity pr. allele. 6 MHC I and 2 MHC II patient specific alleles analysed.
Further Selection Criteria Applied to Shortlist Neo-Epitopes
Through NeoScreen analysis, 200 mutations were predicted to yield stably-binding epitopes. No RNA sequencing had been performed, and therefore the stable epitopes were further filtered using housekeeping genes as a selection criterion, as these are expressed in all cells and their level of expression is known. In the end, 32 epitopes were selected and formulated into a new candidate vaccine that was used to treat the patient.
The vaccine was formulated and administered to the patient at the National Center for Tumor Diseases (NCT) in Heidelberg.
Figure 4. 200 most stable neo-epitopes are further filtered down to 32 according to global tissue expression.
