Abstract
Mutations in CTNNB1, the gene encoding β-catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in β-catenin. Peptides derived from WT β-catenin have previously been shown to be presented on the cell surface as part of major histocompatibility complex (MHC) class I, suggesting an opportunity for targeting this common driver gene mutation with antibody-based therapies. Here, crystal structures of both the WT and S45F mutant peptide bound to HLA-A*03:01 at 2.20 and 2.45 Å resolutions, respectively, confirmed the accessibility of the phenylalanine residue for antibody recognition. Phage display was then used to identify single-chain variable fragment clones that selectively bind the S45F mutant peptide presented in HLA-A*03:01 and have minimal WT or other off-target binding. Following the initial characterization of five clones, we selected a single clone, E10, for further investigation. We developed a computational model of the binding of E10 to the mutant peptide–bound HLA-A3, incorporating data from affinity maturation as initial validation. In the future, our model may be used to design clones with maintained specificity and higher affinity. Such derivatives could be adapted into either cell-based (CAR-T) or protein-based (bispecific T-cell engagers) therapies to target cancer cells harboring the S45F mutation in CTNNB1.
Highlights
Mutations in CTNNB1, the gene encoding -catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in -catenin
Using the COSMIC database and NetMHCv4.0, we identified peptides containing common driver gene mutations from CTNNB1 that were predicted to bind to common human leukocyte antigen (HLA) alleles
We have presented a novel method for potentially targeting tumors harboring mutations in CTNNB1
Summary
Target peptide selection: S45F is exposed upon -catenin peptide binding to HLA-A*03:01. To further assess the specificity of these five phage clones for peptides derived from proteins that might be presented on normal human cells, we identified so-called “BLAST” peptides by comparing the mutant peptide with peptides from the normal human proteome These peptides were identified using the human refseq database and BLASTp, followed by netMHCv4.0 to predict their HLA-A3– binding capacity. We thereby identified seven peptides with similarity to the mutant peptide (Table S1) and found that they each stabilized HLA-A3 on the surface of T2A3 cells to varying degrees (Fig. S3). None of these seven peptides exhibited cross-reactivity with any of the phage clones (Fig. 2B). T2A3 cells pulsed with the WT peptide plus 2-microglobulin or 2-microglobulin alone exhibited marginal cell death (p Ͻ 0.0001)
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