An engineered antibody fragment targeting mutant β-catenin via major histocompatibility complex I neoantigen presentation

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.

Bispecific antibodies targeting mutantRASneoantigens

“…Thus, we turned our attention to two peptides, a 9-mer (VVGAVGVGK) from codons 8 to 16 ("G12V [8][9][10][11][12][13][14][15][16]") and a 10-mer (VVVGAVGVGK) from codons 7 to 16 ("G12V [7][8][9][10][11][12][13][14][15][16]"), containing the G12V mutation that were predicted via NetMHC v4.0 to bind HLA-A*03:01 (henceforth referred to as HLA-A3), one of the most common HLA-A alleles (17,18). Using MANA-SRM, we detected the G12V [7][8][9][10][11][12][13][14][15][16] peptide at 102 copies per cell and the G12V [8][9][10][11][12][13][14][15][16] peptide at 24 copies per cell in COS-7 cotransfected with HLA-A3 and KRAS G12V (fig. S1A and table S1).…”

“…Such peptides are called mutation-associated neoantigens (MANAs). To take advantage of this truly tumor-specific cell surface antigen, our group has developed a class of T cell receptor (TCR)-mimic antibodies called MANA-directed antibodies (MANAbodies) (9,10).…”

Targeting loss of heterozygosity for cancer-specific immunotherapy

“…We therefore turned to identifying an alternative A3-specific scFv. An scFv phage display library, with an estimated complexity of 3.6 × 10 10 , was screened for binders that could selectively target A3 but not other HLA-A alleles ( 103 ). Positive selection was conducted with a different A3 peptide-HLA (pHLA) monomer during each round of panning so as to enforce specificity to the HLA molecule itself and not the associated peptide, while negative selection was performed with a mixture of non-A3 pHLA monomers.…”

“…5 B ). For example, antibodies and TCRs that selectively react to HLA-presented peptides differing by only a single amino acid residue have been described ( 103 , 106 – 109 ). Furthermore, NASCAR inhibitory antigens could also be expanded beyond the direct targeting of polymorphic forms lost through LOH to include other antigen loss mechanisms exposed by LOH ( 110 – 112 ).…”

“…Phage Display. Experimental procedures to isolate phage clones reactive to HLA-A3 followed methods that were previously described (103,106), with the following exceptions. For positive selection, a different HLA-A3 pHLA monomer complex was employed during each round of panning, to enrich for allele-specific phage; conversely, for negative selection, a mixture of non−HLA-A3 pHLA monomer complexes was used to deplete non−allelespecific phage.…”