Abstract
YYB-101 is a humanized rabbit anti-human hepatocyte growth factor (HGF)-neutralizing antibody currently in clinical trial. To test the effect of HGF neutralization with antibody on anti-cancer T cell immunity, we generated surrogate antibodies that are reactive to the mouse homologue of the epitope targeted by YYB-101. First, we immunized a chicken with human HGF and monitored changes in the B cell repertoire by next-generation sequencing (NGS). We then extracted the VH gene repertoire from the NGS data, clustered it into components by sequence homology, and classified the components by the change in the number of unique VH sequences and the frequencies of the VH sequences within each component following immunization. Those changes should accompany the preferential proliferation and somatic hypermutation or gene conversion of B cells encoding HGF-reactive antibodies. One component showed significant increases in the number and frequencies of unique VH sequences and harbored genes encoding antibodies that were reactive to human HGF and competitive with YYB-101 for HGF binding. Some of the antibodies also reacted to mouse HGF. The selected VH sequences shared 98.3% identity and 98.9% amino acid similarity. It is therefore likely that the antibodies encoded by them all react to the epitope targeted by YYB-101.
Highlights
Hepatocyte growth factor (HGF), known as scatter factor, is a ligand for c-MET and was initially identified as a growth factor for fibroblast-derived cell motility factor and hepatocytes [1]
We used the cDNA and specific primers to amplify the variable heavy-chain (VH) gene, which we sequenced on the Illumina MiSeq next-generation sequencing (NGS) platform
To determine the optimal Levenshtein distance (LD) threshold, we focused on the complementary determining region 3 (CDR3) sequence
Summary
Hepatocyte growth factor (HGF), known as scatter factor, is a ligand for c-MET and was initially identified as a growth factor for fibroblast-derived cell motility factor and hepatocytes [1]. High serum levels of HGF were correlated with increasing neutrophil counts and unresponsiveness to anti-PD-1 checkpoint blockade [12] All of those observations suggested that treatment with HGF-neutralizing antibodies might potentiate the efficacy of immune checkpoint inhibitors. The most accurate way to test the combinational therapeutic effects of YYB-101 would be to treat immunologically intact mice with a mouse HGF-neutralizing antibody that binds to the homologous epitope on human HGF. The reactive clones retrieved from the phage-display library contained VH genes that had changed significantly following the immunization. From those reactive clones, we could successfully select antibodies that were reactive to both human and mouse HGF and competitive with YYB-101 for binding to human HGF
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