KRAS is one of the most frequently mutated oncogenes in human cancer, present in up to 30% of solid tumours. While recent advances have been made in small molecule targeting of some KRAS mutations, responses have been short-lived and the unmet need remains high. Immunotherapies targeting KRAS neoantigens naturally processed and presented as peptide-human leukocyte antigen complexes (peptide-HLA) on the cell surface are an alternative strategy. This approach includes T cell-engaging bispecific antibodies, recently gaining clinical approvals for treatment of solid tumours.
Using our antibody discovery platform, Retained Display (ReD), we have isolated single chain variable fragments (scFv) selectively recognising a 10-mer peptide derived from KRAS G12V in the context of HLA-A*11:01, while avoiding the corresponding wildtype KRAS peptide differing only by a single amino acid. Target selectivity was further demonstrated in binding assays using arrays of irrelevant peptide-HLA-A*11:01 complexes, as well as in assays interrogating binding to peptides with sequence homology to the target peptide. Broad footprints on the target complex associated with decreased risk of cross-reactivity were displayed by scFvs using scanning mutagenesis.
Towards the development of T cell-engaging bispecific antibodies, we affinity matured a candidate scFv, identifying new scFvs maintaining high target selectivity with 10-100-fold improvement in target binding affinity. Combined with a clinically validated anti-human CD3 binding domain, tandem scFv T cell engagers mediated potent killing of target peptide-pulsed cells in the presence of donor T cells. A lead scFv converted into a half-life extended T cell-engager format displayed robust killing of HLA-A*11:01-positive tumour models endogenously presenting KRAS G12V in vitro. No to minimal killing of HLA-A*11:01-positive cells lacking the KRAS G12V mutation was observed. The half-life extended engager displayed a favourable pharmacokinetic profile in mice, enabling in vivo evaluation of efficacy, currently in progress. Our data support antibody-mediated targeting of neoantigen peptide-HLA complexes as a powerful precision immunotherapy to combat solid tumours.