BackgroundKRAS is the most frequently mutated oncogene, yet mutant KRAS has historically been a challenging target for conventional small molecule drug development. Tumour specific neoantigen peptides derived from KRAS are presented by cell surface human leucocyte antigens (HLA) and form a class of shared, tumour-specific antigens that are attractive targets for immunotherapy.MethodsA T cell clone that specifically recognizes the most common KRAS G12D mutant presented as a peptide in the context of HLA-A*11:01 was isolated from healthy donor PBMCs. The affinity of the respective T cell receptor (TCR) was enhanced by phage display and the x-ray crystal structures of the affinity-enhanced TCR bound to HLA presenting mutant KRAS G12D and wildtype (KRAS WT) peptides were solved. We used structural, biochemical, and computational approaches to investigate the molecular interactions underlying TCR selectivity for mutant KRAS G12D. Finally, the high affinity TCR was engineered into a soluble T cell engaging ImmTAC (Immune mobilizing monoclonal TCR Against Cancer) molecule, IMC-KRAS-G12D, and in vitro cell-based assays were performed to evaluate its potency and selectivity.ResultsThe affinity of the engineered TCR was enhanced by a million-fold and demonstrated remarkable ability to distinguish between KRAS G12D and KRAS WT peptide presented by HLA-A*11:01. X-ray crystal structures demonstrate that TCR binding is almost identical between KRAS G12D and KRAS WT despite a binding affinity difference of >4000 fold. The mutant residue G12D is buried into the HLA peptide binding groove and acts as a secondary anchor, making it inaccessible to the TCR. Thermodynamic analysis of TCR-HLA interaction combined with molecular dynamics simulations indicates a novel mechanism of peptide selectivity, mediated by an indirect energetic mechanism driven by an induced fit in the peptide upon TCR binding. In functional assays, this molecular differentiation translated into biological specificity with IMC-KRAS-G12D mediating T cell activation in response to cells pulsed with or expressing KRAS G12D but not KRAS WT. Furthermore, IMC-KRAS-G12D was able to redirect T cell cytotoxicity towards target KRAS G12D presenting colon cancer cells, while sparing normal colon epithelial cellsConclusionsWe developed a high affinity TCR bispecific with exquisite specificity towards a common shared neoantigen, KRAS G12D, that is a relevant therapeutic target in a wide range of cancers. These findings reveal a novel molecular mechanism for TCR selectivity for a neoantigen that differs from self-antigen by only a single amino acid, with attendant implications for therapeutic development.
High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-β-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors
