Annabelle Hartt

Abstract Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRAS G12D , presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRAS G12D over KRAS WT . While crystal structures reveal few discernible differences in TCR interactions with KRAS WT versus KRAS G12D , thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRAS G12D . Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen.

<h3>Background</h3> KRAS 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. <h3>Methods</h3> A 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. <h3>Results</h3> The 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 &gt;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 cells <h3>Conclusions</h3> We 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.

This folder contains the starting structures and input scripts required to simulate the wild-type and G12D KRAS peptide bound TCR-pHLA complexes, as performed in this study. <br> Starting_Structures - This folder contains the amber parameter/topology files used to simulate each system (.prmtop) and the coordinates of the starting structure both as amber coordinate file (.rst) and PDB file (.pdb).<br> MD_Inputs - This folder contains the amber MD inputs used to run the md simulations. <br> MMPBSA_inputs - This folder contains the input files for running MMPBSA with the MMPBSA.py script in amber. The mmpbsa.in script was used for calculating overall binding energy whereas the mmpbsa_decomp.in script was used for calculating the per-residue contribution to binding energy.

This folder contains the starting structures and input scripts required to simulate the wild-type and G12D KRAS peptide bound TCR-pHLA complexes, as performed in this study. <br> Starting_Structures - This folder contains the amber parameter/topology files used to simulate each system (.prmtop) and the coordinates of the starting structure both as amber coordinate file (.rst) and PDB file (.pdb).<br> MD_Inputs - This folder contains the amber MD inputs used to run the md simulations. <br> MMPBSA_inputs - This folder contains the input files for running MMPBSA with the MMPBSA.py script in amber. The mmpbsa.in script was used for calculating overall binding energy whereas the mmpbsa_decomp.in script was used for calculating the per-residue contribution to binding energy.