Background TCR-modified T-cell therapy (TCR-T) is emerging as a treatment option for solid tumours containing mutant KRAS,^1–4 an area of great unmet medical need. We have developed a high-precision cell-based analytical platform for scalable TCR-T product development. Herein, we describe use of the platform to perform target quantification and to generate a clinical candidate TCR recognizing the 10mer peptide derived from the KRAS G12V mutant form presented in HLA-A*11:01.

Methods HLA-presented peptides were quantified using mass spectrometry in extracts from mono-allelic engineered antigen presenting cell lines (eAPC) ectopically expressing KRAS G12V and separately in cancer cell lines natively expressing KRAS G12V. TCRs were isolated via paired single cell sequencing following eAPC-stimulated outgrowth of HLA-matched naïve CD8 donor cells. TCRs were characterised in engineered TCR-presenting cells (eTPC) with TCR-linked reporters. Cross-reactivity and allo-reactivity was tested via contact assays using libraries of eAPCs containing epitope and/or HLA variants and eTPC reporters. TCR-T cells were constructed via gene editing of primary donor CD8 cells.

Results A candidate TCR recognizing a 10mer peptide derived from G12V KRAS in HLA-A*11:01 was developed. This TCR recognizes the mutant epitope but not the corresponding wild-type epitope both via peptide loading (mutant EC₅₀1 nM; wild-type not recognised) and via ectopic KRAS-G12V in eAPCs as well as in cancer cell lines expressing KRAS G12V. Allo-reactivity was assessed via a panel of 82 different mono-allelic eAPCs, and cross-reactivity via a library of 200 KRAS G12V peptide variant eAPCs, showing no apparent cross-reactivity or allo-reactivity risk. Primary CD8 cells were transduced by insertional gene editing, generating a TCR-product with high levels of surface TCR and potent cytotoxic action.

Conclusions The TCR-T product is now being advanced to clinical trial and is designed to be the first entry in a broader program covering multiple HLA restrictions and mutant KRAS epitopes.

References

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Ethics Approval Collection of healthy donor peripheral blood and leukapheresis products was undertaken with written informed consent, under ethical approvals 2019–02481/2020–03585 and 2019–02482 granted by the Swedish Ethical Review Authority