T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H

Volker Lennerz ^1* Christoph Doppler ² Martina Fatho ² Anja Droege ³ Sigrid Schaper ³ Kristin Gennermann ³ Nadine Genzel ¹ Stephanie Plassmann ¹ David Weismann ⁴ Sam W. Lukowski ⁴ Dominik Bents ³ Christina Beushausen ⁵ Karen Kriese ⁵ Hermann Herbst ⁵ Volkhard Seitz ³ Rudolf Hammer ³ Paul J. Adam ⁴ Stephan Eggeling ⁵ Catherine Woelfel ² Thomas Woelfel ² Steffen Hennig ³

• ¹ Therycell GmbH, Berlin, Baden-Württemberg, Germany
• ² Johannes Gutenberg University Mainz, Mainz, Rhineland-Palatinate, Germany
• ³ HS Diagnomics GmbH, Berlin, Germany
• ⁴ Boehringer Ingelheim RCV GmbH & Co KG​, Vienna, Austria
• ⁵ Vivantes Klinikum Neukölln, Berlin, Berlin, Germany

Adoptive cell therapy (ACT) with TCR-engineered T-cells represents a promising alternative to TIL- or CAR-T therapies for patients with advanced solid cancers. Currently, selection of therapeutic TCRs critically depends on knowing the target antigens, a condition excluding most patients from treatment. Direct antigen-agnostic identification of tumor-specific T-cell clonotypes and TCR-T manufacturing using their TCRs can advance ACT for patients with aggressive solid cancers. We present a method to identify tumor-specific clonotypes from surgical specimens by comparing TCRβ-chain repertoires of TILs and adjacent tissue-resident lymphocytes. In six out of seven NSCLC-patients analyzed, our selection of tumor-specific clonotypes based on TIL-abundance and high tumor-to-nontumor frequency ratios was confirmed by gene expression signatures determined by scRNA-Seq. In three patients, we demonstrated that predicted tumor-specific clonotypes reacted against autologous tumors. For one of these patients, we engineered TCR T-cells with four candidate tumor-specific TCRs that showed reactivity against the patient’s tumor and HLA-matched NSCLC cell lines. The TCR-T cells were then used to screen for candidate neoantigens and aberrantly expressed antigens. Three TCRs recognized recurrent driver-mutation KRAS Q61H-peptide ILDTAGHEEY presented by HLA-A*01:01. The TCRs were also dominant in a tumor relapse, one was found in cell free DNA. The finding of homologous TCRs in independent KRAS Q61H-positive cancers suggests a therapeutic opportunity for HLA-matched patients with KRAS Q61H-expressing tumors.