Abstract 2789: Development of highly potent T-cell receptor bispecifics with picomolar activity against tumor-specific HLA ligands
Authors: Bunk Sebastian , Hofmann Martin , Unverdorben Felix , Alten Leonie , Hutt Meike , Wagner Claudia , Schoor Oliver , Ferber Mathias , Fritsche Jens , Weinschenk Toni , Singh-Jasuja Harpreet , Maurer Dominik , Reinhardt Carsten 
Published: August 2018

Abstract
T-cell receptor (TCR)-based immunotherapy has emerged as a promising treatment modality for malignant diseases. TCRs naturally recognize human leucocyte antigen (HLA)-bound peptides from foreign and endogenous antigens regardless of their source proteins' extracellular or intracellular location. Using its proprietary discovery engine XPRESIDENT®, Immatics can identify, quantify, and validate Tumor-Associated Peptides (TUMAPs) exclusively presented in cancer tissues. Immatics has established state-of-the-art technology to discover and affinity maturate TUMAP-specific TCRs originating from human T-cell repertoire. The maturated single-chain TCRs (scTv) are used to build a pipeline of highly potent T-cell engaging bispecific TCR molecules directed against TUMAPs. In brief, we use artificial antigen-presenting cells to selectively expand TUMAP-specific T-cells from which the coding TCR sequence is retrieved by 5'RACE after highly sensitive flow cytometry-based single cell sorting. About 50-150 TCRs per TUMAP are transiently re-expressed on human T-cells and extensively characterized for their functional properties. TCRs exhibiting highly active and specific TUMAP recognition are selected for yeast surface display to generate stabilized and affinity maturated scTv. The maturated scTv are engineered into Immatics' proprietary bispecific TCR scaffold comprising a humanized T-cell recruiting antibody domain for potent redirection and activation of T-cells against TUMAPs and an effector function-silenced IgG1 Fc domain. Here we present data of our bispecific TCR program targeting the TUMAP Ag008-01 bound to HLA-A*02. We confirmed the abundant presence of Ag008-01 in several cancer indications and its absence in human normal tissues by using XPRESIDENT® technology combining quantitative mass spectrometry, transcriptomics and bioinformatics. Based on the parental TCR showing highly active and specific recognition of Ag008-01, we generated stabilized and affinity maturated scTv variants with significant improvement in binding affinity towards Ag008-01 in the range of 1000-fold and higher. The maturated scTv variants showed no or minimal binding to off-target peptides selected from the XPRESIDENT® normal tissue database based on the criteria of highest sequence similarity to Ag008-01. By incorporating the maturated scTv into our proprietary bispecific TCR format, which outperformed five alternative TCR bispecific format designs, we obtained highly potent bispecific TCR molecules with picomolar activity. We observed half-maximal lysis of Ag008-01 expressing tumor cell lines at TCR bispecific concentrations below 100 picomolar while no reactivity was observed towards a panel of cell lines lacking Ag008-01 expression. Our data support proof-of-concept for the design of our novel class of T-cell engaging bispecific TCR-antibody fusion proteins.
Citation Format: Sebastian Bunk, Martin Hofmann, Felix Unverdorben, Leonie Alten, Meike Hutt, Claudia Wagner, Oliver Schoor, Mathias Ferber, Jens Fritsche, Toni Weinschenk, Harpreet Singh-Jasuja, Dominik Maurer, Carsten Reinhardt. Development of highly potent T-cell receptor bispecifics with picomolar activity against tumor-specific HLA ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2789.