Abstract 3753: Identification of antibodies against a novel tumor-associated MHC/peptide-target and generation of highly specific and potent HLA-A*02MMP1-003/CD3 TandAbs
Authors: Weinschenk Toni , Rajkovic Erich , Reusch Uwe , Weichel Michael , Ellwanger Kristina , Fucek Ivica , Tesar Michael , Hinz Dominik , Molkenthin Vera , Bunk Sebastian , Hilf Norbert , Schoor Oliver , Maurer Dominik , Mock Kerstin , Reinhardt Carsten , Treder Martin 
Published: July 2017

Abstract
Tumor-associated antigens for effective and safe T-cell engagement are very limited, leaving a need to open up the therapeutic target space. Targeting disease-specific MHC/peptide complexes with bispecific T-cell-recruiting antibodies is a highly attractive strategy to address this need, but so far, generation of antibodies against these peptides has been reported to be challenging. Immatics’ unique target discovery engine XPRESIDENT® holds the promise of identifying novel tumor-associated MHC/peptide complexes by providing direct and quantitative evidence for their presence on a large collection of primary human tumor and normal tissue specimens. By this approach, MMP1-003, an HLA-A*02-binding peptide originating from matrix metallopeptidase 1 (MMP1), was identified as a promising therapeutic target presented by several tumor types, including colorectal and lung cancer, but absent on normal tissues. These findings are underlined by RNAseq analysis of the source antigen which also points to MMP1 being a highly attractive tumor-associated target. Consequently, a fully human antibody phage display library was screened to identify highly specific single chain antibodies, which were shown to recognize the purified HLA-A*02/MMP1-003-complex in ELISA assays as well as on peptide-pulsed HLA-A*02+ T2 cells. The best candidates were reformatted into bispecific tetravalent TandAbs® through Affimed´s proprietary platform using a human/cyno-cross-reactive CD3-binding domain for T-cell engagement. Specific target recognition was confirmed for the TandAbs in binding and cytotoxicity assays on peptide-pulsed T2 cells. HLA-A*02/peptide-complexes selected from the broad normal tissue immunopeptidome with a high degree of sequence similarity to the HLA-A*02/MMP1-003-complex served as controls to confirm the specificity and hence the low risk of off-target binding. The most promising candidates were tested on a panel of endogenously target-expressing cancer cell lines covering MMP1 +/- and HLA-A*02 +/- expression profiles, as well as the source proteins for the most closely related control peptides. The lead TandAb showed excellent target specificity across a wide range of peptide-pulsed and endogenously expressing cell lines as well as potent cytotoxicity with picomolar EC50. In summary, we have identified a tumor-associated MMP1-derived peptide in an HLA-A*02 context by exploiting the knowledge of tumor and healthy tissue immunopeptidomes using XPRESIDENT®. Overcoming the existing barrier of developing antibodies targeting specific MHC/peptide complexes, we generated and characterized highly specific and potent T-cell-recruiting TandAbs. These hold the potential to open up the therapeutic target space for T-cell engagement by providing access to intracellular proteins that are presented in a disease-specific manner as MHC/peptide complexes.
Citation Format: Toni Weinschenk, Erich Rajkovic, Uwe Reusch, Michael Weichel, Kristina Ellwanger, Ivica Fucek, Michael Tesar, Dominik Hinz, Vera Molkenthin, Sebastian Bunk, Norbert Hilf, Oliver Schoor, Dominik Maurer, Kerstin Mock, Carsten Reinhardt, Martin Treder. Identification of antibodies against a novel tumor-associated MHC/peptide-target and generation of highly specific and potent HLA-A*02MMP1-003/CD3 TandAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3753. doi:10.1158/1538-7445.AM2017-3753