Abstract 690: Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes
Authors: Walcer Marcel , Venetz Natalia , Schulte Tim , Fischer Stefanie , Bassler Nicole , Paladino Maria , Pina Nicole , Villemagne Denis , Bruckmaier Sandra , Cornelius Andreas , Hospodarsch Tanja , Duffet Loic , Sandalova Tatjana , Achour Adnane , Levitsky Victor 
Published: September 2020

Abstract
Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. This was manifested as effective activation of T-cells in the presence of relevant DARPin® constructs tested at a range of concentrations and HLA A2 positive cells pulsed with the SLL or irrelevant HLA A2 binding peptides. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of HLA-A2:SLL-specific T-cell engagers. Alanine scanning mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin interactions. Further molecular and cellular analysis of DARPin® protein specificity using additional molecular and cellular approaches will be presented in order to de-risk for potential clinically relevant toxicity which may result from off-target interactions with other pMHC complexes structurally resembling the tumor-associated pMHC complex. In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells.
Citation Format: Marcel Walcer, Natalia Venetz, Tim Schulte, Stefanie Fischer, Nicole Bassler, Maria Paladino, Nicole Pina, Denis Villemagne, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Loic Duffet, Tatjana Sandalova, Adnane Achour, Victor Levitsky. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 690.