Abstract 1349: Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes
Authors: Venetz Natalia , Müller Sandra , Schulte Tim , Fischer Stefanie , Paladino Maria , Pina Nicole , Kadri Nadir , Bruckmaier Sandra , Cornelius Andreas , Hospodarsch Tanja , Sandalova Tatyana , Levitsky Victor , Achour Adnane , Walser Marcel 
Published: July 2021

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. Architectural fine tuning and sequence engineering allowed us to further increase the potency of the selected candidates without compromising the specificity. This was manifested as effective T cell mediated activation by relevant DARPin® constructs in the presence of HLA-A2+/NY-ESO-1+ tumor cells. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of engineered HLA-A2:SLL-specific DARPin® T-cell engagers. The versatility of the DARPin® platform and the engineering experience gained with NY-ESO-1 did allow us to identify highly potent and specific DARPins® proteins for additional clinically attractive pMHC targets.Alanine and X-scan 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® proteins interactions. Further molecular and cellular analysis of DARPin® protein specificity will be evaluated in order to de-risk for potential clinically relevant toxicity.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: Natalia Venetz, Sandra Müller, Tim Schulte, Stefanie Fischer, Maria Paladino, Nicole Pina, Nadir Kadri, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Tatyana Sandalova, Victor Levitsky, Adnane Achour, Marcel Walser. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1349.