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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.

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.

DARPin® molecules are small engineered proteins, derived from natural ankyrin repeat proteins that are selected to bind to specific targets with high affinity. Individual DARPin® molecules can be linked together genetically in order to create multi-specific drug molecules. This versatility of DARPin® molecules makes them an attractive alternative to antibodies for the development of drug conjugates. We have developed DARPin® drug conjugates (DDCs) using a model EGFR multi-specific DARPin® molecule, consisting of four DARPin® domains linked together. DARPin® constructs were conjugated to the indolinobenzodiazepine mono-imine DGN549, a potent DNA alkylating payload. Biophysical characterization showed the DDCs to be well behaved in stability and solubility assays. The in vitro binding and cytotoxicity of these DDCs were evaluated. The DARPin® conjugates displayed in vitro potency in direct cytotoxicity assays across a panel of cell lines expressing EGFR. The impact of conjugation on PK parameters and in vivo efficacy is currently under evaluation. DARPin® drug conjugates combine the potency observed with antibody drug conjugates and the modular design of DARPin® molecules to create designer therapeutics. Citation Format: Laura A. Laviolette, Cynthia J. Guidi, Christian Reichen, Qifeng Qiu, Luke Harris, Patricia Schildknecht, Stefanie Fischer, Zita Arany, Tanja Hospodarsch, Anna Skaletskaya, Megan Fuller, Stephen Abbott, Rebecca McCarthy, Jenny Lee, Katherine Francisco, Kerstin Sinkevicius, Sharlene Adams, Christopher Espelin, Emily Reid, Wei Li, Carla Marashio, Kerry Donahue, Stuart Hicks, Dan Snell. Generation of site-specific DARPin® drug conjugates using EGFR as a model system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 215.