Avidity-Engineered CD3 Engaging DARPin ® Targeting Three Tumor Associated Antigens Induce Strong and Specific T Cell Dependent Killing of AML Cells with Potential for Improved Safety
Authors: Bianchi Matteo , Reschke Nina , Reichen Christian , Fischer Stefanie , Grübler Yvonne , Eggenschwiler Aline , Krieg Jennifer , Ioannou Kyriaki , Ragusa Simone , Looser Thamar , Spitzli Patricia , Herzog Christel , Villemagne Denis , Kaufmann Yvonne , Matzner Mirela , Auge Alienor , Hänggi Martin , Ali Waleed , Franchini Marco , Kirkin Vladimir , Schlereth Bernd , Luethi Ursina , Ochsenbein Adrian , Riether Carsten , Steiner Daniel , Goubier Anne 
Published: November 2021

Abstract
AML is driven by leukemic stem cells (LSC) that resist conventional chemotherapies and remain unaffected in their niche, continually replenishing circulating blast cells. We postulated that an avidity-engineered CD3 engaging DARPin ® (Designed Ankyrin Repeat Protein) able to simultaneously target LSC-specific CD70 as well as CD123 and CD33 could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while preserving a therapeutic window towards healthy cells. Moreover, this simultaneous targeting of three different tumor associated antigens (TAAs) has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each single TAA. To achieve this ambitious goal we used our DARPin ® platform to build a novel class of triple targeting CD3 engaging molecules.
Our DARPin ® libraries contain trillions of molecules allowing the generation of highly diverse binders against target proteins that can be easily combined into multi-specific DARPins ® to elicit desired biological effects. We leveraged this proprietary platform to screen multi-specific CD3 engaging DARPin ® molecules, including serum albumin binding DARPins ® for systemic half-life extension, and identify the optimal target affinity and molecular architecture to ensure potent avidity-driven T cell-mediated killing of AML cells while sparing healthy cells.
This approach allowed the generation of CD3 engaging DARPins ® able to target simultaneously CD33, CD123, and CD70. Such DARPins ® demonstrated, in both allogenic and autologous setting, single digit pM potency against AML cell lines and primary cells expressing any combination of at least 2 of the 3 targeted TAAs, while showing low activity against single TAA-expressing cells, the latter representing cells of the healthy compartment. Higher expression of the selected TAAs on LSCs vs normal hematopoietic stem cells (HSC) can further enhance the selectivity of such an avidity driven molecule, leading to the preferential killing of LSC over HSC.
Moreover, our multi-specific T cell engager (TCE) format resulted in a significant decrease in cytokine release in a whole blood test system for cytokine release syndrome (CRS) when compared to other mono-targeting TCE therapies, confirming its specificity and the potential for an improved safety profile within the normal hematopoietic system. Additionally, while showing similar anti-tumor efficacy in a mouse xenograft model using Molm-13 cell line and human PBMCs, CRS measured in serum 4 h after the initial injection of our multi-specific DARPin ® molecule was drastically reduced compared to a reference CD33 TCE, further strengthening the evidence that our multi targeting DARPins might also exhibit a good safety profile in humans.
In conclusion, we were able to generate multi-specific CD3 engaging DARPin ® molecules with tailored affinities towards different TAAs showing exceptional efficacy and with the potential for superior safety over mono-specific TCE approaches, including systemic half-life extension to avoid a continuous intravenous infusion-based therapy.

Disclosures
Bianchi: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Reschke: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Reichen: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Fischer: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Grübler: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Eggenschwiler: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Krieg: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ioannou: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ragusa: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Looser: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Spitzli: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Herzog: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Villemagne: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Kaufmann: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Matzner: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Auge: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Hänggi: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ali: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Franchini: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Kirkin: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Schlereth: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Luethi: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ochsenbein: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Riether: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Steiner: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Goubier: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company.