Purpose: The medical need due to high mortality in acute myeloid leukemia (AML) remains high, and the treatment of relapsed or refractory AML continues to be therapeutically challenging. MYLOTARG, the only approved anti-CD33 antibody drug conjugate (ADC), has provided proof-of-concept for targeted immunotherapies in AML. Currently, a plethora of ADCs and T-cell engager (TCE) therapies have entered clinical development in AML, but those therapies are often accompanied by dose limiting toxicities, preventing dose escalation to desired anti-tumor efficacy. The biggest challenges seem to be limited target specificity and hyperstimulation of the immune system leading to e.g. myelotoxicities and cytokine release syndrome, respectively. Therefore, more selective therapies are needed to allow for robust anti-tumor activity with a more acceptable safety profile. Experimental design: To address the selectivity challenge, we have generated multi-specific T-cell engaging DARPin® molecules, targeting two different tumor associated antigens (TAAs) with optimized affinity for their targets. In order to find the right target combination, the optimal affinity to increase tumor specificity via avidity, as well as the best molecular architecture, we took advantage of our unique modular DARPin® platform and screened 1000s of combinations of multi-specific DARPin® molecules, binding simultaneously to multiple TAAs in conjunction with our CD3-binding DARPin® molecule. Results: We constructed multi-specific TCEs targeting two different AML antigens with optimized affinity leading to a substantial avidity gain when both targets are co-expressed on tumor cells. The avidity gain resulted in strongly enhanced in vitro potency as shown by activation of both CD8+ and CD4+ T cells and subsequent killing of AML tumor cells, with bioactivities in the range of established TCE benchmark formats (e.g. BiTE® and DART®). In contrast, in an ex vivo whole blood assay the multi-specific DARPin® constructs induced profoundly less cytokine release as compared to benchmark molecules indicating an improved therapeutic window. Finally, we also demonstrated tumor regression in PMBC humanized mouse models bearing MOLM-13 tumors, using both half-life extended (HLE) and non-HLE lead constructs. In conclusion, we have generated TCEs based on multi-specific DARPin® constructs with high potency, selectivity and ultimately with the potential for an improved therapeutic window for the treatment of AML. Citation Format: Nina Reschke, Thamar Looser, Jennifer Krieg, Matteo Bianchi, Patricia Schildknecht, Nicole Bassler, Yvonne Gruebler, Sebastian Grimm, Laura Jeanbart, Tanja Hospodarsch, Alexandra Neculcea, Daniel Steiner, Bernd Schlereth, Christian Reichen. Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies [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 525.
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.
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