Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein–protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

T-cell engagers (TCEs) direct cytotoxic T-cell response towards tumor cells by binding simultaneously to a tumor-associated antigen (TAA) on target cells and to CD3 on T-cells, thereby forming an artificial immune synapse. They have been shown to be very potent anti-tumor drugs, as exemplified by blinatumomab, an α-CD19 x α-CD3 bispecific. However, the development of TCEs for hematological and solid tumors has been hampered by several factors, amongst them severe toxicity, elicited by on-target/off-tumor recruitment of T-cells and cytokine release syndrome (CRS). In order to overcome this challenge, an anti-CD3 Prodrug DARPin® (CD3-PDD) has been developed, consisting of a mouse cross-reactive EGFR-binder and a CD3-binder, linked via a protease-cleavable linker to an anti-idiotypic anti-CD3 binder (termed blocker hereafter). This α-EGFR x α-CD3 x blocker Prodrug is unable to bind and recruit T-cells in its non-cleaved state, but is designed to become activated in the tumor microenvironment upon cleavage of the linker by tumor-associated proteases. A control Prodrug DARPin® with a non-cleavable linker showed neither tumor cell killing, nor T-cell activation at concentrations >1'000-fold over the EC50 of the active, non-blocked TCE DARPin® in in vitro tumor cell killing and T-cell activation assays. In contrast, a CD3-PDD containing a cleavable linker was partially activated by proteases secreted from the tumor cells (HCT 116). Pre-treatment of the CD3-PDD with recombinant protease prior to the in vitro assay fully activated the molecule, with EC50 values comparable to the active, non-blocked TCE. Next, an in vivo proof-of-principle study was performed in a human colon carcinoma xenograft model (HCT 116) using immunodeficient mice humanized with hematopoietic stem cells (CD34+) and optimized for the presence of human myeloid cells. Due to the mouse cross-reactivity of the EGFR-binder, this animal model allowed to assess both anti-tumor efficacy and safety (therapeutic window). The cleavable CD3-PDD demonstrated a robust anti-tumor activity, similar to the one observed with active, non-blocked TCE. Most importantly, while the active, non-blocked TCE elicited strong toxicity, leading to loss of animals and requiring treatment stop, the cleavable CD3-PDD could be dosed without significant safety findings. In summary, a conditionally activated CD3-PDD shows similar efficacy but none of the toxicity of the active, non-blocked TCE. Our approach therefore holds great promise for the development of future CD3-PDD as therapeutics, enabling the utilization of less tumor-specific targets for highly potent TCEs. Ultimately, the ability of the versatile DARPin® technology to generate tailor-made anti-idiotypic DARPin® molecules can unlock novel therapeutic design spaces, which we are exploring beyond the conditionally activated CD3-PDD format. Citation Format: Andreas Bosshart, Julia Katharina Ahlskog, Aline Eggenschwiler, Dieter Schiegg, Yvonne Grübler, Sandra Wandel, Simon Fontaine, Maria Paladino, Susanne Mangold, Tanja Hospodarsch, Alexandra Neculcea, Chloé Iss, Christel Herzog, Bernd Schlereth. A solution to T-cell engager toxicity: An anti-CD3 Prodrug DARPin (CD3-PDD) shows no toxicity, but potent anti-tumor activity in a humanized mouse model [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 1890.

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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.

Background: Background:AML treatment options are generally focused on chemotherapy followed by allogeneic hematopoietic stem cell transplantation. However, many patients are ineligible for these options and often receive palliative treatments with poor long-term survival and there is an urgent need for improved therapeutic solutions. Newer therapies, including T cell engagers (TCE) and chimeric antigen receptor (CAR) T cells that target specific molecules overexpressed on leukemic blasts and stem cells are promising alternative treatment options for AML. However, achieving efficacy and low toxicity remains challenging.MP0533 is a multi-specific, half-life extended, T cell engaging DARPin in development for treatment of patients with AML and higher risk myelodysplastic syndrome (HR-MDS). MP0533 consists of a chain of 6 covalently linked DARPin domains. Three of these domains engage CD33, CD123 and CD70 on the target cells, utilizing an avidity driven approach to ensure that binding occurs preferentially when 2 or more TAAs are present. A fourth DARPin domain targets CD3 for T-cell engagement. Lastly 2 additional HSA binding DARPin domains prolong half-life in circulation. In vitro, MP0533 has demonstrated, in both allogeneic and autologous settings, single to double digit pM potency against AML cell lines and AML primary cells expressing any combination of at least 2 of the 3 targeted TAAs and low cytokine release. Aims:Aims: To evaluate and confirm that the previously established in vitro and ex vivo efficacy and safety profiles of MP0533 are also observed in an in vivo human PBMC xenograft mouse model. Methods:Methods: NXG mice were injected intraperitoneally with hPBMC from healthy donors and xenografted with MOLM-13 cells subcutaneously two days after hPBMC injection. Therapeutic treatment was initiated eight days after tumor implantation once tumors were established. DARPins were injected intravenously three times per week for two weeks. Several readouts such as T cell activation and human immune cell infiltration in tumors were performed by flow cytometry three days after first treatment. Additionally, cytokine and chemokine release were analyzed in serum four hours after the first treatment injection and in tumor supernatant 3 days after the first treatment injection. Results:Results: MP0533 induced AML tumor killing in vivo (P value <0.0001 at day 18 compared to PBS). Three days after the first injection, MP0533 recruited human immune cells in the tumor (around 10% of hCD45+ cells detected by FACS, P value = 0.0044 compared to PBS) and induced T cell activation (around 46% of CD4+/CD25+/CD69+ cells and 38% of CD8+/CD25+/CD69+ cells detected by flow cytometry, P value <0.0001 compared to PBS) which triggered cytokines and chemokines release in the tumor such as IL-6, INFγ and TNFα . However, MP0533 didn't induce significant cytokine release, including at the highest tested dose, in mice serum four hours after the first injection.Summary/Conclusion: Summary/Conclusion: We were able to generate a multi-specif...