Abstract The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell–mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell–mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen–targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Supplementary Fig. S17. TAA expression analysis on AML PDX cells.

Introduction: Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse. Newer therapies, including chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) that target specific tumor associated antigens (TAAs) overexpressed on stem cells and leukemic blasts, are promising options for AML. We postulated that a TCE able to simultaneously target multiple TAAs could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while minimally affecting healthy cells. Moreover, the simultaneous targeting of different TAAs has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each TAA. Methods: Our DARPin (Designed Ankyrin Repeat Protein) 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, to generate MP0533, the first half-life extended avidity-engineered CD3 engaging DARPin capable of targeting CD33, CD123, and CD70 simultaneously. Efficacy and safety of MP0533 was extensively tested in vitro and ex vivo, with cell lines and primary cells in both allogenic and autologous settings, and in vivo in different cell line-derived xenograft mouse models. Results: We demonstrated the avidity effect of the multi-targeting MP0533 in co-culture assays with genetically engineered Molm-13 cells and allogenic T cells, where MP0533 induced up to 10-fold higher T cell mediated killing of cells expressing any combination of at least 2 of the 3 targeted TAAs vs. cells expressing a single TAA. Overall, an EC50 potency ranging from 5 to 25 pM was measured on multiple cell lines showing different levels of expression and co-expression of all TAAs (Molm-13, THP-1, RPMI8226). Efficacy was additionally assessed on primary unsorted AML bone marrow samples: MP0533 demonstrated the ability to induce autologous T cell activation and killing of AML cells in samples from newly diagnosed and previously treated patients with different TAA expression/co-expression levels and E:T ratios ranging from 10:1 to 1:28. MP0533-induced killing of LSC and selectivity over healthy hematopoietic stem cells (HSCs) was analyzed in clonogenic assays in co-cultures of sorted CD34+ cells with allogenic T cells. After 4 days initial killing assay, and 2 weeks colony culture in semi-solid media, MP0533 was able to show depletion of LSC at doses where HSC where only minimally affected. While T-cell activation, proliferation, and cytotoxicity were not affected, the multi-specific TCE format of MP0533 resulted in a significant decrease in cytokine release in both tumor cell killing assays and whole blood test systems when compared to other mono-targeting TCEs, indicating potential for improved safety. Moreover, the avidity approach and the safer profile on single-TAA expressing cells was confirmed by the absence of toxicity measured on CD123-expressing endothelial cells, indicating a low risk of capillary leak syndrome induced by MP0533. Also, potential T cell fratricide induced by CD70 expression on activated T cells was not observed. Finally, efficacy and safety profiles were also confirmed in in vivo xenograft models in human PBMC- or CD34-reconstituted mice. In such models MP0533 induced T cell infiltration in s.c. implanted tumors, leading to anti-tumor activity. Importantly, activation of T cells and release of cytokines were only localized in the tumors, indicating absence of potential systemic on-target off-tumor toxicities induced by MP0533. Conclusion: We generated a multi-specific avidity-driven CD3 engaging DARPin molecule with tailored affinities towards different TAAs showing high efficacy and with the potential for better LSC selectivity and lower cytokine release compared with mono-specific TCE approaches. Further, our approach to achieve systemic half-life extension could ensure a more convenient Q1W IV therapy. A Phase I clinical trial to evaluate safety and dose of MP0533 in humans is imminent.

Abstract 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.

Abstract 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.