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.
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Open Access
May 8, 2024
Maria Grammoustianou + 2
