MP0621 (cKit x CD16a x CD47), a Multi-Specific Switch-Darpin with Conditional Blockade of CD47 Targeting Hematopoietic Stem Cells: Preclinical Evaluation of a Next-Generation Conditioning Agent for Stem Cell Transplantation
Authors: Link Alexander , Frasconi Teresa Maria , Wullschleger Stephan , Venetz Natalia , Ribeiro Alison , Schlegel Anja , Kaufmann Yvonne , Auge Alienor , Ali Waleed , Fic Norbert , Siddiqui Zoya , Eggenschwiler Aline , Jetzer Sarah , Häberle Simon , Prekajski Dragana , De Winter Hilde , Dawson Keith M. , Croset Amélie , Goubier Anne 
Published: December 2024

Current high-intensity myeloablative conditioning for hematopoietic stem cell transplantation (HSCT) is associated with toxicity which limits eligibility for this potentially curative treatment option for patients with acute myeloid leukemia (AML) and other malignant and non-malignant diseases. There is a high unmet need for less toxic conditioning regimens to allow broader access to HSCT for these patients. cKit (CD117) is a critical receptor for stem cell maintenance, and thus an attractive target to selectively eliminate hematopoietic stem cells (HSCs) as well as leukemic stem cells. Preclinical studies in mice showed that cKit targeting with simultaneous blockade of the CD47 “do-not-eat-me” signal can selectively deplete HSCs in the bone marrow niche and thereby enable efficient syngeneic HSCT (Chhabra et al., Sci Transl Med 2016; Persaud et al., Blood Adv 2024). However, anti-CD47 antibodies are limited by toxicity linked to CD47 expression on healthy cells, making it challenging to simply use a combination of cKit-targeting and systemic CD47-blocking antibodies for conditioning in patients.

To achieve effective and more tolerable conditioning for HSCT we used our designed ankyrin repeat protein (DARPin) platform to generate MP0621, a cKit x CD16a x CD47 multi-specific Switch-DARPin candidate. MP0621 contains a masked CD47 blocker that is released only upon binding to cKit and triggers conditional immune cell-mediated killing of HSCs by engaging macrophages and natural killer (NK) cells via CD16a.

In vitro, MP0621 elicited potent macrophage-mediated phagocytosis of cKit+ AML cell lines, comparable to a combination of anti-cKit and anti-CD47 antibodies with functionally active Fc-domain, showing that the anti-CD47 DARPin moiety was engaged in the presence of cKit+ cells. In contrast, no phagocytosis was observed on cKit- CD47+ Raji cells, indicating that the anti-CD47 moiety remained effectively masked in the absence of cKit. Cytotoxicity assays using allogeneic NK cells and human ex vivo samples showed that MP0621 induced killing of CD34+ HSCs from healthy human donors as well as cKit+ tumor blasts from bone marrow samples of patients with AML. In CD34+ humanized NSG mice, MP0621 was able to target human cKit+ cells in the bone marrow and reduce HSCs without direct negative impact on overall frequency of mature human immune cells in the blood. In contrast, the combination of anti-cKit and anti-CD47 antibodies led to a strong reduction in total human blood cells in these mice due to the unconditional engagement of CD47.

In summary, our preclinical results indicate that combining cKit-targeting with conditional blockade of CD47 in a single molecule via the DARPin switch platform might render the combination treatment a viable approach for HSC depletion in patients. The blockade of CD47 exclusively on target cells allows MP0621 to enhance efficacy of cKit-targeting, while reducing off-target effects seen with systemic anti-CD47 blockade. Thus, MP0621 represents a potential novel targeted treatment approach for conditioning that could improve the benefit/risk profile of current HSCT strategies for patients.