Novel CD117 T-cell engagers for targeted bone marrow HSPC depletion

Abstract

Abstract Bone marrow transplantation (BMT), aimed at replacing a defective hematopoietic system with a healthy one, offers curative potential for hematological disorders, such as bone marrow malignancies (e.g., leukemias and lymphomas) and several genetic hematological disorders (e.g., sickle cell disease, b-thalassemia, aplastic anemia etc.). A prerequisite for a successful BMT is effective bone marrow (BM) conditioning. Unfortunately, current BM conditioning regimen rely on genotoxic chemotherapy agents like Busulfan and Cyclophosphamide, alone or in combination with irradiation. Although in use for several decades, these agents are non-specific and lead to numerous side effects, such as prolonged cytopenias, systemic inflammation, fatigue, elevated risk of fatal infections, organ damage, risk of infertility, and risk of developing secondary malignancies, rendering several patients ineligible for BMT (Deeg et al, 1983, Socie et al., 2003, Gyurkocza et al., 2014). Even today over 50% of patient mortality within 100 days post BMT are associated with BM conditioning related complications like infection and organ failure (Spellmann et al., 2025). Antibody (Ab)-based BM conditioning approaches have emerged as a promising alternative (Palchaudhari et al., 2016, Czechowicz et al., 2019, Pang et al., 2019), offering the potential for safer, non-genotoxic and targeted depletion of BM hematopoietic stem progenitor cell (HSPCs) populations. Driven by its expression on BM HSPCs, CD117, a receptor tyrosine kinase, has been the target of choice for many Ab-based BM conditioning programs (Czechowicz et al., 2019, Pang et al., 2019). However, most of these strategies encountered limitations, either lacking sufficient potency as a standalone agent (Andriyana et al., 2021) or exhibiting unacceptable safety profiles most likely due to the selected antibody formats in these programs. Here, we present a CD117 T-cell engager based BM conditioning strategy with a vision to develop a highly potent, safe, and rapidly cleared antibody for BM conditioning via HSPC depletion. We first generated a novel series of CD117 binders by immunizing transgenic rabbits with human CD117 extracellular domain. Extensive biophysical characterization allowed identification of a binder subset with strong binding affinity to both human (hu) and cyno (cy)-CD117 membrane proximal domains. We combined these selected CD117 binders with CD3 engagers of varying affinity in a T-cell bispecific (TCB) format to identify the most efficacious and potent CD117xCD3 combination in functional in vitro killing assays. We performed additional in vitro safety assays, such as inflammatory cytokine response and mast cell degranulation, allowing selection of binder candidates with the most desirable safety profile. Furthermore, in vivo validation studies, in CD34 humanized NSG mouse model, demonstrated that a single administration of the selected CD117-TCBs lead to potent huHSPCs depletion for up to 2 weeks. Additionally, our in vivo analysis revealed that the TCB induced acute inflammatory cytokine response could be significantly attenuated by selecting a favorable CD3 engager in the molecule design without compromising the HSPC depletion potency. Moreover, rapid clearance of the molecule from circulation was achieved through relevant Fc domain engineering, making these molecules highly suitable for bone marrow conditioning approaches in BMT. In summary this innovative strategy aims to overcome the limitations of current and prior antibody-based methods, paving the way for a targeted and less toxic standalone conditioning regimen that could significantly improve patient outcomes in BMT.