CSF1R Targeting T Cell Engaging Bispecific Antibodies Enable Safe and Efficient Immunotherapies in Acute Myeloid Leukemia

Despite recent advancements in the treatment of acute myeloid leukemia (AML) the outcome in the relapsed and refractory setting remains poor (Thol, Dohner et al. 2024). While the use of T cell engaging bispecific antibodies (TCE) targeting B cell lineage antigens such as CD19 (Blinatumomab) or CD20 (Epcoritamab, Glofitamab, Mosuenetuzumab) have induced strong and long-lasting response rates in B cell malignancies (Falchi, Vardhana et al. 2023, Liu, Xi et al. 2023), in AML similar progress is yet to be achieved. Early clinical trials of CD33-TCE (JNJ-67571244, AMG330) or CD123-TCE (Vibecotamab) have shown modest clinical activity (response rates ranging between 0 to 16,6%) and a high degree of treatment-emergent adverse events (TEAE) (Ravandi, Bashey et al. 2023, Short, Bachireddy et al. 2023, Narayan, Pierola et al. 2024). In addition, preclinical work suggests possible on-target-off-tumor toxicity of these antibodies towards hematopoietic stem and progenitor cells (HSPC) (Gill, Tasian et al. 2014), overall questioning the ability of complete hematological recovery after TCE treatment targeting CD33 and CD123.

In previous work, utilizing high-dimensional single-cell RNA sequencing (scRNA-Seq), we identified novel AML-associated antigens with lower off-tumor expression and no apparent toxicities towards HSPC (Gottschlich, Thomas et al. 2023). Chimeric antigen receptor (CAR) T cells targeting one of our lead candidates - the colony-stimulating factor 1 receptor (CSF1R) - demonstrated strong activity in preclinical models including several genetically distinct patient-derived xenograft (PDX) models, despite an overall lower antigen density on AML blasts compared to hallmark AML-associated target antigens CD33 or CD123. Given the lower antigen density on AML blasts, it remains elusive whether CSF1R can be readily targeted through bispecific T cell engagers.

We developed CSF1R targeted TCE (CSF1R-TCB) based on the well-known CrossMAb^® Technology in the 2+1 TCB format, used for the FDA-approved CD20-TCB Glofitamab, and demonstrated its efficacy in preclinical in vitro and in vivomodels. Binding of CSF1R-TCB and its anti-tumor activity was tested in co-cultures with T cells or Peripheral Blood Mononuclear Cells (PBMC) and human AML cell lines (Mv4-11, THP-1, OCI-AML3, PL-21) and primary AML blasts, using fluorescence-associated cell sorting (FACS), luciferase bioluminescence readouts or live cell imaging, respectively. In these assays, CSF1R-TCB demonstrated dose-dependent binding to AML cell lines as well as strong in vitro anti-leukemia activity towards AML cell-lines (Effector to Target (E:T) ratio 1:2 p < 0.0001 for both Mv4-11 and THP-1) and primary blasts (E:T 1:1 p < 0.0001).

In flow cytometry-based co-culture assay or colony-forming unit (CFU) assays of HSPC and T cells, CSF1R-TCB did not induce relevant lysis of HSPC, while CD33-TCB lead to near complete depletion of HSPC (E:T 2:1 p < 0.0001). Importantly, utilizing a CD34⁺ cord blood (CB) stem cell-humanized mouse model, CSF1R-TCB showed significantly lower signs of cytokine release and minimal reduction in HSPC counts compared to CD33-TCB, highlighting the favorable expression profile of CSF1R (p < 0.01 for GMCSF, MIP1a; p < 0.05 for IL2, IL6, MCP1 and MIP1b detected in serum 24 hours after therapy).

In a xenograft-derived cell line model using luciferase-positive Mv4-11 AML cells (Mv4-11-luc⁺), treatment with effector T cells and CSF1R-TCB reduced tumor outgrowth and overall tumor progression compared to control-TCB a germline antigen and CD3-binding TCB (p < 0.0001 at day 38 post tumor inoculation).

In summary, we could show the safety and efficacy of CSF1R-TCB in preclinical in vitro and in vivo models and demonstrate the superior safety profile of CSF1R-TCB compared to CD33-TCB in CB-humanized mouse models. In cell line-derived xenograft models of AML, CSF1R-TCB induced anti-leukemia activity, warranting further preclinical and clinical investigations.