Targeting AML Using an Fc-Engineered BST1/CD157 Monoclonal Antibody
Authors: Krupka Christina , Jansen Anna , Lassmann Irène , Terrett Jon , Aud Dee , Dusek Rachel , Bisht Arnima , Pombo-Villar Esteban , Rohlff Christian , Köhnke Thomas , Lichtenegger Felix S , Altmann Torben , Metzeler Klaus H , Fiegl Michael , Spiekermann Karsten , Hiddemann Wolfgang , Subklewe Marion 
Published: October 2019

Abstract
Antibody-based immunotherapy represents a promising strategy to target and eliminate acute myeloid leukemia (AML) tumor cells. We evaluated BST1/CD157, a novel, slow internalizing surface antigen, for its suitability as immunotherapeutic target in AML. An Fc-optimized antibody against BST1/CD157 was developed (MEN1112/OBT357NF), which binds to the Fc-receptor on natural killer (NK) cells with enhanced affinity. The target antigen expression profile of BST1/CD157 on AML bulk cells as well as on leukemic stem cells (LSC) was analyzed with flow cytometry using a commercially available antibody. BST1/CD157 expression was detected in > 97% of AML patients at primary diagnosis (n=81) and at time of relapse (n=20) using specific fluorescence intensity (SFI, positivity: SFI > 1. 5). Importantly, BST1/CD157 was also expressed on CD34+/CD38-cells (n=20), the compartment with a high percentage of LSCs.
Functional titration experiments of MEN1112/OBT357NF showed antibody-mediated lysis of U937 cells in the low picomolar range (EC50 = 30–140 pM) and demonstrated superior ADCC activity compared to its non Fc-engineered parental analogue. To evaluate the additive effect of the complement cascade, healthy donor (HD) monocytes were co-incubated with autologous NK cells and MEN1112/OBT357NF in the presence or absence of 5% donor serum. After 20 hours of incubation, a significantly enhanced lysis efficacy by MEN1112/OBT357NF through the addition of serum compared to control cultures without serum (p=0.02) was demonstrated.
For further functional characterization of MEN1112/OBT357NF standard chromium release assays using AML cell lines and NK cells from HDs were performed. In this setting, efficient target cell lysis (30–50%) at various effector cell:target (E:T) ratios (50:1–6:1) was observed. Compared to Rituximab-mediated lysis of RAJI cells, MEN1112/OBT357NF triggered more efficient target cell lysis at much lower effector cell concentrations (33% lysis of OCI-AML3 cells vs. 11% lysis of RAJI cells at E:T 6:1).
Furthermore, the cytotoxic potential of NK cells from AML patients after intense double induction chemotherapy on AML cell lines was tested. The results were highly variable with efficient lysis in about one third of samples tested (n=10). As a positive control NK cells from HDs were used which showed superior lysis as compared to patient-derived NK cells. In an autologous set-up using NK cells from either primary diagnosis or at time of remission, we could show efficient lysis of primary AML cells in one of two patient samples (patient in complete remission) in the presence of MEN1112/OBT357NF.
In summary, our analysis demonstrates that BST1/CD157 is a promising target antigen for antibody-based therapy in AML. The effective in-vitro ADCC activity supports further development of MEN1112/OBT35NF as an immunotherapy for patients with AML.

Disclosures
Krupka: Oxford BioTherapeutics Ltd: Research Funding. Jansen:Oxford BioTherapeutics Ltd: Research Funding. Aud:Oxford BioTherapeutics Ltd: Employment. Dusek:Oxford BioTherapeutics Ltd: Employment. Bisht:Oxford BioTherapeutics Ltd: Employment. Pombo-Villar:Oxford BioTherapeutics Ltd: Employment, Equity Ownership. Rohlff:Oxford BioTherapeutics Ltd: Employment. Subklewe:Oxford BioTherapeutics Ltd: Research Funding.