Background Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the treatment of AML, especially monocytic AML subtypes still has a poor outcome. Therapies targeting CD33 and CD123 for the treatment of AML demonstrated hematological toxicity due to the distribution of the targets on hematopoietic stem cells (HSCs). Leukocyte immunoglobulin-like receptor B4 (LILRB4), an inhibitory receptor belonging to the LILR family, is highly expressed on monocytic M4/M5 AML and leukemic stem cells (LSCs), but not on normal HSCs.1 Fc-mediated ADCC/ADCP activity is one of the driving mechanisms of action for therapeutic monoclonal antibodies (mAbs) targeting tumor-associated or specific antigen against malignancies. However, owing to the epitope and/or IgG4 Fc subtype, the ADCC/ADCP effect of current therapeutic mAbs targeting LILRB4 antigen on AML in the clinic is relatively weak.2 Here we report a novel humanized LILRB4 antagonist antibody, ATG-034-S3, binding to a unique epitope that is distinct from the epitopes of other clinical benchmark LILRB4 antibodies, induces strong ADCC/ADCP effects, resulting in a potent anti-tumor efficacy in vitro and in vivo.
Methods The protein-based and cell-based binding affinity of ATG-034-S3 was measured using SPR, ELISA and FACS analysis. Competitive ELISA assay was used to evaluate the ability of ATG-034-S3 to block the interaction of LILRB4 with its ligand ApoE. ADCC assay was PBMC-mediated cytotoxicity and measured by FACS analysis. ADCP assay was M2 macrophage-mediated phagocytosis and measured using FACS analysis. T cell killing assay was CD8+ T cell-mediated cytotoxicity measured by FACS analysis. The in vivo anti-tumor efficacy of ATG-034-S3 was evaluated in the EL4-LILRB4 syngeneic model.
Results Compared with clinical benchmark antibodies, ATG-034-S3 bound to a unique epitope on the human LILRB4 protein (figure 1A). It blocked the interaction between LILRB4 and ApoE and reversed THP-1 (AML)-mediated T cell suppression. ATG-034-S3 triggered strong ADCC and ADCP activity with EC50s of 1.13 nM and 0.22 nM, respectively (figure 1B,C,G), while clinical benchmark antibody demonstrated limited ADCC/ADCP effect. ATG-034-S3 potentiated CD8+ T cell-mediated cytotoxicity (figure 1D,G), and significantly inhibited tumor growth in the EL4-LILRB4 syngeneic model in vivo. 3 mg/kg ATG-034 showed a TGI of 77%.
Conclusions Our data showed that ATG-034-S3 potentiates NK-mediated ADCC, macrophage-mediated ADCP, CD8+ T cell-cytotoxicity and reverses AML-mediated T cell suppress, enhancing anti-tumor immunity (figure 1G), and demonstrates potent in vivo anti-tumor efficacy. ATG-034-S3 may be a promising strategy for the treatment of monocytic M4/M5 AML and other LILRB4 positive hematologic malignancies.
Deng M, Gui X, Kim J, Xie L, Chen W, Li Z, He L, Chen Y, Chen H, Luo W, et al. LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration. Nature. 2018;562:605–09.
Gui X, Deng M, Song H, Chen Y, Xie J, Li Z, He L, Huang F, Xu Y, Anami Y, et al. Disrupting LILRB4/APOE interaction by an efficacious humanized antibody reverses T-cell suppression and blocks AML development. Cancer Immunol Res. 2019;7(8):1244–1257.
Ethics Approval The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the IACUC of CrownBio. All studies were conducted following an approved IACUC protocol. AUP NO.:2004–12-1465, 2004–12-1000; IACUC approval number: IACUC-2021-M-003
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