IMGN779, a CD33-Targeted Antibody-Drug Conjugate (ADC) with a Novel DNA-Alkylating Effector Molecule, Induces DNA Damage, Cell Cycle Arrest, and Apoptosis in AML Cells

IMGN779 is a CD33-targeting ADC consisting of a humanized anti-CD33 antibody, Z4681A, conjugated to DGN462, a novel DNA-alkylating agent, through a cleavable disulfide linker, sulfo-SPDB. CD33 is broadly expressed on leukemic blasts of patients with AML, making it a promising target for AML therapy. DGN462 is a member of the novel IGN class of DNA-acting cytotoxic agents, that consists of an indolino-benzodiazepine dimer containing a mono-imine moiety.

Potent killing of AML tumor cells by DGN462 and IMGN779 has previously been demonstrated in vitro (K. Whiteman et. al, ASH 2014 #2321). Here we describe studies elucidating the mechanism of action of this novel payload in the AML cell lines: MV4-11, HL60, and EOL-1. The ability of DGN462 to alkylate DNA was demonstrated in an AML cell line by isolation and analysis of the genomic DNA from cells. DGN462 was found to co-purify with DNA, demonstrating that it covalently reacts with cellular DNA. A comet assay was performed confirming that DGN462 does not cross-link DNA.

H2AX is known to be phosphorylated on serine 139 in response to DNA damage. Using flow cytometry, we assessed the phosphorylation of H2AX in AML cell lines following exposure to DGN462 and IMGN779. Increases in phosphorylated H2AX levels were detected as early as 4 hours following exposure to DGN462-SMe and 12 hours following exposure to IMGN779. Additional flow cytometry analysis showed that exposure to DGN462-SMe and IMGN779 leads to S-phase accumulation, G2/M arrest, followed by induction of apoptotic markers (cleaved PARP and cleaved Caspase-3) at later time points. Ex vivo studies using AML patient samples also showed elevation of phosphorylated H2AX and an increase in apoptosis in myeloid blasts following exposure to DGN462-SMe and IMGN779.

As a demonstration of proof-of-mechanism in AML disease models, our results suggest that cell killing by IMGN779 is mediated by DNA damage, as a consequence of DNA alkylation. The DNA damage response is accompanied by cell cycle arrest, which leads to apoptosis. This relationship will be further explored in additional AML preclinical models to support the use of phosphorylated H2AX as a pharmacodynamic biomarker for IMGN779 activity in future clinical studies.