Distinguishing on-target efficacy and off-target toxicity with antibody drug conjugates (ADCs) on diseased and normal hematopoietic cells

Abstract

Abstract One of the challenges with ADC treatment is off-target toxicity to hematopoietic cells resulting in neutropenia, severe anemia and thrombocytopenia. It is caused by unintended or premature release of the payload from the construct. Therefore, many ADCs, for example Trastuzumab-vc-MMAE (Enhertu) targeting specific diseased proteins (HER-2) which are not present on hematopoietic cells, may still have hematopoietic liabilities. The additional challenges occur when the ADC is targeting a protein on both diseased and normal hematopoietic cells. Gemtuzumab ozogamicin (Mylotarg), an ADC targeting CD33 was evaluated for on-target efficacy on acute myeloid leukemia (AML) bone marrow clonal growth using a colony forming cell (CFC) assay. The off-target effects of both Mylotarg and Enhertu were assessed on normal bone marrow CFC. CD33 expression was evaluated on all bone marrow samples by flow cytometry. Both ADCs were incubated with normal marrow cells and Mylotarg was additionally incubated with AML marrow for 72 hours. The treated cells were then transferred to methylcellulose containing IL-3, GM-SCF and SCF, known to support normal myeloid progenitor (CFU-GM) and AML-blast colonies and placed in a humidified incubator for 14 days at 37oC. CFU-GM and AML-blast CFC were assessed microscopically and colony numbers enumerated. Flow cytometric analyses of CD33 expression were 16 .4 ± 7.9% of CD45+ cells on normal bone marrow cells (n=5) and 71.3 ± 20.7% of CD45+ cells on AML bone marrow samples (n=6). Mylotarg significantly inhibited AML- blast CFC with an average IC50value of 0.008 µg/mL (on-target efficacy). The IC50 values for AML-blast inhibition were not associated with CD33 expression. Mylotarg additionally significantly inhibited normal hematopoietic myeloid progenitors with an average IC50value of 0.01 µg/mL (off-target toxicity) and once again, was not associated with the CD33 expression. There was no significant difference between the IC50 values on AML versus normal marrow clonal growth. Enhertu also demonstrated toxicity to normal bone marrow progenitors with a mean IC50value of 3.3 µg/mL. Although this value was significantly different to that of Mylotarg on normal marrow progenitors, off-target toxicity was demonstrated. These data suggest that clonal assays with primary normal and diseased bone marrow cells may provide insight as to relative potency of an ADC on a disease-specific target as well as potential off-target toxicity to normal hematopoietic progenitors.