Preclinical Characterization of the Anti-Leukemia Activity of the CD123 Antibody-Drug Conjugate, Pivekimab Sunirine (IMGN632)

Introduction: Pivekimab sunirine (PVEK; IMGN632), a CD123 antibody-drug conjugate carrying an alkylating monoamine indolinobenzodiazepine pseudodimer (IGN), is currently under clinical investigation for acute myeloid leukemia (AML) and other CD123-expressing neoplasms. Here, we studied multiple potential modulators of PVEK's anti-leukemia activity.

Methods: We tested potential variables that might modulate the in vitro cytotoxic effects of PVEK against human acute myeloid and lymphoblastic cells, using genetically and functionally well-defined human acute leukemia cell line models and primary leukemia cells from adults with AML.

Results: We first sought to determine the quantitative relationship between CD123 expression and PVEK-induced cytotoxicity using 2 human AML cell lines (ML-1, MOLM-13) endogenously expressing CD123 and generated sublines in which CD123 was deleted via CRISPR/Cas9. We then used these CD123^KO sublines to lentivirally express human CD123 at different level. Despite very low levels of CD123 expression on parental cells, PVEK induced cell death in a dose dependent manner in both cell lines, as determined flow cytometrically after 3 days of drug exposure. This effect was entirely dependent on the presence of CD123 as indicated by the resistance of the CD123^KO subline to PVEK. However, in both engineered ML-1 and MOLM-13 cells, there was a quantitative dependence of PVEK-induced cytotoxicity on the amount of CD123 displayed on both cell lines, demonstrating the importance not only of CD123 expression per se (with minimal amount of CD123 being required and sufficient for the drug's anti-AML activity), but also of the level of CD123 expression for PVEK-induced cytotoxicity. Across a panel of 13 human acute myeloid and lymphoblastic leukemia cell lines expressing varying levels of CD123, sFGN849 and PVEK were broadly active against TP53 wild-type cell lines and exerted dose-dependent cytotoxicity. sFGN849 and PVEK also dose-dependently induced cell death in cell lines with TP53 alteration. However, they were significantly less active against TP53 mutated/deleted than TP53 wild-type acute leukemia cell lines. When accounting for the varying sFGN849 sensitivity across cell lines, there was a statistically significant relationship between PVEK-induced cytotoxicity and CD123 expression (R²=0.4469, P=0.0125). This association remained in both the TP53 wild-type cell lines (R²=0.7654, P=0.0099) and the TP53mutated/deleted cell lines (R²=0.6709, P=0.0461). A similar relationship was found across 9 primary AML cell samples (R²=0.6724, P=0.0068). Since the above data suggested that TP53 alterations impaired the ability of PVEK to induce cytotoxicity in acute myeloid and lymphoblastic leukemia cells, we used TP53^wild-type EOL-1, MOLM-13, MV4;11, REH, and RS4;11 cells and deleted TP53 from bulk cells via CRISPR/Cas9 and then exposed these engineered cells to idasanutlin to enrich for the population of TP53^KO cells. sFGN849-induced cytotoxicity was significantly lower in TP53^KOcells than parental TP53^wild-type cells in all 5 cell line pairs, demonstrating the importance of functional TP53 for PVEK-induced cytotoxicity. In contrast, overexpression of P-glycoprotein or breast cancer resistance protein (BCRP) only minimally affected the cytotoxic activity of sFGN849 and PVEK in sublines engineered to overexpress these ABC transporter proteins relative to parental cell counterparts. Finally, to study their impact of anti-apoptotic BCL-2 family proteins on PVEK-induced cytotoxicity, we generated sublines EOL-1, MOLM-13, and MV4;11 cells in which we singly over-expressed either BCL-2, BCL-xL, and found sFGN849 activity was reduced in sublines overexpressing one of these proteins relative to parental cells.

Conclusions: PVEK has broad anti-leukemia activity against human acute myeloid and lymphoblastic leukemia cells. Important modulators for the drug's cytotoxic effects we identified include CD123 expression levels, TP53 alterations, and overexpression of anti-apoptotic BCL-2 family proteins but surprisingly not activity of ATP transporter proteins. The clinical relevance of these findings should be explored.

Zweidler-McKay:ImmunoGen, Inc: Current Employment, Current equity holder in publicly-traded company. Lakshmikanthan:ImmunoGen, Inc.: Current Employment, Current equity holder in publicly-traded company. Walter:Aptevo: Research Funding; Wugen, Inc.: Consultancy; VOR: Research Funding; Pfizer: Research Funding; Kura: Research Funding; Kite: Research Funding; ImmunoGen: Research Funding; Celgene/Bristol Myers Squibb: Research Funding; Jazz: Research Funding; Janssen: Research Funding.