Selectivity and Safety of VIP943: A Novel CD123-Targeting Antibody-Drug Conjugate (ADC) Using a Proprietary Linker and Payload Class
Authors: Stelte-Ludwig Beatrix , Schomber Tibor , Izumi Raquel , Wong Harvey , Frigault Melanie M , Rebstock Anne-Sophie , Ludwig Sebastian , Mithal Arushi , Johnson Amy J , Hamdy Ahmed 
Published: December 2023

Introduction

ADCs afford high efficacy though often have shortcomings in their safety profile. Most ADC payloads are cytotoxic agents that can inhibit general processes of cellular homeostasis, including microtubule polymerization or DNA damage repair. While these are good targets for cytotoxicity, they have the drawback of being nonselective for dividing cancer cells. Here, we report the potent, on-target activity and selectivity of our novel CD123-targeting ADC VIP943, which uses a kinesin spindle protein (KSP; also known as KIF11 or Eg5) inhibitor as the payload (VIP716). The kinesin superfamily (KIF) is comprised of motor-proteins involved in various cellular mechanisms, including formation of the bipolar mitotic spindle during cell division. Targeting KSP selectively inhibits dividing cells, while sparing nondividing, differentiated cells thereby increasing the therapeutic index of VIP943.

Methods

VIP716 selectivity and potency: The motor domain of the human KSP and the other KIF family members were incubated with stabilized microtubules in a biochemical assay. The freshly prepared mixture was then incubated VIP716 and ispinesib, a clinical stage KSPi. ATPase activity was directly analyzed by measuring the formed inorganic phosphate.

VIP943 selectivity: The binding of VIP943 to 6,019 human plasma membrane proteins expressed on HEK cells was evaluated (Retrogenix cell microarray).

Cell cycle/death analysis: The effect of VIP943 on the 3 phases of the cell cycle (G0/G1- S- G2/M) was measured with DNA staining and flow cytometry in MOLM-13 and MV-4-11 cell lines. In addition, cell death was measured by flow cytometry after a 4 h incubation with VIP943 in MOLM-13 cells. Isotype ADC was used as a control.

In vitro cytotoxicity: 56 hematologic cell lines were treated in vitro with VIP331 (cell permeable analog of VIP716). IC 50 results were matched with the DNA/RNA profile of relevant oncogenes to determine correlations.

Nonhuman primate safety and toxicokinetics: VIP943 was intravenously administered to Cynomolgus monkeys at 1, 3, or 9 mg/kg once weekly for 4 weeks in a Good Laboratory Practice (GLP) toxicology study. Serial plasma samples were collected on Day 1 and Day 22. Total antibody from VIP943 (as a surrogate for VIP943) and VIP716 concentrations were measured by LC/MS/MS. Toxicokinetics (TK) were assessed using noncompartmental methods.

Results

In a biochemical assay, VIP716 (payload), achieved comparable potency to ispinesib with IC 50 values of 1.4 and 1.6 nM, respectively, for KSP. VIP716 did not inhibit any of the 11 other KIF family members up to 10 µM. In the Retrogenix assay, VIP943 (ADC) had no interaction with any of the analyzed human cell surface proteins except for CD123. Cell cycle analysis in AML cells showed a dose- and time-dependent increase in G2/M arrest with VIP943 treatment, but not with isotype control. An increase in cell death with VIP943 treatment was also observed after a 4-hour incubation. In a broad hematologic cell line panel, the median cytotoxicity IC 50 for VIP331 (cell permeable KSPi) was 2.49 nM. Of note, no genomic alterations correlated with sensitivity, suggesting VIP943 may be effective even in hard-to-treat hematologic malignancies. VIP943 was well tolerated in the GLP monkey toxicology study. No mortality occurred in any monkeys. No neutropenia, thrombocytopenia or anemia were observed. VIP943 and VIP716 exposures increased with increasing dose in a dose-proportional or greater than dose-proportional manner. No consistent differences in TK were observed with sex. Based on area under the concentration curve (AUC), no accumulation was observed. Payload to parent ratios were consistent with a low level of non-specific release of VIP716 from VIP943.

Conclusion

High on-target activity of VIP716, the payload of our new CD123-targeting ADC, VIP943, was demonstrated in a biochemical assay and selectivity against other KIF family members was confirmed. This activity translated into cell cycle arrest in the G2/M phase and subsequent cell death in a dose- and time-dependent manner. Additionally, toxicology in non-human primates and in vivo TK studies confirm safety, favorable drug exposures, and little non-specific release of the payload. Based on these data, evaluation of VIP943 in human clinical trials is warranted.