Design and Evaluation of ZD06519, a Novel Camptothecin Payload for Antibody Drug Conjugates

Abstract

In recent years, the field of antibody drug conjugates (ADC) has seen a resurgence, largely driven by the clinical benefit observed in patients treated with ADCs incorporating camptothecin-based topoisomerase I inhibitor payloads. Herein, we present the development of a novel camptothecin ZD06519 (FD1), which has been specifically designed for its application as an ADC payload. A panel of camptothecin analogs with different substituents at the C-7 and C-10 positions of the camptothecin core was prepared and tested in vitro. Selected compounds spanning a range of potency and hydrophilicity were elaborated into drug-linkers, conjugated to trastuzumab, and evaluated in vitro and in vivo. ZD06519 was selected on the basis of its favorable properties as a free molecule and as an antibody conjugate, which include moderate free payload potency (∼1 nmol/L), low hydrophobicity, strong bystander activity, robust plasma stability, and high-monomeric ADC content. When conjugated to different antibodies using a clinically validated MC-GGFG-based linker, ZD06519 demonstrated impressive efficacy in multiple cell line-derived xenograft models and noteworthy tolerability in healthy mice, rats, and non-human primates.

Graphical abstract

Figure 1.

A, Camptothecin-based ADCs that reached clinical development as of January 1, 2024 (ADCs with an asterisk denote terminated programs). B, Structures of camptothecin payloads utilized in clinically evaluated ADCs are listed in decreasing order of current representation (payload structures for HS-9265, P1021, and P1003 are undisclosed; payloads for DB-1310, IBI129, IBI30, OBI-992, and SGN-CEACAM5C ADCs are undisclosed). DXd and SN-38 (underlined) are payloads employed in approved ADCs. These 72 ADCs utilize more than 15 different camptothecin payloads, more than 21 different linkers, and target 24 different tumor associated antigens.

Figure 2.

Selected SARs for camptothecin derivatives.

Figure 3.

A, General structure of camptothecin library. B, Structures of small molecules (FD1–FD7) evaluated as ADC payloads in vivo. C,In vitro cytotoxicity of FD1–FD7 (orange, blue, and green stars), other novel camptothecin derivatives (orange, blue, and green dots), and camptothecin reference molecules (purple triangles) in the SK-BR-3 tumor cell line expressed as pIC₅₀ (−log[IC₅₀ (M)]) and hydrophobicity (calculated clogD at pH = 7.4) of the drugs. D, Structures of camptothecin-based drug-linkers (DL1–DL14) generated for in vivo studies. See Supplementary Data for detailed information.

Figure 4.

A, pIC₅₀ (−log[IC₅₀ (M)]) of novel camptothecin ADCs compared with DXd-ADC (bystander active) and DXd2-ADC (bystander inactive) benchmarks in both HER2-high SK-BR-3 and HER2-negative MDA-MB-468 cells. Bystander assay at 1.0 and 0.1 nmol/L ADC concentrations: viability of HER2-negative MDA-MB-468 cells treated with either 1.0 nmol/L (B) or 0.1 nmol/L (C) ADC concentration when grown in monoculture (orange bars) or in coculture with HER2-high SK-BR-3 cells (brown bars). D,In vivo efficacy of DAR8 ADCs in CB17.SCID mice implanted with HER2-expressing JIMT-1 cells. At an approximate TV of 150 mm³, mice were treated with single dose of either vehicle or ADC (3 mg/kg). TVs are represented as mean ± SEM on days measured. E, Pharmacokinetic analysis of ADCs highlights comparable exposure of all test articles except T-DL4 which showed more rapid clearance. F, 3D spheroid cytotoxicity assay: JIMT-1 tumor cells were grown in coculture with human dermal fibroblasts in ultra-low attachment plates then treated with ADCs. G,In vitro cytotoxicity in the 3D spheroid assay was well correlated with in vivo efficacy in the JIMT-1 CDX model.

Figure 5.

A and B, Tolerability in 8-week-old non–tumor-bearing BALB/c mice following single intraperitoneal injection of either 60 or 200 mg/kg of ADC. Three animals were included per group and body weight loss is represented as the average % change from baseline. C–E, Tolerability of ADCs in female Sprague Dawley rats following intravenous injection of either 30, 60, or 200 mg/kg on day 0 and day 21 (indicated by the arrows). Six animals were included per group and body weight loss is represented as the percentage change from baseline. In vivo efficacy following single intravenous injection of ZD06519 ADCs in: FRα-expressing OV-90 ovarian cancer CDX, dosed at 6 mg/kg (F); NaPi2b-expressing CTC-0958 ovarian cancer PDX, dosed at 6 mg/kg (G); cMET-expressing HT-29 colorectal cancer CDX, dosed at 6 mg/kg (H); GPC3-expressing HepG2 liver cancer CDX dosed at 8 mg/kg (I).