The development and evaluation of a tublysine-based antibody-drug conjugate with enhanced tumor therapeutic efficacy

Abstract

Antibody-drug conjugates (ADCs) offer targeted cancer therapy by delivering cytotoxic agents directly to tumor cells. However, challenges such as relapse, resistance, and diverse patient needs drive ongoing innovation in ADC development. Exploration of new small-molecule toxins with unique antitumor and toxicity profiles is crucial. Tub114, a novel Tubulysin B analog with a hydrophilic ethylene glycol moiety, has been conjugated to the anti-HER2 antibody DX-CHO9, forming the ADC DX126-262. This study examines the efficacy, pharmacokinetics, and safety profile of DX126-262, with a focus on Tubulysin-associated liver toxicity. In vivo efficacy was assessed using three HER2-positive tumor models, with dose-dependent tumor growth inhibition compared to established treatments. Pharmacokinetic studies were conducted in cynomolgus monkeys across a dosing range (3-30 mg/kg) to compare clearance and stability with Kadcyla and Enhertu. Acute toxicity assays were conducted in mice (75 and 150 mg/kg doses), and repeated-dose toxicity was evaluated over five doses, administered every 3 weeks in rats and cynomolgus monkeys. DX126-262 demonstrated significant and dose-dependent tumor growth inhibition across HER2-positive models, with superior antitumor efficacy compared to Kadcyla and comparable efficacy to Enhertu in vivo studies. In pharmacokinetic studies, DX126-262 exhibited a clearance rate similar to Enhertu and enhanced stability compared to Kadcyla. Acute toxicity assays revealed reduced hepatotoxicity at doses of 75 and 150 mg/kg in mice, with improved tolerance. In repeated-dose toxicity studies, DX126-262 was well tolerated in rats at doses up to 200 mg/kg, with the highest non-severely toxic dose (HNSTD) established at 100 mg/kg. In cynomolgus monkeys, DX126-262 demonstrated superior hepatotoxic tolerability without significant bone marrow suppression, with an HNSTD of 30 mg/kg. DX126-262, incorporating Tub114, a novel Tubulysin B analog, effectively mitigates the inherent hepatotoxicity associated with Tubulysin compounds while maintaining strong antitumor efficacy. These findings suggest that DX126-262 could serve as a safer and more effective option for HER2-targeted cancer therapy, warranting further clinical studies to confirm its therapeutic potential.

Keywords: ADC; DX126-262; HER2; Tub114; target-specific delivery; therapeutic window; tubulysin B analog.

FIGURE 1

The chemical structures of DX126-262. DX-CHO9 is the monoclonal antibody used in the antibody-drug conjugate (DX126-262). Tub114 is the small molecule linked to the antibody with a DAR of 3.5–3.8. Tub114-cys is the primary metabolite of DX126-262 identified in animal plasma.

FIGURE 2

Inhibition of tumor cell proliferation (Cell viability %, mean ± SD of triplicates) plotted as a function of test article concentration (nM). BT-474 (HER2 positive), NCI-N87 (HER2 positive), SK-BR-3 (HER2 positive), MDA-MB-468 (HER2 negtive).

FIGURE 3

Tumor volumes (mm³) were measured biweekly and plotted as a function of time (days). Tumor weights (g) were recorded post-sacrifice in female BALB/c nude mice bearing subcutaneous xenografts of HER2-positive cancers [n = 10 for (A–C); n = 5 for (D); data presented as mean ± SEM]. (A) BT-474 human breast cancer xenografts. Tumor weights were recorded on day 24 post-sacrifice. (B) NCI-N87 human gastric cancer xenografts. (C) SK-OV-3 drug-resistant human ovarian cancer xenografts. (D) NCI-N87 human gastric cancer xenografts. Tumor weights were recorded on day 37 post-sacrifice. One-way analysis of variances (ANOVA) was used. Statistical differences were significant at *p < 0.05 and very significant at **p < 0.01, ***p < 0.001.

FIGURE 4

Concentration (μg/mL)-time (hour) curves of DX126-262 (intact ADC), total antibody (total Ab with and without Tub114-cys) and free Tub114-cys in plasma after a single intravenous injection of DX126-262 at 3, 10, and 30 mg/kg.

FIGURE 5

Comparisons of acute toxicities (mean ± SD, n = 10) in ICR mice after a single intravenous injection of DX126-262 or Kadcyla at the same dose levels of 75 and 150 mg/kg. (A) Body weights (g) measured daily post-dosing. (B) The ratios of liver/brain weights on day 5 and 12 post-dosing. (C) Plasma ALT activities (IU/L) on day 5 and 12 post-dosing. (D) Plasma AST (IU/L) activities on day 5 and 12 post-dosing.

FIGURE 6

Liver pathologic profiles (H&E staining: ×40) in ICR mice on day 5 (A) and day 12 (B) after a single intravenous injection of PBS, 75 and 150 mg/kg of DX126-262 or Kadcyla, respectively.