Development of Novel ADCs: Conjugation of Tubulysin Analogues to Trastuzumab Monitored by Dual Radiolabeling

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug-conjugates (ADCs). For full control over drug-antibody-ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 mAb trastuzumab, are radiolabeled. 131I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 >100nM) and the potent TUB-OMOM (IC50 ~1nM), and their direct covalent conjugation to 89Zr-trastuzumab were established. Radioiodination of tubulysins was 92-98% efficient, conversion to NHS-esters >99%, esters were isolated in an overall yield of 68±5% with radiochemical purity of >99.5%. Conjugation of 131I-tubulysin-NHS-esters to 89Zr-trastuzumab was 45-55% efficient, resulting in ADCs with 96-98% radiochemical purity after size-exclusion-chromatography. ADCs were evaluated for their tumor-targeting potential and anti-tumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM-trastuzumab (DAR 4) showed dose-dependent anti-tumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment.