Radiolabeling of a supertoxic tubulysin analogue and the development of antibody-drug conjugates

Objectives Supertoxic compounds conjugated to monoclonal antibodies (mAbs), i.e. antibody-drug conjugates (ADCs), seem promising new therapeutics for cancer treatment. An appealing novel family of toxins for this purpose are the tubulysins, causing apoptosis in cancer cells at picomolar concentrations. Recently, tubulysin analogues with varying toxicities have been developed*. The aim of this study was to radiolabel a tubulysin analogue with ¹³¹I to enable in vitro and in vivo analysis of the drug in ADC approaches. We developed a stable ADC by coupling ¹³¹I-tubulysin to the anti-HER2 mAb trastuzumab and monitored the pharmacokinetics of this ADC in vivo.

Methods The tubulysin analogue was radiolabeled with ¹³¹I using ChlT and purified by tC2 Seppak. After conversion into the ¹³¹I-tubulysin-NHS ester, the product was again purified by Seppak. Trastuzumab was conjugated with different molar excesses of ¹³¹I-tubulysin-NHS in MeCN at pH 9-9.5. After 30 min, the conjugate was purified by size exclusion chromatography using 0.9% NaCl as eluent. Conjugate stability was monitored in the presence of human serum and analyzed with SDS-PAGE. ¹³¹I-tubulysin-trastuzumab and ¹²⁴I-trastuzumab (as reference) were administered to nude mice, blood was drawn and analysed at various time points.

Results Radiolabeling of tubulysin with ¹³¹I was achieved in 95% yield. Conversion to its NHS-ester was obtained in 75%. Conjugation to trastuzumab with 5 and 10 eq ¹³¹I-tubulysin NHS-ester yielded on average 2±0.3 and 4±0.5 tubulysin groups per mAb molecule. After 7 days in serum at 37°C, 90% of the conjugate was still intact. Studies in nude mice showed that the pharmacokinetics of ¹³¹I-tubulysin-trastuzumab were similar to that of ¹²⁴I-trastuzumab.

Conclusions Tubulysin can be efficiently radiolabeled with ¹³¹I, which facilitates the analysis, imaging and/or quantification of the conjugation in vitro and in vivo. These very promising conjugates will now be evaluated for their efficacy in cancer therapy