title: Development of novel ADCs: conjugation of tubulysin analogues to trastuzumab monitored by dual radiolabeling.
abstract: Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug conjugates (ADC). 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 monoclonal antibody (mAb) trastuzumab, are radiolabeled. (131)I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 > 100 nmol/L) and the potent TUB-OMOM (IC50, ~1 nmol/L), and their direct covalent conjugation to (89)Zr-trastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% ± 5% with radiochemical purity of more than 99.5%. Conjugation of (131)I-tubulysin-NHS esters to (89)Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor 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 antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment.
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Ruth
authors.initials: R
authors.lastname: Cohen
authors.affiliation: Department of Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Danielle J
authors.initials: DJ
authors.lastname: Vugts
authors.affiliation: Department of Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Gerard W M
authors.initials: GW
authors.lastname: Visser
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Marijke
authors.initials: M
authors.lastname: Stigter-van Walsum
authors.affiliation: Department of Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Marije
authors.initials: M
authors.lastname: Bolijn
authors.affiliation: KemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3, Pula, Cagliari, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Spiga
authors.affiliation: KemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3, Pula, Cagliari, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Lazzari
authors.affiliation: Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Sreejith
authors.initials: S
authors.lastname: Shankar
authors.affiliation: KemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3, Pula, Cagliari, Italy. Dipartimento C.M.I.C. del Politecnico di Milano and C.N.R.-I.C.R.M., Milano, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Sani
authors.affiliation: Dipartimento C.M.I.C. del Politecnico di Milano and C.N.R.-I.C.R.M., Milano, Italy. Kosterlitz Centre for Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, United Kingdom.
authors.forename: Matteo
authors.initials: M
authors.lastname: Zanda
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, the Netherlands. Department of Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, the Netherlands. gams.vandongen@vumc.nl.
authors.forename: Guus A M S
authors.initials: GA
authors.lastname: van Dongen
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004355:Drug Stability; D005260:Female; D006801:Humans; D020714:Maximum Tolerated Dose; D008819:Mice, Nude; D009842:Oligopeptides; D014018:Tissue Distribution; D000068878:Trastuzumab; D023041:Xenograft Model Antitumor Assays
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D009842:Oligopeptides; C000593796:TUB-OH-trastuzumab; C000593795:TUB-OMOM-trastuzumab; D000068878:Trastuzumab