dolastatin-15 and Ovarian-Neoplasms

Targeting tubulin binders to cancer cells using antibody-drug conjugates (ADCs) has great potential to become an effective cancer treatment with low normal tissue toxicity. The nature of the linker used to tether the tubulin binder to the antibody and the conjugation sites on the antibody and the small molecule are important factors in the ADC stability and effectiveness.. We explored the use of tubulin-targeting dolastatin 15 derivatives (Dol15) tethered covalently to a representative antibody, trastuzumab, via cleavable and non-cleavable linkers at varying antibody reactive sites (i.e., lysine residues, partially reduced hinge region disulfide bonds) and drug coupling sites (i.e., C-terminus, N-terminus), to investigate which constructs were more effective in killing HER2-positive cells in vitro and in vivo.. We found that Dol15 conjugated to trastuzumab via lysine residues at the drug C-terminus using a non-cleavable linker (trastuzumab-amide-C-term-Dol15) produced target-dependent growth inhibition of cells endogenously expressing high HER2 levels (i.e., SK-BR-3, SK-OV-3) in vitro. This ADC was effective at varying doses (i.e., 10 and 20 mg/kg) in the SK-OV-3 human ovarian cancer xenograft.. Tethering Dol15 via partially reduced disulfide bonds at the drug C-terminus via a non-cleavable linker (trastuzumab-MC-C-term-Dol15) resulted in an equally effective ADC in vitro, showing that site of antibody conjugation did not influence ADC activity. However, tethering Dol15 at the drug N-terminus using non-cleavable and cleavable linkers (trastuzumab-MC-N-term-Dol15 and trastuzumab-MC-VC-PABC-N-term-Dol15, respectively) resulted in ineffective ADCs. Thus, Dol15 tethered at the C-terminus may be a useful tubulin-targeting agent for conjugation at various antibody reactive sites.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Depsipeptides; Dose-Response Relationship, Drug; Drug Delivery Systems; Female; Humans; Mice; Mice, SCID; Ovarian Neoplasms; Receptor, ErbB-2; Trastuzumab; Tubulin; Xenograft Model Antitumor Assays

Dolastatins 10 and 15 are small peptides isolated from the marine sea hare Dolabella auricularia that have been shown to interact with tubulin. Their growth-inhibitory properties were compared using panels of human ovarian and colon-carcinoma cell lines. Both agents were very potent inhibitors of cell growth, with dolastatin 10 being an average of 9.1-fold more potent than dolastatin 15 [mean 50% inhibitory concentrations (IC50 values) 2.3 x 10(-10) and 2.1 x 10(-9) M, respectively; P < 0.05] and more potent than paclitaxel or vinblastine. While neither dolastatin exhibited marked cross-resistance in cisplatin- or etoposide-resistant cell lines, contrasting effects were observed using an acquired doxorubicin-resistant (CH1doxR, 100-fold resistant, P-glycoprotein overexpressing) cell line. Resistance was significantly higher to dolastatin 15 (12.7-fold) than to dolastatin 10 (only 3.2-fold; P < 0.05) and was reversible in both cases by verapamil. In vivo, using a s.c. advanced-stage human ovarian carcinoma xenograft and equitoxic doses, greater activity was observed with dolastatin 10 (6.1-day growth delay) versus 0.4 days for dolastatin 15. A radioimmunoassay for dolastatin 10 (limit of detection in mouse plasma 5 ng/ml) was developed. The rabbit antiserum aslo cross-reacted by 65% with dolastatin 15. Comparative mouse pharmacokinetics following i.v. administration of 1 mg/kg showed that both compounds are rapidly eliminated, but with a shorter second-phase half-life (t1/2 beta) being observed for dolastatin 15 (being detectable for only up to 4 h post-administration), the t1/2 beta being 3 times longer for dolastatin 10. In addition, areas under the plasma concentration-time curve (AUC values) were 1.6-fold higher for dolastatin 10 (333 versus 208 ng ml-1 h). Plasma binding of dolastatin 10 exceeded 90%. The highly sensitive RIA will be useful for pharmacokinetic studies in conjunction with the planned phase I clinical trials of these novel, extremely potent, tubulin-binding agents, of which dolastatin 10 appears to possess the more promising preclinical features.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Division; Colonic Neoplasms; Cross Reactions; Depsipeptides; Drug Resistance, Multiple; Female; Half-Life; Humans; Injections, Intravenous; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Oligopeptides; Ovarian Neoplasms; Rabbits; Radioimmunoassay; Random Allocation; Transplantation, Heterologous; Tumor Cells, Cultured; Verapamil