Abstract
Purpose
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.
Methods
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.
Results
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.
Conclusions
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.
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References
Bai R, Edler MC, Bonate PL, Copeland TD, Pettit GR, Luduena RF, Hamel E (2009) Intracellular activation and deactivation of tasidotin, an analog of dolastatin 15: correlation with cytotoxicity. Mol Pharmacol 75:218–226
Beckwith M, Urba WJ, Longo DL (1993) Growth inhibition of human lymphoma cell lines by the marine products, dolastatins 10 and 15. J Natl Cancer Inst 85:483–488
Bhat KM, Setaluri V (2007) Microtubule-associated proteins as targets in cancer chemotherapy. Clin Cancer Res 13:2849–2854
Bonate PL, Beyerlein D, Crawford J, Roth S, Krumbholz R, Schmid S (2007) Pharmacokinetics in mice implanted with xenografted tumors after intravenous administration of tasidotin (ILX651) or its carboxylate metabolite. Aaps J 9:E378–E387
Bross PF, Beitz J, Chen G, Chen XH, Duffy E, Kieffer L, Roy S, Sridhara R, Rahman A, Williams G, Pazdur R (2001) Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res 7:1490–1496
Carter PJ, Senter PD (2008) Antibody-drug conjugates for cancer therapy. Cancer J 14:154–169
Chari RV (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41:98–107
Chari RV, Jackel KA, Bourret LA, Derr SM, Tadayoni BM, Mattocks KM, Shah SA, Liu C, Blattler WA, Goldmacher VS (1995) Enhancement of the selectivity and antitumor efficacy of a CC-1065 analogue through immunoconjugate formation. Cancer Res 55:4079–4084
Chari RV, Martell BA, Gross JL, Cook SB, Shah SA, Blattler WA, McKenzie SJ, Goldmacher VS (1992) Immunoconjugates containing novel maytansinoids: promising anticancer drugs. Cancer Res 52:127–131
Coombes RC, Buckman R, Forrester JA, Shepherd V, O’Hare MJ, Vincent M, Powles TJ, Neville AM (1986) In vitro and in vivo effects of a monoclonal antibody-toxin conjugate for use in autologous bone marrow transplantation for patients with breast cancer. Cancer Res 46:4217–4220
Cunningham C, Appleman LJ, Kirvan-Visovatti M, Ryan DP, Regan E, Vukelja S, Bonate PL, Ruvuna F, Fram RJ, Jekunen A, Weitman S, Hammond LA, Eder JP Jr (2005) Phase I and pharmacokinetic study of the dolastatin-15 analogue tasidotin (ILX651) administered intravenously on days 1, 3, and 5 every 3 weeks in patients with advanced solid tumors. Clin Cancer Res 11:7825–7833
Doronina SO, Bovee TD, Meyer DW, Miyamoto JB, Anderson ME, Morris-Tilden CA, Senter PD (2008) Novel peptide linkers for highly potent antibody-auristatin conjugate. Bioconjug Chem 19:1960–1963
Doronina SO, Mendelsohn BA, Bovee TD, Cerveny CG, Alley SC, Meyer DL, Oflazoglu E, Toki BE, Sanderson RJ, Zabinski RF, Wahl AF, Senter PD (2006) Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem 17:114–124
Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, Chace DF, DeBlanc RL, Gearing RP, Bovee TD, Siegall CB, Francisco JA, Wahl AF, Meyer DL, Senter PD (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol 21:778–784
Dubowchik GM, Firestone RA, Padilla L, Willner D, Hofstead SJ, Mosure K, Knipe JO, Lasch SJ, Trail PA (2002) Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem 13:855–869
Ebbinghaus S, Rubin E, Hersh E, Cranmer LD, Bonate PL, Fram RJ, Jekunen A, Weitman S, Hammond LA (2005) A phase I study of the dolastatin-15 analogue tasidotin (ILX651) administered intravenously daily for 5 consecutive days every 3 weeks in patients with advanced solid tumors. Clin Cancer Res 11:7807–7816
Garg V, Zhang W, Gidwani P, Kim M, Kolb EA (2007) Preclinical analysis of tasidotin HCl in Ewing’s sarcoma, rhabdomyosarcoma, synovial sarcoma, and osteosarcoma. Clin Cancer Res 13:5446–5454
Griffiths GL, Mattes MJ, Stein R, Govindan SV, Horak ID, Hansen HJ, Goldenberg DM (2003) Cure of SCID mice bearing human B-lymphoma xenografts by an anti-CD74 antibody-anthracycline drug conjugate. Clin Cancer Res 9:6567–6571
Gros O, Gros P, Jansen FK, Vidal H (1985) Biochemical aspects of immunotoxin preparation. J Immunol Methods 81:283–297
Hellstrom I, Hellstrom KE, Senter PD (2001) Development and activities of the BR96-doxorubicin immunoconjugate. Methods Mol Biol 166:3–16
Hu MK, Huang WS (1999) Synthesis and cytostatic properties of structure-simplified analogs of dolastatin 15. J Pept Res 54:460–467
Jeffrey SC, Nguyen MT, Moser RF, Meyer DL, Miyamoto JB, Senter PD (2007) Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker. Bioorg Med Chem Lett 17:2278–2280
Jeffrey SC, Torgov MY, Andreyka JB, Boddington L, Cerveny CG, Denny WA, Gordon KA, Gustin D, Haugen J, Kline T, Nguyen MT, Senter PD (2005) Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates. J Med Chem 48:1344–1358
Johnson IS, Armstrong JG, Gorman M, Burnett JP Jr (1963) The vinca alkaloids: a new class of oncolytic agents. Cancer Res 23:1390–1427
Jordan MA, Thrower D, Wilson L (1991) Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res 51:2212–2222
Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4:253–265
Lambert JM, Senter PD, Yau-Young A, Blattler WA, Goldmacher VS (1985) Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins. J Biol Chem 260:12035–12041
Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blattler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68:9280–9290
Mita AC, Hammond LA, Bonate PL, Weiss G, McCreery H, Syed S, Garrison M, Chu QS, DeBono JS, Jones CB, Weitman S, Rowinsky EK (2006) Phase I and pharmacokinetic study of tasidotin hydrochloride (ILX651), a third-generation dolastatin-15 analogue, administered weekly for 3 weeks every 28 days in patients with advanced solid tumors. Clin Cancer Res 12:5207–5215
Pirker R, FitzGerald DJ, Hamilton TC, Ozols RF, Willingham MC, Pastan I (1985) Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines. Cancer Res 45:751–757
Polgar L (2002) The prolyl oligopeptidase family. Cell Mol Life Sci 59:349–362
Polson AG, Calemine-Fenaux J, Chan P, Chang W, Christensen E, Clark S, de Sauvage FJ, Eaton D, Elkins K, Elliott JM, Frantz G, Fuji RN, Gray A, Harden K, Ingle GS, Kljavin NM, Koeppen H, Nelson C, Prabhu S, Raab H, Ross S, Slaga DS, Stephan JP, Scales SJ, Spencer SD, Vandlen R, Wranik B, Yu SF, Zheng B, Ebens A (2009) Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res 69:2358–2364
Poncet J (1999) The dolastatins, a family of promising antineoplastic agents. Curr Pharm Des 5:139–162
Rasila KK, Verschraegen C (2005) Tasidotin HCl (Genzyme). Curr Opin Investig Drugs 6:631–638
Ray A, Okouneva T, Manna T, Miller HP, Schmid S, Arthaud L, Luduena R, Jordan MA, Wilson L (2007) Mechanism of action of the microtubule-targeted antimitotic depsipeptide tasidotin (formerly ILX651) and its major metabolite tasidotin C-carboxylate. Cancer Res 67:3767–3776
Rowinsky EK, Donehower RC (1996) Antimicrotubule agents. In: Chabner BA, Longo DL (eds) Cancer chemotherapy and biotherapy. Lippincott-Raven, New York, pp 263–296
Scott CF Jr, Goldmacher VS, Lambert JM, Chari RV, Bolender S, Gauthier MN, Blattler WA (1987) The antileukemic efficacy of an immunotoxin composed of a monoclonal anti-Thy-1 antibody disulfide linked to the ribosome-inactivating protein gelonin. Cancer Immunol Immunother 25:31–40
Senter PD (1990) Activation of prodrugs by antibody-enzyme conjugates: a new approach to cancer therapy. FASEB J 4:188–193
Senter PD, Meyer DL (2007) Monoclonal antibody drug conjugates for cancer therapy. Prodrugs: challenges and rewards. Springer, New York, pp 507–524
Sun MM, Beam KS, Cerveny CG, Hamblett KJ, Blackmore RS, Torgov MY, Handley FG, Ihle NC, Senter PD, Alley SC (2005) Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug Chem 16:1282–1290
Supko JG, Lynch TJ, Clark JW, Fram R, Allen LF, Velagapudi R, Kufe DW, Eder JP Jr (2000) A phase I clinical and pharmacokinetic study of the dolastatin analogue cemadotin administered as a 5-day continuous intravenous infusion. Cancer Chemother Pharmacol 46:319–328
Teicher BA (2008) Newer cytotoxic agents: attacking cancer broadly. Clin Cancer Res 14:1610–1617
Teicher BA (2009) Antibody-drug conjugate targets. Curr Cancer Drug Targets 9:982–1004
Tranoy-Opalinski I, Fernandes A, Thomas M, Gesson JP, Papot S (2008) Design of self-immolative linkers for tumour-activated prodrug therapy. Anticancer Agents Med Chem 8:618–637
Wu AM, Senter PD (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 23:1137–1146
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Supplementary data for this article are available online at Cancer Chemotherapy and Pharmacology (http://www.springer.com/medicine/oncology/journal/280).
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Gianolio, D.A., Rouleau, C., Bauta, W.E. et al. Targeting HER2-positive cancer with dolastatin 15 derivatives conjugated to trastuzumab, novel antibody–drug conjugates. Cancer Chemother Pharmacol 70, 439–449 (2012). https://doi.org/10.1007/s00280-012-1925-8
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DOI: https://doi.org/10.1007/s00280-012-1925-8