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Pharmaceutical Research

, Volume 34, Issue 2, pp 352–364 | Cite as

Design and In Vitro Evaluation of Bispecific Complexes and Drug Conjugates of Anticancer Peptide, LyP-1 in Human Breast Cancer

  • Selin Seda Timur
  • Prashant Bhattarai
  • Reyhan Neslihan Gürsoy
  • İmran Vural
  • Ban-An Khaw
Research Paper

Abstract

Purpose

LyP-1, a nine-amino-acid tumor homing peptide, selectively binds to its cognate receptor, p32. Overexpression of p32 in certain tumors should allow use of LyP-1 as a targeting agent for the delivery of therapeutic or diagnostic agents. Peptide conjugates are developed for enhanced pre-targeting of MDA-MB-231 breast cancer cells with peptide-antibody bispecific complexes and targeting with multiple-drug/-fluorophore-conjugated nano-polymers.

Methods

LyP-1-anti-DTPA bispecific antibody complexes (LyP-1-bsAbCx) were generated by conjugation of anti-DTPA antibody and LyP-1. LyP-1–doxorubicin (Dox), Dox-DTPA-succinyl-polylysine (Dox-DSPL), Dox-DSPL-LyP-1, DTPA-Dox-poly glutamic acid (D-Dox-PGA) or DTPA-rhodamine conjugated polylysine (DSPL-RITC) were prepared. In vitro therapeutic efficacy and targeting by immunofluorescence in MDA-MB-231 breast cancer cells were assessed with Dox-LyP-1. Immunofluorescence visualization of cancer cells was evaluated after pretargeting with LyP-1-bsAbCx and targeting with DSPL-RITC.

Results

Cytotoxicity of Dox-LyP-1 conjugates was significantly greater than free doxorubicin (p < 0.0001). For fluorescent-labeled LyP-1, internalization occurred in 30 min in tumor cells. Fluorescence intensity of two-step targeted cells showed that pretargeting with LyP-1-bsAbC, followed by targeting with DSPL-RITC was greater than non-pretargeted DSPL-RITC (p < 0.05).

Conclusions

Peptide-conjugates are effective targeting agents for MDA-MB-231 breast cancer cells in culture. LyP-1-bsAbCx and Dox-LyP-1 conjugates may allow development of novel targeted cancer therapy and diagnosis.

KEY WORDS

peptide-antibody bispecific complexes polymer pro-drug conjugates pre-targeted drug targeting 

ABBREVIATIONS

ADC

Antibody-drug-conjugates

D-Dox-PGA

Doxorubicin conjugated N-terminal DTPA conjugated PGA

DMF

Dimethylformamide

Dox

Doxorubicin hydrochloride-

Dox-DSPL

Dox-DTPA-succinyl-polylysine

Dox-DSPL-LyP-1

LyP-1 conjugated to Dox-DSPL

Dox-LyP-1

LyP-1 conjugated with doxorubicin hydrochloride

D-PGA

N-terminal DTPA conjugated PGA

DPL

DTPA-poly-L-lysine

D-PL-LyP-1-Dox

DTPA conjugated poly-L-lysine conjugated with LyP-1 and Dox

DPL-RITC

RITC conjugated DPL

DSPL-LyP-1

LyP-1 conjugated to DSPL

DSPL-RITC

DTPA-succinyl rhodamine conjugated poly-L-lysine

DTPA

Diethylenetriaminepentaacetic acid

DTPA-BSA

DTPA conjugated bovine serum albumin

GAM-HRP

Goat anti-mouse IgG antibody conjugated with HRP

LyP-1

9 amino acid peptide ligand specific for mitochondrial membrane receptor p32.

LyP-1-bsAbCx

LyP-1 conjugated anti-DTPA bispecific antibody complex

MWCO

Molecular weight cut-off

NHS-Fluorescein

5/6-carboxyfluorescein succinimidyl ester

PDCs

Polymer drug conjugates

PGA

Poly-L-glutamic acid

PL

Poly-L-lysine

RITC

Rhodamine B isothiocyanate

SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TMA

Therapeutic monoclonal antibodies

TNBS

Tri-nitro benzene sulfonic acid

Notes

ACKNOWLEDGMENTS AND DISCLOSURES

This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK 2214-International Research Fellowship Programme).

References

  1. 1.
    Liang XJ, Chen C, Zhao Y, Wang PC. Circumventing tumor resistance to chemotherapy by nanotechnology. Methods Mol Biol. 2010;596:467–88.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Haag R, Kratz F. Polymer therapeutics: concepts and applications. Angew Chem. 2006;45(8):1198–215.CrossRefGoogle Scholar
  3. 3.
    Canal F, Sanchis J, Vicent MJ. Polymer-drug conjugates as nano-sized medicines. Curr Opin Biotechnol. 2011;22(6):894–900.CrossRefPubMedGoogle Scholar
  4. 4.
    Duncan R. Polymer conjugates as anticancer nanomedicines. Nat Rev Cancer. 2006;6(9):688–701.CrossRefPubMedGoogle Scholar
  5. 5.
    Duncan R. Polymer therapeutics as nanomedicines: new perspectives. Curr Opin Biotechnol. 2011;22(4):492–501.CrossRefPubMedGoogle Scholar
  6. 6.
    Greco F, Vicent MJ. Polymer-drug conjugates: current status and future trends. Front Biosci J Virtual Libr. 2008;13:2744–56.CrossRefGoogle Scholar
  7. 7.
    Torchilin VP. Passive and active drug targeting: drug delivery to tumors as an example. Handb Exp Pharmacol. 2010;197:3–53.CrossRefGoogle Scholar
  8. 8.
    Greco F, Vicent MJ. Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Adv Drug Deliv Rev. 2009;61(13):1203–13.CrossRefPubMedGoogle Scholar
  9. 9.
    Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014;6(1):34–45.CrossRefGoogle Scholar
  10. 10.
    Sassoon I, Blanc V. Antibody-drug conjugate (ADC) clinical pipeline: a review. Methods Mol Biol. 2013;1045:1–27.CrossRefPubMedGoogle Scholar
  11. 11.
    Breij EC, de Goeij BE, Verploegen S, Schuurhuis DH, Amirkhosravi A, Francis J, et al. An antibody-drug conjugate that targets tissue factor exhibits potent therapeutic activity against a broad range of solid tumors. Cancer Res. 2014;74(4):1214–26.CrossRefPubMedGoogle Scholar
  12. 12.
    Chang CH, Sharkey RM, Rossi EA, Karacay H, McBride W, Hansen HJ, et al. Molecular advances in pretargeting radioimunotherapy with bispecific antibodies. Mol Cancer Ther. 2002;1(7):553–63.PubMedGoogle Scholar
  13. 13.
    Westerlund K, Honarvar H, Tolmachev V, Eriksson KA. Design, preparation, and characterization of PNA-based hybridization probes for affibody-molecule-mediated pretargeting. Bioconjug Chem. 2015;26(8):1724–36.CrossRefPubMedGoogle Scholar
  14. 14.
    Kuijpers WH, Bos ES, Kaspersen FM, Veeneman GH, van Boeckel CA. Specific recognition of antibody-oligonucleotide conjugates by radiolabeled antisense nucleotides: a novel approach for two-step radioimmunotherapy of cancer. Bioconjug Chem. 1993;4(1):94–102.CrossRefPubMedGoogle Scholar
  15. 15.
    Li X, Huang Q, Xiao J, Liu G, Dou S, Rusckowski M, et al. Novel DNA polymer for amplification pretargeting. ACS Med Chem Lett. 2015;6(9):972–6.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mallikaratchy P, Gardner J, Nordstrom LUR, Veomett NJ, McDevitt MR, Heaney ML, et al. A self-assembling short oligonucleotide duplex suitable for pretargeting. Nucleic Acid Ther. 2013;23(4):289–99.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Patil V, Gada K, Panwar R, Majewski S, Tekabe Y, Varvarigou A, et al. In vitro demonstration of enhanced prostate cancer toxicity: pretargeting with Bombesin bispecific complexes and targeting with polymer-drug-conjugates. J Drug Target. 2013;21(10):1012–21.CrossRefPubMedGoogle Scholar
  18. 18.
    Liu G, Dou S, Pretorius PH, Liu X, Chen L, Rusckowski M, et al. Tumor pretargeting in mice using MORF conjugated CC49 antibody and radiolabeled complimentary cMORF effector. Q J Nucl Med Mol Imaging. 2010;54(3):333–40.PubMedGoogle Scholar
  19. 19.
    Sharkey RM, Chang CH, Rossi EA, McBride WJ, Goldenberg DM. Pretargeting: taking an alternate route for localizing radionuclides. Tumor Biol. 2012;33(3):591–600.CrossRefGoogle Scholar
  20. 20.
    van Duijnhoven SMJ, Rossin R, van den Bosch SM, Wheatcroft MP, Hudson PJ, Robillard MS. Diabody pretargeting with click chemistry in vivo. J Nucl Med. 2015;56(9):1422–8.CrossRefPubMedGoogle Scholar
  21. 21.
    Khaw BA, Gada KS, Patil V, Panwar R, Mandapati S, Hatefi A, et al. Bispecific antibody complex pre-targeting and targeted delivery of polymer drug conjugates for imaging and therapy in dual human mammary cancer xenografts: targeted polymer drug conjugates for cancer diagnosis and therapy. Eur J Nucl Med Mol Imaging. 2014;41(8):1603–16.CrossRefPubMedGoogle Scholar
  22. 22.
    Chen X, Dou S, Liu G, Liu X, Wang Y, Chen L, et al. Synthesis and in vitro characterization of a dendrimer-MORF conjugate for amplification pretargeting. Bioconjug Chem. 2008;19(8):1518–25.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Firer MA, Gellerman G. Targeted drug delivery for cancer therapy: the other side of antibodies. J Hematol Oncol. 2012;5:70.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zhi Jie Li, CHC. Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery. 2012;10(Suppl 1):S1.Google Scholar
  25. 25.
    Laakkonen P, Porkka K, Hoffman JA, Ruoslahti E. A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nat Med. 2002;8(7):751–5.PubMedGoogle Scholar
  26. 26.
    Laakkonen P, Akerman ME, Biliran H, Yang M, Ferrer F, Karpanen T, et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A. 2004;101(25):938–9386.CrossRefGoogle Scholar
  27. 27.
    Laakkonen P, Zhang L, Ruoslahti E. Peptide targeting of tumor lymph vessels. Ann N Y Acad Sci. 2008;1131:37–43.CrossRefPubMedGoogle Scholar
  28. 28.
    Laakkonen P, Vuorinen K. Homing peptides as targeted delivery vehicles. Integr Biol UK. 2010;2(7–8):326–37.CrossRefGoogle Scholar
  29. 29.
    Fogal V, Zhang L, Krajewski S, Ruoslahti E. Mitochondrial/cell– surface protein p32/gC1qR as a molecular target in tumor cells and tumor stroma. Cancer Res. 2008;68(17):7210–8.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Yan F, Li X, Jiang C, Jin Q, Zhang Z, Shandas R, et al. A novel microfluidic chip for assessing dynamic adhesion behavior of cell-targeting microbubbles. Ultrasound Med Biol. 2014;40(1):148–57.CrossRefPubMedGoogle Scholar
  31. 31.
    Gursoy RN, Cevik O. Design, characterization and in vitro evaluation of SMEDDS containing an anticancer peptide, linear LyP-1. Pharm Dev Technol. 2014;19(4):486–90.CrossRefPubMedGoogle Scholar
  32. 32.
    Wang Z, Yu Y, Ma J, Zhang H, Zhang H, Wang X, et al. LyP-1 modification to enhance delivery of artemisinin or fluorescent probe loaded polymeric micelles to highly metastatic tumor and its lymphatics. Mol Pharm. 2012;9(9):2646–57.CrossRefPubMedGoogle Scholar
  33. 33.
    Herringson TP, Altin JG. Effective tumor targeting and enhanced anti-tumor effect of liposomes engrafted with peptides specific for tumor lymphatics and vasculature. Int J Pharm. 2011;411(1–2):206–14.CrossRefPubMedGoogle Scholar
  34. 34.
    Seo JW, Baek H, Mahakian LM, Kusunose J, Hamzah J, Ruoslahti E, et al. (64)Cu-labeled LyP-1-dendrimer for PET-CT imaging of atherosclerotic plaque. Bioconjug Chem. 2014;25(2):231–9.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Miao D, Jiang M, Liu Z, Gu G, Hu Q, Kang T, et al. Co-administration of dual-targeting nanoparticles with penetration enhancement peptide for antiglioblastoma therapy. Mol Pharm. 2014;11(1):90–101.CrossRefPubMedGoogle Scholar
  36. 36.
    Uchida M, Kosuge H, Terashima M, Willits DA, Liepold LO, Young MJ, et al. Protein cage nanoparticles bearing the LyP-1 peptide for enhanced imaging of macrophage-rich vascular lesions. ACS Nano. 2011;5(4):2493–502.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Reichert JM, Dhimolea E. The future of antibodies as cancer drugs. Drug Discov Today. 2012;17(17–18):954–63.CrossRefPubMedGoogle Scholar
  38. 38.
    Peters C, Brown S. Antibody-drug conjugates as novel anti-cancer chemotherapeutics. Biosci Rep. 2015;35.Google Scholar
  39. 39.
    Cao J, Cui S, Li S, Du C, Tian J, Wan S, et al. Targeted cancer therapy with a 2-deoxyglucose-based adriamycin complex. Cancer Res. 2013;73(4):1362–73.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Selin Seda Timur
    • 1
  • Prashant Bhattarai
    • 2
  • Reyhan Neslihan Gürsoy
    • 1
  • İmran Vural
    • 1
  • Ban-An Khaw
    • 2
  1. 1.Pharmaceutical Technology Department, Faculty of PharmacyHacettepe UniversityAnkaraTurkey
  2. 2.Department of Pharmaceutical Sciences, School of Pharmacy, Bouve College of Health SciencesNortheastern UniversityBostonUSA

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