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

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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.

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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

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ACKNOWLEDGMENTS AND DISCLOSURES

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

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Authors and Affiliations

  1. Pharmaceutical Technology Department, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey

    Selin Seda Timur, Reyhan Neslihan Gürsoy & İmran Vural

  2. Department of Pharmaceutical Sciences, School of Pharmacy, Bouve College of Health Sciences, Northeastern University, 360 Huntington Ave, Boston, Massachusetts, USA

    Prashant Bhattarai & Ban-An Khaw

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  1. Selin Seda Timur
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  2. Prashant Bhattarai
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  3. Reyhan Neslihan Gürsoy
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Correspondence to Ban-An Khaw.

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Timur, S.S., Bhattarai, P., Gürsoy, R.N. et al. Design and In Vitro Evaluation of Bispecific Complexes and Drug Conjugates of Anticancer Peptide, LyP-1 in Human Breast Cancer. Pharm Res 34, 352–364 (2017). https://doi.org/10.1007/s11095-016-2066-2

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  • DOI: https://doi.org/10.1007/s11095-016-2066-2

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