Design and In Vitro Evaluation of Bispecific Complexes and Drug Conjugates of Anticancer Peptide, LyP-1 in Human Breast Cancer
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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.
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.
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).
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 WORDSpeptide-antibody bispecific complexes polymer pro-drug conjugates pre-targeted drug targeting
Doxorubicin conjugated N-terminal DTPA conjugated PGA
LyP-1 conjugated to Dox-DSPL
LyP-1 conjugated with doxorubicin hydrochloride
N-terminal DTPA conjugated PGA
DTPA conjugated poly-L-lysine conjugated with LyP-1 and Dox
RITC conjugated DPL
LyP-1 conjugated to DSPL
DTPA-succinyl rhodamine conjugated poly-L-lysine
DTPA conjugated bovine serum albumin
Goat anti-mouse IgG antibody conjugated with HRP
9 amino acid peptide ligand specific for mitochondrial membrane receptor p32.
LyP-1 conjugated anti-DTPA bispecific antibody complex
Molecular weight cut-off
5/6-carboxyfluorescein succinimidyl ester
Polymer drug conjugates
Rhodamine B isothiocyanate
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Therapeutic monoclonal antibodies
Tri-nitro benzene sulfonic acid
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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