Niccolò Ravenni

Modular branched neurotensin peptides for tumor target tracing and receptor-mediated therapy: a proof-of-concept.

The aim of this study was to demonstrate that oligo-branched peptides can be effective either for spotlighting tumor cells that overexpress peptide receptors, or for killing them, simply by exchanging the functional moiety coupled to the conserved receptor-targeting core. Tetra-branched peptides containing neurotensin (NT) sequence are described here as selective targeting agents for human colon, pancreas and prostate cancer. Fluorophore-conjugated peptides were used to measure tumor versus healthy tissue binding in human surgical samples, resulting in validation of neurotensin receptors as highly promising tumor-biomarkers. Drug-armed branched peptides were synthesized with different conjugation methods, resulting in uncleavable adducts or drug-releasing molecules. Cytotoxicity on human cell lines from colon (HT-29), pancreas (PANC-1) or prostate (PC-3) carcinoma indicated branched NT conjugated with MTX and 5-FdU as the most active agents on PANC-1 (EC(50) 4.4e-007 M) and HT-29 (1.1e-007 M), respectively. Tetra-branched NT armed with 5-FdU was used for in vivo experiments in HT-29-xenografted mice and produced a 50% reduction in tumor growth with respect to animals treated with the free drug. An unrelated branched peptide carrying the same drug was completely ineffective. In vitro and in vivo results indicated that branched peptides are valuable tools for tumor selective targeting.

Cancer selectivity of tetrabranched neurotensin peptides is generated by simultaneous binding to sulfated glycosaminoglycans and protein receptors.

In previous papers we demonstrated that tetrabranched peptides containing the sequence of human neurotensin, NT4, are much more selective than native monomeric analogues for binding to different human cancer cells and tissues. We show here that the much higher binding of NT4 peptides, with respect to native neurotensin, to either cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are specifically expressed by different human cancers. We demonstrate that the branched structure provides NT4 with ability to bind heparin and receptors belonging to the low density lipoprotein receptor (LDLR) family, known to be involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides led to identification of a multimeric positively charged motif, which mediates interaction with both heparin and endocytic receptors. Our findings provide the molecular basis for construction of cancer theranostics with high cancer selectivity.

Neurotensin Branched Peptide as a Tumor-Targeting Agent for Human Bladder Cancer

Despite recent advances in multimodal therapy, bladder cancer still ranks ninth in worldwide cancer incidence. New molecules which might improve early diagnosis and therapeutic efficiency for tumors of such high epidemiological impact therefore have very high priority. In the present study, the tetrabranched neurotensin peptide NT4 was conjugated with functional units for cancer-cell imaging or therapy and was tested on bladder cancer cell lines and specimens from bladder cancer surgical resections, in order to evaluate its potential for targeted personalized therapy of bladder cancer. Fluorophore-conjugated NT4 distinguished healthy and cancer tissues with good statistical significance (P < 0.05). NT4 conjugated to methotrexate or gemcitabine was cytotoxic for human bladder cancer cell lines at micromolar concentrations. Their selectivity for bladder cancer tissue and capacity to carry tracers or drugs make NT4 peptides candidate tumor targeting agents for tracing cancer cells and for personalized therapy of human bladder cancer.

Abstract 5625: Targeting different LRP receptors and sulfated proteoglycan by branched neurotensin provide high cancer selectivity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In previous paper we reported on the much higher selectivity toward cancer cells and tissues of tetra-branched neurotensin peptides (NT4) compared to monomeric NT peptide. We also demonstrated that NT4 can be coupled to many different functional units for cancer cell tracing and drug delivery and can induce tumor growth reduction in animal studies. We then proposed NT4 as promising cancer selective theranostics for different human cancers, including CRC, pancreas adenocarcinoma and urinary bladder cancer. Nonetheless, multimeric binding of tetrabranched peptides, together with the chemical modification produced by coupling to the branched core, might have modified receptor selectivity of NT4 with respect to native monomeric NT and actually we had no conclusive indication on which receptor our branched NT4 peptides were binding to. Data reported in the present paper demonstrate that synthesis of neurotensin sequence in a tetra-branched form induce a switching of receptor selectivity, by decreasing affinity to the NT high affinity receptor NTR1 and contemporarily acquiring binding to additional receptors, which produces a much higher cancer cell selectivity of NT4 with respect to monomeric NT peptides. We demonstrate here that NT4 binds sortilin and SorLa and also acquire the ability to bind different receptors belonging to the Low Density Lipoprotein Receptor Related Protein (LRP) family as well as heparin and other Heparan Sulfate Proteoglycans (HSPG). The much higher binding of NT4 in respect to native NT to either cancel cell lines or human cancer surgical samples, as well as the higher selectivity toward human cancer tissues of NT4 is due to binding to different membrane receptors, which are very selectively expressed by many different human cancers. Moreover, our results confirm that sulfated proteoglycan can mimic the ligand binding site of different LRP receptors and indicate that targeting of multiple LRP receptors together with sulfated proteoglycans produce an extremely high selectivity towards many different human cancers. Citation Format: Luisa Bracci, Chiara Falciani, Jlenia Brunetti, Barbara Lelli, Niccolo Ravenni, Luisa Lozzi, Lorenzo Depau, Alessandro Pini. Targeting different LRP receptors and sulfated proteoglycan by branched neurotensin provide high cancer selectivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5625. doi:10.1158/1538-7445.AM2013-5625

Abstract A20: Tumor selective delivery of chemotherapeutics via branched peptides

Oligo-branched peptides, containing the sequence of the human regulatory peptide neurotensin (NT), have been used as specific tumor targeting agents, able to selectively and specifically deliver effector units for cell imaging or killing, to tumor cells that over-express NT receptors. Tetra-branched peptides containing neurotensin (NT) sequence are described here as selective targeting agents for human colon, pancreas and prostate cancer. Fluorophore-conjugated peptides were used to measure tumor versus healthy tissue binding in human surgical samples, resulting in validation of neurotensin receptors as highly promising tumor-biomarkers. Drug-armed branched peptides were synthesized with different conjugation methods, resulting in uncleavable adducts or drug-releasing molecules. Human cell lines from colon (HT-29), pancreas (PANC-1) or prostate (PC-3) carcinoma were challenged with branched NT conjugated with 6-mercaptopurin, combretastain A-4, monastrol and 5-fluoro-deoxyuridine. Results indicated that branched NT conjugated with combretastain A-4 and 5-fluoro-deoxyuridine are the most active agents on HT-29 (EC50 1.1e-007 M) and PANC-1 (EC50 5.0e-007 M) respectively. Tetra-branched NT armed with 5-FdU was used for in vivo experiments in HT-29-xenografted mice and produced a 50% reduction in tumor growth with respect to animals treated with the free drug. An unrelated branched peptide carrying the same drug was completely ineffective. In vitro and in vivo results indicated that branched peptides are valuable tools for tumor selective targeting. The results reported in this presentation tell that branched-armed peptides are very promising pharmacodelivery options. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A20

Abstract 2582: Branched neurotensin peptides for the selective targeting of human colon and pancreas carcinoma

The aim of this study was to validate oligo-branched peptides as selective targeting agents that might be effective either for spotlighting tumor cells that over-express peptide receptors, or for killing them, simply by exchanging the functional moiety coupled to the conserved receptor-targeting core. Tetra-branched peptides containing neurotensin (NT) sequence are described here for selective targeting of human colon, pancreas and prostate cancer. Fluorophore-conjugated peptides were used to measure tumor versus healthy tissue binding in human surgical samples, resulting in validation of neurotensin receptors as highly promising tumor-biomarkers. Drug-armed branched peptides were synthesized with different conjugation methods, resulting in uncleavable adducts or drug-releasing molecules. Cytotoxicity on human cell lines from colon (HT-29), pancreas (PANC-1) or prostate (PC-3) carcinoma indicated branched NT conjugated with MTX and 5-FdU as the most active agents on PANC-1 (EC50 4.4e-007 M) and HT-29 (1.1e-007 M), respectively. Tetra-branched NT armed with 5-FdU was used for in vivo experiments in HT-29-xenografted mice and produced a 50% reduction in tumor growth with respect to animals treated with the same amount of free drug. An unrelated branched peptide carrying the same drug was completely ineffective. In vitro and in vivo results indicated that branched peptides are valuable new tools for tumor selective targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2582.