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BACKGROUND. SEL24/MEN1703 is a first-in-class, orally available, dual PIM/FLT3 kinase inhibitor investigated in unselected AML patients in the First-in-Human, Dose Escalation (DE) and Cohort Expansion (CE) CLI24-001 trial (DIAMOND-01, ClinicalTrials.gov identifier: NCT03008187). The study has completed both the DE and CE part showing an acceptable safety profile up to the recommended dose (RD), with initial evidence of single agent efficacy particularly clustering in patients with IDH mutant disease. Previous studies conducted on a limited number of patients in the DE cohort indicated pS6, a downstream target in the PIM/FLT3 signaling pathway, as a biomarker of target engagement. AIM. To assess the relevance of pS6 as a pharmacodynamic (PD) biomarker in SEL24/MEN1703-treated patients. Moreover, patients were characterized on the basis of genetic profile to identify mutations which might cluster in patients' samples that demonstrated significant target engagement. Finally, longitudinal assessment of Variant Allelic Frequency (VAF) has been implemented. METHODS. S6 phosphorylation has been longitudinally monitored in the DIAMOND-01 study through an optimized assay for multiparametric analysis of phospho-protein activation. The assay allows a quantitative assessment of pS6 at the single cell level among blast cells as well as the identification of blast subpopulations in peripheral blood (PB). CD34+ Blast cells % was monitored to assess whether target engagement translated into blast reduction. Genotype analysis was performed by NGS using the Oncomine Myeloid Research Assay. RESULTS. PB samples from DE and CE patients, treated at 125 mg (RD) were analyzed, for a total of n= 27 assessable patients. At screening, we observed a heterogeneous positivity for pS6 marker in blast cells (range: 0.9%-79%), consistent with the unselected AML patient population recruited in the trial. Overall, 14/27 (51.8%) patients showed pS6 inhibition in blast cells at the end of Cycle 1 in comparison with screening (range: 70%-94%) (Figure 1). When SEL24/MEN1703 treatment continued beyond Cycle 1 (n=11 patients), pS6 inhibition was maintained and long-lasting in 6/11 (54.5%) patients. While pS6 inhibition did not translate into CD34+ blast decrease in all patients, long-lasting pS6 inhibition was observed in 2 out of 3 responding patients (including CRi as best response) evaluable for pS6 levels. Genetic characterization of these two patients treated at 125 mg (RD) showed that they harbored IDH mutations. CONCLUSIONS. The longitudinal PD assessment through the modulation of pS6 activation by flow cytometry confirmed that meaningful target engagement was achieved in more than 50% of patients treated with SEL24/MEN1703 at the RD. In most patients, including 2 out of 3 responders evaluable for pS6, its inhibition was maintained during the entire treatment. Preliminary genotyping showed that responding patients with strong pS6 inhibition harbored IDH mutations. In-depth analysis of genomic profiling and of the longitudinal assessment of VAF is ongoing in order to identify potential correlation with pS6 modulation and blast depletion in specific AML subsets, with the aim to define patient population(s) more likely to respond to SEL24/MEN1703. Figure 1. pS6 (%pS6*MFI) assessment in peripheral blast cells at screening and at C1D14-4H in CE patients treated at RD of 125 mg. Figure 1 Figure 1. Disclosures Paoli: Menarini Group: Current Employment. Bellarosa: Menarini Group: Current Employment. Bressan: Menarini Group: Current Employment. Bertolini: Gilead: Research Funding; Emercell: Research Funding. Montesinos: Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline/Menarini: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Glycomimetics: Consultancy; Tolero Pharmaceutical: Consultancy; Agios: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Mukherjee: Eusa Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Teaching and Speaking; McGraw Hill: Honoraria, Other: Editor of Hematology Oncology Board Review (ongoing); Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research/Independent Contractor, Research Funding; AAMDS in Joint Partnership with Cleveland Clinic Taussig Cancer Institute: Honoraria; Genentech: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; BioPharm: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research/Independent Contractor, Research Funding; Bristol-Myers Squibb Co.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Acceleron: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Partnership for Health Analytic Research: Honoraria. Strickland: AbbVie, ArcherDx, Genentech, Incyte, Kura Oncology, Novartis, Pfizer, and Syros: Consultancy; Sunesis: Research Funding. Santoro: Takeda: Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Arqule: Consultancy, Speakers Bureau; Roche: Speakers Bureau; AbbVie: Speakers Bureau; Eli-Lilly: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Speakers Bureau; Sandoz: Speakers Bureau; Eisai: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Amgen: Speakers Bureau; Novartis: Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Walter: Jazz: Research Funding; Pfizer: Consultancy, Research Funding; Selvita: Research Funding; Amphivena: Consultancy, Other: ownership interests; Agios: Consultancy; Astellas: Consultancy; BMS: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Kite: Consultancy; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding. Fazal: Taiho: Consultancy, Honoraria, Speakers Bureau; Stemline: Consultancy, Honoraria, Speakers Bureau; Sanofi Genzyme: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Agios: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Tolero: Consultancy. Ravandi: Novartis: Honoraria; AstraZeneca: Honoraria; Xencor: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Prelude: Research Funding; Agios: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Brzozka: Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment; Selvita SA: Current Employment. Baldini: Menarini Group: Current Employment. Ravenni: Menarini Group: Current Employment. Binaschi: Menarini Group: Current Employment. Laurent: Menarini Group: Current Employment. Pellacani: Menarini Group: Current Employment.

Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here we describe the isolation affinity maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (Kd) of 10 and 30 nM respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis FACS and immunofluorescence studies using ovarian cancer cell lines and freshly frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors following intravenous administration. Anti PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products armed with cytotoxic drugs radionuclides or cytokines as payloads. © 2014 Landes Bioscience.

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 significanceP<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.

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.

Bladder cancer (BC) ranks ninth in worldwide cancer incidence. It is the seventh most common cancer in men and the 17th most common cancer in women, with more than 330,000 new cases and more than 1...

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.

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, Niccolò 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

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.

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