1. Franz Buchegger, David Viertl, Thierry Stora, Léo Bühler, Helmut R. Maecke, Marek Kosinski, Sébastien Baechler, Raymond Miralbell, John O. Prior and Rosalba Mansi Departments of Nuclear Medicine, Radio-Oncology, Institute of Radiation Physics and Surgical Research Unit, Lausanne and Geneva University Hospitals, European Organization for Nuclear Research, CERN Medicis, Department of Organic Chemistry, Vrije Universiteit Brussels and Department of Nuclear Medicine, University Hospital of Freiburg CERN Medicis - Oct 2014 Targeting receptors of neurotensin and bombesin

2. 1. 68 Ga-DOTA-octreotate (collaboration w. SWAN, Bern), neuroendocrine tumors. 2. 68 Ga-NODAGA-RGDyK, targeting  v  3 integrin, (neovascularization, diverse tumors, atheromatous lesions). 3. 68 Ga-NODAGA-MJ9, bombesin analog with antagonist activity, selected by Prof H.R. Maecke, Freiburg, DE, and Dr R. Mansi, Basel. Study in prostate cancer patients (open) and breast cancer patients (in preparation). 4.Different Neurotensine analogs conjugated to NODAGA/DOTA chelators are studied in vivo in collaboration with Prof D. Tourwé, Bruxelles.

3. Neurotensin and bombesin ligand-receptor systems evolved from a common ancestor The neurotensin and GRP ligand /receptor systems are both G-protein coupled, show similar distributions in the central nervous system and in gastrointestinal tissues and exhibit similar functionalities. Neurotensin (NT) has been shown to induce EGFR-, c-Src- and Stat5b-dependent proliferation of prostate cancer PC-3, while a commercial NT antagonist SR-48692 (Sanofi Research) tested on the breast cancer cell line MDA-MB-231 inhibited its growth. SR-48692 is a radiosensitizer (shown on PC-3 tumor grafts). Bombesin promotes breast cancer cells MDA-MB-231. Bombesin antagonist RC-3095 and RC-3940-II (peptides) inhibit growth of MDA-MB231.

4. « Structure of the agonist-bound neurotensin receptor ». Jim F. White et al N AT U R E ; V O L 4 9 0 ; 2 5 O C T O B E R 2 0 1 2 T. Nakagawa et al. Biochem Pharmacol 69 (2005) 579–593

5. We previously performed a SPECT imaging study with 99m Tc-tricarbonyl-NT-XI in patients scheduled for surgery of ductal pancreatic adenocarcinoma Buchegger et al, J Nucl Med 2003; 44:1649–1654 Tissue activities expressed in %ID/g x10 4, corrected for physical half-life of isotope. Numbers in parentheses represent tumor-to- normal tissue radioactivity ratios.

6. 99m Tc-NT -XI (N  His)Ac-Lys-  (CH 2 NH)-Arg-Pro-Tyr-Tle-Leu 99m Tc-NT -XIX (N  His)Ac-Arg-(NMe)Arg-Pro-Dmt-Tle-Leu V Maes, E Garcia-Garayoa, P Bläuenstein and D Tourwé: J. Med. Chem. 2006, 49, 1833-1836 DOTA-NT-XIX DOTA-Arg-(NMe)Arg-Pro-Dmt-Tle-Leu √ NODAGA-NT20.3 Ac-Lys(NODAGA)-Pro-(NMe)Arg-Arg-Pro-Tyr-Tle-Leu √ DOTA-NT20.3 Ac-Lys(DOTA)-Pro-(NMe)Arg-Arg-Pro-Tyr-Tle-Leu √ DOTA-NT20.3- (modified) Ac-Lys(DOTA)-Pro-(NMe)Arg-Arg-Pro-Tyr-Ile-Leu √ NODAGA-PEG-NT-X NODAGA-PEG-Arg-Arg-Pro-Tyr-Tle-Leu √ NODAGA-PEG-NT-X’ NODAGA-PEG-Arg-Arg-Pro-Dmt-Tle-Leu √ NODAGA-PEG-NT-XII NODAGA-PEG-Arg-(NMe)Arg-Pro-Tyr-Tle-Leu √ (NODAGA-PEG-NT-X’’ ) (NODAGA-PEG-Arg-  (CH 2 NH)-Arg-Pro-Tyr-Tle-Leu ) Sequence of natural NT and NT(8-13) Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu Different NT-chelate conjugates have been studied compared here with previously selected 99m Tc-labeled peptides of neurotensin

7. 68 Ga-NODAGA-NT20.3 Analog to the INSERM : DOTA-NT20.3 (patent) Interesting uptake was observed in reference tumor HT-29 and in co-transplanted prostate cancer PC-3 as well as in normal intestines compared with the 99m Tc-NT-XIX or the 111 In-DOTA-NT20.3 (published by INSERM) and 68 Ga-DOTA-NT20.3 (our results)

8. 68 Ga-NODAGA-PEG-NT-X Looked good, has been prepared in GMP quality ! PA= Phosphoramidon (enzyme inhibitor; B.A. Nock et al JNM 2014;55:121-7)

9. Summary on NT analogs -NODAGA-NT20.3, analog to the French INSERM DOTA-NT20.3 (patented by INSERM) showed good tumor uptake. -NODAGA-PEG-NT-X and derivatives with single further modifications (NT-X’, NT- XII) gave high tumor-to-normal tissue ratios. With 68 Ga-NODAGA-PEG-NT-X we have made a preclinical dosimetry and a tolerance study in mice and produced it in GMP quality (ready for a clinical study). -Prostate cancer PC-3 expresses both bombesin and neurotensin receptors in high amount. -Pancreas tumors Capan-2 and Mia-PaCa-2 will be tested as new targets expressing NT receptors (transferred from Douglas Hanahan’s lab). -Glioblastoma lines LN229, U-87, U-251 are other potential targets of neurotensin

10. Ligands of gastrin releasing peptide (GRP) have been developed as analogs of bombesin. Bombesin was first isolated from skin of the frog “bombina”. Bombesin is very similar to the human GRP. In human, a strong expression of GRP receptors was found in the central nervous system and neuroendocrin gastrointestinal tissues and cancer, particularly in prostate and breast cancer (studies by Prof. J.C. Reubi, US and Bern). Ligands of gastrin releasing peptide (GRP) have been developed as analogs of bombesin. Bombesin was first isolated from skin of the frog “bombina”. Bombesin is very similar to the human GRP. In human, a strong expression of GRP receptors was found in the central nervous system and neuroendocrin gastrointestinal tissues and cancer, particularly in prostate and breast cancer (studies by Prof. J.C. Reubi, US and Bern). A comparative PET/CT study on 18 F-choline and 11 C-acetate has been performed at HUG on 35 patients. A first analysis in 23 patients has been published. It showed a similar behavior of both tracers. A comparative PET/CT study on 18 F-choline and 11 C-acetate has been performed at HUG on 35 patients. A first analysis in 23 patients has been published. It showed a similar behavior of both tracers. A PET/CT study on evolution of tumor hypoxia under radiation therapy of prostate cancer shown by F-MISO is open at HUG. (comparison with F-Choline or 11 C-acétate). A PET/CT study on evolution of tumor hypoxia under radiation therapy of prostate cancer shown by F-MISO is open at HUG. (comparison with F-Choline or 11 C-acétate).

11. Bombesin is a peptide of 14 amino acids while GRP is composed of 27 amino acids. Bombesin is a peptide of 14 amino acids while GRP is composed of 27 amino acids. GRP receptors (GRPr) and its ligand GRP, are frequently co-expressed by the same cells (autocrine loop), notably in prostate, breast and digestif cancers. GRP receptors (GRPr) and its ligand GRP, are frequently co-expressed by the same cells (autocrine loop), notably in prostate, breast and digestif cancers. GRPr belongs together with NMBr (neuromedin receptor), BRS-3, NTr1 and NTR2 to the family of G-protein coupled receptors. GRPr belongs together with NMBr (neuromedin receptor), BRS-3, NTr1 and NTR2 to the family of G-protein coupled receptors. The fragment NTS 8-13 of neurotensin bound to the receptor NTr1 has been resolved by crystallography. The fragment NTS 8-13 of neurotensin bound to the receptor NTr1 has been resolved by crystallography. Similar to NTr1, GRPr has 7 transmembrane domains. The 3 extracellular loops of GRPr form the ligand binding site. Similar to NTr1, GRPr has 7 transmembrane domains. The 3 extracellular loops of GRPr form the ligand binding site. GRP and GRP receptors and bombesin analogs with agonist and antagonist activity - the similarity with neurotensin GRP : Val-Pro-Leu-Pro-Ala-Gly-G ​ ly-Gly-Thr-Val-Leu-Thr-Ly ​ s-Met-Tyr-Pro-Arg-Gly-Asn ​ -His-Trp-Ala-Val-Gly-His- ​ Leu-Met Bombésine : Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met GRP antagonist : D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu DOTA-RM6 : DOTA-PEG 4 -(4-amino-1-carboxymethyl-piperidine)-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu NODAGA-MJ9 NODAGA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu Pyr= pyroglutamyl; Sta=statine

14. Two small PC-3 tumors/mouse (grown over 3 weeks), male SCID mice, DOTA-RM6 as used for Tb-152, 3 mice per group

16. Relative organ contributions to the human effective dose (ED) according to Olinda; The ED predicted from mice was confirmed in 5 male patients with less than 10 % difference. 38.2  Sv/MBq 28.3  Sv/MBq Interval of bladder void : 1 h 0.5 h

18. %ID/g PC-3 tumor10.52 Adrenals0.57 Pancreas0.60 Liver0.72 Kidneys1.29 Lung0.13 Spleen (93 mg)5.61 Heart0.02 Muscle0.01 Bone0.04 Skin0.08 Stomach0.08 Small Intest0.10 Large Intest1.49 Blood0.11 Bladder0.15 SPECT/CT (clinical imaging device) Biodistribution after injection of 8.25 MBq 177 Lu-DOTA-RM6

20. The CHUV Nuclear Medicine Albira PET/SPECT/CT jointly funded by FNS and UNIL should be operational in 2 to 3 months.

21. The clinical study of dual PET/CT foreseen for 60 prostate cancer patients at either initial presentation or recurrence comparing 68 Ga-NODAGA-MJ9 with 18 F-flurocholine, has confirmed the dosimetry projection extrapolated from mice and is currently generally open for inclusion. The clinical study of dual PET/CT foreseen for 60 prostate cancer patients at either initial presentation or recurrence comparing 68 Ga-NODAGA-MJ9 with 18 F-flurocholine, has confirmed the dosimetry projection extrapolated from mice and is currently generally open for inclusion. MJ9 exhibiting antagonist activity was predicted not to provoke side effects such as gastrin release or cellular growth. No side effects were observed in our first 8 patients. MJ9 exhibiting antagonist activity was predicted not to provoke side effects such as gastrin release or cellular growth. No side effects were observed in our first 8 patients. DOTA-RM6 can be radiolabeled with positron emitters for PET such as 152 Tb or 68 Ga, electron emitters for therapy such as 161 Tb or 177 Lu, or the  -particle emitter 149 Tb (the latter co-emitting positrons for PET). DOTA-RM6 radiolabeled with these different radioisotopes should allow approaching therapy studies under well controlled conditions searching for curative approaches. DOTA-RM6 can be radiolabeled with positron emitters for PET such as 152 Tb or 68 Ga, electron emitters for therapy such as 161 Tb or 177 Lu, or the  -particle emitter 149 Tb (the latter co-emitting positrons for PET). DOTA-RM6 radiolabeled with these different radioisotopes should allow approaching therapy studies under well controlled conditions searching for curative approaches. Our first experiments showed that external beam radiation therapy (RT) combines favorably with systemic radiation therapy with DOTA-bombesin labeled with an electron emitter (PRRT). Our first experiments showed that external beam radiation therapy (RT) combines favorably with systemic radiation therapy with DOTA-bombesin labeled with an electron emitter (PRRT). Bombesin in conjunction with neurotensin and neurotensin receptor expression of PC-3 cells can allow double receptor targeting with both peptide analogs for a co-operative therapy study (the neurotensin antagonist SR48692 has been shown to be a radiosensitizer on PC-3 tumors (Cancer Research) or could be used as unlabeled growth inhibitor) or to use neurotensin analogs for imaging independent of the therapy with high amounts of bombesin conjugates. Repeat imaging under therapy requires an on-site micro-PET/SPECT/CT. It will be installed at CHUV this month. Bombesin in conjunction with neurotensin and neurotensin receptor expression of PC-3 cells can allow double receptor targeting with both peptide analogs for a co-operative therapy study (the neurotensin antagonist SR48692 has been shown to be a radiosensitizer on PC-3 tumors (Cancer Research) or could be used as unlabeled growth inhibitor) or to use neurotensin analogs for imaging independent of the therapy with high amounts of bombesin conjugates. Repeat imaging under therapy requires an on-site micro-PET/SPECT/CT. It will be installed at CHUV this month.

22. Thanks for support to : Fondation pour les recherches médicales et biologiques sur le cancer, Genève. Cellex Foundation, Barcelona (Clinical study support). Dr Catherine Servis, Protein and Peptide Chemistry Facility, Department of Biochemistry, UNIL, for the collaboration in peptide design and production. and a great thanks you for your attention !