Spin-off dell'Università di Milano speranze e successi

1. S. Di Giovine Business Development Director Spin-off dell'Università di Milano speranze e successi 29 Novembre 2011 Università degli Studi di Milano Via Golgi 19 aula G10

2. Corporate profile TOP srl, originated in 2006 as a spin-off of the Universityof Milan and has been backed in 2007 by the NEXT fund managed by FinlombardaGestioni SGR. TOP islocated at the Parco Tecnologico Padano in Lodi, near Milan. TOP research team isspecialized in the generation ofvectors, cells and animalsforMolecularImagingusing: • Bioluminescence • PET (PositronEmissionTomography) • NMR (NuclearMagneticResonance)

3. TOP mission • Toprovide the scientific community withnon-invasivetoolsfor the studyof the activityofdrugs and environmental or foodcontaminants • TOP aims at combining the powerofphysics, informatics and molecularbiologyto generate novel models for the bio-medical research and for the control of the environment and alimentary chain. • TOP strives to renew present investigational tools in the respect of animal rights to satisfy two conflicting needs: use of predictive biological models in the absence of animal distress.

4. Ligands Ligand Ligands NR NR NR NR NR NR Reporter green fluorescent dye The Company expertise: MolecularImaging Molecular imaging is based on the appropriate use of REPORTERS: molecules easily detectable and allowing to study spatio-temporally molecular events in living organisms.

5. The Company unique and proprietarytechnology*: the useofinsulatorsequencestoobtaingeneralizedexpressionof the reporter CCD Camera *EU patent No.EP 1298988B1 US patent No. 7,943,815

6. All TOP products are subjectedto intense validationtoensure: constitutive/ inducible PREVENTION OF EPIGENETIC MODIFICATIONS REPRODUCIBLE and REGULATED EXPRESSION OF THE REPORTER REPORTER EXPRESSION MAINTAINED IN TIME AND GENERATIONS ACCURACY SPECIFICITY SATURN VALIDATION TURN-OVER UBIQUITY REPRODUCIBILITY NON-INVASIVENESS

7. E 19.5 E 18.5 18.5 E 17.5 16.5 E 16.5 15.5 E 14.5 E 12.5 TOP productsenabletostudyyour favorite molecularprocessalong the whole life cycle P.N. 1 P.N. 10 DAY 14.5 14.5 3 months Embryonic Dev. & Pregnancy Adulthood 9

8. TOP finds a responseto a varietyofresearchneeds • Pharma: • identification of novel drug targets • speeding of pre-clinical testing • identification of drugs endowed of tissue specificity of action • more predictable toxicological tests • gender pharmacology • pharmacology of infancy and aging • new indications for drugs in the clinic or in the market • Environmental & Food toxicology: • identification of specific activities in mixtures of compounds • threshold effects in short and long term exposures • identification of food contaminants • threshold effects in short and long term exposures • identification of the organs targeted by environmental pollutants • Biomedical research: • understanding the physiological effects of specific molecular events

9. One single tool for the multidimensional analysis of drugs in vivo. • Immediate readout; • Increased prediction power: • - holisticvs. reductionistapproach; • - drug action, not only distribution. • Increased ability to perform multidimensional analysis of drug activity: • - space (organs, PD); • - time (PK, longitudinal studies); • - immediate evidence of undesired activity • (toxicology). • Lower costs; • Decreased research time; • Significantly reduced number of animals. TOP Reporter system

10. Partnership • TOP srl is partner of Caliper Life Sciences (former Xenogen) , the world leader for in vivo optical imaging.

11. TOP reporter models - I • repTOP™ mice available at present:

12. TOP reporter models - II • repTOP™ mice soon available In addition, TOP has generated a vast array of constructs for cell and animal engineering ready to be used and addressing most of current major needs in biological research. All of these constructs contain two or more reporters enabling bimodal and trimodal applications of molecular imaging

13. TOP reporter models - III • Custom- maderepTOP™mice: TOP’s technology platform and the standardized validation process are the guarantee of a rapid and reliable generation of an industry standard-compliant reporter system. Validation Generation Characterization Feasibility 4-5 weeks 15 -18 weeks Founder mouse

14. OUTSOURCING YOUR RESEARCH AT TOP • The expertise of TOP research team in molecular imaging is at our client’s service for advise and testing of compounds in a wide variety of therapeutic areas

15. TOP mice in the world • USA: • University of Kansas Medical Center (KUMC) • University of Miami, University of Chicago • University of Illinois • UT Southwestern Medical Center at Dallas • University of Pennsylvania School of Medicine • University of Tennessee • Dana-Faber Cancer Institute • Baylor College of Medicine. • Leading Pharmaceutical and Biotechnology companies worldwide. • Italy: • Spedali Civili, Brescia • Roma, UniversityTor Vergata • Europe: • Institut de Genetiqueet de Biologie Moleculaireet Cellulare (IGBMC) • KarolinskaInstitute • Max PlanckInstitutfuerPsychiatrieMuenchen • DartmouthMedicalSchool • LeidenUniversityMedical Center. • Australia: • Prince Henry’s Institute of Medical Research

16. TOP newest product:repTOPTMmitoIRE to study proliferation in vivo

17. The repTOPTMmitoIRE mouse tostudyproliferation in vivo Anti – proliferative stimuli 1 u CCD Camera Pro–proliferativestimuli NF-Y promoter Cyclin B2 minimal promoter

18. Photonemission (p/s/cm2/sr) repTOPTMmitoIREvalidation cellproliferationinducedbyapplicationof7,12-dimethylbenz(a)anthraceneto the skinof the leftear can beeasilymeasuredin vivo Time (days)

19. Imaging Ki-67 immunoistochemistry d3 d9 d3 d9 vehicle vehicle Bioluminescence: low background and rapid detection DMBA DMBA Cell Proliferation (p/s/cm2/sr) Cell Proliferation (pos nuclei/tot nuclei) COMPARING the sensitivityof Ki-67 immunohistochemistry and bioluminescence in the MITO mouse

20. 200 100 12 Photon emission (cts/sx103) 4 1 0 repTOPTMmitoIRE following in time the development of chemically induce prostate carcinoma

21. Combining repTOPTM and VivoGloTM technologies repTOP™ Ubi-Luc2-M8 mouse Z-DEVD-Aminoluciferin, Sodium Salt Induction of liver apoptosis 6h i.p. treatment with: D-GalN (800mg/kg) + LPS (100 µg/kg ) Nakama T et al. Hepatology. 2001