1. Herceptin as a Phase 0 Imaging Example. Phase 0 Trials in Oncology Drug Development Steven M. Larson, M.D. Nuclear Medicine Svc, Department of Radiology MSKCC, NY, NY

2. We are experiencing a paradigm shift From anatomic towards biomarker (molecular imaging) as the primary means of assessing treatment response in Oncology

3. Biomarkers Biomarkers are biologic indicators of disease or therapeutic effects, which can be measured through dynamic imaging tests, as well as tests on blood, tissue and other biologic samples. (Oncology Biomarker Qualification Initiative (OBQI ), a joint enterprise of US FDA, NCI and CMS)

4. PET/CT in Cancer Care: Combines 2 high resolution diagnostic imaging methods Positron Emission Tomography (PET) Biochemistry and Function Computerized Tomography (CT) Anatomy and Structure

5. Discovery ST

6. CT

7. PET-CT

8. Why Now, Why Biomarkers? The search for biomarkers as endpoints of clinical trials to help reduce the growing cost of obtaining new drug approvals Dissatisfaction with the classical anatomic based imaging methods for assessing treatment response assess such as WHO criteria, or RECIST Growing evidence the FDG uptake is an important predictor for aggressive behavior of cancers

9. Oncology Biomarker Qualification Initiative (OBQI) “Biomarkers are indicators of disease or therapeutic effects, which can be measured through dynamic imaging tests, as well as tests on blood, tissue and other biologic samples” NCI,FDA, CMS memorandum of understanding Cooperate closely with industry http://www.fda.gov/oc/mous/domestic/FDA-NCI- CMS.html

10. Potential Benefit of Biomarkers in Clinical Trials Determine if patient’s tumor is likely to respond at all to specific treatments Assess after 1 or 2 treatments if a tumor is dying, even if it is not shrinking in size Determine which patients are at high risk for their patient recurring after surgery Efficiently evaluate whether an experimental therapy is effective for tumor treatment

11. PET FDG as Biomarker Larson SM and Schwartz LH: 18F-FDG PET as a candidate for "qualified biomarker": functional assessment of treatment response in oncology. J Nucl Med. 2006 Jun;47(6):901-3.

12. Imaging Treatment Response: tyrosine kinase inhibitors 18 F-FDG in GIST tumors treated with Glivec, an bcr/abl,c-kit, PDGFR kinase inhibitor

13. 686140 Feb 26, 2001 GI Stromal Tumor (Pre-therapy with STI-571)

14. 686140 June 7, 2001 (CT May 4, 2001) GI Stromal Tumor (Post-therapy STI-571)

15. SUV Activity per unit volume Injected Activity/Body Wt* *also, lean body mass, BSA, etc

16. PET imaging and Tumor Response 920 citations in Medline* Breast Cancer NSCLC Lymphoma Esophageal Gastric Head and Neck Brain Tumor Etc. * Dec 3, 2004

17. Combined Anatomic and Molecular Imaging* * Courtesy of Lawrence Schwartz

18. New Candidate Biomarkers (2) Proliferation Markers. – 18 FLT Amino acid transporter – 18 FACBC – 18 F-tyrosine – 18 F-DOPA Hypoxia and aberrant hif-1 alpha expression – 18 FMISO –CA IX, 124 I-cG250 Growth factor expression –Her 2, 68 Ga-Fab’2 Herceptin* Apoptosis – 18 F-annexin Blood Flow –DCE MRI

19. Exploratory IND Phase 0 testing

20. Exploratory IND* Determine whether a mechanism of action defined in experimental systems can also be observed in humans (a binding property of inhibition of an enzyme) Provide important information on PK Select the most promising compound from a group of candidates designed to interact with a particular therapeutic target in humans based on PK or PD properties *Guidance for Industry, Investigators and Reviewers.. Jan 2006 USFDA URL http://www.fda.gov/cder/guidance/7086fnl.pdf

21. Exploratory IND Studies can be performed at “Microdose” levels, i.e., <1/100 th of a pharmacologic dose, or <100  g, for biologicals, <30 nM Toxicity studies in animals should be 100x the dose in humans administered on a BSA basis –observe for 14 days, weight, chem, heme –Necropsy @ 2 days for histopathology

22. 68 Ga (Fab’ 2 ) Herceptin Image target protein Her 2 receptor, non-invasively in vivo

23. HSP 90 : a target for chemo Critical Chaperone Function e.g., Her 2, AR, AKT

24. Ansamycins bind to the ADP\ATP switch site in Hsp90 O N H MeOMeMe O O MeO OH O O NH 2 H 3 CO Geldanamycin (GM) N H MeOMeMe O O O MeO OH O O NH 2 H N 17-AAG

25. Patient 306 17AAG/Herceptin Phase 1 HER2+ Breast cancer with active sites of disease including the lung and bone. Previously treated with 3 different trastuzumab-containing combinations, progressing on bevacizumab plus trastuzumab prior to enrollment on the trial. Confirmed PR by RECIST. 5 April 20051 June 2005 Modi et al., Abstract 501, ASCO 2006

26. Metastatic Breast Cancer 12/28/043/17/05 A B C A – heart B – left adrenal metastasis C – right kidney Solit, Rosen et al

27. Pharmacodynamics Targeted Therapies: Inhibition of signal transduction

29. Imaging Her-2 Expression 15-20% response in Breast Ca with anti-her2 monoclonal antibody, Herceptin Ansamycins, which target HSP-90, induce Her-2 Degradation

30. Imaging her-2 expression in vivo 68 Ga-(Fab’ 2 ) herceptin Virtual Immunohistology (VIH): Non-invasive imaging of tumor antigen expression in the living organism Smith-Jones et al: Nat Biotech.. 2004 Jun;22(6):701-6.

31. Targeted Radiopharmaceutical Therapy Antibody Forms –Intact Antibody –Fab’2 –Fab –sFv –Diabody (sFv)2 –Minibody IntactAb 150kDa F(ab’) 2 120kDa Minibody (scFv-C H3 ) 2 80kDa V L V H C H 3 scFv 28kDa V L V H Diabody (scFv) 2 55kDa V L V H C H 2 C H1 V L V H C k C H 3 Courtesy Anna Wu, City of Hope MedCtr IntactAb 150kDa F(ab’) 2 120kDa Minibody (scFv-C H3 ) 2 80kDa V L V H C H 3 Minibody (scFv-C H3 ) 2 80kDa V L V H C H 3 scFv 28kDa V L V H scFv 28kDa V L V H Diabody (scFv) 2 55kDa V L V H Diabody (scFv) 2 55kDa V L V H C H 2 C H1 V L V H C k C H 3 Courtesy Anna Wu, City of Hope MedCtr IntactAb 150kDa F(ab’) 2 120kDa Minibody (scFv-C H3 ) 2 80kDa V L V H C H 3 scFv 28kDa V L V H Diabody (scFv) 2 55kDa V L V H C H 2 C H1 V L V H C k C H 3 Courtesy Anna Wu, City of Hope MedCtr IntactAb 150kDa F(ab’) 2 120kDa Minibody (scFv-C H3 ) 2 80kDa V L V H C H 3 Minibody (scFv-C H3 ) 2 80kDa V L V H C H 3 scFv 28kDa V L V H scFv 28kDa V L V H Diabody (scFv) 2 55kDa V L V H Diabody (scFv) 2 55kDa V L V H C H 2 C H1 V L V H C k C H 3 Courtesy Anna Wu, City of Hope MedCtr

32. Quantitation of Antigen In-Vivo with  Labeled Antibodies  Uptake =  [Ag] 68 Ga-(Fab’) 2 anti-p185HER2 (humanized 4DT: Herceptin)

33. Pre Treatment T=0 hours MicroPET images obtained 3 hours post injection with 68 Ga- F(ab) 2 -Herceptin in a mouse with a BT 474 breast tumor Post 17 AAG Treatment T=24 hours

34. Gallium-68 herceptin imaging of her-2

35. Western Blot Analysis of HER2 Protein Expression by Tumors Recovered from Control Animals or Animals Treated with 17-AAG.

36. Effect of 17-AAG on HER2 Protein expression by BT474 Breast Cancer Line K d unchanged B max reduced by 80%

38. Example of 68 Ga-F(ab’) 2 -Herceptin imaging of a CWR22 Prostate Tumor in a Mouse

39. ROI Analysis of 68 Ga-F(ab’) 2 -Herceptin Uptake in a CWR22 Prostate Tumor After 17-AAG Treatment. Formation half-life 1.35 days

40. Peter Smith-Jones, Ph.D. Radiochemist

41. Pt. # 1. Her 2 + Breast Cancer, metastatic to skull PET: Her 2+ Breast CT: Temporal lytic lesion Her 2 imaging: IRB #06-090 Akhurst et al. T T T

42. Tumor Response in Skull

43. Pt. # 5. Her 2 + Breast Cancer, metastatic to liver Her 2 imaging: IRB #06-090 Akhurst et al. T FDG 68 Ga Herscan

44. Dosimetry (Olinda) N=5; 10 mCi of 68 Ga (Fab’2) Herceptin Kidney is the critical organ at 0.41 cGy/mCi Effective dose equivalent 0.063 +/-.006 cGy/mCi

45. 64 Cu-herceptin Carrasquillo et al. IRB#

46. Cu-64 Trastuzumab 45 min post injection CT –liver metastasis 24 h post injection

47. Cu-64 Trastuzumab 24 h Reprojected PET

48. Cu-64 Trastuzumab 24 h CT- T9 & rib lesionAxial PET– no visualization of T9 & rib lesion

49. AR FLT FDG CT FDH T HER-2 68Ga-(Fab’2) herceptin Virtual Immunohistology (VIH): Smith-Jones et al: Nat Biotech.. 2004 Jun;22(6):701 PET: Her 2+ Breast CT: Temporal lysis