Membrane-anchored b -glucuronidase

1. Membrane-anchored b-glucuronidase Steve Roffler Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan

2. Glucuronide probe or drug glucuronide glucuronide glucuronide glucuronide ß-glucuronidase B7 TM Transgene Expression MEMzyme Transgene Memzyme activation of imaging or therapeutic agents Potential advantage: Signal amplification due to enzymatic hydrolysis of glucuronide probes or drugs

3. Potential advantages of a ßG reporter ßG in lysosomes – inaccessible to glucuronides Low serum ßG – little systemic activation Low toxicity Fast renal clearance – rapid imaging Endogenous - low immunogenicity Many possible glucuronide probes and drugs

4. In vitro imaging of gene expression red = enzyme expression green = activated probe accumulation Fluorescent probe can be activated and accumulate at cells that express enzyme

5. Imaging of adenovirus memzyme expression Kodak IS2000MM Sites of Ad transgene expression could be imaged

6. In vitro activation of HAMG HAMG Glucuronide prodrug pHAM Active drug COOH O Cl HO Cl O N HO HO N Cl OH Cl HAMG can be activated at EJ/mßG cells

7. HAMG prodrug toxicity to mice HAMG is well tolerated by mice

8. Prodrug therapy of EJ tumors (150 mg/kg HAMG on days 15 and 19) 2000 EJ PBS EJ HAMG EJ/mßG PBS EJ/mßG HAMG Tumor volume (mm3) 1000 0 0 10 20 30 40 50 Days HAMG displayed selective activity against EJ/mßG tumors

9. Summary Surface enzyme allow both imaging and therapy Immunogenicity is low but selectivity is high May possess advantages for cancer gene therapy

10. Surface expression Dr. Tian-Lu Cheng Kaohsiung Medical University T cell activation Tang-Bi Liu Shih-en Chang Wen-Jun Chung Chin-Chuan Chen Pei-Yu Wu Dr. Hsin-Ell Wang National Yang Ming University Yi-Hsuan Chiang Chien-I Su Dr. Yu-Ling Leu Chia-Nan College of Pharmacy and Science Surface scFv Dr. Kuang-Wen Liao Shey-Cherng Tzo Dr. Ji-Wang Chern National Taiwan University Bing-Mae Chen http://www.ibms.sinica.edu.tw/~sroff/sr_refs.html