1. Membrane-bound antibodies for therapy and imaging Steve Roffler Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan

2. Immune cell regulation Prodrug Activation Gene expression imaging Localized cytokine expression Surface expression of biologically-active proteins Metabolic regulation cytokine antibody enzyme TMLS

3. scFv Single-chain antibody receptors Liao et al., Biotechnology & Bioengineering (2001) 73, 313-323

4. Gilliland et al., Tissue Antigens. 1996 47:1-20 Making a scFv from a hybridoma

5. http://www.ibms.sinica.edu.tw/~sroff/protocols/scFv.htm Making a scFV from a hybridoma II

6. pcDNA3 2C11-PDGFR Expression of scFv with commercial pHook-1 vector

7. Enhancing surface expression transmembrane domains

8.  1/2 12.23.8 2.4 1.6 h B7-1 DAF ASGPR PDGFR AFP-B7 is more stable on the cell surface Surface expression: B7 > DAF >> PDGFR = ASGPR Summary of AFP-TM results AFP-B7 is most rapidly transported to the cell surface cyt Import to ERProtein of interestAnchor in plasma membrane Epitope tag B7 TM is good for surface expression B7-1 CD80 DAF decay- accelerating factor ASGPR asialoglycoprotein receptor PDGFRplatelet-derived growth factor receptor

9. Linker domains increase surface expression Effect of linker domains on scFv receptor expression Liao et al., Cancer Gene Therapy (2003) 10: 779-790

10. IP of culture medium Glycosylation controls surface shedding Carbohydrate chains reduce shedding and enhance surface expression protein expression surface expression GPI anchor glycosylated no glycosylation

11. Activation of T cells by surface anti-CD3 scFv Defects or down-regulation TAP-1 TAP-2 Beta 2-microglobulin MHC class I heavy chain proteosome subunits

12. Early events in T-cell activation Direct CD3 ligation can initiate T cell activation

13. T cells can bind to cells that express anti-CD3 scFv

14. . Anti-CD3 scFv activity anti-CD3 scFv can induce CTL activity anti-CD3 scFv with CD86 stimulates IL-2 secretion 0 20000 40000 60000 Day 12345 anti-phOx anti-CD3 anti-CD3 / CD86 IL-2 concentration (pg/mL) no stimulator cells. 0 25 50 75 100 Cytotoxicity (% control) anti-CD3 anti-CD3 + CD86 anti-phOx anti-phOx + CD86 Anti-CD3 scFv is active in vitro

15. B16-F1 010203040 0 1000 2000 Days Tumor size (mm 3 ) 0/6  CD3 010203040 0 1000 2000 Days 0/7 phOx (control scFv) 010203040 0 1000 2000 Days 0/6 phOx + CD86 010203040 0 1000 2000 Days Tumor size (mm 3 ) 0/5 anti-CD3 scFv with CD86 prevented growth of poorly immunogenic tumors in 45% of mice Tumor sizes in mice injected with B16-F1 transfectants

16. Development of systemic anti-tumor immunity in tumor-free mice Long-term protective immunity was established by anti-CD3 and CD86 Original tumor B16/F1 rechallenge (tumor-free/total) B16/  CD3 + CD86 4/4 naive0/4

17. anti-CD28 scFv receptor scFv (  CD28) TM (B7) Linker(eB7) Is anti-CD28 scFv better than CD86? Can bind CD28 but not CTLA-4

18. Tumor sizes after adenoviral therapy anti-CD3 with anti-CD28 delayed tumor growth 0102030 0 500 1000 1500 2000 Days Tumor size (mm 3 ) 0102030 0 500 1000 1500 2000 Days 0102030 0 500 1000 1500 2000 Days 0102030 0 500 1000 1500 2000 Days 0102030 0 500 1000 1500 2000 Days Tumor size (mm 3 ) 0102030 0 500 1000 1500 2000 Days 0102030 0 500 1000 1500 2000 Days control anti-phOx anti-CD3 + anti-CD28 anti-phOx + anti-CD28 anti-phOx + CD86 anti-CD3 + CD86 anti-CD3 1/7

19. Goal: Develop membrane-anchored chimeric proteins that can be employed for both gene-expression imaging and therapy Single-gene for imaging and therapy

20. green fluorescent protein luciferase herpes simplex type 1 virus thymidine kinase cytosine deaminase  –galactosidase dopamine D2 receptor transferrin receptor High selectivity, but immunogenic Low immunogenicity, but less selective Reporter genes Exogenous genes Endogenous genes

21. Antibody-hapten imaging of transgene expression Advantages of antibody/hapten system Low immunogenicity (human or humanized Ab) High specificity and affinity Hydrophilic probes (small volume of distribution) anti-hapten scFv Cell Hydrophilic probe

22. DNS probes for gamma camera imaging dansyl phOx Roffler et al., Gene Ther., 13:412-20, 2006

23. In vivo accumulation of radioactive DNS-probe The DNS probe was retained at tumors that express DNS scFv on their surface

24. In vivo imaging anti-phOX tumor Mouse 1Mouse 2 #1#2 anti-DNS tumo r 1 h24 h48 h #1#2#1#2 Dansyl probe allowed imaging of DNS-positive tumors in mice

25. DNS DNS-PEG-IL-2 DNS DNS-PEG-  G O HO OH COOH O N Cl Glucuronide prodrug HAMG N Cl HO Active drug pHAM Hapten-directed therapy anti-DNS scFv Prodrug: Reduce tumor size/generate antigens IL-2: Stimulate antitumor immunity

26. Binding of hapten-modified proteins to anti-DNS scFv on cells DNS-labeled proteins selectively bound to anti-DNS scFv on CT-26 cells

27. Activity of DNS-PEG-IL-2 DNS-PEG-IL-2 is active when retained by anti-DNS scFv on CT-26 cells

28. O HO OH COOH O N Cl Glucuronide prodrug HAMG N Cl HO Active drug pHAM Prodrug activation by DNS-PEG-  G DNS-PEG-  G can activate HAMG at CT-26/DNS cells

29. In vivo localization of DNS-PEG-  G at CT-26/DNS tumors DNS-labeled  G can accumulate at CT-26/DNS tumors

30. None of the treatments delayed the growth of CT-26/phOx tumors Combination therapy of CT-26/DNS tumors Combined treatment was more effective than single agent therapy Chuang et al., Bioconjugate Chem., 17: 707-714, 2006.

31. Institute of Biomedical Sciences Academia Sinica

32. Dr. Kuang-Wen Liao Tang-Bi Liu Surface scFv T cell activation Surface expression Shih-en Chang Bing-Mae Chen Dr. Yu-Ling Leu Chia-Nan College of Pharmacy and Science Dr. Ji-Wang Chern National Taiwan University Dr. Tian-Lu Cheng Kaohsiung Medical University Chin-Chuan Chen Dr. Hsin-Ell Wang National Yang Ming University Yi-Hsuan Chiang Chien-I Su Joseph Lee Jill Lin