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Membrane-bound antibodies for therapy and imaging

Membrane-bound antibodies for therapy and imaging. Steve Roffler. Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan. LS. TM. Surface expression of biologically-active proteins. enzyme. cytokine. antibody. Localized cytokine expression. Immune cell regulation.

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Membrane-bound antibodies for therapy and imaging

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

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

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

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

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

  6. pcDNA3 2 C 1 1 - P D G F R Expression of scFv with commercial pHook-1 vector

  7. transmembrane domains Enhancing surface expression

  8. Summary of AFP-TM results Epitope tag Epitope tag cyt Import to ER Protein of interest Anchor in plasma membrane B7-1 CD80 DAF decay- accelerating factor ASGPR asialoglycoprotein receptor PDGFR platelet-derived growth factor receptor B7-1DAFASGPRPDGFR t1/2 12.2 3.8 2.4 1.6 h AFP-B7 is most rapidly transported to the cell surface AFP-B7 is more stable on the cell surface Surface expression: B7 > DAF >> PDGFR = ASGPR B7 TM is good for surface expression

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

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

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

  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. no stimulator cells 60000 100 . anti-phOx anti-CD3 75 anti-CD3 / CD86 40000 Cytotoxicity (% control) IL-2 concentration (pg/mL) 50 25 20000 0 anti-CD3 anti-phOx anti-CD3 + CD86 anti-phOx + CD86 0 1 2 3 4 5 Day Anti-CD3 scFv activity anti-CD3 scFv can induce CTL activity anti-CD3 scFv with CD86 stimulates IL-2 secretion . Anti-CD3 scFv is active in vitro

  15. 2000 2000 phOx (control scFv) B16-F1 Tumor size (mm3) 0/6 0/6 1000 1000 0 0 0 10 20 30 40 0 10 20 30 40 Days Days 2000 2000 aCD3 + CD86 2000 aCD3 phOx + CD86 0/7 6/13 0/5 Tumor size (mm3) 1000 1000 1000 0 0 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 Days Days Days Tumor sizes in mice injected with B16-F1 transfectants anti-CD3 scFv with CD86 prevented growth of poorly immunogenic tumors in 45% of mice

  16. Development of systemic anti-tumor immunity in tumor-free mice B16/F1 rechallenge Original tumor (tumor-free/total) naive 0/4 4/4 B16/aCD3 + CD86 Long-term protective immunity was established by anti-CD3 and CD86

  17. anti-CD28 scFv receptor scFv (aCD28) Linker(eB7) TM (B7) Can bind CD28 but not CTLA-4 Is anti-CD28 scFv better than CD86?

  18. Tumor sizes after adenoviral therapy anti-phOx anti-phOx control 2000 2000 anti-phOx 2000 2000 + + anti-CD28 CD86 ) 1500 1500 1500 1500 3 1000 Tumor size (mm 1000 1000 1000 500 500 500 500 0 0 0 0 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 Days Days Days Days 2000 anti-CD3 2000 2000 anti-CD3 anti-CD3 + + anti-CD28 CD86 ) 1500 3 1500 1500 1/7 1000 Tumor size (mm 1000 1000 500 500 500 0 0 0 0 10 20 30 0 10 20 30 0 10 20 30 Days Days Days anti-CD3 with anti-CD28 delayed tumor growth

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

  21. Antibody-hapten imaging of transgene expression Hydrophilic probe 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

  22. DNS probes for gamma camera imaging phOx dansyl 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-DNS tumor anti-phOX tumor Mouse 1 Mouse 2 1 h 24 h 48 h #1 #2 #1 #2 #1 #2 Dansyl probe allowed imaging of DNS-positive tumors in mice

  25. Glucuronide prodrug HAMG Active drug pHAM COOH Cl O Cl HO HO N O N HO Cl Cl OH DNS-PEG-IL-2 DNS-PEG-bG DNS DNS 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. Glucuronide prodrug HAMG COOH O Cl HO O N HO Cl OH Active drug pHAM Cl HO N Cl Prodrug activation by DNS-PEG-bG DNS-PEG-bG can activate HAMG at CT-26/DNS cells

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

  30. Combination therapy of CT-26/DNS tumors None of the treatments delayed the growth of CT-26/phOx 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. Surface expression Dr. Tian-Lu Cheng Kaohsiung Medical University T cell activation Tang-Bi Liu Shih-en Chang Chien-I Su Chin-Chuan Chen Dr. Hsin-Ell Wang National Yang Ming University Yi-Hsuan Chiang Jill Lin Joseph Lee Dr. Yu-Ling Leu Chia-Nan College of Pharmacy and Science Surface scFv Dr. Kuang-Wen Liao Bing-Mae Chen Dr. Ji-Wang Chern National Taiwan University

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