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Slide 1 - Membrane-bound antibodies for therapy and imaging Steve Roffler Institute of Biomedical Sciences Academia Sinica Taipei, Taiwan
Slide 2 - Immune cell regulation Prodrug Activation Gene expression imaging Localized cytokine expression Surface expression of biologically-active proteins Metabolic regulation cytokine antibody enzyme
Slide 3 - scFv Single-chain antibody receptors Liao et al., Biotechnology & Bioengineering (2001) 73, 313-323
Slide 4 - Gilliland et al., Tissue Antigens. 1996 47:1-20 Making a scFv from a hybridoma
Slide 5 - http://www.ibms.sinica.edu.tw/~sroff/protocols/scFv.htm Making a scFV from a hybridoma II
Slide 6 - Expression of scFv with commercial pHook-1 vector
Slide 7 - Enhancing surface expression transmembrane domains
Slide 8 - t1/2 12.2 3.8 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 ER Protein of interest Anchor in plasma membrane Epitope tag Epitope tag B7 TM is good for surface expression B7-1 CD80 DAF decay- accelerating factor ASGPR asialoglycoprotein receptor PDGFR platelet-derived growth factor receptor
Slide 9 - Linker domains increase surface expression Effect of linker domains on scFv receptor expression Liao et al., Cancer Gene Therapy (2003) 10: 779-790
Slide 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
Slide 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
Slide 12 - Early events in T-cell activation Direct CD3 ligation can initiate T cell activation
Slide 13 - T cells can bind to cells that express anti-CD3 scFv
Slide 14 - . 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
Slide 15 - anti-CD3 scFv with CD86 prevented growth of poorly immunogenic tumors in 45% of mice Tumor sizes in mice injected with B16-F1 transfectants
Slide 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/aCD3 + CD86 4/4 naive 0/4
Slide 17 - anti-CD28 scFv receptor scFv (aCD28) TM (B7) Linker(eB7) Is anti-CD28 scFv better than CD86? Can bind CD28 but not CTLA-4
Slide 18 - Tumor sizes after adenoviral therapy anti-CD3 with anti-CD28 delayed tumor growth 0 10 20 30 0 500 1000 1500 2000 Days Tumor size (mm 3 ) 0 10 20 30 0 500 1000 1500 2000 Days 0 10 20 30 0 500 1000 1500 2000 Days 0 10 20 30 0 500 1000 1500 2000 Days 0 10 20 30 0 500 1000 1500 2000 Days Tumor size (mm 3 ) 0 10 20 30 0 500 1000 1500 2000 Days 0 10 20 30 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
Slide 19 - Goal: Develop membrane-anchored chimeric proteins that can be employed for both gene-expression imaging and therapy Single-gene for imaging and therapy
Slide 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
Slide 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
Slide 22 - DNS probes for gamma camera imaging dansyl phOx Roffler et al., Gene Ther., 13:412-20, 2006
Slide 23 - In vivo accumulation of radioactive DNS-probe The DNS probe was retained at tumors that express DNS scFv on their surface
Slide 24 - In vivo imaging anti-phOX tumor Mouse 1 Mouse 2 #1 #2 anti-DNS tumor 1 h 24 h 48 h #1 #2 #1 #2 Dansyl probe allowed imaging of DNS-positive tumors in mice
Slide 25 - Hapten-directed therapy anti-DNS scFv Prodrug: Reduce tumor size/generate antigens IL-2: Stimulate antitumor immunity
Slide 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
Slide 27 - Activity of DNS-PEG-IL-2 DNS-PEG-IL-2 is active when retained by anti-DNS scFv on CT-26 cells
Slide 28 - Prodrug activation by DNS-PEG-bG DNS-PEG-bG can activate HAMG at CT-26/DNS cells
Slide 29 - In vivo localization of DNS-PEG-bG at CT-26/DNS tumors DNS-labeled bG can accumulate at CT-26/DNS tumors
Slide 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.
Slide 31 - Institute of Biomedical Sciences Academia Sinica
Slide 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