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

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

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

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

Making a scFV from a hybridoma II

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

Enhancing surface expression transmembrane domains

 1/ 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

Linker domains increase surface expression Effect of linker domains on scFv receptor expression Liao et al., Cancer Gene Therapy (2003) 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

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

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

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

. Anti-CD3 scFv activity anti-CD3 scFv can induce CTL activity anti-CD3 scFv with CD86 stimulates IL-2 secretion Day anti-phOx anti-CD3 anti-CD3 / CD86 IL-2 concentration (pg/mL) no stimulator cells Cytotoxicity (% control) anti-CD3 anti-CD3 + CD86 anti-phOx anti-phOx + CD86 Anti-CD3 scFv is active in vitro

B16-F Days Tumor size (mm 3 ) 0/6  CD Days 0/7 phOx (control scFv) Days 0/6 phOx + CD 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

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

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

Tumor sizes after adenoviral therapy anti-CD3 with anti-CD28 delayed tumor growth Days Tumor size (mm 3 ) Days Days Days Days Tumor size (mm 3 ) Days Days control anti-phOx anti-CD3 + anti-CD28 anti-phOx + anti-CD28 anti-phOx + CD86 anti-CD3 + CD86 anti-CD3 1/7

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

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

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

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

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

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

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

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

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

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

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

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: , 2006.

Institute of Biomedical Sciences Academia Sinica

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