Membrane-bound antibodies for therapy and imaging

Steve Roffler

Institute of Biomedical SciencesAcademia SinicaTaipei, Taiwan

Immune cell regulation

Prodrug Activation

Gene expression imaging

Localized cytokine expression

Surface expression of biologically-active proteins

Metabolic regulation

cytokineantibody

enzyme

TMLS

TMLS scFv

VL VH

VH VL

VH

VL

CH1

CH2

H

CH3

CL

scFv

Single-chain antibody receptors

Liao et al., Biotechnology & Bioengineering (2001) 73, 313-323

Hybridoma cells

Extract RNA RT-PCR3' 5'

1st strand cDNA

antisense primer100 bp downstreamof J -C junction terminal

transferase

dGTP

3' 5'-GGGGGGGGGGGGG3' 5'GGGGGGGGGGGGGCCCCCCCCCCCCC 3'

extend5'

PCR amplify

3' 5'GGGGGGGGGGGGGCCCCCCCCCCCCC 3'5'

LS VL CK

LS VH CH1

Gilliland et al., Tissue Antigens. 1996 47:1-20

Making a scFv from a hybridoma

VL VH

PCR

subclone into pBluntsequence

VL VH

assembly PCR

VL VH

VL VH

http://www.ibms.sinica.edu.tw/~sroff/protocols/scFv.htm

Making a scFV from a hybridoma II

pcDNA3 2 C 1 1 - P D G F R

Expression of scFv with commercial pHook-1 vector

N N

N C N

C N C C

Type-I Type-II GPI-anchor

Dimer

GPI

1 domain

plasma membrane

hinge

CH2

CH3extracellular

intracellular

Enhancing surface expression transmembrane domains

1/2

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

Sfi I Sal I

LS HA AFP myc TM cyt

Import to ER Protein of interest Anchor inplasma membrane

Epitope tag Epitope tag

B7 TM is good for surface expression

B7-1 CD80

DAF decay- accelerating factor

ASGPR asialoglycoproteinreceptor

PDGFR platelet-derivedgrowth factorreceptor

.

2C11-1-B72C11-B7

2C11-e-B7

101 102 103 104

2C11-BGP-B7

2C11-CD44-B7

101 102 103 104

2C11-mBGP-B7

Fluorescent intensity

Rela

tive c

ell

num

ber

2C11-BGP-B7

2C11-e-B7

2C11-1-B7

2C11-CD44-B7

2C11-B7

2C11-mBGP-B7

HA

scFv B7

Linker domains increase surface expression

Effect of linker domains on scFv receptor expression

Liao et al., Cancer Gene Therapy (2003) 10: 779-790

IP of culture medium

Glycosylation controls surface shedding

Carbohydrate chains reduce sheddingand enhance surface expression

proteinexpression

surfaceexpression

GPI anchor glycosylatedno

glycosylation

SS

tumor cell

T cell

activation

CD3TCR

Activation of T cells by surface anti-CD3 scFv

Defects or down-regulation

TAP-1

TAP-2Beta 2-microglobulin

MHC class I heavy chain

proteosome subunits

Early events in T-cell activation

Direct CD3 ligation can initiate T cell activation

2C11-1-B7 pcDNA3

-2C11 IgG

+2C11 IgG

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

0

20000

40000

60000

Day1 2 3 4 5

anti-phOx

anti-CD3

anti-CD3 / CD86

IL-2

co

nce

ntr

atio

n (

pg

/mL

)

no stimulator cells.

0

25

50

75

100

Cyt

oto

xici

ty (

% c

on

tro

l)

anti-

CD3

anti-

CD3 +

CD86

anti-

phOx

anti-

phOx +

CD86

Anti-CD3 scFv is active in vitro

B16-F1

0 10 20 30 400

1000

2000

Days

Tum

or

size

(m

m3)

0/6

CD3 + CD86

0 10 20 30 400

1000

2000

Days

6/13

CD3

0 10 20 30 400

1000

2000

Days

0/7

phOx (control scFv)

0 10 20 30 400

1000

2000

Days

0/6

phOx + CD86

0 10 20 30 400

1000

2000

Days

Tum

or

size

(m

m3)

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 wasestablished by anti-CD3 and CD86

Original tumor

B16/F1 rechallenge

(tumor-free/total)

B16/CD3 + CD86 4/4

naive 0/4

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

0 10 20 300

500

1000

1500

2000

Days

Tu

mor

siz

e (

mm3)

0 10 20 300

500

1000

1500

2000

Days

0 10 20 300

500

1000

1500

2000

Days

0 10 20 300

500

1000

1500

2000

Days

0 10 20 300

500

1000

1500

2000

Days

Tu

mor

siz

e (

mm3)

0 10 20 300

500

1000

1500

2000

Days

0 10 20 300

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

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 proteinluciferaseherpes simplex type 1 virus thymidine kinasecytosine deaminase –galactosidase

dopamine D2 receptortransferrin 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-haptenscFv

Cell

Hydrophilic probe

DNS probes for gamma camera imaging

dansylphOx

O

C

O

N

N

CH3CH3

SO2 NH C

O

CH2

C

O

CH2HO

N (CH2)2 N

CH2 C OH

O

(CH2)2 N

CH2

CH2

C

O

C

O

OH

NH2

N

CH3CH3

SO2 NH C

O

CH2

C

O

CH2HO

N (CH2)2 N

CH2 C OH

O

(CH2)2 N

CH2

CH2

C

O

C

O

OH

NH SO2

N

CH3CH3

Roffler et al., Gene Ther., 13:412-20, 2006

Blood DNS scFv tumor0.01

0.1

1

10 4 h

24 h

48 h

% i

nje

cted

do

se /

g t

issu

e

In vivo accumulation of radioactive DNS-probe

The DNS probe was retained at tumorsthat express DNS scFv on their surface

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 ofDNS-positive tumors in mice

DNS

DNS-PEG-IL-2

DNS

DNS-PEG-G

OHOHO

OH

COOH

O NCl

Cl

Glucuronide prodrug HAMG

NCl

ClHO

Active drug pHAM

Hapten-directed therapy

anti-DNSscFv

Prodrug: Reduce tumor size/generate antigensIL-2: Stimulate antitumor immunity

Binding of hapten-modified proteins to anti-DNS scFv on cells

DNS-labeled proteins selectively boundto anti-DNS scFv on CT-26 cells

Activity of DNS-PEG-IL-2

DNS-PEG-IL-2 is active when retainedby anti-DNS scFv on CT-26 cells

OHO

HOOH

COOH

O NCl

Cl

Glucuronide prodrug HAMG

N

Cl

ClHO

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: 707-714, 2006.

Institute of Biomedical SciencesAcademia Sinica

Dr. Kuang-Wen Liao

Tang-Bi Liu

Surface scFv

T cell activationSurface 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 ChengKaohsiung Medical

University

Chin-Chuan Chen

Dr. Hsin-Ell WangNational Yang Ming

University

Yi-Hsuan Chiang

Chien-I Su

Joseph Lee

Jill Lin