Mammary gland-specific secretion of biologically active ...

sc.mahidol.ac.th

Mammary gland-specific secretion of biologically active ...

Journal of Immunological Methods 277 (2003) 171–183

Recombinant Technology

Mammary gland-specific secretion of biologically active

immunosuppressive agent cytotoxic-T-lymphocyte

antigen 4 human immunoglobulin fusion protein

(CTLA4Ig) in milk by transgenesis

Vincent C.H. Lui a , Paul K.H. Tam a, *, Michael Y.K. Leung b , James Y.B. Lau b ,

Jacqueline K.Y. Chan a , Vera S.F. Chan a , Margaret Dallman c , Kathryn S.E. Cheah b, *

a Division of Paediatric Surgery, Department of Surgery, The University of Hong Kong Medical Centre, Queen Mary Hospital,

Hong Kong SAR, China

b Department of Biochemistry, The University of Hong Kong, 3/F Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam,

Hong Kong SAR, China.

c Department of Biological Science, Imperial College of Science, Technology, and Medicine, London, UK

Received 7 November 2002; received in revised form 3 January 2003; accepted 10 February 2003

www.elsevier.com/locate/jim

Abstract

A major challenge in the field of transplantation is to prevent graft rejection and prolong graft survival. Tolerance induction

is a promising way to achieve long-term graft survival without the need for potent immunosuppression and its associated side

effects. The recent success of co-stimulatory blockade by the chimeric protein CTLA4Ig in the modulation of the recipient’s

immune system and the prolongation of graft survival in animal models suggests a possible application of CTLA4Ig in clinical

transplantation. To produce sufficient amounts of CTLA4Ig for future clinical application, we sought to use the mammary gland

as a bioreactor and produce CTLA4Ig in the milk of transgenic farm animals. Prior to the generation of transgenic farm animals,

we tested our strategy in mice. Using the promoter of the sheep h-lactoglobulin gene, we expressed our CTLA4Ig chimeric

gene in the mammary gland of transgenic mice. The yield of CTLA4Ig was fivefold higher in transgenic milk than that from

transfected cells. Purified milk-derived CTLA4Ig is biologically active and suppresses T cell activation. We showed that the

production of CTLA4Ig in the milk has no adverse immunosuppression effect on the transgenic animals and the offsprings that

were fed with the transgenic milk. The findings suggest that the approach to produce CTLA4Ig in milk by transgenesis is

feasible; further studies involving farm animals are warranted.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: CTLA4Ig; Milk; Immunosuppression; Transplantation; Transgenesis

Abbreviations: CTLA4Ig, cytotoxic-T-lymphocyte antigen 4 human immunoglobulin fusion protein; BLG, h-lactoglobulin gene; PBS,

phosphate-buffered saline; FCS, fetal calf serum; CHO cells, Chinese hamster ovary cells; MLR, mixed lymphocyte reaction; PCR, polymerase

chain reaction; RT-PCR, reverse transcription and polymerase chain reaction.

* Corresponding authors. P.K.H. Tam is to be contacted at Tel.: +852-285-54850; fax: +852-281-73155. K.S.E. Cheah, Tel.: +852-281-

99240; fax: +852-285-51254.

E-mail addresses: paultam@hkucc.hku.hk (P.K.H. Tam), hrmbdkc@hkusua.hku.hk (K.S.E. Cheah).

0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0022-1759(03)00071-1


172

V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183

1. Introduction

With advances in surgical techniques and perioperative

care, organ transplantation can now be

performed with minimal surgical mortality to save

lives of patients with end-stage organ failure. However,

allograft rejection remains a major obstacle to

the success of clinical transplantation. To prevent

rejection and prolong survival, patients are inevitably

administered lifelong with immunosuppressive drugs

that are associated with many toxic side effects,

including opportunistic infection and cancer development.

Therefore, a major challenge in the field of

transplantation is to induce donor-specific tolerance

and achieve long-term graft survival without a deleterious

immunosuppressive regime.

T cell activation is a key step in the initiation of

graft rejection. Full activation of T cells requires two

signals: one is antigen-specific and based on an

interaction of T cell receptors (TCRs) with an antigen-major

histocompatibility complex (MHC) on

antigen-presenting cells (APCs), and the second is

an antigen-non-specific co-stimulatory signal. The

lack of a co-stimulatory signal after engagement of

the TCR by antigen could result in partial or failed T

cell activation that renders T cells unresponsive to

further antigen challenge, a state known as T cell

anergy. T cell anergy is of prime importance in the

induction of antigen-specific tolerance to prevent graft

rejection. The primary co-stimulatory signal is delivered

by B7 receptors (CD80 and CD86) on APCs after

ligation of CD28 and cytotoxic-T-lymphocyte antigen

4 (CTLA4) on T cells. CTLA4 displays 20-fold

higher affinity than CD28 in binding to B7 receptors

(Linsley et al., 1991, 1994). Unlike CD28, binding of

CTLA4 to CD80 or CD86 delivers an inhibitory

signal, down-regulating T cell activation (Sebille et

al., 2001).

Soluble CTLA4Ig is a fusion protein consisting of

the extracellular domain of CTLA4 on the N-terminus

and the constant regions (CH2 and CH3) and hinge of

the Fc domain of IgG on the C-terminus (Linsley et al.,

1994). The immunoglobulin portion of CTLA4Ig

allows an efficient purification on affinity chromatography

column, production of a dimeric CTLA4 protein

and a long circulating half-life in vivo. CTLA4Ig

functions as a competitive inhibitor of CD28/B7 pathway,

suppressing primary and secondary T celldependent

antibody responses to foreign antigen.

Blockade of CD28/B7 co-stimulation by CTLA4Ig

reduces graft rejection and prolongs graft survival in

rat cardiac transplantation (Guillot et al., 2000; Hayashi

et al., 2000; Turka et al., 1992), in islet-cell xenografts

in mice (Feng et al., 1999; Lenschow et al., 1992), in rat

renal transplantation (Tomasoni et al., 2000), in renal

and islet transplantation in monkeys (Kirk et al., 1997;

Levisetti et al., 1997), and in rat intestine transplantation

(Echizenya et al., 2001; Kurlberg et al., 2000). In

some instances, CTLA4Ig has been shown to induce

transplantation tolerance (Pearson et al., 1994). Successful

use of CTLA4Ig in the prolongation of graft

survival in animal models suggested that CTLA4Ig

might also be an important therapeutic agent for use in

clinical transplantation. Indeed, clinical trials using

CTLA4Ig in the treatment of psoriasis and graft-versus-host

disease (GVHD) in allogenic bone marrow

transplantation have shown promising results (Abrams

et al., 1999, 2000; Guinan et al., 1999).

CTLA4Ig can be delivered to the transplant recipient,

graft or model animals by either direct intravenous

injection of purified protein or by CTLA4-expressing

adenovirus. Adenoviral gene transfer carries its own

disadvantages and adverse effects. Adenovirus in the

host can induce an immune response and results in

inflammatory and toxic reactions in patients. Production

of antibodies in the patients or pre-existing antibody

neutralise the adenovirus and render the gene

delivery unsuccessful (Romano et al., 2000). Intravenous

injection of purified CTLA4Ig can be used to

achieve therapeutic CTLA4Ig levels in the sera of

recipients. Indeed, intravenous infusions of soluble

CTLA4Ig to patients with psoriasis (a T cell-mediated

skin disease) successfully slow down autoimmune

disease progression and improve the patients’ condition

(Abrams et al., 1999, 2000). Multiple injections of

large quantities of purified CTLA4Ig protein are required

to achieve sustained systemic therapeutic levels

of CTLA4Ig in the recipient. Until now, CTLA4Ig is

purified from transfected cells, a method that is inefficient

and, therefore, expensive for the large-scale

production required to supply sufficient quantities for

clinical application. An efficient and cost-effective

method is needed to produce enough CTLA4Ig to cope

with the future demands in clinical applications.

Pigs are frequently used as models to establish and

refine surgical skill and to test the efficacies of immu-


V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183 173

Table 1A

Expression of proteins of pharmaceutical interests by lactationspecific

promoters in farm animals

Species Lactationspecific

promoter

Protein

Yield

Sheep

Pig

Cow

Goat

Rabbit

Sheep betalactoglobulin

Mouse whey

acidic protein

Bovine

alpha-S1

casein

Mouse whey

acidic protein

Goat betacasein

Bovine

alpha-S1

casein

Mouse whey

acidic protein

N.A.: not available.

Human alpha 1-antitrypsin >1 mg/ml

(Wright et al., 1991)

Human factor IX N.A.

(Clark et al., 1989;

Schnieke et al., 1997)

Human factor VIII

(Niemann et al., 1999)

Human protein C

(Velander et al., 1992;

Van Cott et al., 1996)

4 –6 ng/ml

0.1–0.5 mg/ml

Human factor VIII 2.7 Ag/ml

(Paleyanda et al., 1997)

Human lactoferrin 1 mg/ml

(Krimpenfort et al., 1991;

van Berkel et al., 2002)

Human tissue

1 –3 mg/ml

plasminogen activator

(Ebert et al., 1991, 1994)

Human antithrombin

(Edmunds et al., 1998)

Human granulocytemacrophage

colonystimulating

factor

(Ko et al., 2000)

Human insulin-like

growth factor I

(Brem et al., 1994;

Wolf et al., 1997)

Human nerve growth

factor beta

(Coulibaly et al., 1999)

Human growth hormone

(Limonta et al., 1995)

>1 mg/ml

50 Ag/ml

0.05–1 mg/ml

50–250 Ag/ml

50 Ag/ml

nosuppressive regime in transplantation before clinical

application. We sought to produce large amount of pig

CTLA4 human Ig fusion protein and test its efficacy in

the prolongation of graft survival in pig transplantation

models. Simons et al. (1987) first reported the use of

transgenic technology to express foreign proteins in

mouse milk. Thereafter, lactation-specific promoters

have been used to express proteins of pharmaceutical

interests in large quantities in the milk of animals

(Table 1A and 1B). However, the production of immunosuppressive

protein in milk using transgenesis has

not been studied. To investigate the practicality of using

the mammary gland as a bioreactor to produce large

quantity of exogenous ‘‘non-self’’ CTLA4Ig in the

milk of transgenic farm animals and to assess if the

CTLA4Ig in milk will cause detrimental immunosuppression

in transgenic animals, we have tested our

strategy in transgenic mice. In this study, using the

promoter of the sheep h-lactoglobulin gene to direct

the expression of pig CTLA4 human Ig chimeric gene

Table 1B

Expression of proteins of pharmaceutical interests by lactationspecific

promoters in transgenic mice

Species Lactationspecific

promoter

Protein

Yield

Mouse Bovine alpha-

S1 casein

Bovine

beta-casein

Sheep betalactoglobulin

Rat

beta-casein

Rabbit whey

acidic protein

Murine whey

acidic protein

N.A.: not available.

Human factor VIII

(Chen et al., 2002)

Human alpha

1(I) procollagen

(Toman et al., 1999)

Human granulocytemacrophage

colonystimulating

factor

(Uusi-Oukari et al., 1997)

Human acid

alpha-glucosidase

(Bijvoet et al., 1996)

Human lactoferrin

(Platenburg et al., 1994)

Human thrompoietin

(Sohn et al., 1999)

Human fibrinogen

(Prunkard et al., 1996)

Human serum albumin

(Shani et al., 1992)

Human alpha

1-antitrypsin

(Archibald et al., 1990)

Human growth hormone

(Lee et al., 1996)

Bovine folliclestimulating

hormone

(Greenberg et al., 1991)

Bovine growth hormone

(Thepot et al., 1995)

Human erythropoietin

(Rodriguez et al., 1995)

Human growth hormone

(Devinoy et al., 1994)

Human parathyroid

hormone

(Rokkones et al., 1995)

7–50 Ag/ml

8 mg/ml

1 mg/ml

1.5 Ag/ml

36 Ag/ml

1.5 mg/ml

2 mg/ml

2.5 mg/ml

0.5–7 mg/ml

0.02– 5.5 mg/ml

15 Ag/ml

1–16 mg/ml

10 ng/ml

4–22 mg/ml

0.4 Ag/ml


174

V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183

in the mouse mammary gland, we produced pig

CTLA4 human Ig fusion protein in mouse milk.

One would anticipate that the immunosuppressive

agent such as CTLA4Ig in animal milk may induce

detrimental systemic immunosuppression in transgenic

animals and/or animals that have been fed with

the transgenic milk. Our data confirm that the production

of ‘‘non-self’’ CTLA4Ig in animal milk using

transgenesis is safe and transgenic animals do not

develop any symptoms of immunosuppression. The

transgene was transmitted through the germline and

CTLA4Ig is produced in the milk of transgenic

offspring.

2. Materials and methods

2.1. Construction of pig CTLA4 human Ig chimeric

cDNA (pCTLA4Ig)

Lymphocytes were isolated from Duroc pig’s blood

using Ficoll-Paque following the manufacturer’s protocol

(Amersham Pharmacia Biotech, Buckinghamshire,

UK), and were cultured with concanavalin A (5

Ag/ml) for 2 days under standard conditions. Total

RNA was isolated from the lymphocytes using guanidium

thiocyanate–phenol choloroform method

(Chomczynski and Sacchi, 1987). cDNA was prepared

from 2 Ag of total RNA using the SuperScript

II kit (Invitrogen, California, USA) with oligo(dT) as

a reverse primer following the manufacturer’s protocol.

The cDNA encoding the extracellular domain of

the pig CTLA4 was obtained by PCR using primers

which anneal to the first exon and the 5V part of the

third exon of the pig CTLA4 gene (Table 2). The PCR

product was digested with HindIII and BamHI, cloned

into pIG1 vector to give plasmid pMLF8. Splicing

between the CTLA4 cDNA and the IgG genomic

sequence in pIG1 produces the CTLA4Ig fusion transcripts.

To generate a CTLA4Ig expression vector

(pMLF3) for cell transfection, a 2-kb HindIII–NotI

DNA insert of pMLF8 was subcloned into pCDNA3

(Invitrogen).

2.2. Generation of the pBLG-pCTLA4Ig transgenic

construct

To generate the transgenic construct BLG-pCT-

LA4Ig, the SalI–XbaI digested DNA insert of the

sheep h-lactoglobulin minigene construct pBJ41

(kindly provided by Andrew Clark, Roslin Institute,

Roslin, UK) was cloned into the pSC3-ZX DNA vector

to give pBLG. The HindIII–NotI DNA insert from

pMLF8 was end-filled with Klenow and cloned into a

unique EcoRV site of pBLG to give pBLG-pCTLA 4 Ig

(Fig. 1). The DNA insert of the transgenic construct

BLG-pCTLA4Ig was completely sequenced using a

dye terminator cycle sequencing kit to confirm that

there was no mutation introduced in the transgene

during the cloning process (ABI PRISM Big DyeTM

Terminator v 2.0 Cycle sequencing kit, Applied Biosystems,

Foster City, CA, USA) and an ABI 3100

automatic sequencer (Applied Biosystems).

2.3. Cell transfection and purification of CTLA4Ig

fusion protein

The construct pMLF3 was transfected into Cos-1

cells using LIPOFECTAMINEk 2000 (Invitrogen).

Transfected cells were grown in DMEM plus 5% fetal

calf serum (FCS) at 37 jC in atmospheric air supplemented

with 5% CO 2 for 6 days. Culture media were

collected by centrifugation (4800 rpm for 20 min at 4

jC). The CTLA4Ig protein was affinity-purified on a

Table 2

Primers for the amplification of sequence of the pig CTLA4 extracellular domain

Primers

Sequence

5VSIGCTLA4

5V-AATATAAGCTTACTAGTCCGCCATGGCTTGTTCTGGACTCCG-3V

3VCTLA4

5V-ACGGATCCACTTACCTGTAGAATCTGGGCATGGTTCTG-3V

5VSIGCTLA4, primer anneals to the first exon of pig CTLA4 gene. The start codon ATG is indicated in bold, italic, and underlined; the

consensus Kozak sequence for the initiation of translation is double underlined; the HindIII restriction enzyme recognition sequence is

underlined. 3VCTLA4, primer anneals to the third exon of pig CTLA4 gene. Sequence complementary to the 5Vpart of Exon 3 is in italic; the

BamHI restriction enzyme recognition sequence is underlined; the sequence ‘‘GT’’ for the spice acceptor site is double underlined.


V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183 175

Fig. 1. Construction of pBLG-pCTLA4Ig transgene and transgenic mice. Panel a: Construction of pBLG-CTLA4Ig transgene. The vector pBJ41

carried the intronless sheep h-lactoglobulin gene (BLG) in which the exons II to exon IV were removed by linking the exon I directly to the

exon V. The ATG start codon of the BLG gene was removed in pBJ41 and an EcoRV site was created by linkers between exons I and V. The 2.5

kb of 5Vflanking and the 1.7 kb of 3Vflanking sequences of the BLG gene were encoded in pBJ41. The SalI–XbaI DNA insert of pBJ41 was

cloned into a vector pSC-3ZX to give pBLG. The end-filled HindIII – NotI DNA insert of pMLF8 containing the pCTLA4 and human Ig Fc

coding regions was inserted into the EcoRV site of pBLG to generate pBLG-pCTLA4Ig. Exons and introns were indicated with boxes and lines,

respectively. Sizes (kb) of BamHI digested DNA fragments of the pBLG-pCTLA4Ig construct were indicated. Abbreviations: S, SalI; E, EcoRV;

X, XbaI; H, HindIII; N, NotI; B, BamHI. Panel b: Transgenic mice were identified using Southern blotting analysis. DNA of the transgenic

construct pBLG-pCTLA4Ig was digested with BamHI and included as a positive control ( + ctrl) and gave positive hybridised fragments of

expected sizes. Transgenic mice (Tg) DNA showed similar positive hybridised fragments as the positive control. Non-transgenic mouse DNA

(NT) did not show positive hybridisation.

Hitrap protein A column (Amersham Pharmacia Biotech).

The eluate was concentrated under vacuum

before dialysis in phosphate-buffered saline (PBS,

pH 7.2) at 4 jC for 48 h. Protein concentration was

determined by Bradford Microassay method (BioRad,

California, USA). Approximately 3 Ag of CTLA4Ig

could be purified from each milliliter of transfected

medium. Denaturing polyacrylamide gel electrophoresis

was performed on the eluate of the transfected

cells and medium of non-transfected cells. Coomassie

blue staining of the gel revealed a major band of 58

kDa that corresponded to the predicted molecular

weight of the pig CTLA4Ig in the lane of transfected

cells (Fig. 3a). Anti-human IgG Fc antibody recognised

the same 58-kDa protein band in the eluates of

the transfected Cos-1 cells medium only. This confirms

that the major protein band on the gel represents

the pig CTLA4Ig (Fig. 3b).


176

V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183

2.4. Production of BLG-pCTLA4Ig transgenic mice

BLG-pCTLA4Ig transgenic mice were generated

as described previously (Hogan et al., 1994). Transgenic

mice were identified using Southern blotting

analysis of tail DNAs. BamHI digests of DNA were

separated by electrophoresis in 1% agarose gel before

transfer to HybondN membranes (Amersham Pharmacia

Biotech). The DNA insert of pBLG-pCTLA4Ig

was labelled with [a- 32 P]-dCTP by random priming

oligo-labelling (Feinberg and Vogelstein, 1984). The

membrane was hybridised at 60 jC for 18 h in 5 SSC

containing 12% polyethylene glycol 8000 (PEG-8000;

Sigma, UK), 25 mg/ml heparin (Sigma), 0.1% SDS

(Sigma), 50 Ag/ml sheared salmon DNA (Sigma). Posthybridisation

wash was performed at 60 jC at

1 SSC, 0.1% SDS. The membrane was exposed to

Kodak X-ray film with intensifying screens at 70 jC

for 48 h.

2.5. Purification of CTLA4Ig from transgenic mouse

milk

Transgenic mice were milked on day 12 of lactation

by vacuum suction as described previously (Haberman,

1974; Thepot et al., 1995). Milk CTLA4Ig was affinitypurified

using protein A agarose resin (Sigma) as

follows: Milk was diluted with two volumes of PBS

and then incubated with 100 Al of resin at 4 jC for 18 h

on a shaking platform. The resin was allowed to settle

by gravity and the supernatant was discarded. The resin

was washed three times with 500 Al of PBS, then once

with 500 Al of 0.1 M glycine, pH 5.0. Bound CTLA4Ig

was eluted from the resin with 100 Al of 0.1 M glycine,

pH 3.0. The extraction was repeated twice and eluates

pooled. Thirty microliters of 1 M Tris (pH 8.0) was

added to the CTLA4Ig to neutralise the glycine.

CTLA4Ig was dried under vacuum, redissolved in

100 Al of PBS before dialysis in PBS at 4 jC for 18 h.

2.6. SDS-polyacrylamide gel electrophoresis and

Western blotting analysis

Purified CTLA4Ig (1 Ag) was resolved by electrophoresis

in a 10% denaturing polyacrylamide gel as

previously described (Laemmli, 1970). For Western

blotting analysis, proteins were electrotransferred onto

PVDF membrane (Amersham Pharmacia Biotech).

The blot was blocked with PBS + 0.1% Tween 20

(PBST) supplemented with 5% bovine serum albumin

(BSA, w/v) at 4 jC for 16 h. Horseradish peroxidase

(HRP)-conjugated rabbit anti human IgG antibody

(Dako, Glostrup, Denmark) was diluted (1:500) in

PBST + 0.5% BSA (w/v) and incubated with the blot

for 1 h at ambient temperature. After washing in PBST

(3 10 min), the blot was incubated with DAB (3,3Vdiaminobenzidine

tetrahydrochloride; Sigma) to visualise

the pig CTLA4Ig protein on the membrane.

2.7. In vitro binding of CTLA4Ig to CD80

Affinity-purified pig CTLA4Ig or crude transgenic

milk was incubated with Chinese hamster ovary (CHO)

cells (4 10 5 ) that express mouse CD80 on cell surface

in 100 Al onicefor1hinPBS+2%FCS.After

incubation, cell pellets were obtained by centrifugation

(1000 rpm; 5 min). Cells were washed twice in 1 ml of

PBS + 2% FCS. After the final wash, cells were resuspended

in 100 Al of FITC-conjugated human IgG Fcspecific

antibody (Sigma; 1:50 dilution in PBS + 2%

FCS) and incubated at 4 jC for 30 min. Excess

secondary antibody was removed by washing the cell

suspension as before. Finally, the cells were fixed in 0.5

ml of PBS + 2% formalin before analysis as a Becton

Dickinson FACSCalibur (Becton Dickinson Immunocytometry

Systems, California, USA). To construct a

standard binding curve of CTLA4Ig to CD80-expressing

CHO cells, mean fluorescence intensities of the

bindings at different concentrations of CTLA4Ig (Ag/

100 Al of reaction mixture) were determined. Mean

fluorescence intensities were plotted against the logarithm

of the concentrations of CTLA4Ig to construct a

standard binding curve. Transgenic milk (5 Al) was

incubated with CD80-expressing CHO cells and the

mean fluorescence intensity of binding determined.

The CTLA4Ig concentration in the transgenic milk

was determined from the standard binding curve.

2.8. Mixed lymphocyte reaction (MLR)

Two-way MLRs were performed to investigate the

immunosuppressive activity of pig CTLA4Ig fusion

protein in rat, pig and human. Briefly, lymphocytes

from DA and Lewis rat spleens, peripheral blood of

Duroc and Large White pigs, or peripheral blood of

human individuals from different ethnic origins were


V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183 177

prepared using Ficoll-Paque. Cells (10 5 ) of each of

responder and stimulator were co-cultured in a 96-well

plate for 5 days in the absence or presence of CTLA4Ig.

Cultures were pulse-labelled with 1 ACi/well of [ 3 H]-

thymidine (Amersham Pharmacia Biotech) for 18 h

prior to day 5. The cells were transferred onto glass

membrane using cell harvester, and the incorporation

of [ 3 H]-thymidine was determined by scintillation

counting. Incorporation of [ 3 H]-thymidine in the

absence of CTLA4Ig was used for 0% suppression.

The percent suppression in the presence of CTLA4Ig

was calculated using the following formula: (1 [ 3 H]-

thymidine incorporation in the presence of CTLA4Ig/

[ 3 H]-thymidine incorporation in the absence of CT-

LA4Ig) 100%.

At each CTLA4Ig concentration, the mean percentage

of suppression and standard error were determined

from triplicate wells.

3. Results

3.1. Generation of BLG-pCTLA4Ig transgenic mice

The cDNA encoding the extracellular domain of the

pig CTLA4 was obtained by RT-PCR using primers

(Table 2) on total RNA of pig lymphocytes. The PCR

product was cloned into pIG1 vector to give plasmid

pMLF8 (Fig. 1a). Splicing between the CTLA4 cDNA

and the IgG genomic sequence in pIG1 produces the

pig CTLA4 human Ig fusion transcripts. The sheep h-

lactoglobulin gene promoter was fused to the CTLA4Ig

sequences and the 8.6-kb SalI–XbaI DNA insert of the

resulting construct, pBLG-pCTLA4Ig (Fig. 1a), was

microinjected into fertilised eggs. From a total of 1410

injected egg transfers, 85 pups were born and survived

till weaning. Five transgenic mice were identified by

Southern blotting analysis (see example in Fig. 1b)

including two females and three males; the percentage

of transgenesis was therefore 5.9%. Three founders

(two males and one female) transmitted the transgene

through the germline.

3.2. Pig CTLA4Ig is secreted into mouse milk

To investigate if the transgene is properly expressed

in the mammary gland and the pig CTLA4Ig fusion

protein is secreted into the milk, we collected milk from

mice of transgenic lines (#38 and #44), and checked for

binding of transgenic milk to CD80. Milk from transgenic

and non-transgenic mice were incubated with

CHO cells that express the mouse CD80 on the cell

surface. Flow cytometry analysis of cells incubated

with transgenic milk showed a positive shift of the

fluorescence (Fig. 2a). Non-transgenic controls

showed no fluorescence shift, indicating that there

was no non-specific binding of milk proteins to

CD80. Our binding assay confirmed that pig CTLA4Ig

was produced in the mammary gland and secreted into

the milk of the transgenic mice. Incubation of CD80-

expressing CHO cells with FITC-conjugated human

IgG Fc-specific antibody alone showed no binding and

no fluorescence signal (data not shown). Using a standard

binding curve generated from purified CTLA4Ig,

we determined the concentrations of CTLA4Ig in the

milk of transgenic mice #38 and #44 to be 14.8 and

12.5 Ag/ml, respectively (Fig. 2b).

CTLA4Ig was purified from the transgenic milk

using protein A agarose resin. 250–300 Al of milk was

collected from each mouse and approximately 3 Ag of

CTLA4Ig was purified from the milk. This indicated

that percentage yield of CTLA4Ig fusion protein from

the transgenic milk by protein A agarose resin was

about 80.5%. We then analysed the milk CTLA4Ig

preparation by denaturing polyacrylamide gel electrophoresis,

and Western blotting analysis. Protein A

binds preferably to the Fc domain of human immunoglobulin

(Ig) but it may also bind to other milk proteins

such as mouse Ig. To test the non-specific binding of

mouse milk proteins to the resin, we included the

protein A bound fraction of non-transgenic milk as a

negative control. On the Coomassie blue-stained gel, a

major protein band of 58 kDa, which is the predicted

molecular weight of CTLA4Ig, was observed in the

lane of protein A column purified protein fraction of

transfected cells (Fig. 3a, lane 3). However, the 58-kDa

protein band was not observed in the lane of culture

medium of non-transfected cells (Fig. 3a, lane 2). The

58-kDa protein product was specifically recognised on

the Western blot using anti-human IgG antibody as a

developing agent, indicating that the 58-kDa protein

band represents the CTLA4Ig fusion protein (Fig. 3b).

The intense protein band of about 70 kDa in the nontransfected

cells would be the bovine serum albumin in

the culture medium (Fig. 3a, lane 1). A major protein

band of 58 kDa on the Coomassie blue-stained gel was


178

V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183

Fig. 2. Flow cytometry analysis of the binding of transgenic mouse milk to mouse CD80. Panel a: CHO cells expressing CD80 were incubated

with transgenic milk. As a control, the CD80-expressing CHO cell was incubated with non-transgenic milk. Fluorescence profiles of the

incubation with transgenic milk of #38 (thin black line), #44 (thick black line), purified pig CTLA4Ig (black broken line), non-transgenic milk

(grey line) and phosphate-buffered saline (grey broken line) are shown. Panel b: Standard binding curve of pig CTLA4Ig to CD80-expressing

CHO cells. Mean fluorescence intensities of the binding at different concentrations of CTLA4Ig were determined. Mean fluorescence

intensities ( y-axis) were plotted against the logarithm of the concentrations of pCTLA4Ig (Ag purified pCTLA4Ig/100 Al of reaction mixture;

x-axis). Mean fluorescence intensities of binding of transgenic milk (5 Al) of #38 (broken arrow) and #44 (dotted arrow) to CD80-expressing

CHO cells were determined. The respective CTLA4Ig concentrations in the transgenic milk were read from the standard binding curve.

observed in the lanes of protein A affinity-purified

fractions of transfected cells (Fig. 3c, lane 1) and

transgenic milk (Fig. 3c, lane 3), but not in the protein

A resin bound fraction of non-transgenic milk (Fig. 3c,

lane 2). The same 58-kDa protein band in the lanes of

transfected cells and transgenic milk was specifically

recognised by anti-human IgG antibody (Fig. 3d).

3.3. Milk CTLA4Ig is active in suppressing lymphocyte

proliferation

The activity of purified pig CTLA4Ig from transfected

culture medium was tested in rat, pig, and human

mixed lymphocytes reaction (MLR). The purified

CTLA4Ig suppressed the incorporation of [ 3 H]-thymidine

of lymphocytes in human, rat and pig MLRs albeit

at different efficiencies (Fig. 4a–c). The purified

CTLA4Ig from transfected cultures was effective in

suppression of MLRs in pig, but less so in both rat and

human MLR (Fig. 4a–c). The EC 50 of CTLA4Ig on the

suppressionofMLRsinrat,pigandhumanwere

shown to be 0.25, 0.18 and 5.6 Ag/ml, respectively.

Purified CTLA4Ig from transgenic milk was able

to bind to CD80 (data not shown) and was effective in

the suppression of rat lymphocyte proliferation in

MLR (Fig. 4d). The purified CTLA4Ig showed 98%

Fig. 3. SDS-PAGE and Western blotting analysis of purified pig CTLA4Ig protein. Panels a and b: Polyacrylamide gel electrophoresis of

purified pig CTLA4Ig from transfected Cos-1 cells (lane 3), and medium of non-transfected cells (lane 2). The gel was stained with Coomassie

blue to visualise the protein bands on the gel, or for Western blot analysis using HRP-conjugated anti-human immunoglobulin antibody. Panels c

and d: Polyacrylamide gel electrophoresis of purified pig CTLA4Ig from transfected Cos-1 cells (lane 1), protein A resin bound fraction of nontransgenic

milk (lane 2), and affinity-purified pig CTLA4Ig from transgenic milk (lane 3). The gel was stained with Coomassie blue (c) to

visualise the protein bands on the gel, or for Western blot analysis using HRP-conjugated anti-human immunoglobulin antibody (d).


V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183 179

Fig. 4. Immunosuppressive activity of purified pig CTLA4Ig from transgenic milk. Panels a–d: Comparison of immunosuppressive activities of

pig CTLA4Ig in pig, rat, and human mixed lymphocyte reaction. Panels a–c: Mean [ 3 H]-thymidine incorporation (counts per minute, cpm; grey

bar) and standard error in the mixed lymphocyte reactions at different concentrations (Ag/ml) of pig CTLA4Ig in the mixed lymphocyte

reactions are shown. Panel d: Mean [ 3 H]-thymidine incorporation (cpm; grey bar) and standard error in the mixed lymphocyte reactions in

untreated control (UT), in purified CTLA4Ig from transgenic mice (Tg), and protein A bound fraction of non-transgenic milk treated culture

(NTg) are shown. Percentage suppression (% sup) in different treatments was indicated underneath the bar charts.

inhibition of proliferation compared to the untreated

control. The protein A bound fraction of non-transgenic

milk showed no suppression in lymphocytes

proliferation as compared to the untreated culture.

4. Discussion

Proteins of pharmaceutical interests have been

successfully expressed in the milk of pigs, sheep,

goats and cattle using lactation-specific promoters.

In order to test if the immunosuppressive CTLA4Ig

fusion protein can be produced in the milk of animals

using the mammary gland as a bioreactor, we developed

transgenic mice. In this study, we produced pig

CTLA4 human Ig fusion protein in mouse milk using

the promoter of the sheep h-lactoglobulin gene (BLG)

to direct the expression of CTLA4Ig chimeric gene in

the mouse mammary gland.

The promoter of the sheep BLG gene has been

used to drive mammary gland-specific expression of

exogeneous genes in mouse (Archibald et al., 1990;

Whitelaw et al., 1992) and sheep (Clark et al., 1989;

Clark, 1998a,b; Niemann et al., 1999; Schnieke et al.,

1997; Wright et al., 1991) during lactation. We

employed the sheep BLG gene promoter to direct

the expression of a pig CTLA4 human Ig chimeric

gene to the mammary gland of mouse. The fusion

protein CTLA4Ig was secreted into the milk of transgenic

mice. In the two transgenic mice tested, the


180

V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183

yield of CTLA4Ig per ml of milk was 12.5 and 14.8

Ag. This compared favourably with the level of

protein produced in transfected cells which was only

3 Ag/ml (see Materials and methods). This indicates

that the production of CTLA4Ig using the transgenic

milk approach is four to five times more efficient than

that using cell transfection. Protein A agarose resin

was used to affinity purify CTLA4Ig from transgenic

milk. Polyacrylamide gel electrophoresis and Western

blotting analysis using anti-human IgG antibody

revealed a protein product of 58 kDa, which corresponds

to the predicted size of pig CTLA4Ig, in the

protein A bound fraction of transgenic milk only. Data

from the Western blotting analysis confirms successful

purification of CTLA4Ig from transgenic milk.

The activity of purified CTLA4Ig of transgenic milk

was also investigated in rat MLR. The purified milkderived

CTLA4Ig bound CD80 and showed effective

immunosuppressive activity. The protein A bound

fraction of non-transgenic mouse milk displays no

binding activity to CD80 and no immunosuppressive

activity. Our data indicated that the CTLA4Ig could

be effectively purified from transgenic milk by protein

A affinity column chromatography and that the purified

CTLA4Ig retains its immunosuppressive activity.

Using the same sheep BLG gene promoter, a number

of human proteins have been produced in the milk of

transgenic mice and sheep at a wide range of yield

(Tables 1A and 1B). The yield of protein in milk by

transgenesis is dependent on the integration site and

the copy number of the transgene. The yield of

CTLA4Ig in the milk of our transgenic mice can be

increased by crossing the transgenic mice to obtain

mice carrying higher copy number of the CTLA4Ig

transgene. Over 30 therapeutic proteins have been

produced in the milk of transgenic goats, sheeps and

cows by pharmaceutical companies, and few of those

are now in various phases of clinical trial (Das, 2001).

In general, production of therapeutic proteins in transgenic

animal milk are considered to be 5–10 times

more cost-effective than cell culture production

(Betsch, 1995).

The pig CTLA4Ig showed variable activities in

suppressing T cell proliferation in different species.

The EC 50 of pig CTLA4Ig in rat, pig and human were

0.25, 0.18 and 5.6 Ag/ml, respectively. The pig

CTLA4Ig displays strong and comparable suppression

activities in pig, and rat mixed lymphocyte reactions.

The pig protein is 31-fold less effective in the suppression

of T cell proliferation in human than that in

pig and rat. The weaker immunosuppressive activity

of pig CTLA4Ig on human T cells in the present study

is probably due to the weak binding of pig CTLA4Ig

to CD80 and CD86 receptors on human APCs as

shown by Vaughan et al. (2000).

Transgenic females are fertile, transmit the transgene

to their offspring and fed their pups well. This

indicates that expression of the CTLA4 human Ig

chimeric transgene in the mammary gland has no

adverse effect on the fertility and lactation of mice.

Transgenic offspring develop normally and are fertile,

indicating that feeding on CTLA4Ig containing milk

has no deleterious effect on the development of the

neonates. It was possible that the mice that were fed

on the milk containing the immunosuppressant

CTLA4Ig might become immunocompromised and

prone to infections. However, none of the transgenic

mice showed symptoms of immunosuppression such

as diarrhoea or infections. A minute amount of

CTLA4Ig ( < 2Ag/ml) could be detected in the blood

of the transgenic mice and it could be a result of slight

‘‘leakiness’’ of the sheep BLG promoter. Although

this amount is very small when compared to that in

the transgenic milk (12.5–14.8 Ag/ml), this raises the

concern that the long-term presence of CTLA4Ig in

the serum may render the T cell anergic or the intrinsic

properties of T cells may be altered. To address this

issue, we tested the proliferation of T cells of the

transgenic mice using either (a) anti-CD3 antibody,

(b) anti-CD3 together with anti-CD28 antibodies, or

(c) concanavalin A. There was no significant difference

in TCR-mediated response between the transgenic

and the non-transgenic littermate controls (data

not shown), indicating that T cells from the transgenic

mice are not functionally compromised. Furthermore,

in psoriasis vulgaris patients receiving therapeutic

dose of CTLA4Ig, their serum level of CTLA4Ig

could be as high as 104 Ag/ml for a prolonged period

of time, and yet there was no evidence of tolerance

induction towards to a administered control antigen

(Abrams et al., 1999). Therefore, it is unlikely that the

low level of CTLA4Ig in the blood of the transgenic

mice will render them to be immunocompromised.

Using our transgenic strategy, mice can be genetically

engineered to produce ‘‘non-self’’ CTLA4Ig (pig

CTLA4 human Ig fusion protein in present study) in


V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183 181

the milk with no harmful effect on the animal. We

believe that the production of ‘‘non-self’’ CTLA4Ig

such as human CTLA4Ig in the milk of farm animals

would also have no harmful effect to the animals. This

implies that colony of transgenic farm animals could

be maintained indefinitely and used as bioreactors to

produce large quantities of human CTLA4Ig for

clinical application.

Acknowledgements

The study was supported by a grant from the Vice

Chancellor’s Development Fund of the University of

Hong Kong. We also thank A. Clark (Roslin Institute,

UK) for providing us with the sheep h-lactoglobulin

minigene construct pBJ41. We thank J. Lamb (University

of Edinburgh, UK) and J. Ausytn (University

of Oxford, UK) for their comments on the manuscript.

We also thank J. Wu for providing assistance in the

breeding and milking of the transgenic mice.

References

Abrams, J.R., Lebwohl, M.G., Guzzo, C.A., Jegasothy, B.V., Goldfarb,

M.T., Goffe, B.S., Menter, A., Lowe, N.J., Krueger, G.,

Brown, M.J., Weiner, R.S., Birkhofer, M.J., Warner, G.L., Berry,

K.K., Linsley, P.S., Krueger, J.G., Ochs, H.D., Kelley, S.L.,

Kang, S., 1999. CTLA4Ig-mediated blockade of T-cell costimulation

in patients with psoriasis vulgaris. J. Clin. Invest. 103,

1243.

Abrams, J.R., Kelley, S.L., Hayes, E., Kikuchi, T., Brown, M.J.,

Kang, S., Lebwohl, M.G., Guzzo, C.A., Jegasothy, B.V., Linsley,

P.S., Krueger, J.G., 2000. Blockade of T lymphocyte costimulation

with cytotoxic T lymphocyte-associated antigen 4-

immunoglobulin (CTLA4Ig) reverses the cellular pathology of

psoriatic plaques, including the activation of keratinocytes, dendritic

cells, and endothelial cells. J. Exp. Med. 192, 681.

Archibald, A.L., McClenaghan, M., Hornsey, V., Simons, J.P.,

Clark, A.J., 1990. High-level expression of biologically active

human alpha 1-antitrypsin in the milk of transgenic mice. Proc.

Natl. Acad. Sci. U. S. A. 87, 5178.

Betsch, D.F., 1995. Pharmaceutical Production from Transgenic

Animals. Biotechnology Information Series. North Central Regional

Extension Publication NCR #552, 1 – 3.

Bijvoet, A.G., Kroos, M.A., Pieper, F.R., de Boer, H.A., Reuser,

A.J., van der Ploeg, A.T., Verbeet, M.P., 1996. Expression of

cDNA-encoded human acid alpha-glucosidase in milk of transgenic

mice. Biochim. Biophys. Acta 1308, 93–96.

Brem, G., Hartl, P., Besenfelder, U., Wolf, E., Zinovieva, N., Pfaller,

R., 1994. Expression of synthetic cDNA sequences encoding

human insulin-like growth factor-1 (IGF-1) in the mammary

gland of transgenic rabbits. Gene 149, 351.

Chen, C.M., Wang, C.H., Wu, S.C., Lin, C.C., Lin, S.H., Cheng,

W.T., 2002. Temporal and spatial expression of biologically

active human factor VIII in the milk of transgenic mice driven

by mammary-specific bovine alpha-lactalbumin regulation sequences.

Transgenic Res. 11, 257–268.

Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA

isolation by acid guanidinium thiocyanate–phenol–chloroform

extraction. Anal. Biochem. 162, 156.

Clark, A.J., 1998a. Gene expression in the mammary glands of

transgenic animals. Biochem. Soc. Symp. 63, 133.

Clark, A.J., 1998b. The mammary gland as a bioreactor: expression,

processing, and production of recombinant proteins. J. Mammary

Gland Biol. Neoplasia 3, 337.

Clark, A.J., Ali, S., Archibald, A.L., Bessos, H., Brown, P., Harris,

S., McClenaghan, M., Prowse, C., Simons, J.P., Whitelaw, C.B.,

1989. The molecular manipulation of milk composition. Genome

31, 950.

Coulibaly, S., Besenfelder, U., Fleischmann, M., Zinovieva, N.,

Grossmann, A., Wozny, M., Bartke, I., Togel, M., Muller, M.,

Brem, G., 1999. Human nerve growth factor beta (hNGF-beta):

mammary gland specific expression and production in transgenic

rabbits. FEBS Lett. 444, 111.

Das, R.C., 2001. Production of therapeutical proteins from transgenic

animals. BioBussiness, 60–64 (Feb.).

Devinoy, E., Thepot, D., Stinnakre, M.G., Fontaine, M.L., Grabowski,

H., Puissant, C., Pavirani, A., Houdebine, L.M., 1994. High

level production of human growth hormone in the milk of transgenic

mice: the upstream region of the rabbit whey acidic protein

(WAP) gene targets transgene expression to the mammary

gland. Transgenic Res. 3, 79–89.

Ebert, K.M., Selgrath, J.P., DiTullio, P., Denman, J., Smith, T.E.,

Memon, M.A., Schindler, J.E., Monastersky, G.M., Vitale, J.A.,

Gordon, K., 1991. Transgenic production of a variant of human

tissue-type plasminogen activator in goat milk: generation of

transgenic goats and analysis of expression. Biotechnology

(N.Y.) 9, 835.

Ebert, K.M., DiTullio, P., Barry, C.A., Schindler, J.E., Ayres, S.L.,

Smith, T.E., Pellerin, L.J., Meade, H.M., Denman, J., Roberts,

B., 1994. Induction of human tissue plasminogen activator in

the mammary gland of transgenic goats. Biotechnology (N.Y.)

12, 699.

Echizenya, H., Yamashita, K., Takehara, M., Konishi, K., Nomura,

M., Yanagida, N., Kitagawa, N., Kobayashi, T., Furukawa, H.,

Inobe, M., Uede, T., Todo, S., 2001. Adenovirus-mediated

CTLA4-IgG gene therapy in orthotopic small intestinal transplantation

in rats. Transplant. Proc. 33, 183.

Edmunds, T., Van Patten, S.M., Pollock, J., Hanson, E., Bernasconi,

R., Higgins, E., Manavalan, P., Ziomek, C., Meade, H., McPherson,

J.M., Cole, E.S., 1998. Transgenically produced human

antithrombin: structural and functional comparison to human

plasma-derived antithrombin. Blood 91, 4561.

Feinberg, A.P., Vogelstein, B., 1984. ‘‘A technique for radiolabeling

DNA restriction endonuclease fragments to high specific activity’’.

Addendum. Anal. Biochem. 137, 266.

Feng, S., Quickel, R.R., Hollister-Lock, J., McLeod, M., Bonner-


182

V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183

Weir, S., Mulligan, R.C., Weir, G.C., 1999. Prolonged xenograft

survival of islets infected with small doses of adenovirus expressing

CTLA4Ig. Transplantation 67, 1607.

Greenberg, N.M., Anderson, J.W., Hsueh, A.J., Nishimori, K.,

Reeves, J.J., deAvila, D.M., Ward, D.N., Rosen, J.M., 1991.

Expression of biologically active heterodimeric bovine follicle-stimulating

hormone in milk of transgenic mice. Proc. Natl.

Acad. Sci. U. S. A. 88, 8327–8331.

Guillot, C., Mathieu, P., Coathalem, H., Le Mauff, B., Castro, M.G.,

Tesson, L., Usal, C., Laumonier, T., Brouard, S., Soulillou, J.P.,

Lowenstein, P.R., Cuturi, M.C., Anegon, I., 2000. Tolerance to

cardiac allografts via local and systemic mechanisms after adenovirus-mediated

CTLA4Ig expression. J. Immunol. 164, 5258.

Guinan, E.C., Boussiotis, V.A., Neuberg, D., Brennan, L.L., Hirano,

N., Nadler, L.M., Gribben, J.G., 1999. Transplantation of

anergic histoincompatible bone marrow allografts. N. Engl. J.

Med. 340, 1704.

Haberman, B.H., 1974. Mechanical milk collection from mice for

Bittner virus isolation. Lab. Anim. Sci. 24, 935.

Hayashi, S., Guang-Lin, M., Yokoyama, I., Namii, Y., Hamada, H.,

Nakao, A., 2000. Adenovirus-mediated gene transfer of CT-

LA4Ig gene results in prolonged survival of heart allograft.

Transpl. Int. 13 (Suppl. 1), S329.

Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Production

of transgenic mice. Manipulating the Mouse Embryo,

A Laboratory Manual. Cold Spring Harbor Laboratory Press,

New York, USA, pp. 219–251.

Kirk, A.D., Harlan, D.M., Armstrong, N.N., Davis, T.A., Dong, Y.,

Gray, G.S., Hong, X., Thomas, D., Fechner, J.H.J., Knechtle, S.J.,

1997. CTLA4-Ig and anti-CD40 ligand prevent renal allograft

rejection in primates. Proc. Natl. Acad. Sci. U. S. A. 94, 8789.

Ko, J.H., Lee, C.S., Kim, K.H., Pang, M.G., Koo, J.S., Fang, N.,

Koo, D.B., Oh, K.B., Youn, W.S., Zheng, G.D., Park, J.S., Kim,

S.J., Han, Y.M., Choi, I.Y., Lim, J., Shin, S.T., Jin, S.W., Lee,

K.K., Yoo, O.J., 2000. Production of biologically active human

granulocyte colony stimulating factor in the milk of transgenic

goat. Transgenic Res. 9, 215.

Krimpenfort, P., Rademakers, A., Eyestone, W., van der Schans, A.,

van den Broek, S., Kooiman, P., Kootwijk, E., Platenburg, G.,

Pieper, F., Strijker, R., 1991. Generation of transgenic dairy

cattle using ‘in vitro’ embryo production. Biotechnology (N.Y.)

9, 844.

Kurlberg, G., Haglind, E., Schon, K., Tornqvist, H., Lycke, N.,

2000. Blockade of the B7-CD28 pathway by CTLA4-Ig counteracts

rejection and prolongs survival in small bowel transplantation.

Scand. J. Immunol. 51, 224.

Laemmli, U.K., 1970. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680.

Lee, C.S., Kim, K., Yu, D.Y., Lee, K.K., 1996. An efficient expression

of human growth hormone (hGH) in the milk of transgenic

mice using rat beta-casein/hGH fusion genes. Appl. Biochem.

Biotechnol. 56, 211–222.

Lenschow, D.J., Zeng, Y., Thistlethwaite, J.R., Montag, A., Brady,

W., Gibson, M.G., Linsley, P.S., Bluestone, J.A., 1992. Longterm

survival of xenogeneic pancreatic islet grafts induced by

CTLA4lg. Science 257, 789.

Levisetti, M.G., Padrid, P.A., Szot, G.L., Mittal, N., Meehan, S.M.,

Wardrip, C.L., Gray, G.S., Bruce, D.S., Thistlethwaite, J.R.J.,

Bluestone, J.A., 1997. Immunosuppressive effects of human

CTLA4Ig in a non-human primate model of allogeneic pancreatic

islet transplantation. J. Immunol. 159, 5187.

Limonta, J.M., Castro, F.O., Martinez, R., Puentes, P., Ramos, B.,

Aguilar, A., Lleonart, R.L., de la Fuente, J., 1995. Transgenic

rabbits as bioreactors for the production of human growth hormone.

J. Biotechnol. 40, 49.

Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L.S., Damle, N.K.,

Ledbetter, J.A., 1991. CTLA-4 is a second receptor for the B

cell activation antigen B7. J. Exp. Med. 174, 561.

Linsley, P.S., Greene, J.L., Brady, W., Bajorath, J., Ledbetter, J.A.,

Peach, R., 1994. Human B7-1 (CD80) and B7-2 (CD86) bind

with similar avidities but distinct kinetics to CD28 and CTLA-4

receptors. Immunity 1, 793.

Niemann, H., Halter, R., Carnwath, J.W., Herrmann, D., Lemme,

E., Paul, D., 1999. Expression of human blood clotting factor

VIII in the mammary gland of transgenic sheep. Transgenic Res.

8, 237.

Paleyanda, R.K., Velander, W.H., Lee, T.K., Scandella, D.H.,

Gwazdauskas, F.C., Knight, J.W., Hoyer, L.W., Drohan, W.N.,

Lubon, H., 1997. Transgenic pigs produce functional human

factor VIII in milk. Nat. Biotechnol. 15, 971.

Pearson, T.C., Alexander, D.Z., Winn, K.J., Linsley, P.S., Lowry,

R.P., Larsen, C.P., 1994. Transplantation tolerance induced by

CTLA4-Ig. Transplantation 57, 1701.

Platenburg, G.J., Kootwijk, E.P., Kooiman, P.M., Woloshuk, S.L.,

Nuijens, J.H., Krimpenfort, P.J., Pieper, F.R., de Boer, H.A.,

Strijker, R., 1994. Expression of human lactoferrin in milk of

transgenic mice. Transgenic Res. 3, 99–108.

Prunkard, D., Cottingham, I., Garner, I., Bruce, S., Dalrymple, M.,

Lasser, G., Bishop, P., Foster, D., 1996. High-level expression

of recombinant human fibrinogen in the milk of transgenic mice.

Nat. Biotechnol. 14, 867–871.

Rodriguez, A., Castro, F.O., Aguilar, A., Ramos, B., Del Barco,

D.G., Lleonart, R., De la Fuente, J., 1995. Expression of active

human erythropoietin in the mammary gland of lactating transgenic

mice and rabbits. Biol. Res. 28, 141–153.

Rokkones, E., Fromm, S.H., Kareem, B.N., Klungland, H., Olstad,

O.K., Hogset, A., Iversen, J., Bjoro, K., Gautvik, K.M., 1995.

Human parathyroid hormone as a secretory peptide in milk of

transgenic mice. J. Cell. Biochem. 59, 168–176.

Romano, G., Michell, P., Pacilio, C., Giordano, A., 2000. Latest

developments in gene transfer technology: achievements, perspectives,

and controversies over therapeutic applications. Stem

Cells 18, 19.

Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R.,

Ritchie, M., Wilmut, I., Colman, A., Campbell, K.H., 1997.

Human factor IX transgenic sheep produced by transfer of nuclei

from transfected fetal fibroblasts. Science 278, 2130.

Sebille, F., Vanhove, B., Soulillou, J.P., 2001. Mechanisms of tolerance

induction: blockade of co-stimulation. Philos. Trans. R.

Soc. Lond., B Biol. Sci. 356, 649.

Shani, M., Barash, I., Nathan, M., Ricca, G., Searfoss, G.H., Dekel,

I., Faerman, A., Givol, D., Hurwitz, D.R., 1992. Expression of

human serum albumin in the milk of transgenic mice. Transgenic

Res. 1, 195–208.


V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183 183

Simons, J.P., McClenaghan, M., Clark, A.J., 1987. Alteration of the

quality of milk by expression of sheep beta-lactoglobulin in

transgenic mice. Nature 328, 530.

Sohn, B.H., Kim, S.J., Park, H., Park, S.K., Lee, S.C., Hong, H.J.,

Park, Y.S., Lee, K.K., 1999. Expression and characterization of

bioactive human thrombopoietin in the milk of transgenic mice.

DNA Cell Biol. 18, 845–852.

Thepot, D., Devinoy, E., Fontaine, M.L., Stinnakre, M.G., Massoud,

M., Kann, G., Houdebine, L.M., 1995. Rabbit whey acidic

protein gene upstream region controls high-level expression of

bovine growth hormone in the mammary gland of transgenic

mice. Mol. Reprod. Dev. 42, 261.

Toman, P.D., Pieper, F., Sakai, N., Karatzas, C., Platenburg, E.,

de Wit, I., Samuel, C., Dekker, A., Daniels, G.A., Berg, R.A.,

Platenburg, G.J., 1999. Production of recombinant human type I

procollagen homotrimer in the mammary gland of transgenic

mice. Transgenic Res. 8, 415–427.

Tomasoni, S., Azzollini, N., Casiraghi, F., Capogrossi, M.C., Remuzzi,

G., Benigni, A., 2000. CTLA4Ig gene transfer prolongs

survival and induces donor-specific tolerance in a rat renal allograft.

J. Am. Soc. Nephrol. 11, 747.

Turka, L.A., Linsley, P.S., Lin, H., Brady, W., Leiden, J.M., Wei,

R.Q., Gibson, M.L., Zheng, X.G., Myrdal, S., Gordon, D., 1992.

T-cell activation by the CD28 ligand B7 is required for cardiac

allograft rejection in vivo. Proc. Natl. Acad. Sci. U. S. A. 89,

11102.

Uusi-Oukari, M., Hyttinen, J.M., Korhonen, V.P., Vasti, A., Alhonen,

L., Janne, O.A., Janne, J., 1997. Bovine alpha s1-casein

gene sequences direct high level expression of human granulocyte-macrophage

colony-stimulating factor in the milk of transgenic

mice. Transgenic Res. 6, 75–84.

van Berkel, P.H., Welling, M.M., Geerts, M., van Veen, H.A., Ravensbergen,

B., Salaheddine, M., Pauwels, E.K., Pieper, F., Nuijens,

J.H., Nibbering, P.H., 2002. Large scale production of

recombinant human lactoferrin in the milk of transgenic cows.

Nat. Biotechnol. 20, 484.

Van Cott, K.E., Williams, B., Velander, W.H., Gwazdauskas, F.,

Lee, T., Lubon, H., Drohan, W.N., 1996. Affinity purification

of biologically active and inactive forms of recombinant human

protein C produced in porcine mammary gland. J. Mol. Recognit.

9, 407.

Vaughan, A.N., Malde, P., Rogers, N.J., Jackson, I.M., Lechler,

R.I., Dorling, A., 2000. Porcine CTLA4-Ig lacks a MYPPPY

motif, binds inefficiently to human B7 and specifically suppresses

human CD4+ T cell responses costimulated by pig but

not human B7. J. Immunol. 165, 3175.

Velander, W.H., Johnson, J.L., Page, R.L., Russell, C.G., Subramanian,

A., Wilkins, T.D., Gwazdauskas, F.C., Pittius, C., Drohan,

W.N., 1992. High-level expression of a heterologous protein in

the milk of transgenic swine using the cDNA encoding human

protein C. Proc. Natl. Acad. Sci. U. S. A. 89, 12003.

Whitelaw, C.B., Harris, S., McClenaghan, M., Simons, J.P., Clark,

A.J., 1992. Position-independent expression of the ovine betalactoglobulin

gene in transgenic mice. Biochem. J. 286 (Pt 1),

31.

Wolf, E., Jehle, P.M., Weber, M.M., Sauerwein, H., Daxenberger,

A., Breier, B.H., Besenfelder, U., Frenyo, L., Brem, G., 1997.

Human insulin-like growth factor I (IGF-I) produced in the

mammary glands of transgenic rabbits: yield, receptor binding,

mitogenic activity, and effects on IGF-binding proteins. Endocrinology

138, 307.

Wright, G., Carver, A., Cottom, D., Reeves, D., Scott, A., Simons,

P., Wilmut, I., Garner, I., Colman, A., 1991. High level expression

of active human alpha-1-antitrypsin in the milk of transgenic

sheep. Biotechnology (N.Y.) 9, 830.

More magazines by this user
Similar magazines