Journal of Immunological Methods 277 (2003) 171–183
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
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: email@example.com (P.K.H. Tam), firstname.lastname@example.org (K.S.E. Cheah).
0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
V.C.H. Lui et al. / Journal of Immunological Methods 277 (2003) 171–183
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
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
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
Expression of proteins of pharmaceutical interests by lactationspecific
promoters in farm animals
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
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)
1 –3 mg/ml
(Ebert et al., 1991, 1994)
(Edmunds et al., 1998)
(Ko et al., 2000)
growth factor I
(Brem et al., 1994;
Wolf et al., 1997)
Human nerve growth
(Coulibaly et al., 1999)
Human growth hormone
(Limonta et al., 1995)
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
Expression of proteins of pharmaceutical interests by lactationspecific
promoters in transgenic mice
Mouse Bovine alpha-
N.A.: not available.
Human factor VIII
(Chen et al., 2002)
(Toman et al., 1999)
(Uusi-Oukari et al., 1997)
(Bijvoet et al., 1996)
(Platenburg et al., 1994)
(Sohn et al., 1999)
(Prunkard et al., 1996)
Human serum albumin
(Shani et al., 1992)
(Archibald et al., 1990)
Human growth hormone
(Lee et al., 1996)
(Greenberg et al., 1991)
Bovine growth hormone
(Thepot et al., 1995)
(Rodriguez et al., 1995)
Human growth hormone
(Devinoy et al., 1994)
(Rokkones et al., 1995)
0.02– 5.5 mg/ml
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
2. Materials and methods
2.1. Construction of pig CTLA4 human Ig chimeric
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
2.2. Generation of the pBLG-pCTLA4Ig transgenic
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
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
Primers for the amplification of sequence of the pig CTLA4 extracellular domain
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).
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
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-
At each CTLA4Ig concentration, the mean percentage
of suppression and standard error were determined
from triplicate wells.
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
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
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
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.
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
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
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
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.
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