(CANCER RESEARCH 52, 5647-5655. October 15. 1992)
Expression of Carcinoembryonic Antigen and Its Predicted Immunoglobulin-Iike
Domains in HeLa Cells for Epitope Analysis1
Laura J. F. Hefta,2 Fun-Shan Chen, Michael Ronk, Sybille L. Sauter,3 Virender Sarin, Shinzo Oikawa,
Hiroshi Nakazato, Stanley Hefta, and John E. Shively
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 9IOÃŒO
fL. J. F. H., F-S. C., M. R., S. L. S., S. //., J. E. S.J; Abbott
Laboratories, North Chicago, Illinois 60064 [f. S.J; and Suntory Institute for BiomédicalResearch, Osaka, Japan [S. O., H. N.J
ABSTRACT
Carcinoembryonic antigen (CEA) is a member of the immunoglobulin
gene superfamily with one predicted variable domain-like region (N
domain; 108 amino acids) and three sets of constant domain-like regions
(A1B1, A2B2, and A3B3; 92 amino acids for A domains and 86 amino
acids for B domains). In addition, CEA possesses two signal peptides,
one at the amino terminus and one at the carboxyl terminus. Both are
removed during posttranslational
processing, with the one at the car
boxyl terminus being replaced by a glycosylphosphatidylinositol
(GPI)
moiety. We have previously expressed the full length complementary
DNA clone for CEA in Chinese hamster ovary cells and murine L cells,
demonstrating proper processing of nascent polypeptide chains to ma
ture, fully glycosylated CEA including the GPI anchor. Using the same
full length CEA complementary DNA clone and the polyrnerase chain
reaction, we have now constructed expression clones for secreted ver
sions of the N domain, the A3B3 domain, and the A3 and B3 subdomains. The clones were expressed in HeLa cells using the ß-actinpro
moter. A stop codon was introduced at the end of the A3B3 and the A3
and B3 domains to allow secretion instead of retention on plasma mem
branes with the GPI anchor. Expressed products were purified to ho
mogeneity by affinity chromatography using monoclonal antibodies spe
cific for each domain and by reversed phase high pressure liquid
chromatography. Purified domains were characterized by Western blot
ting, antibody binding and inhibition studies, amino-terminal sequence
and amino acid analyses, and laser desorption/time of flight mass spectrometry. These analyses revealed that the monomeric N domain is of
size 15,990, with a glycosylation mass of about 4100, in good agreement
with two .V-li n ki-d glycosyl units of about mass 2100. There is some
evidence that the N domain forms dimers. The N domain reacted with
antibodies specific for this domain with an affinity similar to that of
intact CEA. The A3B3 domain had a mass of 34,462, with a glycosy
lation mass of 14,900, in good agreement with seven /V-linked glycosy
lation sites of average mass 2100. The A3B3 domain reacted only with
antibodies specific for this domain, with a slightly lower affinity than
that of native CEA. The amino-terminal sequences of the N domain and
A3B3 domain proteins demonstrated proper processing of the signal
peptide. We were unable to obtain expression products for the A3 and
B3 subdomains, suggesting that they were unable to fold and be ex
ported properly. We also expressed a secreted form of intact CEA and
nonspecific cross-reacting antigen (related to CEA). These expression
products had molecular sizes similar to those of their native counter
parts which possess the GPI moiety without the diacylglycerol mem
brane anchor.
INTRODUCTION
CEA4 was originally described by Gold and Freedman (1) as
an oncofetal glycoprotein observed in colon carcinomas. Its
Received 4/3/92; accepted 8/5/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported by National Cancer Institute Grant POI
CA43904.
2 To whom requests for reprints should be addressed, at Beckman Research
Institute of the City of Hope. 1450 Duarte Road. Duarte, CA 91010.
3 Current address: Baxter Healthcare. Immunotherapy Division. Santa Ana. CA
92705.
4 The abbreviations used are: CEA. Carcinoembryonic antigen: NCA. nonspe
cific cross-reacting antigen: BGP. biliary glycoprotein; Ig. immunoglobulin: GPI,
glycosylphosphatidylinositol; PCR, polyrnerase chain reaction; HPLC. high pres-
expression has also been found on the apical side of the crypts
in normal adult colon (2). Because of its presence on tumors,
CEA is a useful diagnostic marker for the progression of colon
carcinoma and for localization of primary and metastatic sites
of tumor growth (3). CEA is structurally and immunologically
related to several antigens including NCA (found in normal
lung and spleen and on granulocytes) (4, 5) and BGP I (ex
pressed on the epithelial membranes lining the bile ducts and
also in normal liver) (6-8). Each of these antigens has 30-50%
carbohydrate by weight and has been demonstrated to be a cell
surface glycoprotein. cDNA clones for CEA (9-12), NCA (1214), and BGP (15, 16) have been isolated and sequenced, re
vealing a high degree of amino acid sequence homology to each
other and placing them within the Ig gene superfamily (17-19).
The simplest member of the family, NCA, has an 108-amino
acid amino-terminal domain with no disulfide bonds, resem
bling an Ig variable region-like domain, and two Ig constant
region-like domains with one pair of disulfides each, designated
Al (92 amino acids) and Bl (86 amino acids) (for nomenclature
see Ref. 20). NCA and CEA have an amino-terminal signal
peptide of 34 amino acids and a carboxyl-terminal hydrophobic
peptide of 24-26 amino acids, which is removed during post
translational processing. The carboxyl-terminal peptide is re
placed with a GPI moiety, which anchors these glycoproteins
into the membrane with a diacylglycerol group (21, 22). The
domain structure of CEA resembles that of NCA, but CEA has
four additional Ig-like constant domains designated A2, B2,
A3, and B3. High internal sequence homology is observed for
the Al, A2, and A3 domains and for the Bl, B2, and B3 do
mains. The relationship of these domains to epitopes recog
nized by monoclonal antibodies to CEA is an area of intense
interest.
Use of polyclonal antibodies to CEA in cancer tests has been
problematic due to their possible cross-reaction with antigens
such as NCA and BGP, necessitating their absorption to nor
mal tissues such as spleen, liver, or lung. Monoclonal antibod
ies have solved this problem to a large extent, since they should
react with single epitopes. Recently, we have compared a large
number of monoclonal antibodies to CEA in an international
workshop, using a simple cross-inhibition assay where the bind
ing of one labeled antibody to CEA is tested for inhibition
versus a large panel of antibodies (23). The majority of the
antibodies tested have been shown to recognize epitopes located
on the polypeptide portion of the CEA molecule. Since it was
expected that many of the antibodies would recognize a number
of epitopes close together in position/space, we simplified the
results by dividing the CEA molecule into five major groupings,
designated Gold 1-5 (named after the discoverer of CEA), cor
responding to epitopes which are characterized by complete
inhibitions with a given antibody. Thus, these Gold epitopes
sure liquid chromatography; EIA, enzyme immunoassay; TFA, trifluoroacetic
acid; PBS, phosphate-buffered saline: LD/TOF-MS. laser desorption/time of
flight mass spectrometry: Mah. monoclonal antibody; SDS. sodium dodecyl sul
fate; BSA, bovine serum albumin; cDNA, complementary DNA.
5647
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EPITOPES
IN EXPRESSED
refer to major spatial groupings on the CEA molecule and not
to specific amino acid sequences.
In this report we have begun to refine the epitope analysis of
CEA by expressing several of its domains as secreted molecules
in HeLa cells and testing their binding to monoclonal anti-CEA
antibodies. These studies also test the predicted domain struc
ture of CEA, since only correctly folded domains are expected
to retain antigenic activity (many of the CEA antigenic deter
minants are conformationally dependent) (23). Recently, we
have expressed CEA domains as fusion proteins in Escherichia
coli (24), demonstrating some retention of antigenic activity.
However, CEA domains produced in E. coli may not fold prop
erly and lack the extensive glycosylation found in native CEA.
It is likely that the glycosyl units exert steric effects on antibody
binding, thus necessitating a study involving expressed domains
from eukaryotic cells. Previously we have expressed CEA and
NCA in two rodent cell lines, demonstrating correct posttranslational processing and insertion into the membrane via the
GPI anchor and retention of antigenic activity (25). We found,
however, that the glycosylation, especially for NCA, was dra
matically different in terms of size and extent of glycosylation
in the rodent cell lines, compared with antigen purified from
human colon tumors. We, therefore, chose in this study to
express secreted forms of CEA, NCA, and several domains of
CEA in the human cell line HeLa, which has an epithelial-like
morphology and should glycosylate more normally. The results
demonstrate that the domains are highly glycosylated and are
able to fold properly, as demonstrated by retention of their
antigenic activity.
MATERIALS
AND METHODS
Synthesis of Secreted Forms, Individual Domains, and Subdomains.
The secreted forms of CEA and NCA were made using the Mutagene
phagemid kit from Bio-Rad. The CEA cDNA sequence from clone
pCEA14 (unpublished clone with the same coding region sequence as
that published in Ref. 18) was subcloned as a 2800-base pair EcoRl
fragment into the vector pTZ19U, supplied in the kit, to create clone
CEA/pTZ19U. The oligonucleotide CEA-mut-C3 (see Table 1) was
used according to manufacturer's directions to mutate Gly644 to a stop
codon (GGA to TGA). The process was repeated for NCA by subcloning the Sspl/EcoRl fragment from cDNA clone pNCA13 (13) into
pTZ19U, creating clone NCA/pTZ19U, and then mutagenizing with
oligo-NCA-mut-C2. This caused a glycine to stop mutation (GGA to
TGA) at codon 286. The presence of each mutation was verified by
DNA sequencing. The mutated clones for CEA and NCA were then
subcloned into the expression vector pH/JAPR-l-neo (26) to create
clones CEA-s/pH/3 and NCA-s/pH/3, respectively.
Table 1 Sequence of oligonudeotides used to generate CEA gene constructs
Mutated nucleotides to produce stop codons are indicated in bold type. Re
striction enzyme sites are underlined.
Sequence 5' to 3'
Oligonucleotide
CEA-mut-C3
NCA-mut-C2
CEA-PCR-S
CEA-PCR-A
CEA-N3H
CEA-35A
CEA-33H
CEA-A3H
CEA-B3A
GAAGTTCAAGATGCAGAG
GGAGAAGTTCAAGATGCAGA
CCGTCGACAGAGGAGGACAGAGCAGACAGC
San
CCGACGTCGGCAGTGGTGGGCGGGTTCCAG
Aatll
CCAAGCTTC TATACCCGGAACTGCCCAGTT
Hindm
GGGACGTCATCACAGTCTCTGCGGAGCTGC
Aatll
CCAAGCTTTCA AGATGCAGAGACTGTGATG
Hindm
CTAGCCAAGCTTC TAGAGGACATCCAGGGTCACT
Hintail
GATCGGGACGTCGGGCCGGACACCCCCATCATTT
Aat\\
DOMAINS OF CEA
A secreted form of the CEA N domain was prepared by amplifying
the leader sequence and N domain from cDNA clone CEA/pTZ19U
using PCR (27). The oligonudeotides CEA-PCR-S and CEA-N3H (see
Table 1) were used. CEA-PCR-S contained a Sail site and CEA-N3H a
Hindlll site. CEA-N3H also introduced a stop codon at Tyr107 (TAC to
TAG) of CEA. After 1.5 min at 95°C,40 rounds of amplification were
carried out with the following program: 95°Cfor 1 min, 50°Cfor 2 min,
and 72°Cfor 4 min. During the final cycle the extension time was
lengthened to 8 min. The PCR product was purified on a 1.5% agarose
gel, digested with Sail and Hindlll, cloned into pTZ19U, and sequenced to verify that the entire sequence including the Tyr107 to stop
mutation was correct. The Sall/Hindlll fragment was then subcloned
into pH/3APR-l-neo for expression (N domain/pH^).
In order to be able to express the third repeat domain (A3B3) and the
A3 and B3 subdomains of CEA, these domains had to be fused in-frame
to a leader sequence. This was done by PCR. The leader sequence of
CEA was amplified from clone CEA/pTZ19U using the CEA-PCR-S
and CEA-PCR-A oligonudeotides (see Table 1). CEA-PCR-A con
tained an Aatll site at its 5' end. To amplify the entire A3B3 domain,
oligonudeotides CEA-35A (containing an Aatll site) and CEA-33H
(containing a Hindlll site) were used (see Table 1). CEA-33H also
generated a stop codon by mutating Gly644 (GGA to TGA). Since the
nucleotide sequences at the start of domains Al and A3 are identical for
the first 85 base pairs, the sequence of CEA-53A included four codons
5' of the start of the A3 domain to eliminate competition of Al for the
PCR primer. Following an incubation at 95°Cfor 1.5 min, the leader
and A3B3 domains were amplified for 40 cycles of 1 min at 95°C,2 min
at 60°C,and 4 min at 72°C.As described above, the extension time
during the final cycle was lengthened to 8 min. The PCR products were
purified on a 2% agarose gel, the leader product was digested with
Sall/Aatll, and the A3B3 product was digested with Aatll/Hindlll. The
digested products were ligated into pTZ19U in a three-fragment ligation. A colony hybridization was performed on colonies of E. coli strain
DH5«transformed with the ligation, using separate 32P-labeled oligo
nudeotides internal to the leader and A3B3 domains. Those clones
which hybridized to both oligonudeotides were sequenced to determine
if the entire sequence was correct, especially at the junction of the leader
and A3 domains, and also to confirm the presence of the stop codon
mutation.
The expression construct of the A3 domain alone was prepared by
amplifying the A3B3/pTZ19U construct with oligonudeotides CEAPCR-S and CEA-A3H. CEA-A3H contained a Hindlll site and gener
ated a stop codon from Tyr557. The PCR product was purified on a 2%
agarose gel, digested with Sall/Hindlll, and subcloned into pTZ19U.
The B3 construct was made by amplifying the B3 domain from CEA/
pTZ19U with oligonudeotides CEA-B3A and CEA-33H. The PCR
product was also purified from a 2% agarose gel, digested with Aatll/
Hindlll, and substituted for the Aatll/Hindlll A3B3 domain fragment
in the A3B3/pTZ19U clone. The PCR reaction conditions for ampli
fication of both the A3 and B3 subdomains were carried out as de
scribed above, except that the annealing temperature was 55°C.Both
clones were sequenced in their entirety, to ensure that the sequences of
both constructs were correct. The Sall/Hindlll inserts of the A3B3, A3,
and B3 constructs were subcloned into pH/3APr-l-neo for expression.
The constructs made are outlined in Fig. 1.
All DNA sequencing was done on double-stranded DNA templates
using a Sequenase kit (United States Biochemicals). PCR reactions
were set up using reagents and enzyme from a GeneAmp kit (PerkinElmer/Cetus) and were amplified using an Ericomp thermocycler (San
Diego, CA). All oligonudeotides were synthesized by the DNA Syn
thesis Facility at the City of Hope. All enzymes used were purchased
from Bethesda Research Laboratories, Boehringer Mannheim Biochemicals, or New England Biolabs, Inc.
Transfections. HeLa cells (ATCC CCL 2) were grown in Dulbecco's
modified Eagle medium, 10% calf serum, at 37°Cin 5% CO2. Twenty
Mgeach of CEA-f/pH/3, NCA-f/pH/3 (25), CEA-s/pH/3, NCA-s/pH0,
and N domain/pi M were linearized with Aatll. Twenty micrograms
each of the A3B3/pH/3, A3/pHß,and B3/pH/3 clones were linearized
with Pvul because they contained an internal Aatll site generated during
the construction of those clones.
5648
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EPITOPES
L
L
N
A3
A1
B1
A2
B2
E
A3
B3
L
B3
L
N
TTCTGGAACCCGCCCACCACTGCCSÄCSICATCACAGTCTCTGCGGAGCTGCCCAAGCCC
fwnpptt»
d Y itvsaelpkp
IN EXPRESSED
M
DOMAINS OF CEA
affinity columns were prewashed with elution buffer (0.1 Mglycine HCl,
pH 2.5; 100 ml; 2 ml/min) and application buffer (0.1 M phosphate
buffer, pH 8.0; 300 ml; 2 ml/min) and were incubated overnight with
media from transfected cells, using a recirculation peristaltic pump. The
affinity columns were washed with 240 ml of application buffer until
base-line readings at 280 nm were obtained, washed with 240 ml of
borate-buffered saline (0.1 Mborate, 1 Msodium chloride, 0.1% Tween20, pH 8.3) and 150 ml of application buffer, and eluted with eluting
buffer. Fractions (3 ml) containing protein were neutralized with 3 M
Tris, pH 11, pooled, loaded onto a 0.46-cm (i.d.) x 3.0-cm Brownlee
Aquapore Bu-300 reversed phase column, and eluted with a linear
gradient from 100% solvent A (0.1% TFA in water) to 100% solvent B
(TFA/water/acetonitrile, 0.1/9.9/90) over 60 min, at a flow rate of
1 ml/min. Fractions containing the major peak were pooled and con
centrated to remove acetonitrile. The protein concentration was deter
mined by amino acid analysis.
Amino Acid and Microsequence Analyses. Samples (1-2 Mg)were
hydrolyzed in the vapor phase with 6 N HCl for 24-48 h at 110°C.
Hydrolysates were analyzed on a Beckman 6300 amino acid analyzer.
Microsequence analysis was performed on 20-50 pmol of sample spot
ted on a 1- x 10-mm strip of polyvinylidine difluoride in a continuousFig. I. Strategy for generating CEA fragments by PCR. A full length cDNA
flow reactor microsequencer (30). Phenylthiohydantoin-amino
acids
clone for CEA was used as a template. A, domains are numbered according to
amino acid residue number, using the numbering scheme of Oikawa et al. (IO).
were separated and quantitated with an on-line Beckman System Gold
Primers (see Table 1) were used to generate PCR fragments corresponding to
HPLC with a 0.21-cm (i.d.) x 22-cm phenylthiohydantoin CIS column
CEA-L-A3B3 (leader sequence plus A3B3, ending in stop codon) (B), CEA-L-A3
(O. CEA-L-B3 (D), and CEA-L-N (E). F, sequence of the leader/A3B3 junction
(Applied Biosystems).
for the final product corresponding to CEA-L-A3B3 (B). The last eight amino
Laser Desorption/Time of Flight Mass Spectrometry. Samples
acids of the leader sequence are shown with the alanine residue highlighted to
(10-40 pmol) were dried in a vacuum concentrator, mixed with 3 M'of
indicate where cleavage of the signal peptide should occur. The next two residues
sinapinic acid (0.5 M in TFA/water/acetonitrile, 0.1/29.9/70), air dried
(underlined) were introduced as a result of the cloning strategy. The remainder of
on a 5-mm copper sample stage, and analyzed on a Shimadzu LAMS
the sequence corresponds to the beginning of A3B3. G. sequence of the leader/83
junction for the final product corresponding to CEA-L-B3 (D).
50KS time of flight mass spectrometer equipped with a frequencytripled neodymium-yttrium-aluminum-garnet
laser. The method is sim
The linearized plasmids (CEA-f/pH/3, CEA-s/pH/3, N domain/pH/î, ilar to that described by Beavis and Chait (31). Molecular weights were
A3B3/pH/3, A3/pH0, B3/pH/3, NCA-f/pH/3, and NCA-s/pHß) were calibrated with bovine insulin (5.734), horse apomyoglobin (16,951),
transfected into HeLa cells by lipofection as described previously for
and bovine serum albumin (65,795).
CHO-K1 cells (25), using 20-50 Mgof Lipofectin (Bethesda Research
Enzyme Immunoassays. Domain binding assays were performed as
Laboratories) per transfection. One million HeLa cells were used per
follows: 96-well polyvinyl microtiter plates (Costar) were coated with
transfection. For the membrane-bound constructs CEA-f/pHß and
rabbit anti-CEA antibody (DAKO Al 15) at a concentration of 1 Mg/ml
NCA-f/pH/3, the plating, lipofection, G418 (GIBCO) selection, and
in PBS (0.075 Msodium phosphate, 0.075 Msodium chloride, pH 7.0)
subcloning of high-expressing clones were carried out as described for
for 4 h at 37°C.The plates were washed 3 times with PBS, blocked with
CHO-K1 cells (25). except that 24 h after lipofection the medium was
1% BSA in PBS overnight at room temperature, and washed 3 times
removed and fresh Dulbecco's modified Eagle medium, 10% calf serum,
with PBS. The plates were incubated with 50 Ml/wellof purified antigen
was added, because HeLa cells are more sensitive to Lipofectin than are
(CEA, CEA-S, CEA-N, or CEA-A3B3) in PBS/BSA (50 mg/ml BSA)
CHO-K1 cells. For the remaining constructs which were expected to
at increasing dilutions of antigen, starting from 1 Mg/ml. for 1.5 h at
give secreted products, colonies were assayed using a filter-lift proce
37°Cand were washed 3 times with PBS. The plates were incubated
dure described by Walls and Grinnell (28).
with 100 Ml/wellof monoclonal antibody (T84.66 or T84.1,10 Mg/ml in
Two days after lipofection, G418 was added to each dish at a final
PBS) for 1.5 h at 37°Cand washed 3 times with PBS. The plates were
concentration of 1 mg/ml. Cells were selected for 18-19 days and
incubated with 100 M' of goat anti-mouse-alkaline phosphatase conju
medium was changed every 3-4 days until colonies appeared. The antigate (1/500 dilution; Cappel Labs) for 1.5 h at 37°Cand washed 3 times
CEA monoclonal antibody T84.1 (29), which cross-reacts with NCA,
with ethanolamine-buffered saline (1.5% ethanolamine, 0.15 Msodium
was used to detect CEA N domain- and NCA-secreting colonies. Mon
chloride, 0.5 IHMmagnesium chloride, adjusted to pH 9.3 with 0.1 M
oclonal antibody T84.66 (29), which reacts only with CEA, was used to
HCl). Substrate (100 Ml/well of/»-nitrophenylphosphate, 1 mg/ml in
detect the CEA- and A3B3-secreting colonies. We have evidence from
ethanolamine-buffered saline) was added for l h at 37°C.The reaction
other experiments that T84.66 reacts with the A3B3 domain of CEA
was stopped with 20 M!of 3 Msodium hydroxide. Absorbance at 405 nm
(24). Colonies eliciting dark spots on the filter were picked up through
was read on a MR600 Dynatech microplate reader.
the agarose overlay using sterile, cotton-plugged, Pasteur pipettes and
Inhibition assays were performed by either solid- or solution-phase
were transferred into separate wells of a 24-well microtiter plate. Media
EIA with antibodies T84.66 and H6C8, which were previously shown to
from confluent wells were checked for the presence of secreted product
have high specificity for domain A3B3 (24). In the solid-phase assay,
using an EIA (see below). Those colonies secreting the largest amounts
microtiter plates were coated with CEA (purified from human colon
of the respective product were expanded and subcloned once by limiting
tumors as described in Ref. 39, modified by the use of concanavalin A
dilution. This generated the individual clones designated as CEAchromatography; 1 Mg/ml in PBS) for 4 h at 37°C,washed with PBS,
A3B3-1 to -6.
All media and supplements, as well as Dulbecco's PBS and lx
blocked with 1% BSA in PBS, and washed. Monoclonal antibody
(0.3 Mg/ml for T84.66 and H6C8) was incubated with increasing
trypsin/EDTA, were purchased from J. R. Scientific, GIBCO, or Flow
amounts of inhibitor (CEA, CEA-S, CEA-N, CEA-A3B3, sA3, or sB3)
Laboratories, Inc.
overnight at 4°Cand added to the CEA-coated plates for 2 h at 37°C.
Affinity Purification of Secreted Antigens. Supernatants from trans
The plates were washed, and bound antibody was detected with goat
fected cell lines were pooled and run over affinity columns on a Phar
anti-mouse alkaline phosphatase conjugate as described above. Results
macia fast protein liquid chromatography system. Murine anti-CEA
Mabs (5-10 mg T84.1 or T84.66) were coupled to 3-5 ml of Actigel
were expressed as percentage of inhibition. For a description of the
(Sterogene, Arcadia, CA) according to manufacturer's instructions. The
synthetic peptide inhibitors sA3 and sB3, see below.
5649
TTCTGGAACCCGCCCACCACTGCCSACSICGGGCCGGACACCCCCATCATTTCCCCCCCA
fwnppttm
d j¿ gpdtpiispp
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EPITOPES
IN EXPRESSED
DOMAINS OF CEA
Table 2 HeLa cell transfectants expressing CEA and NCA genes
Expression
productCEA-S
were redissolved in 10% acetic acid at 1-2 mg/ml and diluted into PBS
(1/100) prior to analysis by EIA (see above). Amino acid composition
(/ig/liter)*20.7 and amino-terminal sequence analysis gave results in agreement with
the predicted structures.
level
(Mg/ml)00.20
CEA-N
CEA-N- 1
0.15
22.5
CEA-A3B3
CEA-A3B3-1
0.70
20.0
CEA-A3
CEA-A3-1
None
None
CEA-B3
CEA-B3-1
None
None
NCA-SCloneCEA-S-1
NCA-S- 1Production
0.50Yield
400
" Supernatant production levels in limiting dilution assays in 96-well microtiter
plates.
* Yields of affinity-purified product per liter of cell supernatant.
RESULTS
Cloning and Expression. Full length cDNA clones for CEA
and NCA were converted to secreted forms (CEA-S and
NCA-S) by introducing stop codons at the end of their last
constant region Ig domain (see Fig. 1). Using PCR we also
generated constructs from CEA-S which included the aminoIn the solution-phase inhibition assay, biotinylated CEA [0.3 Mg/ml;
terminal signal peptide plus the N domain (CEA-N), plus the
biotinylated according to the method of Wagener et al. (32)] was incu
A3B3 domains (CEA-A3B3), or plus the A3 (CEA-A3) or B3
bated with antibody (0.6 Mg/ml for T84.66 and H6C8) and increasing
(CEA-B3) domains. Each construct terminated in a stop codon
amounts of inhibitor (CEA, CEA-S, CEA-N, CEA-A3B3, sA3, or sB3)
and utilized the/3-actin promoter expression system of Gunning
and added to goat anti-mouse Ig-coated plates. Bound biotinylated
CEA was detected with avidin-horseradish peroxidase conjugate (dilut
et al. (26). Each of the plasmids was transfected into HeLa cells
ed 1/100; Sigma) as described (32). Results were expressed as percent
and the supernatants were screened for production of antigenage of inhibition, compared to maximum binding (no inhibitor).
ically active products. Previous studies (24) suggested that the
Western Blots. Western blots were performed according to the
CEA-N domain should be recognized by Mab T84.1 (Gold 5)
method of Towbin and Gordon (33). Samples (10-500 ng) were boiled
and the CEA-A3B3 domains by Mab T84.66 (Gold 1). Thus, we
for 5 min in sample buffer containing 1.5% SDS and 2.5% 2-mercapwere able to isolate 5-10 clones each that secreted CEA-N,
toethanol, electrophoresed on 12.5% polyacrylamide gels in a Pharma
CEA-A3B3, CEA-S, or NCA-S. In each case, the highest pro
cia Phast system, and electrotransferred to nitrocellulose membranes
with a Pharmacia Phast transfer unit (30 min, 20 V, in 25 miviTris, 192 ducers were selected for growth in T-25 flasks, supernatants
miviglycine, 20% methanol). Membranes were blocked with 3% BSA in were pooled, and the domains were affinity purified. The results
PBS for 60 min and incubated with anti-CEA Mab T84.66 or T84.1
are summarized in Table 2.
(1 Mg/ml) in PBS/0.05% Tween-20 for l h at room temperature, fol
Although a large number of drug-resistant clones were ob
lowed by detection with goat anti-mouse IgG-alkaline phosphatase con
tained for CEA-A3 and CEA-B3 transfectants, none secreted
jugate (diluted 1/5000; Jackson Immunoresearch), 5-bromo-4-chlorothese domains. In order to verify that the cells transfected with
3-indolylphosphate (50 Mg/ml), and nitro blue tetrazolium (100 Mg/ml) CEA-A3 and CEA-B3 were able to produce message corre
in pH 9.5 buffer (0.1 MTris-HCl, 0.1 MNaCl, 5 miviMgCl2). Prestained
sponding to these domains, we performed PCR analysis on
molecular weight markers (Bethesda Research Labs) were run to cal
several of the transfectants for each domain. These results (data
culate molecular weights.
not shown) demonstrated that message of the correct size was
Peptide Synthesis. Synthetic peptides corresponding to major por
made. Since it was possible that the A3 or B3 domains were
tions of the A3 and B3 domains of CEA were synthesized according to
translated but were not folded or exported properly, we ana
the method of Merrifield (34). Peptide sA3 (57-mer) had the follow
ing sequence, corresponding to residues 487-543 of the published se
lyzed the cell lysates with EIAs using polyclonal antibodies to
quence of CEA (10): FTCEPEAQNTTYLWWVNGQSLPVSPRLQLCEA. In each case, weak but positive signals were observed
SNGNRTLTLFNVTRNDARAYVCGIQNSV.
Peptide sB3 (72-mer)
(data not shown). We conclude that the cells were able to pro
had the following sequence, corresponding to residues 567-637:
duce and translate mRNA corresponding to A3 or B3 domains
PDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIA
but the protein was not folded or secreted properly and may
KITPNNNGTYACFVSNLATGRNNSIVKS1.
Fully protected pephave undergone subsequent proteolytic degradation in the cell.
tides were assembled by stepwise solid-phase synthesis on an automated
Purification and Structural Analysis. High producers were
Applied Biosystems peptide synthesizer. Small amounts of peptide res
selected for the other transfectants, media were collected, and
ins were removed during the synthesis and sequenced to confirm their
antigens were purified on antibody affinity columns. The puri
identities (35). The peptides were deprotected and cleaved from the
resin with anhydrous HF for l h at 0°Cin the presence of scavengers
fied antigens were analyzed by amino acid composition and
microsequence analysis. The results shown in Table 3 demon
(36). Crude peptides were purified by reversed phase HPLC on a C4
strate that, except for CEA-S, all of the secreted antigens agree
column. The major peaks were pooled and lyophilized. The peptides
Table 3 Amino acid analysis of purified expression products
CEA-A3B3
Amino acid
Obtained (mol)
Expected (mol)
CEA-N
Obtained (mol)
CEA-S
Expected (mol)
Obtained (mol)
Expected (mol)
NCA-S
Obtained (mol)
Expected (mol)
AsxThrSerGlxProGly»AlaValMetHeLeuTyrPheHisLysArg2412201313IS1213091454257271322121581213010146425710651353157071064344107514610580
' Substantial glycine background was not subtracted. This background is due to incomplete removal of the eluting buffer, which contains glycine.
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EPITOPES
IN EXPRESSED
DOMAINS OF CEA
Table 4 Amino-îerminalsequence analysis of purified expression products
amount of a second band at A/r 110,000 that reacts with Mab
Samples were spotted onto polyvinylidine difluoride membranes and microseT84.1 (Fig. 3/1) but not with T84.66, thus indicating that the A/r
quenced. Only the first 10 cycles are shown. Amino acids identified and yields are
110,000 fragment lacks the A3B3 domain. A small amount of
shown for each sample analyzed.
the
A3B3 domain (A/r 55,000) was detected in the CEA-S prep
CEA-A3B3"Cycle12345678910AminoacidAspValIleThrValSerAlaGinLeu*Yield(pmol)143024821913914CEA-N*AminoacidLysLeuThrIleGluSerThrProPheAsnMeld(pmol)54264141543678NCA
aration with the T84.66 Mab (Fig. 35). NCA-S has a predicted
molecular weight of 55,000 by Western blot analysis (Fig. 3),
which is slightly higher than the molecular weight of one of the
glycoforms of NCA shown in Fig. 3 [A/r 45,000; corresponding
to that published by Hefta et al. (37)]. NCA and NCA-S react
" 50 pmol were analyzed. The initial yield was 60% (calculated for cycle 2). Cycle
10 (*) was not identified due to a misinjection on the HPLC.
* 50 pmol were analyzed. The initial yield was 52% (calculated for cycle 2). A
secondary sequence corresponding to Tyr-Lys-Gly-Glu-Arg-Val-Asp-Gly-Asn-Arg
was also obtained.
' 40 pmol were analyzed. The initial yield was 42% (calculated for cycle 2).
well with their expected amino acid compositions. The results
shown in Table 4 show that the anticipated amino-terminal
sequence of each of the antigens (except CEA-S) was obtained.
In the case of CEA-N, a minor sequence corresponding to cleav
age at peptide bond Trp33-Tyr34 was observed. This cleavage
appeared to be due to storage of the sample in 0.1% TFA (at
4°C)and was observed to increase with time of storage. Al
though purified by affinity chromatography and reversed phase
HPLC, CEA-S appears to be contaminated with other proteins,
including fragments of CEA (see amino acid analysis; Table 3).
The reasons for this are not clear. Repeated attempts to purify
CEA-S from 0.5 liter of cell supernatant gave similar results.
Laser Desorption
Mass Spectrometry. The molecular
weights of two of the antigens were determined by LD/
TOF-MS (Fig. 2). The N domain gave a molecular weight of
15,990 (±0.5%),which when compared to the expected mass of
the polypeptide chain (11,880) predicts a glycosylation mass of
4110. Since there are two potential /V-glycosylation sites in
CEA-N, the average mass per chain is about 2100. This mass is
very close to the calculated mass for a completely sialylated
biantennary glycosyl unit (2204). It is interesting to note that
dimeric and trimeric forms of CEA-N were detected by LD/
TOF-MS. LD/TOF-MS of CEA-A3B3 gave a molecular weight
of 34,462 (±0.5%),which when compared to the predicted mo
lecular mass of the polypeptide chain (19,580) gives a glycosyl
mass of 14,900. Since the A3B3 domains have seven predicted
/V-glycosylation sites, the average mass per glycosyl unit is
about 2100. Thus, the predicted glycosyl units for this product
are also biantennary. This calculation does not preclude the
possibility of a mixture of more complex forms with the lower
molecular weight high-mannose forms. In the case of A3B3, no
dimeric forms were observed by LD/TOF-MS.
Western Blot Analysis. The molecular weights of the anti
gens were also assessed by Western blot analysis (Fig. 3).
CEA-S had a molecular weight of 140,000, compared to
180,000 for authentic CEA. The decrease in molecular weight is
probably due to lack of the GPI moiety, but in this case the
difference is exaggerated by SDS-gel electrophoresis (the GPI
anchor is predicted to have a mass of <2000). We have recently
obtained a more accurate molecular weight of 125,000 (±0.1 %)
for CEA by LD/TOF-MS5 but have been unsuccessful in ob
taining a good signal for CEA-S. CEA-S has a significant
5 Unpublished observations.
20.000
40,000
40.000
60.000
Fig. 2. Analysis of CEA-A3B3 and CEA-N by laser desorption mass spectromctry. The samples (40 pmol) were dissolved in aqueous trifluoroacetic acid-aceicniu i Mi1(0.1% TFA in water/acetonitrile. .10/70) containing 0.5 Msinapinic acid,
dried on a copper stage, irradiated at 340 nm with a frequency-tripled neodyniumyttrium-aluminum-garnet laser, and analyzed by time of flight mass spectrometry.
A, CEA-N; B, CEA-A3B3.
B
5
228
6
3
4
—¿
1 09
—¿
70
—¿
44
—¿
i
28 —¿
1 8 —¿
1 5 —¿
Fig. 3. Western blot analysis of purified expression products. The samples
(10-500 ng) were boiled in sample buffer containing 2% SDS and 2.5% 2-mercaptoethanol. run on 12.5% SDS-polyacrylamidc gels, and electrotransferred to
nitrocellulose membranes, and protein bands were detected with monoclonal
antibodies T84.1 (A) or T84.66 (A). Lane I. tumor CEA; lane 2. CEA-S; lane 3,
CEA-A3B3; lane 4. CEA-N; lane 5. tumor NCA; lane 6. NCA-S.
5651
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EPITOPES
IN EXPRESSED
only with Mab T84.1 and not with T84.66. The molecular
weights of CEA-N and CEA-A3B3 as determined by Western
blotting are 15,000 and 55,000, respectively. The value for
CEA-N is comparable to that obtained from LD/TOF-MS, but
in the case of CEA-A3B3 the molecular weight obtained from
SDS gels is substantially higher than that from LD/TOF-MS.
CEA-N reacts only with Mab T84.1 and not with Mab T84.66.
CEA-A3B3 reacts only with Mab T84.66 and not with T84.1.
The Mab reactivity profiles indicate that T84.1 reacts with the
N domain of either CEA or NCA, in contrast to T84.66, which
does not react with the N domain of either CEA or NCA.
Antibody Binding and Inhibition Analysis. The purified an
tigens were tested for their ability to bind Mabs T84.1 and
T84.66 in an EIA. As shown in Fig. 4A, T84.1 recognizes CEA,
CEA-S, and CEA-N but not CEA-A3B3. As shown in Fig. 4fi,
T84.66 recognizes CEA, CEA-S, and CEA-A3B3 but not
CEA-N. The concentrations for half-maximal binding for CEA
and CEA-S are more similar for T84.1 (1.5 versus 3.0 nivi)than
for T84.66 (0.5 versus 4.0 HM), suggesting a subtle difference
o
u
C
CO
û
w
O
W
0.001
100
0.01
Nanomolar
oio
C
CO
O
M
io
0.1
100
Nanomolar
Fig. 4. Monoclonal antibody binding lo purified expression products. T84.1
(A) or 184.66 (A) was incubated with antigen and added to microtiter plates
coated with rabbit polyclonal antibody to CEA. Bound antibody was detected with
goat anti-mouse-alkalinc phosphatase conjugate. D, CEA: •¿.
CEA-S: A. CEA-N:
O. CEA-A3B3.
DOMAINS OF CEA
between the two antigens. The half-maximal binding concen
tration for CEA-N to T84.1 is 0.2 nM, compared to 1.5 nisi for
native CEA, suggesting that CEA-N is a better ligand for T84.1
than is native CEA. The half-maximal binding concentration
for CEA-A3B3 to T84.66 is 2.0 nM, compared to 0.5 nvi for
native CEA, suggesting that CEA-A3B3 is a poorer ligand.
Inhibition assays were used to further delineate the epitope
specificity of T84.66 within the A3B3 domains. Since it was not
possible to generate a secreted form of A3 or B3 from transfected HeLa cells, we used synthetic peptides, designated sA3
and sB3, as inhibitors corresponding to these regions. The syn
thetic peptides were designed to include all of the predicted
0-strands of the Ig-like constant domains and the internal disulfide bond. Binding to the monoclonal antibodies T84.66 and
H6C8 was compared, since both antibodies bind to the same
region (24). In preliminary experiments (data not shown),
T84.66 was shown to bind to sA3 but not to sB3, and H6C8 was
shown to bind to sB3 but not to sA3. In a solid-phase inhibition
assay utilizing CEA-coated plates, sB3 but not sA3 was able to
inhibit the binding of H6C8 to CEA (Fig. 5/1). In this assay,
neither sA3 nor sB3 was able to inhibit the binding of T84.66 to
CEA. Since solid-phase inhibition assays may be affected by the
relative affinity constant of the antibody for antigen (T84.66
has a 10-fold higher affinity constant for CEA than does
H6C8), T84.66 was tested in a solution-phase inhibition assay.
In this assay, the antibody was first incubated with biotinylated
antigen and inhibitor and then added to anti-mouse IgG-coated
plates, and bound antigen was detected with avidin-horseradish
peroxidase complex. As shown in Fig. 5B, the binding of
T84.66 to biotinylated CEA was inhibited by sA3 but not by
sB3. The relative binding affinities of T84.66 for CEA and
CEA-A3B3 are similar (2-fold difference), confirming the re
sults shown in the binding curves discussed above. The relative
binding affinity of H6C8 for CEA is 6-fold greater than that for
A3B3.
DISCUSSION
Expression of CEA and NCA Constructs. In this work we
chose to express CEA domains in HeLa cells in order to mimic
the normal glycosylation patterns expected for human cells
expressing CEA in the colon or NCA in the lung or spleen. The
strategy for expressing the domains for epitope mapping in
volved the insertion of stop codons at the end of the expected
domains. Since CEA and NCA normally terminate in hydrophobic signal peptides which are exchanged for GPI anchors,
both antigens were also expressed as secreted forms in which
stop codons were inserted at the end of the last domain (A3B3
for CEA and Al Bl for NCA) and before the hydrophobic carboxyl-terminal signal peptide. As seen from amino-terminal
sequence analysis, cleavage of the amino-terminal signal peptide occurred as expected for each of the expressed antigens. In
the Western blot analysis, CEA-S, NCA-S, CEA-N, and CEAA3B3 reacted strongly with the appropriate antibodies. It is
noteworthy that native CEA has a higher molecular weight than
CEA-S. On the one hand, the native soluble form of CEA
probably has a GPI anchor but lacks the diacylglycerol moiety
(otherwise it would be only detergent soluble). On the other
hand, CEA-S should lack the entire GPI moiety (the carboxylterminal signal peptide was eliminated). Although the GPI moi
ety is expected to contribute only a few thousand to the molec
ular weight of CEA, it may be sufficient to cause the difference
in migration on SDS-gel electrophoresis. It is also possible that
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EPITOPES
IN EXPRESSED
DOMAINS OF CEA
even for highly glycosylated glycoproteins (31). In the case of
CEA and CEA-S, we have been largely unsuccessful in obtain
ing good spectra by LD/TOF-MS, but we obtained excellent
spectra for both CEA-N and CEA-A3B3. As shown in Fig. 2,
80the calculated molecular weights are 15,990 for CEA-N and
34,462 for CEA-A3B3. These accurate molecular weights have
allowed us to deduce estimates of the size of the glycosyl moi
=
60eties attached to the polypeptide chains, based on the assump
tion that each potential site (asparagine linked) is glycosylated.
These calculations are consistent with each glycosyl moiety
40 corresponding to a fully sialylated biantennary glycosyl unit.
O
O
vWe plan to study the glycosylation patterns by sequential gly0»
IL
cosidase treatment, in order to determine if this is indeed the
20case, and to compare these results with those for native CEA.
Interestingly, the molecular weights of CEA-N calculated by
LD/TOF-MS and SDS-gel electrophoresis agree well (15,990
versus 15,000), while those of CEA-A3B3 agree poorly (34,462
0.01
versus 55,000). Since CEA-N has only two predicted glycosy
0.1
10
100
1000
lation sites versus seven for CEA-A3B3, the error in the SDSNanomolar
gel electrophoresis method may be related to the overall per
100
centage of carbohydrate present (25 versus 40%).
CEA-N gave two major peaks by LD/TOF-MS, one corre
sponding to the monomer and the other to a dimer, suggesting
a tendency of this domain to dimerize. On the other hand,
CEA-A3B3 exhibited only a monomer peak. Since molecular
weight studies of CEA reveal dimer formation (38), a feature
o
compatible with its inclusion in the Ig superfamily, it will be of
considerable interest to study the tendency of individually ex
pressed domains to dimerize. The relative peak sharpness of
CEA-N versus CEA-A3B3 is consistent with their relative
amounts of glycosylation, assuming the usual amount of microheterogeneity found in all glycoproteins.
NCA and NCA-S also gave good signals in LD/TOF-MS
(data not shown). Their molecular weights were 46,052, and
49,619, respectively. The molecular weight difference corre
sponds to 3,000, which is consistent with the molecular weights
obtained by SDS-gel electrophoresis (Fig. 3).
100
1000
Attempts to express the subdomains A3 and B3 were unsuc
cessful. Upon analysis of lysed cells transfected with either A3
Nanomolar
or B3 constructs, we were able to detect weak binding activity to
Fig. 5. Inhibition of binding of antigens to monoclonal antibodies specific for
polyclonal anti-CEA antibodies, suggesting that the chains
the A3B3 domain of CEA. H6C8 (A) or T84.66 (B) was tested in inhibition
assays.. I. solid-phase assay on CEA-coated plates. Antibody was incubated with
were present but not secreted. No native epitopes were detected
antigen and added to CEA-coated plates. Bound antibody was detected with goat
with monoclonal antibodies T84.66 and H6C8, which react
anti-mouse Ig-alkaline phosphatase conjugate. B, solution-phase assay on goat
anti-mouse Ig-coated plates. Antibody was incubated with biotinylated CEA and
specifically with the A3B3 domains. Analysis of total RNA
inhibitor and added to goat anti-mouse Ig-coated plates. Bound biotinylated CEA
was delected with avidin-horseradish peroxidase conjugate. D. CEA; A. sA3; •¿. from the cells by PCR using primers specific for either A3 or B3
revealed that the correct size mRNAs were produced. We con
sB3; O, CEA A3B3.
clude that the A3 and B3 domains were not able to fold and be
some other glycosylation differences are responsible for the secreted independently. Although these domains resemble Ig
constant region-like domains, it was not possible to predict
different electrophoretic migration rates.
In the case of NCA, NCA-S migrated more slowly than NCA whether they would be expressed properly as unpaired domains.
In contrast to this, the CEA-N domain, which resembles an Ig
isolated from tumor cells. However, it should be noted that
variable region-like domain, apparently is secreted and is imNCA may be isolated in a variety of glycoforms ranging from
A/r 45,000 to 95,000 (37). In this analysis we compared NCA-S
munologically active.
to the NCA glycoform with the lowest molecular weight (NCAEpitope Specificities of T84.1, T84.66, and H6C8. The
45). In previous studies, we have shown that NCA expressed in Western blot data confirmed our earlier studies suggesting that
T84.1 recognizes an epitope in the N domain shared between
L cells and Chinese hamster ovary cells also has a higher mo
lecular weight than does NCA-45 (25). Indeed, a careful com
CEA and NCA and that T84.66 recognizes an epitope in the
parison of molecular weights can only be made for antigens
A3B3 domain unique to CEA. Direct binding studies in an EIA
confirmed high affinity binding of T84.1 to CEA-N and of
expressed in the same cells under the same conditions.
T84.66 to CEA-A3B3. In order to localize further the subdoThe issue of determining more accurate molecular weights
for glycoproteins is further complicated by the fact that the main specificity of T84.66, we had originally planned inhibition
SDS-gel electrophoresis method is known to give erroneously
studies with A3 and B3 expressed separately, but these domains
high values for glycoproteins. Because of this, we chose LD/
were not properly folded or secreted. We, therefore, performed
TOF-MS, which is known to give accurate molecular weights
inhibition studies on synthetic peptides corresponding to A3
100
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EPITOPES
A3
IN EXPRESSED
DOMAINS OF CEA
a
f
VSAELPKPSISS--NSKPVEDKDAVAFTCEPEA- TTYLWWVNG.QSLPVSPRLQLSNG'.RTLTLF' VTRNDARAY\/CGIQNSVSA'.RSDPVIL
in in in
B3
f
ßVLYGPDIPIISPPaSSYLSGANL' LSCHSASNPSEQYSWRINGIPQQHTQVLFIAKITPNN' GTYACFYSNLATGR'-NSIVKSITV
in i in n i INi n
'l
n11in n
limili
Fig. 6. Epitope model for A3 and B3 subdomains of CEA. Upper, A3 subdomain; lower, B3 subdomain. Predicted (¡-strands(a through g) are shown by bars over
the amino acid sequence. Regions expected to be protected from antibody recognition by glycosylation are shown by shaded bars under the amino acid sequence (the
glycosylated asparagines are outlined). Amino acids differing from their counterparts in the A1B1-A2B2 domains are underlined. Candidate epitopes are shown by
striped bars under the amino acid sequence.
and B3 (designated as sA3 and sB3). These peptides were hydrophobic (no glycosylation) and required prior solubili/alimi
in acetic acid and dilution into PBS prior to analysis. The
antibody H6C8, which binds the A3B3 domain but does not
inhibit the binding of T84.66 to CEA, was included as a control.
When inhibition analysis was performed on CEA-coated
plates, sB3 but not sA3 was able to inhibit the binding of H6C8
to CEA. The relative affinities of H6C8 for CEA, CEA-A3B3,
and SB3 were 0.1 n\i, 2.0 n.M,and 700 n\i, respectively. The
large difference in affinity observed for the synthetic peptide,
compared to CEA or CEA-A3B3, is probably due to poor fold
ing into a native state (Ig constant region-like domain with
disulfide bond), a problem commonly encountered with syn
thetic peptides. CEA-A3B3, but neither sA3 nor sB3, was able
to inhibit the binding of T84.66 to CEA-coated plates. This
problem may be related to the high affinity of T84.66 for anti
gen bound to the solid phase (10-fold higher for T84.66, com
pared to H6C8). This problem was circumvented by performing
a solution-phase assay in which antibody, antigen, and inhibitor
were coincubated prior to separation of free from bound anti
body (or antigen). In order to perform this assay, CEA was
biotinylated and the complex (antibody, antigen, and inhibitor)
was added to anti-Ig-coated plates. Bound biotinylated CEA
was detected with avidin-horseradish peroxidase conjugate. In
this assay biotinylated CEA binding was inhibited by CEA,
CEA-A3B3, or sA3 but not by sB3. Again, the relative affinity
of T84.66 was much higher for CEA or CEA-A3B3 than for
sA3.
From these studies we conclude that the epitope for T84.66
resides on the A3 subdomain and the epitope for H6C8 is on the
B3 subdomain and that glycosylation is not required for bind
ing. In earlier work, we had shown that T84.66 and H6C8 bind
to A3B3 when expressed as a nonglycosylated fusion product in
E. coli (24). The results presented here extend this analysis,
fixing epitope locations to subdomains, and demonstrate that
domains such as CEA-N or CEA-A3B3 can be expressed as
correctly folded (as determined by immunological analysis) do
mains in the absence of a fusion peptide. Since a major portion
of each domain must be masked or shielded from antibody
recognition by glycosylation, we are now in a position to select
regions most likely to contain the amino acid sequence involved
in the epitope. The candidate regions can also be narrowed
down by comparing amino acid sequences between the A1B1,
A2B2, and A3B3 domains [T84.66 and H6C8 do not react with
A1B1 or A2B2; see Haas et al. (24)]. Candidate regions are
shown in Fig. 6. Candidate regions were chosen as contiguous
amino acid sequences differing from counterpart domains by at
least one amino acid and not including a glycosylation site or a
disulfide bond. Only regions covered by the sA3 or sB3 peptides
were considered, since these peptides were shown to contain the
T84.66 and H6C8 epitopes, respectively. Because the A3B3
domain is expected to fold like an Ig constant domain, this
model also accounts for the loss of antibody binding activity for
incorrectly folded domains (expression of A3 and B3 as indi
vidual domains). This model does not take into account the
possibility of an antibody recognizing noncontiguous amino
acids. The lack of three-dimensional structural information pre
cludes predictions of this type. Site-directed mutagenesis exper
iments are in progress to test the potential role of amino acids
in the epitopes predicted by our model.
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Expression of Carcinoembryonic Antigen and Its Predicted
Immunoglobulin-like Domains in HeLa Cells for Epitope
Analysis
Laura J. F. Hefta, Fun-Shan Chen, Michael Ronk, et al.
Cancer Res 1992;52:5647-5655.
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