Transcript anti- IIb 3

DISSECTING THE EPITOPE FOR THE 7E3 mAb BY GENERATING HUMAN/MOUSE
CHIMERIC C177-C184 DISULFIDE-LOOPS IN THE HUMAN 3 INTEGRIN
DEBORAH L. FRENCH, JIHONG LI, JIAN RUAN, BARRY S. COLLER
DEPARTMENT OF MEDICINE, MOUNT SINAI SCHOOL OF MEDICINE, NEW YORK, NY 10029
Figure 1: Background
Integrin IIb3 and V3 receptors are key components in the maintenance of
normal hemostasis and in pathogenic events such as thrombosis after rupture
of an atherosclerotic plaque. Murine 7E3, the parent antibody of the antiplatelet
abciximab, inhibits ligand binding to both human IIb3 and V3 receptors.
Puzon-McLaughlin et al. demonstrated that substitution of the human 3C177C184 disulfide-bonded loop with the murine sequence eliminates the binding
of 7E3. These data implicate the C177-C184 disulfide-bonded loop of human 3
as being part of, and/or affecting, the epitope of this mAb. This loop is located
near or in the MIDAS-like domain of 3, which has been implicated in both
cation and ligand binding. The C177-C184 disulfide loops of human
(CYDMKTTC) and mouse (CYNMKNAC) 3 differ by 3 amino acids (underlined)
and these amino acids were interchanged to generate human-mouse chimeric
3 subunits. Chimeric receptors were expressed in mammalian cells and
analyzed for: 1) protein expression by immunoprecipitation and SDS-PAGE, 2)
antibody binding by flow cytometry, and 3) 7E3-mediated inhibition of cell
adhesion to immobilized fibrinogen. Substitution of the human C177-C184 loop
with mouse residues essentially eliminated 7E3 binding and 7E3-mediated
inhibition of cell adhesion to immobilized fibrinogen. In SDS-PAGE under nonreduced conditions, the chimeric 3 subunit migrated as a doublet with the
majority of protein migrating at a higher apparent Mr. Analysis of the chimeric
subunits in which single amino acids were interchanged showed that the
3T182N substitution uniquely caused the change in SDS-PAGE migration
pattern. Immunoprecipitation studies demonstrate that 7E3 does not react with
receptors containing the 3 subunit of higher apparent Mr. Consistent with this
finding, adhesion of 3T182N cells to fibrinogen was not inhibited by 7E3. Low
level 7E3 binding was detectable, presumably reflecting binding to the 3
species of lower apparent Mr. Affinity analyses showed that the binding
affinities of 7E3 to cells expressing normal and chimeric receptors were the
same, but the number of chimeric receptors recognized by 7E3 was
significantly reduced. We conclude that the 3T182N chimeric subunit causes
a change in conformation and/or glycosylation that is either part of the binding
epitope for 7E3 or affects the epitope located elsewhere in the molecule.
7E3
Mouse 3:
177CYNMKNAC184

Human 3: 177CYDMKTTC184
IIb (V)
+
217
3H-H-H 3H-D179N-H 3H-T182N-H 3H-T183A-H 3H-M-H
IIb
IIb
3
123
3
10E5
IP 10E5 mAb (anti- IIb3)
NON-REDUCED
IIb3
IP 10E5 mAb (anti- IIb3)
REDUCED
CONCLUSION: The T182N substitution causes a change in the migration pattern
of the 3 subunit resulting in a doublet of normal and higher Mr. Upon reduction,
the doublets disappear, but the 3 band continues to migrate at a slightly higher
Mr suggesting the possibility of additional glycosylation.
FIGURE 3: Immunoprecipitation of Chimeric IIb3 Receptors
Containing Single Amino Acid Substitutions using 7E3 mAb
Cells (2-6 X
were incubated with mAbs (5 g/ml/100 l of PBS containing 2%
FBS) for 1 hour, washed, and incubated with FITC-labeled sheep anti-mouse IgG
(Jackson ImmunoResearch, West Grove, PA). Cells were analyzed with a
FACScan flow cytometer using Lysis II software (Bectin Dickinson).
Fibrinogen Adhesion Assay:
Wells of NUNC flat bottom 96-well plates were precoated with 100 l/well of Trissaline buffer (50mM Tris-HCl, 100mM NaCl, pH 7.4) containing human fibrinogen
(Enzyme Research Laboratories, South Bend, IN) at a concentration of 10 g/ml
for 1 hour at room temperature followed by incubation with 100 l/well of DMEM
(GIBCO-BRL Life Technologies, Gaithersburg, MD) containing 0.5% BSA.
Transiently transfected human kidney 293T cells or CHO cells (105 cells/well)
expressing IIb3 receptors were preincubated with mAbs (7E3 and 10E5 at 50
g/ml) in 100 l of DMEM containing 0.5% BSA for 30 minutes on ice. Cells were
added to the wells and incubated at 370C for 1 hour. After rinsing the wells with
PBS (100 l/well), the attached cells were fixed and stained with a solution (50
l/well) containing 1% formaldehyde, 0.5% crystal violet and 20% methanol for
30 minutes at room temperature. The wells were washed and 100 l/well of 10%
acetic acid was added to solubilize the dye which was read at OD550 nm.
Immunoprecipitation Analysis:
Cells were lysed in buffer containing 1% Triton X-100, separated by SDS-PAGE,
and electrophoresed onto polyvinylidene difluoride (PVDF) membranes
(Millipore, Burlington, MA). Immunoprecipitations were performed using whole
cell lysates (buffer containing 1% Triton X-100) from cells biosynthetically
labeled with 35S-methionine (300 Ci/plate for 2-3 hours). Pre-cleared lysates
(~2x106 counts/tube) were incubated with the 10E5 or 7E3 mAbs (anti-IIb3 or IIb3+V3, respectively) (kindly provided by Dr. Barry Coller) or AP3 mAb
(anti-human 3) (kindly provided by Dr. Peter Newman) for 2 hours or overnight
at 4C with gentle rocking. Protein G was added to the tubes and incubated for
1 hour followed by 3 washes. Samples were separated by SDS-PAGE,
electrophoresed onto PVDF membranes, and membranes were exposed to film.
0.98
0.60
0.92
0.30
1.00
0.90
0.73
0.80
0.89
IIb3 surfac e rece ptors w ere norm alized to human  3 ex pression
Values are ca lculated as % cells ex pressing chim eric receptors/% of cells e xpress ing wildtype receptors
CONCLUSION: Low level 7E3 binding was detectable on receptors
expressing 3H-M-H and3H-182N-H subunits, presumably reflecting
binding to the 3 species of lower apparent M r.
Figure 6: Fibrinogen Adhesion of Cells Expressing
Chimeric IIb3 Receptors Containing Single Amino Acid
Substitutions
IIb3H-H-H H-179N-H
120
Human 3
Chimeric cDNA Constructs for Mammalian Cell
Expression Studies to Analyze 7E3 Binding
IP 7E3 mAb (anti-IIb3 + V3)
NON-REDUCED
H-182N-H
H-183A-H
3
N E E 5 7E
O
N
10
3
N E E 5 7E
O
N
10
IIb3H-M-H
100
217
3
71
105)
1.00
71
3
Flow Cytometric Analysis:
7E3
IIb3+V3
123
METHODS
Figure 5: Flow Cytometry Analyzing 7E3 and 10E5 mAb
Binding to Chimeric IIb3 Receptors Containing Single
Amino Acid Substitutions
IP 7E3 mAb (anti-IIb3 + V3)
REDUCED
% Adhesion
INTRODUCTION
By flow cytometry, Puzon-McLaughlin et al. (JBC 275:7795,
2000) showed that the 7E3 mAb does not bind to cells
expressing chimeric receptors containing the mouse
C177-C184 disulfide-loop in the human 3 subunit.
FIGURE 2: Immunoprecipitation of Chimeric IIb3 Receptors
Containing Single Amino Acid Substitutions using 10E5 mAb
80
60
40
20
0
E 5 E3
E 5 E3
N
N
7
E
E 7
N O 10
N O 10
cDNA Constructs:
3H-M-H
CONCLUSION: The band migrating at a higher Mr due to the T182N amino acid
substitution, as shown in Figure 2 is not recognized by the 7E3 mAb.
pcDNA3/3179N182N183A
3H-D179N-H
3H-T182N-H
3H-T183A-H
pcDNA3/3179N
pcDNA3/3182N
pcDNA3/3183A
3H-D179N/T182N-H
3H-D179N/T183A-H
3H-T182N/T183A-H
pcDNA3/3179N182N
pcDNA3/3179N183A
pcDNA3/3182N183A
By PCR mutagenesis, human-mouse chimeric 3 cDNA
constructs were generated in pcDNA3 (Invitrogen Corp).
Mammalian cells were transfected with cDNA constructs using
Lipofectamine (Life Technologies, Gaithersburg, MD) and
analyzed 48 hours later.
INHIBITORS
CONCLUSION: The inability of 7E3 to inhibit the adhesion of cells
expressing 3H-M-H and3H-182N-H subunits presumably reflects
the absence of binding of 7E3 to the 3 species of higher apparent Mr.
FIGURE 4: Immunoprecipitation of Chimeric IIb3 Receptors
Containing Two Amino Acid Substitutions: 10E5 & 7E3 mAbs
SUMMARY AND CONCLUSIONS
• 7E3 epitope recognition and inhibition of
adhesion is lost by substitution of human
3D179 T182T183 residues by mouse 3N179 N182A183
residues
205
H and blue color = Human sequence
M and red color = Mouse sequence
3
N E E 5 7E
O
N
10
116
3
3
81
IP 10E5 mAb (anti- IIb3)
NON-REDUCED
IP 7E3 mAb (anti-IIb3 + V3)
NON-REDUCED
CONCLUSION: Amino acid substitutions containing residue 182N cause a
change in the migration pattern of the 3 subunit as shown in Figure 2. The
band migrating at a higher Mr is not recognized by the 7E3 mAb while the band
migrating at the normal Mr is recognized by the 7E3 mAb.
• This loss of epitope recognition appears to be
due to a conformational change and/or
glycosylation in the 3 subunit caused by the
T182N substitution
• These data suggest that the 3C177-C184 loop is
either part of the binding epitope for 7E3 or
affects the epitope located elsewhere in the
molecule.