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 METHODS Flow Cytometric Analysis: Cells (2-6 X 105) 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 Tris-saline 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. INTRODUCTION 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 3C177-C184 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 non-reduced 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. Chimeric cDNA Constructs for Mammalian Cell Expression Studies to Analyze 7E3 Binding cDNA Constructs: 3H-M-H pcDNA3/3179N182N183A 3H-D179N-H pcDNA3/3179N 3H-T182N-H pcDNA3/3182N 3H-T183A-H pcDNA3/3183A 3H-D179N/T182N-H pcDNA3/3179N182N 3H-D179N/T183A-H pcDNA3/3179N183A 3H-T182N/T183A-H pcDNA3/3182N183A H and blue color = Human sequence M and red color = Mouse sequence 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. 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 1: Background 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. Mouse 3: 177CYNMKNAC184  Human 3: 177CYDMKTTC184 7E3 - + Human 3 FIGURE 2: Immunoprecipitation of Chimeric IIb3 Receptors Containing Single Amino Acid Substitutions using 10E5 mAb IP 10E5 mAb (anti- IIb3) NON-REDUCED REDUCED 217 123 71 3 3H-H-H 3H-M-H 3H-179N-H 3H-182N-H 3H-183A-H IIb 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. IP 7E3 mAb (anti-IIb3 + V3) FIGURE 3: Immunoprecipitation of Chimeric IIb3 Receptors Containing Single Amino Acid Substitutions using 7E3 mAb 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. FIGURE 4: Immunoprecipitation of Chimeric IIb3 Receptors Containing Two Amino Acid Substitutions: 10E5 & 7E3 mAbs 205 116 81 3H-182N183A-H 3H-179N182N-H 3H-179N183A-H 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.