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• W2167390304 abstract "Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc γ receptors (FcγRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcγR interactions are hence important parameters for understanding FcγR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcγRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 × 103, 8.2 × 103, 1.1 × 104 and 1.8 × 104 m–1 s–1, off-rates of 4.7 × 10–3, 5.7 × 10–3, 5.9 × 10–3, and 1.9 × 10–2 s–1, and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 μm, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcγR interactions. Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc γ receptors (FcγRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcγR interactions are hence important parameters for understanding FcγR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcγRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 × 103, 8.2 × 103, 1.1 × 104 and 1.8 × 104 m–1 s–1, off-rates of 4.7 × 10–3, 5.7 × 10–3, 5.9 × 10–3, and 1.9 × 10–2 s–1, and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 μm, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcγR interactions. Fc γ receptors (FcγRs) 7The abbreviations used are: FcγR, Fc γ receptor; SPR, surface plasmon resonance; BSA, bovine serum albumin; CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; IgG, immunoglobulin G; h, human; mAb, monoclonal antibody; NA1 and NA2, neutrophil antigen 1 and 2; PBS, phosphate-buffered saline; Rb, rabbit; MFI, mean fluorescence intensity; TM, transmembrane. are a family of cell surface glycoproteins with varying affinities for the Fc region of immunoglobulins G (IgG). Three classes of human FcγRs have been described: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), which are widely distributed in hematopoietic cell lineages. These include at least 12 different isoforms many of which are polymorphic. For example, CD16 has two isoforms, a and b, that differ by six amino acids in the extracellular domain and the presence (CD16a) or absence (CD16b) of the transmembrane and cytoplasmic domains (1van de Winkel J.G.J. Capel P.J.A. Human IgG Fc receptors. R. G. Landes, Austin, TX1996Google Scholar). The IgG-FcγR interaction-mediated binding of antibody-opsonized pathogens to leukocytes is a key event by which antibody effector functions are initiated. Kinetic rates and binding affinity are critical determinants of an IgG-FcγR interaction, as they control how likely and how rapidly such binding will occur, how many bonds will be formed, and how long the bonds last. In addition, it has been hypothesized that these parameters are related to the signaling events following the initial binding (2Matsui K. Boniface J.J. Steffner P. Reay P.A. Davis M.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12862-12866Crossref PubMed Scopus (371) Google Scholar, 3McKeithan T.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5042-5046Crossref PubMed Scopus (726) Google Scholar, 4van der Merwe P.A. Bodian D.L. Daenke S. Linsley P. Davis S.J. J. Exp. Med. 1997; 185: 393-403Crossref PubMed Scopus (430) Google Scholar). Using a micropipette method, we have measured the kinetic rates and binding affinities of several IgG-FcγR interactions when both interacting molecules are anchored on apposing surfaces. We found that in this assay the half-lives of these IgG-FcγR bonds are in the order of seconds (5Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 6Chesla S.E. Li P. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2000; 275: 10235-10246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Williams T.E. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1858-1866Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Williams T.E. Nagarajan S. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1867-1875Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 9Huang J. Chen J. Chesla S.E. Yago T. Mehta P. McEver R.P. Zhu C. Long M. J. Biol. Chem. 2004; 279: 44915-44923Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In addition to interaction parameters of surface-linked molecules, the kinetic rates and affinities of FcγRs for soluble IgG are also of interest, because in vivo interactions of immobilized IgG to leukocyte FcγRs are subject to competitive binding of soluble antibodies in sera. Of the three FcγRs, CD64 binds to monomeric IgG with high affinity (Kd ∼ tens of nm) (10Miller K.L. Duchemin A.M. Anderson C.L. J. Exp. Med. 1996; 183: 2227-2233Crossref PubMed Scopus (70) Google Scholar), CD32 and CD16b are of low affinities (Kd, several and several tens of μm, respectively) (6Chesla S.E. Li P. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2000; 275: 10235-10246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Sondermann P. Jacob U. Kutscher C. Frey J. Biochemistry. 1999; 38: 8469-8477Crossref PubMed Scopus (36) Google Scholar, 12Vance B.A. Huizinga T.W. Wardwell K. Guyre P.M. J. Immunol. 1993; 151: 6429-6439Crossref PubMed Google Scholar), and CD16a is considered as an intermediate affinity receptor (Kd ∼ hundreds of μm) for monomeric IgG (6Chesla S.E. Li P. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2000; 275: 10235-10246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 10Miller K.L. Duchemin A.M. Anderson C.L. J. Exp. Med. 1996; 183: 2227-2233Crossref PubMed Scopus (70) Google Scholar, 12Vance B.A. Huizinga T.W. Wardwell K. Guyre P.M. J. Immunol. 1993; 151: 6429-6439Crossref PubMed Google Scholar). In addition to the above results, which were obtained by conventional assays using FcγR-expressing cells, there were two studies using the surface plasmon resonance (SPR) technology and soluble FcγRs. Galon et al. (13Galon J. Robertson M.W. Galinha A. Mazieres N. Spagnoli R. Fridman W.H. Sautes C. Eur. J. Immunol. 1997; 27: 1928-1932Crossref PubMed Scopus (45) Google Scholar) reported half-lives of the order of 10 min and equilibrium dissociation constants of several micromolar for sCD16bNA2 binding to human (h) IgG1 and IgG3. By comparison, Maenaka et al. (14Maenaka K. van der Merwe P.A. Stuart D.I. Jones E.Y. Sondermann P. J. Biol. Chem. 2001; 276: 44898-44904Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) found a much faster off-rate (half-lives of the order of seconds) but similar Kd for IgG-sCD16bNA2 binding. Here, we report kinetic and affinity measurements of a soluble dimeric human FcγRIIIa (sCD16a) interacting with various IgG ligands in both monomeric and multimeric forms using SPR. We found that sCD16a interacted with monomeric rabbit (Rb) IgG, hIgG, hIgG1, and hIgG3 with on-rates of 1.8 × 104, 6.5 × 103, 8.2 × 103, and 1.1 × 104 m–1 s–1, off-rates of 1.9 × 10–2, 4.7 × 10–3, 5.7 × 10–3, and 5.9 × 10–3 s–1, and equilibrium dissociation constants of 1.1, 0.72, 0.71, and 0.56 μm, respectively. sCD16a bound with aggregated ligands with lower apparent equilibrium dissociation constants and slower apparent off-rates. To ensure that the relatively slow kinetics is not artifactual, various controls were performed in both the experiment and analysis steps. The slow kinetics was not found to be artifacts because of the mass transport limitation, ligand aggregation, or dimeric binding of the sCD16a molecule. The kinetic rates and affinities were also measured by real-time flow cytometry and a competition inhibition binding experiment using CD16 expressed on the cell surface, which were found to be in agreement with those obtained by SPR. Cells, Soluble CD16a, and Antibodies—Chinese hamster ovary (CHO) cells transfected to express human CD16a™, CD16bNA1, CD16bNA2, and B7-1GPI as well as untransfected CHO cells were cultured as previously described (15Nagarajan S. Chesla S. Cobern L. Anderson P. Zhu C. Selvaraj P. J. Biol. Chem. 1995; 270: 25762-25770Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16McHugh R.S. Ahmed S.N. Wang Y.C. Sell K.W. Selvaraj P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8059-8063Crossref PubMed Scopus (104) Google Scholar). sCD16 was generated by attaching the extracellular domain of CD16a to the Fc domain of IgG1. The mutated IgG1 CH2-CH3 Fc domains were obtained from Dr. Peter Linsley, Bristol-Meyers Squibb, as the hB7-1-Ig construct and the extracellular domain of hCD16a was cloned in the place of hB7-1 as described (17Li P. Nagarajan S. Zhu C. Selvaraj P. Mol. Immunol. 2002; 38: 527-538Crossref PubMed Scopus (7) Google Scholar). Mutations in the Fc domain of hCD16a-Ig are L267F, L268E, G270A, and A363T (numbered as in accession number AAH69020.1). These mutations were shown to abolish the binding of FcγRs (18Chappel M.S. Isenman D.E.M.E. Xu Y.Y. Dorrington K.J. Klein M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9036-9040Crossref PubMed Scopus (119) Google Scholar, 19Chappel M.S. Isenman D.E. Oomen R. Xu Y.Y. Klein M.H. J. Biol. Chem. 1993; 268: 25124-25131Abstract Full Text PDF PubMed Google Scholar). The anti-CD16 nonblocking monoclonal antibody (mAb) 214.1 (murine IgG1) was a generous gift from Dr. Howard Fleit (State University of New York, Long Island). The anti-CD16 adhesion blockade mAb CLBFcgran-1 (murine IgG2a) was purified in house from hybridomas as previously described (20Selvaraj P. Rosse W.F. Silber R. Springer T.A. Nature. 1988; 333: 565-567Crossref PubMed Scopus (283) Google Scholar). Cleavage of CLBFcgran-1 into Fab fragments was done by Lampire (Pipersville, PA). The rabbit anti-mouse Fc polyclonal antibody was purchased from BIAcore (Piscataway, NJ). Total hIgG and subtypes (hIgG1, hIgG2, and hIgG3) as well as RbIgG were purchased from Sigma, except hIgG1 used for the real time flow cytometry experiment, which was a generous gift from Dr. Adrian Whitty (Biogen Inc., Boston, MA). Fab of CLBFcgran-1 and hIgG1 was labeled with fluorescein isothiocyanate (Molecular Probes, Eugene, OR) following the manufacturer’s instructions. Size Exclusion Chromatography—Monomeric and multimeric IgG ligands were separated by size exclusion chromatography. 7.5 g of Sephadex G-200 (Amersham Biosciences) was swelled in 200 ml of PBS/EDTA (containing 5 mm EDTA, pH 7.4) at 90 °C for 5 h and then cooled at 4 °C overnight. The supernatant was decanted and the gel was resuspended in 150 ml of PBS/EDTA and poured into a column. Two columns were used, with respective diameters of 1.7 and 1.0 cm and respective volumes of 150 and 103 ml (Bio-Rad). The columns were rinsed with 300 ml of PBS/EDTA at 0.3 ml/min between each run. Two sets of gel filtration standards were used to calibrate the columns. The first set included 4 mg each in 1 ml of PBS/EDTA of cytochrome c (molecular mass 12 kDa), blue dextran (2,000 kDa), and BSA (88 kDa) (Sigma). The second set included 2.5 mg of thyroglobulin (670 kDa), 2.5 mg of bovine γ-globulin (158 kDa), 2.5 mg of chicken ovalbumin (44 kDa), 1.25 mg of equine myoglobin (17 kDa), and 0.25 mg of vitamin B12 (1.4 kDa). After adding 5–20 mg of IgG in 1 ml of PBS/EDTA, the column was connected to a 1-liter reservoir of PBS/EDTA. Setting the flow rate at 0.25–0.75 ml/min, the effluent was collected sequentially in fractions of 1.5 ml each. The optical density at 280 nm was measured to monitor the protein concentration in each fraction. Monomeric or multimeric IgG fractions were used immediately after separation, either directly or further diluted to lower concentrations for SPR or real time flow cytometry experiments. In some cases where higher concentrations were desired, chromatographed IgG was reconcentrated by using protein concentrators (Amicon, Beverly, MA). The concentrations of monomeric and multimeric IgG were determined by a protein estimation kit (Bio-Rad). SPR Measurement—SPR experiments were conducted in a BIAcore™ 1000 instrument using CM5 sensor chips (BIAcore) at 25 °C. Samples were prepared in PBS/EDTA and perfused over the sensor chip at 30 μl/min (unless otherwise stated) for kinetic measurements. For other control experiments, the flow rate was 5 μl/min. The running buffer was BIA-certified HBS buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.005% Surfactant p20) filtered and degassed using 0.2-μm bottle top filter (Millipore, Bedford, MA). Several approaches were tried in preliminary experiments to immobilize sCD16a on the sensor chip, including the standard amine coupling procedure to coat either sCD16a directly or a nonblocking anti-CD16 mAb 214.1, which in turn captured sCD16a. Unfortunately, the former adversely affected the ability of sCD16a to bind ligands and the latter did not allow satisfactory regeneration of the sensor chip. The approach that worked best with our reagents appeared to be coupling first a rabbit anti-mouse Fc antibody to the sensor chip with the amine coupling procedure then binding mAb 214.1, followed by capturing sCD16a. This approach also ensured the consistent orientation of the sCD16a molecule on the sensor chip. The sensor chip could be regenerated with three washes of 10 μlof 1 m formic acid (Sigma), which completely removed noncovalently bound sCD16a and 214.1 without impairing the reactivity of the anti-mouse Fc antibody. The binding of mAb 214.1 was ≥95% of the initial level after 40 times of regeneration. Using the full rabbit anti-mouse Fc antibody yielded negligible sCD16a binding to its Fc region, as confirmed by a control experiment in which sCD16a was injected over the sensor chip coupled with the full rabbit anti-mouse Fc antibody alone without mAb 214.1 followed by subsequent injection of IgG ligands. The kinetic rates of IgG-sCD16a binding were derived by globally fitting the Langmuir (1:1) model (cf. Equations 1 and 2) to the family of association and dissociation curves collected at different ligand concentrations, using BIAevaluation 3.0 software provided by the manufacturer. The affinity of CLBFcgran-1 Fab-sCD16a binding was derived by Scatchard analysis. Kinetic Measurement of Cell Surface CD16 by Real Time Flow Cytometry—5 × 106 cells were washed three times in 5 ml of binding buffer (RPMI, 1% IgG-free BSA, 0.02% sodium azide). Cells were resuspended in 0.5 ml of binding buffer and transferred to a tube. Fluorescein isothiocyanate-labeled ligands in binding buffer were added and quickly mixed with the cells to obtain 2.5 × 106 cells/ml cell concentration and the desired final ligand concentration. A range of ligand concentrations was selected based on separate affinity measurements using other techniques, such that the receptors could be saturated at high concentrations yet the ligands would not be depleted in low concentrations. To assay for association, the sample was immediately run in a FACS Vantage machine (BD Biosciences) and a total of 15 fluorescence intensity histograms were measured sequentially in predetermined time points over a 12-min period. The earliest time point a histogram could be measured was ∼10 s. Each histogram included >2,000 events and took ∼2 s to acquire. The sample was vortexed between two consecutive time points to ensure cells and ligands were well mixed. The right-shift of the histogram toward higher fluorescence intensity was monitored in real time. To assay for dissociation, the remaining sample from the association assay was incubated for another 20 min to ensure equilibrium was achieved. 1 ml of the sample was centrifuged, the supernatant was decanted, and the tube was vortexed to disperse the cell pellet. 2 ml of plain medium was added to resuspend the cells. The sample was immediately run in the FACS machine to measure another 15 fluorescence intensity histograms sequentially in predetermined time points over a 12-min period. The background-subtracted mean fluorescence intensity (MFI) versus time data of the association and dissociation assays were, respectively, fit to equations, C=konLRTkonL+koff{1−exp[−(konL+Koff)t]} (Eq. 1) and C=ηkonLRTkonL+koffexp(−kofft) (Eq. 2) where kon and koff are the respective on- and off-rates and t is time. The concentration of bound ligands C is proportional to the measured MFI. Neglecting depletion due to binding, the free ligand concentration L is assumed to be the same as that added to the reaction mixture. RT is the concentration of total receptors. The proportionality constant η accounts for the difference between the equilibrium MFI from the association assay and the initial MFI from the dissociation assay. The standard free energy of binding, ΔG° (kcal mol–1), was calculated from the Kd measured at various temperatures, ΔG° = RTln(Kd), where R is the universal gas constant (1.987 × 10–3 kcal mol–1 K–1), T is the absolute temperature, and Kd is expressed as mole liter–1. Affinity Measurement for Cell Surface CD16—The affinities of CLBFcgran-1 Fab for CHO cell CD16a™, CD16bNA1, and CD16bNA2 were determined by Scatchard analysis. The affinities of monomeric IgG to CD16 were measured by a competitive inhibition binding assay as previously described (6Chesla S.E. Li P. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2000; 275: 10235-10246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, CHO cells were grown in flasks until near confluence. Cells were rinsed once in PBS and then removed from the flask using PBS/EDTA. After washing, they were resuspended at 106 cells/ml in PBS/EDTA and added to V-bottom 96-well plates at 100 μl/well. The wells were precoated with 1% IgG-free BSA (Sigma) in PBS by incubating at room temperature for 2 h. They were rinsed with PBS/EDTA and kept on ice until the cells were added. After adding cells, the plates were spun at 2000 rpm for 2 min. The supernatant was removed and 50 μl of IgG in PBS/EDTA at the titrated concentrations were added to each well with mixing. Then, 50 μlof 125I-CLBFcgran-1 Fab in PBS/EDTA at a concentration of 0.25–0.50 μg/ml was added to each well, followed by a 45-min incubation on a shaker at 5 °C. After washing 3 times, the cell pellets were removed and counted in a γ counter. In the presence of increasing concentrations of the low affinity ligand (IgG, concentration cll), the binding of the high affinity ligand (125I-CLBFcgran-1 Fab, concentration chl) to the cell surface receptor (CD16) is gradually reduced, or displaced. The displaced fraction, defined as the bound fraction (f) of CLBF-cgran-1 normalized by the value when no IgG was present (f0), can be expressed by Equation 3. f/f0=[cr+crh+(cllKal+1)/Kal]/2cr−{[cr+clh+(cllKal+1)/Kal]2−4crclh}1/2/2cr (Eq. 3) Because the affinity to CD16 of 125I-CLBFcgran-1 Fab (Kah) and the receptor concentration (cr) were predetermined from a separate experiment by Scatchard analysis, the only unknown in Equation 3 is the affinity of IgG (Kal). Therefore, (Kal) can be calculated from a single measurement of F without the experimental displacement curve to include data at the IC50 point. To increase the accuracy of the (Kal) value, however, the predicted displaced fraction (Equation 3) was nonlinearly fit to the entire f/f0 versus cll data set. Demonstration of Binding Specificity—As shown in Fig. 1, monomeric hIgG did not bind to a CM5 chip surface only coated with rabbit anti-mouse Fc antibody and mAb 214.1. However, after further functionalization with sCD16a, a similar injection of monomeric hIgG resulted in a time-dependent binding of 370 resonance units in 150 s. Switching the perfusate from hIgG solution to plain buffer resulted in dissociation of the bound hIgG. The chip was regenerated by washing off the 214.1-sCD16a-HIgG complex with three injections of 1 m formic acid at 2 min each, and then re-capturing mAb 214.1 and sCD16a. Binding resulted when the saturating concentration of CLBFcgran-1 Fab (anti-CD16 blocking mAb) was injected; however, subsequent injection of monomeric hIgG did not yield further binding (not shown), indicating nearly complete blockade of the sCD16a sites for hIgG binding. These results indicate that the binding of hIgG to the surface coated with sCD16a was mediated by the specific hIgG-sCD16a interaction. Separation of Monomeric and Multimeric IgG—The presence of multimers in commercial IgG even after high speed centrifugation was revealed by size exclusion chromatography as exemplified in Fig. 2A for RbIgG. Results for hIgG, hIgG1, and hIgG3 were similar (data not shown). The first peak, which appeared at fraction 28, was eluted at nearly the same time as the peak fraction of blue dextran (indicated by arrow). Blue dextran has a molecular mass (2,000 kDa) much higher than that of monomeric IgG (∼150 kDa) and was excluded by Sephadex G-200. This suggests that the first peak represents multimeric RbIgG. A second peak of RbIgG, which appeared at fraction 40, was eluted before BSA (peaked at fraction 51). The relative elution volumes (Ve/Vo) of cytochrome c, BSA, and IgG (2nd peak, assumed to be monomeric) are plotted against their molecular weights in Fig. 2B and compared with published results (21Andrews P. Biochem. J. 1965; 96: 595-606Crossref PubMed Scopus (2430) Google Scholar). Good agreement is seen between our data and those of Andrews (21Andrews P. Biochem. J. 1965; 96: 595-606Crossref PubMed Scopus (2430) Google Scholar), indicating that the second IgG peak in Fig. 2A indeed represents monomers. The small difference in the relative volume of cytochrome c between the results of Andrews (21Andrews P. Biochem. J. 1965; 96: 595-606Crossref PubMed Scopus (2430) Google Scholar) and ours may be attributed to the difference in how long the column had been used, which was noted to influence the relative position of low molecular weight protein. As shown in Fig. 2A, the multimeric RbIgG comprised a significant fraction of RbIgG from the commercial source, which would have adversely affected the SPR measurement if not removed. To determine whether the chromatographed IgG would form aggregates in experiments where reconcentrated IgG was used, the monomeric RbIgG fractions were pooled, reconcentrated to 5 mg/ml, and passed through the column again in the time scale of SPR experiments. It is evident from Fig. 2A that only a single, symmetric peak of monomeric IgG was seen, apparently free of any detectable multimers. This suggests that re-aggregation of IgG is a relatively slow process and did not occur under our experimental conditions, which is consistent with a previous report (12Vance B.A. Huizinga T.W. Wardwell K. Guyre P.M. J. Immunol. 1993; 151: 6429-6439Crossref PubMed Google Scholar). Kinetics of Monomeric IgG-sCD16a Interactions—Monomeric IgG solutions of increasing concentrations were perfused over the sCD16a-derivitized sensor chip and the interaction time courses were measured, as exemplified in Fig. 3. It can be seen that both the association and dissociation were relatively slow, requiring a few minutes to achieve equilibrium or reach half-dissociation. It is also apparent that RbIgG bound to and dissociated from sCD16a faster than human IgGs. sCD16a was immobilized to the sensor chip via a nonblocking anti-CD16 mAb 214.1, which in turn was captured by a rabbit anti-mouse Fc antibody. Although at rates much slower than those of IgG ligands dissociating from sCD16a, sCD6a and 214.1 also dissociated from their respective capturing antibodies over time, manifested as a negatively drifting baseline. To correct for this effect, the curve resulting from an injection of plain buffer alone, in which the slow dissociation of 214.1 and sCD16a was monitored, was subtracted from all the measured binding curves. The refraction index difference between protein samples and the running buffer, manifested as an instantaneous resonance unit change, was also determined in a control experiment (on the same rabbit anti-mouse Fc antibody-coated chip but without sCD16a immobilization) and subtracted from raw data in all sensorgrams presented here. A Langmuir (1:1) model was fit simultaneously to the entire family of data curves in both association and dissociation phases. Such a global fitting procedure accentuates deviation at any one concentration but ensures more robust kinetic constants overall (22Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (268) Google Scholar). The kinetic rates so evaluated are summarized in Table 1; and the model predictions based on these parameters are exemplified in Fig. 4 along with the data. It can be seen that the model fits both the association and dissociation data of the hIgG-sCD16a interaction reasonably well. Similarly good fits were obtained for the hIgG1- and RbIgG-sCD16a interactions (data not shown). By comparison, the fit for the hIgG3-sCD16a interaction is not as good (data not shown). Despite its low concentration in serum (23Allansmith M. McClellan B.H. Butterworth M. Maloney J.R. J. Pediatr. 1968; 72: 276-290Abstract Full Text PDF PubMed Scopus (187) Google Scholar), hIgG3 is known to comprise at least 12 allotypes (24Propert D. Exp. Clin. Immunogenet. 1995; 12: 198-205PubMed Google Scholar). In this study, no attempt was made to further separate the various hIgG3 allotypes from the purchased heterogeneous mixture, which, we believe, is the reason why the homogeneous Langmuir model does not fit the hIgG3-sCD16a interaction data well.TABLE 1Kinetic rates and equilibrium dissociation constants of sCD16a for IgG measured by SPRLigandkonkoffKd103 m-1 s-110-3 s-1μmMonomerichIgG (n = 6)6.51 ± 0.264.71 ± 0.240.72 ± 0.02hIgG1 (n = 4)8.18 ± 0.285.74 ± 0.360.71 ± 0.04hIgG3 (n = 4)10.64 ± 1.345.89 ± 0.330.56 ± 0.09RbIgG (n = 5)17.62 ± 3.7518.6 ± 2.521.07 ± 0.16MultimerichIgG (n = 3)12.67 ± 0.931.98 ± 0.140.16 ± 0.02hIgG1 (n = 3)15.59 ± 1.131.19 ± 0.020.077 ± 0.006hIgG3 (n = 3)13.19 ± 0.240.67 ± 0.020.051 ± 0.001RbIgG (n = 3)9.27 ± 0.151.03 ± 0.020.11 ± 0.002 Open table in a new tab Kinetics of Multimeric IgG-sCD16a Interactions—Compared with monomers, multimeric IgG dissociated from sCD16a much more slowly and required much lower concentrations to achieve the same level of binding, indicating a much higher avidity (Fig. 5). For the purpose of comparing our results with data in the literature, the same Langmuir model was used to fit the association and dissociation curves of IgG multimers. The kinetic mechanism for binding of multimeric ligands must not be 1:1; as such, a more involved model is needed to extract the intrinsic kinetic constants. Fitting the simple Langmuir model to the multimeric ligand binding data returns the apparent rates of a surrogate monomeric ligand whose kinetics would generate approximately the same time courses as the multimeric ligand. The apparent on- and off-rates so obtained nevertheless reveal the much higher binding capacities of the IgG multimers than those of the IgG monomers. The apparent rates obtained from the multimeric ligand experiments are listed in Table 1. Affinity of mAb CLBFcgran-1 Fab-sCD16a Interactions—It should be instructive to compare receptor-ligand and antibody-antigen interactions. CLBFcgran-1 Fab solutions at various concentrations were perfused over a sCD16a-derivitized surface. It is evident from Fig. 6A that CLBFcgran-1 Fab bound sCD16a with a much higher affinity and a much slower off-rate than any of the sCD16a ligands tested. Antibody typically binds to antigen with a rather high onrate. In such a case mass transfer may be the limiting step (4van der Merwe P.A. Bodian D.L. Daenke S. Linsley P. Davis S.J. J. Exp. Med. 1997; 185: 393-403Crossref PubMed Scopus (430) Google Scholar); fitting a transient model to the association and dissociation time courses would likely yield erroneous results. To circumvent this problem, Scatchard analysis (Fig. 6B) was used to derive the binding affinity of CLBFcgran-1 Fab for sCD16a. The estimated equilibrium dissociation constant is in the order of nanomolar (Table 2). In comparison, the Kd values for IgG-sCD16a interactions are in the order of micromolar.TABLE 2Equilibrium dissociation constants of monomeric IgG ligands and mAb to sCD16a measured by SPR as well as to CHO cell CD16 isoforms measured by Scatchard plot and competitive inhibitionsCD16aCD16atmCD16bna1CD16bna2RIgG (μm)1.07 ± 0.16 (n = 5)1.86 ± 0.19 (n = 3)29.9 ± 3.2 (n = 3)36.4 ± 12.4 (n = 3)hIgG (μm)0.72 ± 0.02 (n = 6)1.37 ± 0.30 (n = 3)31.1 ± 9.5 (n = 3)53.0 ± 11.0 (n = 3)CLBFcgran-1 Fab (nm)2.37 ± 0.07 (n = 2)4.59 ± 0.09 (n = 2)6.46 ± 0.76 (n = 2)12.1 ± 4.9 (n = 2) Open table in a new tab The sCD16a-IgG Binding Kinetics Were Not Affected by the Injection Time—The presence of a significant amount of multimers in the chromatographed IgG could give rise to a multiexponential appearance in both association and dissociation sensorgrams. Such a diagnostic feature was not observed (e.g. Fig. 3). However, this effect could be amplified by the long injection time because multimeric ligands would continue to bind the receptors after the binding of monomeric ligands had achieved equilibrium (25van der Merwe P.A. Brown M." @default.
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