Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

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 J. A. R. C. J. Immunol. 1997; PubMed Scopus (45) Google half-lives of the order of and equilibrium dissociation constants of several for binding to human IgG1 and K. der Merwe P.A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google found a of the order of for we kinetic and affinity of a soluble human FcγRIIIa (sCD16a) interacting with IgG in both monomeric and using We found that sCD16a interacted with monomeric rabbit IgG, and with on-rates of 1.8 × 6.5 × 103, 8.2 × 103, and 1.1 × 104 m–1 s–1, off-rates of 1.9 × 4.7 × 10–3, 5.7 × 10–3, and 5.9 × s–1, and equilibrium dissociation constants of 0.72, 0.71, and μm, respectively. sCD16a with with equilibrium dissociation constants and that the kinetics is were in both the and The kinetics found to be because of the or binding of the sCD16a The kinetic rates and affinities were also measured by flow cytometry and a competition inhibition binding using CD16 on the cell which were found to be in with those obtained by and hamster to human and as well as were as S. Chesla S. Anderson P. Zhu C. Selvaraj P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Selvaraj P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). by the extracellular domain of CD16a to the Fc domain of The IgG1 Fc domains were obtained from as the and the extracellular domain of in the of as P. Nagarajan S. Zhu C. Selvaraj P. Immunol. 38: PubMed Scopus Google Scholar). in the Fc domain of are and as in These were to the binding of FcγRs Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). The monoclonal antibody a from of Long The in from as P. R. PubMed Scopus Google Scholar). of by The rabbit Fc antibody from and subtypes and as well as were from used for the real time flow cytometry which a from of and with following the and IgG were by of in of at for and at The and the in of and a were with of and and of and The were with of at of were used to the The in 1 of of 12 and The of of bovine of of and of of IgG in 1 of the to a of the flow at the in of The at measured to the in or IgG were used or to for or real time flow cytometry In were IgG by using The of monomeric and IgG were by a experiments were in a using at were in and the at for kinetic For control the flow The and using were in experiments to sCD16a on the the to sCD16a or a which in the the of sCD16a to and the of the The that with our to be a rabbit Fc antibody to the with the binding by also the of the sCD16a on the The be with three of 1 which sCD16a and the of the Fc The binding of of the initial of Using the rabbit Fc antibody sCD16a binding to its Fc as by a control in which sCD16a the with the rabbit Fc antibody by of IgG The kinetic rates of binding were by the 1 and to the family of and dissociation at different using by the The affinity of binding by Kinetic of CD16 by × were three in of binding BSA, were in of binding and to a in binding were and with the to × cell and the of on affinity using such that the receptors be at high the be in low assay for the in a and a of fluorescence were measured in time a The time a be measured events and to The two time to and were well The of the fluorescence in real assay for the from the assay for to equilibrium 1 of the the and the to the cell of to the The in the to fluorescence in time a The mean fluorescence time data of the and dissociation assays to and and are the and off-rates and is The of is to the measured to the is to be the as that to the is the of receptors. The for the the equilibrium from the assay and the initial from the dissociation The of from the measured at is the × is the and is as for affinities of for cell and were by The affinities of monomeric IgG to CD16 were measured by a competitive inhibition binding assay as (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). were in were in and from the using they were at in and to at The were with in by at for were with and on the were cells, the were at for The and of IgG in at the were to well with in at a of to by a on a at the cell were and in a γ In the presence of of the low affinity the binding of the high affinity to the cell surface receptor is or The as the of by the when IgG be by the affinity to CD16 of and the receptor were from a by the in is the affinity of IgG be from a of the to include data at the the of the the to the data of Binding in monomeric to a surface with rabbit Fc antibody and with a of monomeric in a binding of resonance in the from to in dissociation of the The by the with three of 1 at and and Binding when the of of monomeric binding of the sCD16a for These that the binding of to the surface with sCD16a by the of and presence of in IgG high by as in for for and were The which at at the time as the of by has a that of monomeric IgG and by that the of which at at The of BSA, and IgG to be are in and with P. J. PubMed Scopus Google Scholar). is our data and those of P. J. PubMed Scopus Google Scholar), that the IgG in The in the of the of P. J. PubMed Scopus Google and be to the in how long the been which to the of low in the a of from the which have the the IgG in experiments IgG the monomeric were to and the in the time of is from that a of monomeric IgG of that of IgG is a and our which is with a B.A. Huizinga T.W. Wardwell K. Guyre P.M. J. Immunol. 1993; 151: 6429-6439Crossref PubMed Google Scholar). of IgG of were the and the interaction time were as in be that both the and dissociation were a to equilibrium or is also that to and from sCD16a human sCD16a immobilized to the a which in by a rabbit Fc at rates those of IgG from and also from antibodies as a for this the from an of in which the dissociation of and sCD16a from the measured binding The and the as an resonance also in a control the rabbit Fc sCD16a and from data in to the family of data in both and dissociation a at kinetic constants 1998; PubMed Scopus Google Scholar). The kinetic rates are in and the on these parameters are in with the be that the both the and dissociation data of the interaction were obtained for the and interactions the for the interaction is as its low in serum M. M. J. Full Text PDF PubMed Scopus Google Scholar), is to at least 12 Exp. 1995; Google Scholar). In this to the from the we is the the the interaction data rates and equilibrium dissociation constants of sCD16a for IgG measured by in a of with IgG from sCD16a and to the of a For the of our with data in the the used to the and dissociation of IgG The kinetic for binding of be as a is to the kinetic the to the binding data the rates of a monomeric kinetics the time as the The and off-rates obtained the binding of the IgG those of the IgG The rates obtained from the experiments are in of be to and interactions. at were a is from that sCD16a with a affinity and a of the sCD16a binds to antigen with a high In such a be the 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 a to the and dissociation time likely this used to the binding affinity of for The equilibrium dissociation is in the order of In the for interactions are in the order of dissociation constants of monomeric IgG and to sCD16a measured by as well as to cell CD16 isoforms measured by and competitive in a The Binding by the presence of a of in the IgG to a in both and dissociation a this be by the long time because to the receptors the binding of monomeric equilibrium der Merwe P.A. Davis S.J. J. 1993; PubMed Scopus Google Scholar). to high in in the the to an The the the the as the on the surface time der Merwe P.A. Davis S.J. J. 1993; PubMed Scopus Google Scholar). these of were immobilized sCD16a in an with long time and low flow and respectively) to the The kinetic rates obtained × m–1 and × from the data in were with those in which were from experiments that the by using a time and flow and The of of kinetic rates on the time and flow as by the the two of a of the in The Binding by the to the of and were in a kinetic has been that in this monomeric by which in the kinetic der Merwe P.A. Davis S.J. J. 1993; PubMed Scopus Google Scholar). this to a those used in kinetic which used in an with long time and flow and respectively) to The and off-rates × m–1 and × s–1, respectively) obtained are to those measured from experiments using and with time and flow that the IgG have of an on the kinetic Binding to sCD16a in Kinetic as the two binding of the sCD16a to binding and the of immobilized sCD16a were by of a of to the binding by at were to the as in The and off-rates × m–1 and × s–1, respectively) are to those obtained from experiments using sCD16a the of the two binding of the on Binding and to sCD16a on of experiments were to on has binding with anchored on the cell In the the binding kinetics of monomeric for the cell were measured by real time flow time of to to binding of of the dissociation time The low affinity of binding (Kd in of to which in high of The high in the which also with in to the presence of a high of fluorescence molecules in the binding as in the dissociation assay the that such were by binding that the time for the data binding were obtained by of from that of measured at at time and 1 and were to the and dissociation data for with a of parameters and the of an because the equilibrium from the assay the initial of the dissociation assay and to the of the in the dissociation data that were used in the to the of on the The found to with the of as by The the and for × × 104 m–1 and equilibrium dissociation (Kd which are with from the × s–1, × 104 m–1 s–1, were at and and the kinetic parameters are in is that they are with those of the binding measured by The of binding at these were also The of on found to with K. der Merwe P.A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Biochemistry. 1995; PubMed Scopus Google Scholar). of of the above results, the affinities of and monomeric IgG for cell surface CD16 isoforms were measured in the of 12 of the binding of to and The affinities are in the of monomeric and for three CD16 The from to the data (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), are in is that for three and the affinities of on are in with those of sCD16a immobilized on the also be that and with affinities and and have affinities for and of monomeric and with for CD16 to or on in the presence of varying of monomeric or mean were by binding in the absence of and by which the affinity The in and three with The of the to the kinetic rates of interactions in the technology these studies have several of that be by the of this P. Biophys. 1997; PubMed Scopus Google Scholar). We to are the kinetic rates measured in the to monomeric this several were to in of the include P. Biophys. 1997; PubMed Scopus Google Scholar), the presence of to of the or the of der Merwe P.A. Davis S.J. J. 1993; PubMed Scopus Google Scholar), the the two binding of the the of kinetic of the and in receptor P. Biophys. 1997; PubMed Scopus Google Scholar). These are in the the have been by the of and have been the dissociation to The for a in the is the of of to that of such that when the is considered to be is the binding of the surface and is the on the of and for the b, and are the and of the flow is the flow and is the of the the in our experiments were the were and in be at the low flow the were of for IgG a which the for the to The of kinetic rates to the flow and × m–1 and and × for and respectively) and to the of immobilized sCD16a and × m–1 and and × for and resonance respectively) for the of of on our the that to be a were in both and data The of sCD16a immobilized on the low resonance The flow used for the kinetic the in our the data in the to the used for the dissociation to of these and the it is that the measured off-rates are by the of monomeric binding in experiments with two flow in a The interaction and sCD16a likely by the kinetic rates from the in × m–1 and × be to the low for the is to be a and the affinity measured be The Kinetic of by the of used to and from the IgG the monomeric IgG used in have of a to the dissociation and to this monomeric IgG by to the used in the and the in the time of the be from the IgG as a at the as that of the monomeric IgG in the were in the is in with a which that monomeric IgG monomeric in for at least B.A. Huizinga T.W. Wardwell K. Guyre P.M. J. Immunol. 1993; 151: 6429-6439Crossref PubMed Google Scholar). this low IgG were used in the which were These that the of IgG be the of the of in experiments were with different flow rates and has been that when monomeric were with time in binding der Merwe P.A. Davis S.J. J. 1993; PubMed Scopus Google Scholar). Using we also found binding of IgG with a time in the kinetic rates of monomeric binding The found when IgG used the kinetic rates were the for both and monomeric or the high or low for the kinetic rates to be to the of The two be Kinetic of binding on-rates and off-rates experiments were to in the of in the IgG, its have been low to the kinetic rates of the monomeric interactions. The of of in our is to the low of monomeric used in the experiments In the of K. der Merwe P.A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), by the used were as high as sCD16a Binding as is that two IgG molecules that are to the sCD16a to a which the sCD16a because there are two for the IgG of in an this sCD16a in an by a of of an which binds to an the binding A. J. Immunol. 1996; PubMed Google Scholar). and binding of to antigen of the on the sCD16a is or it be that when of Fc binding of those sCD16a that have at least have both for IgG In of sCD16a molecules have a for IgG the sCD16a a have been in this the kinetic rates in this were found to those measured using sCD16a and × m–1 and and × for experiments using and respectively) that the of sCD16a the kinetics of its interaction with Binding Kinetic and used a to the data to kinetic The for the interaction is by a R. Biochemistry. 1995; PubMed Scopus Google and by a P. R. Jacob U. 2000; PubMed Scopus Google Scholar, S. S. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). kinetic we both a and a and IgG, intermediate and using the in to the the and the data were to the with two The of surface is to the with antibodies likely in and of the immobilized sCD16a for sCD16a this a surface for because time sCD16a from which also from the rabbit Fc of the surface is 1998; PubMed Scopus Google Scholar), the dissociation of interactions that of interactions the to be an for the of the dissociation on the measured kinetic rates of the The IgG in the are for those of For example, at least 12 Exp. 1995; Google Scholar). to the of the to the data of or human IgG in serum is to of with of and for C. of R. G. Landes, Austin, Google Scholar). experiments that binds sCD16a with an affinity that of The of the to in the of its with that monomeric interactions have been the sCD16a the binding of cell surface are our with from These are because which by the extracellular domain of CD16a with the Fc of P. Nagarajan S. Zhu C. Selvaraj P. Immunol. 38: PubMed Scopus Google Scholar), the transmembrane and cytoplasmic domains and the γ signaling the sCD16a by affinity and immobilized on the the (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 and the γ (10Miller K.L. Duchemin A.M. Anderson C.L. J. Exp. Med. 1996; 183: 2227-2233Crossref PubMed Scopus (70) Google have been to kinetic the two of binding studies were using flow cytometry kinetic off-rates to that measured by SPR, the on-rates and hence dissociation from the two experiments by a that to cell with a ∼ of the in the using The of for monomeric and measured from the competitive inhibition binding experiments were also of those of the sCD16a for the These data are with the of a of which that the of CD16 on its the on the of the of CD16 on the cell surface or from the cell and immobilized on a P. and C. the different the cell and the sCD16a it is to the in the binding sCD16a on the IgG and a with affinities to those of on the cell of and binding to off-rates and × s–1, respectively) and for or for equilibrium dissociation constants J. A. R. C. J. Immunol. 1997; PubMed Scopus (45) Google with the found for the and interactions our competitive inhibition binding experiments using found that and IgG both human and rabbit with several tens those of cell the a using an monomeric immobilized found off-rates of a K. der Merwe P.A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). has been found at this and studies are to these We measured the kinetic rates of cell and and cell and cell interacting with IgG to the cell surface using a micropipette (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). The off-rates for the and and and interactions were and s–1, respectively. the the and surface the binding off-rates have the of s–1, they have been as PubMed Scopus Google Scholar). the and binding is of by in the molecules in interactions are by to which they are are the of the and the of the molecules to binding Zhu C. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The the measured the and the measured by the micropipette has been in a Selvaraj P. Zhu C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The has such These a for the and kinetic