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High-affinity binding measurements of antibodies to cell-surface-expressed antigens
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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 373 (2008) 52–60 www.elsevier.com/locate/yabio
High-affinity binding measurements of antibodies to cell-surface-expressed antigens Palaniswami Rathanaswami *, John Babcook, Michael Gallo Amgen Inc., Burnaby, BC, Canada V5A 1V7 Received 7 July 2007 Available online 16 August 2007
Abstract A simple method that allows affinity measurements of antibodies to integral membrane proteins is described. Kinetic Exclusion Assay was used to determine the concentration of free antibody that remains in solution after equilibrium has been established between the antibody and the cell-surface-expressed antigen, from which the equilibrium dissociation constant (Kd) was determined. It eliminates the requirement for soluble antigen and modifications such as radio-labeling or fluorescent labeling of the antibody. For one of the cell-surface-expressed antigens, it was determined that the affinity of the antibody to the cell-surface-expressed antigen was similar to that of the purified, soluble form of the antigen. In addition to the simplicity of the approach, the method provides a true measure of the affinity/avidity of the antibody to the native form of cell-surface-expressed targets, including antigens that cannot be produced in soluble forms, and to unknown cell surface antigens. 2007 Elsevier Inc. All rights reserved. Keywords: Affinity measurement; On-cell binding; Membrane-expressed antigen; High affinity; KinExA; Cell-surface-expressed molecule; Transmembrane-expressed molecule; Human antibodies; Unmodified antibodies; Native binding
Monoclonal antibodies (mAbs)1 represent a new and increasingly important therapeutic modality in treating many diseases such as cancer [1,2]. When choosing the best antibody for therapeutic purposes, in addition to specificity, the affinity of an antibody to the target is also important. A direct correlation between the affinity and the potency of an antibody exists [3,4] and, for therapeutic purposes, antibodies with binding constants greater than 10 nM are not likely to be useful for drug targeting or tumor imaging [5]. High-affinity mAbs, in contrast to low-affinity mAbs, target tumors better [6] and enhance the antibody-dependent cellular-cytotoxicity-mediated killing of tumor cells even at low antigen expression levels [7]. *
Corresponding author. Fax: +1 (604) 676 8349. E-mail address: [email protected] (P. Rathanaswami). 1 Abbreviations used: mAbs, monoclonal antibodies; FACS, fluorescence-activated cell sorting; KinExA, Kinetic Exclusion Assay; ELISA, enzyme-linked immunosorbent assay; NHS, N-hydroxy succinimide; CHO, chinese hamster ovary; AgM, antigen multiplier. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.08.014
There are several established technologies/methodologies available to experimentally determine the affinity of antibodies to soluble antigens. If the antigen is expressed on a cell membrane, the measured affinity of the antibody to the soluble antigen counterpart, typically the extracellular domain, may not correlate with the antibody’s binding affinity to the native form of the antigen on the cell. In addition, purification of membrane proteins is difficult and may result in a loss of stability or in partially functional protein [8]. Studies characterizing the affinity measurements of antibodies to cell-surface-expressed molecules typically modify the antibodies with fluorescent or radioactive labels so that they can be detected [9,10]. However, it has been shown that chemical modification of an antibody by 125I [11], flourescein isothiocyanate, and RD1 labeling can lead to binding constants that are 10 to 100-fold lower than those of the native antibodies [12]. Some of the methods using radiolabeled antibodies require extensive cell washing after the equilibrium is reached [10] which might lead to the dissoci-
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
ation of the antibody from the cell membrane and disturb the equilibrium. Bator and Reading [13] described a simple ELISA method to measure the apparent affinity of an antibody for cell-surface antigens using Scatchard analysis. The sensitivity of determination of the affinity by this method is in the nanomolar range. Fluorescence-activated cell sorting (FACS) methods also have been used to measure the affinity of cell-membrane-expressed antigens [14,15]. However, these methods used a linear transformation of the data to calculate the affinity. Scatchard and other linear transformations of the binding isotherm data can sometimes conceal deviations from simple single-site binding patterns that can be recognized by nonlinear analysis [16]. A recent report [17] describes the affinity measurement of an antibody to cell-surface receptors and overcomes many of the above problems by using Kinetic Exclusion Assay (KinExA). However, this reported method requires the use of a soluble antigen as a probe for measuring the affinity of the antibody to cell-surfaceexpressed molecules. Here we describe a modified method in which soluble antigen is not required for measuring the affinity of a purified antibody to its cell-membraneexpressed target.
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ose beads were prepared by adsorbing the GST-TT-TNFa onto glutathione–Sepharose beads. Cell culture CHO cells expressing a mutant noncleavable form of TNFa on the surface of the membrane were used. Briefly, the cDNA of TNFa was mutated at various positions to prevent cleavage of TNFa from the surface of cells and cloned into an expression vector. CHO cells were transfected with this mutated form of TNFa and cells expressing TNFa in stable form on the membrane were placed under drug selection to generate a DTNFa cell line. The cells were cultured in standard tissue culture conditions in an incubator with 5% CO2. Ham-F12 medium was used to grow the cells with 10% fetal calf serum (Gibco), 2 mM L-glutamine, 1 mM sodium pyruvate, and penicillin/streptomycin (100 IU/ml/100 mg/ml (Gibco). Cells were treated with 8 mM sodium butyrate overnight before use. CEM cells expressing CD45RB were cultured in RPMI 1640 medium with 2 mM L-glutamine and adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM Hepes, and 1.0 mM sodium pyruvate. Fetal bovine serum was added to this to a final concentration of 10% V/V.
Materials and methods Cell-binding equilibrium assays Azlactone beads were obtained from Sapidyne Instruments (Idaho, U.S.A). Cy5-conjugated affinity-purified antibodies (all have minimal cross-reactivity to bovine, horse, and mouse serum proteins) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, U.S.A). Goat anti-human Fcc fragment-specific IgG antibody was obtained from Immunodiagnostics (Bellingham, WA, U.S.A). Protein A–Sepharose, NHS-activated Sepharose 4 Fast Flow, and glutathione–Sepharose beads were purchased from Amersham Biosciences, Inc. (Quebec, Canada). ImmunoPure Fab preparation kit was purchased from Pierce (Rockford, IL, U.S.A.). The rhTNFa was obtained from R & D Systems (Minneapolis, MN, U.S.A.). Human TNFa was also expressed as a fusion protein with GST and a T-cell epitope from tetanus toxin (GST-TT-TNFa). Anti-CD45RB mAb 6G3 [18] was produced from a hybridoma. Anti-human CD45RB mAb 487 and anti-human TNFa mAb 263 were developed by Amgen 0 s XenoMax technology [4] and produced as recombinant antibodies. Protein A–Sepharose affinity–chromatography-purified mAbs were used in all experiments. The purity was assessed and the quantity was calculated by nonreducing SDS–PAGE. The concentration was further confirmed by UV analysis at A280 and ELISA. Preparation of antigen- or Ig-coated beads Goat anti-human Fcc fragment-specific IgG antibody (100 lg) was coupled to Azlactone beads (300 mg) as recommended by the manufacturer or to NHS-activated Sepharose beads as described [4]. GST-TT-TNFa-Sephar-
Cells were serially diluted 1 in 2 in 11 falcon tubes using the culture medium and containing a final concentration of 0.05% NaN3. The 12th tube contained just the medium. An appropriate concentration of purified mAb was made in cell culture medium and an equal volume of the mAb was mixed with the serially diluted cells. The cells with the mAb were then rotated at 4 C for 36 h. At the end of 36 h, the cells were centrifuged at 2400 rpm for 4 min and the free mAb present in the supernatant was measured by KinExA using appropriate beads and Cy5-conjugated anti-human secondary antibodies as described [4]. The equilibrium dissociation constant (Kd) [17] was obtained using KinExA software [19,20] and by ‘‘n-curve analysis’’ which fits all of the given curves to a single Kd value simultaneously [4,17]. Cell-binding affinity measurements with a corresponding Fab fragment The Fab fragment was prepared as per the manufacturer 0 s recommendation. The completeness of digestion and the purity were assessed by running on an acrylamide gel. Cells were titrated, mixed with an equal volume of Fab fragment at defined concentrations, and continued as described for the full mAb. The free Fab fragment present in the supernatant was measured by KinExA using antigencoated Sepharose beads to absorb the Fab fragment and detected using Cy5-conjugated affinity-purified goat antihuman (H+L)-specific IgG. The Kd was obtained as described above.
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High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Affinity measurements with soluble TNFa and anti-human TNFa mAb or Fab Briefly, a constant amount of anti-human TNFa mAb or Fab 263 was incubated with titrating concentrations of rhTNFa antigen in sample buffer (phosphate-buffered saline with 0.1% bovine serum albumin and 0.05% NaN3). Antigen/antibody complexes were incubated at room temperature for 36 h to allow equilibrium to be reached. The mixture was drawn through the GST-TT-TNFa-Sepharose beads to accumulate unbound antibody. The captured antihTNFa mAb or Fab is directly proportional to the remaining free-binding sites [4] and was detected using solutions containing Cy5-conjugated anti-human secondary antibody in sample buffer. The Kd was determined as described above. Scatchard analysis A constant number of cells was added to titrating concentrations of anti-hTNFa mAb 263 and incubated for 36 h at 4 C. At the end of 36 h, the cells were centrifuged at 2400 rpm for 4 min and the signal generated by the free mAb present in the supernatant was measured by KinExA using either goat anti-human Fcc fragment-specific IgG antibody-coupled azlactone beads or GST-TT-TNFaSepharose beads to absorb the mAb and detected using solutions containing Cy5-conjugated affinity-purified goat anti-human Fcc fragment-specific IgG secondary antibodies. The signal corresponding to the total antibody in each dilution was also measured in KinExA. From the signal obtained for the total antibody, the amount of free antibody present in the supernatant and the amount of antibody bound to cells were calculated. The Scatchard plot was done by plotting B/F vs B [21] and the Kd was calculated from the slope.
site concentration of mAb was used as a determinant for the KinExA software to calculate the antigen multiplier (AgM) and the Kd using the unknown antigen method [17]. The antigen multiplier is a multiplication constant used in conjunction with the assumed antigen concentration (1 · 106 cells = 1 nM antigen) variable to determine an active molar concentration of the receptor. The calculated antigen multiplication constant will be a fraction if 1 · 106 cells contain a molar concentration of receptors less than the arbitrarily assumed concentration (1 nM). It will be greater than one if 1 · 106 cells contain more than the assumed antigen concentration. As shown in Fig. 1, the Kd calculated for the anti-hCD45RB mAb 487 was 670 fM (Fig. 1B) with 95% confidence interval of 2.30 pM (Kd high) to 140 fM (Kd low). The % error was only 2.9. Even though the active site concentration of antibody used in the incubation was higher than the calculated Kd, the determined Kd bounds were reasonably narrow [4]. Based on the bounds and the antigen concentration that binds to 50% of the mAb (Fig. 1A), it can be concluded that the Kd should be in the low picomolar range. An antigen multiplier of 0.0185 was determined by the software (Fig. 1C), which translates into the presence of 11,470 antibody-binding antigen molecules/cell as described by Xie et al. [17]. The Kd measurements for the anti-CD45RB mouse/ human chimeric mAb 6G3 using the same CEM cells expressing CD45RB are shown in Figs. 1D, 1E, and 1F. The determined Kd for the mouse/human chimeric mAb is 1.56 pM. The AgM was 0.0323, which is equal to 20,000 antibody-binding antigen molecules/cell. The variation seen in the number of receptors on the CEM cells between mAb 487 and 6G3 may be due to the possible difference in available epitope concentration, actual differences in the active binding site concentration for each mAb, or day to day variation of receptor expression levels.
Results Affinity of membrane-expressed TNFa Affinity of CD45RB expressed on CEM cells CEM cells expressing CD45RB antigen were harvested, resuspended in fresh medium (2.5 · 106 cells/ml), titrated 1 in 2, incubated with a constant amount (10 pM) of antihCD45RB mAb in the presence of 0.05% NaN3, and allowed to reach equilibrium. At equilibrium, the free mAb left in the supernatant was measured in KinExA using Protein A–Sepharose beads and Cy5-conjugated affinity-purified Fab fragment of goat anti-human (H+L)-specific IgG. The percentage free mAb left in solution was plotted against the concentration of antigen (arbitrarily defined as 1 nM of antigen per 1 million cells, see below for more details) by the KinExA software and a sigmoidal curve was generated, as shown in Fig. 1. A theoretical curve was fit to the data (Fig. 1A) and 95% confidence intervals were calculated by the KinExA software. The mAb was assumed to be 100% active. The active binding
Using goat-anti human Fc-coupled beads. The DTNFaCHO cells were dissociated from plates using cell dissociation buffer. Cells (25 · 106/ml) were resuspended in culture media, titrated 1 in 3, incubated with a constant amount (10 or 100 pM) of anti-hTNFa mAb 263, and allowed to reach equilibrium. After equilibrium, the free mAb left in the supernatant was measured using goat anti-human Fcc fragment-specific IgG antibody-coupled Azlactone beads and the Kd was calculated by the KinExA software using n-curve analysis [4]. A representative experiment is shown in Fig. 2. The Kd calculated for the human anti-hTNFa mAb 263 was 13.64 pM with Kd high of 26.8 pM and Kd low of 7.2 pM as 95% confidence intervals. The % error was only 2.8. AgM of 0.0828 was calculated by this method which translates into 49,870 receptors per cell. The experiment was repeated several times and the average Kd calculated from four experiments was 18.3 pM (Table 1).
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
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Fig. 1. Measurement of the Kd of anti-hCD45RB mAbs for the CD45RB expressed on CEM cells. CEM cells (2.5 · 106/ml) were serially diluted and incubated with 10 pM active binding site concentration of (A) anti-hCD45RB mAb 487 or (D) anti-CD45RB mouse/human chimeric mAb 6G3, in the presence of 0.05% NaN3 and allowed to equilibrate. The free mAb left in the supernatant is measured as explained in the text and the % free mAb is plotted against the antigen concentration (taking arbitrarily each million cells to be equal to 109 M antigen). The unknown antigen method was performed to determine optimal values for Kd and the antigen multiplier. (B and E) Best fit error analysis was performed by the software for the Kd by iteratively changing the value for the Kd while reoptimizing the other parameters of the curve fit for each iteration. The 95% confidence intervals were determined by plotting the change in best fit for each value of Kd. (C and F) Best fit error analysis for antigen multiplier was performed similarly by iteratively changing the value for antigen multiplier while holding the remaining parameters at their optimized values.
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High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Fig. 2. Determination of Kd of anti-hTNFa mAb 263 for cell-membrane-expressed TNFa. DTNFa-CHO cells (25 · 106/ml) were serially diluted and incubated with 10 pM (triangles) or 100 pM (diamonds) active binding site concentration of anti-hTNFa mAb 263, in the presence of 0.05% NaN3, and allowed to equilibrate. The free mAb left in the supernatant was measured as explained in the text. (A) The % free mAb is plotted against the antigen concentration (taking arbitrarily each million cells to be equal to 109 M antigen). Multiple curve analysis using the unknown antigen method was performed to determine optimal values for Kd and the antigen multiplier. (B and C) The 95% confidence intervals were determined by changing iteratively the optimized value for Kd or AgM while keeping other parameters at their optimal values.
Table 1 Kd measurement of anti-hTNFa mAb 263 for TNFa expressed on the cell membrane and soluble secreted TNFa mAb
Target
Kd (pM)
SEM
Anti-TNFa mAb 263a Anti-TNFa mAb 263b Anti-TNFa mAb 263c Fab fragment of anti-TNFa mAb 263d Fab fragment of anti-TNFa mAb 263e Anti-TNFa mAb 263f Anti-TNFa mAb 263g
Membrane-expressed TNFa Membrane-expressed TNFa Membrane-expressed TNFa Membrane-expressed TNFa Secreted soluble TNFa Secreted soluble TNFa Membrane expressed TNFa
18.4 13.4 15.9 79.0 31.6 9.1 63.3
3.14 1.05 1.75 0.05 Kd high = 42.8 Kd low = 22.8 Kd high = 13.4 Kd low = 3.5 5.45
a b c d,e,f g
Kd was measured using goat-anti human Fc-coupled beads. Kd was measured using antigen-coated (TT-TNFa-GST-Sepharose) beads. Average Kd was measured using both goat-anti human Fc-coupled and antigen coated beads. Kd was determined using antigen-coated beads. Kd was determined by Scatchard plot.
Using antigen-coated beads. Next we determined whether there would be a difference in the calculated Kd, if antigencoated beads are used in the bead column instead of the goat anti-human Fcc fragment-specific antibody capture. Titrated cells were incubated with mAb as before and
allowed to reach equilibrium. After equilibrium, the free mAb left in the supernatant was measured using GST-TTTNFa-Sepharose beads and the Kd was calculated by the KinExA software using n-curve analysis [4]. The average Kd calculated from four experiments was 13.4 pM (Table 1).
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Comparison of the affinity of full mAb with Fab. To determine whether the on-cell binding affinity determined for the full mAb in KinExA is the true affinity or whether there is an avidity effect involved, the experiment was repeated using the same batch of DTNFaCHO cells, which was used in experiments with full mAb, but replacing with Fab molecule. The cells (20 · 106/ml) were titrated 1 in 2, incubated with a constant amount (20 or 300 pM) of Fab fragment of mAb 263, and allowed to reach equilibrium. After equilibrium, the free Fab left in the supernatant was measured using GST-TT-TNFa-Sepharose beads and the Kd was calculated by the KinExA software using n-curve analysis. The antigen concentration on the cells which was determined using the full-length mAb was used as a determinant for calculating the Kd of the Fab using standard analysis. The affinity determined for the Fab molecule from several experiments is given in Table 1. It is clear from this table that the affinity of the Fab to the cellsurface-expressed TNFa is about 4 to 5 · lower than that of the full mAb, 79 pM for Fab VS 15.9 pM for full mAb. Comparison of affinity of full mAb and its Fab to membraneexpressed TNFa and secreted soluble TNFa To determine whether the lower affinity of the Fab fragment of anti-hTNFa mAb 263, compared to full mAb, to the cell-surface-expressed TNFa is due to a possible difference in the structure of the Fab or the affinity determined for the full mAb to cell-surface-expressed TNFa may be the result of an avidity effect, the affinities of full mAb and the Fab were determined for secreted soluble TNFa. A constant amount of anti-hTNFa mAb 263 (5 or 50 pM) or the Fab fragment (30 or 300 pM) was incubated with titrating concentrations of rhTNFa antigen starting at 100 nM and allowed to reach equilibrium and the Kd was determined [4]. Table 1 shows that the affinity of Fab to the secreted soluble TNFa also was about 4 times lower than the affinity of the full mAb, 31.6 pM for Fab VS 9.1 pM for full mAb. Scatchard analysis of membrane-expressed TNFa Since one conventional means of measuring affinity to a cellular target is done by Scatchard plot, we also did Scatchard experiments. Anti-hTNFa mAb 263 (200 pM) was titrated 1:2 in medium and incubated with 500,000 DTNFa cells/ml for 36 h at 4 C. The free antibody left in solution was measured and the amount of antibody bound to cells was calculated using KinExA software and used in Scatchard analysis. Fig. 3 shows a representative experiment and the Kd for full mAb was determined as 64 pM. The number of antigen molecules present on the cells was calculated to be 84,300 antigens/cell. Several experiments were done and the mean Kd was calculated to be 63.3 pM with SEM of 5.45.
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Fig. 3. Affinity determination of anti-hTNFa mAb 263 for cell-membrane-expressed TNFa by Scatchard analysis. Serially diluted 200 pM anti-hTNFa mAb 263 was incubated with 0.5 · 106/ml of DTNFa-CHO cells and allowed to reach equilibrium. The free mAb left in solution was measured in a KinExA instrument. The data from a representative experiment are presented as a Scatchard plot. B is the fraction of the bound mAb and F is the concentration of the free antibody at equilibrium. The slope of the straight line represents KA (=1/Kd) from which Kd was calculated. The x axis intercept of the line gives the amount of mAb bound/cell from which the binding site concentration of the receptor (TNFa) was calculated as 84,300/cell.
Discussion The affinity of an antibody to a soluble molecule or a ligand can be determined easily and precisely by several techniques such as KinExA and surface plasmon resonance using Biacore [4,20]. When the molecule is expressed on a cell surface, the conventional method for measuring the cell-binding affinity of an antibody is by Scatchard analysis [22]. This requires labeling the antibody with a radio nucleotide or some other label. Competitive inhibition methods to measure the cell-binding affinity of mAbs, such as CNTO 95 [10], also used labeled antibody and the determined affinity ranged from nanomolar to subnanomolar. Modifications to the antibody may result in a change in the intrinsic affinity of the antibody [11,12]. ELISA-based methods to measure the affinity of an antibody to cell-surfaceexpressed antigens albeit in the nanomolar range have been described [13]. When the affinity of the antibody is in the low picomolar to subpicomolar range these methods may lack the sensitivity to measure such a high affinity. Recently, Xie et al [17] have published an approach using KinExA to measure the affinity of an antibody to a cell membraneantigen. Although the report describes a method to measure the affinity of an antibody to membrane-expressed molecules without the need for labeling one of the binding components, the method still requires soluble antigen to coat beads to measure the free antibody. For the measurement of the affinity of an antigen present on the cell membrane, a soluble form of the antigen for coating the beads, might not be available, particularly in the case of integral membrane proteins with multiple transmembrane domains. We have independently also developed a method using KinExA to measure directly the affinity of three antibodies to two different antigens as presented on the cell surface. In
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High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
addition, we measured the affinity of one of the mAbs using Scatchard analysis for comparison. The method that we describe uses beads coated with an anti-species antibody, which in turn captured the free antibody left in solution and allows one to measure the affinity of the antibody as precisely as it could have been measured by coating beads with soluble antigen. This is especially important for membrane-bound antigens that are hard to purify or for those antigens that lose their native confirmation after being purified from the membrane. One can even use the Protein A beads to capture the free antibody if the secondary detection antibody is a Fab fragment and does not contain the Protein A binding domain. With the modifications to KinExA technology, we measured the affinity of antibodies to cell-membrane-expressed molecules with precision ranging from subpicomolar to low picomolar. The binding strength of a univalent antigen to a single combining site on a divalent IgG antibody is defined as affinity. In solution, the binding of each combining site of an IgG antibody to a univalent antigen is independent. However, when the antigen is immobilized on a solid phase or its movement is partially restricted, as on a cell membrane, epitopes on several antigens may become spatially proximal to both IgG combining sites and the binding of one site may increase the binding strength of the other combining site. The sum of the strength of all binding sites between an antibody and an antigen is defined as avidity. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity can be more than the sum of the individual affinities. To determine whether the cell-binding affinity measured by this method is the true affinity or the result of an avidity effect, we measured the affinity of the Fab fragment of one of the antibodies (Fab molecule of the anti-hTNFa mAb 263) to DTNFaCHO cell-surface-expressed TNFa. The affinity of the Fab to the antigen present on the cell surface was about 5· lower than that of the full mAb. Since soluble TNFa exists as a trimer [23], it is possible that the cell-surfaceexpressed TNF is in a multimeric form and the Kd determined for the full mAb may be an avidity measurement. This would be in contrast to the affinity of anti-hIGFR IgG and its Fab fragment to hIGFR expressed on cells [17]. For the anti-hIGFR mAb there was no difference in affinity of the Fab molecule to that of bivalent IgG and it was argued that the affinity determined was the true affinity and not due to an avidity effect. We have also shown that the Fab, compared to the full mAb, has about 4 to 5· lower affinity to soluble TNFa. If the full-length mAb is binding to soluble TNFa in a monovalent manner, then the lower Fab affinity could be a result of the Fab 0 s antigen binding site conformation being altered during its preparation. In this situation the Kd measured for the full mAb would reflect its true affinity. Another important finding in this study is that the affinity determined by Scatchard analysis is not the same as that was determined using KinExA. Scatchard plots are conventionally performed by labeling the antibody and mea-
suring the cell-bound antibody after the equilibrium is reached. With the KinExA instrument, however, we measured the free antibody left in the supernatant after equilibrium was reached and not the antibody bound to the cells. From the measured free antibody we calculated the cellbound antibody, and used this information in the Scatchard plot. Even though we used unmodified antibody in the experiment, the Kd calculated by Scatchard analysis was about 4· lower (15.9 pM by KinExA vs 63.3 pM by Scatchard) than was determined by the KinExA method. Although close, the Scatchard result is outside the confidence interval of the KinExA result. Since there is no appropriate way to obtain a confidence interval for the Scatchard result, we cannot determine whether the KinExA result falls within the uncertainty of the Scatchard result. It was shown that the Scatchard plot introduces a severe compression of the data at the higher concentration end, resulting in a distortion that may affect the result [16]. In addition, it is often overlooked that measurements made at antibody concentrations above the Kd are less influenced by Kd than concentration, and therefore the uncertainty of a Kd measurement is much greater [4]. Due to these reasons, the uncertainties of the Scatchard results are much larger than that for KinExA. Scatchard experiments attempted at lower concentrations of cells, to limit the amount of antigen, were too noisy to analyze (data not shown), whereas the KinExA experiments were not. With the greater uncertainty in the Scatchard experiments, the KinExA results are probably not in disagreement but provide a tighter, and more importantly, a defined confidence interval. While we performed both types of experiments in this case for comparison purposes, only the KinExA experiments resulted in precise Kd determinations. We have often observed that proteins expressed as soluble extracellular domains do not retain their complete native conformation compared to the full-length membrane-associated molecule. When the affinity is measured by Biacore using soluble extracellular domains, the calculated affinity frequently does not correlate with the biological potency of the antibodies, requiring affinity determinations to be performed by a cell binding assay. Measuring the affinity using FACS [14,15] also might result in irrelevant affinity measurements due to the linear transformation of the data, which may mathematically skew the affinity. The on-cell binding affinity measurements using KinExA potentially overcome these problems. Another basic difference between FACS- and KinExA-based methods is that with the FACS method the antibody is titrated with a constant number of cells and the signal of the bound antibody corresponding to each concentration of mAb is plotted to calculate the Kd. Additionally, a minimum number of cells are required to perform FACS analysis, which may result in the antigen concentration being higher than Kd leading to irrelevant results. A recent report [24] explains how important it is to set up FACS-based experiments in a Kd-controlled fashion to obtain satisfactory results. Moreover, with the FACS method, a significant
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
dissociation of mAb from cells could occur during the incubation period with the secondary antibody, effecting the equilibrium, whereas, with KinExA, the cells are titrated with a constant amount of mAb and the free mAb present at equilibrium is measured. This allows one to keep the mAb at a Kd-controlled concentration. In addition, performing multiple experiments using the same amount of titrated cells in each experiment and varying only the mAb concentration allows one to globally analyze the data by n-curve analysis, adding further precision to the measurement. KinExA methodology is suitable for measuring the affinity of soluble antigens and antibodies over a broad range (micromolar to subpicomolar) [4,25]. Here we show that it can also be used to measure the affinity/avidity of antibodies to cell-surface-expressed molecules, ranging from picomolar to subpicomolar. However, if the affinity/avidity is in the nanomolar to micromolar range, this method may warrant further optimizations. For example, to measure a 1 nM affinity, the starting antigen concentration present on cells should be in the range of hundreds of nanomolar to provide saturation and obtain the lower end of the Kd curve. For cells expressing 1 · 105 antigens/cell, to provide 100 nM of antigen, one would need approximately 6 · 108 cells/ml. This requirement would be further exasperated if the affinity is lower than 1 nM or the number of antigens expressed is <1 · 105/cell. In addition to determining the affinity of antibodies to cell-surface-expressed antigens, this method could be easily extended to measure the affinity of a protein to its receptor or an adaptor. However, one would need to have a sensitive method to measure the free protein left in the supernatant after the protein is equilibrated with the receptor or adaptor present on a cell. For such determinations a pair of sandwiching antibodies to the protein would be required, one to couple to the bead and the other one to use as a detecting antibody. Monoclonal antibodies offer great promise to fulfill the role of ‘‘magic bullets’’ in the treatment of malignancy due to their high specificity and their ability to affect an anti-tumor response. When the tumor antigen is expressed in low levels on malignant cells, high-affinity antibodies play an important role in improving the mAb potency [7]. This report describes a sensitive method using KinExA technology that determines the Kd of antibodies having high affinity to membrane-associated antigens that are difficult to purify or often unknown. Acknowledgments We thank Steve Lackie for critical reading of the manuscript and Desiree Lim and Karen Richmond for technical assistance. References [1] M.L. Veronese, P.J. O 0 Dwyer, Monoclonal antibodies in the treatment of colorectal cancer, Eur. J. Cancer 40 (2004) 1292–1301.
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[2] G.P. Adams, L.M. Weiner, Monoclonal antibody therapy of cancer, Nat. Biotechnol. 23 (2005) 1147–1157. [3] L.S. Zuckier, E.Z. Berkowitz, R.J. Sattenberg, Q.H. Zhao, H.F. Deng, M.D. Scharff, Influence of affinity and antigen density on antibody localization in a modifiable tumor targeting model, Cancer Res. 60 (2000) 7008–7013. [4] P. Rathanaswami, S. Roalstad, L. Roskos, Q.J. Su, S. Lackie, J. Babcook, Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8, Biochem. Biophys. Res. Commun. 334 (2005) 1004–1013. [5] S.J. Kennel, L.J. Foote, P.K. Lankford, M. Johnson, T. Mitchell, G.R. Braslawsky, Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging, Hybridoma 2 (1983) 297–310. [6] S.M. Andrew, R.W. Johnstone, S.M. Russell, I.F. McKenzie, G.A. Pietersz, Comparison of in vitro cell binding characteristics of four monoclonal antibodies and their individual tumor localization properties in mice, Cancer Res. 50 (1990) 4423–4428. [7] M.P. Velders, C.M. van Rhijn, E. Oskam, G.J. Fleuren, S.O. Warnaar, S.V. Litvinov, The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas, Br. J. Cancer 78 (1998) 478–483. [8] P.J. Booth, The trials and tribulations of membrane protein folding in vitro, Biochim. Biophys. Acta 1610 (2003) 51–56. [9] S.H. Tam, P.M. Sassoli, R.E. Jordan, M.T. Nakada, Abciximab (ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and alpha(v)beta3 integrins, Circulation 98 (1998) 1085–1091. [10] M. Trikha, Z. Zhou, J.A. Nemeth, Q. Chen, C. Sharp, E. Emmell, J. Giles-Komar, M.T. Nakada, CNTO 95, a fully human monoclonal antibody that inhibits alphav integrins, has antitumor and antiangiogenic activity in vivo, Int. J. Cancer 110 (2004) 326–335. [11] M. Debbia, P. Lambin, Measurement of anti-D intrinsic affinity with unlabeled antibodies, Transfusion 44 (2004) 399–406. [12] O. Siiman, A. Burshteyn, Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies, Cytometry 40 (2000) 316–326. [13] J.M. Bator, C.L. Reading, Measurement of antibody affinity for cell surface antigens using an enzyme-linked immunosorbent assay, J. Immunol. Methods 125 (1989) 167–176. [14] T.A. Barr, A.W. Heath, Functional activity of CD40 antibodies correlates to the position of binding relative to CD154, Immunology 102 (2001) 39–43. [15] C.A. Benedict, A.J. MacKrell, W.F. Anderson, Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay, J. Immunol. Methods 201 (1997) 223–231. [16] I.M. Klotz (Ed.), Ligand-Receptor Energetics: A guide for the perplexed, John Wiley and Sons, Inc., New York, 1997. [17] L. Xie, R. Mark Jones, T.R. Glass, R. Navoa, Y. Wang, M.J. Grace, Measurement of the functional affinity constant of a monoclonal antibody for cell surface receptors using kinetic exclusion fluorescence immunoassay, J. Immunol. Methods (2005) 1–14. [18] G. Chen, P.P. Luke, H. Yang, L. Visser, H. Sun, B. Garcia, H. Qian, Y. Xiang, X. Huang, W. Liu, G. Senaldi, A. Schneider, S. Poppema, H. Wang, A.M. Jevnikar, R. Zhong, Anti-CD45RB monoclonal antibody prolongs renal allograft survival in cynomolgus monkeys, Am. J. Transplant 7 (2007) 27–37. [19] R.M. Jones, H. Yu, J.B. Delehanty, D.A. Blake, Monoclonal antibodies that recognize minimal differences in the three-dimensional structures of metal-chelate complexes, Bioconjug. Chem. 13 (2002) 408–415. [20] A.W. Drake, D.G. Myszka, S.L. Klakamp, Characterizing highaffinity antigen/antibody complexes by kinetic- and equilibrium-based methods, Anal. Biochem. 328 (2004) 35–43.
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[21] E.N. Kaufman, R.K. Jain, Effect of bivalent interaction upon apparent antibody affinity: experimental confirmation of theory using fluorescence photobleaching and implications for antibody binding assays, Cancer Res. 52 (1992) 4157–4167. [22] G. Scatchard, The attractions of proteins for small molecules and ions, Ann. N.Y.Acad. Sci. 51 (1949) 660–672. [23] R.A. Smith, C. Baglioni, The active form of tumor necrosis factor is a trimer, J. Biol. Chem. 262 (1987) 6951–6954.
[24] A.W. Drake, S.L. Klakamp, A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants, J. Immunol. Methods 318 (2007) 147–152. [25] D.A. Blake, P. Chakrabarti, M. Khosraviani, F.M. Hatcher, C.M. Westhoff, P. Goebel, D.E. Wylie, R.C. Blake 2nd, Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes, J. Biol. Chem. 271 (1996) 27677– 27685.
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 373 (2008) 52–60 www.elsevier.com/locate/yabio
High-affinity binding measurements of antibodies to cell-surface-expressed antigens Palaniswami Rathanaswami *, John Babcook, Michael Gallo Amgen Inc., Burnaby, BC, Canada V5A 1V7 Received 7 July 2007 Available online 16 August 2007
Abstract A simple method that allows affinity measurements of antibodies to integral membrane proteins is described. Kinetic Exclusion Assay was used to determine the concentration of free antibody that remains in solution after equilibrium has been established between the antibody and the cell-surface-expressed antigen, from which the equilibrium dissociation constant (Kd) was determined. It eliminates the requirement for soluble antigen and modifications such as radio-labeling or fluorescent labeling of the antibody. For one of the cell-surface-expressed antigens, it was determined that the affinity of the antibody to the cell-surface-expressed antigen was similar to that of the purified, soluble form of the antigen. In addition to the simplicity of the approach, the method provides a true measure of the affinity/avidity of the antibody to the native form of cell-surface-expressed targets, including antigens that cannot be produced in soluble forms, and to unknown cell surface antigens. 2007 Elsevier Inc. All rights reserved. Keywords: Affinity measurement; On-cell binding; Membrane-expressed antigen; High affinity; KinExA; Cell-surface-expressed molecule; Transmembrane-expressed molecule; Human antibodies; Unmodified antibodies; Native binding
Monoclonal antibodies (mAbs)1 represent a new and increasingly important therapeutic modality in treating many diseases such as cancer [1,2]. When choosing the best antibody for therapeutic purposes, in addition to specificity, the affinity of an antibody to the target is also important. A direct correlation between the affinity and the potency of an antibody exists [3,4] and, for therapeutic purposes, antibodies with binding constants greater than 10 nM are not likely to be useful for drug targeting or tumor imaging [5]. High-affinity mAbs, in contrast to low-affinity mAbs, target tumors better [6] and enhance the antibody-dependent cellular-cytotoxicity-mediated killing of tumor cells even at low antigen expression levels [7]. *
Corresponding author. Fax: +1 (604) 676 8349. E-mail address: [email protected] (P. Rathanaswami). 1 Abbreviations used: mAbs, monoclonal antibodies; FACS, fluorescence-activated cell sorting; KinExA, Kinetic Exclusion Assay; ELISA, enzyme-linked immunosorbent assay; NHS, N-hydroxy succinimide; CHO, chinese hamster ovary; AgM, antigen multiplier. 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.08.014
There are several established technologies/methodologies available to experimentally determine the affinity of antibodies to soluble antigens. If the antigen is expressed on a cell membrane, the measured affinity of the antibody to the soluble antigen counterpart, typically the extracellular domain, may not correlate with the antibody’s binding affinity to the native form of the antigen on the cell. In addition, purification of membrane proteins is difficult and may result in a loss of stability or in partially functional protein [8]. Studies characterizing the affinity measurements of antibodies to cell-surface-expressed molecules typically modify the antibodies with fluorescent or radioactive labels so that they can be detected [9,10]. However, it has been shown that chemical modification of an antibody by 125I [11], flourescein isothiocyanate, and RD1 labeling can lead to binding constants that are 10 to 100-fold lower than those of the native antibodies [12]. Some of the methods using radiolabeled antibodies require extensive cell washing after the equilibrium is reached [10] which might lead to the dissoci-
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ation of the antibody from the cell membrane and disturb the equilibrium. Bator and Reading [13] described a simple ELISA method to measure the apparent affinity of an antibody for cell-surface antigens using Scatchard analysis. The sensitivity of determination of the affinity by this method is in the nanomolar range. Fluorescence-activated cell sorting (FACS) methods also have been used to measure the affinity of cell-membrane-expressed antigens [14,15]. However, these methods used a linear transformation of the data to calculate the affinity. Scatchard and other linear transformations of the binding isotherm data can sometimes conceal deviations from simple single-site binding patterns that can be recognized by nonlinear analysis [16]. A recent report [17] describes the affinity measurement of an antibody to cell-surface receptors and overcomes many of the above problems by using Kinetic Exclusion Assay (KinExA). However, this reported method requires the use of a soluble antigen as a probe for measuring the affinity of the antibody to cell-surfaceexpressed molecules. Here we describe a modified method in which soluble antigen is not required for measuring the affinity of a purified antibody to its cell-membraneexpressed target.
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ose beads were prepared by adsorbing the GST-TT-TNFa onto glutathione–Sepharose beads. Cell culture CHO cells expressing a mutant noncleavable form of TNFa on the surface of the membrane were used. Briefly, the cDNA of TNFa was mutated at various positions to prevent cleavage of TNFa from the surface of cells and cloned into an expression vector. CHO cells were transfected with this mutated form of TNFa and cells expressing TNFa in stable form on the membrane were placed under drug selection to generate a DTNFa cell line. The cells were cultured in standard tissue culture conditions in an incubator with 5% CO2. Ham-F12 medium was used to grow the cells with 10% fetal calf serum (Gibco), 2 mM L-glutamine, 1 mM sodium pyruvate, and penicillin/streptomycin (100 IU/ml/100 mg/ml (Gibco). Cells were treated with 8 mM sodium butyrate overnight before use. CEM cells expressing CD45RB were cultured in RPMI 1640 medium with 2 mM L-glutamine and adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM Hepes, and 1.0 mM sodium pyruvate. Fetal bovine serum was added to this to a final concentration of 10% V/V.
Materials and methods Cell-binding equilibrium assays Azlactone beads were obtained from Sapidyne Instruments (Idaho, U.S.A). Cy5-conjugated affinity-purified antibodies (all have minimal cross-reactivity to bovine, horse, and mouse serum proteins) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, U.S.A). Goat anti-human Fcc fragment-specific IgG antibody was obtained from Immunodiagnostics (Bellingham, WA, U.S.A). Protein A–Sepharose, NHS-activated Sepharose 4 Fast Flow, and glutathione–Sepharose beads were purchased from Amersham Biosciences, Inc. (Quebec, Canada). ImmunoPure Fab preparation kit was purchased from Pierce (Rockford, IL, U.S.A.). The rhTNFa was obtained from R & D Systems (Minneapolis, MN, U.S.A.). Human TNFa was also expressed as a fusion protein with GST and a T-cell epitope from tetanus toxin (GST-TT-TNFa). Anti-CD45RB mAb 6G3 [18] was produced from a hybridoma. Anti-human CD45RB mAb 487 and anti-human TNFa mAb 263 were developed by Amgen 0 s XenoMax technology [4] and produced as recombinant antibodies. Protein A–Sepharose affinity–chromatography-purified mAbs were used in all experiments. The purity was assessed and the quantity was calculated by nonreducing SDS–PAGE. The concentration was further confirmed by UV analysis at A280 and ELISA. Preparation of antigen- or Ig-coated beads Goat anti-human Fcc fragment-specific IgG antibody (100 lg) was coupled to Azlactone beads (300 mg) as recommended by the manufacturer or to NHS-activated Sepharose beads as described [4]. GST-TT-TNFa-Sephar-
Cells were serially diluted 1 in 2 in 11 falcon tubes using the culture medium and containing a final concentration of 0.05% NaN3. The 12th tube contained just the medium. An appropriate concentration of purified mAb was made in cell culture medium and an equal volume of the mAb was mixed with the serially diluted cells. The cells with the mAb were then rotated at 4 C for 36 h. At the end of 36 h, the cells were centrifuged at 2400 rpm for 4 min and the free mAb present in the supernatant was measured by KinExA using appropriate beads and Cy5-conjugated anti-human secondary antibodies as described [4]. The equilibrium dissociation constant (Kd) [17] was obtained using KinExA software [19,20] and by ‘‘n-curve analysis’’ which fits all of the given curves to a single Kd value simultaneously [4,17]. Cell-binding affinity measurements with a corresponding Fab fragment The Fab fragment was prepared as per the manufacturer 0 s recommendation. The completeness of digestion and the purity were assessed by running on an acrylamide gel. Cells were titrated, mixed with an equal volume of Fab fragment at defined concentrations, and continued as described for the full mAb. The free Fab fragment present in the supernatant was measured by KinExA using antigencoated Sepharose beads to absorb the Fab fragment and detected using Cy5-conjugated affinity-purified goat antihuman (H+L)-specific IgG. The Kd was obtained as described above.
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High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Affinity measurements with soluble TNFa and anti-human TNFa mAb or Fab Briefly, a constant amount of anti-human TNFa mAb or Fab 263 was incubated with titrating concentrations of rhTNFa antigen in sample buffer (phosphate-buffered saline with 0.1% bovine serum albumin and 0.05% NaN3). Antigen/antibody complexes were incubated at room temperature for 36 h to allow equilibrium to be reached. The mixture was drawn through the GST-TT-TNFa-Sepharose beads to accumulate unbound antibody. The captured antihTNFa mAb or Fab is directly proportional to the remaining free-binding sites [4] and was detected using solutions containing Cy5-conjugated anti-human secondary antibody in sample buffer. The Kd was determined as described above. Scatchard analysis A constant number of cells was added to titrating concentrations of anti-hTNFa mAb 263 and incubated for 36 h at 4 C. At the end of 36 h, the cells were centrifuged at 2400 rpm for 4 min and the signal generated by the free mAb present in the supernatant was measured by KinExA using either goat anti-human Fcc fragment-specific IgG antibody-coupled azlactone beads or GST-TT-TNFaSepharose beads to absorb the mAb and detected using solutions containing Cy5-conjugated affinity-purified goat anti-human Fcc fragment-specific IgG secondary antibodies. The signal corresponding to the total antibody in each dilution was also measured in KinExA. From the signal obtained for the total antibody, the amount of free antibody present in the supernatant and the amount of antibody bound to cells were calculated. The Scatchard plot was done by plotting B/F vs B [21] and the Kd was calculated from the slope.
site concentration of mAb was used as a determinant for the KinExA software to calculate the antigen multiplier (AgM) and the Kd using the unknown antigen method [17]. The antigen multiplier is a multiplication constant used in conjunction with the assumed antigen concentration (1 · 106 cells = 1 nM antigen) variable to determine an active molar concentration of the receptor. The calculated antigen multiplication constant will be a fraction if 1 · 106 cells contain a molar concentration of receptors less than the arbitrarily assumed concentration (1 nM). It will be greater than one if 1 · 106 cells contain more than the assumed antigen concentration. As shown in Fig. 1, the Kd calculated for the anti-hCD45RB mAb 487 was 670 fM (Fig. 1B) with 95% confidence interval of 2.30 pM (Kd high) to 140 fM (Kd low). The % error was only 2.9. Even though the active site concentration of antibody used in the incubation was higher than the calculated Kd, the determined Kd bounds were reasonably narrow [4]. Based on the bounds and the antigen concentration that binds to 50% of the mAb (Fig. 1A), it can be concluded that the Kd should be in the low picomolar range. An antigen multiplier of 0.0185 was determined by the software (Fig. 1C), which translates into the presence of 11,470 antibody-binding antigen molecules/cell as described by Xie et al. [17]. The Kd measurements for the anti-CD45RB mouse/ human chimeric mAb 6G3 using the same CEM cells expressing CD45RB are shown in Figs. 1D, 1E, and 1F. The determined Kd for the mouse/human chimeric mAb is 1.56 pM. The AgM was 0.0323, which is equal to 20,000 antibody-binding antigen molecules/cell. The variation seen in the number of receptors on the CEM cells between mAb 487 and 6G3 may be due to the possible difference in available epitope concentration, actual differences in the active binding site concentration for each mAb, or day to day variation of receptor expression levels.
Results Affinity of membrane-expressed TNFa Affinity of CD45RB expressed on CEM cells CEM cells expressing CD45RB antigen were harvested, resuspended in fresh medium (2.5 · 106 cells/ml), titrated 1 in 2, incubated with a constant amount (10 pM) of antihCD45RB mAb in the presence of 0.05% NaN3, and allowed to reach equilibrium. At equilibrium, the free mAb left in the supernatant was measured in KinExA using Protein A–Sepharose beads and Cy5-conjugated affinity-purified Fab fragment of goat anti-human (H+L)-specific IgG. The percentage free mAb left in solution was plotted against the concentration of antigen (arbitrarily defined as 1 nM of antigen per 1 million cells, see below for more details) by the KinExA software and a sigmoidal curve was generated, as shown in Fig. 1. A theoretical curve was fit to the data (Fig. 1A) and 95% confidence intervals were calculated by the KinExA software. The mAb was assumed to be 100% active. The active binding
Using goat-anti human Fc-coupled beads. The DTNFaCHO cells were dissociated from plates using cell dissociation buffer. Cells (25 · 106/ml) were resuspended in culture media, titrated 1 in 3, incubated with a constant amount (10 or 100 pM) of anti-hTNFa mAb 263, and allowed to reach equilibrium. After equilibrium, the free mAb left in the supernatant was measured using goat anti-human Fcc fragment-specific IgG antibody-coupled Azlactone beads and the Kd was calculated by the KinExA software using n-curve analysis [4]. A representative experiment is shown in Fig. 2. The Kd calculated for the human anti-hTNFa mAb 263 was 13.64 pM with Kd high of 26.8 pM and Kd low of 7.2 pM as 95% confidence intervals. The % error was only 2.8. AgM of 0.0828 was calculated by this method which translates into 49,870 receptors per cell. The experiment was repeated several times and the average Kd calculated from four experiments was 18.3 pM (Table 1).
High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
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Fig. 1. Measurement of the Kd of anti-hCD45RB mAbs for the CD45RB expressed on CEM cells. CEM cells (2.5 · 106/ml) were serially diluted and incubated with 10 pM active binding site concentration of (A) anti-hCD45RB mAb 487 or (D) anti-CD45RB mouse/human chimeric mAb 6G3, in the presence of 0.05% NaN3 and allowed to equilibrate. The free mAb left in the supernatant is measured as explained in the text and the % free mAb is plotted against the antigen concentration (taking arbitrarily each million cells to be equal to 109 M antigen). The unknown antigen method was performed to determine optimal values for Kd and the antigen multiplier. (B and E) Best fit error analysis was performed by the software for the Kd by iteratively changing the value for the Kd while reoptimizing the other parameters of the curve fit for each iteration. The 95% confidence intervals were determined by plotting the change in best fit for each value of Kd. (C and F) Best fit error analysis for antigen multiplier was performed similarly by iteratively changing the value for antigen multiplier while holding the remaining parameters at their optimized values.
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High-affinity binding measurements of antibodies to antigens / P. Rathanaswami et al. / Anal. Biochem. 373 (2008) 52–60
Fig. 2. Determination of Kd of anti-hTNFa mAb 263 for cell-membrane-expressed TNFa. DTNFa-CHO cells (25 · 106/ml) were serially diluted and incubated with 10 pM (triangles) or 100 pM (diamonds) active binding site concentration of anti-hTNFa mAb 263, in the presence of 0.05% NaN3, and allowed to equilibrate. The free mAb left in the supernatant was measured as explained in the text. (A) The % free mAb is plotted against the antigen concentration (taking arbitrarily each million cells to be equal to 109 M antigen). Multiple curve analysis using the unknown antigen method was performed to determine optimal values for Kd and the antigen multiplier. (B and C) The 95% confidence intervals were determined by changing iteratively the optimized value for Kd or AgM while keeping other parameters at their optimal values.
Table 1 Kd measurement of anti-hTNFa mAb 263 for TNFa expressed on the cell membrane and soluble secreted TNFa mAb
Target
Kd (pM)
SEM
Anti-TNFa mAb 263a Anti-TNFa mAb 263b Anti-TNFa mAb 263c Fab fragment of anti-TNFa mAb 263d Fab fragment of anti-TNFa mAb 263e Anti-TNFa mAb 263f Anti-TNFa mAb 263g
Membrane-expressed TNFa Membrane-expressed TNFa Membrane-expressed TNFa Membrane-expressed TNFa Secreted soluble TNFa Secreted soluble TNFa Membrane expressed TNFa
18.4 13.4 15.9 79.0 31.6 9.1 63.3
3.14 1.05 1.75 0.05 Kd high = 42.8 Kd low = 22.8 Kd high = 13.4 Kd low = 3.5 5.45
a b c d,e,f g
Kd was measured using goat-anti human Fc-coupled beads. Kd was measured using antigen-coated (TT-TNFa-GST-Sepharose) beads. Average Kd was measured using both goat-anti human Fc-coupled and antigen coated beads. Kd was determined using antigen-coated beads. Kd was determined by Scatchard plot.
Using antigen-coated beads. Next we determined whether there would be a difference in the calculated Kd, if antigencoated beads are used in the bead column instead of the goat anti-human Fcc fragment-specific antibody capture. Titrated cells were incubated with mAb as before and
allowed to reach equilibrium. After equilibrium, the free mAb left in the supernatant was measured using GST-TTTNFa-Sepharose beads and the Kd was calculated by the KinExA software using n-curve analysis [4]. The average Kd calculated from four experiments was 13.4 pM (Table 1).
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Comparison of the affinity of full mAb with Fab. To determine whether the on-cell binding affinity determined for the full mAb in KinExA is the true affinity or whether there is an avidity effect involved, the experiment was repeated using the same batch of DTNFaCHO cells, which was used in experiments with full mAb, but replacing with Fab molecule. The cells (20 · 106/ml) were titrated 1 in 2, incubated with a constant amount (20 or 300 pM) of Fab fragment of mAb 263, and allowed to reach equilibrium. After equilibrium, the free Fab left in the supernatant was measured using GST-TT-TNFa-Sepharose beads and the Kd was calculated by the KinExA software using n-curve analysis. The antigen concentration on the cells which was determined using the full-length mAb was used as a determinant for calculating the Kd of the Fab using standard analysis. The affinity determined for the Fab molecule from several experiments is given in Table 1. It is clear from this table that the affinity of the Fab to the cellsurface-expressed TNFa is about 4 to 5 · lower than that of the full mAb, 79 pM for Fab VS 15.9 pM for full mAb. Comparison of affinity of full mAb and its Fab to membraneexpressed TNFa and secreted soluble TNFa To determine whether the lower affinity of the Fab fragment of anti-hTNFa mAb 263, compared to full mAb, to the cell-surface-expressed TNFa is due to a possible difference in the structure of the Fab or the affinity determined for the full mAb to cell-surface-expressed TNFa may be the result of an avidity effect, the affinities of full mAb and the Fab were determined for secreted soluble TNFa. A constant amount of anti-hTNFa mAb 263 (5 or 50 pM) or the Fab fragment (30 or 300 pM) was incubated with titrating concentrations of rhTNFa antigen starting at 100 nM and allowed to reach equilibrium and the Kd was determined [4]. Table 1 shows that the affinity of Fab to the secreted soluble TNFa also was about 4 times lower than the affinity of the full mAb, 31.6 pM for Fab VS 9.1 pM for full mAb. Scatchard analysis of membrane-expressed TNFa Since one conventional means of measuring affinity to a cellular target is done by Scatchard plot, we also did Scatchard experiments. Anti-hTNFa mAb 263 (200 pM) was titrated 1:2 in medium and incubated with 500,000 DTNFa cells/ml for 36 h at 4 C. The free antibody left in solution was measured and the amount of antibody bound to cells was calculated using KinExA software and used in Scatchard analysis. Fig. 3 shows a representative experiment and the Kd for full mAb was determined as 64 pM. The number of antigen molecules present on the cells was calculated to be 84,300 antigens/cell. Several experiments were done and the mean Kd was calculated to be 63.3 pM with SEM of 5.45.
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Fig. 3. Affinity determination of anti-hTNFa mAb 263 for cell-membrane-expressed TNFa by Scatchard analysis. Serially diluted 200 pM anti-hTNFa mAb 263 was incubated with 0.5 · 106/ml of DTNFa-CHO cells and allowed to reach equilibrium. The free mAb left in solution was measured in a KinExA instrument. The data from a representative experiment are presented as a Scatchard plot. B is the fraction of the bound mAb and F is the concentration of the free antibody at equilibrium. The slope of the straight line represents KA (=1/Kd) from which Kd was calculated. The x axis intercept of the line gives the amount of mAb bound/cell from which the binding site concentration of the receptor (TNFa) was calculated as 84,300/cell.
Discussion The affinity of an antibody to a soluble molecule or a ligand can be determined easily and precisely by several techniques such as KinExA and surface plasmon resonance using Biacore [4,20]. When the molecule is expressed on a cell surface, the conventional method for measuring the cell-binding affinity of an antibody is by Scatchard analysis [22]. This requires labeling the antibody with a radio nucleotide or some other label. Competitive inhibition methods to measure the cell-binding affinity of mAbs, such as CNTO 95 [10], also used labeled antibody and the determined affinity ranged from nanomolar to subnanomolar. Modifications to the antibody may result in a change in the intrinsic affinity of the antibody [11,12]. ELISA-based methods to measure the affinity of an antibody to cell-surfaceexpressed antigens albeit in the nanomolar range have been described [13]. When the affinity of the antibody is in the low picomolar to subpicomolar range these methods may lack the sensitivity to measure such a high affinity. Recently, Xie et al [17] have published an approach using KinExA to measure the affinity of an antibody to a cell membraneantigen. Although the report describes a method to measure the affinity of an antibody to membrane-expressed molecules without the need for labeling one of the binding components, the method still requires soluble antigen to coat beads to measure the free antibody. For the measurement of the affinity of an antigen present on the cell membrane, a soluble form of the antigen for coating the beads, might not be available, particularly in the case of integral membrane proteins with multiple transmembrane domains. We have independently also developed a method using KinExA to measure directly the affinity of three antibodies to two different antigens as presented on the cell surface. In
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addition, we measured the affinity of one of the mAbs using Scatchard analysis for comparison. The method that we describe uses beads coated with an anti-species antibody, which in turn captured the free antibody left in solution and allows one to measure the affinity of the antibody as precisely as it could have been measured by coating beads with soluble antigen. This is especially important for membrane-bound antigens that are hard to purify or for those antigens that lose their native confirmation after being purified from the membrane. One can even use the Protein A beads to capture the free antibody if the secondary detection antibody is a Fab fragment and does not contain the Protein A binding domain. With the modifications to KinExA technology, we measured the affinity of antibodies to cell-membrane-expressed molecules with precision ranging from subpicomolar to low picomolar. The binding strength of a univalent antigen to a single combining site on a divalent IgG antibody is defined as affinity. In solution, the binding of each combining site of an IgG antibody to a univalent antigen is independent. However, when the antigen is immobilized on a solid phase or its movement is partially restricted, as on a cell membrane, epitopes on several antigens may become spatially proximal to both IgG combining sites and the binding of one site may increase the binding strength of the other combining site. The sum of the strength of all binding sites between an antibody and an antigen is defined as avidity. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity can be more than the sum of the individual affinities. To determine whether the cell-binding affinity measured by this method is the true affinity or the result of an avidity effect, we measured the affinity of the Fab fragment of one of the antibodies (Fab molecule of the anti-hTNFa mAb 263) to DTNFaCHO cell-surface-expressed TNFa. The affinity of the Fab to the antigen present on the cell surface was about 5· lower than that of the full mAb. Since soluble TNFa exists as a trimer [23], it is possible that the cell-surfaceexpressed TNF is in a multimeric form and the Kd determined for the full mAb may be an avidity measurement. This would be in contrast to the affinity of anti-hIGFR IgG and its Fab fragment to hIGFR expressed on cells [17]. For the anti-hIGFR mAb there was no difference in affinity of the Fab molecule to that of bivalent IgG and it was argued that the affinity determined was the true affinity and not due to an avidity effect. We have also shown that the Fab, compared to the full mAb, has about 4 to 5· lower affinity to soluble TNFa. If the full-length mAb is binding to soluble TNFa in a monovalent manner, then the lower Fab affinity could be a result of the Fab 0 s antigen binding site conformation being altered during its preparation. In this situation the Kd measured for the full mAb would reflect its true affinity. Another important finding in this study is that the affinity determined by Scatchard analysis is not the same as that was determined using KinExA. Scatchard plots are conventionally performed by labeling the antibody and mea-
suring the cell-bound antibody after the equilibrium is reached. With the KinExA instrument, however, we measured the free antibody left in the supernatant after equilibrium was reached and not the antibody bound to the cells. From the measured free antibody we calculated the cellbound antibody, and used this information in the Scatchard plot. Even though we used unmodified antibody in the experiment, the Kd calculated by Scatchard analysis was about 4· lower (15.9 pM by KinExA vs 63.3 pM by Scatchard) than was determined by the KinExA method. Although close, the Scatchard result is outside the confidence interval of the KinExA result. Since there is no appropriate way to obtain a confidence interval for the Scatchard result, we cannot determine whether the KinExA result falls within the uncertainty of the Scatchard result. It was shown that the Scatchard plot introduces a severe compression of the data at the higher concentration end, resulting in a distortion that may affect the result [16]. In addition, it is often overlooked that measurements made at antibody concentrations above the Kd are less influenced by Kd than concentration, and therefore the uncertainty of a Kd measurement is much greater [4]. Due to these reasons, the uncertainties of the Scatchard results are much larger than that for KinExA. Scatchard experiments attempted at lower concentrations of cells, to limit the amount of antigen, were too noisy to analyze (data not shown), whereas the KinExA experiments were not. With the greater uncertainty in the Scatchard experiments, the KinExA results are probably not in disagreement but provide a tighter, and more importantly, a defined confidence interval. While we performed both types of experiments in this case for comparison purposes, only the KinExA experiments resulted in precise Kd determinations. We have often observed that proteins expressed as soluble extracellular domains do not retain their complete native conformation compared to the full-length membrane-associated molecule. When the affinity is measured by Biacore using soluble extracellular domains, the calculated affinity frequently does not correlate with the biological potency of the antibodies, requiring affinity determinations to be performed by a cell binding assay. Measuring the affinity using FACS [14,15] also might result in irrelevant affinity measurements due to the linear transformation of the data, which may mathematically skew the affinity. The on-cell binding affinity measurements using KinExA potentially overcome these problems. Another basic difference between FACS- and KinExA-based methods is that with the FACS method the antibody is titrated with a constant number of cells and the signal of the bound antibody corresponding to each concentration of mAb is plotted to calculate the Kd. Additionally, a minimum number of cells are required to perform FACS analysis, which may result in the antigen concentration being higher than Kd leading to irrelevant results. A recent report [24] explains how important it is to set up FACS-based experiments in a Kd-controlled fashion to obtain satisfactory results. Moreover, with the FACS method, a significant
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dissociation of mAb from cells could occur during the incubation period with the secondary antibody, effecting the equilibrium, whereas, with KinExA, the cells are titrated with a constant amount of mAb and the free mAb present at equilibrium is measured. This allows one to keep the mAb at a Kd-controlled concentration. In addition, performing multiple experiments using the same amount of titrated cells in each experiment and varying only the mAb concentration allows one to globally analyze the data by n-curve analysis, adding further precision to the measurement. KinExA methodology is suitable for measuring the affinity of soluble antigens and antibodies over a broad range (micromolar to subpicomolar) [4,25]. Here we show that it can also be used to measure the affinity/avidity of antibodies to cell-surface-expressed molecules, ranging from picomolar to subpicomolar. However, if the affinity/avidity is in the nanomolar to micromolar range, this method may warrant further optimizations. For example, to measure a 1 nM affinity, the starting antigen concentration present on cells should be in the range of hundreds of nanomolar to provide saturation and obtain the lower end of the Kd curve. For cells expressing 1 · 105 antigens/cell, to provide 100 nM of antigen, one would need approximately 6 · 108 cells/ml. This requirement would be further exasperated if the affinity is lower than 1 nM or the number of antigens expressed is <1 · 105/cell. In addition to determining the affinity of antibodies to cell-surface-expressed antigens, this method could be easily extended to measure the affinity of a protein to its receptor or an adaptor. However, one would need to have a sensitive method to measure the free protein left in the supernatant after the protein is equilibrated with the receptor or adaptor present on a cell. For such determinations a pair of sandwiching antibodies to the protein would be required, one to couple to the bead and the other one to use as a detecting antibody. Monoclonal antibodies offer great promise to fulfill the role of ‘‘magic bullets’’ in the treatment of malignancy due to their high specificity and their ability to affect an anti-tumor response. When the tumor antigen is expressed in low levels on malignant cells, high-affinity antibodies play an important role in improving the mAb potency [7]. This report describes a sensitive method using KinExA technology that determines the Kd of antibodies having high affinity to membrane-associated antigens that are difficult to purify or often unknown. Acknowledgments We thank Steve Lackie for critical reading of the manuscript and Desiree Lim and Karen Richmond for technical assistance. References [1] M.L. Veronese, P.J. O 0 Dwyer, Monoclonal antibodies in the treatment of colorectal cancer, Eur. J. Cancer 40 (2004) 1292–1301.
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[2] G.P. Adams, L.M. Weiner, Monoclonal antibody therapy of cancer, Nat. Biotechnol. 23 (2005) 1147–1157. [3] L.S. Zuckier, E.Z. Berkowitz, R.J. Sattenberg, Q.H. Zhao, H.F. Deng, M.D. Scharff, Influence of affinity and antigen density on antibody localization in a modifiable tumor targeting model, Cancer Res. 60 (2000) 7008–7013. [4] P. Rathanaswami, S. Roalstad, L. Roskos, Q.J. Su, S. Lackie, J. Babcook, Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8, Biochem. Biophys. Res. Commun. 334 (2005) 1004–1013. [5] S.J. Kennel, L.J. Foote, P.K. Lankford, M. Johnson, T. Mitchell, G.R. Braslawsky, Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging, Hybridoma 2 (1983) 297–310. [6] S.M. Andrew, R.W. Johnstone, S.M. Russell, I.F. McKenzie, G.A. Pietersz, Comparison of in vitro cell binding characteristics of four monoclonal antibodies and their individual tumor localization properties in mice, Cancer Res. 50 (1990) 4423–4428. [7] M.P. Velders, C.M. van Rhijn, E. Oskam, G.J. Fleuren, S.O. Warnaar, S.V. Litvinov, The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas, Br. J. Cancer 78 (1998) 478–483. [8] P.J. Booth, The trials and tribulations of membrane protein folding in vitro, Biochim. Biophys. Acta 1610 (2003) 51–56. [9] S.H. Tam, P.M. Sassoli, R.E. Jordan, M.T. Nakada, Abciximab (ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and alpha(v)beta3 integrins, Circulation 98 (1998) 1085–1091. [10] M. Trikha, Z. Zhou, J.A. Nemeth, Q. Chen, C. Sharp, E. Emmell, J. Giles-Komar, M.T. Nakada, CNTO 95, a fully human monoclonal antibody that inhibits alphav integrins, has antitumor and antiangiogenic activity in vivo, Int. J. Cancer 110 (2004) 326–335. [11] M. Debbia, P. Lambin, Measurement of anti-D intrinsic affinity with unlabeled antibodies, Transfusion 44 (2004) 399–406. [12] O. Siiman, A. Burshteyn, Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies, Cytometry 40 (2000) 316–326. [13] J.M. Bator, C.L. Reading, Measurement of antibody affinity for cell surface antigens using an enzyme-linked immunosorbent assay, J. Immunol. Methods 125 (1989) 167–176. [14] T.A. Barr, A.W. Heath, Functional activity of CD40 antibodies correlates to the position of binding relative to CD154, Immunology 102 (2001) 39–43. [15] C.A. Benedict, A.J. MacKrell, W.F. Anderson, Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay, J. Immunol. Methods 201 (1997) 223–231. [16] I.M. Klotz (Ed.), Ligand-Receptor Energetics: A guide for the perplexed, John Wiley and Sons, Inc., New York, 1997. [17] L. Xie, R. Mark Jones, T.R. Glass, R. Navoa, Y. Wang, M.J. Grace, Measurement of the functional affinity constant of a monoclonal antibody for cell surface receptors using kinetic exclusion fluorescence immunoassay, J. Immunol. Methods (2005) 1–14. [18] G. Chen, P.P. Luke, H. Yang, L. Visser, H. Sun, B. Garcia, H. Qian, Y. Xiang, X. Huang, W. Liu, G. Senaldi, A. Schneider, S. Poppema, H. Wang, A.M. Jevnikar, R. Zhong, Anti-CD45RB monoclonal antibody prolongs renal allograft survival in cynomolgus monkeys, Am. J. Transplant 7 (2007) 27–37. [19] R.M. Jones, H. Yu, J.B. Delehanty, D.A. Blake, Monoclonal antibodies that recognize minimal differences in the three-dimensional structures of metal-chelate complexes, Bioconjug. Chem. 13 (2002) 408–415. [20] A.W. Drake, D.G. Myszka, S.L. Klakamp, Characterizing highaffinity antigen/antibody complexes by kinetic- and equilibrium-based methods, Anal. Biochem. 328 (2004) 35–43.
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[21] E.N. Kaufman, R.K. Jain, Effect of bivalent interaction upon apparent antibody affinity: experimental confirmation of theory using fluorescence photobleaching and implications for antibody binding assays, Cancer Res. 52 (1992) 4157–4167. [22] G. Scatchard, The attractions of proteins for small molecules and ions, Ann. N.Y.Acad. Sci. 51 (1949) 660–672. [23] R.A. Smith, C. Baglioni, The active form of tumor necrosis factor is a trimer, J. Biol. Chem. 262 (1987) 6951–6954.
[24] A.W. Drake, S.L. Klakamp, A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants, J. Immunol. Methods 318 (2007) 147–152. [25] D.A. Blake, P. Chakrabarti, M. Khosraviani, F.M. Hatcher, C.M. Westhoff, P. Goebel, D.E. Wylie, R.C. Blake 2nd, Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes, J. Biol. Chem. 271 (1996) 27677– 27685.