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Conformational isomerism of IgG antibodies
Biochimica et Biophysica Acta 1340 Ž1997. 53–62
Conformational isomerism of IgG antibodies Ulla-Britt Hansson b
a,)
, Christer Wingren a , Ulf Alkner
b
a Department of Biochemistry, Lund UniÕersity, Lund, Sweden Department of Bioanalytical Chemistry, Astra Draco AB, Lund, Sweden
Received 20 November 1996; accepted 28 January 1997
Abstract The purpose of this study was to determine why apparently homogeneous IgG antibodies were, in some cases, fractionated into at least two components by liquid–liquid partition chromatography ŽLLPC. in an aqueous two-phase system. Four mouse monoclonal IgG antibodies, two against albumin, one against IgG and one against thyroxine, were shown to adopt different conformational isomeric forms. The four antibodies existed in an equilibrium between two or three conformational forms, the proportion of which could also be estimated by LLPC. Since LLPC detects mainly conformational differences within the antigen-binding sites of IgG antibodies, it could be concluded that the conformational forms differed with respect to their combining sites. Moreover, the isomeric forms of an antibody directed against a protein antigen, formed antigen–antibody complexes with almost identical surface properties. In contrast, complexes with different surface properties were formed when the hapten or hapten conjugated to BSA was bound. Thus, both the conformational isomers could bind antigen, at least when the antigen was a small hapten or a hapten conjugated to a carrier protein. Our results suggest that six out of 57 monoclonal IgG antibodies exist in equilibrium between at least two conformational forms and the biological significance of this isomerism is discussed. Keywords: Antigen binding; Conformational isomerism; IgG antibody; Liquid-liquid partition chromatography; Phase partition; Surface property
1. Introduction Partitioning in aqueous PEGrdextran two-phase systems is useful for separation of biomolecules w1x. The distribution of a molecule in these systems depends on its three-dimensional structure and general surface properties and is described by its partition
Abbreviations: CDR, complementarity-determining region; LLPC, liquid–liquid partition chromatography; PEG, polyethylene glycol; T4, thyroxine ) Corresponding author. Fax: q46 46 222 4534; e-mail: [email protected]
coefficient w2–5x. The selectivity and sensitivity of the two-phase technique can be considerably increased by adopting a column chromatographic approach, liquid–liquid partition chromatography ŽLLPC., in which the bottom phase of the two-phase system is adsorbed onto a support and packed into a column which is eluted with the corresponding top phase w6–8x. LLPC has been shown to be a unique tool for protein analysis in that it can be used to separate and fractionate proteins w9–11x, to detect conformational changes occurring upon binding of a ligand w11–13x and to relate surface properties of proteins to their biological specificities w6,12,14x. We have recently shown that the surface properties of
0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 2 8 - 9
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IgG1, -2 and -4 antibodies are dominated by those of their antigen-binding regions and that isolated Fc fragments derived from these three IgG subclasses displayed similar surface properties w14,15x. Hence, the constant parts of the IgG1, -2 and -4 isotypes may form similar scaffoldings, on which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties. However, our studies on immunoglobulins also showed that some monoclonal antibodies were fractionated into at least two components by LLPC, in spite of the fact that they were homogeneous with respect to their immunochemical and physicochemical properties w12,14x. This phenomenon was observed not only for IgG antibodies but also for IgA and IgM myeloma proteins w15x. Consequently, the data gave rise to the question of whether these antibodies, as many enzymes, occurred in different conformational forms. We have previously explored the capacity of LLPC to detect different Žisomeric. forms of a protein by analysing a large set of well-characterized enzymes known to exist in equilibrium between an open and a closed conformation. In agreement with data obtained by several methods, including X-ray crystallography, the equilibrium between two forms of unliganded alcohol dehydrogenase and lactate dehydrogenase was detected by LLPC, as was the ligand-dependent equilibrium between two forms of citrate synthase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, glutamate–oxaloacetate transaminase, hexokinase and 3-phosphoglycerate kinase w11,13x. Hence, the results clearly indicated that isomeric forms of enzymes could be detected and in some cases even separated by LLPC. Like enzymes, immunoglobulins can have a catalytic function w16–18x and they are generally produced in vitro w16,18–20x, but have also in some cases been detected in vivo in the sera of some patients with autoimmune diseases w21–23x. Since antibodies and enzymes can share some functional properties, one may further speculate on whether they can also share the structural feature and thus exist in conformational isomeric forms. Indeed, studies on several myeloma proteins have demonstrated kinetically distinct conformational isomers w24–28x. These examples do, however, leave an
uncertainty as to whether conformational antibody isomerism is an oddity or a general and important factor in an immune response. Evidence that conformational polymorphism may be more general came in 1994 when Foote and Milstein observed biphasic or triphasic reactions in stopped-flow fluorescence experiments of monoclonal IgGs against 2-phenyl-5 oxazolone ŽOx. w29x. Three out of 40 Ox-specific antibodies seemed to exist in equilibrium between at least two antibody conformations. The authors concluded that one-tenth of all antibodies may display conformational isomerism but, since the phenomenon cannot always be detected by kinetic techniques, the isomerism detected by kinetic data may represent only the tip of an iceberg. The purpose of this study was to determine why apparently homogeneous IgG antibodies in some cases were fractionated into at least two components by LLPC. To this end, we analysed a large set of well-characterized human and murine monoclonal IgG antibodies of all subclasses. Our results suggest that six out of 57 monoclonal IgGs exist in equilibrium between two components with different surface properties. The ligand-binding properties of these antibodies and their subfractions are also examined and discussed. 2. Materials and methods 2.1. Materials LiParGel 650 was obtained from Merck AG ŽDarmstadt, Germany. . Protein G-Sepharose 4 Fast Flow, Ultropac TSK G 3000 SW column Ž600 = 7.5 mm I.D.., PhastGel ŽIEF 5-8., Ampholin PAGplate Ž3.5–9.0. and dextran T 500 Ž Mr s 500 000. were purchased from Pharmacia Biotech Norden ŽUppsala, Sweden.. Polyethylene glycol 8000 Ž PEG. Ž Mr s 6000–7500. was supplied by Union Carbide ŽNew York.. 2.2. Proteins Human albumin was obtained from Kabi Diagnostica ŽStockholm, Sweden.. Thyroxine ŽT4. conjugated to bovine serum albumin ŽBSA. was purchased from Biodesign International ŽKennebunk, ME.. Horseradish peroxidase and whale myoglobin were
U.-B. Hansson et al.r Biochimica et Biophysica Acta 1340 (1997) 53–62
obtained from Merck AG and Sigma ŽSt. Louis, MO.. The four mouse monoclonal IgG antibodies against human albumin, human IgGFc and T4 analysed in this study were purchased as purified preparations from Biodesign International Ž anti-albumin: IgG1–clone 3001 and IgG2b–clone 3002; anti-T4: IgG2b–clone 9101. and The Binding Site ŽBirmingham, UK. Žanti-IgGFc: IgG1–clone MK1A6.. The characterization of the additional 54 monoclonal IgG antibodies included in this study has been published elsewhere wU.-B. Hansson, C. Wingren, and U. Alkner, unpublished data; w12,14,15xx. These monoclonal antibodies consist of 20 human myeloma IgG1, -2, -3 and -4 proteins purified from human myeloma sera by affinity chromatography on Protein G-Sepharose, 5 chimeric mouserhuman IgG1, -2, -3 and -4 antibodies against 5-iodo-4-hydroxy-3-nitrophenacetyl produced as described elsewhere w15x, 12 human m onoclonal IgG 1 antibodies against cy tolomegalovirus and tetanus toxoid prepared as previously described w30x and 17 purified murine monoclonal IgG1, -2a and -2b antibodies against a variety of haptens and proteins supplied by The Binding Site, Biodesign International, Cortex Biochem ŽSan Leandro, CA., OEM Concepts ŽToms River, NJ., Sigma and Zymed Laboratories ŽSan Francisco, CA.. 2.3. Control of the homogeneity of the IgG antibodies The IgG preparations contained more than 95% IgG and were homogeneous, as determined by SDSPAGE w31x, isoelectric focusing Ž IEF. on Ampholin PAGplate and Phastgel, size-exclusion chromatography ŽHPLC-SE. on a Ultropac TSK G 3000 SW column and by immunoelectrophoresis w32x and double immunodiffusion w33x against appropriate antisera. The protein concentration was determined by absorbency at 280 nm Ž E280 nm , 1% s 12.5. . The affinity constants of the four monoclonal IgG antibodies used in this investigation Ž10 6 y 10 10 My1 . were in the same range as those of the other antibodies with known specificities ww14,30x, data sheets from manufacturersx. 2.4. Preparation of antigen–antibody complexes Antigen–antibody complexes were prepared in 50 mM sodium phosphate, 0.1 M NaCl, 0.1 M glycine,
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pH 7.0 Žtotal antibody populations. or in the top phase Žfractions I and II.. Identical results were obtained using LLPC irrespective of in which of these two solvents the complexes were prepared. The antigen–antibody complexes were prepared at the lowest molar ratio of antigen to antibody at which the maximum amount of antibody had reacted and no free ligand could be detected by LLPC. With the exception of T4-BSA-a-T4 Ž10:1., all the complexes were prepared at a molar ratio of antigen to antibody of 2:1. In all cases, but for anti-IgGFc ŽG 70%., HPLC-SE showed that 100 " 3% of the IgG had formed antigen–antibody complexes. The antigen– antibody mixtures were first incubated for 15 min at 20.08C. The concentration of PEG was then adjusted to that of the top phase by adding appropriate volumes of a 40% Ž wrw in distilled water. PEG 8000 stock solution, whereafter the top phase of the phase system was added, making a final volume of 220 ml. A 200-ml volume of this mixture was then immediately applied to the LLPC column. All the LLPC analyses were performed in such a way that free ligand did not interfere with the chromatogram of the complexes, i.e. free ligand could not be detected at 280 nm in the concentrations used here andror it was well separated from the complexes. The elution profiles obtained for complexes made at identical molar ratios were reproducible both with respect to the proportions between the components and to their partition coefficients Žsee below.. 2.5. Liquid–liquid partition chromatography All experiments were performed in a 4.4% Žwrw. PEG 8000r6.2% Žwrw. dextran T 500 two-phase system containing 50 mM sodium phosphate, 0.1 M NaCl and 0.1 M glycine at pH 7.0. The two-phase system was prepared by thoroughly mixing 110 g 40% PEG Ž wrw in distilled water. and 248 g 25% dextran Žwrw in distilled water. with about 500 g water. Dry NaCl, glycine and sodium phosphate were added and the pH was adjusted with 4 M HCl before the rest of the water was added to make up a 1000 g two-phase system. The system was equilibrated at 208C for 72 h and the clear phases were separated. The matrix, LiParGel 650, was equilibrated with the dextran-rich bottom phase Ž stationary phase. overnight. The coated matrix was rinsed with the
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PEG-rich top phase Žmobile phase. on a glass funnel in order to remove excess stationary phase, suspended in mobile phase and poured into a thermostated Ž208C. steel column Ž300 = 8.0 mm I.D.. with a filling reservoir. The column was first packed under gravitational sedimentation ŽF 30 h. and then at flow rates F 1.0 mlrmin ŽF 25 MPa. using an HPLC pump Ž 2248, Pharmacia. w8x. Protein was applied to the column, 1–40 mg, in 0.2 ml of mobile phase at a flow rate of 0.12 mlrmin. The first day each antibody preparation was analysed is denoted as Day 0. Eluates were continuously monitored at 280 nm. The performance and capacity of the LLPC columns were determined by daily application of two reference proteins, peroxidase and myoglobin Ž20–40 mg., to the columns. The partition coefficients of the reference proteins, K batch , were determined in batch experiments w34x. 1rK batch was used as K C , the ratio of the concentration of a molecule in the two phases: K C s Cstationary phaserCmobile phase
Ž1.
The volumes of the stationary and mobile phases, VS and VM , were calculated from the retention volumes of the references, VR , using the relationship V R s V M q K C VS
Ž2.
deviationrmean.. of K C was F 3%. All LLPC analyses were run 2–5 times. 2.6. Statistical analysis of data A two-tailed t-test was used to determine whether the difference between two K C values was statistically significant at the 95% significance level Ž P s 0.05.. Essentially the same result was obtained using a non-parametric test, i.e. a two-tailed Mann-Whitney U-test, at the 95% significance level.
3. Results In order to determine the reason why apparently homogeneous IgG antibodies are in some cases fractionated into at least two components by LLPC in an aqueous PEGrdextran two-phase system, a large set of well-characterized human and murine monoclonal IgG antibodies of all subclasses were analysed. Six of 57 monoclonal IgGs were fractionated into two components by LLPC, in spite of the fact that they were all homogeneous with respect to their immunochemical and other physicochemical properties Žas determined by HPLC-SE, SDS-PAGE and IEF. Ž Fig.
The plate number, N, was calculated from the peak width at half height Ž w h . of the myoglobin peak according to N s 5.54 Ž VRrw h .
2
Ž3.
The resolution of the peroxidase and myoglobin peaks, R S , was calculated as R S s Ž 'N r4 . Ž kr Ž 1 q k . . Ž a y 1 .
Ž4.
where k is the capacity factor Ž k s Ž VSrVM . K C . and a is the ratio of the partition coefficients of the references Ž a s K batch, peroxidaserK batch, myoglobin .. The parameters of the LLPC columns used were VSrVM s 1.6 " 0.2, VSrVC s 0.40 " 0.03, VMrVC s 0.25 " 0.03, N s 650 " 100 and R S s 3.5 " 0.3, where VC is the column volume. In order to facilitate the comparison of chromatograms obtained from columns with different parameters, retention volumes were expressed as K C , according to Eq. Ž2. . The relative standard deviation Ž 100 Ž standard
Fig. 1. LLPC of 57 monoclonal IgG antibodies that all contained )95% IgG and were homogeneous with respect to their physico-chemical properties as determined by HPLC-SE, SDSPAGE and IEF. The LLPC chromatograms are schematically illustrated in ŽA. which represents 51 of the IgGs, and ŽB. which represents 6 of the IgGs. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2 Žsee Section 2..
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Fig. 2. LLPC of a mouse monoclonal IgG1 antibody against human IgGFc and collected fractions thereof Žfractions I and II., before and after binding antigen. The monoclonal IgG antibody was chromatographed on Day 0 Žsee Section 2. and Day 5. Fractions I and II, collected on Day 0, were reinjected separately on Days 0 and 5. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 2:1 on Day 0 for both fractions I and II. Unbound antigen Ža myeloma IgG1 protein, K C s1.07. did not interfere with the chromatogram of the complexes. Protein Ž1–40 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.02.
1.. HPLC-SE analysis of the LLPC fractions collected showed that all fractions contained monomeric molecules Ždata not shown. . The four IgGs in which the minor component constituted G 20% were selected for further analysis ŽFigs. 2–5.. Fig. 2 shows the LLPC chromatograms of the first antibody, a mouse monoclonal IgG1 antibody against human IgGFc, which was fractionated into two components, fractions I and II, by LLPC on Day 0. When fractions I and II were collected and reinjected separately on Day 0, a two-peak chromatogram was obtained for fraction I, while fraction II was eluted as three components. In both cases, the major compo-
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nent was eluted with a K C value identical to that of the component applied and a new minor component Ž K C s 1.72. was also detected. Moreover, the third component in fraction II had a K C value Ž 1.32. identical to that of the major component in fraction I. On day 5, however, the elution profile of fraction I had shifted, apparently via the new minor component, towards that of fraction II and vice versa. Thus, there was an equilibrium between three forms with different surface properties ŽK C values. . It may also be of interest to observe that the new minor component ŽK C s 1.72. could not be detected in the unfractionated IgG, indicating that fractions I and II were at equilibrium when the unfractionated antibody was analysed the first time ŽDay 0.. Upon addition of antigen to both fractions I and II, a new component with a K C value significantly different from those of the free components Ž K C s 1.64. was detected ŽFig. 2. . HPLC-SE showed that more than 70% of the IgG in both fractions I and II had formed antigen–antibody complexes Ždata not shown.. It should be noted that fractions I and II, in spite of having different surface properties Ž K C values. , formed antigen–antibody complexes which had identical K C values Ž1.64.. Two mouse monoclonal IgG antibodies against human albumin ŽIgG1 and IgG2b. were also fractionated into two components by LLPC ŽFigs. 3 and 4. . The proportion between fractions I and II changed with time Ž 30 days. for both antibodies, but in opposite directions, i.e. fractions I and II were not at equilibrium when the unfractionated antibodies were analysed on Day 0. Fraction II of the IgG2b antibody ŽFig. 3. and fractions I and II of the IgG1 antibody ŽFig. 4. were then collected Ž see figure legends. and reinjected separately on Day 30. Because fraction I of the IgG2b antibody was not available in sufficient amount, it was not further analysed. A two-peak chromatogram with K C values identical to that of the total antibody population was then obtained for each of the reinjected fractions. Hence, our results demonstrated an equilibrium between two fractions with different surface properties Ž K C values. in both cases. The ligand-binding properties of the two anti-albumin antibodies and their subfractions were then examined ŽFigs. 3 and 4.. The fact that a single peak with identical value of K C Ž1.74. was in all cases detected by LLPC whether antigen was added to the
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total antibody population or its subfractions is noteworthy. In this connection, it should also be pointed out that 100 " 3% of the IgG in all fractions had formed antigen–antibody complexes, as determined by HPLC-SE Ždata not shown. . As in the previous case where the antigen was also a large protein, fractions I and II, despite having different surface properties Ž K C values., formed antigen–antibody complexes which had identical K C values. Interestingly, the anti-albumin antibodies, in spite of having different heavy chain isotypes, formed antigen–antibody complexes which exhibited identical partition properties. Finally, a mouse monoclonal IgG2b antibody against a hapten ŽT4. was also eluted as two components by LLPC Ž Fig. 5.. The proportion between fractions I and II did not change with time Ž30 days.. When fractions I and II were collected and reinjected separately on Day 30, a two-peak chromatogram Fig. 4. LLPC of a mouse monoclonal IgG1 antibody against human albumin and collected fractions thereof Žfractions I and II., before and after binding antigen. The monoclonal IgG antibody was chromatographed on Day 0 Žsee Section 2. and Day 30. Fractions I and II, collected on Day 30, were reinjected separately on Day 30. It should be noted that fraction I was collected to the left and fraction II to the right of their peak maximum Žobserved on Day 0. in order to minimize any cross-contamination. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 2:1 on Day 0 for the total antibody population and on Day 30 for both fractions I and II. Unbound antigen Žalbumin, K C s1.85. could not be detected in the chromatograms at 280 nm at the concentrations used here. Protein Ž1–30 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.02. Fig. 3. LLPC of a mouse monoclonal IgG2b antibody against human albumin and a collected fraction thereof Žfraction II., before and after binding antigen. The monoclonal IgG2b antibody was chromatographed on Day 0 Žsee Section 2. and Day 30. Fraction II, collected on Day 30, was reinjected on Days 30 and 34. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 2:1 on Day 0 for the total antibody population and on Day 30 for fraction II. Unbound antigen Žalbumin, K C s1.85. could not be detected in the chromatograms at 280 nm at the concentrations used here. Protein Ž1–30 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.03.
similar to that of the total IgG population was obtained for fraction I, while fraction II was eluted as a single peak with a K C value identical to that of the component applied. Contrary to fraction I, the elution profile of fraction II changed with time Ž2 days. and two components with K C values identical to those of the total IgG population could then be detected. Hence, also for this antibody an equilibrium between two fractions with different surface properties Ž K C values. could be demonstrated.
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different surface properties Ž K C values. formed antigen–antibody complexes which had different surface properties. In order to investigate whether the carrier molecule, BSA, affected the results, antigen–antibody complexes formed by unfractionated anti-T4 and T4 were analysed on Day 30 Ž Fig. 5.. Also in this case a two-peak chromatogram was obtained by LLPC. The proportion between the components was similar to that obtained when T4-BSA was used as antigen, although the K C values of the two components differed slightly. Thus, the carrier molecule apparently did not affect the results.
4. Discussion Fig. 5. LLPC of a mouse monoclonal IgG2b antibody against T4 and collected fractions thereof Žfractions I and II., before and after binding T4-BSA. The monoclonal IgG2b antibody was chromatographed on Day 0 Žsee Section 2. and Day 30. Fractions I and II, collected on Day 30, were reinjected separately on Days 30 and 32. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 10:1 on Day 0 for the total antibody population and on Day 30 for both fractions I and II. Unbound antigen ŽT4-BSA, K C s1.89. did not interfere with the chromatogram of the complexes. Protein Ž2–30 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.02.
On addition of antigen ŽT4-BSA. to unfractionated anti-T4 or fraction I, similar two-peak chromatograms with K C values significantly different from those of the free components were obtained by LLPC ŽFig. 5.. It should be noted that ligand-binding had no detectable affect on the proportion between the two components. Moreover, the complexes formed by fraction II Žon Day 30. were eluted as a single peak with a K C identical to that of the second component in the other complex mixtures. Again, it should be pointed out that 100 " 3% of the IgG in all fractions had formed antigen–antibody complexes, as determined by HPLC-SE Ždata not shown.. Thus, in contrast to the previous cases, the two fractions with
Prior to this study, conformational isomerism of antibodies had been detected by kinetic techniques ww29x and references thereinx. Our results showed that also LLPC can be used to detect different conformational forms of IgG antibodies. Six of 57 monoclonal IgG antibodies existed as two or three conformational forms in equilibrium. These findings agree with kinetic data which state that about one-tenth of all IgG antibodies show conformational isomerism w29x. Methods like stopped-flow w29x measures fast reactions in real-time, in this case the antigen–antibody reaction itself. LLPC, on the other hand, measures the end products at equilibrium, separating the components according to their surface properties. The reaction kinetics itself is not analysed in LLPC, the results of which merely give indications of reaction patterns. However, information about the rate of the equilibria reactions of the unliganded antibody isomers can be obtained by LLPC. Our results indicated that the antibody isomers interconverted slowly, from F 2 h ŽFig. 5. to several days ŽFigs. 3 and 4.. Thus, LLPC can be used to detect and even separate the conformational isomers of unliganded antibodies. The partition properties of unliganded IgG antibodies are determined by the structure of their antigenbinding regions w14x, i.e. implying that the isomeric forms of IgG antibodies differ with respect to the surface properties of their antigen-binding sites. NMR and X-ray crystallography experiments on Fab frag-
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ments from monoclonal IgG antibodies support the idea of conformational heterogeneity in the antibody combining sites w35–37x. These studies have presented evidence that a single tyrosine residue can assume two distinct conformations which interconvert when the binding site is empty. LLPC detects differences in K C Žsurface properties. of about 5% Žsee Section 2. . Assuming that the differences observed by LLPC between the conformational isomers are located only within the antigen-binding sites and that each site involves about 20 amino acid residues w38–42x, LLPC would thus be able to detect a difference of about one amino acid per binding site. However, even if the observed differences in the K C values cannot be used to identify the type of residueŽs. and in what way they differ, the complexity of the parameters determining K C provides a selectivity which may not readily be obtained by any other method or combination of methods. We have previously shown that homogeneous monoclonal IgG antibodies are eluted as single homogeneous peaks by LLPC even after binding of antigen at molar ratios ranging from antibody excess to antigen excess ww11x; U.-B. Hansson, C. Wingren, and U. Alkner, unpublished datax. Assuming that the components in the equilibria studied here could all bind antigen, one would expect the antigen–antibody complexes to be eluted as two or three Žfor the anti-IgGFc antibody. components by LLPC. This was also shown to be the case for the complexes formed by the anti-T4 antibody and T4-BSA or T4, i.e. both the unliganded antibody and its complexes with T4BSA or T4 gave two components in LLPC analyses. Thus, the isomeric forms of the anti-T4 antibody, which display different surface properties, also formed hapten–antibody complexes with different surface properties. Moreover, the binding of ligand had no detectable effect on the proportions of the isomers. Thus, our results indicated that both isomeric forms making up the equilibrium can bind antigen. However, the heterogeneity observed for the T4 complexes may also be related to a conformational isomerism in the antibody similar to that observed for some anti-dinitrophenyl antibodies w43x. Their results indicated the co-existence of two forms of hapten– antibody complexes due to isomerism of a tryptophane residue in the antibody combining site. We have recently shown that there is a linear relationship
between the K C values of unliganded IgG antibodies and their hapten–antibody complexes, even when the hapten is conjugated to BSA wU.-B. Hansson, C. Wingren, and U. Alkner, unpublished datax. Thus, parts of the antigen-binding sites are still exposed after binding hapten or hapten-BSA. Taken together, our results also locate the observed differences in surface properties between the T4–anti-T4 complexes to the antibody combining sites. In the initial discovery of antibody isomerism, the following reaction mechanism, based on kinetic evidence, was proposed by Lancet and Pecht w25x:
In this model the antibody molecule exists in an equilibrium between two conformations, Ab and Ab ). Different reactivity of the two isomers towards ligand L gives rise to complex kinetics which should be triphasic. However, Foote and Milstein w29x observed biphasic reactions and suggested that only one of the antibody conformational isomers bound antigen. The mechanism proposed by Foote and Milstein does not apply to our antibody–hapten system, since both the isomeric forms of the antibodies formed complex with hapten Žthe equilibrium appears to be frozen. ŽFig. 5.. Therefore, our data concerning haptens fits into the model suggested by Lancet and Pecht w25x, with the possible exception for the transition between the two complexes. The antigen–antibody complexes were eluted as single peaks when the isomeric forms of the antibodies were directed against a large protein instead of a hapten ŽFigs. 2–4.. The shape of the antigen-binding sites has been shown to correlate with the type of antigen recognized w39,44,45x. Antibodies specific for haptens have concave antigen-binding sites while those binding proteins tend to have flattened combining sites w44–46x. We have recently shown that the antigen-binding regions of antibodies are concealed when binding a large protein antigen wU.-B. Hansson, C. Wingren, and U. Alkner, unpublished datax, i.e. antibodies with different K C values, but directed against the same protein antigen, formed complexes with the same value of K C . Thus, we cannot deter-
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mine whether only one or both of the conformational isomers could bind antigen when the antigen was a large protein. Therefore, we cannot anticipate whether any of the suggested kinetic models w25,29x is relevant for these antibody–protein systems. In agreement with Foote and Milstein w29x, we think that conformational isomerism may allow antibodies to cross-react and recognize dissimilar antigens. Such an effect may be of great importance if the antigen rapidly changes its surface properties or has a very flexible structure. In addition, conformational isomerism may be a way to modify the affinity of antibodies, i.e. may be an alternative way to obtain affinity maturation. In this connection it may be of interest to note that even the variable regions of antibodies have been demonstrated to influence their effector functions w47–49x. Furthermore, we have recently shown that antibodies with catalytic activity wU.-B. Hansson, C. Wingren, and D.S. Tawfik, unpublished datax as well as immunoglobulins of isotypes other than IgG w15x display the same elution patterns as the IgG antibodies analysed here. Thus, conformational isomerism of antibodies can be of great importance for the biological functionŽs. of the molecules. References ˚ Albertsson, Partition of Cell Particles and Macrow1x P.-A. molecules, John Wiley, New York, 1986. ˚ Albertsson, J. Chromatogr. 159 Ž1978. 111–122. w2x P.-A. w3x A.D. Diamond, J.T. Hsu, J. Chromatogr. 513 Ž1990. 137– 143. w4x G. Johansson, in: H. Walter, E.D. Brooks ŽEds.., Partitioning in Aqueous Two-Phase Systems, Academic Press, Orlando, FL, 1985, pp. 161-226. w5x H. Walter, G. Johansson, D.E. Brooks, Anal. Biochem. 197 Ž1991. 1–18. ˚ Albertsson, w6x U.-B. Hansson, K. Andersson, Y. Liu, P.-A. Anal. Biochem. 183 Ž1989. 305–311. w7x W. Muller, Eur. J. Chrom. 155 Ž1986. 213–222. ¨ w8x C. Wingren, B. Persson, U.-B. Hansson, J. Chromatogr. 668 Ž1994. 65–73. w9x W. Muller, Bioseparation 1 Ž1990. 265–282. ¨ w10x U.-B. Hansson, C. Wingren, in: M. Kastner ŽEd.., Protein Liquid Chromatography, Elsevier, Oxford, 1997. w11x C. Wingren, U.-B. Hansson, Biochim. Biophys. Acta 1244 Ž1995. 209–215. w12x K. Andersson, C. Wingren, U.-B. Hansson, Scand. J. Immun. 38 Ž1993. 95–101.
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w13x C. Wingren, U.-B. Hansson, J. Chromatogr. 680 Ž1996. 113–122. w14x C. Wingren, U.-B. Hansson, C.G.M. Magnusson, M. Ohlin, Mol. Immun. 32 Ž1995. 819–827. w15x C. Wingren, T.E. Michaelsen, C.G.M. Magnusson, U.-B. Hansson, Scand. J. Immun. 44 Ž1996. 430–436. w16x A. Tramantano, K.D. Janda, R.A. Lerner, Science 234 Ž1986. 1566–1569. w17x D.S. Tawfik, Towards Antibody-Mediated Peptide Hydrolysis. Thesis, Weizmann Institute of Science, Israel, 1995. w18x S.J. Pollack, J.W. Jacobs, P.G. Schultz, Science 234 Ž1986. 1570–1573. w19x V. Raso, B.D. Stollar, Biochemistry 14 Ž1975. 591–599. w20x F. Kohen, J.B. Kim, H.R. Lindner, Z. Eshar, B.S. Green, FEBS Lett. 111 Ž1980. 427–431. w21x S. Paul, D.J. Volle, C.M. Beach, D.R. Johnson, M.J. Powell, R.J. Massey, Science 244 Ž1989. 1158–1162. w22x A.M. Shuster, G.V. Gololobov, O.A. Kvashuk, A.E. Bogornolova, I.V. Smirnov, A.G. Gabibov, Science 256 Ž1992. 665–667. w23x L. Li, S. Paul, S. Tyutyulkova, M. Katzachkine, S. Kaveri, J. Immun. 154 Ž1995. 3328–3332. w24x D.M. Kranz, J.N. Herron, E.W. Voss, J. Biol. Chem. 257 Ž1982. 6987–6995. w25x D. Lancet, I. Pecht, Proc. Natl. Acad. Sci. USA 73 Ž1976. 3548–3553. w26x I. Pecht, in: M. Sela ŽEd.., The Antigens, Vol. IV, Academic Press, San Diego, CA, 1982, pp. 1–68. w27x S. Vuk-Pavlovic, Y. Blatt, C.P.J. Glaudemans, D. Lancet, I. Pecht, Biophys. J. 24 Ž1978. 161–174. w28x R. Zidovetski, Y. Blatt, C.P.J. Glaudemans, B.N. Manjula, I. Pecht, Biochemistry 19 Ž1980. 2790–2795. w29x J. Foote, C. Milstein, Proc. Natl. Acad. Sci. USA 91 Ž1994. 10370–10374. w30x M. Ohlin, V.-A. Sundqvist, M. Mach, B. Wahren, C.A.K. Borrebaeck, J. Virol. 67 Ž1993. 703–710. w31x U.K. Laemmli, Nature 227 Ž1970. 680–685. w32x J.J. Scheidegger, Int. Archs. Allergy Appl. Immun. 7 Ž1955. 103–110. w33x O. Ouchterlony, Prog. Allergy 6 Ž1962. 30–154. w34x C. Wingren, U.B. Hansson, K. Andersson, J. Chromatogr. 603 Ž1992. 73–81. w35x I.S. Mian, A.R. Bradwell, A.J. Olson, J. Mol. Biol. 217 Ž1991. 133–151. w36x T.P. Theriault, D.J. Leahy, M. Levitt, H.M. Mconnell, G.S. Rule, J. Mol. Biol. 221 Ž1991. 257–270. w37x T.P. Theriault, H.M. McConnell, A.K. Singhal, G.S. Rule, J. Phys. Chem. 97 Ž1993. 3034–3039. w38x C. Chothia, Curr. Opin. Struct. Biol. 1 Ž1991. 53–57. w39x D.R. Davies, E.A. Padlan, S. Sheriff, Ann. Rev. Biochem. 59 Ž1990. 439–473. w40x D.R. Davies, G.H. Cohen, Proc. Natl. Acad. Sci. USA 93 Ž1996. 7–12. w41x J. Foote, H.N. Eisen, Proc. Natl. Acad. Sci. USA 92 Ž1995. 1254–1256. w42x E.A. Padlan, Mol. Immun. 31 Ž1994. 169–217.
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w43x M. Martinez-Yamout, H.M. McConnell, J. Mol. Biol. 244 Ž1994. 301–318. w44x D.M. Webster, A.H. Henry, A.R. Rees, Curr. Opin. Struct. Biol. 4 Ž1994. 123–129. w45x I.A. Wilson, R.L. Stanfield, Curr. Opin. Struct. Biol. 3 Ž1993. 113–118. w46x E. Vargas-Madrazo, F. Lara-Ochoa, J.C. Almagro, J. Mol. Biol. 254 Ž1995. 497–504.
w47x C. Horgan, K. Brown, S.H. Picus, J. Immun. 145 Ž1990. 2527–2532. w48x I. Yokoyama, F. Waxman, Immunology 80 Ž1993. 168–176. w49x K.D. White, M.B. Frank, S. Foundling, F.J. Waxman, Mol. Immun. 33 Ž1996. 759–768.
Conformational isomerism of IgG antibodies Ulla-Britt Hansson b
a,)
, Christer Wingren a , Ulf Alkner
b
a Department of Biochemistry, Lund UniÕersity, Lund, Sweden Department of Bioanalytical Chemistry, Astra Draco AB, Lund, Sweden
Received 20 November 1996; accepted 28 January 1997
Abstract The purpose of this study was to determine why apparently homogeneous IgG antibodies were, in some cases, fractionated into at least two components by liquid–liquid partition chromatography ŽLLPC. in an aqueous two-phase system. Four mouse monoclonal IgG antibodies, two against albumin, one against IgG and one against thyroxine, were shown to adopt different conformational isomeric forms. The four antibodies existed in an equilibrium between two or three conformational forms, the proportion of which could also be estimated by LLPC. Since LLPC detects mainly conformational differences within the antigen-binding sites of IgG antibodies, it could be concluded that the conformational forms differed with respect to their combining sites. Moreover, the isomeric forms of an antibody directed against a protein antigen, formed antigen–antibody complexes with almost identical surface properties. In contrast, complexes with different surface properties were formed when the hapten or hapten conjugated to BSA was bound. Thus, both the conformational isomers could bind antigen, at least when the antigen was a small hapten or a hapten conjugated to a carrier protein. Our results suggest that six out of 57 monoclonal IgG antibodies exist in equilibrium between at least two conformational forms and the biological significance of this isomerism is discussed. Keywords: Antigen binding; Conformational isomerism; IgG antibody; Liquid-liquid partition chromatography; Phase partition; Surface property
1. Introduction Partitioning in aqueous PEGrdextran two-phase systems is useful for separation of biomolecules w1x. The distribution of a molecule in these systems depends on its three-dimensional structure and general surface properties and is described by its partition
Abbreviations: CDR, complementarity-determining region; LLPC, liquid–liquid partition chromatography; PEG, polyethylene glycol; T4, thyroxine ) Corresponding author. Fax: q46 46 222 4534; e-mail: [email protected]
coefficient w2–5x. The selectivity and sensitivity of the two-phase technique can be considerably increased by adopting a column chromatographic approach, liquid–liquid partition chromatography ŽLLPC., in which the bottom phase of the two-phase system is adsorbed onto a support and packed into a column which is eluted with the corresponding top phase w6–8x. LLPC has been shown to be a unique tool for protein analysis in that it can be used to separate and fractionate proteins w9–11x, to detect conformational changes occurring upon binding of a ligand w11–13x and to relate surface properties of proteins to their biological specificities w6,12,14x. We have recently shown that the surface properties of
0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 2 8 - 9
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IgG1, -2 and -4 antibodies are dominated by those of their antigen-binding regions and that isolated Fc fragments derived from these three IgG subclasses displayed similar surface properties w14,15x. Hence, the constant parts of the IgG1, -2 and -4 isotypes may form similar scaffoldings, on which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties. However, our studies on immunoglobulins also showed that some monoclonal antibodies were fractionated into at least two components by LLPC, in spite of the fact that they were homogeneous with respect to their immunochemical and physicochemical properties w12,14x. This phenomenon was observed not only for IgG antibodies but also for IgA and IgM myeloma proteins w15x. Consequently, the data gave rise to the question of whether these antibodies, as many enzymes, occurred in different conformational forms. We have previously explored the capacity of LLPC to detect different Žisomeric. forms of a protein by analysing a large set of well-characterized enzymes known to exist in equilibrium between an open and a closed conformation. In agreement with data obtained by several methods, including X-ray crystallography, the equilibrium between two forms of unliganded alcohol dehydrogenase and lactate dehydrogenase was detected by LLPC, as was the ligand-dependent equilibrium between two forms of citrate synthase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, glutamate–oxaloacetate transaminase, hexokinase and 3-phosphoglycerate kinase w11,13x. Hence, the results clearly indicated that isomeric forms of enzymes could be detected and in some cases even separated by LLPC. Like enzymes, immunoglobulins can have a catalytic function w16–18x and they are generally produced in vitro w16,18–20x, but have also in some cases been detected in vivo in the sera of some patients with autoimmune diseases w21–23x. Since antibodies and enzymes can share some functional properties, one may further speculate on whether they can also share the structural feature and thus exist in conformational isomeric forms. Indeed, studies on several myeloma proteins have demonstrated kinetically distinct conformational isomers w24–28x. These examples do, however, leave an
uncertainty as to whether conformational antibody isomerism is an oddity or a general and important factor in an immune response. Evidence that conformational polymorphism may be more general came in 1994 when Foote and Milstein observed biphasic or triphasic reactions in stopped-flow fluorescence experiments of monoclonal IgGs against 2-phenyl-5 oxazolone ŽOx. w29x. Three out of 40 Ox-specific antibodies seemed to exist in equilibrium between at least two antibody conformations. The authors concluded that one-tenth of all antibodies may display conformational isomerism but, since the phenomenon cannot always be detected by kinetic techniques, the isomerism detected by kinetic data may represent only the tip of an iceberg. The purpose of this study was to determine why apparently homogeneous IgG antibodies in some cases were fractionated into at least two components by LLPC. To this end, we analysed a large set of well-characterized human and murine monoclonal IgG antibodies of all subclasses. Our results suggest that six out of 57 monoclonal IgGs exist in equilibrium between two components with different surface properties. The ligand-binding properties of these antibodies and their subfractions are also examined and discussed. 2. Materials and methods 2.1. Materials LiParGel 650 was obtained from Merck AG ŽDarmstadt, Germany. . Protein G-Sepharose 4 Fast Flow, Ultropac TSK G 3000 SW column Ž600 = 7.5 mm I.D.., PhastGel ŽIEF 5-8., Ampholin PAGplate Ž3.5–9.0. and dextran T 500 Ž Mr s 500 000. were purchased from Pharmacia Biotech Norden ŽUppsala, Sweden.. Polyethylene glycol 8000 Ž PEG. Ž Mr s 6000–7500. was supplied by Union Carbide ŽNew York.. 2.2. Proteins Human albumin was obtained from Kabi Diagnostica ŽStockholm, Sweden.. Thyroxine ŽT4. conjugated to bovine serum albumin ŽBSA. was purchased from Biodesign International ŽKennebunk, ME.. Horseradish peroxidase and whale myoglobin were
U.-B. Hansson et al.r Biochimica et Biophysica Acta 1340 (1997) 53–62
obtained from Merck AG and Sigma ŽSt. Louis, MO.. The four mouse monoclonal IgG antibodies against human albumin, human IgGFc and T4 analysed in this study were purchased as purified preparations from Biodesign International Ž anti-albumin: IgG1–clone 3001 and IgG2b–clone 3002; anti-T4: IgG2b–clone 9101. and The Binding Site ŽBirmingham, UK. Žanti-IgGFc: IgG1–clone MK1A6.. The characterization of the additional 54 monoclonal IgG antibodies included in this study has been published elsewhere wU.-B. Hansson, C. Wingren, and U. Alkner, unpublished data; w12,14,15xx. These monoclonal antibodies consist of 20 human myeloma IgG1, -2, -3 and -4 proteins purified from human myeloma sera by affinity chromatography on Protein G-Sepharose, 5 chimeric mouserhuman IgG1, -2, -3 and -4 antibodies against 5-iodo-4-hydroxy-3-nitrophenacetyl produced as described elsewhere w15x, 12 human m onoclonal IgG 1 antibodies against cy tolomegalovirus and tetanus toxoid prepared as previously described w30x and 17 purified murine monoclonal IgG1, -2a and -2b antibodies against a variety of haptens and proteins supplied by The Binding Site, Biodesign International, Cortex Biochem ŽSan Leandro, CA., OEM Concepts ŽToms River, NJ., Sigma and Zymed Laboratories ŽSan Francisco, CA.. 2.3. Control of the homogeneity of the IgG antibodies The IgG preparations contained more than 95% IgG and were homogeneous, as determined by SDSPAGE w31x, isoelectric focusing Ž IEF. on Ampholin PAGplate and Phastgel, size-exclusion chromatography ŽHPLC-SE. on a Ultropac TSK G 3000 SW column and by immunoelectrophoresis w32x and double immunodiffusion w33x against appropriate antisera. The protein concentration was determined by absorbency at 280 nm Ž E280 nm , 1% s 12.5. . The affinity constants of the four monoclonal IgG antibodies used in this investigation Ž10 6 y 10 10 My1 . were in the same range as those of the other antibodies with known specificities ww14,30x, data sheets from manufacturersx. 2.4. Preparation of antigen–antibody complexes Antigen–antibody complexes were prepared in 50 mM sodium phosphate, 0.1 M NaCl, 0.1 M glycine,
55
pH 7.0 Žtotal antibody populations. or in the top phase Žfractions I and II.. Identical results were obtained using LLPC irrespective of in which of these two solvents the complexes were prepared. The antigen–antibody complexes were prepared at the lowest molar ratio of antigen to antibody at which the maximum amount of antibody had reacted and no free ligand could be detected by LLPC. With the exception of T4-BSA-a-T4 Ž10:1., all the complexes were prepared at a molar ratio of antigen to antibody of 2:1. In all cases, but for anti-IgGFc ŽG 70%., HPLC-SE showed that 100 " 3% of the IgG had formed antigen–antibody complexes. The antigen– antibody mixtures were first incubated for 15 min at 20.08C. The concentration of PEG was then adjusted to that of the top phase by adding appropriate volumes of a 40% Ž wrw in distilled water. PEG 8000 stock solution, whereafter the top phase of the phase system was added, making a final volume of 220 ml. A 200-ml volume of this mixture was then immediately applied to the LLPC column. All the LLPC analyses were performed in such a way that free ligand did not interfere with the chromatogram of the complexes, i.e. free ligand could not be detected at 280 nm in the concentrations used here andror it was well separated from the complexes. The elution profiles obtained for complexes made at identical molar ratios were reproducible both with respect to the proportions between the components and to their partition coefficients Žsee below.. 2.5. Liquid–liquid partition chromatography All experiments were performed in a 4.4% Žwrw. PEG 8000r6.2% Žwrw. dextran T 500 two-phase system containing 50 mM sodium phosphate, 0.1 M NaCl and 0.1 M glycine at pH 7.0. The two-phase system was prepared by thoroughly mixing 110 g 40% PEG Ž wrw in distilled water. and 248 g 25% dextran Žwrw in distilled water. with about 500 g water. Dry NaCl, glycine and sodium phosphate were added and the pH was adjusted with 4 M HCl before the rest of the water was added to make up a 1000 g two-phase system. The system was equilibrated at 208C for 72 h and the clear phases were separated. The matrix, LiParGel 650, was equilibrated with the dextran-rich bottom phase Ž stationary phase. overnight. The coated matrix was rinsed with the
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PEG-rich top phase Žmobile phase. on a glass funnel in order to remove excess stationary phase, suspended in mobile phase and poured into a thermostated Ž208C. steel column Ž300 = 8.0 mm I.D.. with a filling reservoir. The column was first packed under gravitational sedimentation ŽF 30 h. and then at flow rates F 1.0 mlrmin ŽF 25 MPa. using an HPLC pump Ž 2248, Pharmacia. w8x. Protein was applied to the column, 1–40 mg, in 0.2 ml of mobile phase at a flow rate of 0.12 mlrmin. The first day each antibody preparation was analysed is denoted as Day 0. Eluates were continuously monitored at 280 nm. The performance and capacity of the LLPC columns were determined by daily application of two reference proteins, peroxidase and myoglobin Ž20–40 mg., to the columns. The partition coefficients of the reference proteins, K batch , were determined in batch experiments w34x. 1rK batch was used as K C , the ratio of the concentration of a molecule in the two phases: K C s Cstationary phaserCmobile phase
Ž1.
The volumes of the stationary and mobile phases, VS and VM , were calculated from the retention volumes of the references, VR , using the relationship V R s V M q K C VS
Ž2.
deviationrmean.. of K C was F 3%. All LLPC analyses were run 2–5 times. 2.6. Statistical analysis of data A two-tailed t-test was used to determine whether the difference between two K C values was statistically significant at the 95% significance level Ž P s 0.05.. Essentially the same result was obtained using a non-parametric test, i.e. a two-tailed Mann-Whitney U-test, at the 95% significance level.
3. Results In order to determine the reason why apparently homogeneous IgG antibodies are in some cases fractionated into at least two components by LLPC in an aqueous PEGrdextran two-phase system, a large set of well-characterized human and murine monoclonal IgG antibodies of all subclasses were analysed. Six of 57 monoclonal IgGs were fractionated into two components by LLPC, in spite of the fact that they were all homogeneous with respect to their immunochemical and other physicochemical properties Žas determined by HPLC-SE, SDS-PAGE and IEF. Ž Fig.
The plate number, N, was calculated from the peak width at half height Ž w h . of the myoglobin peak according to N s 5.54 Ž VRrw h .
2
Ž3.
The resolution of the peroxidase and myoglobin peaks, R S , was calculated as R S s Ž 'N r4 . Ž kr Ž 1 q k . . Ž a y 1 .
Ž4.
where k is the capacity factor Ž k s Ž VSrVM . K C . and a is the ratio of the partition coefficients of the references Ž a s K batch, peroxidaserK batch, myoglobin .. The parameters of the LLPC columns used were VSrVM s 1.6 " 0.2, VSrVC s 0.40 " 0.03, VMrVC s 0.25 " 0.03, N s 650 " 100 and R S s 3.5 " 0.3, where VC is the column volume. In order to facilitate the comparison of chromatograms obtained from columns with different parameters, retention volumes were expressed as K C , according to Eq. Ž2. . The relative standard deviation Ž 100 Ž standard
Fig. 1. LLPC of 57 monoclonal IgG antibodies that all contained )95% IgG and were homogeneous with respect to their physico-chemical properties as determined by HPLC-SE, SDSPAGE and IEF. The LLPC chromatograms are schematically illustrated in ŽA. which represents 51 of the IgGs, and ŽB. which represents 6 of the IgGs. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2 Žsee Section 2..
U.-B. Hansson et al.r Biochimica et Biophysica Acta 1340 (1997) 53–62
Fig. 2. LLPC of a mouse monoclonal IgG1 antibody against human IgGFc and collected fractions thereof Žfractions I and II., before and after binding antigen. The monoclonal IgG antibody was chromatographed on Day 0 Žsee Section 2. and Day 5. Fractions I and II, collected on Day 0, were reinjected separately on Days 0 and 5. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 2:1 on Day 0 for both fractions I and II. Unbound antigen Ža myeloma IgG1 protein, K C s1.07. did not interfere with the chromatogram of the complexes. Protein Ž1–40 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.02.
1.. HPLC-SE analysis of the LLPC fractions collected showed that all fractions contained monomeric molecules Ždata not shown. . The four IgGs in which the minor component constituted G 20% were selected for further analysis ŽFigs. 2–5.. Fig. 2 shows the LLPC chromatograms of the first antibody, a mouse monoclonal IgG1 antibody against human IgGFc, which was fractionated into two components, fractions I and II, by LLPC on Day 0. When fractions I and II were collected and reinjected separately on Day 0, a two-peak chromatogram was obtained for fraction I, while fraction II was eluted as three components. In both cases, the major compo-
57
nent was eluted with a K C value identical to that of the component applied and a new minor component Ž K C s 1.72. was also detected. Moreover, the third component in fraction II had a K C value Ž 1.32. identical to that of the major component in fraction I. On day 5, however, the elution profile of fraction I had shifted, apparently via the new minor component, towards that of fraction II and vice versa. Thus, there was an equilibrium between three forms with different surface properties ŽK C values. . It may also be of interest to observe that the new minor component ŽK C s 1.72. could not be detected in the unfractionated IgG, indicating that fractions I and II were at equilibrium when the unfractionated antibody was analysed the first time ŽDay 0.. Upon addition of antigen to both fractions I and II, a new component with a K C value significantly different from those of the free components Ž K C s 1.64. was detected ŽFig. 2. . HPLC-SE showed that more than 70% of the IgG in both fractions I and II had formed antigen–antibody complexes Ždata not shown.. It should be noted that fractions I and II, in spite of having different surface properties Ž K C values. , formed antigen–antibody complexes which had identical K C values Ž1.64.. Two mouse monoclonal IgG antibodies against human albumin ŽIgG1 and IgG2b. were also fractionated into two components by LLPC ŽFigs. 3 and 4. . The proportion between fractions I and II changed with time Ž 30 days. for both antibodies, but in opposite directions, i.e. fractions I and II were not at equilibrium when the unfractionated antibodies were analysed on Day 0. Fraction II of the IgG2b antibody ŽFig. 3. and fractions I and II of the IgG1 antibody ŽFig. 4. were then collected Ž see figure legends. and reinjected separately on Day 30. Because fraction I of the IgG2b antibody was not available in sufficient amount, it was not further analysed. A two-peak chromatogram with K C values identical to that of the total antibody population was then obtained for each of the reinjected fractions. Hence, our results demonstrated an equilibrium between two fractions with different surface properties Ž K C values. in both cases. The ligand-binding properties of the two anti-albumin antibodies and their subfractions were then examined ŽFigs. 3 and 4.. The fact that a single peak with identical value of K C Ž1.74. was in all cases detected by LLPC whether antigen was added to the
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total antibody population or its subfractions is noteworthy. In this connection, it should also be pointed out that 100 " 3% of the IgG in all fractions had formed antigen–antibody complexes, as determined by HPLC-SE Ždata not shown. . As in the previous case where the antigen was also a large protein, fractions I and II, despite having different surface properties Ž K C values., formed antigen–antibody complexes which had identical K C values. Interestingly, the anti-albumin antibodies, in spite of having different heavy chain isotypes, formed antigen–antibody complexes which exhibited identical partition properties. Finally, a mouse monoclonal IgG2b antibody against a hapten ŽT4. was also eluted as two components by LLPC Ž Fig. 5.. The proportion between fractions I and II did not change with time Ž30 days.. When fractions I and II were collected and reinjected separately on Day 30, a two-peak chromatogram Fig. 4. LLPC of a mouse monoclonal IgG1 antibody against human albumin and collected fractions thereof Žfractions I and II., before and after binding antigen. The monoclonal IgG antibody was chromatographed on Day 0 Žsee Section 2. and Day 30. Fractions I and II, collected on Day 30, were reinjected separately on Day 30. It should be noted that fraction I was collected to the left and fraction II to the right of their peak maximum Žobserved on Day 0. in order to minimize any cross-contamination. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 2:1 on Day 0 for the total antibody population and on Day 30 for both fractions I and II. Unbound antigen Žalbumin, K C s1.85. could not be detected in the chromatograms at 280 nm at the concentrations used here. Protein Ž1–30 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.02. Fig. 3. LLPC of a mouse monoclonal IgG2b antibody against human albumin and a collected fraction thereof Žfraction II., before and after binding antigen. The monoclonal IgG2b antibody was chromatographed on Day 0 Žsee Section 2. and Day 30. Fraction II, collected on Day 30, was reinjected on Days 30 and 34. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 2:1 on Day 0 for the total antibody population and on Day 30 for fraction II. Unbound antigen Žalbumin, K C s1.85. could not be detected in the chromatograms at 280 nm at the concentrations used here. Protein Ž1–30 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.03.
similar to that of the total IgG population was obtained for fraction I, while fraction II was eluted as a single peak with a K C value identical to that of the component applied. Contrary to fraction I, the elution profile of fraction II changed with time Ž2 days. and two components with K C values identical to those of the total IgG population could then be detected. Hence, also for this antibody an equilibrium between two fractions with different surface properties Ž K C values. could be demonstrated.
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different surface properties Ž K C values. formed antigen–antibody complexes which had different surface properties. In order to investigate whether the carrier molecule, BSA, affected the results, antigen–antibody complexes formed by unfractionated anti-T4 and T4 were analysed on Day 30 Ž Fig. 5.. Also in this case a two-peak chromatogram was obtained by LLPC. The proportion between the components was similar to that obtained when T4-BSA was used as antigen, although the K C values of the two components differed slightly. Thus, the carrier molecule apparently did not affect the results.
4. Discussion Fig. 5. LLPC of a mouse monoclonal IgG2b antibody against T4 and collected fractions thereof Žfractions I and II., before and after binding T4-BSA. The monoclonal IgG2b antibody was chromatographed on Day 0 Žsee Section 2. and Day 30. Fractions I and II, collected on Day 30, were reinjected separately on Days 30 and 32. The antigen–antibody complexes were prepared at a molar ratio of antigen to antibody of 10:1 on Day 0 for the total antibody population and on Day 30 for both fractions I and II. Unbound antigen ŽT4-BSA, K C s1.89. did not interfere with the chromatogram of the complexes. Protein Ž2–30 mg. was applied to the LLPC column Ž300=8 mm I.D.. in 200 ml of mobile phase at a flow rate of 0.12 mlrmin. The retention volumes are expressed as partition coefficients Ž K C . according to Eq. 2. The relative standard deviation of K C was F 3%. The 95% confidence limit of K C was "0.02.
On addition of antigen ŽT4-BSA. to unfractionated anti-T4 or fraction I, similar two-peak chromatograms with K C values significantly different from those of the free components were obtained by LLPC ŽFig. 5.. It should be noted that ligand-binding had no detectable affect on the proportion between the two components. Moreover, the complexes formed by fraction II Žon Day 30. were eluted as a single peak with a K C identical to that of the second component in the other complex mixtures. Again, it should be pointed out that 100 " 3% of the IgG in all fractions had formed antigen–antibody complexes, as determined by HPLC-SE Ždata not shown.. Thus, in contrast to the previous cases, the two fractions with
Prior to this study, conformational isomerism of antibodies had been detected by kinetic techniques ww29x and references thereinx. Our results showed that also LLPC can be used to detect different conformational forms of IgG antibodies. Six of 57 monoclonal IgG antibodies existed as two or three conformational forms in equilibrium. These findings agree with kinetic data which state that about one-tenth of all IgG antibodies show conformational isomerism w29x. Methods like stopped-flow w29x measures fast reactions in real-time, in this case the antigen–antibody reaction itself. LLPC, on the other hand, measures the end products at equilibrium, separating the components according to their surface properties. The reaction kinetics itself is not analysed in LLPC, the results of which merely give indications of reaction patterns. However, information about the rate of the equilibria reactions of the unliganded antibody isomers can be obtained by LLPC. Our results indicated that the antibody isomers interconverted slowly, from F 2 h ŽFig. 5. to several days ŽFigs. 3 and 4.. Thus, LLPC can be used to detect and even separate the conformational isomers of unliganded antibodies. The partition properties of unliganded IgG antibodies are determined by the structure of their antigenbinding regions w14x, i.e. implying that the isomeric forms of IgG antibodies differ with respect to the surface properties of their antigen-binding sites. NMR and X-ray crystallography experiments on Fab frag-
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ments from monoclonal IgG antibodies support the idea of conformational heterogeneity in the antibody combining sites w35–37x. These studies have presented evidence that a single tyrosine residue can assume two distinct conformations which interconvert when the binding site is empty. LLPC detects differences in K C Žsurface properties. of about 5% Žsee Section 2. . Assuming that the differences observed by LLPC between the conformational isomers are located only within the antigen-binding sites and that each site involves about 20 amino acid residues w38–42x, LLPC would thus be able to detect a difference of about one amino acid per binding site. However, even if the observed differences in the K C values cannot be used to identify the type of residueŽs. and in what way they differ, the complexity of the parameters determining K C provides a selectivity which may not readily be obtained by any other method or combination of methods. We have previously shown that homogeneous monoclonal IgG antibodies are eluted as single homogeneous peaks by LLPC even after binding of antigen at molar ratios ranging from antibody excess to antigen excess ww11x; U.-B. Hansson, C. Wingren, and U. Alkner, unpublished datax. Assuming that the components in the equilibria studied here could all bind antigen, one would expect the antigen–antibody complexes to be eluted as two or three Žfor the anti-IgGFc antibody. components by LLPC. This was also shown to be the case for the complexes formed by the anti-T4 antibody and T4-BSA or T4, i.e. both the unliganded antibody and its complexes with T4BSA or T4 gave two components in LLPC analyses. Thus, the isomeric forms of the anti-T4 antibody, which display different surface properties, also formed hapten–antibody complexes with different surface properties. Moreover, the binding of ligand had no detectable effect on the proportions of the isomers. Thus, our results indicated that both isomeric forms making up the equilibrium can bind antigen. However, the heterogeneity observed for the T4 complexes may also be related to a conformational isomerism in the antibody similar to that observed for some anti-dinitrophenyl antibodies w43x. Their results indicated the co-existence of two forms of hapten– antibody complexes due to isomerism of a tryptophane residue in the antibody combining site. We have recently shown that there is a linear relationship
between the K C values of unliganded IgG antibodies and their hapten–antibody complexes, even when the hapten is conjugated to BSA wU.-B. Hansson, C. Wingren, and U. Alkner, unpublished datax. Thus, parts of the antigen-binding sites are still exposed after binding hapten or hapten-BSA. Taken together, our results also locate the observed differences in surface properties between the T4–anti-T4 complexes to the antibody combining sites. In the initial discovery of antibody isomerism, the following reaction mechanism, based on kinetic evidence, was proposed by Lancet and Pecht w25x:
In this model the antibody molecule exists in an equilibrium between two conformations, Ab and Ab ). Different reactivity of the two isomers towards ligand L gives rise to complex kinetics which should be triphasic. However, Foote and Milstein w29x observed biphasic reactions and suggested that only one of the antibody conformational isomers bound antigen. The mechanism proposed by Foote and Milstein does not apply to our antibody–hapten system, since both the isomeric forms of the antibodies formed complex with hapten Žthe equilibrium appears to be frozen. ŽFig. 5.. Therefore, our data concerning haptens fits into the model suggested by Lancet and Pecht w25x, with the possible exception for the transition between the two complexes. The antigen–antibody complexes were eluted as single peaks when the isomeric forms of the antibodies were directed against a large protein instead of a hapten ŽFigs. 2–4.. The shape of the antigen-binding sites has been shown to correlate with the type of antigen recognized w39,44,45x. Antibodies specific for haptens have concave antigen-binding sites while those binding proteins tend to have flattened combining sites w44–46x. We have recently shown that the antigen-binding regions of antibodies are concealed when binding a large protein antigen wU.-B. Hansson, C. Wingren, and U. Alkner, unpublished datax, i.e. antibodies with different K C values, but directed against the same protein antigen, formed complexes with the same value of K C . Thus, we cannot deter-
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mine whether only one or both of the conformational isomers could bind antigen when the antigen was a large protein. Therefore, we cannot anticipate whether any of the suggested kinetic models w25,29x is relevant for these antibody–protein systems. In agreement with Foote and Milstein w29x, we think that conformational isomerism may allow antibodies to cross-react and recognize dissimilar antigens. Such an effect may be of great importance if the antigen rapidly changes its surface properties or has a very flexible structure. In addition, conformational isomerism may be a way to modify the affinity of antibodies, i.e. may be an alternative way to obtain affinity maturation. In this connection it may be of interest to note that even the variable regions of antibodies have been demonstrated to influence their effector functions w47–49x. Furthermore, we have recently shown that antibodies with catalytic activity wU.-B. Hansson, C. Wingren, and D.S. Tawfik, unpublished datax as well as immunoglobulins of isotypes other than IgG w15x display the same elution patterns as the IgG antibodies analysed here. Thus, conformational isomerism of antibodies can be of great importance for the biological functionŽs. of the molecules. References ˚ Albertsson, Partition of Cell Particles and Macrow1x P.-A. molecules, John Wiley, New York, 1986. ˚ Albertsson, J. Chromatogr. 159 Ž1978. 111–122. w2x P.-A. w3x A.D. Diamond, J.T. Hsu, J. Chromatogr. 513 Ž1990. 137– 143. w4x G. Johansson, in: H. Walter, E.D. Brooks ŽEds.., Partitioning in Aqueous Two-Phase Systems, Academic Press, Orlando, FL, 1985, pp. 161-226. w5x H. Walter, G. Johansson, D.E. Brooks, Anal. Biochem. 197 Ž1991. 1–18. ˚ Albertsson, w6x U.-B. Hansson, K. Andersson, Y. Liu, P.-A. Anal. Biochem. 183 Ž1989. 305–311. w7x W. Muller, Eur. J. Chrom. 155 Ž1986. 213–222. ¨ w8x C. Wingren, B. Persson, U.-B. Hansson, J. Chromatogr. 668 Ž1994. 65–73. w9x W. Muller, Bioseparation 1 Ž1990. 265–282. ¨ w10x U.-B. Hansson, C. Wingren, in: M. Kastner ŽEd.., Protein Liquid Chromatography, Elsevier, Oxford, 1997. w11x C. Wingren, U.-B. Hansson, Biochim. Biophys. Acta 1244 Ž1995. 209–215. w12x K. Andersson, C. Wingren, U.-B. Hansson, Scand. J. Immun. 38 Ž1993. 95–101.
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w13x C. Wingren, U.-B. Hansson, J. Chromatogr. 680 Ž1996. 113–122. w14x C. Wingren, U.-B. Hansson, C.G.M. Magnusson, M. Ohlin, Mol. Immun. 32 Ž1995. 819–827. w15x C. Wingren, T.E. Michaelsen, C.G.M. Magnusson, U.-B. Hansson, Scand. J. Immun. 44 Ž1996. 430–436. w16x A. Tramantano, K.D. Janda, R.A. Lerner, Science 234 Ž1986. 1566–1569. w17x D.S. Tawfik, Towards Antibody-Mediated Peptide Hydrolysis. Thesis, Weizmann Institute of Science, Israel, 1995. w18x S.J. Pollack, J.W. Jacobs, P.G. Schultz, Science 234 Ž1986. 1570–1573. w19x V. Raso, B.D. Stollar, Biochemistry 14 Ž1975. 591–599. w20x F. Kohen, J.B. Kim, H.R. Lindner, Z. Eshar, B.S. Green, FEBS Lett. 111 Ž1980. 427–431. w21x S. Paul, D.J. Volle, C.M. Beach, D.R. Johnson, M.J. Powell, R.J. Massey, Science 244 Ž1989. 1158–1162. w22x A.M. Shuster, G.V. Gololobov, O.A. Kvashuk, A.E. Bogornolova, I.V. Smirnov, A.G. Gabibov, Science 256 Ž1992. 665–667. w23x L. Li, S. Paul, S. Tyutyulkova, M. Katzachkine, S. Kaveri, J. Immun. 154 Ž1995. 3328–3332. w24x D.M. Kranz, J.N. Herron, E.W. Voss, J. Biol. Chem. 257 Ž1982. 6987–6995. w25x D. Lancet, I. Pecht, Proc. Natl. Acad. Sci. USA 73 Ž1976. 3548–3553. w26x I. Pecht, in: M. Sela ŽEd.., The Antigens, Vol. IV, Academic Press, San Diego, CA, 1982, pp. 1–68. w27x S. Vuk-Pavlovic, Y. Blatt, C.P.J. Glaudemans, D. Lancet, I. Pecht, Biophys. J. 24 Ž1978. 161–174. w28x R. Zidovetski, Y. Blatt, C.P.J. Glaudemans, B.N. Manjula, I. Pecht, Biochemistry 19 Ž1980. 2790–2795. w29x J. Foote, C. Milstein, Proc. Natl. Acad. Sci. USA 91 Ž1994. 10370–10374. w30x M. Ohlin, V.-A. Sundqvist, M. Mach, B. Wahren, C.A.K. Borrebaeck, J. Virol. 67 Ž1993. 703–710. w31x U.K. Laemmli, Nature 227 Ž1970. 680–685. w32x J.J. Scheidegger, Int. Archs. Allergy Appl. Immun. 7 Ž1955. 103–110. w33x O. Ouchterlony, Prog. Allergy 6 Ž1962. 30–154. w34x C. Wingren, U.B. Hansson, K. Andersson, J. Chromatogr. 603 Ž1992. 73–81. w35x I.S. Mian, A.R. Bradwell, A.J. Olson, J. Mol. Biol. 217 Ž1991. 133–151. w36x T.P. Theriault, D.J. Leahy, M. Levitt, H.M. Mconnell, G.S. Rule, J. Mol. Biol. 221 Ž1991. 257–270. w37x T.P. Theriault, H.M. McConnell, A.K. Singhal, G.S. Rule, J. Phys. Chem. 97 Ž1993. 3034–3039. w38x C. Chothia, Curr. Opin. Struct. Biol. 1 Ž1991. 53–57. w39x D.R. Davies, E.A. Padlan, S. Sheriff, Ann. Rev. Biochem. 59 Ž1990. 439–473. w40x D.R. Davies, G.H. Cohen, Proc. Natl. Acad. Sci. USA 93 Ž1996. 7–12. w41x J. Foote, H.N. Eisen, Proc. Natl. Acad. Sci. USA 92 Ž1995. 1254–1256. w42x E.A. Padlan, Mol. Immun. 31 Ž1994. 169–217.
62
U.-B. Hansson et al.r Biochimica et Biophysica Acta 1340 (1997) 53–62
w43x M. Martinez-Yamout, H.M. McConnell, J. Mol. Biol. 244 Ž1994. 301–318. w44x D.M. Webster, A.H. Henry, A.R. Rees, Curr. Opin. Struct. Biol. 4 Ž1994. 123–129. w45x I.A. Wilson, R.L. Stanfield, Curr. Opin. Struct. Biol. 3 Ž1993. 113–118. w46x E. Vargas-Madrazo, F. Lara-Ochoa, J.C. Almagro, J. Mol. Biol. 254 Ž1995. 497–504.
w47x C. Horgan, K. Brown, S.H. Picus, J. Immun. 145 Ž1990. 2527–2532. w48x I. Yokoyama, F. Waxman, Immunology 80 Ž1993. 168–176. w49x K.D. White, M.B. Frank, S. Foundling, F.J. Waxman, Mol. Immun. 33 Ž1996. 759–768.