TY - JOUR
AU - Notkins, A L
AB - SUMMARY Human monoclonal antibody 63 (mAb63) is an IgM/λ polyreactive antibody that binds to multiple self and non-self antigens. The molecular basis of polyreactivity is still unclear. The present study was initiated to prepare a recombinant Fab of mAb63 and use it to study the determinants involved in polyreactivity. The baculovirus system was employed to express large amounts of mAb63 Fab in Sf9 cells. Our experiments showed that infected Sf9 cells secreted a soluble 50-kD Fab heterodimer that bound to multiple self and non-self antigens. The antigen-binding activity of mAb63 Fab was inhibited by both homologous and heterologous antigens. To study in more detail the molecular determinants involved in polyreactivity, the heavy chain complementarity-determining region 3 (HCDR3), which is known to play a key role in the binding of monoreactive antibodies to antigens, was subjected to site-directed mutagenesis. A single substitution, alanine for arginine, at position 100A resulted in complete loss of antigen-binding activity. The 19 amino acids comprising the HCDR3 of mAb63 were then synthesized and a cyclic peptide prepared. The cyclic peptide showed the same antigen-binding pattern as the parental mAb63 and the recombinant mAb63 Fab. A five amino acid motif (RFLEW), present in the HCDR3 of mAb63, was found by searching the GenBank in three of 50 other human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. It is concluded that HCDR3 plays a major role in polyreactivity and that in some cases cyclic peptides comprising the HCDR3, by themselves, may be polyreactive. polyreactive antibody, Fab fragment, baculovirus expression system site-directed mutagenesis INTRODUCTION Monoclonal polyreactive antibody molecules, in contrast to monoclonal monoreactive antibody molecules, are capable of binding a variety of self and non-self antigens [1–6]. Although polyreactive antibodies are predominantly of the IgM isotype, polyreactive IgG and IgA have also been reported. Polyreactive antibodies are generally of low affinity (dissociation constant, 10−5–10−7 M) and the VH and VL genes are often, but not always, of germ-line or near germ-line configuration [7–9]. In the circulation, these antibodies are usually masked because they are bound to self-antigens. Purification and dissociation of the circulating complexes unmasks the polyreactive properties of these antibodies [10]. Originally it was thought that polyreactive antibodies were made by CD5+ B cells, but recent studies have shown that polyreactive antibodies also can be made by CD5− B cells [11–14]. Cells capable of making these antibodies express the polyreactive immunoglobulin receptor on their surface and can bind, as evaluated by FACS analysis, a variety of different antigens. These polyreactive antigen-binding B (PAB) cells represent 10–20% of the cells in the normal B cell repertoire [14, 15]. In the newborn, PAB cells are the predominant cell B type [16]. The role of polyreactive antibodies and the cells that make them is still not clear. Originally it was thought that polyreactive antibodies might represent a first line of defence against foreign invaders, but the demonstration that polyreactive antibodies are present in low concentrations and masked as a result of binding to self antigens has weakened this argument [10]. Alternatively, it has been suggested that it is the PAB cells themselves, rather than the secreted polyreactive antibodies, that have the more important function. According to this argument, the B cell repertoire consists of two components [14–16]. The first is B cells that make the conventional high-affinity monoreactive antibodies that are secreted and involved in defence against foreign invaders. The second is PAB cells with low-affinity polyreactive receptors on their surface. These cells are present in high numbers and can bind and process self-antigens. It has been speculated, but still not proven, that presentation of processed self-antigens by PAB cells to T cells may contribute to the establishment and maintenance of peripheral immunological tolerance [14–16]. The precise molecular and structural determinants that allow a polyreactive antibody to bind a variety of different antigens also is still unresolved. The possibility that antigen binding may take place at a site other than the conventional antigen-binding site has been largely ruled out by showing that the Fc sequence of polyreactive antibody is identical to that of monoreactive antibody and that the deglycosylated polyreactive antibody molecule shows the same antigen-binding pattern as the non-deglycosylated parental antibody [17–19]. Sequence analysis of the VH and VL regions of polyreactive antibodies has failed, thus far, to show unique differences or identifiable motifs compared with monoreactive antibodies, except for the fact that many of the polyreactive antibodies are of germ-line or near germ-line configuration [7–9]. Within the VH of monoreactive antibodies, CDR3 is known to be a critical region in antigen binding and recent studies indicate that this region may be equally important in the binding of antigens to polyreactive antibodies [20–24]. To elucidate the molecular determinants by which a polyreactive antigen-binding site can accommodate a variety of different antigens, the baculovirus system was employed to express large amounts of recombinant polyreactive Fab. Site-directed mutagenesis was then used to alter individual amino acids in the heavy chain complementarity-determining region 3 (HCDR3) of the Fab and cyclic peptides comprising the HCDR3 sequence were synthesized and tested for polyreactivity. MATERIALS AND METHODS Cell culture Monolayer cultures of Sf9 cells from Spodoptera frugiperda were maintained at 27°C in Grace’s insect medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS), fugizone and gentamycin (Life Technologies, Rockville, MD) [25]. Suspension cultures of Sf9 cells were maintained at 27°C in Sf 900II serum-free medium (Life Technologies) supplemented with fugizone and gentamycin. The human mAb63 cell line that expresses an IgM/λ polyreactive antibody [6] was maintained in RPMI 1640 culture medium (Life Technologies) supplemented with 10% FBS, 2 mml-glutamine, 10 μmβ-mercaptoethanol and antibiotics. RNA isolation, cDNA synthesis and DNA amplification Total RNA was isolated from 108 mAb63 hybridoma cells as described [26]. mAb63 μFd and λ cDNA were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The primers for the heavy chain were 5′-GGCGAGATCTACAGGTGCAGCT ACAGCA-3′ and 5′-GGCGGCTAGCTTACTAGGGAGGCAGC TCAGCAATCA-3′; the primers for the λ-chain were 5′-GGCGA GATCTATCTGAGCTGACTCAGGA-3′ and 5′-GGCGGAAT TCTTACTATGAACATTCTGTAGGGGCCACCTG-3′. Amplification of mAb63 μFd and λ cDNA were performed in separate reactions containing 200 ng each of the 5′ and 3′ primers. Reverse transcriptase reaction mixture (10 μl) was incubated with the primers together with 1.0 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) in a buffer containing Tris–HCl pH 8.3, KCl MgCl and dNTP at 0.2 mm. The reaction mixtures were subjected to 35 cycles of amplification using 94°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min. The amplified PCR products were purified using the Wizard PCR purification system (Promega, Madison, WI). Construction of baculovirus transfer vector The purified μFd PCR product was digested with BlgII and NheI and the purified λ light chain PCR product was digested with BlgII and EcoRI. These digests were then individually cloned into a pVT-Bac transfer vector [27], kindly provided by Dr T. Vernet (National Research Council of Canada, Montreal, Quebec) to produce two separate recombinant transfer vectors, pBac-mAb63 μFd and pBac-mAb63 λ. To facilitate purification of recombinant mAb63 Fab secreted by Sf9 cells, an oligo-histidine tail (6xHis) was introduced at the 3′ end of μFd cDNA, using pBac-mAb63 μFd as template and 5′-GGCGAGATCTACAGGTGCAGCTACAGC A-3′ and 5′-GGCGGCTAGCTTACTAGTGATGGTGATGGTGA TGGGGAGGCAGCTCAGCAATCA-3′ as primers. The PCR product was digested with BlgII and NheI and cloned into pVT-Bac that had been cut with BamHI and NheI, to generate the final μFd transfer vector, pBac-mAb63 μFdhis, which is illustrated in Fig. 1, along with pBac-mAb63 λ. DNA sequence analysis was performed by the dideoxy-chain termination method [28]. Fig. 1 Open in new tabDownload slide Baculovirus transfer vectors used to express recombinant human polyreactive IgM mAb63 Fab. The pBac-mAb63 μFdhis vector was used to express the mAb63 μFd chain (a) and the pBac-mAb63 λ vector was used to express the mAb63 λ-chain (b). Heterodimers of these chains produced mAb63 Fab. MCS, Multiple cloning site; HBM signal, honeybee melittin secretion signal. Fig. 1 Open in new tabDownload slide Baculovirus transfer vectors used to express recombinant human polyreactive IgM mAb63 Fab. The pBac-mAb63 μFdhis vector was used to express the mAb63 μFd chain (a) and the pBac-mAb63 λ vector was used to express the mAb63 λ-chain (b). Heterodimers of these chains produced mAb63 Fab. MCS, Multiple cloning site; HBM signal, honeybee melittin secretion signal. Splice overlap extension PCR was used to construct mAb63 Fab mutants, one containing a Ser to Arg substitution at amino acid residue 97 in HCDR3 and another containing an Arg to Ala substitution at amino acid residue 100A. The numbering system was according to Kabat et al. [29]. These mutations were introduced using the primer 5′-GCGAGAGGGGGAAGGGTATTACGATTTTTGGAGTGGTTA-3′ for 97 S/R Fab and the primer 5′-GCGAGAGGGGATCGGTATTAGCATTTTTGGAGTGGTTA-3′ for 100A R/A Fab. The PCR products were cloned into pVT-Bac vector as described above. The complete DNA sequences of all PCR-generated products were verified by the dideoxy-chain termination method. Co-transfection and isolation of recombinant baculoviruses Co-transfection of Sf9 cells was carried out with 4 μg of either pBac-mAb63 μFdhis or pBac-mAb63 λ plasmid DNA and 1 μg of BaculoGold baculovirus DNA (Pharmingen, San Diego, CA), according to the manufacturer’s protocols. Plaques were purified, recombination was verified by PCR screening [30], and the recombinant baculoviruses were amplified and titred by plaque assay. Expression, analysis and purification of recombinant Fab63 Sf9 suspension cultures in Sf900II serum-free medium were infected with recombinant baculoviruses at a multiplicity of infection of 3. At 24-h intervals, the supernatant and cells were harvested, lysed and analysed by SDS–PAGE for expression of recombinant mAb63 Fab. Proteins were transferred to nitrocellulose membranes for Western blot. Blotting reactions were performed with an affinity-purified goat anti-human μ-chain (Zymed, San Francisco, CA) and/or goat anti-human λ-chain (Sigma, St Louis, MO) and detected with rabbit anti-goat IgG conjugated to horseradish peroxidase (HRP). To purify Fab, supernatants from Sf9 cells were passed through an Ni-NTA column (Qiagen, Valencia, CA), and the bound Fab was eluted with 100 mm imidazole in PBS. Protein concentration was determined by the BCA method (Pierce, Rockford, IL). Synthesis of cyclic HCDR3 peptide Two cyclic peptides, one with the amino acid sequence GGCRGGSVLRFLEWLLYPAFDYCG derived from HCDR3 of polyreactive mAb63, another with the amino acid sequence GGCRLGPDDYTLDYFDYCG derived from HCDR3 of monoreactive mAb61 which reacts specifically with IgG Fc [6, 7], were synthesized, cyclized and biotinylated (Biosythesis, Lewisville, TX). Functional analysis of recombinant mAb63 Fab Binding of recombinant mAb63 Fab to self and exogenous antigens was analysed by ELISA. Microtitre plates were coated with 100 μl/well of IgG Fc fragment, ssDNA, insulin (Ins), thyroglobulin (Tg) and tetanus toxoid (TT) at a concentration of 10 μg/ml in 0.05 m sodium carbonate buffer pH 9.5. Following overnight incubation at 4°C the wells were blocked with 150 μl/well of 1% gelatin (Sigma) in PBS at room temperature for 2 h. The recombinant mAb63 Fab at varying concentrations was added at 100 μl/well and the plates were incubated at room temperature for 2 h. After four washings with PBS containing 0.05% Tween 20 (Sigma), goat anti-human λ coupled to HRP (Southern Biotechnology, Birmingham, AL) (1:3000 dilution) was added at 100 μl/well and incubated at room temperature for 1 h. After washing six times with 0.05% Tween 20 PBS, 3,3′,5,5′-tetramethylbenzidine (TMB) (KLP, Gaithersburg, MD) was added at 100 μl/well and incubated at room temperature for 5 min, then 1:8 diluted H2SO2 at 50 μl/well was added to stop the reactions. The plates were read with a microtitre reader at 450 nm. Competitive inhibition assays of the binding of recombinant mAb63 Fab to solid-phase antigens by homologous or heterologous soluble antigens were carried out as described [4]. Briefly, 2 μg of recombinant mAb63 Fab, mAb63 (97 S/R) Fab or cyclic peptide 63 were incubated with increasing concentrations (1–200 μg) of Ins, IgG Fc, ssDNA, Tg, or TT or cyclic peptides 63 and 61. After 18 h, the mixtures were transferred to ELISA plates coated with solid-phase antigens. The amount of mAb63 Fab or cyclic peptides that bound to the solid-phase antigens in the presence or absence (100% binding) of the competing soluble antigens was determined. For the cyclic peptide studies, the avidin–biotin–peroxidase complex detection system (Vector Labs, Burlingame, CA) was used to measure the binding of biotinylated peptides. RESULTS Baculovirus-infected Sf9 cells express and secrete mAb63 Fab Sf9 cells were infected with recombinant baculovirus containing cDNA encoding either the heavy (Fd) or light (λ) chain of mAb63. As seen in Fig. 2a,b, recombinant Fd and λ were expressed in Sf9 cell lysates, but not secreted into the cell culture supernatants. Secretion into the cell culture supernatant requires the presence of heterodimers consisting of the heavy and light chains [31]. When Sf9 cells were co-infected with mAb63 Fd and λ recombinant baculovirus, both Fd and λ-chains were detected in cell culture supernatants: one at 31 kD, representing the recombinant Fd heavy chain (Fig. 2) and other at 29 kD, representing the recombinant λ light chain (Fig. 2). Maximum secretion of Fd and λ occurred between 72 h and 96 h post-infection. When the co-infected Sf9 cell supernatant was applied to a Ni-NTA column, a single immunoreactive band was detectable at 50 kD on a non-reducing gel (Fig. 2). Reduction of the 50-kD band by dithiothreitol (DTT) generated two distinct bands, one at 31 kD and the other at 29 kD. This argues that the co-infected Sf9 cells in fact secrete mAb63 Fab heterodimers. The yield of purified mAb63 Fab was approximately 0.4–0.6 mg per litre of cell culture supernatant. Fig. 2 Open in new tabDownload slide Expression and characterization of recombinant mAb63. Recombinant baculoviruses containing cDNA encoding mAb63 Fd and/or λ were used to infect Sf9 cells. Uninfected Sf9 cells and Sf9 cells infected with wild-type baculovirus (AcNPV) served as negative controls. Cell lysates and culture supernatants were subjected to 12% SDS–PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotting was carried out using goat anti-human μ- or λ-chain. (a,b) Cells were infected for 72 h with either mAb63 Fd or λ or (c,d) co-infected with mAb63 Fd and λ recombinant baculovirus for 24–96 h. (e) Secreted recombinant mAb63 Fab (2 μg) was subjected under reducing (R) and non-reducing (NR) conditions to 12% SDS–PAGE, transferred to nitrocellulose and immunoblotted with goat antibodies to human μ- and λ-chain. Fig. 2 Open in new tabDownload slide Expression and characterization of recombinant mAb63. Recombinant baculoviruses containing cDNA encoding mAb63 Fd and/or λ were used to infect Sf9 cells. Uninfected Sf9 cells and Sf9 cells infected with wild-type baculovirus (AcNPV) served as negative controls. Cell lysates and culture supernatants were subjected to 12% SDS–PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotting was carried out using goat anti-human μ- or λ-chain. (a,b) Cells were infected for 72 h with either mAb63 Fd or λ or (c,d) co-infected with mAb63 Fd and λ recombinant baculovirus for 24–96 h. (e) Secreted recombinant mAb63 Fab (2 μg) was subjected under reducing (R) and non-reducing (NR) conditions to 12% SDS–PAGE, transferred to nitrocellulose and immunoblotted with goat antibodies to human μ- and λ-chain. Recombinant mAb63 Fab maintains polyreactivity To see whether the recombinant Fab maintained the binding pattern of the parental polyreactive IgM, both molecules were tested for their antigen-binding capacity. As seen in Fig. 3a,b, the recombinant Fab and the parental IgM displayed the same general pattern of polyreactive antigen binding. Fig. 3 Open in new tabDownload slide Dose-dependent binding of (a) parental mAb63 (IgM), (b) recombinant mAb63 Fab, (c) recombinant mAb63 (97 S/R) Fab, (d) recombinant mAb63 (100A R/A) Fab, (e) cyclic peptide 63, and (f) cyclic peptide 6l to solid-phase ssDNA, insulin (Ins), IgG Fc, thyroglobulin (Tg), and tetanus toxoid (TT). The molecular weights of mAb63 (IgM), mAb63 Fabs, cyclic peptide 61, and cyclic peptide 63 are 900 000, 50 000, 2130 and 2680, respectively. Fig. 3 Open in new tabDownload slide Dose-dependent binding of (a) parental mAb63 (IgM), (b) recombinant mAb63 Fab, (c) recombinant mAb63 (97 S/R) Fab, (d) recombinant mAb63 (100A R/A) Fab, (e) cyclic peptide 63, and (f) cyclic peptide 6l to solid-phase ssDNA, insulin (Ins), IgG Fc, thyroglobulin (Tg), and tetanus toxoid (TT). The molecular weights of mAb63 (IgM), mAb63 Fabs, cyclic peptide 61, and cyclic peptide 63 are 900 000, 50 000, 2130 and 2680, respectively. Polyreactivity of recombinant mAb63 Fab is dependent on the structure of HCDR3 Site-directed mutagenesis was used to alter the structure of the HCDR3 segment of recombinant mAb63 Fab. Because of the known importance of positively charged arginine in the binding site of monoreactive antibodies [32, 33], in our experiments with polyreactive mAb63 Fab, arginine was substituted for serine at position 97 in one of the constructs (97 S/R) and alanine was substituted for arginine at position 100A in the other construct (100A R/A) (Fig. 4). Both Fab mutants then were expressed in Sf9 cells. The recombinant mAb63 Fab mutants were tested for binding activity with a panel of antigens. As seen in Fig. 3c, whereas substitution of arginine for serine at position 97 had little or no effect on binding activity (Fig. 3), substitution of alanine for arginine at position 100A resulted in complete loss of binding activity (Fig. 3). Competitive inhibition experiments revealed that soluble IgG Fc, Tg, ssDNA, TT and Ins could inhibit the binding of both mAb63 Fab and mAb63 (97 S/R) Fab to solid-phase insulin (Fig. 5). Fig. 4 Open in new tabDownload slide Site-directed mutagenesis of residues within heavy chain complementarity-determining region 3 (HCDR3). The substituted residues are shown within boxes. Fig. 4 Open in new tabDownload slide Site-directed mutagenesis of residues within heavy chain complementarity-determining region 3 (HCDR3). The substituted residues are shown within boxes. Fig. 5 Open in new tabDownload slide Dose-dependent inhibition of binding of recombinant mAb63 Fab (a) and recombinant mAb63 (97 S/R) Fab (b) to solid-phase insulin (Ins) in the presence of different concentrations of soluble antigen (i.e. Ins, IgG Fc, thyroglobulin (Tg), ssDNA, or tetanus toxoid (TT)). Dose-dependent inhibition of binding of mAb63 Fab to solid-phase antigens (i.e. Ins, IgG Fc, ssDNA, or TT) in the presence of different concentrations of soluble cyclic peptide 63 (c) and cyclic peptide 61 (d). Dose-dependent inhibition of binding of cyclic peptide 63 to solid-phase insulin in the presence of different concentrations of soluble antigens (i.e. Ins, IgG Fc, ssDNA or TT) (e). The molecular weights of ssDNA, Ins, IgG Fc, Tg, TT, cyclic peptide 61, and cyclic peptide 63 are 500 000, 6000, 25 000, 640 000, 110 000, 2130 and 2680, respectively. Fig. 5 Open in new tabDownload slide Dose-dependent inhibition of binding of recombinant mAb63 Fab (a) and recombinant mAb63 (97 S/R) Fab (b) to solid-phase insulin (Ins) in the presence of different concentrations of soluble antigen (i.e. Ins, IgG Fc, thyroglobulin (Tg), ssDNA, or tetanus toxoid (TT)). Dose-dependent inhibition of binding of mAb63 Fab to solid-phase antigens (i.e. Ins, IgG Fc, ssDNA, or TT) in the presence of different concentrations of soluble cyclic peptide 63 (c) and cyclic peptide 61 (d). Dose-dependent inhibition of binding of cyclic peptide 63 to solid-phase insulin in the presence of different concentrations of soluble antigens (i.e. Ins, IgG Fc, ssDNA or TT) (e). The molecular weights of ssDNA, Ins, IgG Fc, Tg, TT, cyclic peptide 61, and cyclic peptide 63 are 500 000, 6000, 25 000, 640 000, 110 000, 2130 and 2680, respectively. Analysis of a cyclic peptide derived from the HCDR3 of mAb63 To see whether HCDR3 itself was polyreactive, a cyclic peptide derived from HCDR3 of mAb63 and a control cyclic peptide derived from HCDR3 of mAb61 (a monoreactive antibody specific for IgG Fc) were synthesized, cyclized and biotinylated (Fig. 6). Cyclic peptide 63 contains the entire HCDR3 of mAb63, an N-proximal Arg, two Cys for cyclization and a Gly tail for biotinylation of the peptide. The binding of cyclic peptide 63 to a panel of solid-phase antigens is shown in Fig. 3 was polyreactive and displayed an antigen-binding pattern that was in general similar to mAb63 Fab. In contrast to cyclic peptide 63, cyclic peptide 61 did not bind to solid-phase antigens (Fig. 3). Figure 5c,d show that cyclic peptide 63, but not cyclic peptide 61, competitively inhibits, in a dose-dependent fashion, the binding of mAb63 Fab to a panel of solid-phase antigens. Conversely, the binding of cyclic peptide 63 to solid-phase human insulin is competitively inhibited in a dose-dependent fashion, by both soluble homologous (i.e. Ins) and heterologous antigens (i.e. ssDNA, IgG Fc, or TT) antigens (Fig. 5). Fig. 6 Open in new tabDownload slide Schematic drawing of cyclized and biotinylated peptides derived from the heavy chain complementarity-determining region 3 (HCDR3) of mAb63 and mAb61. Fig. 6 Open in new tabDownload slide Schematic drawing of cyclized and biotinylated peptides derived from the heavy chain complementarity-determining region 3 (HCDR3) of mAb63 and mAb61. DISCUSSION In the experiments reported here, we show that the cDNA encoding the Fd and λ-chains of a polyreactive antibody was expressed as a Fab in the baculovirus system, that large amounts of protein (0.4–0.6 mg/l) were produced and that the Fab retained the polyreactive antibody-binding pattern of the parental antibody molecule. These findings support and extend earlier studies in bacteria showing that the Fab fragment, but not the Fc or carbohydrate moiety of the antibody molecule, is required for polyreactivity [17–19]. Recent studies suggest that HCDR3 plays a pivotal role in polyreactivity [20–24]. Polyreactivity was lost when HCDR3 from a polyreactive antibody was replaced with HCDR3 from a monoreactive antibody, and conversely polyreactivity was gained when HCDR3 from a monoreactive antibody was replaced with HCRD3 from a polyreactive antibody [22–24]. The importance of HCDR3 in polyreactivity is further illustrated in the present study by site-directed mutagenesis. The fact that replacement of a positively charged arginine with a neutral alanine at position 100A resulted in loss of polyreactivity, but that replacement of serine with arginine at position 97 did not affect polyreactivity, argues that position 100A is directly or indirectly required for the conformation of the mAb63 Fab polyreactive antigen-binding site. Similarly, substitution of other amino acids within the HCDR3 region have been shown to suppress or enhance polyreactivity [34, 35]. Presumably, these changes may also alter the electrostatic, hydrophobic and van der Waals’ forces and/or alter flexibility of the antigen-binding site. Still another approach for demonstrating the importance of HCDR3 is through the use of conformationally constrained synthetic peptides consisting of the HCDR3 region. Cyclic HCDR3 synthetic peptides have been shown to mimic the binding characteristics of intact monoreactive antibodies [36–43]. In this context, Levi et al. showed that a cyclic peptide derived from the HCDR3 sequence of a MoAb specific for the V3 region of HIV-1, inhibited HIV-1 replication [44]. Similarly, Burton and colleagues synthesized a cyclic peptide based on the HCDR3 sequence of a polyreactive antibody and showed that it mimicked the binding pattern of the parental antibody [24]. Our studies extend these observations to a second polyreactive antibody by showing that a synthetic peptide consisting of the HCDR3 sequence of mAb63 retained the polyreactive antigen-binding pattern of the parental antibody. Precisely how short conformationally constrained synthetic peptides bind to antigens is not known. Short peptides are thought to have a disordered solution conformation and may not have a dominant-folding pattern. Thus, for binding to occur the peptides must assume the appropriate conformation. It has been suggested that cyclic peptides may overcome the entropic cost of binding, orientate the contact residues in appropriate conformations and more accurately mimic binding configurations than linear analogues [38]. Comparison of the peptide sequence of the HCDR3 of polyreactive mAb63 with other VH genes in the GenBank and Kabat databases [29, 45] revealed a five amino acid motif (RFLEW) (Table 1) that was present in three of 50 other known human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. This motif was not found in 3560 mouse antibodies or in 53 shark antibodies. The germ-line D segment, DXP4, which encodes the RFLEW motif, may very well be found in the genes of other antibodies, but because of N(D)N diversity different reading frames are used and the RFLEW motif is not usually translated. A search for still other motifs, which when incorporated into a cyclic configuration result in polyreactivity, may prove rewarding. Table 1 RFLEW motif in the heavy chain complementarity-determining region 3 (HCDR3) segment of four human polyreactive antibodies* ([24], [13], [13]) * The HCDR3 sequences of 2497 human immunoglobulin VH genes were analysed from the GenBank and Kabat database [29, 45].† Antigens with which polyreactive antibodies bound. LEG30: dsDNA, MHC class II, CD14, epidermal growth factor (EGF), and ganglioside GD2; mAb10 and mAb426.4.2F20: insulin, ssDNA, tetanus toxoid and tumour necrosis factor-alpha. Open in new tab Table 1 RFLEW motif in the heavy chain complementarity-determining region 3 (HCDR3) segment of four human polyreactive antibodies* ([24], [13], [13]) * The HCDR3 sequences of 2497 human immunoglobulin VH genes were analysed from the GenBank and Kabat database [29, 45].† Antigens with which polyreactive antibodies bound. LEG30: dsDNA, MHC class II, CD14, epidermal growth factor (EGF), and ganglioside GD2; mAb10 and mAb426.4.2F20: insulin, ssDNA, tetanus toxoid and tumour necrosis factor-alpha. Open in new tab Taken together, our studies and those from other laboratories show the importance of HCDR3 for polyreactivity. Precisely how the individual amino acid residues in HCDR3 make one antibody polyreactive and another monoreactive is still not clear. One possibility is that conformational flexibility at the antigen-binding site of a polyreactive antibody is greater than that of a monoreactive antibody [46, 47], thereby allowing a greater number of antigens to bind, albeit at low affinity. This possibility is supported by two recent studies. The first study, using a synthetic peptide combinatorial library, showed that a single antibody molecule could bind to a number of different peptides possessing little if any sequence relatedness [48]. The second study showed by crystallography that a single germ-line Fab fragment could adopt more than one configuration when complexed with a hapten [49]. The same situation may apply to polyreactive antibodies, many of which have germ-line or near germ-line sequences [7–9] and may be able to adopt multiple configurations. Antigen-driven somatic mutations may result in the stabilization of a single configuration, resulting in monoreactivity. Computer-assisted homology models of the combining site of three polyreactive Fv suggest other possibilities such as a diverse array of binding site structures [50]. Crystallographic studies involving the binding of different antigens to the same polyreactive Fab molecule should help resolve the precise mechanism of polyreactivity. Acknowledgements We thank Drs Paul Zhou, Michael Lan and George Chen for helpful discussion, and Will Blackburn and Ben Smith for excellent editorial assistance. 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TI - Molecular determinants of polyreactive antibody binding: HCDR3 and cyclic peptides
JF - Clinical & Experimental Immunology
DO - 10.1046/j.1365-2249.2000.01096.x
DA - 2002-04-05
UR - https://www.deepdyve.com/lp/oxford-university-press/molecular-determinants-of-polyreactive-antibody-binding-hcdr3-and-XxyGPo2E7y
SP - 69
EP - 76
VL - 119
IS - 1
DP - DeepDyve
ER -