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The Journal of Immunology, 1999, 163: 4604-4611.
Copyright © 1999 by The American Association of Immunologists

Isolation, Characterization and Sequence Analysis of Five IgG Monoclonal Anti-ß2-Glycoprotein-1 and Anti-Prothrombin Antigen-Binding Fragments Generated by Phage Display1

Reginald U. Chukwuocha2,*, Elie T. Hsiao{dagger}, Peter Shaw{ddagger}, Joseph L. Witztum{ddagger} and Pojen P. Chen*

* Department of Medicine, Division of Rheumatology, University of California, Los Angeles, CA 90095; {dagger} Department of Biology, Johns Hopkins University, Baltimore, MD 21205; and {ddagger} Department of Medicine, University of California at San Diego, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have isolated five monoclonal IgG anti-ß2-glycoprotein-1 (anti-ß2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three ß2GP-1-enriched mAbs (B14, B22, B27) reacted with ß2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with ß2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 x 10-6 M for ß2GP-1 vs 3.2 x 10-6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between ß2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to ß2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to ß2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-ß2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antiphospholipid Abs (aPL),3 which include anticardiolipin Abs (aCL) detected by ELISA and lupus anticoagulant (LAC) Abs detected by in vitro blood clotting assays, are associated with thrombosis, recurrent fetal loss, and thrombocytopenia in patients with antiphospholipid syndrome (APS) (1, 2, 3). It is generally considered that the binding targets of aPLs include negatively charged phospholipids (PLs), various plasma proteins or complexes formed by PLs, and plasma proteins (4, 5, 6, 7). These plasma proteins include ß2-glycoprotein-1 (ß2GP-1), prothrombin, annexin V, protein C, and its cofactor protein S (5, 8, 9). In 1990, two studies showed that the binding of aPL to cardiolipin (CL) was enhanced by ß2GP-1, suggesting that aPLs recognized a complex of CL and ß2GP-1 (7, 10). Other studies have reported that aPL reacted with ß2GP-1 alone (5, 11, 12). Over the last few years, the consensus is that anti-ß2GP-1 Abs make up a significant percentage of aPL found in APS patients (5, 7, 12, 13, 14). In addition, recent studies show that anti-ß2GP-1 Abs are more closely associated with APS thrombosis (5, 12, 15).

In contrast, increasing attention is being paid to anti-prothrombin Abs and the role they may play in thrombosis in APS patients. The prevalence of these Abs in patients varies greatly, ranging from 20 to 60% when detected via ELISA using immobilized human prothrombin on activated poly(vinyl chloride) plates (8, 16). Importantly, it was reported that affinity-purified IgG anti-prothrombin Abs bound to immobilized phosphatidylserine (PS) in the presence of Ca2+ and prothrombin, suggesting that IgG anti-prothrombin Abs bound to prothrombin and then is transported onto PS as a "passenger" upon prothrombin binding to PS (17, 18). In this context, it is conceivable that anti-prothrombin IgG may cross-link prothrombin molecules and thus increase the valency of interactions between prothrombin and PS. This implies that the anti-prothrombin IgG may enhance the binding of prothrombin to PS and to PL surface on endothelial cells and thus increase thrombin generation and promote thrombosis.

The oxidative modification of low-density lipoproteins (Ox-LDL) is thought to play an important role in various disease states including atherosclerosis (19, 20). Studies have shown that PLs are structurally similar to LDL and circulating lipoproteins contain various amounts of PLs and ß2GP-1 (21). In addition, aPL may be directed against epitopes of oxidized PLs and cross-react with Ox-LDL (22, 23). Together, these data suggest that there is an overlap between aPL and anti-Ox-LDL Abs.

Although significant progress has been made in understanding the binding specificities of aPL, little is known about the structures and genetic basis of these potentially pathogenic autoantibodies. For reasons that are connected with the low efficiency of generating IgG Abs by conventional methods, few IgG aPL have been generated and studied. As a result, structure analysis of the potentially pathogenic Abs in APS has been difficult. Here we describe the isolation of five IgG monoclonal anti-ß2GP-1 and anti-prothrombin Abs by phage display method from a patient with anti-Ox-LDL Abs. We report a detailed characterization of their binding specificities and sequence analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient

The patient is a 72-year-old male who underwent coronary artery bypass surgery despite having low total plasma cholesterol levels for many years. His plasma was screened for the presence of autoantibodies to epitopes of Ox-LDL as part of a study being conducted in patients seen in the Lipid Research Clinic at the University of California at San Diego. Because anti-Ox-LDL Abs overlap substantially with aPL, we analyzed his sera for aCL and reactivities with ß2GP-1 and prothrombin. The results showed that he had significant titers of anti-ß2GP-1 Abs at one in one hundred (1:100, in borate-buffered saline (BBS), 0.2 M boric acid, 0.15 M NaCl, pH 8.2, containing 0.25% gelatin) and anti-prothrombin Abs (1:50, in calcium buffer, 50 mM Tris-HCl, 150 mM NaCL, 50 mM CaCl2, pH 7.5, containing 0.25% gelatin) (data not shown). Accordingly, his monocytes were isolated and used to prepare a combinatorial library.

The control library was prepared from a normal individual whose plasma did not contain anti-DNA, anti-ß2GP-1, and anti-prothrombin Abs (data not shown).

Construction of combinatorial libraries

An IgG1 {kappa} and {lambda} libraries were constructed according to published protocols (24, 25). Briefly, lymphocytes from the patient and control subjects were isolated and used as the source of total RNA for the phage library construction. PCR was then used to amplify and clone Fab genes from isolated cells into the phage display vector, pComb3H. Phage Fab clones are then selected based on Ab-binding specificity on ß2GP-1 or prothrombin-coated plates. We obtained a library of 108 members with an insert frequency of >80% as determined by restriction endonuclease analysis.

Selection of aPL Fab clones and initial characterization of the isolated clones

The selection of aPL clones was performed as previously described (24). Briefly, microtiter plates (3690; Costar, Cambridge, MA) were coated overnight at 4°C with either ß2GP-1 at 10 µg/ml in BBS or prothrombin (Enzyme Research Laboratories, South Bend, IN) at 10 µg/ml in calcium buffer (50 mM Tris, 150 mM NaCl, and 50 mM calcium chloride, pH 7.5). After washing, ß2GP-1-coated plates were blocked with 3% of BSA in BBS while prothrombin-coated plates were blocked with 0.25% gelatin for 1 h at room temperature. Then, freshly prepared phage particles (1012 phage particles) were added and incubated for 2 h at room temperature. Thereafter, wells were washed extensively with TBS containing 0.5% Tween 20 (TBST) and bound phage particles were eluted with 50 µl of 0.1 M HCl/glycine (pH 2.2)/0.1% BSA. Following the third round of panning, phagemid DNA was recovered and used to generate soluble Fab as previously described (24).

Then, each isolated clone was lysed and analyzed for soluble Fab by ELISA. Wells were coated with goat anti-human IgG Fab mAb (Cappel Research Products, Durham, NC) overnight at 4°C and blocked with 0.25% BSA. Bacterial lysates containing soluble Fab were added to wells in duplicates, and the bound Fab were detected with enzyme-labeled goat anti-human IgG. Fab from each positive clone were affinity purified with the goat anti-human Fab column and analyzed for their binding property.

Ab-binding ELISA

The binding specificity of Fab clones were determined as previously described for anti-ß2GP-1 and anti-prothrombin Abs (24). For anti-ß2GP-1, microtiter plates were precoated with 10 µg/ml ß2GP-1 in BBS overnight at 4°C. After blocking with 0.25% BSA, serial dilutions of purified Fab were distributed to wells in duplicates, and plates were incubated for 2 h at room temperature. The plates were then washed four times with BBS and incubated for 1 h at room temperature with affinity-purified enzyme-labeled goat anti-human IgG.

ELISA for the detection of anti-prothrombin activity was similar to the anti-ß2GP-1 ELISA assay with some modification. Briefly, wells were coated with 10 µg/ml prothrombin (Enzyme Research Laboratories) in calcium buffer and blocked with 0.25% gelatin in calcium buffer.

The purified Fab were also used to study the binding specificities of the mAbs to a panel of unrelated Ags, including: chicken OVA, collagen, and ssDNA. All Ags were used at 10 µg/ml, and ELISA was performed as described for ß2GP-1 and prothrombin.

SDS-PAGE analysis of Fab

Purified mAb Fab and control proteins (150 ng except for mAb P13, which was 100 ng) were loaded into 7% Tris-acetate gel for nonreducing SDS-PAGE analysis (NOVEX, San Diego, CA). After electrophoresis, the gel was analyzed by silver staining for the detection of Fab protein bands according to the manufacturer’s instructions (Pierce, Rockford, IL).

Determination of Fab-binding affinity

The binding affinity of each mAb was determined by Ag inhibition, with each mAb used at a concentration that gave 50% maximal binding. Diluted mAbs (mAbs P11, 20 µg/ml; B14, 10 µg/ml; B22, 20 µg/ml; B27, 10 µg/ml; P13, 30 µg/ml) were then preincubated for 2 h at room temperature with an equal volume of buffer or increasing concentration of ß2GP-1 or prothrombin (100, 200, 400, 600, and 800 µg/ml). The amount of free mAb in the Ab inhibitor mixtures were then measured in an anti-ß2GP-1 or anti-prothrombin ELISA using Ag-precoated plates. The average mAb affinity was calculated according to previously described method (26).

Sequencing of Fab V region genes

Sequencing was performed on purified dsDNA by using previously published sequencing primers (25). Sequence data were compiled and analyzed using the Basic Local Alignment Search Tool (BLAST) (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Panning of the aPL phage clones

After panning against ß2GP-1, three clones were isolated from the {kappa} library while none was isolated from the {lambda} library. For prothrombin, two clones with anti-prothrombin activity were recovered from the {kappa} library alone. Restriction analysis of their DNA revealed that all contained both the H and L chain inserts.

Characterization of the anti-ß2GP-1 and anti-prothrombin Fab

To study the binding property of these clones, affinity-purified Fab were analyzed against ß2GP-1, prothrombin, and other unrelated Ag. As shown in Fig. 1Go, all three ß2GP-1-enriched clones (termed mAbs B14, B22, B27) reacted with ß2GP-1. Of these, two (mAbs B14 and B22) reacted with CL when complexed with ß2GP-1 (data not shown). All three clones did not react with four unrelated Ags, including collagen, OVA, prothrombin, and ssDNA (data not shown). In Fig. 2Go, both prothrombin-selected clones (termed mAb P13 and P11) reacted with prothrombin. When tested against four unrelated Ags (ß2GP-1, collagen, OVA, and ssDNA), P11 reacted strongly with ß2GP-1 (Fig. 1Go), weakly with OVA and ssDNA, but not at all with collagen (data not shown). Fig. 3Go shows that each of the mAb Fab displayed the expected 47-kDa Fab band on silver staining of SDS-PAGE gel.



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  		FIGURE 1. Binding specificity of affinity-purified monoclonal Fab mAbs B14, B22, B27, and P11. mAbs were analyzed against ß2GP-1. Bars represent mean net OD ± SD readings of duplicate samples.

 


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  		FIGURE 2. Binding specificity of affinity-purified monoclonal Fab P11 and P13. mAbs were analyzed against prothrombin. Bars represent mean net OD ± SD readings of duplicate samples.

 


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  		FIGURE 3. SDS-PAGE of mAbs, in silver staining. Lane 1, m.w. marker; lane 2–7, nonreduced mAbs B14, B22, B27, P11, P13, and a control Fab clone isolated from a normal human lymphocyte library; lane 8–9, nonreduced human Fab and IgG1 standard used as positive controls. Arrow indicates the position of the 47-kDa Fab band.

 
Competitive inhibition ELISA

Binding specificity was confirmed by demonstration that soluble ß2GP-1 and/or prothrombin inhibited the mAb interactions with immobilized ß2GP-1/prothrombin as previously described (26). As shown in Figs. 4Go and 5, four Fab clones were specific for ß2GP-1 while two clones were reactive with prothrombin. The reactivity of mAb P11 to ß2GP-1 and prothrombin was inhibited by soluble Ags, suggesting that mAb P11 may recognize an epitope shared by prothrombin and ß2GP-1. The inhibitions range from 23 to 61% and are consistent with the low affinity of anti-ß2GP-1 Abs, which remains free in the presence of 200 µg/ml of ß2GP-1 in the plasma (28). The binding affinities (Kd) were estimated from the inhibition ELISAs and are shown in Table IGo.



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  		FIGURE 4. Competitive inhibition of binding of mAbs B14, B22, and B27 to ß2GP-1 and representative mean OD readings of duplicate samples.

 

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  		Table I. Ig gene usage of five aPL monoclonal Fab from a patient with Ox-LDL

 
Sequence analysis of the aPL Fab clones

Sequence analysis of the H and L chain V regions of all Fab clones (Figs. 6Go and 7) revealed that B22, B27, and P13 shared an almost identical rearranged VH6-D-J gene segment termed Humha622. It consists of VH6, D5, and D21/10 in the reverse orientation, and JH4 (Fig. 6GoB). The putative D5-encoded segment may derive from DN4. The VH sequences for B22 and B27 are identical and are represented by ha622 (Fig. 6GoB); they differed from that of P13, denoted as ha613 in Fig. 6GoB, by one silent change in the framework (FR) 1. However, these three clones used different L chains termed Humka122, Humka127, and Humka113, respectively (denoted as ka122 [B], ka127 [C], and ka113 [E] in Fig. 7Go); each employed different members of the Vk1 family.



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  		FIGURE 6. The nucleotide and deduced amino acid sequences of Ab H chain V regions B14 (A), B22 and P13 (B), and P11 (C), which are designated Humha314, Humha622, Humha613, and Humha411 and are abbreviated ha314, ha622, ha613, and ha411, respectively. The H chain of B27 is identical with that of B22 and thus is represented by ha622; ha613 differs from ha622 by a single silent base and is given for this region only. The putative corresponding germline gene sequences are included for comparison (29 30 31 ). In each panel, the complete nucleotide and amino acid sequences of a H chain are given, while the corresponding germline sequences (and other related H chain sequence) are given only at the positions where they differ from VH cDNA in the overlapping regions. Dashes denote the identities, the PCR primers are underlined. The CDRs are indicated, and D region and JH genes of all clones are included.

 


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  		FIGURE 7. The nucleotide and deduced amino acid sequences of Ab L chain V regions: (A) B14, (B) B22, (C) B27, (D) P11 and (E) P13 which are designated Humka114, Humka122, Humka127, Humka111 and Humka113 and are abbreviated ka114, ka122, ka127, ka111 and ka113. The putative corresponding germline L chain V gene sequences are included for comparison (32 33 34 35 ). In each panel, the complete nucleotide and amino acid sequences of a L chain are given, while the germline sequence is given only at the positions where it differs from the L chain in the overlapping regions. Bars denote the identities, the PCR primers are underlined, and the CDRs are indicated.

 
The mAb B14 employed the VH-26 VH3 gene termed Humha314 and the A20 Vk1 gene termed Humka114 (Figs. 6GoA and 7A). Of note, the invariant tryptophan residue that represents the beginning of the fourth FR in ha314 is absent (Fig. 6AGo). To the best of our knowledge, this is the first VH sequence of a functional Ab that has no invariant tryptophan. Finally, clone P11 used the VH4.33 gene, termed Humha411, and the A30 Vk1 gene, termed Humka111 (Figs. 6GoC and 7D).

It was difficult to discern the closest germline D gene used in all five clones because of extensive modification, but there appears to be certain germline genes that could have been employed by these clones as shown in Fig. 6Go and Table IGo. As for the JH gene usage, the mAbs P11 and B14 employed JH1 while others used JH4 gene segments (Fig. 6Go).

There was no restriction in Jk usage in the clones analyzed (Table IGo). ka113 used Jk1, ka114 used Jk2, ka122 and ka111 employed Jk3, and ka127 used Jk4. The Jk2 employed by ka114 and the Jk3 in ka111 each contained two mutations (Fig. 7Go, A and D).

A comparison of the V gene-encoded regions in all five clones with both GenBank and EMBL databases as well as all published sequences revealed that the expressed VH showed a range of 92 to 94% homology with their nearest germline genes for an average of 93.4% (Table IGo) (29, 30, 31, 32, 33, 34, 35). When compared with these putative germline counterparts, the replacement to silent changes (R/S) in the complementarity-determining regions (CDRs) was 5.0 for mAbs B14, B22, B27, and P13 and <1.0 for mAb P11. In contrast, the R/S ratios in the FRs was 4.0 for mAb B14 and <1.4 for mAbs B22, B27, P11 and P13.

Sequence analysis of the L chains of all five clones revealed significant mutations that range from 6 to 20 nt per L chain V region, which results in a mutation frequencies of 2.1–7.0%. In Fig. 7Go, the germline nucleotide sequences are included for comparison. When compared with their germline counterparts, the R/S ratios in the CDRs was 2.0 for mAb B22, 4.0 for mAbs B14 and B27. This ratio was 8.0 and 6.0, respectively, for mAbs P11 and P13. In contrast, the R/S ratios in the FRs was 3.0 for mAb P11 and <0.5 for mAbs B14, B22, B27, and P13.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In an attempt to define the structural basis of anti-ß2GP-1 and anti-prothrombin activities obtained from a coronary artery bypass patient, we employed the phage display method to isolate and analyze the structural features of five monoclonal IgG aPL Fab. The results show that all three ß2GP-1-enriched clones, mAbs B14, B22, and B27, specifically recognized the plasma cofactor, ß2GP-1 (Fig. 1Go). The affinities of these three clones range from 7 x 10-5 to 1.5 x 10-6 M and are comparable to the reported Kd values of serum anti-ß2GP-1 Abs ranging from 10-5 to 3.4 10-6 M (14, 36). The mAb B27 had the highest affinity of the three ß2GP-1 binding clones and was the most specific Ab.

Both prothrombin-enriched clones (mAbs P11 and P13) reacted with prothrombin (Fig. 2Go). Intriguingly, mAb P11 also reacted strongly with ß2GP-1, suggesting that P11 may recognize an epitope shared by prothrombin and ß2GP-1. It is possible that similar dual-reactive autoantibodies may be present in APS patients. In the future, it would be important to study the presence of mAb P11-like aPL in APS patients and the role of such aPL in APS pathogenesis. To this end, it will be required to first define the epitope recognized by mAb P11. The conversion of clone P11 into intact IgG secretor would allow the screening of ß2GP-1 and prothrombin peptide libraries to identify the shared epitope. Subsequently, the peptide representing this shared epitope can then be used to screen patients serum samples for the putative P11-like aPL.

The mAbs B22, B27, and P13 shared identical VH6-D-J H chain but different L chains. The first two mAbs recognized ß2GP-1, while P13 bound to prothrombin, suggesting that while VH6-D-J gene may have an intrinsic binding affinity for ß2GP-1, certain L chain pairings apparently influence that binding. To decipher the role of L chains in binding to ß2GP-1, we compared their amino acid sequences. Fig. 8Go shows that the two ß2GP-1-reactive L chains, ka122 and ka127, are not more similar to each other than to the prothrombin-reactive ka13.



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  		FIGURE 8. The deduced amino acid sequence of clone P13, B22, and B27 L chain cDNA V regions, designated Humka113, abbreviated ka113; Humka122, abbreviated ka122; and Humka127, abbreviated ka127, respectively. The complete amino acid sequence of ka113 are given, while all others are given only at the positions where they differ from the sequence of ka113. Dashes denote identities, while the CDRs are indicated. The underlined amino acid sequence is coded by the PCR primer.

 
The H chains of mAbs B22, B27, P13, and B14 derive from VH6/V6–1 and VH26/V3–23 genes segments, respectively. These VH gene segments belong to a set of VH genes that have been shown to be preferentially expressed in ontogeny (37, 38). Previously, analysis of several natural autoantibodies derived from normal individuals revealed usage of the same restricted set of V genes. In this context, the natural IgM autoantibody Kim 13.1, which is encoded by the 51P1 gene in germline configuration, displays anti-CL and rheumatoid factor activity (39). Taken together, these data suggest that some aPL autoantibodies may arise from natural autoantibodies.

The mAb P11 used the A30 Vk1 L chain, which is rarely employed in the functional Ig Vk gene repertoire. In contrast, it was reported recently that in systemic lupus erythematosus (SLE) patients A30 is rearranged to the Jk2 gene and encodes the nephritogenic anti-DNA Ab L chain (35, 40). In addition, several of the presently characterized aPL-associated V genes have been shown to encode diagnostic anti-DNA autoantibodies in SLE (Table IIGo) (40, 41, 42, 43, 44). In particular, VH26 and VH6 encode, respectively, the H chains of the 18/2 and the A10 anti-DNA Ab; A20, A30, and L5 encode the L chain of the III-2R, SC17, and NE-3 anti-DNA autoantibodies, respectively. The meaning of these findings are not clear. However, the extensive overlap of the V gene usage in the aPL with that of characteristic anti-DNA autoantibodies in SLE suggests that some aPL in SLE patients may arise as the byproducts of receptor editing of autoreactive B cells, in which one of the original H and L chain V regions is replaced with a newly rearranged V gene (45, 46). In support of this, sequence analysis of three monoclonal LAC Abs revealed extensive overlap in the Ig V genes with anti-DNA Abs found in lupus patients (47).


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  		Table II. aPL share Ig V genes with other anti-DNA autoantibodies in SLE

 
In conclusion, we isolated five mAbs aPL representing two of the major groups of autoantibodies found in APS patients. The structural features of these Abs revealed that certain H and L chain combinations may be important in the development of aPL reactivity.



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  		FIGURE 5. Competitive inhibition of binding of mAbs P11 and P13 to prothrombin and to ß2GP-1 (mAb P11) and representative mean OD readings of duplicate samples.

 

   Footnotes
 
1 This work is supported by National Institutes of Health Grant 7K14HL03523-03. Back

2 Address correspondence and reprint requests to Dr. Reginald U. Chukwuocha, Department of Medicine, Division of Rheumatology, University of California School of Medicine, 32-59 Rehabilitation Center, Los Angeles, CA 90095-1670. E-mail address: Back

3 Abbreviations used in this paper: aPL, antiphospholipid Abs; aCL, anti-cardiolipin Abs; APS, antiphospholipid syndrome; LAC, lupus anticoagulant;; SLE, systemic lupus erythematosus; ß2GP-1, ß2-glycoprotein-1; CL, cardiolipin; LDL, low density lipoprotein; Ox-LDL, oxidized low density lipoprotein; PL, phospholipid; PS, phosphatidylserine; BBS, borate-buffered saline; FR, framework region; R/S, replacement to silent; CDR, complementarity-determining region. Back

Received for publication January 5, 1999. Accepted for publication August 2, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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