Characteristics and pathogenic role of anti-ß2-glycoprotein I single-chain Fv domains: induction of experimental antiphospholipid syndrome

Miri Blank1, Ari Waisman2, Edna Mozes2, Takao Koike3 and Yehuda Shoenfeld1

1 Research Unit of Autoimmune Diseases, Department of Medicine `B', Sheba Medical Center, Tel-Hashomer 52621, Israel, affiliated to Sackler Faculty of Medicine, Tel-Aviv University.
2 Department of Immunology, The Weizmann Institute of Sciences, Rehovot 76100, Israel
3 Department of Medicine II, Hokkaido University School of Medicine, Sapporo 060, Japan

Correspondence to: Y. Shoenfeld, Department of Medicine `B', Sheba Medical Center, Tel-Hashomer 52621, Israel


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antiphospholipid syndrome is characterized by the presence of high titers of anti-ß2-glycoprotein I (ß2GPI) antibodies, lupus anticoagulant associated with thromboembolic phenomena, thrombocytopenia and recurrent fetal loss. Single-chain Fv (scFv) were prepared from four anti-ß2GPI mAb, CAM, CAL, CAR and 2C4C2, and one anti-ssDNA. All five scFv showed the same antigen binding properties as the original mAb. Replacement of the pathogenic CAM VH domain with the non-pathogenic CAL VH or anti-ssDNA VH decreased the binding affinity of the scFv to ß2GPI and completely abrogated the anticoagulant activity. Exchanging the CAM VH with anti-DNA VH resulted in a shift from anti-ß2GPI to anti-ssDNA binding of the scFv. Replacement of the CAM VL with CAL VL did not affect the binding and activity. BALB/c mice were immunized with the anti-ß2GPI scFv, and the scFv resulting from the substitution of the heavy (H) and light (L) chains. The mice which were immunized with CAM, 2C4C2 and CAR scFv developed clinical manifestations of experimental anti-phospholipid syndrome. Elevated titers of mouse anti-cardiolipin (aCL), anti-ß2GPI, associated with lupus anticoagulant activity, thrombocytopenia, prolonged activated partial thromboplastin time and a high percentage of fetal resorptions were detected, in the CAM scFv group and in the scFv composed of CAM VH groups. High titers of aCL, anti-ß2GPI, anti-ss/dsDNA and anti-histone associated with lupus findings were observed in the sera of the 2C4C2 scFv-immunized mice. Immunization with CAL scFv did not lead to any clinical findings. The current study shows that scFv of pathogenic antibodies are capable of inducing the same clinical manifestations as the whole antibody molecule upon active immunization. Replacement of H/L chains point to the importance of the VH domains in the pathogenic potential of anti-ß2GPI.

Keywords: anti-ß2-glycoprotein I, anti-cardiolipin antibodies, anti-phospholipid syndrome, single-chain Fv, autoimmunity


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The `Hughes Syndrome' [anti-phospholipid syndrome (APS)] is characterized by the presence of anti-cardiolipin antibodies (aCL), ß2-glycoprotein (ß2GPI)-dependent antibodies and/or lupus anticoagulant associated with thromboembolic phenomena, thrombocytopenia and recurrent fetal loss (13).

It is widely accepted that aCL antibodies, purified from patients with primary or secondary APS, bind anionic phospholipid through ß2GPI (25). ß2GPI (50 kDa), a member of the complement control protein, is the target antigen for many of the autoimmune anti-ß2GPI antibodies, composed of five respective consensus repeats (2,6). ß2GPI exhibits several properties in vitro which define it as an anticoagulant (e.g. inhibition of prothrombinase activity, ADP-induced platelet aggregation, platelet factor IX production) (78). It binds negatively charged phospholipids through a lysine-rich locus (Cys281–Cys288) located in the fifth domain (9).

It has been postulated that anti-ß2GPI antibodies exert a direct pathogenic effect by interfering with homeostatic reactions occurring on the surface of platelets or vascular endothelial cells (1012). Passive transfer of these antibodies into naive mice results in induction of experimental APS (13,14).

We developed aCL/ß2GPI-dependent mAb from the mice with experimental systemic lupus erythematosus (SLE) (designated CAL, 2C4C2) (1517) from experimental SLE with secondary APS (designated CAM) (15) and from mice immunized with human anti-cardiolipin ß2GPI-dependent mAb which developed primary APS (designated CAR) (18). CAM and CAR anti-ß2GPI mAb were found to be pathogenic and to induce APS when infused or injected into naive mice (15,18). Immunization of naive mice with 2C4C2 (aCL/anti-DNA) mAb resulted in experimental SLE with secondary APS (16,17), while CAL failed to induce a disease (15). The mice which received CAM, 2C4C2 and CAR anti-ß2GPI mAb developed persistent titers of anti-phospholipid antibodies in the sera, prolonged activated partial thromboplastin time (aPTT), thrombocytopenia and a high percentage of fetal resorptions (equivalent to human recurrent fetal loss seen in APS) (1518).

The analysis of the pathogenic potential of an autoantibody should entail in addition to its binding and functional properties also its V gene usage. Various reports indicated the importance of the Ig H and L chains contribution to the specificity of the autoimmune response (1922). Furthermore, V gene analysis of aCL antibodies from MRL-lpr/lpr and NZWxBXSB F1 (W/B F1) mice of human SLE and of primary APS patients suggested the possibility that the usage of VH/V{kappa} genes in aCL is not random and that aCL consists of somatically mutated Ig genes (2327).

In the current study, we delineated the contribution of the binding properties and V gene usage of anti-ß2GPI mAb (CAM, CAR, 2C4C2 and CAL) from different sources to their pathogenic potential in vivo. The contribution of specific heavy (H) or light (L) chains to the pathogenic potential of anti-ß2GPI can be analyzed by employing expression systems for cloning of Ig genes (2831). In order to study the contribution of the H or L chain of the Ig molecules to the binding and pathogenic properties of the anti-ß2GPI, we converted the anti-ß2GPI mAb into single-chain (sc) Fv, replaced H and L chains between the pathogenic and non-pathogenic anti-ß2GPI scFv, and studied their contribution in vitro and in vivo.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
BALB/c mice were obtained from the animal facility of the Tel-Aviv University, Israel. Mice were used at the age of 10–12 weeks.

mAb
CAM is a mouse aCL/ß2GPI-dependent mAb (IgG) that was derived from a mouse with experimental SLE associated with secondary APS (15). This mAb has the potential to induce primary APS by passive administration (15) or by active immunization of naive mice (15).

CAL is a mouse aCL/ß2GPI-dependent mAb (IgG) that was derived from a mouse with experimental SLE (15). CAL mAb did not induce experimental APS (15).

N40 is a mouse IgG mAb that was derived from the same hybridoma as CAM, but does not bind cardiolipin. It was employed as a negative control (15).

CAR is a mouse aCL/ß2GPI-dependent mAb (IgM) that was derived from a mouse with experimental primary APS and is able to induce experimental APS in naive mice (18).

C4C2 is a mouse anti-cardiolipin ß2GPI-dependent/ssDNA (IgM) with lupus anti-coagulant activity. Immunization of naive mice with the mAb resulted in induction of experimental APS (16,17). V gene analysis of 2C4C2 showed usage of the J558 VH family (32).

16/6 is a human anti-ssDNA (IgG) mAb. Immunization of naive mice with this antibody led to induction of experimental SLE (33). V gene analysis of 16/6 showed usage of the J558 VH family (34).

aCL/ß2GPI-dependent ELISA
Detection of aCL/ß2GPI-dependent binding was performed by ELISA. ELISA plates were coated with 50 µg/ml of cardiolipin overnight at 4°C. Evaporated plates were blocked with 3% BSA for 1 h at 37°C. ß2GPI was added to the plates at different concentrations (0–10 µg/ml) for 2 h at room temperature. CAM, CAR and CAL mAb and scFv were added at different concentrations (0–10 µg/ml) for 2 h at room temperature, and probed with rabbit IgG 1 µg/ml to detect the Protein A portion of the scFv molecules and anti-rabbit IgG alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA) was employed later (28). 2C4C2 scFv was detected by adding specific mAb 9E10 (35). Extensive washings were performed between each step. Specificity of the binding was confirmed by competition assays as previously detailed (15). For direct detection of anti-ß2GPI binding, we used {gamma}-irradiated plates coated with 10 µg/ml ß2GPI and blocked with 3% BSA. The ELISA was performed as described above.

For detection of the tested antibodies for direct binding to ß2GPI, irradiated plates were coated with 10 µg/ml ß2GPI and blocked with 3% BSA. The rest of the assay was performed as described above.

Determination of antibody affinity
The affinity of the various anti-ß2GPI mAb and the corresponding scFv to cardiolipin/ß2GPI bound to solid phase was determined using the method described by Pullen et al. (36) and MacDonald et al. (37). The affinity of the mAb and the corresponding scFv was tested on cardiolipin-coated plates blocked with 10% FCS. The concentration of the Ig was 10 µg/ml. The affinity index (AI), which is the molar concentration of NH4SCN required to reduce the initial optical density by 50%, was determined.

RNA preparation
Total RNA was isolated from hybridoma cells as previously described (32). Briefly, 108 cells were incubated in high salt mixture in the presence of 0.5% NP-40 (Sigma, St Louis, MA). After the nuclei were precipitated, the supernatant which contains total RNA was mixed with an equal volume of 7 M urea Tris buffer. The proteins were removed by phenol–chlorophorm extraction, following ethanol precipitation at -80°C.

Cloning and sequencing of the antibody V regions
cDNA were synthesized from 20 µg of total mRNA in 20 µl of reaction mixture containing oligo(dT) primer by AMV reverse transcriptase (Promega, Madison, WI). The cDNA was then subjected to PCR amplification using TaqI polymerase (Promega) with forward primers located in the constant region or the 3' region and backward primers located in the V regions close to the leader sequences in a total volume of 50 µl. Denaturation was done at 94°C for 11 min, annealing at 67°C for 12 min and extension at 73°C for 13 min for 30 cycles. PCR products were separated on a 2% agarose gel and amplified DNA bands encoding VHDJHCH or V{kappa}J{kappa}C{kappa} genes were cleaned by GeneClean (Bio101, La Jolla, CA). PCR products were cloned into the EcoRI site of TAPCR DNA vector (Invitrogen, Groningen, the Netherlands). The ligated DNA was transformed into Escherichia coli DH5a strain and sequenced using Sequenase version II sequencing system (United Stated Biochemical, Cleveland, OH) with [{alpha}-35S]dATP. For homology searches we used GenBank and EMBL libraries during December 1998.

Bacterial expression, purification and characterization of anti-ß2GPI scFv
The plasmid vector PIgl6 was constructed for expression of scFv domain by B. D. Stollar et al. (2830), and was kindly provided by him and used in the current study. The pIgl6 vector is under the control of the T7 RNA polymerase promoter. It includes gene segments for a leader peptide sequence of the bacterial alkaline phosphatase gene, followed by the VH domain, a flexible polypeptide linker (GGGGS)3, the VL domain, His5 sequence and Protein A domain. The CAM, CAL and 16/6 VH and VL domains were cut employing EcoRI restriction site from DNA preparations from specific DH5{alpha} colonies and amplified by PCR employing specific primers for scFv. The VH regions were amplified with primers containing XmaI (5' upstream primer) and XbaI (3' downstream primer) sites. The VL were amplified employing primers containing BglII (upstream primers) and NcoI (downstream primer) restriction sites. The downstream primers were complementary to the 5' end of the C{kappa} and one 3' codon of the J{kappa} segment. The PCR products were digested with XmaI–XbaI for insertion of the H chain and BglII–NcoI for insertion of the L chain, and ligated into plasmid pIgl6 that was digested with the same enzymes.

Bacterial expression of the recombinant proteins was previously described (30). Culture media were mixed with periplasmatic extracts and loaded onto a column of IgG–Sepharose (Pharmacia, Uppsala, Sweden) to purify scFv as previously described (2830).

Products used for serologic assays and immunizations were >95% pure-length Fv, as judged from Coomassie blue staining. Antigen binding and competitive ELISA were performed as described above.

The 2C4C2 scFv was prepared according to a method described by Clackson et al. detailed elsewhere (31). Amplified DNA was cloned for expression into a special M13 phagemid, pHEN1, a vector which incorporates a C-terminal peptide tag to facilitate detection of expressed H and L chains V regions (31,35), that was used for transfection into bacteria. 2C4C2 scFv molecules were isolated from the supernatant of the bacteria using a scFv-specific mAb 9E10 coupled to Protein A–sepharose column (35). Antigen binding and competitive ELISA were performed as described above.

Induction of experimental APS by anti-ß2GPI scFv domains
BALB/c mice were immunized intradermally in the hind footpads with 10 µg of the following scFv (20 mice in each group): CAM (H + L) scFv, CAL(H + L) scFv, or CAM(H) + CAL(L) scFv, 16/6(H) + CAM(L) scFv, CAL(H) + CAM(L) scFv, 2C4C2(H + L) scFv, or 16/6(H + L) scFv in complete Freund's adjuvant. Three weeks later a booster injection of the appropriate scFv in PBS was given at the same sites. The mice were bled every month and the levels of autoantibody titers were determined in the sera by ELISA. We looked for experimental APS and SLE parameters (increased fetal loss, thrombocytopenia, aPTT, leukopenia and enhanced erythrocyte sedimentation rate) as detailed by us previously (1517).

Statistical analysis
Student's t-test was used to evaluate differences between the binding properties of the various antibodies. P < 0.05 was considered as a statistically significant result.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Properties of the mouse anti-ß2GPI mAb
Mouse anti-ß2GPI mAb (CAM, CAL, 2C4C2 and CAR) from different sources bound to negatively charged phospholipids (cardiolipin and phosphatidylserine) in the presence of ß2GPI or ß2GPI alone. Employing cardiolipin-modified ELISA we could show that the binding of the mAb was ß2GPI dependent as presented in Fig. 1Go (e.g. 1.453 ± 0.104 OD at 405 nm for CAR mAb in the presence of 10 µg/ml ß2GPI in comparison to 0.204 ± 0.034 OD at 405 nm in the absence of ß2GPI ). Furthermore, all the anti-ß2GPI mAb showed direct binding to ß2GPI (P < 0.001) for all the four mAb when compared to N40 and 16/6 irrelevant anti-ssDNA control mAb, as documented in Fig. 2Go(a and b).



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Fig. 1. Binding of aCL/ß2GPI-dependent mAb in a modified aCL-ELISA: dose-dependent enhancement of CAR, CAL, CAM and 2C4C2 mAb binding to cardiolipin in the presence of different concentrations of ß2GPI. Each point represents mean ± SD of three separate experiments.

 




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Fig. 2. Binding properties of the anti-ß2GPI mAb and their corresponding scFv. (a) Anti-ß2GPI binding of CAM, CAR, 16/6 mAb and the following scFv: CAM(H + L), CAM(H) + CAL(L), CAM(H) + 16/6(L), CAR(H + L), 16/6(H + L) and 16/6(H) + CAM(L). (b) Anti-ß2GPI binding of CAL, 2C4C2 mAb and the following scFv: CAL(H + L), CAL(H) + CAM(L) and 2C4C2(H + L), 16/6(H + L). (c) Anti-ssDNA binding of 16/6, CAM mAb and the following scFv: 16/6(H + L), 16/6(H) + CAM(L), CAM(H) + 16/6(L) and CAM(H + L).

 
The binding affinity of the mouse anti-ß2GPI mAb to cardio- lipin/ß2GPI was studied and described in Fig. 3Go. The AI of 2C4C2 and CAM to ß2GPI was relatively higher than that of mAb CAR and CAL (AI of 6.8 and 6.4 respectively in comparison to 5.6 and 3.9 respectively).



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Fig. 3. Affinity determination of anti-ß2GPI mAb and their corresponding scFv. AI was determined for the CAR, CAM, 2C4C2 and CAL anti-ß2GPI mAb, and their corresponding scFv: CAR(H + L), CAM(H + L) CAM(H) + CAL(L), CAL(H + L) and CAL(H) + CAM(L) 2C4C2(H + L). Each point represent mean ± SD of three separate experiments.

 
The CAR, 2C4C2 and CAM mAb had lupus anticoagulant (LAC) activity (the mAb prolonged the aPTT), prolonging the aPTT of a normal plasma from 22 to 84, 77 and 87 s respectively (P < 0.001 to P < 0.004) when added to the complex. The anticoagulant activity behaved in a dose-dependent manner (Fig. 4Go). CAL anti-ß2GPI mAb had less ability to cause an increase in clotting time (P < 0.09) when compared to N40 mAb.



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Fig. 4. Anti-coagulant activity of anti-ß2GPI mAb and their corresponding scFv. CAR, CAM, 2C4C2, CAL mAb and their corresponding scFv: CAR(H + L), CAM(H + L), CAM(H) + CAL(L), CAM(H) + 16/6(L), CAL(H + L), CAL(H) + CAM(L) 16/6(H) + CAM(L) and 2C4C2(H + L) were studied for their ability to prolong clotting time. aPTT was measured in seconds when different amounts of mAb (0–10 µg/ml) were added to a normal plasma. Each point represent mean ± SD of three separate experiments.

 
Usage of the VH genes in the anti-ß2GPI clones
Sequence analysis of the three hybridomas producing the pathogenic anti-ß2GPI mAb (CAM and CAR) and of the non-pathogenic CAL anti-ß2GPI mAb shows that CAM and CAR are encoded by the 3558 VH gene family, and CAL is encoded by the 7183 VH gene family (Table 1Go). mAb 2C4C2 has been shown previously to be encoded by the 3558 VH gene family (32). The sequences of CAM, CAR and CAL mAb are available in the GenBank using the following accession nos: CAM-heavy U16182, CAM-light U16181, CAR-heavy U16183, CAR-light U16184, CAL-heavy U 16179 and CAL-light U16180.


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Table 1. Summary of sequence data of anti-ß2GPI-producing hybridoma
 
Comparative analyses of the nucleotide sequences of the H chain of CAM, CAR 2C4C2 and CAL mAb showed 83% homology in the whole chain between CAM and CAR, and 90% homology in the V gene segment of the latter (Table 2Go). Low homology in the sequence of the whole chain or V gene segments was demonstrated when the sequence of CAL was compared to that of CAM and CAR (60 and 59% respectively). mAb CAM and CAL were originally derived from BALB/c mice immunized with anti-DNA (16/6 Id+) mAb and eventually developed experimental SLE.


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Table 2. Homology comparison (%) between the sequence analyses of anti-ß2GPI antibodies and antibodies isolated from mice with experimental SLEa
 
It was interesting to compare the sequences of mAb CAM, CAR and CAL VH to the sequences of other mAb derived from C3H.SW mice with experimental SLE induced by immunization with anti-16/6 Id (32). Comparative analyses of the nucleotide sequences demonstrate that mAb CAM and CAR share 92 and 88% sequence homology in the VH segment respectively with 3B12-1 mAb which bind 110 and 90 kDa of HeLa NE, 86 and 84% sequence homology in the VH segment to 5G12 an anti-DNA (16/6 Id+) mAb, and 85 and 83% sequence homology respectively to aCL mAb 2C4C2 (32).

The VH sequence of mAb CAL is 87% homologous in the whole chain and 93% homologous in the V gene segment to 3F7-8 mAb which is encoded by the same 7183 VH gene family as CAL (38). It carries the 16/6 Id and is not pathogenic (i.e. CAL is unable to induce experimental APS).

Usage of the V{kappa} gene by the anti-ß2GPI clones
The nucleotide sequences of the V{kappa} of the four aCL mAb show that CAM and CAR are encoded by the V{kappa} gene group 5, 2C4C2 is encoded by the V{kappa} gene group 2 (32), while CAL is encoded by the V{kappa} gene group 9 (Table 1Go).

Comparative analyses of the V{kappa} nucleotide sequences of CAM, CAR and CAL show 92% sequence homology in the whole chain between CAM and CAR, and 66 and 67% sequence homology between CAM and CAR to CAL respectively (Table 2Go). The V gene segment homology between CAM and CAR is 93%, while the comparison to CAL yields 64 and 65% sequence homology respectively. Comparative analyses of CAM, CAR and CAL VH gene sequences to other autoantibody sequences derived from experimental SLE (25) show that CAM and CAR share ~81–84% sequence homology in the whole chain and in the V gene segments to 2D12, an anti-La (SS-B) mAb which has the pathogenic potential to induce experimental SLE in naive mice, associated with high titers of aCL antibodies in the sera (39,40) and 3BR-1 which binds to 110 and 90 kDa of HeLa NE (32).

Usage of JH, D and J{kappa} genes
mAb CAL and CAM use the JH3 and J{kappa}2 genes, CAR uses the JH4 and J{kappa}4 genes, and 2C4C2 as previously described (32) uses the JH3 and J{kappa}1 genes. CAM and CAR use the D region 5P2.2 and SP 2.3 respectively, CAL uses the FL-16.1, and 2C4C2 uses the D region NA (32).

Characterization of the anti-ß2GPI scFv with and without replacement of H and L chains
BL21 carrying plasmid pIgl6 produced soluble rFv protein, identified in culture medium. Active rFv was purified directly from the culture medium by affinity chromatography with IgG–Sepharose, with a yield of up to 400 µg/l. The rFv migrated as a doublet in PAGE corresponding to molecular mass of ~ 36 and 34 kDa. All the corresponding scFv bind ß2GPI as compared to negative control scFv 16/6 (single chain generated for human anti-DNA mAb named 16/6) (Fig. 2a and bGo). The 16/6 scFv bind specifically ssDNA (P < 0.001) when compared to anti-ssDNA binding of CAM scFv (Fig. 2cGo). Substitution of CAM(H) into 16/6(H) in the construct 16/6(H) + CAM(L) resulted in a switch between anti-ß2GPI binding of CAM(H + L) scFv into anti-ssDNA binding of the new construct. Exchanging the CAM(L) into 16/6(L) did not affect either the ß2GPI binding of CAM(H) + 16/6(L) scFv (Fig. 2aGo) or targeting the ssDNA (Fig. 2cGo).

The binding affinity of the ß2GPI scFv to ß2GPI was studied (Fig. 3Go). All the scFv bound less effectively to the ß2GPI target molecule at least by an order of magnitude less than the whole antibodies. The ß2GPI binding AI of CAM(H + L) scFv and CAM(H) + CAL(L) scFv was relatively higher (4.4 and 4.8 AI respectively) than CAL(H + L) scFv (3.7 AI) or CAL(H) + CAM(L) scFv (3.6 AI) (P < 0.05).

Lupus anticoagulant characterization demonstrated that 10 µg of each CAM mAb, or scFv constructed with CAM(H) such as CAM(H + L) scFv, CAM(H) + CAL(L) scFv and CAM(H) + 16/6(L) scFv, caused prolongation of aPTT to 84, 78, 71 and 74 s respectively (P < 0.001) when compared to the effect of CAL, 16/6 or 16/6(H) + CAM(L) scFv (Fig. 4Go). 2C4C2 mAb at a concentration of 10 µg prolonged aPTT to 78 s and its corresponding scFv prolonged the aPTT to 72 s (P < 0.01 when compared to CAL or 16/6 mAb).

Cassettes composed of CAL(H), i.e. CAL(H + L) scFv and CAL(H) + CAM(L) scFv, did not have the ability to prolong clotting time (32 and 29 s respectively, P > 0.05, data not shown).

Immunization of naive mice with anti-ß2GPI scFv with and without replacement of H and L chains
BALB/c mice were immunized intradermally in the hind footpads with 10 µg of the following scFv, 20 mice in each group: CAM(H + L) anti-ß2GPI LAC, CAM(H) + CAL(L) anti-ß2GPI LAC, CAM(H) + 16/6(L) anti-ß2GPI LAC, 2C4C2 anti-ß2GPI/anti-ssDNA LAC, CAL(H + L) anti-ß2GPI, CAL(H) + CAM(L) anti-ß2GPI and 16/6(H) + CAM(L) anti-ssDNA, and 16/6(H + L) anti-ssDNA.

The mice which were immunized with CAM scFv and 2C4C2 scFv developed high titers of antibodies to negatively charged ß2GPI-dependent phospholipids (cardiolipin, phosphatidylserine) (e.g. aCL: 1.284 ± 0.123 and 1.208 ± 0.201 OD at 405 nm respectively), and not to positively charged phospholipids such as phosphatidylcholine (e.g. aPC: 0.235 ± 0.059 and 0.364 ± 0.065 OD respectively). ß2GPI direct binding was documented in the sera of these mice (1.322 ± 0.127 and 1.195 ± 0.152 OD respectively). Only the mice injected with 2C4C2 scFv had elevated titers of anti-DNA and anti-histone antibodies (1.004 ± 0.175 and 0.987 ± 0.093 OD respectively at a sera dilution of 1:200). CAL scFv-immunized mice did not develop significant titers the above-mentioned autoantibodies (Table 3Go). However, the mice subjected to 16/6(H + L) scFv or 16/6(H) + CAM(L) scFv developed primary experimental lupus-like disease characterized by high titers of anti-DNA and anti-histone antibodies, leukopenia, elevated erythrocyte sedimentation rate, and proteinuria (Table 3Go).


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Table 3. Induction of experimental APS by immunization of BALB/c mice with anti-ß2GPI and control scFv
 
Mice that had high titers of anti-ß2GPI or anti-ß2GPI- dependent antibodies (CAM scFv- and 2C4C2 scFv-injected mice) developed clinical parameters of experimental APS such as: (i) increased percentage of fetal loss (67 and 48 respectively versus 4% in CAL scFv immunized mice) (Table 3Go), (ii) thrombocytopenia (326,000 and 604,000 respectively in comparison to 1,129,000 cells/mm3 in CAL scFv-immunized mice) and (iii) prolonged aPTT (74 and 61 in comparison to 32 s in CAL scFv-immunized mice). The 2C4C2 scFv-immunized mice also developed manifestations of experimental lupus (in addition to anti-DNA and anti- histone antibodies, the mice showed an accelerated erythrocyte sedimentation rate of 14 versus 2 or 3 mm/6 h in the other two groups of mice) and leukopenia (3089 versus 6139 cells/mm3).

Mice immunized with CAM(H) + CAL(L) scFv or with CAM(H) + 16/6(L) following H/L chain replacement developed experimental APS as described in Table 3Go. The APS manifestations were documented by high titers of mouse anti-ß2GPI and anti-ß2GPI-dependent antibodies (P < 0.02 to P < 0.006 when compared to anti-DNA antibodies), thrombocytopenia (P < 0.02 when compared to CAL scFv immunized mice), prolonged aPTT (82 ± 14 and 67 ± 4 s ) and a high percentage of fetal resorption (59 ± 7 and 39 ± 5 compared to 4 ± 2 in CAL scFv immunized mice). CAL(H) + CAM(L) scFv- or 16/6(H) + CAM(L) scFv-immunized mice did not develop any clinical manifestations of experimental APS.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During the last few years it has been demonstrated that anti-ß2GPI antibodies exert a direct pathogenic effect by interfering with homeostatic reactions occurring on the surface of platelets or vascular endothelial cells (1012). Activation of endothelial cells upon exposure to anti-ß2GPI antibodies was associated with elevated expression of adhesion molecules (ICAM-1, VCAM-1 and E-selectin), as well as enhanced monocyte adhesion to the endothelial cells, as documented in vitro (14,41,42) and in vivo (42). Passive transfer of these antibodies into naive mice resulted in induction of experimental APS (13,14). Furthermore, the pathogenic activity of the anti-ß2GPI antibodies could be prevented in vitro and in vivo by ß2GPI corresponding synthetic peptides (43).

In our study we assessed the contribution of the Ig variable region to the binding and biological functions of the anti-ß2GPI mAb. We converted four anti-ß2GPI mAb named CAM, CAR, 2C4C2 and CAL into scFv. The binding properties of the above mAb correlated well with aCL antibodies found in patients with SLE and APS (46,44). Previously, we have shown that immunization of naive mice with CAM, CAR and 2C4C2 anti-ß2GPI mAb resulted in induction of experimental APS, while CAL mAb did not cause development of APS clinical manifestations in the CAL-treated mice (1517). In addition we have demonstrated that CAR, 2C4C2 and CAM were found to have anticoagulant activity, whereas CAL differed in this respect.

We addressed the question of whether the nucleotide sequences of H and L chains of the above antibodies could contribute more to our understanding of the pathogenic potential of CAM, 2C4C2 and CAR anti-ß2GPI mAb in comparison to the non-pathogenic mAb CAL. Nucleotide sequence analyses of the V region genes encoding both the H and L chains of anti-ß2GPI mAb was performed, three of which have pathogenic potential in vivo (CAM, CAR and 2C4C2) and one (CAL) which is non-pathogenic. Our data show that CAM and CAR use the VH gene of the J558 family as 2C4C2 (32), and CAL uses the VH gene of the 7183 family. Previous studies have shown that the J558 VH gene family is used at a higher frequency by anti-DNA antibodies (2225,32,33) by aCL antibodies from MRL-lpr/lpr mice (23,25) and by pathogenic anti-mouse red blood cells in NZB mice which had the potential to induce anemia in normal BALB/c mice (45). The J558 VH gene family was also predominantly used by anti-88 kDa splicing factor I mAb (32).

The major difference at the level of VHJV{kappa} gene sequence analysis between CAM, CAR, 2C4C2 and CAL is the use of the 7183 VH gene family by the non-pathogenic CAL. A low frequency in the use of the 7183 VH gene family was described also in NZB, MRL-lpr/lpr and in mice with experimental SLE (23,25,32). CAL has 93% homology in the V gene segment to 3F7-8 mAb (16/6 Id+) which also uses the VH 7183 family (38) and does not induce experimental SLE in vivo, while the anti-DNA 16/6 Id+ mAb, which uses the VH J558, are pathogenic in vivo (33,34). No pathogenic autoantibody which has a high sequence homology to the VH 7183 gene family has been described until now.

To evaluate the direct contribution of H and L chain of anti-ß2GPI to its pathogenic potential, we took the approach of constructing anti-ß2GPI scFv domains. Single-chain antibodies tend to exhibit greater stability and activity than their Fv counterparts (4648). In addition, the use of the scFv expression vector permits us to exchange the H and L chains between the pathogenic and non-pathogenic anti-ß2GPI molecules, and to study their role and contribution to the binding and function of the anti-ß2GPI mAb.

scFv were prepared from three anti-ß2GPI and one anti-ß2GPI/anti-DNA. All four scFv showed less antigen binding properties than the original mAb. Replacement of the pathogenic CAM VH domain with the non-pathogenic CAL VH or with 16/6 VH decreased the binding affinity of the scFv to cardiolipin/ß2GPI and completely abrogated the anti- coagulant activity (no prolongation of the aPTT). Replacement of the CAM VL with CAL VL or with 16/6 VL did not affect significantly the affinity for cardiolipin/ß2GPI or the anti- coagulant activity.

BALB/c mice were immunized with the scFv domains of the three anti-ß2GPI scFv (CAM, CAL and 2C4C2) and the scFv resulting from the replacement of the H and the L chains. The mice which were immunized with CAM and 2C4C2 scFv developed the same clinical manifestations as the original mAb (e.g. elevated titers of mouse aCL, anti-phosphatidylserine and anti-ß2GPI associated with lupus anticoagulant activity, thrombocytopenia, prolonged aPTT, and a higher percentage of fetal resorptions), while anti-ß2GPI CAL scFv-immunized mice failed to do so. High titers of aCL, anti-ß2GPI, anti-ss/dsDNA and anti-histone were observed in the sera of the 2C4C2 scFv-immunized mice, and the APS picture was associated with lupus findings, e.g. leukopenia, enhanced erythrocyte sedimentation rate and Ig deposition in the kidneys.

The mice immunized with scFv following the H/L chain replacements showed the following: (i) mice immunized with CAM(VH) + CAL(VL) scFv LAC or with CAM(H) + 16/6(L) scFv LAC develop experimental APS and (ii) mice immunized with CAL(VH) + CAM(VL) scFv or with 16/6(H) + CAM(L) did not develop any clinical manifestations of APS.

One may assume that Protein A may play a role in the process of induction of the experimental disease, either by a carrier effect or through binding to IgG. The assumption is very unlikely since the scFv 2C4C2 (which does not have Protein A and was constructed with cMyc-tag), CAM(H + L)–Protein A and CAM(H) + CAL(L)–Protein A were found to be pathogenic in vivo, while CAL(H + L) and CAL(H) + CAM(L) were non-pathogenic in vivo. The last two did contain Protein A.

The current study shows that scFv of pathogenic antibodies are capable of inducing the manifestations of the whole antibody molecule and points to the importance of the VH domain in the pathogenic potential of anti-ß2GPI antibodies.

In summary, it can be concluded that the pathogenic properties of anti-ß2GPI inducing in vivo experimental APS may be influenced by multiple factors: (i) affinity to ß2GPI, (ii) anticoagulant activity and (iii) VH domain. Presumably T cells together with specific pathogenic anti-ß2GPI antibodies are involved in the selection and affinity maturation of B cells, producing a panel of anti-phospholipid antibodies that are responsible for the clinical manifestations of APS.


    Acknowledgments
 
We thank Professor David Stollar (Tufts University, Boston, MA) for teaching and advice throughout the project. This study was supported by the Japanese–Israeli Binational grant for research, the Israel Ministry of Science, and the Freda and Leon Schaller Fund for Research in Autoimmunity.


    Abbreviations
 
ß2GPI ß2-glycoprotein I
AI activity index
aCL anti-cardiolipin antibodies
APS anti-phospholipid syndrome
aPTT activated partial thromboplastin time
ds double-stranded
LAC lupus anticoagulant
scFv single-chain Fv
SLE systemic lupus erythematosus
ss single-stranded

    Notes
 
Transmitting editor: G. Klein

Received 1 April 1999, accepted 17 August 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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