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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 496-506
By
From the Center for Molecular and Vascular Biology, Katholieke
Universiteit Leuven, Leuven, Belgium; and the Department of
Hematology-Immunology, Vrije Universiteit Brussels, Brussels, Belgium.
The development of an immune response towards factor VIII (fVIII)
remains a major complication for hemophilia A patients receiving fVIII
infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody.
Molecular analysis of the specific immune response is therefore
required. To this end, we have characterized an fVIII-specific human
IgG4
HEMOPHILIA A IS AN X-linked bleeding
disorder characterized by the absence or an insufficient amount of
functional factor VIII (fVIII). The deficiency affects 1 in 10,000 males and results in bleeding in joints, muscles, and soft tissues.
fVIII replacement therapy relies on the use of concentrates prepared
from plasma or of recombinant fVIII (rfVIII). However, in both cases,
such a treatment can trigger the production of specific
antibodies,1,2 in particular in patients with severe forms
of the disease.3-5 Circulating antibodies often preclude
further use of human fVIII, because they immediately neutralize its
function. Such patients, therefore, find themselves in a
life-threatening situation for which there is no current specific
treatment.
fVIII is a 330-kD glycoprotein produced by the liver as a single
polypeptide chain of 2,332 amino acids that subsequently undergoes
proteolytic processing.6 The circulating molecule consists
of two chains. The heavy chain is constituted of the A1 and A2 domains
and variable lengths of the B domain; the light chain contains the A3
domain and the C1 and C2 domains. fVIII contains a phospholipid (PL)
binding site in the C2 domain, between amino acids 2302 and
2332.7,8 Within the same fVIII region, there is also a von
Willebrand factor (vWF) binding site, which acts in conjunction with
amino acid residues 1649 through 1689 in the A3 domain.9-11
fVIII circulates complexed to vWF that protects fVIII from rapid
degradation in plasma.12 Upon cleavage by thrombin, activated fVIII (fVIIIa) dissociates from vWF,13 binds to
negatively charged PL, and participates as a cofactor to factor IXa in
the factor X activating (tenase) complex.
The human immune response towards fVIII is highly
heterogeneous.14,15 It is made of antibodies belonging to
almost any isotype, with a preferential involvement of IgG4
antibodies.16 However, three major clusters of B-cell
epitopes have been delineated using native or rfVIII fragments or mouse
monoclonal antibodies (MoAbs) of defined specificity. Thus, heavy
chain-specific inhibitors react primarily with the 18.3-kD
amino-terminal segment of the A2 domain.15,17 Light
chain-specific inhibitors recognize epitopes in the A3
domain18 and a major antigenic region in the C2
domain.9,10,19,20 Binding of polyclonal human fVIII
antibodies to the C2 domain inhibits the fVIII procoagulant activity by
either preventing the binding of fVIII to PL7 or reducing
the dissociation rate of fVIII from vWF.21 In addition, antibodies binding to the carboxy-terminal end of the C2 domain can
prevent fVIII binding to vWF.9-11 Interestingly, a
proportion of anti-fVIII antibodies do not inhibit the procoagulant
function of the molecule,18 but could possibly play a role
in the clearance of fVIII from the circulation.
The complex interactions between fVIII, PL, vWF, and specific
antibodies are far from being elucidated due to the heterogeneous nature of the human anti-fVIII antibody response. However,
understanding these interactions in more detail may provide the
molecular basis for the development of new forms of therapy. Thus, we
and others have proposed that specific suppression of the production of
certain anti-fVIII antibodies could possibly be achieved by
anti-idiotypic antibodies.22-25 It has also been suggested
that PL or vWF could efficiently compete with inhibitors for fVIII
binding26-28 and that such a property could be exploited
for replacement therapy with fVIII concentrates.
To get further insight into the mechanisms of fVIII neutralization and
to establish rational grounds for a specific therapy of fVIII
inhibitors, it was deemed necessary to analyze the human immune
response to fVIII at the clonal level. We have therefore generated
human MoAbs (hu-MoAbs) from cell lines derived from the B-memory cell
repertoire of hemophilia A patients with inhibitors. We report here on
the first of such antibodies, selected on the basis of its capacity to
interfere with fVIII binding to both vWF and PL.
Reagents and Buffers
Human Peripheral Blood Lymphocytes and Cell Lines
Immortalization of Human PBMCs PBMCs were immortalized according to described procedures.30,33 Briefly, 107 PBMCs were resuspended in 2 mL culture medium and incubated for 2 hours at 37°C with 200 µL Epstein-Barr virus (EBV) supernatant (B95-8 strain). Cells were then seeded at 300 to 24,000 cells/well in 96-well microtiter plates (Nunc, Roskilde, Denmark) containing LCD32 cells and 0.5 µg/mL anti-CD40 MoAb89. LCD32 cells had been irradiated (7,000 rads) or treated with mitomycin C (50 µg/mL) for 1 hour at 37°C and seeded in culture wells the day before EBV infection of PBMCs. Alternatively, mitomycin C-treated 3T6-TRAP cells were used as feeders instead of LCD32 cells. One hundred fifty microliters of culture supernatant was replaced every week by fresh culture medium. After 4 to 8 weeks, depending on growth rate in individual wells, culture supernatants were tested in enzyme-linked immunosorbent assay (ELISA) for the presence of anti-fVIII antibodies. Positive cell lines were transferred to 24-well plates and immediately cloned at 60 cells per 96-well plate without feeder cells.Sequencing of Ig Rearranged Genes The isolation of RNA from EBV-immortalized human B-cell lines was performed using TRIzol Reagent according to the manufacturer's instructions (Life Technologies). cDNA was synthesized with the SuperScript preamplification system for first-strand cDNA synthesis. The cDNA encoding the heavy chain variable region genes (VH) was amplified by polymerase chain reaction (PCR) using primers specific for the leader sequence of the VH families and for the first exon of the C region, as described.34
Annealing was performed at 60°C for 40 PCR cycles. PCR products of
the appropriate size (460 bp) were isolated from 1.5% agarose gel and
cloned using the TA Cloning Kit (Invitrogen BV, Leek, The Netherlands).
A PCR screening using couples of primers corresponding to the
VH gene family of interest was performed on cultures of
randomly selected colonies. Plasmid DNA from positive colonies was
isolated using Wizard Plus Minipreps (Promega, Menlo Park, CA) and
sequenced in both directions with Sequenase (US Biochemical, Cleveland,
OH), according to the manufacturer's instructions. Analysis of the
variable gene sequences was made using the V BASE Sequence Directory
(Tomlinson et al, MRC Centre for Protein Engineering, Cambridge, UK).
The complete sequences of the VH and VL were
submitted to the EMBL Nucleotide Sequence Database under the accession
numbers AJ224083 and AJ224084, respectively.
Purification of Human IgG Human MoAbs were purified by adsorption on immobilized protein A. One hundred milliliters of cell culture supernatant was passed through a high-TRAP protein A (Pharmacia, Uppsala, Sweden) at a flow rate of 1 mL/min. Bound IgG was eluted with citric acid 100 mmol/L, pH 3. After pH neutralization with Tris, pH 9, IgG was dialyzed against 150 mmol/L NaCl. The concentration of proteins was determined with the Bio-Rad assay (Bio-Rad, Hercules, CA). The purity of the final preparation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 8% gel and unreduced proteins followed by Coomassie-blue staining, showing one major band corresponding to unreduced IgG and two minor bands at 45 and 60 kD, respectively. The IgG band represented 80% of the total protein content as determined using a Du-68 Spectrophotometer (Beckman-Coulter Inc, Fullerton, CA). Human polyclonal anti-fVIII antibodies were prepared from plasma by salt precipitation and gel filtration chromatography, as previously described.18Recombinant DNA Fragments DNA fragments encoding fVIII amino acid residues 1981-2222 (exons 19-24) and 2125-2332 (exons 23-26) were generated by PCR using primers bound by the restriction sites BamHI and Not I.10 Primers corresponding to fVIII amino acid residues 1981-2222 were 5 -CGTGGTGGATCCTCTGCAGACGGTGTTTTTGAGACAGTGGAAATG-3 and
5-TTCTCGACTTGCGGCCGCCTGAGGTCTCCAGGCATTACTCCTC-3. Those corresponding to fVIII amino acid residues 2125-2332 were 5-CGTCGTGGATCCTCTGCAGACGTCTTCTTTGGCAATGTGGATTCA-3 and
5-TTCTCGACTTGCGGCCGCGTAGAGGTCCTGTGCCTCGCAGCC-3. Primers for fVIII 2125-2303 amino acid residues were
5-CGTCGTGGATCCTCTGCAGACGTCTTCTTTGGCAATGTGGATTCA-3 and
5-TTCTCGACTTGCGGCCGCAGTCAGTAACGGTGGGTCTAGAGA-3. The
5 primer for the fragment corresponding to residues 2216-2332 was
5-TCGAAACTAATACGACTCACTATAGGGAAGATGAAGCTTGGATCCAGTAATGCCTGGAGACCTCAGGTG-3. The 5 primer for the C2 domain gene (residues 2170-2332) with a
site directed mutation coding for a glycine residue in place of the
cysteine residue 2174 was
5-TCGAAACTAATACGACTCACTATAGGGAAGATGAAGCTTGGATCCGATTTAAATAGTGGCAGCATGCCA-3. The fragments were cloned in frame in pGEX4T-2 (Pharmacia) and controlled by sequencing in both directions with the T7 Sequencing kit
(Pharmacia). The glutathione S-transferase (GST) fusion proteins were
expressed in the DH5 Escherichia coli strain. Fusion
proteins were solubilized with 1.5% Sarkosyl (Sigma) and 2% Triton
X-100 and purified on a glutathione column, as described.35
Purified proteins were evaluated by ELISA, SDS-PAGE, and Western
blotting. The presence in the chimeric proteins of amino acid residues
2108-2121 or 2248-2312 was controlled using a rabbit polyclonal
antiserum directed to fVIII amino acid residues 2108-2121 of the C1
domain (kind gift of M. Di Giambattista, Belgian Red Cross, Brussels, Belgium) or the anti-C2 MoAbESH8,21,29
respectively.
Immunoassays Detection of anti-fVIII antibodies.
Plasma-derived fVIII was insolubilized on microtitration plates through
binding on a specific MoAb. Thus, plates were incubated overnight with
50 µL GBS containing 5 µg/mL of MoAb7.18 The plates
were washed 4 times with PBS-Tween before the addition of 50 µL of
plasma-derived fVIII diluted at 5 µg/mL in Tris-casein. Alternatively, 50 µL of rfVIII (1 µg/mL) was used as a substitute to plasma-derived fVIII. In some experiments, rfVIII was insolubilized by incubating plates for 2 hours at 4°C directly with 50 µL of rfVIII (1 µg/mL) diluted in GBS. The plates were washed as described above and 50 µL of culture supernatant was added for a further incubation of 2 hours at 4°C. After washing, 50 µL
peroxidase-labeled antihuman Fc IgG subclass determination.
IgG subclasses in cell line supernatants were determined as
described.18 Briefly, polystyrene microtitration plates
(Nunc) were incubated for 2 hours at RT with 50 µL of an antimouse
IgG1 rat MoAb (UCL, Brussels, Belgium) at a concentration of 5 µg/mL in GBS. After each step, the plates were washed four times with Tris-Tween. Fifty microliters of a mouse IgG1 MoAb diluted to 0.5 µg/mL in PBS-BSA and specific for either human IgG1 (Oxoid Ltd,
Basingstoke, UK), IgG2, or IgG3 (Calbiochem, La Jolla, CA) was added
and the plates were incubated for 2 hours at RT. For the evaluation of
IgG4, 50 µL of a specific mouse MoAb (Calbiochem), diluted at a
concentration of 5 µg/mL in GBS, was incubated directly on the plate
for 2 hours at RT. The plates were washed and 50 µL of culture
supernatant or of a calibrated reference serum diluted in PBS-BSA were
added for an incubation of 2 hours at RT. Human antibody binding was
detected by the addition of 50 µL peroxidase-labeled antihuman Fc
Inhibition of fVIII binding to PS. Antibodies interacting with the binding of fVIII to PS were detected using an assay system adapted from described methods.7,36 Polystyrene plates (Maxisorb; Nunc) were incubated with 100 µL of PS dissolved at 5 µg/mL in methanol and allowed to dry overnight at RT. The plates were then saturated by incubation with 200 µL PBS-BSA 5% for 1 hour at RT. Fifty microliters of a 4 µg/mL rfVIII solution made in Tris-BSA were mixed with 50 µL of purified hu-MoAb diluted in the same buffer. The mixture was incubated for 30 minutes at 37°C and then added to the plate in 50-µL aliquots for a further incubation of 2 hours at RT. After washing with Tris-Tween, horseradish peroxidase (HRP)-coupled anti-A2 MoAbF15B12 was added at a 1/1,000 dilution in Tris-BSA for a further 1 hour of incubation. OPD was then added and the OD was measured at 492 nm. Negative controls included an hu-MoAb of irrelevant specificity. The concentration of fVIII used in this assay was just below the plate-saturating concentration and gave the highest signal to noise ratio. Inhibition of fVIII binding to vWF. Microtitration plates were incubated overnight at 4°C with the anti-vWF MoAb4H1D7 diluted at 4 µg/mL in GBS. The plates were then blocked with 120 µL of PBS-BSA 5% for 1 hour at RT. After washing the plates with Tris-Tween, 50 µL of a normal human plasma pool diluted 1/20 in the same buffer was added for 1 hour at RT. The plates were washed 3 times with PBS-Tween and then incubated for 30 minutes with 50 µL of 400 mmol/L CaCl2 to detach fVIII from vWF. Fifty microliters of rfVIII diluted at 0.4 µg/mL in PBS-BSA was mixed with 50 µL of purified hu-MoAb made at different dilutions in the same buffer. The mixture was incubated for 30 minutes at 37°C before adding a 50-µL aliquot to the plate for 2 hours of incubation at RT. After washing with PBS-Tween, bound fVIII was detected as in the PS assay described above. In preliminary experiments, fVIII bound to insolubilized vWF was detected by the addition of HRP-coupled mouse MoAb F15B12 before and after treatment of the plates with 400 mmol/L CaCl2. Such a treatment reduced absorbency by more than 90%, showing effective removal of fVIII from vWF. Effect of vWF on fVIII binding to BO2C11. Microtiter plates were coated with BO2C11 (2 µg/mL) in GBS and blocked as in previously described assays. rfVIII (0.2 µg/mL) in Tris-BSA 5% was preincubated for 30 minutes at 37°C in the presence or absence of vWF (1 to 20 µg/mL) before addition to BO2C11-coated plates. After an incubation of 5 minutes at 37°C, the wells were washed and the binding of fVIII to BO2C11 was detected as described above for inhibition of fVIII binding to PS by addition of the anti-A2 MoAb F15B12 for 1 hour at 4°C. The amount of fVIII bound to BO2C11 was determined by comparing the OD with those of a calibration curve established with known amounts of fVIII. To establish a calibration curve, 50 µL of different concentrations of rfVIII (0.2 to 200 ng/mL) were incubated at 4°C in microtitration plates coated with BO2C11. Supernatants were removed after 2 hours of incubation and the anti-A2 MoAb F15B12 was added to the wells for 1 hour at 4°C followed by OPD. Residual fVIII activity in the supernatants was determined in an fVIII chromogenic assay as described below. The quantity of bound fVIII was calculated by subtracting unbound fVIII from the total amount of fVIII added to wells. The low dissociation rate constant of fVIII from BO2C11 (see below) allowed us to relate the levels of fVIII captured by BO2C11, determined in the fVIII chromogenic assay, to the optical densities, recorded in ELISA after the addition of the anti-A2 MoAb F15B12. Antibody-dependent dissociation of fVIII-vWF complex. Microtitration plates were coated with anti-vWF MoAb4H1D7 and blocked by incubation with TBS-BSA. Fifty microliters of plasma-derived fVIII-vWF (15:1 vWF to fVIII wt/wt ratio), diluted at 1 IU/mL in Tris-BSA, was mixed with 50 µL of the same buffer containing or not containing human IgG (1 to 10 µg/mL). The mixture was preincubated for 1 hour at 37°C and a 50-µL aliquot was added to the plate for a further incubation of 2 hours at 4°C. After washing with Tris-Tween, bound fVIII was detected as in the inhibition of fVIII binding to PS. ODs recorded after the addition of OPD were used to determine the amount of fVIII bound to vWF, by comparison with a calibration curve obtained with known amounts of fVIII bound to vWF. To establish the calibration curve, 50 µL of different concentrations of plasma-derived fVIII-vWF (0.2 to 200 ng/mL) was incubated at 4°C in microtitration plates coated with MoAb4H1D7. After 2 hours of incubation at 4°C, the supernatants were removed and the anti-A2 MoAb F15B12 was added to the wells for 1 hour at 4°C. Bound fVIII was detected by the addition of OPD or in a functional assay. In the latter case, 30 µL of imidazole-BSA was added to the wells, and fVIII was measured in the fVIII chromogenic assay described below. The reference curve of the fVIII chromogenic assay was made with known amounts of soluble fVIII diluted in 30 µL imidazole-BSA. Preliminary experiments indicated that MoAb F15B12 did not interfere with fVIII activity in fVIII functional assays. A calibration curve was then established by plotting the amounts of fVIII bound to vWF, as measured in the fVIII chromogenic assay, and the OD obtained in ELISA. Functional Assays Kinetics of fVIII inactivation by BO2C11. The functional inhibitory capacity of anti-fVIII hu-MoAbs was evaluated by using a modification of the DADE fVIII chromogenic assay (Dade AG, Switzerland), as described.18 In this assay, thrombin-activated fVIII accelerates the conversion of factor X into factor Xa in the presence of factor IXa, PL, and calcium ions; factor Xa activity is then assessed by hydrolysis of a p-nitroanilide substrate. Reagents, which were reconstituted according to the manufacturer's instruction, comprised bovine factor X (1 mmol/L), factor IXa (0.3 mmol/L), thrombin (0.3 mmol/L), CaCl2 (30 mmol/L), PL (60 mmol/L), a chromogenic factor Xa substrate (CH3OCO-D-CHG-gly-Arg-pNA.AcOH; 3.4 mmol/L), and a thrombin inhibitor (L-amidinophenylalanine piperidine). One vol of rfVIII (240 ng/mL) in imidazole-HSA was mixed with 1 vol of purified vWF (12 µg/mL) or buffer and incubated for 30 minutes at 37°C. The mixtures were then kept at 4°C. Aliquots containing rfVIII or rfVIII-vWF complexes were mixed with an equal volume of purified hu-MoAb (concentration varying from 170 ng to 170 µg/mL) and incubated during 2 to 60 minutes at 37°C. Aliquots of 30 µL were then retrieved and displayed in microtitration plates; 30 µL of the factor X and factor IXa/thrombin reagents were added sequentially. After 90 seconds, 60 µL of the chromogenic substrate was added and the incubation was extended for 10 minutes at 37°C. The reaction was then blocked by the addition of 30 µL citric acid (1 mol/L), and OD was measured at 405 nm. The residual fVIII activity was determined by comparing the OD405nm of test samples with that obtained with rfVIII solutions of known concentrations. Preliminary experiments had shown that there was no significant difference between the OD405nm of rfVIII as compared with rfVIII-vWF complexes. The rfVIII concentration (120 ng/mL) used for mixing experiments with hu-MoAb was selected as the highest concentration that could still be measured in the chromogenic assay without further diluting the sample. The residual fVIII activity was expressed as the percentage of activity measured in rfVIII aliquots handled and diluted exactly as test samples throughout the entire experiment. Inhibition of activated fVIII by hu-MoAbs. fVIII was activated by thrombin by mixing 1 vol fVIII (10 IU/mL) with 1 vol thrombin (0.2 IU/mL) at 37°C. After 30 seconds of incubation, thrombin activity was inhibited by the addition of 1 vol of a 60 µg/mL hirudin solution. fVIIIa was measured in the chromogenic assay described above, except that hirudin was added to the reagents before the assay. Thus, a 30-µL aliquot of thrombin-activated fVIII was added to a microtitration plate and incubated with 80 µL of a solution made by mixing 30 µL factor X and factor IXa reagents, 10 µL hirudin (60 µg/mL), and 10 µL imidazole-HSA containing or not containing inhibitor antibody. After 90 seconds, the generation of factor Xa was evaluated by the addition of a chromogenic substrate for a further 5 minutes of incubation. The OD was read at 405 nm and the results are expressed as percentages of the OD measured in the absence of inhibitor antibody. Coagulation assays. fVIII inhibitor titers were measured by the Bethesda method in which normal pooled plasma was used as an fVIII source. After an incubation of 2 hours with antibody, the residual fVIII activity was measured by a one-stage clotting assay according to Kasper et al37 with the modifications of Verbruggen et al.38 Measurement of Surface Plasmon Resonance (SPR) Real-time kinetic interaction between fVIII and hu-MoAbs was analyzed using a Pharmacia Biosensor BIAcore TM instrument (Pharmacia Biosensor AB). Purified BO2C11 (20 µg/mL in 10 mmol/L sodium acetate buffer, pH 5.0) was immobilized on the activated surface of a CM5 sensor chip, according to the manufacturer's instructions. All binding experiments were performed in HBS at a constant flow rate of 10 µL/min. fVIII in HBS was infused at various concentrations over the ECR-immobilized sensor chip surface. At the end of each cycle, the surface was regenerated by flushing HCl, pH 2, for 36 seconds. The amount of fVIII molecule bound per BO2C11 molecule was determined by assuming that the molecular weight of FVIII and BO2C11 are 330.000 and 170.000, respectively, and that the response in RU corresponding to the binding of one molecule is proportional to the molecular weight.
Production and Characterization of the Anti-fVIII BO2C11 hu-MoAb PBMCs of a hemophilia A patient with inhibitor (BO) were immortalized by EBV transformation. Five hundred cell line supernatants were screened by ELISA for the presence of antibodies towards fVIII and rfVIII C2 domain. Five supernatants contained anti-fVIII antibodies. Two supernatants containing anti-C2 antibodies were further tested for their ability to inhibit fVIII activity in a chromogenic assay and to prevent the binding of fVIII to PS and/or to vWF in an ELISA system. The BO2C11 cell line was selected because it produced an IgG antibody that fulfilled these characteristics. BO2C11 was then cloned by limiting dilution. Clonality was verified by two independent reverse transcription-PCR amplifications of mRNA coding for the variable part of the antibody heavy chain: a single sequence was obtained from the 12 clones of PCR products (data not shown). The complete sequences of the VH genes of BO2C11 were determined. BO2C11 VH gene was most homologous to DP-5, a member of the VH-1 gene family, and the J segment was most homologous to JH3b. Sequencing of the cloned light chain gene identified the VL as a V III and the J segment as a
J5. Purified antibodies were obtained by passage of the
BO2C11 cell culture supernatant on protein-A Sepharose. An ELISA
performed with IgG subclass- and light chain-specific antibodies
identified BO2C11 as an IgG4 .
Inhibition of fVIII Function by BO2C11
BO2C11 inhibits fVIII functional activity.
BO2C11 capacity to inhibit plasma fVIII activity was tested in a
coagulation assay. As shown in Fig 2, when
a solution containing 0.14 µg/mL BO2C11 was incubated with plasma as
in a conventional Bethesda assay, fVIII activity was reduced by 50%.
The BO2C11 specific activity is therefore approximately 7,000 BU/mg
protein.
BO2C11 inhibits fVIII binding to PS.
The capacity of BO2C11 to inhibit the binding of fVIII to PS-coated
plates was investigated by ELISA. As shown in
Fig 3, a dose-dependent inhibition was
observed. The BO2C11 concentration yielding 50% inhibition
(IC50) of fVIII binding is 1.6 µg/mL.
BO2C11 inhibits the binding of fVIII to vWF.
The capacity of BO2C11 to inhibit the binding of fVIII to vWF was
assessed in ELISA. Figure 3 shows that BO2C11 inhibits the binding of
fVIII to vWF in a dose-dependent manner. The concentration of BO2C11
required to achieve 50% inhibition (IC50) of fVIII binding is 0.12 µg/mL. The difference in BO2C11 concentrations required to
inhibit the binding of fVIII to PS or to vWF is related to the
difference in fVIII concentrations in the two assay systems. Thus, on a
molar basis, the ratios of BO2C11 IC50 to fVIII
concentration in the PS- and vWF-binding assays are 0.8 and 0.6, respectively.
Inhibition of BO2C11-fVIII Interaction by vWF
vWF inhibits the binding of fVIII to BO2C11.
The inhibitory property of BO2C11 on the binding of fVIII to vWF
prompted us to determine whether physiological concentrations of vWF
could compete with BO2C11 for fVIII binding. This was assessed in an
ELISA system in which the binding of free or vWF-bound fVIII to BO2C11
was measured. As shown in Fig 4, when fVIII
was complexed to vWF at a ratio similar to that found in plasma (50:1
vWF to fVIII wt/wt ratio) and incubated for 5 minutes at 37°C with
insolubilized BO2C11, the binding to fVIII was only 10% of that
obtained in the absence of vWF. The protective effect of vWF was
concentration-dependent. However, when the incubation time in the above
system was prolonged, the protective effect of vWF progressively
disappeared (data not shown). This suggested that BO2C11 could displace
fVIII from its vWF-binding site.
BO2C11 displaces fVIII from vWF.
To test this hypothesis, we incubated plasma-derived fVIII (0.5 U/mL)
complexed to vWF (15:1 vWF to fVIII wt/wt ratio) for 60 minutes at
37°C in the presence or absence of BO2C11 (5 µg/mL). We then
evaluated the proportion of fVIII that remained bound to vWF by
capturing fVIII-vWF complexes on a microtiter plate onto which an
anti-vWF MoAb had been insolubilized. fVIII was then detected by the
addition of the anti-A2 MoAbF15B12. BO2C11 reduced the amount of fVIII
bound to vWF by more than 90% within the time frame of the experiment,
whereas the addition of a pool of normal donor gammaglobulins had no
effect (Fig 5). To determine whether this
BO2C11 activity was representative of the inhibitory activity of the
patient's inhibitor antibodies, polyclonal IgG antibodies were purifed
from the plasma of the patient from whom the BO2C11 cell line was
derived. The latter polyclonal IgG antibodies also strongly reduced the
amount of fVIII bound to vWF (Fig 5). Similar results were obtained
when complexes made of biotinylated fVIII and vWF were used and the
binding of biotinylated fVIII was evaluated by the addition of
HRP-labeled avidine instead of anti-A2 MoAbF15B12 (data not shown).
vWF Prevents fVIII Inactivation by BO2C11 in a Functional
Assay
Kinetics of fVIII-BO2C11 Association
BO2C11 Inhibits fVIIIa
The emergence of an immune response towards fVIII remains a major
complication for hemophilia A patients treated by infusions of fVIII.
Available therapies to restore hemostasis in those patients are based
on the administration of porcine fVIII or of fVIII bypassing agents,
such as activated prothrombin complex concentrates or activated
recombinant factor VII.44,45 Alternatively, restoring fVIII
unresponsiveness can be attempted by administering high doses of fVIII,
in conjunction or not in conjunction with extracorporeal removal of
IgG, cytostatic agents, or IV infusions of pooled human gammaglobulins.23 However, all of these methods have shown
disadvantages, such as inconstant results and high costs. The
development of a novel therapy has been hampered by the lack of
understanding of the mechanisms by which antibodies interact with
fVIII, which is mainly due to the diversity of the human antibody
response to this coagulation factor.
Submitted October 15, 1997;
accepted March 17, 1998.
The authors thank Jean-Jacques Pin and Drs Serge Lebecque and Jacques
Banchereau for their invaluable help in the production of human MoAbs.
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Abstract
Introduction
1 s
1 × 10
Abstract
(kdisst),
2-mercaptoethanol in the
presence of 8 mol/L urea, followed by alkylation with iodoacetamide, did not significantly reduce BO2C11 binding in ELISA (data not shown);
(2) BO2C11 also recognized reduced fVIII in Western blot (data not
shown), and (3) in addition, a mutant chimeric GST-fVIII C2 domain,
corresponding to residues 2170 to 2332 and in which cysteine 2174 was
replaced by a glycine, was recognized by BO2C11 (data not shown). Taken
together, these results suggest that the formation of the epitope
recognized by B02C11 requires residues located between amino acid
residues 2303 and 2332 and between residues 2170 and 2216. However,
this does not exclude the fact that amino acid residues located
elsewhere in the fVIII C2 domain may contribute to the constitution of
the BO2C11 epitope.
) or to vWF (
), rfVIII at 2 µg/mL or 0.2 µg/mL final concentration, respectively, was mixed for 30 minutes at 37°C
with different concentrations of BO2C11 before addition to PS- or
vWF-coated plates, as appropriate. In both cases, the plates were then
incubated for 2 hours at RT and the binding of fVIII was
detected by the addition of the anti-A2 MoAbF15B12.
) rfVIII with vWF
at 3 µg/mL (12 nmol/L); (
) rfVIII + BO2C11 at 85 ng/mL (0.57 nmol/L); (
) rfVIII-vWF + BO2C11 at 85 ng/mL (0.57 nmol/L); (
) rfVIII-vWF + BO2C11 at 85 µg/mL (570 nmol/L).
2 = 50 to 300).
