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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 540-545
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Plasma Proteins, CLB, the Laboratory for
Experimental and Clinical Immunology, Academic Medical Centre,
University of Amsterdam, and the Emma Children's Hospital AMC,
Amsterdam, The Netherlands.
One of the major binding sites for factor VIII inhibitors is located
within the A2 domain. In this study, phage display technology was used
to isolate 2 human monoclonal antibodies, termed VK34 and VK41,
directed toward the heavy chain of factor VIII. The VH
domain of a single-chain variable domain antibody fragment (scFv) VK34
is encoded by germline gene segment DP-10. Epitope-mapping studies
revealed that scFv VK34 is directed against amino acid residues
Arg484-Ile508 , a previously identified
binding site for factor VIII inhibitors in the A2 domain. ScFv VK34
inhibited factor VIII activity with a titer of 280 BU/mg. The
VH domain of VK41 was encoded by germline gene segment
DP-47. A phage corresponding to VK41 competed with a monoclonal
antibody for binding to amino acid residues
Asp712-Ala736 in the acidic region adjacent to
the A2 domain. Reactivity of VK41 with a factor VIII variant in which
we replaced amino acid residues Asp712-Ala736
for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 for
Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit
factor VIII activity. This study not only defines the primary structure
of human anti-factor VIII antibodies reactive with the A2 domain, it
also describes an antibody with an epitope not previously identified in
the antibody repertoire of hemophilia patients with an inhibitor.
(Blood. 2000;96:540-545)
Factor VIII is an essential cofactor in the intrinsic
pathway of blood coagulation that enhances the activation of factor X
by factor IXa in the presence of Ca++ ions and
phospholipids. Based on internal sequence homology, the factor VIII
molecule can be defined by the domain structure A1-a1-A2-a2-B-a3-A3-C1-C2 (for review, see
Lenting et al).1 In plasma, factor VIII circulates as a
heterodimer composed of a heavy chain (A1-a1-A2-a2-B
domains) and a light chain (a3-A3-C1-C2 domains). The
functional absence of factor VIII is associated with the X-linked
bleeding disorder hemophilia A. In patients with hemophilia A, the
bleeding tendency can be corrected by the administration of factor VIII
concentrates. After multiple infusions, some patients with hemophilia A
develop antibodies that neutralize the procoagulant activity of factor
VIII.2
These antibodies, commonly termed factor VIII inhibitors, are
directed against epitopes present in the A2, A3, and C2 domains of
factor VIII.3 More detailed mapping of anti-C2 antibodies revealed a common binding site consisting of residues
Val2248-Ser2312.4
Using a series of active human/porcine factor VIII hybrids, a second
determinant of the anti-C2 inhibitor epitope has been attributed to the
region Glu2181-Val2243.5 Anti-C2
inhibitors prevent factor VIII from binding to phospholipids and von
Willebrand factor.6,7 Two independent studies identified a
binding site for factor VIII inhibitors in the A3 domain of factor
VIII, which overlaps a previously identified binding site for factor
IXa.8-10 Binding of these inhibitors interferes with assembly of the factor IXa-factor VIIIa complex.
Within the A2 domain, residues Arg484-Ile508
have been shown to constitute a binding site for factor VIII
inhibitors.11 Alanine scanning mutagenesis within this
region indicated that amino acid residue Tyr487 is
essential for binding most human inhibitors to the A2
domain.12 Anti-A2 inhibitors block the activation of factor
X by the phospholipid bound factor VIIIa-factor IXa
complex.13 Recently, it was shown that these antibodies
abrogate the stimulatory effect of isolated A2 domain on factor IXa
activity.14 These data indicate that anti-A2 inhibitors
prevent the interaction of the A2 domain with factor IXa.
Previously, we have used phage display technology to isolate
anti-C2 antibodies from the immunoglobulin repertoire of a patient with
acquired hemophilia.15 Anti-C2 antibodies were
characterized by an unusually long CDR3 of 20-23 amino acids and
extensive somatic hypermutation. Surprisingly, the immunoglobulin heavy
chain variable (VH) domains of all these antibodies were
encoded by VH gene segments derived from the
VH1 gene family. These findings suggest that a subset of
VH gene segments is used to generate human anti-C2 antibodies. Here, we have used phage display technology to further define anti-A2 antibodies. The current study defines the molecular characteristics of a human antibody reactive with factor VIII sequence
Arg484-Ile508, the major inhibitor binding
site located within the A2 domain. Moreover, we provide evidence for
the existence of an additional epitope for human anti-factor VIII
antibodies located between residues
Asp712-Ala736 in the a2 region.
Materials
FVIII assays
Construction of a hybrid FVIII/FV recombinant A2 domain Plasmid pCLB-GP67B-A221 and factor V cDNA served as templates for the construction of a plasmid encoding the A2 domain and the a2 region (residues Ser373-Arg740) in which residues Arg484-Ile508 were replaced by the corresponding sequence of coagulation factor V. Primer combinations A2-1-484FV AS (5'-TCT TCA TAA GGG ACA TCA GTG ATT CCG-3'), 484FV S (TGA TGT CCC TTA TGA AGA TGA AGT, C-3')-508FV AS (5'-TAT TTG AAT GTT TCC CCT GGT TGA AC-3'), and 508FV S (5'-CAG GGG AAA CAT TCA AAT ATA AAT GG-3')-A2-2 were used to amplify 3 DNA fragments that were reassembled by overlap extension polymerase chain reaction using outer primers A2-1 and A2-2 in a second round of amplification.21 The final product was cloned as NcoI-NotI fragment in pAc-GP67B to yield pCLB-GP67B-A2-FV484-508. Expression in insect cells and labeling of recombinant factor VIII fragments was performed as described previously.21Phage display library construction and selection In this study, peripheral blood mononuclear cells were used as a source of RNA for generation of the patient's IgG4-specific VH gene repertoire essentially as described previously.15 The obtained repertoire was combined with a VL gene repertoire of nonimmune origin in pHEN-1-VLrep and displayed as scFv on the surface of filamentous phage.23 Phage were selected for binding to the factor VIII heavy chain using the following methods: microtiter wells were coated overnight at 4°C with 100 µL mAb CLB-CAg 9 (35 nmol/L in 50 mmol/L NaHCO3, pH 9.6). Subsequently, wells were blocked with Tris-buffered saline (150 mmol/L NaCl, 50 mmol/L Tris, pH 7.4) and 3% (wt/vol) human serum albumin (HSA) for 2 hours at 37°C. To reduce nonspecific binding, phage in Tris-buffered saline, 3% (wt/vol) HSA, and 0.5% (vol/vol) Tween-20 were preincubated for 2 hours at room temperature in blocked CLB-CAg 9-coated microtiter wells. Meanwhile, CLB-CAg 9-coated microtiter wells were incubated for 2 hours at 37°C with human factor VIII heavy chain (16 nmol/L in 1 mol/L NaCl, 50 mmol/L Tris, pH 7.4, 2% [wt/vol] HSA). Wells were blocked with HSA as outlined above and incubated for another 2 hours at room temperature with nonbound phage, which were transferred from the preincubations. After intensive washing, bound phage were eluted and rescued by reinfection of Escherichia coli TG1.24 Alternatively, phage were selected on factor VIII heavy chain (40 nmol/L in 50 mmol/L NaHCO3, pH 9.6) immobilized to immunotubes (Maxisorp; Nunc, Breda, The Netherlands), thereby allowing the selection of phage directed toward epitopes blocked by antibody CLB-CAg 9. The library was subjected to 3 rounds of selection using the 2 procedures outlined above.Screening and sequencing of selected clones After 3 rounds, phages obtained from 20 single infected colonies of both selections were tested for binding to the factor VIII heavy chain immobilized to mAb ESH5. Bound phages were detected by anti-M13 antibody peroxidase conjugate (Pharmacia-LKB, Woerden, The Netherlands). VH and VL genes of factor VIII heavy chain binding clones were sequenced using the BigDye Terminator sequencing kit on a 377XL automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were compared with a database of germline V genes as compiled in the V-BASE sequence directory.25Expression and purification of scFv To facilitate the purification of scFv, a His-tag was introduced into the expressed protein by subcloning the V gene cassettes into the vector pUC119-Sfi/Not-His6.26 ScFv expression in E coli was induced with isopropyl -D-thiogalactoside for 3 hours at 25°C. Purification of scFv by immobilized metal chelate affinity chromatography was performed as described previously.27
Eluted fractions were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis under nonreducing
conditions; protein concentrations were determined
spectrophotometrically at A280.
Characterization of isolated clones Immunoprecipitation of metabolically labeled factor VIII fragments by scFv was performed as described previously.15 Reactivity of phage derived from the isolated clones with plasma-derived factor VIII heavy chain, recombinant A2 domain, factor VIII heavy chain, rFVIII, rFVIII-HCII, and rFVIII-Tyr718,719,723 Phe
was determined by enzyme-linked immunosorbent assay (ELISA). Factor
VIII antigen was immobilized at a concentration of 1 nmol/L to
ESH5-coated microtiter wells. Microtiter wells were incubated for 2 hours at room temperature with recombinant phage in 500 mmol/L NaCl, 50 mmol/L Tris, pH 7.4, 3% (wt/vol) HSA, and 0.5% (vol/vol) Tween-20.
Bound phages were detected by anti-M13 antibody peroxidase
conjugate.28 Experiments were performed in duplicate, and
values were expressed as percentages of maximum binding.
Characterization of anti-factor VIII antibodies in patient's plasma Previously, we reported on the domain specificity of anti-factor VIII antibodies in a patient (AMC-67) with mild hemophilia A caused by an Arg593Cys substitution.22 The
patient had a transient inhibitor with a maximum titer of 250 BU/mL.
Plasma and peripheral blood mononuclear cells were isolated from blood
samples collected when the inhibitor reached its peak value. Most
factor VIII inhibitory antibodies in the patient's plasma were
directed against the A2 domain. Here, we evaluated binding of these
antibodies to a hybrid factor VIII/factor V recombinant A2 domain in
which residues Arg484-Ile508 were substituted
for the corresponding sequence of factor V. Immunoprecipitation
analysis revealed that antibodies in the patient's plasma did not
react with A2-FV484-508 (Figure 1A).
Subsequently, an inhibitor neutralization assay using this fragment was
performed. Limited neutralization was observed with the addition of
A2-FV484-508, whereas the A2 domain almost completely neutralized
factor VIII inhibitory activity (Figure 1B). These findings suggest
that approximately 70% of the factor VIII inhibitory antibodies in the
plasma of this patient are directed toward an epitope consisting of
residues Arg484-Ile508.
Isolation and sequence analysis of antibodies directed toward the factor VIII heavy chain V gene phage display was used to isolate human antibodies reactive with the factor VIII heavy chain from the immunoglobulin repertoire of the patient. Isotyping revealed that the factor VIII heavy chain-specific antibodies in the patient's plasma consisted predominantly of subclass IgG4 (data not shown). Therefore, a subclass-specific oligonucleotide primer was used for amplification of the patient's IgG4 VH gene repertoire. The IgG4-enriched VH gene repertoire was recombined with a nonimmune VL gene repertoire in pHEN-1-Vlrep, resulting in a library of 1.9 × 107 clones. To isolate anti-A2 antibodies, the library was selected for binding to the factor VIII heavy chain. After the third round of selection, phages derived from 40 single clones were analyzed for binding to the factor VIII heavy chain. Twenty-six of 40 clones reacted with factor VIII heavy chain (data not shown).
Biochemical characterization of VK34 and VK41
Epitope mapping studies revealed that a significant portion of
factor VIII inhibitors binds to the A2 domain of factor
VIII.3 Within the A2 domain, residues
Arg484-Ile508 constitute a major determinant
of the epitope of factor VIII inhibitors.11,12 In this
study, we selected a phage display library of the IgG4-restricted
VH gene repertoire derived from a patient with anti-A2
inhibitor for binding to the heavy chain of factor VIII. Two different
antibodies (VK34 and VK41) reactive with the factor VIII heavy
chain were isolated. Epitope mapping revealed that clone VK34 was
directed toward the amino acid residues Arg484-Ile508 in the A2 domain. Antibodies
directed toward this region account for most factor VIII inhibitory
activity in the patient's plasma (Figure 1B). Furthermore, our study
provides evidence for an additional binding site for anti-factor VIII
antibodies in the a2 region, which comprises amino acid
residues Asp712-Ala736. So far, anti-A2
antibodies are predominantly directed toward a major binding site that
has been attributed to the region
Arg484-Ile508.11,12,14 Anti-A2
inhibitors have been studied in functional assays, which only detect
inhibitory anti-factor VIII antibodies.11,12 Because scFv
VK41 does not inhibit factor VIII activity, antibodies in patient
plasma corresponding to VK41 may have escaped detection using these
assays. This may explain why amino acid region
Asp712-Ala736 has not been identified
previously as a binding site for anti-factor VIII antibodies.
Alternatively, the plasma concentration of IgG corresponding to VK41
may be low in patients with an inhibitor. Competition experiments
indicated that IgG in the patient's plasma was able to compete for
binding to factor VIII heavy chain by scFv VK41 (data not shown). These
findings suggest that IgG corresponding to VK41 is present in
significant amounts in the plasma of patient AMC-67.
The authors thank G. van Stempvoort and P. H. N. Celie for providing
the purified factor VIII variants and factor VIII heavy chain. They
also thank R. C. Aalberse, W. G. van Aken, J. A. van Mourik, and K. Mertens for critical review of the manuscript.
Submitted January 14, 2000; accepted March 6, 2000.
Reprints: Jan Voorberg, Department of Plasma Proteins, CLB,
Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: j_voorberg{at}clb.nl.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Presented in part at the 41st Annual Meeting of the American Society of
Hematology, December 3-7, 1999, New Orleans, LA.
1.
Lenting PJ, van Mourik JA, Mertens K.
The life cycle of coagulation factor VIII in view of its structure and function.
Blood.
1998;92:3983-3996
2.
Hoyer LW.
Hemophilia A.
N Engl J Med.
1994;330:38-47
3.
Prescott R, Nakai H, Saenko EL, et al.
The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies.
Blood.
1997;89:3663-3671
4.
Scandella D, Gilbert GE, Shima M, et al.
Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site.
Blood.
1995;86:1811-1819
5.
Healey JF, Barrow RT, Tamim HM, et al.
Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.
Blood.
1998;92:3701-3709
6.
Arai M, Scandella D, Hoyer LW.
Molecular basis of factor-VIII inhibition by human antibodies-antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid.
J Clin Invest.
1989;83:1978-1984.
7.
Shima M, Scandella D, Yoshioka A, et al.
A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine.
Thromb Haemost.
1993;69:240-246[Medline]
[Order article via Infotrieve].
8.
Fijnvandraat K, Celie PHN, Turenhout EAM, et al.
A human allo-antibody interferes with binding of factor IXa to the factor VIII light chain.
Blood.
1998;91:2347-2352
9.
Zhong D, Saenko EL, Shima M, Felch M, Scandella D.
Some human inhibitor antibodies interfere with factor VIII binding to factor IX.
Blood.
1998;92:136-142
10.
Lenting PJ, Van de Loo JWP, Donath MJSH, van Mourik JA, Mertens K.
The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX.
J Biol Chem.
1996;271:1935-1940
11.
Healey JF, Lubin IM, Nakai H, et al.
Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII.
J Biol Chem.
1995;270:14505-14509
12.
Lubin IM, Healey JF, Barrow RT, Scandella D, Lollar P.
Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis.
J Biol Chem.
1997;272:30191-30195
13.
Lollar P, Parker ET, Curtis JE, et al.
Inhibition of human factor VIIIa by anti-A2 subunit antibodies.
J Clin Invest.
1994;93:2497-2504.
14.
Fay PJ, Scandella D.
Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and IXa.
J Biol Chem.
1999;274:29826-29830
15.
van den Brink EN, Turenhout EAM, Davies J, et al.
Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes.
Blood.
2000;95:558-563
16.
Lenting PJ, Donath MJSH, van Mourik JA, Mertens K.
Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.
J Biol Chem.
1994;269:7150-7155
17.
Mertens K, Donath MJSH, van Leen RW, et al.
Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.
Br J Haematol.
1993;85:133-142[Medline]
[Order article via Infotrieve].
18.
Voorberg J, van Stempvoort G, Klaasse Bos JM, Mertens K, van Mourik JA, Donath MJSH.
Enhanced thrombin sensitivity of a factor VIII-heparin cofactor II hybrid.
J Biol Chem.
1996;271:20985-20988
19.
Veltkamp JJ, Drion EF, Loeliger EA.
Detection of the carrier state in hereditary coagulation disorders. II.
Thromb Diath Haemorrh.
1968;19:403-422[Medline]
[Order article via Infotrieve].
20.
Kasper CK, Aledort LM, Counts RB, et al.
A more uniform measurement of factor VIII inhibitors.
Thromb Diathes Haemorrh.
1975;34:869-872[Medline]
[Order article via Infotrieve].
21.
Fijnvandraat K, Turenhout EAM, van den Brink EN, et al.
The missense mutation Arg593
22.
van den Brink EN, Timmermans SMH, Turenhout EAM, et al.
Longitudinal analysis of factor VIII inhibitors in a previously untreated mild haemophilia A patient with an Arg593
23.
Griffin HM, Ouwehand WH.
A human monoclonal antibody specific for the leucine-33 (p1A1, HPA-1a) form of platelet glycoprotein IIIa from a V gene phage display library.
Blood.
1995;86:4430-4436
24.
Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G.
By-passing immunization: human antibodies from V-gene libraries displayed on phage.
J Mol Biol.
1991;222:581-597[Medline]
[Order article via Infotrieve].
25.
Tomlinson IM, Williams SC, Ignatovitch O, Corbett SJ, Winter G.
V Base Sequence Directory. Cambridge, UK: MRC Centre for Protein Engineering; 1999.
26.
Griffiths AD, Williams SC, Hartley O, et al.
Isolation of high affinity human antibodies directly from large synthetic repertoires.
EMBO J.
1994;13:3245-3260[Medline]
[Order article via Infotrieve].
27.
Schier R, Marks JD, Wolf EJ, et al.
In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library.
Immunotechnology.
1995;1:73-81[Medline]
[Order article via Infotrieve].
28.
McCafferty J, Griffiths AD, Winter G, Chiswell DJ.
Phage antibodies: filamentous phage displaying antibody variable domains.
Nature.
1990;348:552-554[Medline]
[Order article via Infotrieve].
29.
Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C.
Sequences of Immunological Interest. 5th ed. Bethesda, MD: US Department of Health and Human Services; 1991.
30.
Pittman DD, Wang JH, Kaufman RJ.
Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.
Biochemistry.
1992;31:3315-3325[Medline]
[Order article via Infotrieve].
31.
Foster PA, Fulcher CA, Houghten RA, de Graaf Mahoney S, Zimmerman TS.
Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain.
J Clin Invest.
1988;82:123-128.
32.
Barrow RT, Healey JF, Gailani D, Scandella D, Lollar P.
Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules.
Blood.
2000;95:564-568
33.
de Wildt RMT, Hoet RMA, van Venrooij WJ, Tomlinson IM, Winter G.
Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire.
J Mol Biol.
1999;285:895-901[Medline]
[Order article via Infotrieve].
34.
Wu TT, Johnson G, Kabat EA.
Length distribution of CDRH3 in antibodies.
Proteins.
1993;16:1-7[Medline]
[Order article via Infotrieve].
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Top
Abstract
Introduction
) and A2-FV484-508
(
). After incubation for 1 additional hour at 37°C in the
presence of normal plasma, residual factor VIII activity was determined
relative to a sample that was incubated in the absence of an
inhibitor.
3 gene family (Figure 
