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Blood, 1 July 2001, Vol. 98, No. 1, pp. 13-19
PLENARY PAPER
Structure of a factor VIII C2
domain-immunoglobulin G4 Fab complex: identification
of an inhibitory antibody epitope on the surface of factor
VIII
Paul Clint Spiegel Jr,
Marc Jacquemin,
Jean-Marie R. Saint-Remy,
Barry L. Stoddard, and
Kathleen P. Pratt
From the Graduate Program in Biomolecular Structure and
Design, University of Washington, and Division of Basic Sciences, Fred
Hutchinson Cancer Research Center, Seattle, WA; and Center for
Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus
Gasthuisberg, Leuven, Belgium.
 |
Abstract |
The development of an immune response to infused factor VIII is a
complication affecting many patients with hemophilia A. Inhibitor
antibodies bind to antigenic determinants on the factor VIII molecule
and block its procoagulant activity. A patient-derived inhibitory
immunoglobulin G4 antibody (BO2C11) produced by an immortalized
memory B-lymphocyte cell line interferes with the binding of factor
VIII to phospholipid surfaces and to von Willebrand factor. The
structure of a Fab fragment derived from this antibody complexed with
the factor VIII C2 domain was determined at 2.0 Å resolution. The Fab
interacts with solvent-exposed basic and hydrophobic side chains that
form a membrane-association surface of factor VIII. This atomic
resolution structure suggests a variety of amino acid
substitutions in the C2 domain of factor VIII that might prevent the
binding of anti-C2 inhibitor antibodies without significantly
compromising the procoagulant functions of factor VIII.
(Blood. 2001;98:13-19)
© 2001 by The American Society of Hematology.
| |
Introduction |
Factor VIII is a large, 2332-residue plasma
glycoprotein that acts as a regulatory cofactor in the process of blood
coagulation.1-3 It binds to activated factor IX (factor
IXa) in the presence of calcium and negatively charged phospholipids
that are presented at the surface of activated platelets to form a
membrane-associated, proteolytically active complex. Upon complex
formation, the Vmax (maximum velocity) of factor
IXa is increased by approximately 200 000-fold, promoting the rapid
activation of its substrate, the serine protease factor X. The
proteolytic conversion of factor X to its active form, Xa, is a central
control point in the coagulation cascade, leading to activation of
thrombin, formation of a fibrin mesh, and establishment of a stable
blood clot. The binding of factor VIIIa and other activated proteins to
these membrane surfaces allows for localization of procoagulant
processes to sites of vascular damage.
The factor VIII sequence contains 6 sequential domains arranged in the
order A1-A2-B-A3-C1-C2.4-6 The A domains are homologous to
one another and display sequence similarity to the copper-binding protein ceruloplasmin. They are flanked by short spacer sequences that
are highly acidic. The C domains are also homologous to each other and
have a weak homology to the discoidin protein fold family (eg, the
lipid-binding domain of galactose oxidase).7,8 The circulating form of the factor VIII protein is a metal bridged heterodimer consisting of a heavy chain (A1-A2-B) and a light chain
(A3-C1-C2). This form of factor VIII is bound tightly to von Willebrand
factor (vWF). Factor VIII is processed further by specific thrombin
cleavages into a heterotrimeric form. This active form, factor
VIIIa, dissociates from vWF and binds to negatively charged
phospholipids on activated platelet surfaces. The carboxyl terminal C2
domain of factor VIII contains binding sites for vWF and for negatively
charged phospholipids. The binding of factor VIIIa to membranes
involves stereoselection for O-phospho-L-serine, the
negatively charged head group of phosphatidylserine
(PS).9 The binding of factor VIII or VIIIa to vWF or PS is
mutually exclusive, even though the activation of factor VIII involves
cleavages outside the C2 domain. This implies that the binding sites
for vWF and PS overlap on the C2-domain surface.
Hemophilia A is a congenital bleeding disorder that is due to
deleterious factor VIII gene mutations. These mutations may block
factor VIII expression or secretion and may involve premature truncations, sequence rearrangements, or single-residue substitutions. To date, a total of 28 missense mutations at 21 different amino acids
within the C2 domain have been associated with hemophilia A.10 The crystal structure of the factor VIII C2 domain
was reported recently.11 A mechanism for the interaction
of factor VIII with negatively charged phospholipid surfaces, as well
as explanations for the molecular basis of hemophilia A point mutations localized to this domain, were proposed on the basis of this
study.10,11
Current therapy for patients with hemophilia A involves therapeutic
infusions of factor VIII. Inhibitory antibodies against factor VIII,
which may occur transiently or may persist as a serious long-term
complication, are generated in up to 35% of patients with severe
hemophilia A.12-16 These patients generally have very low
or undetectable factor VIII antigen. Patients who have mild or moderate
hemophilia A, which is usually associated with missense mutations, can
also develop inhibitory antibodies. This is less common and occurs in
3% to 13% of patients.17,18 Antibody inhibitor development has been associated with the mutations R593C and W2229C (single-letter amino acid code), with about a 40% inhibitor incidence for each mutation. Autoantibodies to factor VIII can also develop in
postpartum women or in individuals with various underlying disease
states, but this is very rare, as it occurs in one per million
individuals.19,20
The primary antigenic epitopes on factor VIII have been localized to
the A2 and C2 domains.21-26 The A3 and C1 domains and the
acidic region between A1 and A2 have also been implicated, although
epitopes in these regions occur less frequently.27,28 Inhibitory antibodies against the A2 domain generally allow factor VIII
to form complexes with vWF or factor IXa, but the proteolytic activation of factor X is blocked.13 In contrast,
inhibitory antibodies specific to the C2 domain have been shown to
prevent the binding of factor VIII to vWF and to membrane surfaces that expose PS. Two studies have localized a C2-domain epitope to the regions between residues 2181 to 224322 and 2248 to
2312.22,29 Blocking the interaction between factor VIII
and vWF greatly reduces the half-life of factor VIII in the
circulation, while antibodies that prevent the binding of factor VIIIa
to membrane surfaces abolish its procoagulant cofactor function.
To gain further insight into the interaction of autoantibodies and
alloantibodies against factor VIII at the molecular level, a factor
VIII-specific human IgG4 antibody (BO2C11) was produced by a cell
line derived from the memory B-cell repertoire of a patient with
hemophilia A with a strong inhibitor response.30 Biosensor
measurements indicated that although the association rate of factor
VIII with BO2C11 is slower than that measured for factor VIII binding
to vWF, the dissociation rate of the factor VIII:BO2C11 complex is
100-fold slower than that of the factor VIII:vWF
complex.30 Thus, the current working model for the inactivation of factor VIII by BO2C11 is that the antibody forms a
tight, stable complex with factor VIII (or factor VIIIa) as it
dissociates from vWF. The antibody blocks the C2-domain
membrane-binding site, thus effectively sequestering factor VIII and
neutralizing its procoagulant effects. The antibody complex may also
lead to accelerated clearance of the bound factor VIII, but this has
not been measured directly.
| |
Materials and methods |
The recombinant C2 domain was expressed and purified as
described previously.11 The factor VIII
C2-domain-specific human IgG4 monoclonal antibody (BO2C11)
was digested with papain, and the Fab fragment was purified using
standard methods. Approximately equimolar amounts of Fab and C2 protein
were combined in 10 mM HEPES, 0.1 M NaCl, pH 7.5, at approximately 10 mg/mL total protein concentration. Crystals were grown at room
temperature in 16% polyethylene glycol (PEG) 8000, 0.1 M HEPES
(pH 7.0), 0.2 M NaCl. The crystals were flash frozen in the same
solution, plus a final concentration of 25% vol/vol PEG 400 as
cryoprotectant. Data were collected to 2.0 Å resolution at the
Advanced Light Source (Berkeley, CA) beamline 5.0.2 and processed using
Denzo/Scalepack program suite.31 The space group
was P212121 with unit cell
parameters a = 64.5 Å, b = 73.7 Å, and
c = 112.4
Å. The structure was solved by molecular replacement with the use of
EPMR version 2.1.32 The models used for molecular
replacement during independent, iterative searches were the recombinant
factor VIII C2 domain,11 the constant domain of a human
IgG4 Fab (pdb acc.: 1BBJ), and a molecular model of the variable
domain that used optimal CDR loop length and sequence alignment (pdb:
1GC1 and 1AD9). The constant-domain search model was found by
searching the Protein Data Bank for a structure of a Fab of homologous
classification. The heavy and light variable-chain sequences were used
independently to locate optimal search models, and the program Blast
(Basic local alignment search tool) was used to search the Protein Data
Bank (the VL and VH sequences are in the
European Molecular Biology Laboratory (EMBL) Nucleotide Sequence
Database under the accession numbers AJ224084 and AJ224083,
respectively). As each phase solution was determined, it was
fixed in orientation as subsequent rounds were performed. Model
building of all deleted loops and side chains was performed with
XFIT33 version 3.7, and the structure was refined using
CNS34 after removing 10% of the data for cross-validation purposes. The final model consists of 564 amino acids and 477 water molecules. The Rcryst and Rfree both
decreased at every step of the refinement, and the final
Rcryst was 20.4% and Rfree was
25.5%. The stereochemical validity of the final model was verified using Procheck.35 Data and refinement
statistics are shown in Table 1.
| |
Results |
The structure of the C2-domain-BO2C11 Fab complex was
solved to 2.0 Å resolution (Table 1). The factor VIII C2-domain
construct consists of residues 2171 to 2332. The heavy and light chains of the Fab fragment consist of residues 1 to 211 and 1 to 212, respectively. Electron density of the entire complex was of excellent quality except for 3 residues at the C2-domain amino and carboxyl termini, 4 residues in the light chain, and 13 residues in the heavy
chain. None of these residues was involved in the binding interface.
The crystallographic model contains a total of 477 water molecules, 41 of which reside in the binding region of the complex. The core
structure of the C2 domain is completely conserved, displaying an
8-stranded  -sandwich fold. The amino and carboxyl terminal regions
of the C2 domain are linked by an internal disulfide bond joining C2174
to C2326. Two exposed hydrophobic turns and an underlying pair of
basic residues (R2215 and R2220), all previously hypothesized to
participate in the C2-membrane-binding site, formed critical
interactions at the antibody interface (Figures
1 and 2;
Table 2). An area of approximately
1200 Å2 on the surface of each molecule is buried in the
protein interface. The C2-epitope surface is basic in character,
whereas the Fab surface is quite acidic at the interface, indicating a
favorable overall charge complementarity (Figure
3).
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| Figure 1.
Stereo ribbon diagram of the
factor VIII C2-domain/BO2C11 Fab complex.
The light chain, heavy chain, and C2 domain are displayed in blue,
magenta, and green, respectively. The BO2C11 Fab displays a typical
immunoglobulin fold with 18 strands in the light chain and 19 strands in the heavy chain. Two hairpins from the C2 domain project
into the CDR loops of the Fab fragment.
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| Figure 2.
Electron density at residues involved in critical interactions of the
binding interface.
The first antigenic peptide of C2 (residues 2250 to 2253),
corresponding to an exposed hydrophobic turn containing L2251 and
2252, forms primarily hydrophobic interactions with residues from
CDR-H1, CDR-H3, and the amino terminus of the heavy chain (A). The
second antigenic region from C2 (residues 2197 to 2203), corresponding
to a second exposed hydrophobic turn containing M2199 and F2200,
exhibits more extensive van der Waals contact with the Fab surface, and
there are more polar interactions than in the first epitope (B). Two R
residues from the C2 domain, both of which are proposed to interact
with anionic lipid head groups when factor VIII binds to platelet
membranes, form salt links with D residues on the Fab surface. R2220
lies within a cleft between the 2 hydrophobic hairpin turns (C) and
interacts with D102 of the heavy chain. R2215 resides on the third loop
at this end of the C2 molecule and interacts with D52 of the heavy
chain (D). An adjacent loop contains residues Q2222 and V2223, which
contact the Fab surface directly (E). V2223 is another hydrophobic
residue that was solvent-exposed in the free C2 structure, and it was
proposed that it may make additional contacts with membrane surfaces.
Finally, residues H2315 and Q2316 participate in polar interactions
with specific residues and buried water molecules at the C2-Fab
interface (F). See Table 2 for a list of all contacts in this
interface.
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| Figure 3.
Molecular surface
representation of the surfaces that interact in the BO2C11-C2-domain
complex.
The individual proteins have been separated and rotated to allow the
reader to view the complementary, buried protein interfaces. The
predominantly negative charge (red) of the Fab fragment interacts
favorably with the positively charged patches (blue) on the surface of
the C2 domain.
|
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The fold of the C2 domain is similar in the free and Fab-bound forms,
with an all-atom rms difference of 0.33 Å. However, there is a shift
in the position of the hydrophobic -hairpin loop containing M2199
and F2200. The backbone of this loop region twists significantly,
allowing the side chains to form specific interactions at the binding
interface (Figure 4). The analogous region of the factor V C2 domain has been visualized in 2 different conformations,36 indicating that the membrane-binding
region has some inherent flexibility.
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| Figure 4.
Stereo diagram of superimposed
structures of the recombinant factor VIII C2 domain in the free and
antibody-bound crystal forms.
(A) The free and Fab-complexed C2 proteins are shown in blue and green,
respectively. The structure of the core is virtually identical in
the 2 structures. Most of the deviations between the structures occur
at or near the 2 hydrophobic -hairpin turns at the bottom of the
figure. The plane formed by the 2 strands of the second hairpin
(residues 2197 to 2203) moves to a position perpendicular to that seen
in the unbound C2-domain structure. The  angle of M2199 rotates from
 11° to +122°, whereas in F2200, the  and angles rotate
from 99° to +56° and from 23° to +26°, respectively. This
region interacts with residues in CDR-H1, CDR-H2, and CDR-H3 of the
heavy chain as well as with sites on CDR-L1 and CDR-L3 of the light
chain. (B) Rotations about backbone dihedral angles in the hairpin
containing residues N2198 through A2201 shift the orientation of these
side chains by up to 5 Å.
|
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Epitope mapping using fragments of the factor VIII C2 domain indicated
that BO2C11 does not recognize a linear epitope corresponding to a
single stretch of amino acid residues.30 Rather, it
interacts with a conformational epitope composed of side chains from
various regions within the C2 sequence that are adjacent to each other in the folded protein. These results are in agreement with the interactions present in this crystal structure, as illustrated in
Figure 2 and listed in Table 2. Two -turn regions (residues 2250 to
2253 and 2197 to 2203) and an additional loop containing residues Q2222
and V2223 present hydrophobic residues at the protein surface. These
residues interact with residues from multiple CDRs on the heavy and
light chains of the Fab and the amino terminus of the heavy chain.
These loop regions of factor VIII represent the largest amount of
surface area buried within the antibody interface. Two basic residues
from the C2 protein, R2215 and R2220, are postulated to interact with
phospholipid head groups. These residues form salt links with D52 and
D102 of the heavy chain, respectively. Finally, residues H2315 and
Q2316 participate in polar interactions with specific residues and
buried water molecules at the antibody interface.
| |
Discussion |
The BO2C11 IgG antibody binds to the C2 domain of factor VIII,
inhibiting its ability to bind negatively charged phospholipids and
vWF.30 The evidence strongly suggests that the BO2C11
antibody masks the membrane-binding surface of the C2 domain and thus
prevents binding to negatively charged phospholipids. The
membrane-binding surface of factor VIII has been proposed to consist of
2 -hairpins turns and an adjacent loop. This region displays 5 exposed hydrophobic residues that are available to participate in
membrane binding (L2251, L2252, M2199, F2200, and possibly V2223). Four
of these hydrophobic residues are buried in the Fab complex, whereas
V2223 is located immediately adjacent to the interface and is in
contact with the Fab molecule. A ring of positively charged basic
residues is positioned to interact with charged phospholipid head
groups upon burial of the hydrophobic side chains in the lipid bilayer. Two of these residues, R2215 and R2220, are buried in the Fab interface, where they form salt bridges with D residues in BO2C11 (Figure 2).
The large contact surface between the C2 domain and BO2C11 is in
agreement with the high affinity of factor VIII binding to BO2C11, as
measured by surface plasmon resonance. The latter showed an
antibody-factor VIII association rate constant of
7.4 × 105 · M 1 · s1 and dissociation rate constant of less than or equal
to 1 × 105 · s1, with a
calculated apparent dissociation constant of
1.4 × 1011 · M1.30
Anti-factor VIII antibodies were initially distinguished according to
the kinetics of factor VIII inactivation.37,38 In 1982, Gawryl and Hoyer39 delineated 2 populations of such
antibodies: type I antibodies inactivate factor VIII completely,
following second-order kinetics; whereas type II inhibitors inactivate
factor VIII only partially, even when added in large excess over factor VIII, and follow more complex kinetics. Later, Biggs40
observed that even antibodies completely inactivating plasma factor
VIII frequently follow complex kinetics of interaction with factor VIII, thereby identifying an intermediate category between type I and
type II inhibitor antibodies. As a consequence of their particular
kinetics of factor VIII inhibition, such antibodies can be difficult to
quantify in plasma using the conventional Bethesda
method.41
Competition with vWF for factor VIII binding was first described as the
mechanism responsible for the type II kinetic pattern of many type II
inhibitor antibodies, which in the absence of vWF completely inhibited
factor VIII activity following second-order kinetics, much like type I
inhibitors.39 However, some type II inhibitors inactivate
factor VIII only when the latter is bound to vWF,21,42 and
recent observations have indicated that some type II antibodies inhibit
factor VIII partially even in the absence of vWF.27 The
population of type II inhibitors therefore appears to be heterogeneous.
The data on the crystal structure of the C2 domain in combination with
BO2C11 presented here shed light on the actual mechanisms by which the
antibody inhibits factor VIII function. BO2C11 inactivates factor VIII
following a kinetic pattern intermediate between that of type I and
type II inhibitors. With excess of antibody, factor VIII inactivation
by BO2C11 is complete.30 However, when analyzed as a
function of time, the kinetics of the reaction is different from that
of type I inhibitor antibodies; that is, the relation between the
logarithm of residual factor VIII activity and time is not linear
(M.J., unpublished results, September 1997). The likely explanation for
this observation is that BO2C11 is in competition with vWF for factor
VIII binding.30 In plasma, factor VIII is complexed to
vWF, which prevents BO2C11 binding. Over time, however, the factor
VIII/vWF complex dissociates, and BO2C11 binds to factor VIII in a
nearly irreversible manner.30 Indeed, although the association rate constant of BO2C11 is only 8-fold lower than that of
vWF for factor VIII, the dissociation rate constant of BO2C11 from
factor VIII is 100-fold lower than that of the vWF-factor VIII complex
as estimated by Vlot et al.43 Therefore, given the rapid
spontaneous dissociation of the factor VIII/vWF complex, the protection
toward BO2C11-mediated inactivation provided by vWF is significant only
for a short time.44 Moreover, BO2C11 can bind and
inactivate factor VIIIa upon its dissociation from vWF, thereby
preventing the binding of factor VIIIa to phospholipids. This
phenomenon was previously shown to allow rapid inactivation of factor
VIII when BO2C11 was present at concentrations of several hundred
Bethesda units.30,44 In addition, given the importance of
vWF for factor VIII stability in plasma, it is possible that factor
VIII bound to BO2C11 is cleared more rapidly from the
circulation.45-47 It is noteworthy that polyclonal
anti-factor VIII antibodies of the patient from whom the cell line
producing BO2C11 was derived inactivate factor VIII following a type I
pattern (M.J., unpublished data, September 1997). This probably occurs
because in addition to antibodies toward the C2 domain, this patient's
plasma contains antibodies recognizing antigenic determinants in other
regions of the factor VIII molecule, including the heavy
chain.48
The observed competition between the BO2C11 antibody and vWF for
binding to factor VIII suggests the straightforward explanation that
the C2 domain visualized here either includes or overlaps with the
factor VIII binding site for vWF. However, caution must be exercised in
proposing structural explanations for the various binding experiments
because there are as yet no direct data on the structure of the
vWF-factor VIII complex. Two additional hypotheses, which do not
exclude the possibility that the C2-domain epitope identified here may
overlap the binding site for vWF, can be presented to account for the
competition between BO2C11 and vWF for binding to factor VIII. The
bound BO2C11 antibody molecule may cause steric interference with the
binding of vWF, which is itself an extremely large, multimeric protein.
In other words, occlusion of the binding of 2 large proteins may not
involve a direct competition for the same surface on the C2 domain, but
could be due to interference elsewhere between the factor VIII or vWF
proteins. Alternatively, the binding of BO2C11 to the C2 domain may
prevent a conformational change within factor VIII that is necessary
for vWF binding. In support of the first possibility, indirect steric
interference between bound vWF and bound BO2C11, we note that the
binding of vWF involves other regions of factor VIII in addition to the
C2 domain. An acidic stretch of 41 amino acid residues at the amino terminal region of the light chain (A3-C1-C2) increases the affinity of
factor VIII binding to vWF. This peptide is removed during the
proteolytic activation of factor VIII. The proximity of this acidic
stretch to regions on the C2-domain surface is not yet known.
Several C2-domain missense mutations associated with hemophilia A
appear to affect vWF binding, indicating that these residues may
represent part of the vWF-binding surface. However, the effects of
these mutations, some of which affect bulky side chains (W2229, R2307,
and R2304), on the structure of factor VIII have not yet been
characterized. Therefore, it is not yet known whether the substitutions
alter merely the surface characteristics of the C2 domain or whether
they result in more profound effects on folding or secretion of the
factor VIII protein. The value of these mutations as a tool for mapping
the association surface of C2 with vWF awaits a more complete
characterization of the mutations themselves, and it may be possible to
investigate this further using recombinant factor VIII constructs. The
picture of the C2-domain epitope presented here can now be used to
inform additional, detailed mutational and biochemical studies that
will test the possible involvement of the epitope region in the binding
of vWF to factor VIII.
It is likely that the complex described here is similar to those formed
by the C2 domain with other anti-C2 inhibitor antibodies. The
equivalence of the epitope with the proposed membrane-binding region is
completely consistent with the observed blockage of membrane binding by
other anti-C2 inhibitor antibodies. Studies of unrelated antibody
complexes have shown that antigenic determinants on protein surfaces
may correspond to peptide regions having dynamic flexibility,
hydrophobicity, and/or significant solvent accessibility of side
chains.49-51 The C2 residues in contact with BO2C11 meet all of these criteria. Crystal structures of the C2 domains of factors
V and VIII have shown unequivocally that the -hairpin turns can
assume various orientations under different
conditions,11,36 and the crystal structures set only a
lower limit on the accessible range of motion available to these loops
as they interact with various surfaces. The epitope region is also
highly hydrophobic and presents a side-chain surface area that is
larger than that of other surface loops of similar length on the C2
domain. The membrane-binding surface of the C2 domain thus appears to
represent an antigenic "hotspot" on the factor VIII surface. A
sequence alignment of human and porcine factor VIII supports this
conclusion (Figure 5). Chimeric
human/porcine factor VIII molecules have been constructed that exhibit
less antigenicity against a variety of patient-derived antibody
inhibitors. Such chimeric proteins represent a promising approach to
improve treatment options for patients who develop inhibitory
antibodies.52 Inspection of the sequence alignment reveals
that there are significant differences between the human and porcine
sequences at and around the -hairpin turn containing M2199 and
F2200. Indeed, recent experiments using recombinant factor VIII
proteins incorporating point mutations in this region have shown that
the substitutions affected the antigenicity of factor VIII against both
polyclonal and monoclonal antibodies. These results are consistent with
the BO2C11 epitope identified here.53
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| Figure 5.
Sequence alignment of the C2 domains of human and
porcine factor VIII.
The regions corresponding to those in the C2-domain epitope in contact
with the BO2C11 Fab are underlined.
|
|
Only 2 point mutations associated with hemophilia A, A2201P and V2223M,
have been identified within the antibody interface identified here. The
patients carrying these mutations suffered from relatively mild
bleeding disorders.10 It was proposed previously that the
relative dearth of deleterious point mutations near the exposed
hydrophobic surface of the C2 domain reflected a redundant, protective
evolution of the membrane-binding region.10,11 The binding
energy to membrane surfaces was proposed to derive from a combination
of favorable solvation changes upon insertion of hydrophobic residues
into a nonpolar lipid environment. Favorable electrostatic interactions
were also proposed between several basic residues and polar
phospholipid head groups. At least 10 to 12 amino acid side chains were
deemed likely to contribute to the interface with the membrane. Because
the free energy of solvation changes involving hydrophobic moieties is
relatively insensitive to precise side-chain orientation, the
attachment of the C2 domain to membranes should readily accommodate
minor structural changes due to movement of the loops or substitutions of individual side chains. Similarly, the large number of residues involved in the membrane-protein interface would tend to minimize the
energetic penalty of mutating any individual side chain in this region.
In contrast, the binding of this C2 surface to protein ligands,
including vWF or inhibitor antibodies, may be more vulnerable to
conformational changes and to disruptions caused by point mutations. This may have positive implications for patients with hemophilia A with
an antibody inhibitor response. It should be possible to introduce
minor modifications to these loops that preclude an effective
antibody/antigen interaction but that result in a molecule that is
still competent to bind membranes and to carry out the critical
cofactor function of factor VIII in coagulation. It is well established
that effective hemostasis is possible even with a plasma concentration
of factor VIII as low as 10% of average levels. Below this level, an
increase in factor VIII concentration of even a few percentage points
can have a profound impact on the quality of life for the patient.
Thus, the design of a "hobbled" factor VIII, which may sacrifice
some membrane-binding capacity to decrease the antigenicity of the
infusion, may be a desirable and achievable goal. The identification
here of specific amino acid residues mediating an inhibitor antibody
response presents a compelling opportunity to further fine tune the
production of improved, recombinant "designer" factor VIII proteins
that are tolerated by a larger fraction of the patient population.
| |
Acknowledgments |
We thank Betty W. Shen for assistance at all stages of structure
determination; Eric Galburt, Roland Strong, Kam Zhang, and Adrian
Ferre-D'Amare for advice and discussion; Earl Davie and Kazuo Fujikawa
for assistance with the C2-domain purification and analysis;
Benoît Desqueper for production of BO2C11 Fab fragment; and
Thomas Earnest and staff at ALS beamline 5.0.2 for assistance with data collection.
| |
Footnotes |
Submitted December 19, 2000; accepted March 6, 2001.
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.
Reprints: Barry L. Stoddard, Fred Hutchinson Cancer Research
Center, Division of Basic Sciences, 1100 Fairview Ave North A3-023,
Seattle, WA 98109; e-mail: bstoddar{at}fhcrc.org.
| |
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Abstract
Introduction