Haematologica 2000;85:supplement to no. 10 - Supplements ...

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M.D. Kazatchkine et al.
normal limits because the concentration of natural
antibodies with inhibitor activity is low, and
because of an effective peripheral regulation of
autoantibody activity by natural anti-idiotypes.
There is also some suggestion that natural anti-
FVIII antibodies with neutralizing properties are
more effective against allogeneic than autologous
FVIII.
The presence of anti-anti-FVIII anti-idiotypes
in normal plasma is evidenced by the characterization
of such antibodies in IVIg. 3,4 The antiidiotypes
may be readily isolated from IVIg by
affinity-chromatography on Sepharose-bound
affinity-purified anti-FVIII antibodies. The presence
of anti-idiotypes in normal plasma was also
directly evidenced by the observation by Gilles et
al., that IgG purified from the plasma of healthy
individuals is recognized by mouse monoclonal
anti-FVIII antibody when the plasma is depleted
of natural anti-FVIII IgG by affinity-chromatography
on insolubilized FVIII. 5 The anti-FVIII
depleted fraction of IgG was further shown to
inhibit the binding of both mouse monoclonal
and human polyclonal anti-FVIII antibodies to
FVIII.
Idiotypic regulation of FVIII inhibitors
FVIII inhibitors arise as alloantibodies in the
plasma of patients with hemophilia A in response
to multiple infusions of FVIII or develop spontaneously
as autoantibodies in non-hemophilic
patients. FVIII inhibitors block functional epitopes
of the FVIII molecule by steric hindrance.
FVIII inhibitors that bind to the heavy-chain of
FVIII prevent the cleavage of the FVIII molecule by
thrombin. Light-chain-specific inhibitors prevent
the interaction of FVIII with activated factor IX or
phospholipids and with von Willebrand factor
(vWF). FVIII inhibitors may also bind to epitopes
expressed by the complex of FVIII and vWF, and
reduce the dissociation rate of FVIII from vWF.
We have recently shown that inhibitors may also
neutralize FVIII activity by proteolytic cleavage of
the FVIIII molecule. In addition to antibodies with
neutralizing properties, the plasma of inhibitorpositive
patients contains anti-FVIII antibodies to
non-functional determinants of the FVIII molecule.
Mapping of the epitopes recognized by anti-
FVIII antibodies has not revealed any particular
restriction in the number and location of FVIII
epitopes recognized by FVIII inhibitors. 6 Clusters
of B-cell epitopes have been delineated: a 18.3-
kD amino-terminal segment of the A2 domain on
the heavy chain (amino acids 373-740) , epitopes
in the A3 domain, in the C1 domain and in the
C2 domain. Antibodies to FVIII vary from one
patient to another with regard to epitope specificity,
with anti-FVIII antibodies in a single patient’s
plasma that react with epitopes in the heavy
chain, the light chain, or both chains. In some
patients, FVIII inhibitors show a change of specificity
over time, which may be a consequence of
determinant spreading, as has been described in
other autoimmune conditions. We have found
that the patterns of reactivity of FVIII inhibitors
with FVIII, as analyzed by immunoblotting, do
not differ significantly from those of natural
autoantibodies to FVIII isolated from normal
plasma. Immunoprecipitation experiments confirmed
the heterogeneity of both natural anti-FVI-
II antibodies and FVIII inhibitors and did not
allow natural anti-FVIII antibodies to be discriminated
from inhibitors isolated from patients'
plasma.
Several years ago we demonstrated that the
spontaneous recovery of anti-FVIII autoimmune
disease is associated with the generation of antiidiotypic
antibodies capable of neutralizing the
inhibitory activity of autologous anti-FVIII
autoantibodies from the acute phase of the disease.
7 F(ab')2 fragments prepared from a patient's
post-recovery IgG were also shown to neutralize
the FVIII inhibitory activity of IgG of two
other patients with anti-FVIII autoimmune disease,
suggesting that anti-FVIII autoantibodies
share recurrent idiotypes. The presence of private
idiotypes on anti-FVIII autoantibodies was
evidenced by the higher capacity of Sepharosebound
post-recovery anti-idiotypic antibodies
to retain autologous anti-FVIII inhibitors as
compared with inhibitors of other patients.
Post-recovery anti-idiotypes were not, however,
capable of retaining the inhibitory activity of
alloantibodies from a patient with hemophilia
A, suggesting that allo- and autoantibodies do
not share cross-reactive idiotypes.
Tolerance induction is successful in 60 to 80%
of hemophilic patients with FVIII inhibitors.
Gilles et al. elegantly demonstrated that successful
desensitization in hemophilia patients is
not associated with a reduction in anti-FVIII IgG
alloantibodies but that tolerization with FVIII is
primarily eliciting anti-idiotypic antibodies capable
of neutralizing the inhibitory activity of autologous
anti-FVIII alloantibodies. 8 Anti-idiotypic
antibodies, generated in hemophilia patients
undergoing tolerance induction, were further
shown to inhibit the binding of autologous anti-
FVIII alloantibodies to FVIII in competitive binding
assays.
IVIg has been shown to result in a rapid and
prolonged suppression of FVIII inhibitory activity
in patients with anti-FVIII autoimmune disease.
Several lines of evidence demonstrated that
IVIg contains anti-idiotypic antibodies to anti-
FVIII autoantibodies: F(ab')2 fragments of IVIg
dose-dependently inhibited anti-FVIII activity in
Haematologica vol. 85(supplement to n. 10):October 2000