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

Immune Tolerance and the Treatment of Hemophilacs with an Inhibitor 27
differing immune settings that give rise to FVIII
alloantibodies and autoantibodies.
The C2 epitope
The hybrid human/porcine FVIII approach has
been used to map the C2 inhibitor epitope. 10 A
hybrid containing a substitution of porcine
sequence for Glu2181-Val2243 in the human C2
domain, designated HP24, is less antigenic than
human FVIII toward five C2-specific human antibodies
and a murine anti-FVIII monoclonal antibody,
NMC-VIII/5. Thus, a major FVIII inhibitor
epitope determinant is bounded by Glu2181-
Val2243 at the NH 2 -terminal end of the C2
domain.
This result was unexpected because earlier
studies had indicated that the COOH-terminal
end of the C2 domain contains the inhibitory
antigenic site. 11-14 However, the NH 2 -terminal
and COOH-terminal sequences of the C2
domain may be close to one another in space
and form a single epitope. This may be the case
because residues Cys2174-Cys2326, which are
at opposite ends of the C2 domain, are disulfide
bonded. 15
Coagulation factor V is homologous to FVIII.
Hybrid factor V/FVIII proteins that contain substitutions
of FVIII in the factor V C2 domain have
been used to map the epitope of an inhibitory
anti-factor V antibody that blocks phospholipid
binding to the NH 2 -terminal third of the C2
domain. 16 This result is consistent with FVIII
inhibitor epitope mapping studies and suggests
that the NH 2 -terminal region of the C2 domain
of both factors V and VIII contains an immunodominant
epitope that is important for phospholipid
binding.
Only one human monoclonal FVIII inhibitor
has been characterized, a human IgG4k antibody
produced by an immortalized B-cell line. 17 This
antibody recognizes the C2 domain of FVIII and
inhibits FVIII binding to both vWF and phospholipid.
The epitope recognized by this inhibitor has
not been mapped. Further analysis of monoclonal
inhibitors will be an important way to
determine whether there is heterogeneity in the
epitopes recognized within given FVIII domains.
The A3 epitope
An anti-A3 inhibitor was recently identified in
a patient with hemophilia A by deletion mapping,
which placed the epitope within an A3 segment
bounded by amino acids 1778-1823. 18
The antibody also inhibited the binding of factor
IXa to the FVIII light chain, which is necessary
for assembly of the intrinsic factor X activation
complex. 19 The FVIII light chain binding site for
factor IXa has been localized to amino acids
1811-1818 in the A3 domain. Consistent with
these observations, three inhibitor IgGs have
been identified that prevented the binding of
factor IXa to the FVIII light chain. 20 In that study,
the binding of these inhibitors to the FVIII light
chain was competed by a synthetic peptide corresponding
to A3 amino acids 1804-1819.
The ar3 epitope
We have recently identified an additional epitope
in the ar3 region using hybrid
human/porcine FVIII molecules. 21 We identified
patient plasmas that had activity against the
ar3-A3 region and tested their activities against
two hybrid human/porcine FVIII molecules designated
HP35 and HP41. Both molecules contained
porcine substitutions encompassing the
A2 and C2 epitopes to eliminate effects of antibodies
to these regions. HP35 contains the
human ar3 region and porcine A3 domain. Conversely,
HP41 contains the human A3 domain
and porcine ar3 region. The cross-reactivity of
seven patient plasmas against HP35 and HP41
were compared to that of human FVIII. In four
of the seven plasmas, HP35 was significantly
more cross-reactive than HP32. Conversely,
HP41 was less cross-reactive than HP33 in six of
the seven plasmas. These results are consistent
with the presence of an epitope in the human
ar3 region that is recognized by some inhibitor
plasmas.
Therapeutic implications
Plasma-derived porcine FVIII concentrates have
been used extensively as a low antigenicity product
to treat FVIII inhibitor patients. 22,23 A hybrid
human/porcine FVIII molecule containing
porcine substitutions at known antigenic sites
might provide a useful therapeutic alternative.
Anti-porcine FVIII antibodies that frequently
accompany porcine therapy 24 may be directed
toward epitopes that are distinct from the common
human FVIII epitopes. 25 Hybrid FVIII might
be superior to porcine FVIII because it avoids
exposure to porcine neoepitopes. Recombinant
hybrid FVIII would also provide a better alternative
because of potential infectious problems
inherent in the use of plasma-derived material. In
addition to reducing the antigenicity of FVIII, it
would be desirable to reduce its immunogenicity,
that is, to reduce its ability to be recognized
by the immune system. The immunogenicity of
a molecule depends on the B-cell repertoire, T-
cell help and suppression, and the major histocompatibility
complex, which together determine
the concentration and binding affinity of
antibodies for an antigenic site. An important
question regarding FVIII B-cell epitopes is
whether they are intrinsically immuno-dominant.
If so, then mapping and modification of
Haematologica vol. 85(supplement to n. 10):October 2000