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Chapter 135 Hemophilia A and B 2019

inhibitors, much less is known about the basic science of FIX inhibi- Every factor concentrate’s immunogenicity is initially evaluated in
tors than about FVIII inhibitors. There is, as previously described, a previously treated patients (PTPs, i.e., those with a minimum of 100
relationship between FIX inhibitors and anaphylaxis to FIX that to 150 prior exposure days to a FVIII or FIX concentrate). In studies
generally does not occur with FVIII inhibitors. of PTPs exposed to new factor concentrates, very few inhibitors have
FIX inhibitors are primarily IgG4 subclass antibodies although so far occurred. This suggests that in widespread clinical use, inhibi-
some are IgG2 subclass. tors will occur only rarely, if at all, in PTPs who switch to new
Epitopes for FIX inhibitors have been identified in the concentrates.
γ-carboxyglutamic acid domain, which is involved in binding to There is less experience using new concentrates in hemophilia
phospholipid surfaces, the serine protease domain, and possibly in patients that have not yet been exposed to a factor concentrate
the activation peptide region, at which FIX is activated by activated (PUPs). Since the risk of both FVIII and FIX inhibitors is greatest
FXI. As is the case with inhibitory FVIII antibodies, the specificity early in a patient’s treatment course, the immunogenicity of new
of these antibodies for epitopes in functionally important regions of concentrates in PUPs may differ from what occurs in PTPs.
the FIX molecule explains their ability to inhibit FIX’s coagulant There are some reasons to suspect that immunogenicity of new
activity. concentrates may not be worse than that of existing concentrates,
Because FIX inhibitors are antibodies of the IgG isotype, they are despite the presence of additional modifications such as Pegylation
products of interaction between FIX-specific B cells and helper T or fusion to Fc or albumin. For example, pegylated versions of other
cells, but this process is less well described than is the case for FVIII protein therapeutics (e.g., asparaginase, granulocyte-colony stimulat-
inhibitors. The roles of co-stimulatory molecules, immunoregulatory ing factor) have not proven to be more immunogenic than their
genes, and Tregs in FIX inhibitors have not been demonstrated. nonpegylated counterparts, and there is some animal evidence to
suggest that FVIII and FIX concentrates that have been fused to the
Fc region of IgG may be less immunogenic than standard FVIII or
Detection of Inhibitors and Antibodies FIX, perhaps because of interactions with Fc receptors present on
cells involved in immune responses. In addition to developing EHL
Inhibitors are detected using the Bethesda assay (see Chapter 136). factor concentrates some manufacturers are developing FVIII con-
The Bethesda assay will not, by definition, detect nonneutralizing centrates that are produced in human cell lines, rather than in cells
antibodies (i.e., those antibodies to FVIII or FIX that do not inhibit obtained from other mammalian species, with the hope that factor
the coagulant function of these proteins). New modifications of the produced in human cell lines will show reduced immunogenicity
Bethesda assay, including an “ultrasensitive” Bethesda assay that uses because of more “native” posttranslational modifications of the factor
concentrated patient plasma, are being developed and may improve protein.
the reliable identification of low-titer inhibitors (Fig. 135.12). Of course all of this remains speculative and the results of studies
Both neutralizing and nonneutralizing antibodies to FVIII or FIX of the immunogenicity of new factor concentrates in PUPs are eagerly
may also be detected by ELISA. This assay can only detect and awaited.
quantify the antibodies present and cannot determine their inhibitory
activity, but assays that identify the epitopes to which antibodies bind
can discriminate between antibodies that interfere with important Nonclotting Factor Concentrate Treatments
functional domains of the FVIII or FIX protein and those that do for Hemophilia
not. Techniques for measuring binding affinity of antibodies may also
be of clinical use in the future. Since about 2010, there has been significant interest in developing
novel strategies for treating hemophilia that do not rely upon clotting
factor replacement. In 2017 some of these therapies are just begin-
Immunogenicity of New Factor Concentrates ning to enter the clinic.
There are two principal approaches that are being used to enhance
Many new FVIII and FIX concentrates are in different stages of hemostasis through these alternative strategies: (1) through “rebalanc-
development for clinical use. In view of recent research that suggests ing hemostasis” by inhibiting natural anticoagulant pathways (e.g.,
the possibility of different rates of immunogenicity between different through tissue factor pathway inhibitor [TFPI] or AT inhibition) and
FVIII products that are currently in use, the immunogenicity of any (2) through the delivery of a bispecific antibody that mimics FVIII
new products will be of interest and importance. cofactor activity (Table 135.9).
The studies involving anticoagulant pathway inhibition have, to
date, focused on TFPI and AT, and have used contrasting methods
to reduce production or inhibit the function of these proteins. The
Patient Buffered FVIII- anti-AT approach has used the subcutaneous delivery of small inter-
31
plasma normal deficient fering RNA (siRNA) to reduce synthesis of the protein. The mag-
plasma plasma nitude of AT knock down is dose dependent, and the effect persists
pH, 7.4

Test Control TABLE Alternative, Nonclotting Factor Concentrate Therapies
mixture 50/50 mix mixture
135.9 for Treating Hemophilia
Incubate 2 hr @ 37° C 1. Rebalancing hemostasis strategies:
a. TFPI Inhibition approaches: anti-TFPI aptamer, antibody and
peptide.
b. Antithrombin siRNA biosynthesis inhibition.
One-stage FVIII assay with Both approaches can be delivered by infrequent (weekly or less
FVIII-deficient substrate plasma
often) subcutaneous injections.
2. Factor VIII mimetic molecule: bispecific antibody to FIXa and FX.
Calculate residual FVIII activity; derive Bethesda units Can be administered by subcutaneous injection and has shown
activity in FVIII inhibitor patients.
Fig. 135.12 THE NIJMEGEN-MODIFIED BETHESDA ASSAY. This is
the currently recommended methodology for quantifying anti–factor VIII FX, Factor X; FIXa, Factor IXa; FVIII, factor VIII; siRNA, small interfering
ribonucleic acid; TFPI, tissue factor pathway inhibitor.
(anti–FVIII) inhibitory antibodies.