Synthetic sorbents for removal of factor VIII inhibitors from haemophilic A plasma

Human factor VIII (FVIII) is a protein of the blood coagulation system that is absent or defective in patients with haemophilia A. A most serious complication following replacement therapy in 10–15% of patients treated with available FVIII concentrate is the development of inhibitors to FVIII (anti-FVIII). Some polymers functionalized with suitable chemical substituents which mimic part of the epitope of FVIII recognized by the inhibitors might be used in extracorporeal circulation to reduce the concentration of antibodies to FVIII. For this purpose, insoluble polystyrene bearing sulfonate and l-tyrosine methyl ester sulfamide groups have been synthesized. The in vitro removal of anti-FVIII inhibitors from plasmas of patients with haemophilia A was performed. Different chromatographic parameters were studied and optimized.     

Designed Ankyrin Repeat Proteins: A New Approach to Mimic Complex Antigens for Diagnostic Purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.     

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4⁺ T cell-dependent process. Activation of FVIII-specific CD4⁺ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.     

Factor VIII-hydrolyzing IgG in acquired and congenital hemophilia

Anti-factor VIII (FVIII) inhibitory IgG may arise as alloantibodies to therapeutic FVIII in patients with congenital hemophilia A, or as autoantibodies to endogenous FVIII in individuals with acquired hemophilia. We have described FVIII-hydrolyzing IgG both in hemophilia A patients with anti-FVIII IgG and in acquired hemophilia patients. Here, we compared the properties of proteolytic auto- and allo-antibodies. Rates of FVIII hydrolysis differed significantly between the two groups of antibodies. Proline-phenylalanine-arginine-methylcoumarinamide was a surrogate substrate for FVIII-hydrolyzing autoantibodies. Our data suggest that populations of proteolytic anti-FVIII IgG in acquired hemophilia patients are different from that of inhibitor-positive hemophilia A patients.     

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.     

Catalytic IgG from patients with hemophilia A inactivate therapeutic factor VIII

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.     

Factor VIII hydrolysis mediated by anti-factor VIII autoantibodies in acquired hemophilia

Acquired hemophilia is a rare hemorrhagic disorder caused by the spontaneous appearance of inhibitory autoantibodies directed against endogenous coagulation factor VIII (FVIII). Inhibitory Abs also arise in patients with congenital hemophilia A as alloantibodies directed to therapeutic FVIII. Both autoimmune and alloimmune inhibitors neutralize FVIII by steric hindrance. We have described FVIII-hydrolyzing IgG in 50% of inhibitor-positive patients with severe hemophilia A that inactivate therapeutic FVIII. In this study, we investigated the presence of autoimmune FVIII-hydrolyzing IgG in patients with acquired hemophilia. Pooled IgG from healthy donors demonstrated moderate FVIII-hydrolyzing activity (56 +/- 26 micromol/min/mol). Purified IgG from 21 of 45 patients with acquired hemophilia demonstrated FVIII hydrolysis rates (mean 219 +/- 94 micromol/min/mol) significantly greater than that of control IgG. Three of four patients followed over the course of the disease had rates of FVIII hydrolysis that co-evolved with inhibitory titers in plasma, suggesting that IgG-mediated FVIII hydrolysis participates, in part, in FVIII inactivation. The present work extends the scope of the diseases associated with FVIII proteolysis and points toward the importance of FVIII as a key target substrate for hydrolytic immunoglobulins. Our data suggest that elevated levels of FVIII-hydrolyzing IgG in acquired hemophilia result from the exacerbation of a physiological catalytic immune response.     

A state of acquired IL-10 deficiency in 0.4% of Danish blood donors

Autoantibodies against a variety of growth factors and cytokines are present in preparations of pooled normal human IgG, such as IVIg. The present study demonstrated that healthy Danish blood donors produced high concentrations of anti-IL-10 IgG antibodies that bound IL-10 with extremely high avidity. The antibodies were of IgG class, polyclonally derived and acted as competitive IL-10 inhibitors in vitro, substantially inhibiting cellular IL-10 receptor binding and neutralizing IL-10 activity in vitro. The antibodies failed to bind viral forms of IL-10 or other members of the human IL-10 family (IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, IL-29). The production of anti-IL-10 antibodies was stable from months to years, and high positive donors were likely to acquire a state of IL-10 deficiency in the circulation during this period. Anti-IL-10 antibodies were readily measurable even in highly diluted plasma samples, providing the explanation for the fact that relatively low antibody activity can be detected in normal human pooled IgG, derived from the plasma of over 1000 blood donors.     

Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis

Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL). The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60. In this study, B-cell epitopes on P. gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach. To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed. Deletion mutants were constructed from the cloned PgGroEL gene (P. gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants. Sera from periodontitis patients reacted with all truncated PgGroEL used in this study. The results suggest that the B-cell epitopes were overlaid throughout PgGroEL. To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined. Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies. Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects. IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients. Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.     

An immunogenic region within residues Val1670-Glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand factor

We have identified a monoclonal anti-factor VIII (FVIII) antibody, C4, which inhibits the binding of purified human FVIII to purified human von Willebrand factor (vWF). Both whole immunoglobulin C4 and its Fab fragment demonstrated dose-dependent inhibition of FVIII binding to vWF immobilized on the surface of polystyrene beads. Synthetic peptides based on the amino acid sequence of FVIII were tested for the ability to block the binding of C4 to FVIII in an enzyme-linked immunosorbent assay system. A single synthetic FVIII pentadecapeptide, consisting of residues Val1670-Glu1684, was able to inhibit C4 binding to FVIII. Under the conditions used, the Val1670-Glu1684 peptide demonstrated total inhibition of C4 binding at a concentration of 1 microM. Synthetic FVIII peptides flanking and overlapping the Val1670-Glu1684 peptide had no significant inhibitory activity on C4 binding in concentrations up to 100 microM. A polyclonal antibody made to the Val1670-Glu1684 peptide also demonstrated inhibition of FVIII binding to vWF. Polyclonal antibodies made to synthetic FVIII peptides flanking and partially overlapping the Val1670-Glu1684 sequence did not demonstrate such inhibition. Localization of the binding region of the monoclonal anti-FVIII antibody C4 to residues Val1670-Glu1684 suggests that this site is at, or near, a major vWF binding domain of FVIII.     

Covalent inactivation of factor VIII antibodies from hemophilia A patients by an electrophilic FVIII Analog

The antigen-binding sites of antibodies (Abs) can express enzyme-like nucleophiles that react covalently with electrophilic compounds. We examined the irreversible and specific inactivation of antibodies (Abs) to Factor VIII (FVIII) responsible for failure of FVIII replacement therapy in hemophilia A (HA) patients. Electrophilic analogs of FVIII (E-FVIII) and its C2 domain (E-C2) were prepared by placing the strongly electrophilic phosphonate groups at surface-exposed Lys side chains of diverse antigenic epitopes. IgG Abs to FVIII from HA patients formed stable immune complexes with E-FVIII and E-C2 that were refractory to dissociation by SDS treatment and boiling, procedures that dissociate noncovalent Ab-antigen complexes. The rate-limiting step in the reaction was formation of the initial noncovalent complexes. Conversion of the initial complexes to the irreversible state occurred rapidly. The antigenic epitopes of E-FVIII were largely intact, and most of the Abs were consumed covalently. E-FVIII expressed poor FVIII cofactor activity in clotting factor assays. Nonspecific interference by E-FVIII in clotting factor function was not evident. Treatment with E-FVIII, and to a lesser extent E-C2, irreversibly relieved the FVIII inhibitory effect of HA IgG in clotting factor assays. Small FVIII peptides did not display useful reactivity, highlighting the diverse epitope specificities of the Abs and the conformational character of FVIII epitopes. E-FVIII is a prototype reagent able to attain irreversible and specific inactivation of pathogenic Abs.     

Initial B-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia

A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.     

Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects,patients with coronary heart disease and inflammatory bowel disease

Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.     

Durable engraftment of genetically modified FVIII‐secreting autologous bone marrow stromal cells in the intramedullary microenvironment

Genetically modified FVIII‐expressing autologous bone marrow‐derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture‐expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII‐secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture‐expanded primary canine BMSCs. FVIII‐secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti‐human FVIII antibody having negligible cross‐reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti‐human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII‐secreting BMSCs.     

Immunological Studies of Factor VIII (Antihaemophiliac Globulin) in Haemophilia A

PATIENTS with haemophilia A have recently been divided into two groups; one characterized by a functionally defective factor VIII (AHG) molecule in the plasma, which is immunologically similar to normal factor VIII but lacks procoagulant activity^1–3, while the other seems to represent a true deficiency of factor VIII, for both procoagulant and immunological properties of factor VIII are absent. These conclusions are based on the ability of normal and some haemophiliac plasma to neutralize human antibodies from patients with spontaneously occurring inhibitors against factor VIII or from multi-transfuscd haemophiliacs who have developed circulating inhibitors directed against factor VIII. These studies have divided haemophilia A into those with cross reactive material (CRM +) and those without (CRM ?). Other methods of characterizing this genetic polymorphism in haemophilia have not been reported. We have used immunoelectrophoresis and antibody neutralization with a heterologous antibody to show a similar division of haemophilia A into a small group of CRM+ (15%) and a larger group of CRM? (85%). Immunoelectrophoresis and antibody neutralization studies have also been described in factor X polymorphism⁴.     

Heterogeneity of the immune response to adenovirus-mediated factor VIII gene therapy in different inbred hemophilic mouse strains

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies (inhibitors) is a critical concern when considering gene therapy as a potential treatment modality for hemophilia A. We used a hemophilia A mouse model bred on different genetic backgrounds to explore genetically controlled differences in the immune response to FVIII gene therapy. METHODS: C57BL/6 FVIII knockout (C57-FVIIIKO) mice were bred with normal BALB/c (BAL) mice, to generate a recombinant congenic BAL-FVIIIKO model of hemophilia A. Early generation adenoviral (Ad) vectors containing the canine FVIII B-domain-deleted transgene under the control of either the CMV promoter or a tissue-restricted (TR) promoter were administered to C57-FVIIIKO, C57xBAL(F1)-FVIIIKO crosses, and BAL-FVIIIKO mice. FVIII expression, inhibitor development, inflammation, and vector-mediated toxicity were assessed. RESULTS: In response to administration of Ad-CMV-cFVIII, C57-FVIIIKO mice attain 3-fold higher levels of FVIII expression than BAL-FVIIIKO. All strains injected with Ad-CMV-FVIII displayed FVIII expression lasting only 2 weeks, with associated inhibitor development. C57-FVIII-KO mice that received Ad-TR-FVIII expressed FVIII for 12 months post-injection, whereas FVIII expression was limited to 1 week in C57xBAL(F1)-FVIIIKO and BAL-FVIIIKO mice. This loss of expression was associated with anti-FVIII inhibitor development. BAL-FVIIIKO mice showed increased hepatotoxicity with alanine aminotransferase levels reaching 4-fold higher levels than C57-FVIIIKO mice. However, C57-FVIIIKO mice initiate a more rapid and effective cell-mediated clearance of virally transduced cells than BAL-FVIIIKO, as evidenced by real-time PCR analysis of transduced tissues. Overall, strain-dependent differences in the immune response to FVIII gene delivery were only noted in the adaptive response, and not in the innate response. CONCLUSIONS: Our results indicate that the genetic background of the murine model of hemophilia A influences FVIII expression levels, the development of anti-FVIII inhibitors, clearance of transduced cells, and the severity of vector-mediated hepatotoxicity.     

Low molecular weight peptides restore the procoagulant activity of factor VIII in the presence of the potent inhibitor antibody ESH8

Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the (2231)QVDFQKTMKV(2240) sequence of the C(2) domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence (2267)DGHQ(2270). Upon inspection of the C(2) domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivo in a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.