Switching clotting factor concentrates: considerations in estimating the risk of immunogenicity.

The development of neutralizing antibodies to factor VIII (FVIII) is the most serious complication of therapy for haemophilia A. There is now excellent documentation that a large number of both genetic and environmental factors contribute to the risk of FVIII inhibitor incidence. One of the environmental factors that has been proposed as an influence on this complication is the occurrence of FVIII product switching. There are only a small number of clinical studies that have addressed this question, and thus, the amount of objective information available to assess this association is limited. In this review, in addition to summarizing past evidence pertinent to this subject, we present the results of a complementary strategy, a Delphi analysis, to add to the considerations of product switching and FVIII immunogenicity. With the imminent arrival in the clinic of several new FVIII products, the haemophilia community must be prepared to collect prospectively controlled data to better address this important management issue.

Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3.

During antigen-induced immune responses, human B cells switch isotype from immunoglobulin M (IgM)-IgD to IgG1-4, IgA1-2, or IgE. In the human, no cytokines have yet been demonstrated to act as switch factors for IgG1, IgG2, and IgG3. In this paper, we report that in response to interleukin 10 (IL-10), anti-CD40 activated tonsillar surface IgD+ (sIgD+) B cells are induced to secrete large amounts of IgM, IgG1, and IgG3 but neither IgG2 nor IgG4. Cord blood purified B cells and lymphocytes from Hyper-IgM patients also produced IgG1 and IgG3 after culture with anti-CD40 and IL-10. In contrast, sIgD- isotype-committed B cells produce IgG1, IgG2, and IgG3 when activated through CD40 in the presence of IL-10. Thus, in addition to its growth-promoting and differentiating activities on human B cells, IL-10 may represent a switch factor for IgG1 and IgG3.

Autoantibodies to coagulation factors.

Tolerance to autoantigens such as coagulation factors is the result of censoring mechanisms occurring at the level of the thymus and bone marrow for autoreactive T and B cells, respectively. In addition, peripheral mechanisms, both intrinsic and extrinsic further control activation of autoreactive cells that have escaped central deletion. Emergence of autoimmunity can occur from disturbances of these control mechanisms by a number of events, many of which are incompletely understood. Insight into this clinically important field is expected from exploitation of recent animal models.

Comparative enzymology of native and recombinant house dust mite allergen Der p 1.

BACKGROUND: The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. METHODS: Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. RESULTS: Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. CONCLUSION: The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens.

The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high-titre inhibitors: association of clinical outcome with inhibitor epitope profile.

A retrospective chart review of 11 subjects with severe haemophilia A and high-titre inhibitors who received a von Willebrand factor-containing FVIII concentrate (VWF/FVIII) for immune tolerance induction (ITI) was accompanied by B cell inhibitor epitope mapping during 10/11 treatment courses. ITI was successful or partially successful in all seven subjects who received VWF/FVIII for initial ITI, and failed in all four subjects whose ITI with this product was initiated following treatment failure using recombinant factor VIII. Variables including age at inhibitor development and age at ITI initiation, interval between inhibitor detection and ITI initiation, titre at start of ITI, and peak historical titres prior to and during ITI were not statistically significant outcome predictors in this cohort. However, the B cell epitope specificity in all four successful and in one of two partially successful ITI subjects for whom information was available included the C2 and excluded the A2 domains. Conversely, FVIII B cell epitopes in one partially successful ITI and in all three failed ITI subjects for whom data were available mapped to both the C2 and the A2 domains. The FVIII B cell epitope profile was associated with ITI outcome in this VWF/FVIII-treated cohort. Its role in predicting ITI outcome and guiding choice of FVIII product for ITI requires further study.

Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies.

Anti-Factor VIII (FVIII) antibodies were prepared by a combination of salt precipitation, gel filtration chromatography, and specific adsorption over insolubilized FVIII from the serum of 10 healthy subjects with normal levels of FVIII. Antibody specificity was confirmed by the capacity to recognize soluble and insolubilized FVIII and to neutralize FVIII cofactor activity in FX activation. Epitope mapping was carried out using a competition ELISA in which affinity-purified human antibodies inhibited the binding of labeled monoclonal antibodies. In most cases, a single region of the A3 domain of the FVIII light chain was recognized by the antibodies, while the reactivity toward heavy chain epitopes differed from one antibody preparation to the other. Sera or IgG fractions of the serum before immunoadsorption over insolubilized FVIII did not bind to FVIII. The IgG fraction that was not retained on the FVIII immunosorbent contained IgG that bound to the variable part of anti-FVIII mouse monoclonal antibodies and inhibited the binding of labeled FVIII; in addition, the IgG fraction inhibited the binding of affinity-purified human antibodies to FVIII, thereby strongly suggesting the presence of anti-idiotypic antibodies. These findings indicate that the presence of anti-FVIII antibodies is a more universal phenomenon than previously thought and that anti-idiotypic antibodies capable of inhibiting the binding of anti-FVIII antibodies to FVIII are produced spontaneously.

Neutralizing antiidiotypic antibodies to factor VIII inhibitors after desensitization in patients with hemophilia A.

Hemophilia A patients producing antibodies towards FVIII are usually treated with infusions of high doses of FVIII in an attempt to "desensitize" them. To examine the mechanisms by which such desensitization operates, sequential plasma samples of two unrelated inhibitor patients were analyzed for anti-FVIII and antiidiotypic antibodies before and during infusions of high doses of FVIII. Anti-FVIII antibodies were separated from antiidiotypic antibodies by immunoaffinity chromatography before analysis. We show in the present study that the concentration of anti-FVIII antibodies did not change during a successful desensitization and that antibodies maintained their capacity to inhibit the procoagulant function of FVIII, even though the number of Bethesda units in plasma was reduced to undetectable levels. Using a competition assay with mAbs, we further show that the specificity of human antibodies did not vary significantly during therapy. Finally, we show that the treatment elicited antiidiotypic antibodies, which neutralized the inhibitory capacity of anti-FVIII antibodies. Inhibitor antibodies can therefore not be accurately evaluated in plasma, as their function appears to be neutralized by antiidiotypic antibodies. These findings could have implications for the design of new therapies for hemophilia A patients with inhibitors.

Allergic bronchial asthma due to Dermatophagoides pteronyssinus hypersensitivity can be efficiently treated by inoculation of allergen-antibody complexes.

Antigen-antibody complexes were made from allergens of the common house dust mite, Dermatophagoides pteronyssinus (Dpt) and an excess of purified autologous specific antibodies. These complexes have been used to treat Dpt-hypersensitive patients who suffered from chronic bronchial asthma. Clinical symptoms and medication intake were followed by filling in diary cards. Peak expiratory flow, measured four times a day, was also followed. Intradermal skin tests and bronchial challenge tests were performed with allergen together with an evaluation of nonspecific bronchial reactivity. Specific IgE and IgG antibodies were assayed after separation from the bulk of serum immunoglobulins by immunoadsorption. The study was carried out over two years according to a double-blind protocol. Intradermal inoculation of antigen-antibody complexes resulted in a marked reduction of both clinical and medication scores. No systemic side-effects were observed and only mild wheal and flare reactions were noted at the injection site. The treatment showed a drastic reduction of specific skin and bronchial reactivities with only marginal effects on nonspecific bronchial reactivity. Concentrations of specific IgE antibodies decreased significantly during the first weeks of treatment and remained at these lower values throughout the study. Specific IgG antibodies actually decreased in the majority of treated patients. The total amount of allergen used in this study was less than 1% of the amount currently used for conventional hyposensitization with the same allergen. These findings show that antigen-antibody complex inoculation is an efficient and safe means of treating allergic bronchial asthma and that the mechanism of action is likely to differ from conventional hyposensitization.

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody.

BACKGROUND: N-glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood. OBJECTIVES: We investigated the function of N-glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb-LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation. METHODS AND RESULTS: Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N-glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N-glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies. CONCLUSIONS: Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.

The use of antibodies to coagulation factors for anticoagulant therapy.

Current treatments for preventing thrombotic diseases are associated with a significant risk of bleeding. Improved anticoagulant agents are therefore still required. The specificity and pharmacokinetics properties of monoclonal antibodies to coagulation factors allow novel anticoagulation approaches. Treatment with human antibodies or humanized mouse monoclonal antibodies should avoid unacceptable side effects due to immune response to the drug. Such antibodies were developed against three coagulation factor: Tissue factor (TF), Factor IX (FIX) and Factor VIII (FVIII). A fully humanized antibody was successfully derived from a mouse monoclonal antibodies to TF. In vivo studies with monoclonal antibodies to TF demonstrated efficient antithrombotic activity. Anti-TF antibodies may also prove useful in cardiovascular disorders and cancer, given the role of TF in these diseases. Mouse and human monoclonal antibodies to FIX were also efficient to prevent thrombosis in animal models of venous and arterial thrombosis and in stroke. A humanized anti-FIX antibody was tested in phase I study in healthy volunteers. The pharmacokinetics of the antibody were determined by the rapid formation of stable complexes with newly synthesised FIX. Human anti-FVIII antibodies inhibiting only partially FVIII activity were recently described. Investigations in mice have established that treatment with such anti-FVIII antibodies is efficient to prevent deep vein thrombosis. Given the low concentration of FVIII in plasma and the long half-life of antibody, treatment with anti-FVIII antibody could be very convenient, allowing one administration every month. Altogether, monoclonal antibodies to coagulation factor appear as promising novel antithrombotic drugs.

Inhibition of factor VIII with a partially inhibitory human recombinant monoclonal antibody prevents thrombotic events in a transgenic model of type II HBS antithrombin deficiency in mice.

Venous thromboembolic disease is a major cause of morbidity and mortality, necessitating antithrombotic therapy. A human monoclonal anti-factor (F)VIII antibody, LCL-mAb-LE2E9, produced by a lymphoblastoid cell line derived from a hemophilia A patient with inhibitor to wild-type but not mutant self FVIII, was previously reported to achieve efficient inhibition of thrombosis in an experimental vena cava thrombosis model in mice. Here, the antithrombotic efficacy of a recombinant DNA-derived version of this anti-FVIII antibody (rec-mAb-LE2E9) was tested in mice which carry a type II heparin binding site antithrombin deficiency mutation and display spontaneous chronic thrombosis in several sites including the penile vein of sexually active males. The recombinant anti-FVIII antibody (100 microg, repeated after 3 days) prevented thrombotic priapism in all treated males, whereas all control animals treated with saline (group of four animals) developed priapism within 6 days after mating (P < 0.05 for treated vs. saline). The rec-mAb-LE2E9 and the original LCL-mAb-LE2E9 were equally effective (five and seven males/group, respectively). These results confirm that FVIII inhibition represents a potent antithrombotic strategy, and show that both LCL-mAb-LE2E9 and rec-mAb-LE2E9 efficiently prevent thrombosis in a physiological model representative of thrombosis in patients with a severe prothrombotic risk.

Molecular mechanisms of mild and moderate hemophilia A.

Mutations responsible for mild/moderate hemophilia A were extensively characterized over the last 15 years and more than 200 mutations have been identified. However, most of the molecular mechanisms responsible for the reduced factor (F)VIII levels in patients' plasma were determined only recently. Recent progresses in the study of the FVIII molecule three-dimensional structure provided a major insight for understanding molecular events leading to mild/moderate hemophilia A. This allowed prediction of mutations impairing FVIII folding and intracellular processing, which result in reduced FVIII secretion. Mutations potentially slowing down FVIII activation by thrombin were also identified. A number of mutations were also predicted to result in altered stability of activated FVIII. Biochemical analyses allowed identification of mutations reducing FVIII production. Mutations impairing FVIII stability in plasma, by reducing FVIII binding to von Willebrand factor (VWF) were also characterized. Defects in FVIII activity, notably slow activation by thrombin, or abnormal interaction with FIXa, were also recently demonstrated. Biochemical analysis of FVIII variants provided information regarding the structure/function relationship of the FVIII molecule and validated predictions of the three-dimensional structure of the molecule. These observations also contributed to explain the discrepant activities recorded for some FVIII variants using different types of FVIII assays. Altogether, the study of the biochemical properties of FVIII variants and the evaluation of the effects of mutations in three-dimensional models of FVIII identified molecular mechanisms potentially explaining reduced FVIII levels for a majority of patients with mild/moderate hemophilia A. It is expected that these studies will improve diagnosis and treatment of this disease.

An M(r) 145,000 low-density lipoprotein (LDL)-binding protein is conserved throughout the Kinetoplastida order.

In view of the importance of the low-density lipoprotein (LDL)-receptor in Trypanosoma brucei, we have examined whether other bloodstream trypanosomes of medical and veterinary importance (T.b. rhodesiense, T. equiperdum, T. vivax, T. congolense), but also related parasites developing in mammalian (Leishmania donovani) and non-mammalian hosts (Crithidia luciliae and Phytomonas sp. isolated from Euphorbia), would possess an LDL-receptor of their own. (1) All these parasites specifically accumulate human 125I-LDL with a relatively 2.5-fold higher rate for bloodstream trypanosomes. (2) A mixture of monoclonal antibodies raised against T.b. brucei LDL-receptor inhibit binding of LDL to all species but with different efficiency. (3) A single glycoprotein of similar M(r) (gp145) is isolated by LDL-affinity chromatography from all the above species, as well as from both human serum-resistant and sensitive strain of T.b. rhodesiense, and from the bodonid member of the Kinetoplastida Trypanoplasma borelli. (4) Several control experiments including 35S-metabolic labeling of procyclic T.b. brucei and of C. luciliae followed by LDL-affinity chromatography or immunoprecipitation demonstrate that gp145 is indeed synthesised by the parasites and is not a contaminant of the experimental system. (5) In immunoblots and ELISA, these gp145 cross-react with the polyclonal and monoclonal antibodies raised against the LDL-receptor of T.b. brucei, the highest degree of cross-reactivity being found among the members of the Trypanozoon subgroup. (6) Finally, immunisation of mice with the purified LDL-receptor from one strain of T.b. brucei is not sufficient to confer durable protection against another strain of this parasite.

Identification of a specific epitope on the extracellular domain of the LDL-receptor of Trypanosoma brucei brucei.

We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4 degrees C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30 degrees C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.

Humanized severe combined immunodeficient mice as a potential model for the study of tolerance to factor VIII.

A Severe Combined Immunodeficient (SCID) mouse model has been established to evaluate experimental conditions leading to the production of factor VIII (FVIII) autoantibodies. To this end, we humanized 10 groups of 7 mice with peripheral blood mononuclear cells of 10 unrelated healthy blood donors (15 x 10(6) cells/mouse). Mice were injected with saline or immunized i.p. with 50 IU of a plasma derived human FVIII 24 h after reconstitution. Further immunization was made with 25 IU of FVIII every fortnight during 6 weeks and animals were sacrificed after 8 weeks. All reconstituted mice showed a spontaneous production of anti-FVIII antibodies in the absence of immunization with the corresponding antigen. However, no differences were observed regarding the quantity or the quality of these antibodies produced in the immunized or the saline group, indicating that tolerance to FVIII had been transferred with cell reconstitution. Affinity purified FVIII specific antibodies were capable of inhibiting FVIII activity and preventing the binding of FVIII to phospholipids in a dose-dependent manner. Immunoprecipitation experiments showed that the antibodies recognized only the C1 and C2 light chain domains. Since antibodies of interest can be found in the SCID mouse model and, moreover, since they are qualitatively comparable with the source donor's antibodies, this model provides a tool to study the regulation of tolerance against self antigens in normal subjects and in acquired haemophilia patients.

A higher than expected incidence of factor VIII inhibitors in multitransfused haemophilia A patients treated with an intermediate purity pasteurized factor VIII concentrate.

In May 1990, 218 patients with haemophilia A regularly attending the Leuven Haemophilia Center were randomly assigned to a group receiving either of two newly introduced factor VIII concentrates: factor VIII-P, an intermediate purity pasteurized concentrate, or factor VIII-SD, a high purity concentrate treated with solvent-detergent for viral inactivation. Patients were followed from May 1990 until October 1991. Between August 1991 and October 1991 a clinically important factor VIII inhibitor was detected in five out of the 109 patients receiving factor VIII-P while none of the 109 patients receiving factor VIII-SD developed such antibodies. All patients acquiring an inhibitor had previously been clinically tolerant to transfused factor VIII with 200 to more than 1,000 days of exposure to factor VIII prior to May 1990. Patients with inhibitors were transfused daily with 30 U factor VIII-SD per kg body weight, which was associated with a gradual decline of the inhibitor level. In all patients the antibodies were relatively slow-acting and predominantly directed towards the light chain of factor VIII. This study demonstrates a higher than expected incidence of factor VIII inhibitors associated with the use of a specific factor VIII concentrate in multitransfused haemophilia A patients. It indicates the usefulness of evaluating newly introduced concentrates in prospective, randomized trials.

Restricted epitope specificity of anti-FVIII antibodies that appeared during a recent outbreak of inhibitors.

We recently described an outbreak of anti-factor VIII (FVIII) antibodies in a population of haemophilia A patients non-responsive to FVIII (1). To find out what part of the FVIII molecule had been altered, we purified specific anti-FVIII antibodies from the plasma of the five patients showing high titres of inhibitors. An average of 100 micrograms antibodies per ml of initial plasma was recovered by immunoadsorption on insolubilised FVIII. The antibodies followed the normal isotypic distribution, including the presence of specific IgG2 antibodies; the relative increase in IgG4 that is usually observed in patients with long-standing inhibitors, was not present. The regions of FVIII to which human antibodies bound were determined by a competition assay using a panel of murine monoclonal antibodies: two major regions were identified, one located in the A2 heavy chain domain, and the other made of determinants of both the A3 and C2 light chain domains. Affinity-purified antibodies inhibited the function of FVIII as determined in a chromogenic assay. However, variations existed in the affinities with which antibodies bound to soluble FVIII. This study shows that the immunogenicity of two particular regions of FVIII has been altered. A screening for alterations located in these two regions should possibly be included in the preclinical evaluation of FVIII concentrates.

Factor VIII inhibitors in previously treated haemophilia A patients with a double virus-inactivated plasma derived factor VIII concentrate.

Antibodies to factor VIII (inhibitors) are usually produced at the beginning of treatment with factor VIII and are rare in multitransfused patients. Such antibodies are deemed to be patient-related, as supported by the description of a number of associated risk factors. However, a second category of inhibitors has recently been identified, namely antibodies occurring in multitransfused patients as a result of exposure to a particular factor VIII concentrate. A first outbreak of product-related inhibitors was recently described. The present paper describes the second well-documented occurrence of such inhibitors. Eight out of 140 multitransfused patients with severe haemophilia A developed an inhibitor to factor VIII shortly after changing treatment to a double-virus inactivated plasma-derived factor VIII concentrate. In addition to solvent-detergent treatment, this concentrate was pasteurised at 63 degrees C for 10 hours. Exposure to the pasteurised product before inhibitor detection ranged from 9 to 45 days. Inhibitor titers varied between 2.2 and 60 Bethesda Units and recovery of transfused factor VIII ranged from 0.21 to 0.68 (expressed as i.u./dl factor VIII rise per i.u./kg administered). In contrast to usual inhibitors in haemophilia A patients, these product-related inhibitors showed complex inhibition kinetics. They were found specific for the factor VIII light chain. The inhibitors gradually declined when exposure to the pasteurised product was stopped, despite further treatment with other factor VIII concentrates. The present data stress the importance of carefully monitored clinical studies, both in previously treated and previously untreated patients, before introduction of a new or modified clotting factor concentrate.

Some factor VIII (FVIII) inhibitors recognise a FVIII epitope(s) that is present only on FVIII-vWF complexes.

A mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wildtype from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.

Epitope mapping: a new method for biological evaluation and immunotoxicology.

The extraordinary capacity of the immune system to recognise and distinguish between molecules that are almost identical can be exploited to generate reagents useful for the identification of specific determinants on drugs, blood-derived products, hormones, receptors or micro-organisms. This manuscript provides a rationale for the use of epitope mapping and a brief outline of its multiple applications.