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.

Successful immune tolerance induction by FVIII in hemophilia A patients with inhibitor may occur without deletion of FVIII-specific T cells.

BACKGROUND: The development of an inhibitor is the major complication facing patients with hemophilia A treated by administration of factor (F) VIII concentrates. Restoration of tolerance to FVIII can be achieved by prolonged administration of FVIII (immune tolerance induction, ITI). Although ITI has been used for more than 30years in patients with hemophilia A and inhibitor, its mechanism of action is still poorly understood. OBJECTIVES: As administration of high doses of antigen can induce the apoptosis of the T cells recognizing the antigen, a potential mechanism of action of ITI may be the deletion of FVIII-specific T cells. PATIENTS/METHODS: We studied the CD4+ T-cell response to FVIII in five (one mild, one moderate and three severe) patients successfully desensitized by administration of FVIII and in control subjects. RESULTS: Following repeated stimulation with autologous dendritic cells loaded with FVIII, FVIII-specific T oligoclonal cell lines were expanded from the blood of one of the successfully desensitized patients. The FVIII-specific T cells produced IL-5, IL-13 and IL-2. By contrast, FVIII-specific T-cell lines could not be derived from three patients with mild hemophilia A without inhibitor or from four normal control subjects. CONCLUSIONS: These data represent the first analysis of the cellular mechanisms regulating the induction of tolerance to FVIII. They demonstrate that successful tolerance induction may occur without deletion of FVIII-specific T cells.

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.

A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF-binding site distant from its epitope.

SUMMARY BACKGROUND: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab(BO2C11)) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. OBJECTIVES: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. METHODS: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab(BO2C11)-complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. RESULTS: Binding of Fab(BO2C11) to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. CONCLUSIONS: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.

A human monoclonal antibody inhibiting partially factor VIII activity reduces thrombus growth in baboons.

BACKGROUND: The inhibitory activity of an anti-factor VIII (FVIII) antibody can be modulated through glycosylation of the antigen binding site, as has recently been described. This offers the opportunity to develop an optimized anticoagulant agent targeting partial FVIII inhibition. OBJECTIVES: We investigated in non-human primates the antithrombotic activity, pharmacokinetics,and pharmacodynamics of a human monoclonal antibody, Mab-LE2E9Q, inhibiting FVIII activity partially. METHODS: The ability of Mab-LE2E9Q to prevent thrombosis was evaluated in baboons after administration of 1.25 and 5 mg kg(-1) antibody or saline as a single intravenous (i.v.) bolus. Thrombus development was recorded in expansion ('venous') and in Dacron ('arterial') thrombosis chambers incorporated in an extracorporeal arteriovenous shunt implanted between the femoral vessels 1 h, 24 h and 7 days after the administration of Mab-LE2E9Q. RESULTS: Mab-LE2E9Q reduced thrombus growth to a similar extend 1 h, 1 day and 1 week after administration of the antibody. Ex vivo pharmacodynamic analysis indicated that the evaluation of the residual FVIII activity was strongly dependent on the type of FVIII assay and on the phospholipid concentration in the assay. No significant difference in bleedings was observed between animals treated with Mab-LE2E9Q or with saline. CONCLUSIONS: Understanding the role of glycosylation in FVIII inhibition by a human monoclonal antibody allowed selection of an antibody inhibiting only moderately FVIII activity while significantly reducing thrombus development in a baboon extracorporeal model. As that antibody did not increase the bleeding tendency, it may represent a novel type of a long-acting antithrombotic agent with an optimal safety/efficacy profile.

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.

Efficient factor VIII affinity purification using a small synthetic ligand.

BACKGROUND: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. OBJECTIVE: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. METHODS: Peptide ligands were screened for binding of (125)I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. RESULTS: A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. CONCLUSIONS: Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.

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.

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.

New high-technology products for the treatment of haemophilia.

This review will focus on new technologies in development that promise to lead to further advances in haemophilia therapeutics. There has been continued interest in the bioengineering of recombinant factor VIII (rFVIII) and factor IX (rFIX) with improved function to overcome some of the limitations in current treatment, the high costs of therapy and to increase availability to a broader world haemophilia population. Bioengineered forms of rFVIII, rFIX or alternative haemostatic molecules may ultimately have an impact on improving the efficacy of therapeutic strategies for the haemophilias by improving biosynthesis and secretion, functional activity, half-life and immunogenicity. Preventing and suppressing inhibitors to factor (F) VIII remain a challenge for both clinicians and scientists. Recent experiments have shown that it is possible to obtain anti-idiotypic antibodies with a number of desirable properties: (i) strong binding avidity to FVIII inhibitors; (ii) neutralization of inhibitory activity both in vitro and in vivo; (iii) cross-reactivity with antibodies from unrelated patients, and (iv) no interference with FVIII function. An alternative, although complementary approach, makes use of peptides derived from filamentous-phage random libraries. Mimotopes of FVIII can be obtained, which bind to the paratope of inhibitory activity and neutralize their activity both in vitro and in vivo. In this paper, we review advanced genetic strategies for haemophilia therapy. Until recently the traditional concept for gene transfer of inherited and acquired haematological diseases has been focused on how best to obtain stable insertion of a cDNA into a target-cell genome, allowing expression of a therapeutic protein. However, as gene-transfer vector systems continue to improve, the requirement for regulated gene transcription and hence regulated protein expression will become more critical. Inappropriate protein expression levels or expression of transferred cDNAs in non-intended cell types or tissues may lead to target-cell toxicity or activation of unwanted host immune responses. Regulated protein expression requires that the transferred gene be transferred with its own regulatory cassette that allows for gene transcription and translation approaching that of the normal gene in its endogenous context. New molecular techniques, in particular the use of RNA molecules, now allow for transcription of corrective genes that mimic the normal state.

Inhibitors in haemophilia: pathophysiology.

Development of inhibitors to coagulation factors is one of the major problems faced by people with haemophilia. Up to a third of patients, following treatment with factor concentrates, will develop an antibody (inhibitor) to that factor, rendering it inactive, and leaving the patient at risk from life-threatening bleeding. Evidence shows that this immune response is T-cell-dependent, but as yet, the epitopes responsible have not been identified. Risk for inhibitor development is highest within the first 50 days of treatment, with reactions being rare after 200 days. The risk is mediated by the major histocompatibility complex class of the patient, and by mutations in the factor VIII genotype, with large deletions conferring greatest risk.

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.

Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII.

The development of an immune response to infused factor VIII is a complication affecting many patients with hemophilia A. Inhibitor antibodies bind to antigenic determinants on the factor VIII molecule and block its procoagulant activity. A patient-derived inhibitory immunoglobulin G4kappa antibody (BO2C11) produced by an immortalized memory B-lymphocyte cell line interferes with the binding of factor VIII to phospholipid surfaces and to von Willebrand factor. The structure of a Fab fragment derived from this antibody complexed with the factor VIII C2 domain was determined at 2.0 A resolution. The Fab interacts with solvent-exposed basic and hydrophobic side chains that form a membrane-association surface of factor VIII. This atomic resolution structure suggests a variety of amino acid substitutions in the C2 domain of factor VIII that might prevent the binding of anti-C2 inhibitor antibodies without significantly compromising the procoagulant functions of factor VIII.

Antigenicity of putative phospholipid membrane-binding residues in factor VIII.

Most inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of fVIII to phospholipid. The x-ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors frequently target the Met2199/Phe2200 and Leu2251/Leu2252 beta-hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.

Factor VIII Inhibitors. Natural autoantibodies and anti-idiotypes.

Natural antibodies to factor VIII are present in the normal antibody repertoire as other self-reactive antibodies to soluble proteins. The question as to whether they represent just a chance occurrence linked to the huge diversification of the antibody repertoire or whether these antibodies have an actual physiological relevance is not entirely settled. Evidence is in favor of a role in the maintenance of immune homeostasis, however, namely self-reactive antibodies are required to maintain the capacity of the immune system to distinguish self from nonself. Anti-factor VIII antibodies pose an interesting case in point because they exhibit the capacity to inhibit the function of factor VIII. Such a property is neutralized at least in part by the production of corresponding anti-idiotypic antibodies. Normal homeostasis can therefore be viewed as a network of interacting molecules, idiotypes, and anti-idiotypes; disruption of this equilibrium leads to the development of autoimmunity. A question that remains open for the time being is whether this network of interactions can be modulated in a defined way for the treatment of autoimmune reactions. This would mean either passive administration of anti-idiotypic antibodies or active immunization with idiotypes. The former has proved to be efficient, and the latter has still to be demonstrated. Further, and probably most importantly, is the question of the possible application of the idiotypic network concept to the treatment of hemophilia patients producing inhibitors. This essentially requires that an analysis of the anti-factor VIII immune response be carried out at the clonal level. Such work is ongoing in our laboratory.

Natural antibodies to factor VIII.

Anti-factor VIII antibodies represent a unique model to study the relationship between natural autoreactivity (natural antibodies to factor VIII of healthy individuals), disease-associated autoimmunity ("spontaneous" factor VIII inhibitors of patients with anti-factor VIII autoimmune disease) and antigen-driven immune responses (immune inhibitors in multitransfused patients with hemophilia A) to a single human protein antigen. Although natural and disease-associated anti-factor VIII antibodies are not readily distinguished based on the comparison of their isotypic distribution and epitope mapping, available studies of cross-reacting idiotypes suggest that factor VIII inhibitors in patient's plasma encompass two populations of anti-factor VIII antibodies. Some antibodies result from the clonal expansion of B lymphocytes that exist before treatment with factor VIII and secrete anti-factor VIII antibodies with properties similar to those of natural anti-factor VIII antibodies present in healthy individuals; other inhibitors are produced by B cell clones that have undergone affinity maturation and hypermutation of the V regions of the antibodies they produce. The implications for the treatment of patients with anti-factor VIII inhibitors are discussed.