Jan Voorberg, PhD, Department of Plasma Proteins, CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: J_Voorberg@CLB.nl
Summary. We characterized anti-factor VIII antibodies in a mild haemophilia A patient with an Arg593→Cys mutation in the A2 domain, using V gene phage-display technology. All isolated single-chain variable-domain antibody fragments were directed against residues Arg484-Ile508, a binding site for factor VIII inhibitors in the A2 domain. After a further period of replacement therapy, a transient rise in inhibitor titre was observed. These antibodies were directed against the A2 domain. Activation of a pre-existing pool of B cells, which express antibodies against residues Arg484-Ile508, could explain the rapid anamnestic response.
A serious complication in haemophilia A treatment is the development of antibodies that neutralize factor VIII activity (inhibitors). The prevalence of factor VIII inhibitors in patients with severe haemophilia A is approximately 25% (Mannucci & Tuddenham, 2001). Factor VIII inhibitors are infrequently observed in patients with mild or moderate haemophilia A (Sultan, 1992). The lower risk of inhibitor formation in this group of patients can be explained by the presence of tolerizing amounts of circulating endogenous factor VIII. Inhibitor development in these patients commonly arises after intensive factor VIII replacement therapy and is usually a transient event (Hay et al, 1998).
Interestingly, some genetic defects are frequently observed in patients with mild haemophilia A who developed inhibitors. Amino acid substitutions located at the junction between the C1 and C2 domain or located in the A2 domain of factor VIII may predispose towards the development of factor VIII inhibitors (Hay et al, 1998). We have previously reported on the presence of human alloantibodies in a patient with an Arg593→Cys mutation (Fijnvandraat et al, 1997). In this patient, the anti-factor VIII antibodies were directed to wild-type A2 domain and not to the A2 domain containing the Arg593→Cys mutation when analysed at a late stage of inhibitor formation. These findings indicate that the missense mutation Arg593→Cys was related to antibody development.
In the present study, phage display technology was used for the characterization of anti-factor VIII antibodies in the patient. Our findings showed that peripheral IgG+ B cells can express human antibodies reactive with residues Arg484-Ile508. These antibodies are distinct from the alloantibodies found in plasma. Following a period of intensive treatment with FVIII concentrate after a surgical intervention, the patient developed a transient low-titre inhibitor that cross-reacted with endogenous factor VIII. We propose that activation of pre-existing factor VIII-specific B cells underlies the rapid appearance of cross-reactive antibodies.
Materials and methods
Expression, metabolic labelling and immunoprecipitation of A2 domain variants. The wild-type factor VIII A2 domain, the A2 domain containing the Arg593→Cys mutation (A2-R593C) and the A2 domain in which the residues Arg484-Ile508 were replaced by the corresponding sequence of factor V (A2-FV484-508) have been described previously (Fijnvandraat et al, 1997; van den Brink et al, 2000). Recombinant factor VIII fragments were expressed and metabolically labelled in insect cells and immunoprecipitation was performed as described (Fijnvandraat et al, 1997).
Phage display library construction, selection and characterization of selected clones. Peripheral blood mononuclear cells were used from a mild haemophilia A patient with a factor VIII inhibitor. Approximately half of the anti-factor VIII antibodies in plasma of this patient were of subclass IgG4 (Fijnvandraat et al, 1997). RNA was isolated from the peripheral blood cells and used for the construction of a phage display library as described previously (van den Brink et al, 2000). The patient's IgG4-specific VH gene repertoire was amplified and combined with a VL gene repertoire of non-immune origin in pHEN-1-VLrep, and displayed as scFv on the surface of filamentous phage.
Phages from the library were selected for binding to the factor VIII heavy chain essentially as described previously (van den Brink et al, 2000). VH and VL genes of clones reactive with factor VIII heavy chain were sequenced and compared with non-mutated germline V genes in the V-BASE sequence database (Tomlinson et al, 1999). To produce scFvs, V genes were subcloned into the vector pUC119-Sfi/Not-His6. ScFvs were expressed and subsequently purified by immobilized metal chelate-affinity chromatography as described (van den Brink et al, 2000). Inhibition of factor VIII activity by scFv was measured in the one-stage clotting assay.
Results and discussion
Longitudinal analysis of factor VIII inhibitors in patient AMC-92
The mild haemophilia A patient with an Arg593→Cys substitution in factor VIII had a baseline factor VIII activity of 0·20 IU/ml. He developed an inhibitor after a period of peri-operative replacement therapy in 1980 (Fijnvandraat et al, 1997). At that time, an inhibitor titre of 22 BU/ml was observed and the patient's endogenous factor VIII level dropped below 0·01 IU/ml. The inhibitor titre gradually declined. A low level (< 1 BU/ml) of inhibitor persisted for 15 years. Epitope mapping studies revealed that antibodies present in plasma samples of 1992 and 1995 bound exclusively to wild-type A2 domain and not to A2 containing the Arg593→Cys mutation (A2-R593C) (Fig 1B, lane 1 and 2). These findings suggested that antibodies were present that solely react with exogenous, administered factor VIII. In 1999, the patient underwent vasectomy under the protection of factor VIII and desmopressin (DDAVP). Following surgery, he developed a transient rise in inhibitor titre, which resulted in a decrease of plasma levels of factor VIII. Initially, the patient's factor VIII level decreased below 0·02 IU/ml and an inhibitor titre of 2·6 BU/ml was measured. Within 3 months, the inhibitor titre gradually decreased and the circulating factor VIII level returned to a value of 0·15 IU/ml (Fig 1A). The transient decline in factor VIII suggests cross-reactivity of the inhibitor with endogenous factor VIII. To address this issue, we evaluated the reactivity of the inhibitor with recombinant fragments corresponding to the A2 and variant A2-R593C domains. Epitope mapping studies were performed using plasma samples collected at different time points during inhibitor development. Five days following surgery, an increase in reactivity of the inhibitor towards the A2 domain was detected (Fig 1B, lanes 3 and 4); this persisted for 6 months. At later time points, lower reactivity was observed (Fig 1B, lanes 13–15). Immediately following peri-operative treatment, binding of antibodies to A2-R593C was observed (Fig 1B, lanes 3 and 4). Concomitantly, the factor VIII plasma level of the patient decreased. After 10 weeks, factor VIII plasma levels returned to 0·15 IU/ml, which coincided with a decrease in antibodies directed against A2-R593C (Fig 1B, lanes 11–13).
Our data indicated the existence of two populations of antibodies directed against the A2 domain in this patient. The first class of antibodies bound to a B-cell epitope that overlaps with Arg593 and persisted for an extended period of time. These antibodies did not bind to endogenous factor VIII and consequently plasma factor VIII levels were not affected. Following peri-operative factor VIII replacement therapy, a transient rise in a second class of antibodies was observed that cross-reacted with endogenous factor VIII. Cross-reactive antibodies directed towards residues Arg484-Ile508, a major binding site for factor VIII inhibitors in the A2 domain, have been described in two other unrelated inhibitor patients with the same genetic defect (Thompson et al, 1997; van den Brink et al, 1999). In these patients, antibodies reactive with Arg593 were not observed. These data suggest that the region Arg484-Ile508 constitutes an immunodominant epitope in patients with the Arg593→Cys mutation.
Isolation and characterization of antibodies specific for the heavy chain of factor VIII, employing phage display
Complementary to the characterization of factor VIII inhibitors from plasma, the anti-factor VIII repertoire of the patient was analysed by phage display. A V gene phage display library was constructed with a blood sample taken from the patient in 1993 (see Fig 1). The patient's IgG4-specific VH gene repertoire was combined with a VL gene repertoire of non-immune source (Van den Brink et al, 2000). The resulting library consisted of 4·7 × 106 clones and was used to isolate antibodies binding to the factor VIII heavy chain. After four rounds of selection, phages derived from 23 of 45 clones were specific for factor VIII (data not shown). The nucleotide sequences of VH and VL genes of the 23 clones were analysed, and it appeared that the sequences of 22 clones were completely identical. The VH domain of these clones was encoded by germline gene segment DP-47, a member of the VH3 family. The deduced protein sequence of the heavy chain of one of these clones (92–102) is shown in Fig 2A. One clone (92–137) isolated from the library was encoded by the VH-germline gene segment DP-58, also belonging to the VH3 family (Fig 2A). Phages expressing scFv 92–102 and 92–137 were tested for binding to variant and wild-type factor VIII A2 domain. Both clones bound to wild-type A2 domain as well as to the mutated A2-R593C domain. Phages 92–102 and 92–137 did not react with the A2-FV484-508 (Fig 2B). Similar results were obtained when epitope mapping studies were performed using purified scFv (data not shown). The inhibitory capacity of scFv 92–102 and 92–137 was evaluated in a Bethesda assay. Only slight inhibition of factor VIII was observed at elevated levels of scFv (specific activity < 5 BU/mg).
Our analysis revealed that the patients' B cells expressed antibodies directed against Arg484-Ile508 at a time when antibodies with this specificity were not observed in plasma. Phage display provides information on the immunoglobulin repertoire expressed by peripheral B lymphocytes whereas antibodies in plasma are derived from antibody-secreting plasma cells present in bone marrow and spleen. Only a limited number of plasma cells circulate in the periphery. Virtually all peripheral blood B lymphocytes can be phenotypically divided into naive and memory B cells (Klein et al, 1998). In view of the large number of somatic hypermutations present in the scFvs, it is likely that these antibody fragments originated from the memory B-cell pool. Long-living memory B cells differentiate into plasma cells upon stimulation with antigen (MacLennan et al, 1997; Ochsenbein et al, 2000). The rapid anamnestic response observed in this patient may be due to stimulation of factor VIII-specific memory B cells. Alternatively, antibodies may originate from anergic self-reactive B cells that are activated following a strong immunogenic stimulus during peri-operative treatment (Goodnow, 1996). Future studies should address whether human antibodies isolated from peripheral blood lymphocytes by phage display originate from the memory B-cell pool.
This study was financially supported by a grant from the Netherlands Organization of Scientific research (NWO) (grant 902-26-204). We thank Professor Dr K. Mertens, Dr K. Fijnvandraat and Professor Dr R. A. W. van Lier for helpful suggestions and critical reading of the manuscript.