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.

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 recognition of hypothalamo-neurohypophysial functions by developing T cells.

Neuropeptide signals and specific neuropeptide receptors have been described in the thymus supporting the concept of a close dialogue between the neuroendocrine and the immune systems at the level of early T-cell differentiation. In this paper, we review recent data about neurohypophysial (NHP)-related peptides detected in the thymus from different species. We suggest that we are dealing in fact with other member(s) of the NHP hormone family, which seems to exert its activity locally through a novel model of cell-to-cell signaling, that of cryptocrine communication. This model involves exchange of signals between thymic epithelial cells and developing thymocytes. The NHP-related peptides have been shown to trigger thymocyte proliferation and could induce immune tolerance of this highly conserved neuroendocrine family.

The human genome contains hundreds of genes coding for finger proteins of the Krüppel type.

Our aim was to identify new human proteins with potential DNA binding activity, related to the Krüppel protein which regulates Drosophila segmentation. We screened a human placenta cDNA library and a human genomic DNA library with a synthetic oligonucleotide probe corresponding to the H/C link region that connects finger loops in the multifingered Krüppel protein. We found more than 100 different mRNAs encoding Krüppel multifingered proteins in the human placenta. In the whole human genome, the number of genes encoding such proteins reaches about 300. Sequence analysis of 14 cloned cDNAs indicated that they code for at least nine undescribed human finger proteins. The sequences of the 106 finger repeats present in these nine proteins are highly homologous. Most of the variability lies in a limited number of positions located in their postulated alpha-helical structure, and therefore could be implicated in their DNA-binding specificity.

Development and evolutionary aspects of thymic T cell education to neuroendocrine self.

Thymic epithelial cells, including nurse cells (TECs/TNCs), from various species synthesize neuroendocrine-related precursors belonging to neurohypophysial, tachykinin and insulin hormone families. The thymic repertoire of neuroendocrine-related polypeptides illustrates at the molecular level the paradoxical role of the thymus in both T cell positive and negative selection. On the one hand, these precursors are a source of signals which interact with neuroendocrine-type receptors expressed by target pre-T cells according to the cryptocrine type of cell-to-cell signaling. On the other hand, the same precursors constitute a source of self-antigens which are presented to pre-T cells by the thymic major histocompatibility complex system. Basically, the model of thymic T cell education to neuroendocrine self was established by the identification in TECs/TNCs of immunoreactive (ir) oxytocin as the self-antigen of the neurohypophysial family. Nevertheless, through the expression in TECs/TNCs of ir-neurokinin A and ir-insulin-like growth factor-II, the model also applies to the tachykinin and insulin hormone families.

Membrane translocation and relationship with MHC class I of a human thymic neurophysin-like protein.

Thymic epithelial and nurse cells (TEC/TNC) synthesize an oxytocin (OT)-like peptide in association with a neurophysin (NP)-related protein in a way similar to in the hypothalamo-neurohypophysial (NHP) system. The central T-cell tolerance of the NHP neuroendocrine functions have been proposed to be mediated through these thymic NHP-related peptides due to their close homology with the NHP neurohormones OT and vasopressin (VP). In order to investigate their putative presentation by proteins of the major histocompatibility complex (MHC), human thymic membranes were purified and passed through an immunoaffinity column using mAb B9.12 directed to the monomorphic determinant of human MHC class I proteins. This methodology provided the following observations: (1) a NP-like protein is translocated in human thymic membranes and is retained by B9.12 on the column; (2) the MW of this NP-like material (50-55 kD) is quite different from the MW of hypothalamic NP proteins (10 kD), and (3) this thymic NP-like protein could be identified on Western blots with mAb B9.12. The precise extent of this relationship between the thymic NP-like protein and the Ig/MHC superfamily is actually investigated through the characterization of the genetic mechanisms responsible for the thymic expression of NHP-related peptides. Given the physiological importance of OT and of its binding to NP for transport along the axonal processes of the NHP tract, we postulate that, somewhat analogously, the thymic NP-/MHC class I-related protein could be involved in the presentation of the OT-like peptide to immature T-cells.

A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor.

The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4kappa antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor.

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4kappa monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass approximately 7.4 x 10(5) M-1 s-1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss < or = 1 x 10(-5) s-1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor.

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.