Low Molecular Weight Peptides Restore the Procoagulant Activity of Factor VIII in the Presence of the Potent Inhibitor Antibody ESH8

Abstract

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.Coagulation defects attributed to the absence or dysfunction of blood coagulation factor VIII (FVIII) 1 are observed in 0.01-0.02% of the male population (1). This recessive X-linked bleeding disorder is known as hemophilia A. The adequate treatment of this disease relies on infusions of human FVIII concentrates. However, a serious treatment complication is the development of a humoral immune response to FVIII in ϳ25% of patients with hemophilia A. These antibodies (Abs), also called inhibitors, block the FVIII procoagulant activity and complicate seriously the medical care of the patients by precluding further FVIII injections (2). This immune response is not only polyclonal but also heterogeneous in its specificity (3). Nevertheless, epitope mapping studies have shown that inhibitors recognize restricted binding sites, predominantly on the A 2 , C 2 , and A 3 domains on the FVIII molecule (4). The incidence of inhibitor occurrence in hemophilia A patients is difficult to estimate and can vary from 18 to 28% (5). This variation can be the result of a number of parameters, which needs to be considered, including quantitative criteria of inhibitor dosages, the elapsed time from the first apparition, the origin, the viral inactivation procedure applied, the purity of the administered product, and, of course, the patient.A number of different therapeutic approaches have been developed to circumvent complications arising from inhibitors, such as the administration of porcine FVIII, which is less antigenic than its human counterpart (6), the administration of activated human FVII to bypass the intrinsic pathway (7), and the activation of ...

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

“…The comparison of the decapeptides that overlap the 45 cleavage sites based on hydropathicity profiles and of the frequency of occurrence of amino acids, did not allow us to identify a consensus linear peptide that could account with certainty for a majority of the identified scissile bonds. However, it may be that some of the cleavage activity would only be neutralized by peptides that mimic conformational rather than linear epitopes, as previously shown for several of the conventional noncatalytic FVIII inhibitors (36,37). Based on the observations that almost 35% of cleavages occurred after an Arg, that the hydropathicity profile of PFR correlated with 16 of the 45 identified cleavage sites (data not shown) and that MCA-coupled PFR is an appropriate surrogate substrate for FVIIIhydrolyzing Abs (10), it may be speculated that a single or a restricted number of PFR-based tripeptides would be of relevance to block FVIII-specific Ab catalysts.…”

In vivo neutralization of a C2 domain–specific human anti–Factor VIII inhibitor by an anti-idiotypic antibody

“…20 The capacity of mAb14C12 to restore the function of FVIII was examined by using various concentrations of mAb14C12. Preliminary experiments have established that the t 1/2 of mAbBO2C11 and mAb14C12 in FVIII Ϫ/Ϫ mice were of 3 and 5 days, respectively.…”

“…The assay was run essentially as described, 20 except for the use of mAb14C12 instead of peptides. Briefly, C57BL/6 FVIII Ϫ/Ϫ mice were reconstituted by intravenous injection of 1 IU rFVIII, which gives a concentration of 0.5 IU/mL and a reaction half time (t 1/2 ) of 180 minutes.…”

Desenvolvimento de inibidores do fator VIII na hemofilia A

“…6 Esta variação na produção de inibidores pode ser o resultado de vários parâmetros que incluem critérios quantitativos da dosagem do inibidor, tempo decorrido da primeira aparição, a origem, pureza do produto administrado e o próprio paciente. 7 Desta forma, fatores de risco podem ser estabelecidos para o desenvolvimento desta resposta imune contra o FVIII.…”

“…Recentemente foram descritas experiências baseadas no bloqueio da atividade deletéria dos anticorpos por peptídeos de baixo peso molecular construídos para mimetizar os epítopos reconhecidos pelos anticorpos anti-FVIII na tentativa de restaurar a atividade procoagulante normal por impedir a ligação dos anticorpos ao FVIII. 7 A respeito da possibilidade de mapear os diferentes epítopos reconhecidos pelos inibidores do FVIII dos pacientes, vários estudos revelaram que o padrão de reatividade do anticorpo é policlonal, direcionado contra múltiplos locais situados no FVIII e único para cada inibidor do plasma investigado. 6 Todavia, estudos mostraram que os inibidores reconhecem sítios de ligação restritos, predominantemente, nos domínios A2, C2 e A3 da molécula de FVIII.…”