anti-FVIII Antibodies Research Articles

Somatic Mutations in Factor VIII Inhibitors Determine Their Factor VIII Binding and Inhibitory Properties.

The formation of inhibitors against Factor VIII (FVIII) is one of the major adverse effects of FVIII substitution therapy in hemophilia A patients. Previously we have cloned a panel of antibodies from a phage display library from the VH gene repertoire of a single hemophilia A patient (Van den Brink et al, Blood 2002). One single-chain variable fragment (scFv), designated KM33, was found to display remarkably high affinity FVIII binding and FVIII nhibition. The epitope of this scFV was located in the FVIII light chain, in particular within the C1 domain. Using computational epitope prediction methods we identified three putative antibody binding C1 loops. By mutagenesis we found one of these (K2092-S2094) critical for KM33 binding. This region is the counterpart of one of the lipid-binding loops in the FVIII C2 domain. This suggest that KM33 inhibits FVIII activity by interfering in FVIII with membrane binding. Sequence analysis revealed that the scFV fragment KM33 was derived from the germ-line sequence VH 3–30 with a number of substitutions. As somatic mutations have the potential of contributing to affinity maturation and antigen neutralization, we addressed the contribution of individual mutations to the inhibitory properties of scFV KM33. Somatic mutations were reversed to germ-line residues, and purified KM33 variants were analyzed by Surface Plasmon Resonance (SPR) and by solid-phase binding and inhibition assays. Somatic mutations in positions H56 (S to N) and H57 (N to D) within the Complementarity Determining Region H2 (CDR H2) proved neutral with regard to affinity for FVIII. In contrast, the somatic mutations near the CDR H1 (H23 A to V, H24 A to D, and H27 F to L) proved of major impact. The equilibrium binding constant (Kd) of mutant VH-D24A (4.4 nM) and the triple mutant VH-(V23A-D24A-L27F) (10 nM) were 2–3 orders of magnitude higher than that of the wild-type KM33 (Kd = 0.05 nM). The SPR sensorgrams revealed that the difference in affinity resulted mainly from higher dissocation rates for the mutants. Molecular modeling and molecular dynamics suggested that Asp in position H24 plays a key role in that it forms a hydrogen bond network that stabilizes the CDR H1 in the mature KM33 antibody. The stabilization of CDR H1 in KM33 proved essential for its inhibitory properties and its detection as an anti-FVIII antibody. First, the KM33 D24A variant failed to display any inhibitory activity in the classical Bethesda assay, irrespective whether residual FVIII activity was assessed by one-stage assay or by chromogenic assay. Second, this variant tested negative in ELISA based assays for FVIII-binding, but non-inhibitory antibodies, presumably due to its relatively high dissociation rate from immobilized FVIII. This finding may have major implications for the detection of FVIII inhibitors. First, anti-FVIII antibodies may exist that are directed against functional epitopes on the FVIII molecule that escape detection in functional Bethesda assays. The same may hold for binding assays that are designed to detect typical non-inhibitory antibodies. While patients having such antibodies seem virtually inhibitor-free, a single somatic mutation within a pre-existing non-inhibitory antibody may result in the a strong FVIII inhibitory response. This may be particularly relevant in linking FVIII inhibitor development in individual patients to exposure to specific FVIII products.

T-Cell-Independent Re-Stimulation of FVIII-Specific Memory B Cells Requires Help from Activated Plasmacytoid Dendritic Cells.

Memory B cells are essential for maintaining FVIII inhibitors in patients with hemophilia A. Using the murine E-17 model of hemophilia A, we showed previously that re-exposure to FVIII re-stimulates memory B cells very rapidly and drives their differentiation into antibody-producing plasma cells. Furthermore, we presented evidence that the re-stimulation of FVIII-specific memory B cells is regulated by the dose of FVIII used. Low doses re-stimulate memory B cells whereas high doses of FVIII inhibit this process and prevent the differentiation into anti-FVIII antibody-producing plasma cells. Both the re-stimulation and the inhibition can be modulated by triggering toll-like receptors (TLR) 7 and 9 with specific ligands that are typically found in microbial components derived from viruses or bacteria. Re-stimulation of FVIII-specific memory B cells in the presence of TLR ligands can even be observed in the absence of CD4+ helper T cells that are otherwise absolutely essential for this process. Based on these previous observations we asked whether the re-stimulation of FVIII-specific memory B cells in the absence of CD4+ helper T cells requires interaction with alternative “helper” cells that provide co-stimulatory signals to memory B cells. To address this question we used spleen cells obtained from hemophilic mice treated with FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity of the different cell populations was achieved by a combination of magnetic bead separation and multi-color flow cytometric cell sorting. The memory B cell compartment was specified by the expression of CD19 together with surface IgG and the absence of surface IgM and IgD. Memory B cells were single-cell sorted and cultivated in micro-well cultures in the presence of FVIII to stimulate the in vitro differentiation into anti-FVIII antibody- producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 or 9 and dendritic cells were added to the micro-well cultures to find out which of the additives were required for the re-stimulation and differentiation of memory B cells. Neither FVIII alone nor any combination of FVIII and ligands for TLR 7 and 9 were able to re-stimulate highly purified memory B cells to differentiate into anti-FVIII antibody-producing plasma cells. The re-stimulation strictly depended on the presence of additional cells that could provide co-stimulation. These additional cells could be either activated CD4+ T cells or, alternatively, plasmacytoid dendritic cells activated by ligands for TLR 7 or 9. Some re-stimulation in the presence of activated plasmacytoid dendritic cells was even observed in the complete absence of FVIII. Based on our results we conclude that plasmacytoid dendritic cells that are activated by TLR ligands such as those expressed by infectious agents can replace CD4+ T cells in triggering the re-stimulation of memory B cells and their differentiation into antibody-producing plasma cells. Our findings provide important new insights into the regulation of memory-B-cell re-stimulation that need to be considered in the development of new therapeutic strategies for treating patients with FVIII inhibitors. Furthermore, our findings underscore the importance of environmental factors in the regulation of FVIII inhibitor development.

FVIII Tolerance Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Encode a High-Expression Factor VIII Transgene in Hemophilia A Mice.

Clinical gene therapy trials for hemophilia A have failed due to insufficient target cell transduction or expression of factor VIII (fVIII). Animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII from recombinant viral vectors without eliciting an anti-fVIII humoral response. We previously demonstrated curative fVIII activity levels in hemophilia A mice following a nonmyeloablative, genetically-modified bone marrow transplantation (Ide et al., Blood 2007). In these studies recipient hemophilia A mice received 3×105 bone marrow-derived sca-1+ cells transduced with a retrovirus encoding B-domain-deleted high-expression porcine fVIII (BDDpfVIII) after pre-transplantation conditioning with reduced-intensity TBI or busulfan followed by minimal immunosuppression with either costimulation blockade or antithymocyte serum (ATS). Using our high-expression fVIII transgene and busulfan or 3Gy TBI coupled with ATS resulted in sustained fVIII activity of >100% normal human levels. The main hematopoietic toxicity observed was transient T-cell suppression. High-level fVIII activity (>2 units/ml) was achieved without induction of anti-BDDpfVIII humoral immune responses. Furthermore because the development of inhibitory antibodies directed against fVIII remains a significant clinical complication associated with the treatment of hemophilia A, we tested the effectiveness of genetically-modified cells encoding BDDpfVIII in the setting of pre-existing fVIII immunity. Transplantation of genetically-modified HSCs into 5.5 Gy TBI/ATS or busulfan/ATS-conditioned hemophilia A mice that had significant anti-human fVIII inhibitory titers induced sustained, high-level fVIII activity. Following transplantation and stable engraftment, both naïve and pre-immunized mice were challenged with six weekly injections of 10 units human fVIII, an immunization regimen that results in antibody formation in 100% of naïve hemophilia A mice. All mice sustained their pre-challenge BDDpfVIII activity levels, and anti-fVIII antibodies were not detected. Surface markers of T-cell activation were the same between naïve hemophilia A mice and transplanted mice, for both naïve and preimmunized animals. In a mixed lymphocyte reaction, T-cells were found to be equally capable of reacting to allogenic antigens (i.e. irradiated Balb/C splenocytes) when isolated from mice with stable fVIII expression compared to nontransduced controls, demonstrating that the lack of anti-fVIII inhibitors is not due to a lack of immunocompetent T-cells. Further studies are underway assessing T-cell nonresponsiveness to BDDpfVIII in these transplanted mice. By demonstrating that transplanted mice are tolerant to BDDpfVIII, even in the context of pre-existing inhibitory antibodies and nonmyeloablative conditioning, this data further supports our previous studies showing that retroviral gene transfer using high-expression porcine fVIII elements is a compelling treatment for hemophilia A.

Enhanced Anticoagulant Activity of Factor VIII Inhibitors Due to Positive Cooperativity between Two Classes of Anti-Factor VIII C2 Antibodies.

We recently identified 5 distinct structural classes of anti-human factor VIII (fVIII) C2 domain monoclonal antibodies (MAbs) in a murine model of hemophilia A. Three classes, Groups A, B, and C, consist of MAbs with non-overlapping epitopes. Group AB MAbs overlap epitopes of Group A and B MAbs, and Group BC MAbs overlap epitopes of Group B and C MAbs. Concentration dependent binding of biotinylated Group A MAbs to fVIII bound to a microtiter plate was studied in the presence and absence of Group BC MAbs by ELISA. Surprisingly the binding of all 3 Group A antibodies tested, ESH4, 3E6, and I54, was increased in the presence of Group BC antibodies. Group A antibodies block the binding of fVIII to von Willebrand factor (VWF) and phospholipid. Group BC antibodies inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to VWF and phospholipid. To establish functional relevance, the effect of Group BC MAbs on the ability of Group A MAbs to block fVIII binding to VWF was determined by ELISA. Group A MAbs were serially diluted into fixed concentrations of Group BC MAbs and then incubated with fVIII in solution for 2 hours. The fVIII - antibody mixtures were added to microtiter wells coated with VWF, and fVIII binding to VWF was measured. Group BC MAbs increased the ability of ESH4 and 3E6 to inhibit the binding of fVIII to VWF, consistent with the observation that the binding of Group A and Group BC antibodies is positively cooperative. We previously have shown that antibody-mediated inhibition of fVIII binding to VWF is correlated with inhibition of fVIII binding to phospholipid and with fVIII inhibitor titers. Although most Group BC MAbs have high inhibitory titers against fVIII, Group BC MAb D105 is only minimally inhibitory at 0.8 Bethesda units/mg IgG. Because D105 increased the binding of ESH4 to fVIII like other Group BC MAbs, the effect of D105 on the inhibitor titer of ESH4 was investigated using the Bethesda assay. The inhibition of fVIII was significantly increased by the presence of D105 (Fig. 1). This study identifies increased binding of Group A MAbs to fVIII in the presence of Group BC MAbs using 3 different assays. Our results suggest that the cooperative interaction of fVIII antibody epitopes affects the overall inhibitor patient phenotype and that inhibitors with very low inhibitor titers can significantly increase the inhibitory effects of other anti-fVIII antibodies. [Display omitted]

In the Presence of Pre-Existing Factor VIII (FVIII) Immunity, Hematopoietic Stem Cells (HSC) That Are Genetically Modified To Express FVIII in Platelets Were Successfully Transplanted into Hemophilic Mice under Myeloablative and Various Non-Myeloablative Conditions.

While genetic induction of FVIII expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of pre-existing FVIII inhibitory antibodies, and whether such antibodies would affect therapeutic engraftment. The development of inhibitors to exogenous FVIII is considered a severe and important complication of FVIII infusion in hemophilia A patients. The clinical hallmark of inhibitor development in hemophilia A patients is failure to response to routine replacement therapy for bleeding episodes. In this study, we wanted to explore whether syngeneic transplantation of HSC that are genetically modified to express FVIII in platelets would restore hemostasis to hemophilic mice with pre-existing FVIII immunity. We generated a line of transgenic mice that express FVIII only in platelets using the platelet-specific αIIb promoter (2bF8) and bred this 2bF8 transgene into a FVIIInull background. Bone marrow (BM) from immunized or non-immunized heterozygous 2bF8 transgenic mice was transplanted into immunized FVIIInull mice following lethal or sub-lethal irradiation. The recipients were analyzed by PCR, quantitative real-time PCR, platelet-lysate FVIII activity assay, inhibitor assay, and tail clip survival test to assess engraftment and phenotypic correction. Our results demonstrated that pre-existing anti-FVIII inhibitory antibodies in recipients did not inhibit successful engraftment of BM cells with 2bF8 genetic modification into hemophilia A mice. When we looked at the effect of inhibitor titer, following one month BM reconstitution, all recipients with inhibitor titers of 50 – 400 BU/ml (n = 9) survived tail clipping and 80% of recipients with a titer of 401 – 20,000 BU/ml (n = 25) also survived tail clipping when 1100 R (myeloablative) or 660 R (non-myeloablative) regimens were employed. When comparing conditioning regimens, in immunized recipients conditioned with 1100 R that received BM from 2bF8 donors the tail clip survival rate was 85% (n = 20). In the group of recipients in which 660R was employed 85.7% (n = 14) recipients survived tail clipping. When immunized recipients were conditioned with 440 R, 60% (n = 5) survived. In the group of recipients in which 220 R was employed, none (n = 4) survived tail clipping. The tail clip survival rate was not significantly different between the groups that received BM from immunized 2bF8 donors and from non-immunized donors. Different proportions of mixed BM from 2bF8 transgenic mice and FVIIInull mice were transplanted into lethally irradiated FVIIInull recipients to address what fraction of platelets would need to express FVIII in order to restore hemostasis to hemophilic mice with inhibitors. Our results showed that transplantation with only 5% 2bF8 BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not alter engraftment of 2bF8 genetically modified HSC and transplantation of these HSC can efficiently restore hemostasis to hemophilic mice with pre-existing inhibitory antibodies under either myeloablative or non-myeloablative regimens.

Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.

Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.

Epitope Identification and Generation of an Antigen-Specific T-Cell Clone in a Mild Hemophilia A Inhibitor Subject with Missense Genotype R593C.

The development of inhibitory anti-FVIII antibodies is a major clinical problem in hemophilia A. While less common in mild/moderately severe patients, the relative risk of inhibitor formation is elevated in patients with missense mutations in the FVIII A2 domain, especially those with an R593C genotype. In this study, T-cell responses to 14 FVIII A2 domain peptides with predicted DRB1*1101 MHC binding motifs were investigated using MHC class II tetramer reagents. CD4+ T cells were isolated from a hemophilic subject (DRB1*1101, 1302) with FVIII missense genotype R593C and stimulated with pooled peptides. This subject had developed a longstanding low titer inhibitor after receiving multiple FVIII infusions. Staining with fluorescent, peptide-loaded tetramers revealed that the hemophilic subject, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide A2589–608, which contained the wild-type R593 sequence. MHC class II tetramers with bound A2589–608 were used to sort antigen-specific T cells and then generate a T-cell clone recognizing this high avidity DRB1*1101-restricted epitope. FVIII residues 594–602 (FLPNPAGVQ) comprise a predicted high-affinity binding motif. Peptide binding assays confirmed that A2589–608 bound to recombinant, monomeric DRB1*1101 protein with high affinity. A peptide with the corresponding hemophilic sequence (A2589–608, 593C) bound with affinity that was threefold lower, but in a range that should be sufficient for presentation to T cells. The other 12 A2 peptides had a wide spectrum of binding affinities for the DRB1*1101 protein. Interestingly, prediction algorithms suggest that the peptides containing residue 593 occupy the MHC Class II peptide-binding groove in a register that places this residue just outside the p1 anchor position. Thus we anticipate that residue 593 modulates T-cell recognition of peptide A2589–608. This provides a potential explanation for inhibitor development in mild hemophilia A patients with a FVIII R593C genotype who are HLA-DRB1*1101. Immunogenicity of this FVIII region with respect to other HLA types is under investigation.

The Relevance of the Inhibitory Receptor FcγIIB for the Regulation of Anti-Factor VIII Antibody Responses in Murine Models.

The prevention of neutralizing anti-factor VIII (FVIII) antibodies remains the major challenge in the treatment of hemophilia A patients with FVIII products. Therefore, it is important to understand how B cells differentiate into antibody-producing plasma cells, how this process is regulated and how it can be inhibited. A number of inhibitory receptors have been identified that are expressed on B cells and might provide targets for the prevention of antibody development against FVIII. The inhibitory receptor FcγIIB has emerged as a key regulator of B cell responses in the later phase of antibody responses. It has been described as a potent inhibitor of B-cell-receptor (BCR) signaling when co-ligated to the BCR by engagement of antigen-containing immune complexes. Based on these findings we asked whether the inhibitory receptor FcγIIB is involved in the natural regulation of anti-FVIII antibody responses in murine models. If this receptor were an important negative regulator of anti-FVIII antibody responses, mice that do not express this receptor should develop higher titers of anti-FVIII antibodies than mice that express the receptor. We treated wildtype mice and FcγRIIB knockout mice, both on a C57BL/6J background, with four intravenous doses of 200ng of human FVIII (80U/kg) and analyzed titers of anti-FVIII antibodies after the second, the third and the fourth dose. Surprisingly, we did not see any significant difference in antibody titers between both strains of mice. We extended our study and asked under which conditions FcγRIIB knockout mice would develop higher titers of anti-FVIII antibodies than wildtype mice. We tested different application routes (i.v. and i.p.) of FVIII, compared low doses (200ng) and high doses (80μg) and combined the i.p. application with or without Freund's adjuvant. Furthermore, we included KLH-TNP (i.p. application of 100μg with Freund's adjuvant) as a positive control that was previously shown to induce different levels of antibodies in wildtype mice compared with FcγRIIB knockout mice. We found significant differences in the development of specific antibodies between both strains of mice when we used the positive control (i.p. KLH-TNP with adjuvants) and when we applied high dose FVIII (80μg) with Freund's adjuvant i.p. We did not see any differences in antibody titers between both strains of mice with any of the other treatment schedules. Our results indicate that the inhibitory receptor FcγIIB is only active as a negative regulator for antibody responses against foreign proteins in vivo when a certain threshold level of antibodies is reached in the circulation. This critical threshold level is obviously not reached when mice are treated with clinically relevant doses of FVIII. Based on our results we conclude that the inhibitory receptor FcγIIB is not involved in the regulation of antibody responses against therapeutically relevant doses of human FVIII in murine models. Whether the same situation is true for patients with hemophilia A remains to be shown. Furthermore, the relevance of the inhibitory receptor FcγIIB might differ between patients who react to the FVIII product as a foreign protein and patients who react to FVIII as an altered self protein or self protein.

ACQUIRED HEMOPHILIA IN OLDER PEOPLE: A POOR PROGNOSIS DESPITE INTENSIVE CARE

ACQUIRED HEMOPHILIA IN OLDER PEOPLE: A POOR PROGNOSIS DESPITE INTENSIVE CARE

Assessment ofIn VitroCytokine Response in Hemophilia A Patients With or Without Factor VIII Inhibitory Antibody

Factor VIII (FVIII) inhibitor antibodies are produced in a proportion of hemophilia A patients. Development of anti-FVIII inhibitor antibodies is a T cell-dependent response, mediated by FVIII specific CD4(+) T cells. This study was performed to investigate the contribution of T helper (Th) cell-mediated cytokine response in inhibitor production. Peripheral blood mononuclear cells (PBMCs) were obtained from hemophilia A patients with (n = 14) or without inhibitor (n = 14) and from normal individuals (n = 14). Following stimulation of PBMCs with rFVIII and phytohemagglutinin (PHA) mitogen, the secreted cytokines, interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-beta1), in culture supernatant and the proliferative response were assessed using sandwich ELISA and (3)H-thymidine incorporation, respectively. No significant proliferative response to FVIII was observed, whereas PHA induced a strong response in all groups. No cytokine secretion was observed in response to FVIII stimulation. Although PHA induced IL-10, TGF-beta1 and IFN-gamma secretion in all groups, the level of IFN-gamma was significantly lower in hemophilia A patients than in normal individuals (p < 0.0001). The levels of TGF-beta1 and IL-10 were similarly higher in patients compared with normal subjects, but the difference was not statistically significant. Lack of FVIII-induced proliferative response and cytokine production together with reduced secretion of PHA-induced IFN-gamma in both groups of patients suggest involvement of nonspecific immunosuppression possibly due to hepatitis C virus (HCV) infection observed in the majority of patients.

Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A. Disclosure of potential conflicts of interest is found at the end of this article.

A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes.

Several therapeutic self-proteins elicit immune responses when administered to patients. Such adverse immune responses reduce drug efficacy. To induce an immune response, a protein must interact with different immune cells, including antigen-presenting cells, T cells, and B cells. Each cell type recognizes distinct immunogenic patterns on antigens. Mannose-terminating glycans have been identified as pathogen-associated molecular patterns that are essential for internalization of microbes by antigen-presenting cells, leading to presentation. Here, we have investigated the importance of exposed mannosylation on an immunogenic therapeutic self-protein, procoagulant human factor VIII (FVIII). Administration of therapeutic FVIII to hemophilia A patients induces inhibitory anti-FVIII antibodies in up to 30% of the cases. We demonstrate that entry of FVIII into human dendritic cells (DC) leading to T cell activation, is mediated by mannose-terminating glycans on FVIII. Further, we identified macrophage mannose receptor (CD206) as a candidate endocytic receptor for FVIII on DC. Saturation of mannose receptors on DC with mannan, and enzymatic removal of mannosylated glycans from FVIII lead to reduced T cell activation. The interaction between FVIII and CD206 was blocked by VWF, suggesting that, under physiological conditions, the intrinsic mannose-dependent immunogenicity of FVIII is quenched by endogenous immunochaperones. These data provide a link between the mannosylation of therapeutic self-proteins and their iatrogenic immunogenicity. Such a link would be of special relevance in the context of replacement therapy where mechanisms of central and peripheral tolerance have not been established during ontogeny because of the absence of the antigen.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

Molecular characterization of human B domain-specific anti-factor VIII monoclonal antibodies generated in transgenic mice

The development of antibodies directed against factor VIII (FVIII) represents a major hurdle in the treatment of hemophilia A. Most anti-FVIII antibodies are identified through their ability to inhibit the FVIII procoagulant activity. Many of them, however, do not interfere with the functional properties of FVIII. Antibodies directed against the B domain belong to this latter category. Here, we characterized B domain-specific human monoclonal Abs (mAbs) at the molecular level. A series of human mAbs directed against FVIII was produced upon immunization of transgenic XenoMouse mice with human recombinant FVIII (rFVIII). Selection of the hybridoma with epitope specificity for the B domain was performed by differential recognition of full-length and B domain-deleted rFVIII. None of the anti-B domain mAbs demonstrated inhibitory activity against FVIII. Three of the mAbs recognized linear epitopes: mAb 25H3 bound to the (1014)HIDGPSLLIEN(1024) sequence; mAbs 8E3 and 22B6 shared the same epitope, composed of residues (1534)KWNEANR(1540). The corresponding soluble peptides inhibited the binding of their respective mAbs to FVIII. mAbs 8E3 and 22B6 displaced the binding of FVIII to vonWillebrand factor. Moreover, some of them (in particular mAbs 4G6 and 8E3) were able to compete for binding to the B domain with the anti-FVIII Abs from hemophilia A patients without inhibitor or with low Bethesda titers. Further investigation will allow to better characterize their clinical relevance.

In silico prediction of FVIII epitopes recognised by natural autoantibodies in polyvalent immunoglobulin concentrates

Inhibitory antibodies directed against blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic and autoimmune patients. Identifying B-cell FVIII epitopes and mapping them on the molecule remain important challenges. Using a combination of different algorithms, more than 30 hypothetical linear epitopes were predicted on the FVIII molecule surface. We selected several major predicted sequences, spanning all FVIII domains, for specific antibody induction in rabbits. All peptides tested successfully induced production of specific anti-FVIII rabbit antibodies, supporting the relevance of our approach. To investigate the presence of FVIII-reactive antibodies in the healthy donor population, a pooled fraction rich in all IgG subclasses was purified on peptide-Sepharose columns. Substantial amounts of Ig, specific for each FVIII peptide, were purified with yields ranging from 8 to 223 ng/mg immunoglobulins. Our results confirm the diversity of FVIII epitopes recognised by natural human anti-FVIII autoantibodies. All IgG subclasses were found in the affinity-isolated anti-peptide material, with overrepresentation of IgG2 and IgG4. Evidence was also found for new FVIII epitopes. Five human anti-peptide preparations displayed FVIII-neutralising activity, ranging from 1.3 to 5.3 BU/mg. Although the presence of naturally occurring anti-FVIII antibodies in healthy donors has been previously described, our methodology has allowed, for the first time, a fine mapping of several inhibitory and non-inhibitory epitopes. Our observations support the hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.

Comparable Long-Term Persistence of Anti-FVIII Antibodies in Hemophilic E17 Mice on Different Genetic Backgrounds Despite Significant Differences in the Amplitude of the Immune Responses.

About 30% of patients with severe hemophilia A develop neutralizing antibodies against FVIII (FVIII inhibitors) following replacement therapy. The type of FVIII gene mutation as well as other predisposing genetic factors contribute to the inhibitor phenotype. Based on these findings, we asked if the genetic background modulates the long-term persistence of anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells in the E17 murine hemophilia model. Furthermore, we asked if the recently described inhibition of memory-B-cell re-stimulation by high doses of FVIII is influenced by the genetic background of the murine model.E17 mice on two different genetic backgrounds (C57Bl/6J and Balb/c) were treated with four doses of 200 ng human FVIII at weekly intervals. Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were followed up to 12 months after the last dose of FVIII. Antibody titers and subclasses of antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) were measured by ELISA. Antibody secreting plasma cells in spleen and bone marrow were detected by ELISPOT as described (Hausl et al., Thromb Haemost 2002). The re-stimulation of FVIII-specific memory B cells was studied as described recently (Hausl et al., Blood 2005). Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were first detectable in E17 Balb/c mice. IgM antibodies in the circulation and IgM secreting plasma cells in the spleen were observed after the first dose of FVIII, IgG antibodies and IgG secreting plasma cells after the second dose. No anti-FVIII antibodies after the first dose of FVIII were observed in E17 C57BL/6J mice but both IgM and IgG antibodies as well as IgM and IgG producing plasma cells were detectable after the second dose of FVIII. The antibody response involved all IgG subclasses in both mouse strains. However, IgG1 was dominant in E17 Balb/c mice whereas IgG2a was dominant in E17 C57BL/6J mice.When the in vitro restimulation of FVIII-specific memory B cells was examined, similar patterns were observed for both mouse strains. Low concentrations of FVIII between 10 and 100 ng/ml FVIII restimulated memory B cells and induced their differentiation into antibody secreting plasma cells whereas high concentrations of FVIII between 1,000 and 20,000 ng/ml FVIII inhibited memory-B-cell-restimulation. These results indicate that the dose-dependent effect of FVIII on the restimulation of FVIII-specific memory B cells does not depend on the genetic background.The major difference between both hemophilic mouse strains was the amplitude of the anti-FVIII immune response. Peak titers of anti-FVIII antibodies and peak concentrations of anti-FVIII antibody secreting plasma cells in spleen and bone marrow were significantly higher in E17 C57BL/6J mice than in E17 Balb/c mice. Whether or not higher ELISA titers correlate with higher Bethesda titers of neutralizing antibodies is currently being investigated. Despite the substantial differences in the amplitude of the immune response, anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells persisted for the whole observation period of 12 months after the last dose of FVIII in both mouse strains.We conclude that the amplitude of the anti-FVIII immune response in hemophilic mice is significantly different between E17 C57BL/6J and E17 Balb/c mice. However, the persistence of the immune response is comparable.

A Limited Number of Anti-Idiotypic Antibodies Is Required To Maintain FVIII Function in Presence of Polyclonal Anti-FVIII Antibodies of Haemophilia A Patients with Inhibitors.

Replacement therapy using either plasma-derived or recombinant FVIII elicits FVIII-specific Abs in about 25% of the patients. The majority of such antibodies are directed towards specific FVIII regions on which major epitopes have been identified. Most of these epitopes are located in the carboxy-terminal end of the C2 domain of the FVIII light chain and in the amino-terminal end of the A2 domain of the heavy chain, whereas, antibodies directed towards the C1 domain have been described in cases of mild/moderate haemophilia A. From a functional point of view, epitopes have been mapped to the phospholipid and von Willebrand binding sites in the C1 and C2 domains and to the FIX binding site in A2.We have derived human mabs (mabRhd5, mabBo2C11 and mabBoIIB2) that react with high affinity to the FVIII C1, C2 and A2 domains, respectively, and are representative of most of the specific inhibitors observed in haemophilia A patients.Recently, we have generated mouse anti-Id mab (mab14C12) against mabBo2C11 and provided the first demonstration that an anti-Id mab directed towards a C2-specific inhibitor restored FVIII activity both in vitro and in vivo in the presence of the inhibitor. Futher more, we demonstrated that FVIII inhibition obtained by a mixture of two anti-FVIII mabs (mabBo2C11 (anti-C2) and mabBoIIB2 (anti-A2)) was neutralized up to 100% when a mixture of the corresponding anti-Ids (mab14C12 and mab30D1) was added to the assay establishing the proof of concept that combination of anti-Id mabs will be able to neutralize the anti-FVIII immune response of haemophilia A patients. Based on this observation, We have also generated an anti-idiotypic antibody (mab18B6) towards an anti-C1 domain inhibitor (mabRHD5). In ELISA, mab18B6 specifically prevents, in a dose-dependent manner, mabRHD5 binding to C1 and fully neutralized mabRHD5 inhibitory properties in a coagulation assay. With a mixture of anti-Ids (mab14C12, mab30D1 and mab18B6) we were able to completely neutralized the cumulative anti-FVIII activity obtained in functional assay by a mixture of the corresponding monoclonal anti-FVIII antibodies. We next evaluated whether a combination of anti-Ids (anti-anti-A2,-C1,-C2) had the ability to neutralize the inhibitory properties of polyclonal antibodies obtained from haemophilia A patients. To this end, eight patients with severe haemophilia A and inhibitor were tested. An average of 70% neutralization of the inhibition was obtained in 6 out of 8 cases. These data allow to conclude that potent polyclonal high-affinity FVIII inhibitors can be neutralized with corresponding anti-idiotypic antibodies and that only a limited number of anti-idiotypic antibodies is required for inhibitor neutralization in a majority of patients.

Factor VIII-Specific Memory B-Cells in Patients with Hemophilia A.

The quick anamnestic antibody response seen after recurrent exposure to antigen involves memory B- cells that, helped by T-cells, undergo rigid proliferation and subsequent differentiation into antibody producing cells. The presence of a pool of memory B-cells allows for a rapid response to antigens which quickly eliminates incoming pathogens. In the context of immune responses to therapeutic agents such as blood coagulation factor VIII (FVIII), antigenic re-stimulation of specific memory B-cells is undesirable. In approximately 25% of hemophilia A patients replacement therapy is hampered by inhibitory antibodies that bind to FVIII. Currently, the FVIII-specific memory B-cell compartment in patients with hemophilia A has remained poorly characterized. We have developed a protocol that allows for identification and quantification of circulating memory B-cells in patients with hemophilia A. CD19+ B-cells were sorted on a layer of irradiated EL4B5 thymoma cells expressing CD40L in the presence of the supernatant of mitogen-stimulated T-cells. These experimental conditions, that mimic the interaction of B-cells with an activating helper CD4+ T-cell, induce proliferation of memory B-cells and allow them to differentiate into antibody secreting cells (ASC) in an antigen-independent manner. After 9–10 days of culture, total IgG and FVIII-specific IgG was determined by ELISA and number of ASC was determined by ELISpot. We analyzed blood samples of five multi-transfused patients (>50 FVIII administrations) who never experienced any inhibitor episode, five patients who experienced inhibitory antibodies in the past but were successfully treated with immune tolerance induction and 6 patients with an inhibitor at the time of blood sampling. The ELISA set-up appeared to be more sensitive than ELISpot showing ASC producing anti-FVIII antibodies varying from 0.2–50 ng/ml. In contrast, ELISpot analysis only allowed for detection of B-cell clones producing over ~4 ng/ml of FVIII-specific IgG. Frequencies of FVIII-specific memory B-cells varied from 0–0.027% of total number of circulating peripheral B-cells. The relative amount of circulating memory B-cells did not correspond to inhibitor titers as measured in a Bethesda assay. The highest frequencies were observed in patients suffering from anamnestic response to FVIII suggesting the importance of antigenic stimulation for maintenance of memory B-cell levels. This is further supported by the low frequency that was observed in a high-titer inhibitor patient who had not been treated with FVIII for several months prior to blood sampling. Surprisingly, we detected FVIII-specific memory B-cells in two multi-transfused patients who did not experience any inhibitor episode in the past. These B-cells were present in a low frequency however and developed into ASC producing only limited amounts of anti-FVIII antibodies. These observations suggest that peripheral blood memory B-cells can develop in the absence of clinically relevant inhibitors.

Inactivation-Resistant Recombinant Factor VIII Exhibits Superior Thrombin Generation Capacity in Comparison to Wild-Type and B Domain-Deleted Factor VIII.

Activated factor VIII (FVIIIa) functions as a cofactor in the intrinsic hemostatic pathway leading to thrombin generation. Recombinant FVIII (rFVIII) has proven effective in replacement therapy for patients with hemophilia A (FVIII deficiency). The activity of FVIIIa in plasma is limited by both spontaneous dissociation of the A2 subunit and by cleavage by activated protein C (APC). Inactivation resistant FVIII (IR8) has been bioengineered to be resistant to both mechanisms of inactivation. The specific activity of purified IR8, as determined by one-stage clotting (aPTT) and two-stage chromogenic assays, was significantly higher (~7 to 20-fold) than that of wild-type (WT)-FVIII and B domain deleted (BDD)-FVIII. The specific activity was calculated based on ELISA antigen results and complemented by Western blots using commercial anti-FVIII antibodies. Since bioengineered IR8 may have altered immunoreactivity with anti-FVIII antibodies, an alternative functional assay was investigated to better characterize its potency. We evaluated WT-FVIII, BDD-FVIII and IR8 via the Calibrated Automated Thrombogram (CAT), a global assay of hemostasis, in platelet-free plasma (PPP) from 6 severe hemophilia A patients (<1 IU/dl FVIII) without inhibitors. The CAT test was chosen because of its ability to offer more valuable insight into the potential clinical value of IR8 than traditional clotting and chromogenic assays. Blood samples were taken into Corn trypsin inhibitor (CTI) to block contact activation and ensure that thrombin generation was triggered exclusively by tissue factor (TF) via the extrinsic hemostatic pathway. In an effort to demonstrate the dose dependency of each concentrate on its thrombin generating capacity, all 3 proteins were added to PPP along with a low TF concentration (1 pM) at varying FVIII activities (0, 25, 50 & 100 IU/dl). At each protein concentration, IR8 showed a significantly higher endogenous thrombin potential (ETP, the area under the thrombin generation curve) and peak height of the thrombin burst compared to either WT-FVIII or BDD-FVIII. The mean ETP values (nM*min) at 100 IU/dl, were WT-FVIII 650 and BDD-FVIII 725 (Mann Whitney test, p=0.69) and IR8 1107 (Mann Whitney test, p=0.04) with a mean ETP for FVIII <1% of 315 used as a control. Similar results were obtained in the presence of 1 nM thrombomodulin, which was added to sensitize the system to the action of APC. Consistent with the increased specific activity of IR8, the mean ETP of IR8 at 25 IU/dl was comparable to that of WT-FVIII at a concentration 4 times greater (100 IU/dl). Furthermore, no significant difference was found between the lag times of IR8 versus WT-FVIII and BDD-FVIII indicating that the advantage of IR8 does not lie in its ability to activate the initiation phase of thrombin generation, but rather in its persistent cofactor activity during the propagation phase of coagulation. These results are encouraging because the development of a rFVIII with markedly increased potency would potentially allow for reduced protein requirements in replacement therapy, thereby reducing costs and possibly decreasing inhibitor antibody development and would improve the efficacy of hemophilia A gene therapy without necessitating large improvements in genetic transfer strategies.

Characterization of an Anti-Factor VIII Monoclonal Antibody with A1 and A3 Epitopes Which Increases Factor VIII Activity.

Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.