Ernest T. Parker

Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors

The antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa. Group BC antibodies are the most common and display high specific inhibitory activity and type II kinetics. The C2 epitope groups recognized by 26 polyclonal human anti-fVIII inhibitor plasmas were identified by a novel competition enzyme-linked immunosorbent assay using group-specific murine monoclonal antibodies. Most of the anti-C2 inhibitor plasmas inhibited the binding of both classical and nonclassical antibodies. These results suggest that nonclassical anti-C2 antibodies contribute significantly to the pathogenicity of fVIII inhibitors.

The humoral response to human factor VIII in hemophilia A mice

Background: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. Objectives: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. Methods: Splenic B‐cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti‐FVIII domain‐specific enzyme‐linked immunosorbent assay were compared to Ab isotype and anti‐FVIII inhibitory activity. Results: The robust immune response resulted in the production of ∼300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti‐A2 and anti‐C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti‐A2 and anti‐C2 Abs were significantly associated with IgG1 and IgG2a isotypes, respectively. Because the IgG2a isotype is associated with enhanced Fc receptor‐mediated effector mechanisms, this result suggests that anti‐C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. Conclusion: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B‐cell epitopes.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Comparative immunogenicity of recombinant B domain‐deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

Summary.  Hyate is a commercial plasma‐derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI‐1 is a recombinant B domain‐deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI‐1 or Hyate:C in a comparative immunogenicity trial. OBI‐1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg−1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI‐1 or Hyate:C at doses of 1 or 10 U kg−1, but were identified in 81% and 94% of mice given 100 U kg−1 of OBI‐1 or Hyate:C, respectively. There was no significant difference between OBI‐1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI‐1‐treated mice at 10 U kg−1 (P = 0.09). Total anti‐FVIII antibodies to Hyate:C and OBI‐1 were also measured by ELISA using immobilized purified plasma‐derived porcine FVIII and OBI‐1, respectively, as antigens. At the 10 and 100 U kg−1 doses, the mean anti‐FVIII response was higher in Hyate:C‐treated‐mice than in OBI‐1‐treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI‐1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model.

Summary.  Objective: The pathogenicity of anti‐human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 nm, followed by injection of human B domain‐deleted FVIII at 180 U kg−1, producing peak plasma concentrations of ∼2 nm. At 2 h, blood loss following a 4‐mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti‐A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti‐C2 inhibitors, (iii) 2–77 and B45, non‐classical type II anti‐C2 inhibitors, and (iv) 2–117, a non‐classical anti‐C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg−1 overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL−1 and 3 U mL−1 FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non‐classical anti‐C2 antibodies both the in vivo and in vitro results suggest that treatment with high‐dose FVIII rather than bypassing agents may be warranted.

Factor VIII inhibitors.

The differential diagnosis in the bleeding patient includes inhibitory antibodies to blood coagulation proteins. Factor VIII (fVIII) is the most commonly targeted coagulation protein by the immune system. FVIII inhibitors arise as alloantibodies in transfused hemophiliacs and as autoantibodies in nonhemophiliac populations (1, 2, 3, 4). They develop in response to fVIII infusions in approximately 25% of patients with hemophilia A (5). In nonhemophiliacs, fVIII autoantibodies develop in a variety of clinical settings, including the postpartum period, systemic erythematosus, and chronic lymphocytic leukemia. Interestingly, autoantibody patients tend to present with more severe bleeding than hemophilia A patients with a de novo inhibitor. In both settings, fVIII inhibitors are polyclonal IgG populations directed against multiple epitopes.

The diversity of the immune response to the A2 domain of human factor VIII

Approximately 30% of patients with severe hemophilia A develop inhibitory anti-factor VIII (fVIII) antibodies (Abs). We characterized 29 anti-human A2 monoclonal Abs (mAbs) produced in a murine hemophilia A model. A basis set of nonoverlapping mAbs was defined by competition enzyme-linked immunosorbent assay, producing 5 major groups. The overlapping epitopes covered nearly the entire A2 surface when mapped by homolog-scanning mutagenesis. Most group A mAbs recognized a previously described epitope bounded by Arg484-Ile508 in the N-terminal A2 subdomain, resulting in binding to activated fVIII and noncompetitive inhibition of the intrinsic fXase complex. Group B and C mAbs displayed little or no inhibitory activity. Group D and E mAbs recognized epitopes in the C-terminal A2 subdomain. A subset of group D mAbs inhibited the activation of fVIII by interfering with thrombin-catalyzed cleavage at Arg372 at the A1-A2 domain junction. Other group D mAbs displayed indeterminate or no inhibitory activity despite inhibiting cleavage at Arg740 at the A2-B domain junction. Group E mAbs inhibited fVIII light-chain cleavage at Arg1689. Inhibition of cleavages at Arg372 and Arg1689 represent novel mechanisms of inhibitor function and, along with the extensive epitope spectrum identified in this study, reveal hitherto unrecognized complexity in the immune response to fVIII.

A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, Kd, were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant.

The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice

Inhibitory antibodies to factor VIII (FVIII inhibitors) are the most significant complication in the management of haemophilia A. The immunogenicity of FVIII may be driven in part by structural determinants within the FVIII molecule itself. Regions of nonidentity between human and porcine FVIII possibly could drive differential immune responses. The goal of this study was to compare the overall antibody response and levels of antibodies to the individual FVIII domains in naïve haemophilia A mice immunised with human or porcine FVIII. Haemophilia A mice were immunised with human or porcine FVIII using a protocol that mimics human clinical use. Inhibitor and total anti-FVIII antibody titers were measured and the domain-specificity of antibodies from 1,759 anti-FVIII hybridomas was determined. The overall immunogenicity of human and porcine FVIII was similar but significant differences in domain recognition were discovered. Anti-A2 and anti-C2 antibodies constituted the majority of inhibitors in both the human and porcine FVIII groups, similar to inhibitors that develop in humans. The proportions of anti-A2 or anti-C2 antibodies were not significantly different between the two groups. However, the specific inhibitory activity of anti-A2 antibodies was higher in the human FVIII group. Additionally, proportion of anti-C1 antibodies was significantly higher in the human FVIII group. In contrast, anti-A3 antibodies were more common in the porcine FVIII group. The differential immune response to human and porcine FVIII suggests that it may be possible to reduce the immunogenicity of FVIII by mutagenesis of the A2, A3 and C1 domains.

Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein

A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII. The specific activity of intracellular N1922S-fVIII was 10% of that of wt-fVIII, indicating the presence of large amounts of a nonfunctional N1922S-fVIII-folding intermediate. wt-fVIII colocalized with both endoplasmic reticulum (ER)- and Golgi-resident proteins. In contrast, N1922S-fVIII colocalized only with ER-resident proteins, indicating a block in transit from the ER to the Golgi. A panel of conformation-dependent monoclonal antibodies was used to determine native or nonnative folding of N1922S-fVIII. Intracellular N1922S-fVIII but not secreted N1922S-fVIII displayed abnormal folding in the A3 and C1 domains, indicating that the A1, A2, and C2 domains fold independently into antigenically intact tertiary structures, but that folding is stalled in the mutant A3 and its contiguous C1 domain. In summary, the N1922S substitution results in poor secretion of a functional protein, and the domain-specific defect in folding and intracellular trafficking of N1922S-fVIII is a novel mechanism for secretion defects leading to hemophilia A.