Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody

A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.     

Immunochemical characterization of a polyclonal human antibody to factor IX.

Inhibitors of clotting factors occuring in humans are often antibody molecules synthesized in response to exogeneous proteins used in replacement therapy. Extensive studies of inhibitors to factor VIII indicate such antibodies may be monoclonal or polyclonal in nature. To date, only one factor IX inhibitor has been subjected to detailed immunochemical analysis and it appears to be a monoclonal IgGA lambda antibody. We have discovered a second inhibitor of factor IX in a patient with severe hemophilia B and have subjected it to immunochemical analysis. Studies on this second inhibitor have been carried out before and after an anamnestic response. Column chromatography, preparative zone electrophoresis, and specific inhibitor neutralization assays using monospecific heterologous antisera to human immunoglobulin classes, subclasses, and light-chain types indicate that the antibody is of the IgG class and contains both kappa and lambda light chains and probably all four IgG subclasses. Thus, the inhibitor appears to be polyclonal by immunochemical and structural criteria. In addition, preparative isoelectric focusing of pre- and postanamnestic inhibitor samples indicates that recruitment of new clones of IgG antibody occurs as a result of anamnesis. It is conceivable that an antibody initially restricted in immunoglobulin subclass became polyclonal following an anamnestic response.     

B-domain deleted recombinant factor VIII preparations are bioequivalent to a monoclonal antibody purified plasma-derived factor VIII concentrate: a randomized, three-way crossover study

BACKGROUND: Deletion of the B-domain of recombinant blood coagulation factor VIII (BDDrFVIII) increases the manufacturing yield of the product but does not impair in vitro or in vivo functionality. BDDrFVIII (ReFacto) has been developed with the additional benefit of being formulated without human albumin. OBJECTIVE: The primary objective of this three-way crossover-design study was to compare the pharmacokinetic (PK) parameters of two BDDrFVIII formulations (one reconstituted with 5 mL of sterile water, the other reconstituted with 4 mL sodium chloride 0.9% USP) with those of a plasma-derived, full-length FVIII preparation (Hemofil M) in patients with haemophilia A to determine bioequivalence. METHODS: A series of blood samples were collected over a period of 48 h after i.v. administration of each of the FVIII preparations. Plasma FVIII activity was determined using a validated chromogenic substrate assay. Plasma FVIII activity vs. time curves was characterized for a standard set of PK parameter estimates. Two parameter estimates, the maximum plasma concentration (Cmax) and the area under plasma concentration vs. time curves (AUCs), were used to evaluate bioequivalence. The two preparations were considered bioequivalent if the 90% confidence intervals for the ratio of geometric means for Cmax and AUCs fell within the bioequivalence window of 80% to 125%. RESULTS/CONCLUSION: Results show that each BDDrFVIII formulation is bioequivalent to Hemofil M and the two formulations of BDDrFVIII are bioequivalent to each other.     

Characterization and biological activity of a rat monoclonal antibody to rat/human corticotropin-releasing factor

To produce a rat monoclonal antibody directed to rat CRF (rCRF), female Wistar rats were actively immunized with a rCRF-bovine thyroglobulin conjugate. Immunization resulted in the formation of CRF antibodies, as indicated by binding of [125I]iodo-rCRF and attenuation of the plasma corticosterone responses to ether stress. Rat spleen cells were fused with mouse myeloma P3 cells, and a hybridoma clone (PFU 83) was selected according to its capacity to bind [125I]iodo-rCRF and inhibit rCRF-induced ACTH release from cultured rat pituitary cells. PFU 83 antibodies (IgG2a subclass) are directed to the extreme C-terminal part (amino acids 38-39) of rCRF and bind with an affinity constant of 21 nM. In vitro, PFU 83 causes a parallel shift to the right of the rCRF dose-ACTH response curve, with an apparent affinity of 10 nM. PFU 83 neither binds nor blocks the ACTH-releasing activity of oCRF. Intravenous administration of PFU 83 ascites (generated in nude mice) to Wistar rats caused a dose-dependent inhibition of ether-induced ACTH secretion. Full blockade of the ACTH response to ether was found at a dose of 10 nmol PFU 83/rat. Based on the dynamics of rCRF binding and its in vitro and in vivo effects, we conclude that the rCRF-blocking bioactivity of PFU 83 is due to binding of PFU 83 to native rCRF and formation of a biologically inactive complex. Finally, we found that PFU 83 did not affect resting or ether-induced alpha MSH secretion, indicating that CRF does not play a major role in the control of alpha MSH secretion.     

Macrophage modifying factor secreted by the tetrathyridia of Mesocestoides corti (cestoda): monoclonal antibody to the modifying factor antagonizes its immunological activity

Immunomodulation of macrophage activity by in vitro secretions of Mesocestoides corti has been previously demonstrated. The modifying activity secreted by M. corti had the effect of reducing the normal accessory function of macrophages in a Con-A-activated lymphocyte proliferation assay. This paper describes the purification of the modifying activity by FPLC techniques and the generation of a monoclonal antibody (MoAb) to this molecule in mice. The MoAb bound immunomodulatory FPLC fractions of M. corti in an ELISA. When MoAb was applied in conjunction with immunomodulatory parasite secretions to macrophages in vivo or in vitro , the modifying effect of the secretions was abolished. This profound effect of the MoAb should help to elucidate the mechanisms by which metacestode parasites avoid host immune responses and may enable therapeutic intervention.     

Enhanced pharmacokinetic properties of a glycoPEGylated recombinant factor IX: a first human dose trial in patients with hemophilia B

Replacement therapy with factor IX (FIX) concentrates is the recommended treatment for patients with hemophilia B, an X-linked bleeding disorder occurring in 1:25,000 male births. N9-GP is a recombinant FIX molecule with a prolonged half-life which is obtained by site-directed glycoPEGylation where a 40-kDa polyethylene glycol molecule is attached to the activation peptide of FIX. This first human dose trial in patients with hemophilia B investigated the safety and pharmacokinetic properties of a single IV dose of N9-GP. Sixteen previously treated patients received one dose of their previous FIX product followed by one dose of N9-GP at the same dose level (25, 50, or 100 U/kg). None of the patients developed inhibitors. One patient developed transient hypersensitivity symptoms during administration of N9-GP and was excluded from pharmacokinetic analyses. In the remaining 15 patients, N9-GP was well-tolerated. The half-life was 93 hours, which was 5 times higher than the patient's previous product. The incremental recovery of N9-GP was 94% and 20% higher compared with recombinant and plasma-derived products, respectively. These results indicate that N9-GP has the potential to reduce dosing frequency while providing effective treatment of bleeding episodes with a single dose. The trial was registered at www.clinicaltrials.gov as NCT00956345.     

Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an important mediator of tumor-induced angiogenesis and represents a potential target for anticancer therapy. Therefore, we prepared a panel of monoclonal antibodies (mAb) against both the VEGF₁₂₁ and VEGF₁₆₅ isoforms. Three of them completely neutralized the mitogenic stimulation by VEGF of human umbilical vein endothelial cells at mAb concentrations below 0.1 μg/ml. The most potent one, with a dissociation constant (K _d) of 8 pM, inhibited, in a dose-dependent manner, VEGF-induced angiogenesis in a growth factor implant model in mice. A complete inhibition of the angiogenic response was obtained by daily intraperitoneal injections of 10 μg mAb/mouse. Angiogenesis induced by basic fibroblast growth factor was not inhibited by the mAb. Epitope mapping of the mAb, performed by competitive enzyme-linked immunosorbent assay and Western blot analysis, showed that it did not bind to the reduced and denatured monomer of VEGF. Substitutions of three residues (Q87R, G88K, Q89K), located on the major surface loop β5 to β6 of VEGF, resulted in the complete loss of binding (more than 400-fold reduction). The results suggest that the mAb binds primarily to a conformation-dependent epitope on the VEGF dimeric form, encompassing one of the loop regions involved in KDR receptor binding. The mAb with its strong neutralizing properties represents a useful agent for effective blocking of VEGF-mediated tumor neovascularization. Received: 11 November 1998 / Accepted: 16 December 1998     

Capture of recombinant ricin A chain by a bispecific anti-RTA: anti-CEA monoclonal antibody pre-targeted to a human gastric carcinoma xenograft in nude mice

Summary A bispecific antibody against carcinoembryonic antigen (CEA) and ricin A chain has been shown to localise in a CEA-producing human tumour xenograft. When labelled recombinant ricin A chain was subsequently injected there was uptake into tumours, indicating that this bispecific antibody can capture or carry toxin into tumour deposits.Abbreviations CEA carcinoembryonic antigen - r-RTA recombinant ricin toxin A chain     

Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen.

The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 10(3.5) chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B approximately 1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant of HBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.     

Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.     

Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.     

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 10⁹ members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.     

Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.

A method is presented whereby antigenic determinants recognized by specific monoclonal antibodies can be mapped to specific sites on a protein sequence with high resolution. Short DNase I-generated DNA fragments encoding portions of the protein of interest are molecularly cloned into the EcoRI site of the beta-galactosidase gene of phage lambda Charon 16 so as to obtain expression of random protein fragments as fusion proteins. The monoclonal antibody is used to screen the phage library to isolate phage expressing the specific antigenic determinant. DNA of immunoreactive phage can be analyzed rapidly and subcloned to allow DNA sequence determination. The method is generally applicable and permits antigenic determinants of functionally interesting monoclonal antibodies to be mapped and related to specific protein sequences. We have used this procedure to determine the region of the feline leukemia virus envelope protein gp70 recognized by a virus-neutralizing monoclonal antibody, cl.25. Antibody binding was mapped to a 14-amino acid region in the amino-terminal half of gp70. This region may be directly involved in an essential function of the gp70 protein, perhaps in gp70-mediated host recognition functions. Synthetic peptides derived from this region may provide useful vaccine antigens for the prevention of feline leukemia virus-associated disease in cats.     

Reactivity with erythroid and non-erythroid tissues of a murine monoclonal antibody to a synthetic peptide having amino acid sequence common to cytoplasmic domain of human glycophorins C and D

Three synthetic peptides encompassing the entire cytoplasmic polypeptide sequence (amino acid residues 82-128) of glycophorin C (GPC) and glycophorin D (GPD) were used to immunize mice for the production of monoclonal antibodies (MoAbs). Only the synthetic peptide (GPC-peptidel) corresponding to C-terminal residues 112-128 elicited a MoAb (named BGRL-100) which could react with native and denatured GPC and GPD. We characterized BGRL-100 by inhibition using GPC-peptide 1 and red cell sialoglycoproteins. The ability of BGRL-100 to interact with native GPC and GPD was assessed by immunoprecipitation with normal red cells (RBCs), and with denatured GPC and GPD by Western blotting of both normal RBCs and RBCs carrying GPC variants. Immunohistochemical staining of human tissue sections was performed using both BGRL-100 and a rat MoAb (named BRAC-1), which is specific for an extracellular domain of GPC and GPD. Both antibodies showed strong staining of erythroid lineage haemopoietic cells in fetal liver, sinusoids of adult liver and RBCs in the blood vessels of all tissues tested. Neither antibody reacted with epithelia from a range of human tissues. However, both MoAbs stained neural tissue in a distinctive fibrillar pattern. This suggests the presence of an analogue of erythroid GPC in neural tissues.     

Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa.

Among snake venom procoagulant proteins, group II prothrombin activators are functionally similar to blood coagulation factor Xa. We have purified and partially characterized the enzymatic properties of trocarin, the group II prothrombin activator from the venom of the Australian elapid, Tropidechis carinatus (rough-scaled snake). Prothrombin activation by trocarin is enhanced by Ca2+, phospholipids, and factor Va, similar to that by factor Xa. However, its amidolytic activity on peptide substrate S-2222 is significantly lower. We have determined the complete amino acid sequence of trocarin. It is a 46,515-Dalton glycoprotein highly homologous to factor Xa and shares the same domain architecture. The light chain possesses an N-terminal Gla domain containing 11 gamma-carboxyglutamic acid residues, followed by two epidermal growth factor (EGF)-like domains; the heavy chain is a serine proteinase. Both chains are likely glycosylated: the light chain at Ser 52 and the heavy chain at Asn 45. Unlike other types of venom procoagulants, trocarin is the first true structural homologue of a coagulation factor. It clots snake plasma and thus may be similar, if not identical, to snake blood coagulation factor Xa. Unlike blood factor Xa, it is expressed in high quantities and in a nonhepatic tissue, making snake venom the richest source of factor Xa-like proteins. It induces cyanosis and death in mice at 1 mg/kg body weight. Thus, trocarin acts as a toxin in venom and a similar, if not identical, protein plays a critical role in hemostasis.     

A monoclonal antibody to human insulin-like growth factor-I: characterization, use in radioimmunoassay and effect on the biological activities of the growth factor

A monoclonal antibody (BPL-M23) to insulin-like growth factor-I (IGF-I) was obtained following immunization of BALB/c mice with human IGF-I conjugated to ovalbumin. The affinity constant of BPL-M23 for IGF-I was 10.5 litres/nmol and the cross-reactivities of IGF-II, multiplication-stimulating activity III-2 and insulin were 0.8, 0.03 and less than 0.0001% respectively. Porcine, bovine, ovine and rabbit sera, but not rat or mouse sera, showed substantial reactivity with the antibody. Comparison of radioimmunoassay analyses of 54 human serum samples from normal subjects and acromegalic and GH-deficient patients using BPL-M23 and a polyclonal rabbit antiserum (R557A) to human IGF-I showed a high correlation, indicating the usefulness of the monoclonal antibody in radioimmunoassay. Monoclonal antibody BPL-M23 was capable of abolishing the sulphation, mitogenic and insulin-like activities of IGF-I in in-vitro bioassays, suggesting that these activities may rely upon the same receptor-binding site which is near to the antibody-binding site.     

Assessment of antibodies to double-stranded DNA induced in rheumatoid arthritis patients following treatment with infliximab, a monoclonal antibody to tumor necrosis factor alpha: findings in open-label and randomized placebo-controlled trials

OBJECTIVE: To compare the incidence of anti-double-stranded DNA (anti-dsDNA) antibodies in rheumatoid arthritis (RA) patients receiving either single or multiple doses of a chimeric anti-tumor necrosis factor alpha (anti-TNFalpha) antibody or placebo infusions, with or without methotrexate, in open-label, randomized, placebo-controlled trials. METHODS: Multiple sera obtained from 156 patients before and after treatment with infliximab and from 37 patients treated with placebo infusions were tested for anti-dsDNA antibodies by 3 methods: Crithidia luciliae indirect immunofluorescence test (CLIFT), a commercial Farr assay (Ortho Diagnostics radioimmunoassay [RIA]) in which the antigen source is mammalian DNA, and a Farr assay employing 125I-labeled circular plasmid DNA (Central Laboratory of The Netherlands Red Cross Blood Transfusion Service [CLB] RIA). Patients with positive findings on the CLIFT were also tested for antibodies to histones (H1-H5) and chromatin and for IgM rheumatoid factors (IgM-RFs). RESULTS: None of the RA patients had a serum sample that was positive for anti-dsDNA antibodies by the CLIFT prior to infliximab therapy. Of the 22 patients who developed a positive CLIFT result, 11 (7% of 156 exposed to infliximab) also had positive findings on the Ortho RIA at a concentration of >10 units/ml and another 8 (5%) were positive at a concentration of >25 units/ml. In all but 1 patient, the anti-dsDNA antibodies were solely of the IgM isotype. Only 1 patient had detectable anti-dsDNA antibodies by the CLB RIA. All sera containing anti-dsDNA by the CLIFT contained antibodies to chromatin, and sera from 2 patients also contained antibodies to histones. IgM-RF titers showed a significant reduction following infliximab therapy in these 22 patients. One patient developed anti-dsDNA antibodies of IgG, IgA, and IgM isotype and had positive results on both Farr assays (peaking at 22 weeks and resolving by 54 weeks); this was associated with a reversible lupus syndrome. CONCLUSION: Anti-dsDNA antibodies of IgM class are induced by infliximab therapy; the frequency is dependent on the assay method used. Only 1 of the 156 patients who were treated with infliximab developed a self-limiting clinical lupus syndrome; that patient developed high titers of anti-dsDNA antibodies of IgG, IgM, and IgA class, as detected by the CLIFT and by 2 different Farr assays.     

The glycoprotein Ib-IX complex-specific monoclonal antibody SZ1 binds to a conformation-sensitive epitope on glycoprotein IX: implications for the target antigen of quinine/quinidine-dependent autoantibodies

The monoclonal antibody SZ1 is of interest for two reasons: it was used to define complex formation between glycoprotein (GP) Ib and GP IX, and its epitope is likely to be identical to that recognized by most quinine- and quinidine-dependent autoantibodies that cause thrombocytopenia. To determine the location of the epitope for SZ1 within the GP Ib-IX complex (which consists of three subunits: GP Ib alpha, GP Ib beta, and GP IX), we tested the ability of the antibody to bind transfected cells that expressed different combinations of complex subunits, and compared this binding to the binding of antibodies of known specificity. SZ1 bound to cells that expressed the entire GP Ib- IX complex in the same pattern as did AN51 (an antibody specific for GP Ib alpha). However, unlike AN51, SZ1 did not bind alpha beta cells (ie, cells that express GP Ib alpha and GP Ib beta, but not GP IX), but did bind to beta IX and alpha IX cells. We then compared the binding patterns of SZ1 and FMC25, an antibody specific for GP IX. Both bound virtually identically to cell lines that expressed every combination of two of the three GP Ib-IX complex subunits. However, the epitopes of the two antibodies were not identical, because fixation with 4% paraformaldehyde of cells that expressed GP IX destroyed the SZ1 epitope while maintaining the FMC25 epitope. Because of the ability of SZ1 to block the binding of many quinine- and quinidine-dependent antibodies, these data strongly suggest that GP IX is the component of the GP Ib-IX complex recognized by those antibodies.