Recombinant factor VIII Fc fusion protein for immune tolerance induction in patients with severe haemophilia A with inhibitors-A retrospective analysis.

INTRODUCTION: Immune tolerance induction (ITI) is the gold standard for eradication of factor VIII inhibitors in severe haemophilia A; however, it usually requires treatment for extended periods with associated high burden on patients and healthcare resources. AIM: Review outcomes of ITI with recombinant factor VIII Fc fusion protein (rFVIIIFc) in patients with severe haemophilia A and high-titre inhibitors. METHODS: Multicentre retrospective chart review of severe haemophilia A patients treated with rFVIIIFc for ITI. RESULTS: Of 19 patients, 7 were first-time ITI and 12 were rescue ITI. Of 7 first-time patients, 6 had at least 1 high-risk feature for ITI failure. Four of 7 first-time patients were tolerized in a median of 7.8 months. The remaining 3 patients continue on rFVIIIFc ITI. Of 12 rescue patients, 7 initially achieved a negative Bethesda titre (≤0.6) in a median of 3.3 months, 1 had a decrease in Bethesda titre and continues on rFVIIIFc ITI and 4 have not demonstrated a decrease in Bethesda titre. Of these 4, 3 continue on rFVIIIFc ITI and 1 switched to bypass therapy alone. Two initially responsive patients transitioned to other factors due to recurrence. Overall, 16 of 19 patients remain on rFVIIIFc (prophylaxis or ITI). For those still undergoing ITI, longer follow-up is needed to determine final outcomes. No adverse events reported. CONCLUSIONS: Recombinant factor VIII Fc fusion protein demonstrated rapid time to tolerization in high-risk first-time ITI patients. For rescue ITI, rFVIIIFc showed therapeutic benefit in some patients who previously failed ITI with other products. These findings highlight the need to further evaluate the use of rFVIIIFc for ITI.

Improved joint health in subjects with severe haemophilia A treated prophylactically with recombinant factor VIII Fc fusion protein.

INTRODUCTION: Joint arthropathy is the long-term consequence of joint bleeding in people with severe haemophilia. AIM: This study assessed change in joint health over time in subjects receiving recombinant factor VIII Fc fusion protein (rFVIIIFc) prophylaxis. METHODS: ALONG is the phase 3 pivotal study in which the benefit of rFVIIIFc as a prophylactic treatment for bleeding control was shown in previously treated severe haemophilia patients ≥12 years of age (arm 1: 25-65 IU/kg every 3-5 days, arm 2: 65 IU/kg weekly and arm 3: episodic). After completing ALONG, subjects had the option to enrol into the extension study (ASPIRE). This interim, post hoc analysis assessed changes in joint health over ~2.8 years in these patients. RESULTS: Forty-seven subjects had modified Haemophilia Joint Health Score (mHJHS) data at A-LONG baseline, ASPIRE baseline and ASPIRE Year 1 and Year 2. Compared with A-LONG baseline (23.4), mean improvement at ASPIRE Year 2 was -4.1 (95% confidence interval [CI], -6.5, -1.8; P = .001). Regardless of prestudy treatment regimen, subjects showed continuous improvement in mHJHS from A-LONG baseline through ASPIRE Year 2 (prestudy prophylaxis: -2.4, P = .09; prestudy episodic treatment: -7.2, P = .003). Benefits were seen in subjects with target joints (-5.6, P = .005) as well as those with severe arthropathy (-8.8, P = .02). The mHJHS components with the greatest improvement at ASPIRE Year 2 were swelling (-1.4, P = .008), range of motion (-1.1, P = .03) and strength (-0.8, P = .04). CONCLUSIONS: Prophylaxis with rFVIIIFc may improve joint health over time regardless of prestudy prophylaxis or episodic treatment regimens.

Treatment of bleeding episodes with recombinant factor VIII Fc fusion protein in A-LONG study subjects with severe haemophilia A.

INTRODUCTION: The Phase 3 A-LONG study demonstrated the safety and efficacy of rFVIIIFc for the control and prevention of bleeding episodes in severe haemophilia A. AIM: To describe the treatment of bleeding episodes with rFVIIIFc in the A-LONG study. METHODS: A-LONG subjects (<1 IU dL-1 endogenous FVIII) were treated with individualized prophylaxis (Arm 1), weekly prophylaxis (Arm 2) or episodic treatment (Arm 3). Information recorded for each bleeding episode included type, location and dose to treat the episode. RESULTS: During A-LONG, 757 bleeding episodes occurred during the efficacy period; the majority [456 (60%)] occurred in Arm 3 (episodic treatment). Of 93 subjects in the prophylaxis arms who entered the study with target joints, 43 (60%) in Arm 1 and 11 (52%) in Arm 2 did not experience a target joint bleed. Overall, 98% of bleeding episodes (and 98% of bleeds involving a target joint) resolved with one or two infusions; the median dose per infusion to treat a bleed was 27 IU kg-1 (27 IU kg-1 for target joints). Using population pharmacokinetic simulations, FVIII activity levels were predicted to be below the upper limit of normal (150 IU dL-1 ) in most patients in the event that rFVIIIFc is used to treat a bleeding episode in close proximity to a prophylactic dose. CONCLUSIONS: These findings demonstrate the efficacy of rFVIIIFc for the treatment of acute bleeding episodes in subjects with severe haemophilia A, regardless of treatment regimen.

Change in clinician-assessed measures of multiple sclerosis and subject-reported quality of life: results from the IMPACT study.

BACKGROUND: The IMPACT study demonstrated the benefit of interferon beta-1a (IFNbeta-1a, Avonex) two-year change in disability measured by the Multiple Sclerosis Functional Composite (MSFC) in secondary progressive multiple sclerosis (SP-MS) and health-related quality of life (HRQoL) measured by the Multiple Sclerosis Quality of Life Inventory (MSQLI). The IMPACT data permit a detailed assessment of the relation between clinical and self-reported measures. METHODS: IMPACT was an international randomized, double-blind, placebo-controlled trial of SP-MS patients. As the MSQLI is only in English, this report includes US and Canadian subjects. Subjects were randomized to weekly intramuscular (im) injections of INbeta-1a (60 microg) or placebo for 24 months. RESULTS: At baseline and follow-up, MSQLI correlations were generally stronger with the EDSS than with the MSQLI, MSFC but comparable with MSFC components. Combining the two groups, MSQLI changes for those in the best and worst MSFC change quartiles demonstrated a statistical difference for six of the 11 MSQLI scales. Linear regression demonstrated that EDSS change from baseline to month-24 scores was correlated with change in two MSQLI components. CONCLUSION: These data support the appropriateness of using the MSQLI with individuals who have SP-MS.

A randomized, double-blind, dose-comparison study of weekly interferon beta-1a in relapsing MS.

BACKGROUND: Interferon beta-1a (IFNbeta-1a; Avonex) is effective for the treatment of relapsing MS; however, the optimal dose of IFNbeta-1a is not known. OBJECTIVE: To determine whether IFNbeta-1a 60 micro g IM once weekly is more effective than IFNbeta-1a 30 micro g IM once weekly in reducing disability progression in relapsing MS. METHODS: In a double-blind, parallel-group, dose-comparison study, 802 patients with relapsing MS from 38 centers in Europe were randomized to IFNbeta-1a 30 micro g (n = 402) or 60 micro g (n = 400) IM once weekly for >/=36 months. The primary endpoint was disability progression, defined as time to a sustained increase of >/=1.0 point on the Expanded Disability Status Scale (EDSS) persisting for 6 months. Additional endpoints included relapses, MRI, safety, immunogenicity, and subgroup analyses of disability progression. RESULTS: Both groups showed equal rates of disability progression (hazard ratio, 0.96; 95% CI, 0.77 to 1.20; p = 0.73). In both groups the proportion of subjects with progression of disability by 36 months estimated from Kaplan-Meier curves was 37%. No dose effects were observed on any of the secondary clinical endpoints. Only one MRI measure at one time point, number of new or enlarging T2 lesions at month 36 compared with month 24, showed a difference favoring the 60- micro g dose. Both doses were well tolerated; however, slightly higher incidences of flulike symptoms and muscle weakness were observed in the 60- micro g group. The incidences of neutralizing antibodies (titers >/= 20) were 2.3% in the 30- micro g group and 5.8% in the 60- micro g group. CONCLUSION: There was no difference between IFNbeta-1a 30 micro g and 60 micro g IM in clinical or MRI measures.

Benefit of interferon beta-1a on MSFC progression in secondary progressive MS.

BACKGROUND: Interferon beta-1a (IFNbeta-1a, Avonex) is efficacious in relapsing forms of MS. Studies of other IFNbeta preparations in secondary progressive MS (SPMS) yielded conflicting results. This study was undertaken to determine whether IFNbeta-1a slowed disease progression in SP-MS. METHODS: A total of 436 subjects with SPMS and Expanded Disability Status Scale (EDSS) score 3.5 to 6.5 were randomized to receive IFNbeta-1a (60 micro g) or placebo by weekly intramuscular injection for 2 years. The primary outcome measure, used for the first time in a large-scale MS trial, was baseline to month 24 change in the MS Functional Composite (MSFC), comprising quantitative tests of ambulation (Timed 25-Foot Walk), arm function (Nine-Hole Peg Test [9HPT]), and cognition (Paced Auditory Serial Addition Test [PASAT]). RESULTS: Median MSFC Z-score change was reduced 40.4% in IFNbeta-1a subjects (-0.096 vs -0.161 in placebo subjects, p = 0.033), an effect driven mainly by the 9HPT and PASAT. There was no discernible benefit on the EDSS, which in this range principally reflects walking ability. IFNbeta-1a subjects had 33% fewer relapses (p = 0.008). There was significant benefit on eight of 11 MS Quality of Life Inventory subscales. New or enlarging T2-hyperintense brain MRI lesions and gadolinium-enhancing lesions were reduced at months 12 and 24 (both p < 0.001). IFNbeta-1a was well tolerated by the majority of subjects. Neutralizing antibodies developed in 3.3% of IFNbeta-1a-treated subjects. CONCLUSIONS: IFNbeta-1a demonstrated benefit on MSFC progression, relapses, quality of life, and MRI activity in SPMS.

A recombinant lipoprotein antigen against Lyme disease expressed in E. coli: fermentor operating strategies for improved yield.

Decorin-binding lipoprotein, lpp-DBP, a bacterial surface adhesin, shows promise as a vaccine against Lyme disease. It is expressed in recombinant E. coli as an undesirable 20.5 KDa apoprotein that is subsequently lipidated in vivo to the desired 22 KDa lpp-DBP form. This study defines fermentation conditions for maximizing lpp-DBP yield. Super broth medium, a low post-induction temperature (30 degrees C), and a glucose feed based on dissolved oxygen resulted in high lpp-DBP yield and minimized apoprotein formation. Since cells lysed within 2-3 h after induction, the cell yield was maximized by growing cells to high cell density prior to induction. Compared to a glucose feed based on maintaining a constant fermentor glucose concentration (Glucose-Stat), feeding based on maintaining a constant dissolved oxygen level (DO-Stat) improved yields. Also, a dissolved oxygen level of 60% (air saturation) was best, as no product degradation was detected by Western blotting and SDS-PAGE. Acetic acid levels under both modes of glucose feed were sufficiently low, and no adverse growth effects were observed.

Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing.

Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development. Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent. The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure. Studies were also performed to support cell line stability assessment and cell culture process validation. Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product. In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range. Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture. No other changes in microheterogeneity were apparent for the other culture conditions studied. In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product.

Immunocytochemical localization of gamma-aminobutyric acid plasma membrane transporters in the tiger salamander retina.

Gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) play an important role in regulating GABA neurotransmission in the nervous system. The distribution of two GATs, GAT 1 and GAT 3, in salamander retina was investigated by using affinity-purified polyclonal antisera directed to the predicted C-terminals of rat GAT 1 and rat GAT 3. GAT 1-immunoreactivity (-IR) was found in type IB and IIB orthotopic bipolar cells (BCs) located in the distal and middle of the inner nuclear layer (INL), respectively; in type IIA and IA amacrine cells (ACs) located in the middle and proximal INL, respectively; and in interplexiform cells and cells in the ganglion cell layer (GCL). No detectable staining was found in horizontal cells (HCs) or in structures resembling Müller cells. GAT 1-immunoreactive fibers were present in the outer plexiform layer (OPL) and inner plexiform layer (IPL) in three bands corresponding to the three bands previously reported to be GABA-IR. GAT 3 antibodies labeled fewer cells and cell types than GAT 1 antibodies. GAT 3-IR was localized to type IIA and IA ACs and cells in the GCL, but not to BCs, HCs, or Müller cell-like structures. There was weak labeling of the OPL and stronger labeling of the IPL, with three distinct bands at the same depth as observed with GAT 1-IR. Double-labeling showed that the majority of GAT 1-IR BCs (88%), ACs (88%), and cells in the GCL (78%) colocalized with GABA-IR. The present study provides the first direct evidence of the expression of two GAT subtypes in neurons of nonmammalian retinas. These transporters could regulate GABA neurotransmission by reuptake and termination of GABA's action and, perhaps, by GABA release mechanisms. The presence of GAT 1-IR/GABA-IR bipolar cells further supports our earlier observations that a subgroup of orthotopic bipolar cells are likely to be GABAergic.

Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus.

Neutralizing polyclonal antibody to respiratory syncytial virus (RSV) has been shown to be an effective prophylactic agent when administered intravenously in high-risk infants. This study describes the generation of a humanized monoclonal antibody, MEDI-493, that recognizes a conserved neutralizing epitope on the F glycoprotein of RSV. The affinity of MEDI-493 was found to be equal to or slightly better than an isotype-matched chimeric derivative of the parent antibody. In plaque reduction, microneutralization, and fusion-inhibition assays, MEDI-493 was significantly more potent than the polyclonal preparation. Broad neutralization of a panel of 57 clinical isolates of the RSV A and B subtypes was demonstrated. Pretreatment of cotton rats with MEDI-493 resulted in 99% reduction of lung RSV titers at a dose of 2.5 mg/kg, corresponding to a serum concentration of 25-30 microg/mL. Further, MEDI-493 did not induce increased RSV infection or pathology in either a primary or a secondary challenge.

Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli.

The extracellular domain of the human erythropoietin receptor (EPO binding protein (EBP)) has been expressed and overproduced in Escherichia coli. Regardless of the presence ofpelB or ompT signal sequences the recombinant protein produced in this fashion appears, as with many other recombinant eukaryotic proteins produced in E. coli as an insoluble product in laboratory scale fermentations. The induction product of the pelB protein expression system appears as two protein forms with slightly different molecular weights. Based on N-terminal sequence analysis of recovered protein, these forms represent two variants, one with the signal sequence properly processed to yield the expected "native" amino terminus and another which retains the signal sequence. Both forms appear as insoluble fermentation products. Control of oxygen levels and pH during high density fermentation allows the production of only the protein variant with the native amino terminus. Methods reported here permit the efficient recovery of purified EBP which quantitatively binds EPO in solution as determined by high performance size exclusion chromatography. A long-lived refolding intermediate was observed which penultimately collapses into an active conformation. The active purified protein competes with membrane associated EPO receptor for binding [125I]EPO and neutralizes EPO-dependent stimulation in a cell based proliferation assay. Further, the radioligand equilibrium binding constant for this interaction has been determined by immobilizing EBP on agarose gel via a free cysteine. The production of EBP by these methods should facilitate the structural determination of the protein by NMR or crystallography and may serve as a guide for the refolding of other hematopoietic receptors.

Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses.

Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. (c) 1996 John Wiley & Sons, Inc.

Generalized clustering networks and Kohonen's self-organizing scheme.

The relationship between the sequential hard c-means (SHCM) and learning vector quantization (LVQ) clustering algorithms is discussed. The impact and interaction of these two families of methods with Kohonen's self-organizing feature mapping (SOFM), which is not a clustering method but often lends ideas to clustering algorithms, are considered. A generalization of LVQ that updates all nodes for a given input vector is proposed. The network attempts to find a minimum of a well-defined objective function. The learning rules depend on the degree of distance match to the winner node; the lesser the degree of match with the winner, the greater the impact on nonwinner nodes. Numerical results indicate that the terminal prototypes generated by this modification of LVQ are generally insensitive to initialization and independent of any choice of learning coefficient. IRIS data obtained by E. Anderson's (1939) is used to illustrate the proposed method. Results are compared with the standard LVQ approach.

Effects of heat shock on the production of human erythropoietin from recombinant CHO cells.

The production of recombinant proteins by mammalian cells demands a highly controlled environment for cell cultivation. Temperature stress represents a commonly encountered disturbance in both research and process environments. In this study, we examined the effects of heat shock on the expression of recombinant human erythropoietin (EPO) in a Chinese hamster ovary (CHO) cell line. Biosynthetic radiolabeling experiments indicated that the cells exposed to a 42 degrees C/1-hour heat shock exhibit a transient reprogramming of transcription and translation characterized by the inhibition of protein synthesis and induction of heat shock proteins. The rate of protein synthesis decreased by 50% after the heat shock, while the rate of RNA synthesis increased by 50% initially and then quickly reduced to 80% of the control level. The protein and RNA synthesis rates were fully recovered in approximately 48 hours after the heat shock. However, we found that the expression of EPO was not arrested by the heat shock. The glycosylation patterns, as examined by isoelectric focusing, of either the culture supernatant or the purified EPO were not affected by the heat shock. In contrast, a 45 degrees C/1-hour heat shock terminated RNA and protein synthesis immediately and caused culture death in 12 hours. Cellular responses to temperature stress were affected by the metabolic state of the cells; cells maintained in serum-free medium were more sensitive than cells growing exponentially in the presence of serum. We have also examined the kinetics of metabolic responses of the cells to heat shock with respect to nutrient uptake and metabolite accumulation.