Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE.

Clinical impact of peripheral attacks in hereditary angioedema patients.

BACKGROUND: Episodes of acute subcutaneous angioedema affecting the extremities in patients with known hereditary angioedema are called peripheral attacks. These attacks are considered to be of limited clinical importance. OBJECTIVE: To evaluate the impact of peripheral attacks in patients with hereditary angioedema and to assess the response to treatment with recombinant human C1-inhibitor (rhC1INH). METHODS: Hereditary angioedema patients with a peripheral attack included in a clinical database of rhC1INH were analyzed. Visual analog scale (VAS) scoring was used to evaluate symptom severity and response to therapy. RESULTS: Sixty-five patients with a peripheral attack were identified. VAS scores for 64 patients were available. Twenty-nine (45%) patients reported a single peripheral location of the attack, the others multiple locations. Eight patients (13%) indicated moderate (VAS 20-50 mm) and 55 (86%) severe (VAS ≥50 mm) swelling, 17 (27%) had moderate and 35 (55%) severe pain, while 8 (13%) patients reported moderate and 51 (80%) severe dysfunction for the peripheral attack. Symptom VAS scores decreased over time more rapidly in patients treated with rhC1INH than in patients treated with placebo. Onset of relief was achieved in 95% of the rhC1INH-treated patients within 4 hours, whereas only 21% of saline-treated patients had relief in the same time period. CONCLUSION: Peripheral attacks in hereditary angioedema patients often are located at multiple anatomical locations and frequently have associated pain and dysfunction, in addition to swelling, as dominant symptoms. The medical need for treatment of these attacks may be underestimated. Treatment with rhC1INH constitutes a therapeutic option for acute peripheral hereditary angioedema attacks.

Recombinant human C1-inhibitor for the treatment of acute angioedema attacks in patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) results from a genetic deficiency of C1-inhibitor. Two similar independent, randomized, saline controlled, double-blind studies were conducted to evaluate the efficacy and safety of recombinant human C1-inhibitor (rhC1INH) as a treatment of acute angioedema attacks in patients with HAE. OBJECTIVE: Analysis of pooled study results. METHODS: Patients with an eligible attack were randomized to a single intravenous dose of rhC1INH or saline. Efficacy was assessed by using patient-reported visual analog scale outcomes, and safety was assessed by using adverse events and immunogenicity of rhC1INH. RESULTS: rhC1INH at 100 (n = 29) and 50 (n = 12) U/kg body weight resulted in a significant reduction for both the primary endpoint time to the beginning of relief of symptoms compared with saline (n = 29): median, 66 (95% CI, 61-122) minutes, 122 (72-136) minutes, and 495 (245-520) minutes, P < .001 and P = .013, respectively; and for the secondary endpoint time to minimal symptoms, median, 266 (242-490) minutes, 247 (243-484) minutes, and 1210 (970-1500) minutes, P < .001 and P = .001, respectively. Therapeutic failure occurred in 59% (17/29) of the saline group compared with 0% (0/12) of the 50 U/kg group and 10% (3/29) of the 100 U/kg group. Treatment-emergent adverse events were unremarkable and tended to be reported more frequently in the saline group. No postexposure antibody responses against rhC1INH or host-related impurities were observed. CONCLUSION: Administration of rhC1INH at 100 or 50 U/kg was highly effective as a treatment of acute attacks in patients with HAE and appeared to be safe and well tolerated.

Recombinant human C1-inhibitor in the treatment of acute angioedema attacks.

BACKGROUND: Patients with hereditary C1-inhibitor deficiency have recurrent attacks of angioedema, preferably treated with C1-inhibitor concentrate. A recombinant human C1-inhibitor (rHuC1INH) was developed, derived from milk from transgenic rabbits. This study was undertaken to investigate the effects of rHuC1INH in the treatment of acute angioedema attacks in patients with a hereditary C1-inhibitor deficiency. STUDY DESIGN AND METHODS: Patients with hereditary C1-inhibitor deficiency were treated with rHuC1INH (at a dose of 100 U/kg) within 8 hours after onset of an acute attack. Time to initiation of relief and time to minimal symptoms were reported by both the patient and the treating physician. RESULTS: Thirteen severe angioedema attacks in 9 patients with hereditary C1-inhibitor deficiency were treated with rHuC1INH. There was rapid improvement of clinical conditions for all attacks, with approximately 80 percent of treated patients experiencing symptom relief within 2 hours and minimal symptoms within 12 hours. There were no drug-related adverse events or immunogenic reactions to C1-inhibitor or rabbit proteins. CONCLUSION: The transgenically produced rHuC1INH was successfully used in the treatment of acute angioedema attacks in patients with hereditary C1-inhibitor deficiency.

[Recombinant human C1-inhibitor is effective in the treatment of acute attacks of hereditary angioedema--case report]. / Rekombinowany C1-inhibitor jest skuteczny w leczeniu ostrych napadów wrodzonego obrzeku naczynioruchowego--opis przypadków.

Hereditary angioedema (HAE) is a rare condition, resting on attacks of edema in various localizations, potentially life-threatening if in facial, laryngeal, pharyngeal or gastrointestinal area. The disease is caused by deficiency or impaired activity of C1 inhibitor, therefore C1 inhibitor infusion is the the essential treatment and the only efficient method in an acute attack of HAE. Nowadays C1 inhibitor applied in our patients is obtained from human plasma, what restricts the availability of the drug and carries relevant risks. After many years of research it came to the synthesis of the recombinant protein with features of human C1 inhibitor. Its first usage in Poland took place-in two HAE patients with severe edema in 2004. The course and efficiency of this treatment is reported in the paper. Recombinant human C1 inhibitor occurred efficient and safe in presented case of severe angioedema.

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 A. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 A for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.

A phase I study of recombinant human C1 inhibitor in asymptomatic patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a congenital disorder with recurrent attacks of localized swelling of submucosal tissue, subcutaneous tissue, or both caused by a deficiency of the plasma protein C1 inhibitor (C1 esterase inhibitor [C1INH]). OBJECTIVE: We sought to evaluate the effects of recombinant human C1INH (rhC1INH) isolated from the milk of transgenic rabbits in 12 asymptomatic patients with HAE. METHODS: rhC1INH was intravenously administered at doses of 6.25 to 100 U/kg on 2 occasions. RESULTS: rhC1INH appeared safe and was well tolerated. The course of functional C1INH in plasma showed a full initial recovery (dose-normalized maximum concentration of about 0.02 U/mL/U/kg) and a dose-dependent clearance of rhC1INH. After infusion of rhC1INH at 100 U/kg, a clearance of approximately 13 mL/min, a half-life of approximately 3 hours, and a volume of distribution of approximately 3 L were observed. Infusion at this dose led to functional C1INH levels in plasma of at least twice the normal level for about 2 hours and greater than 0.4 U/mL for about 9 hours. rhC1INH displayed dose-dependent biologic activity by increasing the C4 level, which was about 2-fold at 12 hours after rhC1INH at 100 U/kg, and decreasing levels of cleaved C4. CONCLUSION: The observed safety profile and biologic activity of rhC1INH warrants further clinical studies to assess its efficacy in treating HAE attacks.

Hereditary and acquired angioedema: problems and progress: proceedings of the third C1 esterase inhibitor deficiency workshop and beyond.

Hereditary angioedema (HAE), a rare but life-threatening condition, manifests as acute attacks of facial, laryngeal, genital, or peripheral swelling or abdominal pain secondary to intra-abdominal edema. Resulting from mutations affecting C1 esterase inhibitor (C1-INH), inhibitor of the first complement system component, attacks are not histamine-mediated and do not respond to antihistamines or corticosteroids. Low awareness and resemblance to other disorders often delay diagnosis; despite availability of C1-INH replacement in some countries, no approved, safe acute attack therapy exists in the United States. The biennial C1 Esterase Inhibitor Deficiency Workshops resulted from a European initiative for better knowledge and treatment of HAE and related diseases. This supplement contains work presented at the third workshop and expanded content toward a definitive picture of angioedema in the absence of allergy. Most notably, it includes cumulative genetic investigations; multinational laboratory diagnosis recommendations; current pathogenesis hypotheses; suggested prophylaxis and acute attack treatment, including home treatment; future treatment options; and analysis of patient subpopulations, including pediatric patients and patients whose angioedema worsened during pregnancy or hormone administration. Causes and management of acquired angioedema and a new type of angioedema with normal C1-INH are also discussed. Collaborative patient and physician efforts, crucial in rare diseases, are emphasized. This supplement seeks to raise awareness and aid diagnosis of HAE, optimize treatment for all patients, and provide a platform for further research in this rare, partially understood disorder.

The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis.

Lactoferrin (LF) is an iron-binding glycoprotein of the innate host defence system. To elucidate the role of N-linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130-->Thr and Gly404-->Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404. Hence, N-linked glycosylation is not involved in protection of hLF against tryptic proteolysis. Apparently, the previously reported protection by N-linked glycosylation of hLF [van Berkel, P.H.C., Geerts, M.E.J., van Veen, H.A., Kooiman, P.M., Pieper, F., de Boer, H.A. & Nuijens, J.H. (1995) Biochem. J. 312, 107-114] is restricted to rhLF containing the Thr130 and Cys404. Comparison of the tryptic proteolysis of hLF and bovine LF (bLF) revealed that hLF is about 100-fold more resistant than bLF. Glycosylation variants A and B of bLF differed by about 10-fold in susceptibility to trypsin. This difference is due to glycosylation at Asn281 in bLF-A. Hence, glycosylation at Asn281 protects bLF against cleavage by trypsin at Lys282.

N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.

Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants.

Characterization of monoclonal antibodies against human lactoferrin.

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.

Large scale production of recombinant human lactoferrin in the milk of transgenic cows.

The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.