Nanometer-Sized Aggregates Generated Using Short Solubility Controlling Peptide Tags Do Increase the In Vivo Immunogenicity of a Nonimmunogenic Protein.

Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 µm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

A novel coagulation inhibitor from Schistosoma japonicum.

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

Postoperative complications following the Fontan procedure: the role of aprotinin.

OBJECTIVE: To determine how the anti-inflammatory properties of aprotinin impact on postoperative complications in children undergoing the Fontan procedure. METHODS: We included all patients between 14 months and 18 years (n=56) undergoing a Fontan operation at our institution between January 2005 and June 2009. The study group (n=29) included patients from January 2005 through December 2007 all of whom received aprotinin. The control group (n=27) included all patients from January 2008 through June 2009 who did not receive aprotinin. We reviewed all medical records and collected preoperative, intraoperative and postoperative data. Duration and volume of chest tube drainage were the primary outcome measures. RESULTS: Of the 20% of patients who had postoperative arrhythmias, multivariate logistic regression analysis demonstrated only aprotinin was associated with significantly decreased postoperative arrhythmias (P=0.01). Renal function and fenestration or Fontan thrombosis did not differ significantly; there was no statistically significant difference in volume or duration of chest tube drainage. Median duration of chest tube drainage was 7 days in the aprotinin group and 8 days for patients who did not receive aprotinin (P=0.36). CONCLUSION: The anti-inflammatory properties of aprotinin may be protective against postoperative arrhythmias. Aprotinin does not confer increased risks of prolonged chest tube drainage, renal dysfunction or thrombosis in patients undergoing the Fontan procedure.

Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems.

Plants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell-based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high purity, specific activity and promising preclinical outcomes. Adaptation of transient plant virus systems to large-scale manufacturing formats required development of virus particle and Agrobacterium inoculation methods. One transient plant system case study illustrates the properties of greenhouse and field-produced recombinant aprotinin compared with an US Food and Drug Administration-approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A second transient plant system case study demonstrates a fully functional monoclonal antibody conforming to release specifications. In conclusion, the production capacity of large quantities of recombinant protein offered by transient plant expression systems, coupled with robust downstream purification approaches, offers a promising solution to recombinant protein production that compares favourably to cell-based systems in scale, cost and quality.

A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein.

Poor immunogenicity of small proteins is a major hurdle in developing vaccines or producing antibodies for biopharmaceutical usage. Here, we systematically analyzed the effects of 10 solubility controlling peptide tags (SCP-tags) on the immunogenicity of a non-immunogenic model protein, bovine pancreatic trypsin inhibitor (BPTI-19A; 6 kDa). CD, fluorescence, DLS, SLS, and AUC measurements indicated that the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. ELISA results indicated that the 5-proline (C5P) and 5-arginine (C5R) tags unexpectedly increased the IgG level of BPTI-19A by 240- and 73-fold, respectively, suggesting that non-oligomerizing SCP-tags may provide a novel method for increasing the immunogenicity of a protein in a highly specific manner.

Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

Systemic allergic reactions to aprotinin injection around the Achilles tendon.

Local aprotinin injections are used in the treatment of chronic tendinopathy. Severe allergic reactions, although uncommon, have been reported. We highlight two instances of systemic allergic reaction, and discuss the potential side effects of local aprotinin injections in the orthopaedic setting as well as the evidence base for its use.

[Anaphylactic shock related to aprotinin induced by anti-aprotinin immunoglobulin G antibody alone; report of a case].

Anaphylactic shock related to aprotinin has been reported to be induced exclusively in the presence of IgE antibody. And the possibility of anaphylactic shock induced by anti-aprotinin IgG antibody alone was controversial. In this paper, we describe the first case of anaphylactic shock induced by aprotinin-specific IgG antibody alone. A 55-year-old man underwent surgical repair of the descending aorta with the use of aprotinin at 2 months after first aprotinin usage. Immediately after initiation of cardiopulmonary bypass with the continuous infusion of aprotinin, clinical symptoms of anaphylactic reaction were found. Postoperative drug lymphocyte stimulation test for aprotinin and aprotinin-specific IgE antibody were negative, but aprotinin-specific IgG antibody was 163 mg/l and positive.

Safety of aprotinin use and re-use in pediatric cardiothoracic surgery.

BACKGROUND: Hypersensitivity reactions to aprotinin have been reported in adult cardiac surgical patients undergoing initial and re-exposure to the medication. This study describes the incidence and impact of aprotinin hypersensitivity reactions in children undergoing cardiothoracic surgery. METHODS AND RESULTS: In this retrospective review of our entire experience with aprotinin (n=865), 681 first exposures, 150 second exposures, and 34 third or higher exposures were examined. Reactions were classified as mild (generalized cutaneous erythema, Type A) or severe (unexplained cardiopulmonary instability after aprotinin exposure, Type B). Records of patients sustaining a reaction were reviewed to assess the impact of the reaction on outcome and to survey reaction management strategies. Reactions occurred in 7 of 681 first exposures (1.0%), of which 2 were Type A and 5 were Type B. In second exposures, there were reactions in 2 of 150 (1.3%), of which both were Type B. In 34 third or higher exposures, there was only 1 reaction (2.9%), which was Type B. Reactions were no more likely on second, third, or higher exposure than on initial exposure. Skin testing had a negative predictive value of 98.9% and a positive predictive value of 20%. Anti-aprotinin IgE was undetectable in 7 of 8 reactor cases tested. No adverse sequelae were attributed to aprotinin reaction. CONCLUSIONS: The risk of hypersensitivity reactions to aprotinin is low in children undergoing cardiothoracic surgery, even with multiple exposures to the medication. Reactions are more likely with re-exposure, and risk increases with multiple exposures. Neither skin testing nor assays for IgE identified reactors.

Inhibition of cytotoxic T-lymphocyte-triggered apoptosis by target cell surface-coupled aprotinin.

A variety of recent investigations have implicated granzymes A and/or B in the target cell nuclear injury which accompanies cytotoxic T-lymphocyte-mediated cytolysis. Since soluble antiproteases have had limited efficacy in inhibiting CTL-mediated lysis, we developed a method to couple aprotinin, a peptide inhibitor of serine proteases, to the surface of target cells. Aprotinin modified by N-succinimidyl 3-(2-pyridyldithio)propionate retained trypsin-inhibitory activity, and target cells modified with aprotinin had demonstrable cell surface trypsin-inhibitory activity. Flow cytometry demonstrated that aprotinin was detectable on the target cell surface but underwent modulation at a rather rapid rate. When radiolabeled, aprotinin-coupled target cells were studied in 1-2 hr CTL assays, 51Cr release was little affected, but 125IUdR release was reduced up to 75% compared to controls. Corresponding apoptosis analysed by agarose gel electrophoresis and direct cytologic visualization was similarly reduced. Thus, aprotinin bound to the surface of target cells selectively protected target cells against CTL-mediated nuclear injury, and may serve as a model for the development of novel inhibitors of CTL-mediated lysis.

Aprotinin in fibrin tissue adhesives induces specific antibody response and increases antibody response of high-dose intravenous application.

BACKGROUND: In cardiac operations, aprotinin therapy is used either locally as a component of commercially available fibrin tissue adhesives, intravenously, or combined. Our aim was to examine the formation of aprotinin-specific antibodies with regard to the application mode. METHODS: Sera of 150 patients who had undergone cardiac operations and were receiving aprotinin therapy for the first time were sampled before the operation and at medians of 3.5 and 13.3 months after the operation. Aprotinin-specific IgG including all subgroups and aprotinin-specific IgE were analyzed. Aprotinin was given locally (as contained in fibrin sealant; n = 45; median dose, 6000 KIU), intravenously (n = 46; 2.000 x 10(6) KIU), and combined (n = 59; 2.012 x 10(6) KIU). RESULTS: At 3.5 months, the prevalence of aprotinin-specific IgG antibodies was 33% (15/45 patients) after local, 28% (13/46 patients) after intravenous, and 69% (41/59 patients) after combined exposure (P =.0001). At 13.3 months, the prevalence of aprotinin-specific IgG antibodies was 10% (4/41 patients) after local, 31% (13/42 patients) after intravenous, and 49% (28/57 patients) after combined exposure. Total aprotinin dose was similar in patients who were antibody positive and negative. Before the operation, no aprotinin-specific antibodies were detected. Aprotinin-specific IgE were not found after the operation. CONCLUSION: Local aprotinin contact induces a specific immune response and reinforces that of intravenous exposure. The antibody spectrum is identical to the immune response induced by intravenous exposure. Any exposure should be documented. For use in cardiac operations as a hemostyptic, the necessity itself and alternatives for aprotinin as a stabilizing agent merit consideration.

Fibrin sealant, aprotinin, and immune response in children undergoing operations for congenital heart disease.

OBJECTIVE: Most commercially available fibrin sealants contain aprotinin in doses of 1500 kallikrein inactivator units per milliliter. They are used in many operative disciplines. An elevated risk of hypersensitivity reactions exists at reexposure to aprotinin. Our aim was to examine the immunogenic potency of aprotinin as a fibrin sealant content. METHODS: We investigated 49 children with operatively treated congenital heart disease. All patients received aprotinin only topically as contained in fibrin sealant. Serum samples were drawn preoperatively, 1 week, 2 weeks, 6 weeks, and approximately 1 year after operation. They were analyzed for aprotinin-specific immunoglobulin G antibodies with a standard enzyme-linked immunosorbent assay and a fluorescence enzyme immunoassay for aprotinin-specific immunoglobulin E antibodies. RESULTS: At 1 week, 2 weeks, 6 weeks, and 1 year, we found prevalences of 8% (2 of 26), 8% (2 of 24), 6% (3 of 49), and 0% for aprotinin-specific Immunoglobulin E, and for aprotinin-specific immunoglobulin G 8% (2 of 26), 17% (4 of 24), 39% (19 of 49), and 12% (5 of 41). The doses of aprotinin given did not differ significantly in antibody-negative and antibody-positive patients; no significant factors could predict the immune response. CONCLUSIONS: Our findings show the existence of a subgroup of patients who had aprotinin-specific antibodies develop after topical aprotinin application. Any use of aprotinin must be carefully documented. If aprotinin use is planned in patients who previously underwent a surgical procedure, preexposure to aprotinin in any form must be sought to avoid unexpected anaphylactic reactions. The necessity itself and alternatives for aprotinin as a stabilizing agent in fibrin sealants merit consideration.

Anaphylactic shock after aprotinin reexposure: time course of aprotinin-specific antibodies.

We report a case of severe anaphylactic shock during a cardiac operation that occurred as a consequence of aprotinin readministration in the presence of preformed aprotinin-specific antibodies. Both immunoglobulin G (3 hours) and immunoglobulin E (5 minutes) antibody levels dropped early after the clinical event. Despite their possibly limited clinical significance, we still recommend the conductance of specific antibody screening tests before readministration of aprotinin.

Routine intraoperative application of high-dose aprotinin in open heart surgery in adults: antibody formation after first exposure.

High-dose aprotinin is now routinely used in cardiac surgery to reduce postoperative blood loss and transfusion requirements, although several cases of anaphylactic reactions to the proteinase inhibitor have been reported. As part of a multi-centre study to evaluate the immunological response to aprotinin after first exposure 61 cardiac surgical patients were treated with the Hammersmith regimen. Patients with previous aprotinin exposure were excluded from the study. To determine specific IgG and IgE antibodies blood samples were taken pre-operatively, within 3 to 4 weeks and 6 to 7 months after operation. Determinations were made by using Western Blot and ELISA methods. Fifty-six patients were followed up for a 6-month period, 26 (46.4%) of them developed IgG antibodies to aprotinin determined by Western Blot, whereas only 14 (26.8%) patients with IgG antibodies were found by the ELISA. IgE antibodies were not found in any of the patients. On hospital admission and 6 months post-operatively additional intradermal prick tests were performed. No clear-cut positive reaction to the skin test was found in any patient.

Separation of two fractions from rabbit anti-BPTI antibody by affinity chromatography.

Non-competitive enzyme-linked immunosorbent assay (ELISA) for rabbit anti-BPTI antibody is described. The antibody was purified by affinity chromatography on Sepharose-BPTI column and then separated into two antibody fractions on Sepharose-trypsin (BPTI) column. The fraction I antibody, not retarded on the second biosorbent column, efficiently inactivated BPTI, but the fraction II antibody, eluted from the column with glycine buffer, pH 2.2 was firmly bound to active BPTI. This proves that BPTI has at least two epitopes.

Bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from the isolated rat heart: the effect of aprotinin and forskolin.

Aprotinin has been reported to reduce plasma levels of inflammatory cytokines associated with cardiopulmonary bypass (CPB). Because CPB is also associated with elevated levels of bacterial lipopolysaccharide (LPS) and LPS stimulates release of inflammatory cytokines from the heart we tested the hypothesis that aprotinin would inhibit cardiac release of tumor necrosis factor-alpha (TNF) provoked by LPS. Isolated rat hearts were perfused Langendorf style. After 30 minutes of equilibration LPS (100 ng/mL) was infused for 60 minutes. Timed samples of coronary effluent were collected at 0, 30, 60, 90, 120, and 150 minutes after the initiation of LPS for the measurement of coronary flow and the determination of TNF and cyclic AMP. Other hearts were perfused with buffer containing aprotinin [137 kallikrein-inhibiting units (KIU)/mL or 250 KIU/mL] and then infused with LPS. An additional group received forskolin (10 microM) and LPS. In hearts perfused as controls with buffer alone no TNF was detected in the coronary effluent. In hearts perfused with LPS TNF was reliably detected in the coronary effluent at 60 minutes (606 +/- 450 pg/min) and increased with time to a level of 1792 +/- 650 pg/min at 150 minutes. The addition of aprotinin had no significant effect on LPS-stimulated TNF release. For instance in hearts perfused with 137 KIU/mL aprotinin LPS-stimulated release at 150 minutes was 2141 +/- 732 pg/min and in hearts perfused with 250 KIU/mL LPS-stimulated TNF release was 2049 +/- 789 pg/min. Forskolin administration was associated with release of cyclic AMP from the heart and completely inhibited LPS-stimulated TNF release. We conclude that LPS stimulated release of TNF from the heart. Adding aprotinin to the perfusion buffer in either high or low concentrations did not attenuate LPS-stimulated cytokine release. Elevating myocardial cyclic AMP with forskolin completely attenuated LPS-stimulated TNF release.

Characterisation of Ag1, the major species-specific contaminant of bovine crude heparin, and its identification as an aprotinin/heparin complex.

Heparin is a potent anticoagulant polysaccharide purified for decades from ruminants or porcine tissues. However, with the emergence of bovine spongiform encephalopathy (BSE), the source of pharmaceutical heparin is currently restricted to porcine intestinal mucosa. A major species-specific contaminant, called Ag1, has recently been identified in bovine crude heparin [Rivera et al., J. Pharm. Biomed. Anal., submitted] and used to develop an enzyme-linked immunosorbent assay (ELISA) for the species origin control of crude heparins [Levieux et al., J. Immunoassay, submitted]. In this report, we describe the different investigations, which were carried out to identify Ag1. This antigen was first localised by immunohistological studies essentially in the connective tissue of the bovine small intestine. After extraction from an intestinal extract by immuno-affinity chromatography, Ag1 was isolated as a single band by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ag1 was then partly sequenced and identified as an aprotinin/heparin complex. Aprotinin, also known as the bovine pancreatic trypsin inhibitor (BPTI), is present with heparin in mast cells, and is very resistant to heat, pH, chemical treatments and proteolytic digestion. The stability of Ag1 towards the different treatments performed during heparin extraction process allows this protein to remain in sufficient amounts in crude heparin and makes it an ideal target for the immunochemical control of the absence of bovine material in crude heparins.

Aprotinin and hemostasis monitoring concerns during cardiac surgery.

Aprotinin (Trasylol) is a serine protease inhibitor, isolated from bovine lung that initially was marketed for the treatment of pancreatitis. In the mid 1980s, reports of its ability to decrease hemorrhaging after cardiopulmonary bypass surgery introduced the drug to the realm of cardiac surgery. Unfortunately, its introduction into this arena was followed by the publication of multiple studies and case reports that blamed aprotinin for poor outcomes in the form of early graft closure. More than 17 years have passed since the initial article describing the use of aprotinin during cardiopulmonary bypass, and with time there has been a significant increase in scientific knowledge and clinical experience. Interestingly, modern literature does not support the dogma that aprotinin is a procoagulant. Aprotinin increases the activated partial thromboplastin time (aPTT), as well as the kaolin- and celite-activated clotting time (ACT), regardless of heparin. Aprotinin, because of its ability to inhibit kallikrein, has been found to decrease thrombin antithrombin III complexes, fibrin-split products, fibrinopeptide 1+2, prothrombin fragments, and all markers of thrombin formation. Some authors have suggested that it may have a synergistic effect with heparin to ensure graft patency. Anticoagulation monitoring during the use of aprotinin also has been developed based on early studies. Aprotinin administration does influence the results of various ACT tests, and consequently different methods of testing anticoagulation have been developed. Researchers have demonstrated that the celite ACT is not "artificially" prolonged in the presence of heparin and aprotinin, rather the kaolin ACT is "artificially" shortened. This article will review the scientific literature with regard to aprotinin's anticoagulatory effects and review the current recommendations for hemostasis monitoring during the use of aprotinin.

Monoclonal antibodies against protease inhibitors: the case of the bovine pancreatic trypsin inhibitor.

A monoclonal antibody (MoAb) directed against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a small protein made up by 58 aminoacids, has been produced. Mice were immunized with the native form of the protein and gave low antibody response. After somatic hybridization of spleen cells from immunized mice, few clones secreting antibodies against BPTI have been found and just two of them were stable. Both secreted IgM. One (ICI) produces a monoclonal antibody which binds to BPTI with an equilibrium dissociation constant, Kd, of 6.1 x 10(-7) M at pH 7.4. Competition experiments demonstrated that ICI recognizes an epitope close to the reactive site of BPTI. Furthermore, Kd of ICI with an isoinhibitor similar to BPTI (S.I. II) but with few differences at the active site is higher, confirming specificity of binding.

[Adsorption on synthetic membranes and immunogenic properties of aprotinin cross-linked by glutaraldehyde]. / Adsorbtsiia na sinteticheskikh membranakh i immunogennye svoistva aprotinina, kon''iugirovannogo s pomoshch'iu glutarovogo al'degida.

A procedure for the intermolecular crosslinking of aprotinin (natural polypeptide, an inhibitor of serine proteases) by glutaraldehyde was proposed. This autoconjugation of aprotinin increased its immunogenicity and the efficiency of sorption on nitrocellulose and polyvinylidene difluoride membranes. The immunization of animals by the autoconjugated aprotinin induced the production of antibodies that reacted with both conjugated and monomeric aprotinin. The properties of the crosslinked aprotinin are promising for the enhancement of sensitivity through its use in EIA and immunoblotting membrane methods that employ aprotinin and antiaprotinin antibodies.