UA77385C2 - Use of anti-hbp monoclonal antibody for therapeutic agent intended for treating or preventing disorders resulting from release of bradykinin in mammals, method for assaying monoclonal antibody, assay kit, method for assaying hbp production in mammalian cells and tissues - Google Patents

Abstract

The invention is related to a method for treating or preventing a disorder resulting from the release of bradykinin in said mammal, particularly a mammal which produces an HBP which binds to an HBP antagonist, e.g. a monoclonal antibody that binds to at least one epitope of human HBP, said disorder selected from the group consisting of systemic inflammatory response syndrome, ischemia reperfusion, adult respiratory distress syndrome, anaphylaxis and allograft rejection, comprising administering to said mammal an HBP antagonist in an amount effective to decrease the release of bradykinin in a mammal. Furthermore, the invention is directed to methods and kits for determining if a mammal produces HBP that binds to an HBP antagonist, e.g., a monoclonal antibody that binds to at least one epitope of human HBP.

Description

Description of the invention

The invention relates to a method of treatment or prevention of diseases caused by the release of bradykinin 2 in mammals, in particular mammals that produce heparin-bound protein, and this protein binds to an antagonist of heparin-bound protein, this method consists in administering to the aforementioned mammal an appropriate dose of a heparin-bound protein antagonist to reduce bradykinin release in mammals. In addition, the invention relates to methods and kits for determining the ability of a mammal to produce a heparin-bound protein that binds to a heparin-bound protein antagonist, and a method for detecting a heparin-bound protein antagonist.

Inflammation

An acute inflammatory reaction has certain features, which include changes in the diameter and tone of blood vessels, namely a violation of the function of the permeability of blood vessels, which as a result leads to the formation of exudate rich in protein (Sem, Meadiaugv oi IpPyattaiiop, MUUgidni, VgizioIi, O.K., 1986. Therefore, as soon as neutrophils (NMMs) receive 72 Chemotaxic signals, they border and adhere to vascular endothelial cells through specific adhesive molecules that are synthesized both on the endothelial surface and on the surface of neutrophils. After the attachment of neutrophils to endothelial cells, the opening of endothelial gaps occurs , which cause impaired vascular permeability and allow migration of neutrophils into the interstitial interstitium.

The contact phase system includes three enzyme factors, factor XI (E XI), factor XI! (E XII) and plasma prokallikrein (prekallikrein) (RK) and a non-enzymatic cofactor, H-kininogen (NK), which forms equimolar complexes with E

XI and RK, respectively. The presence of the contact phase is observed in monocytes, fibroblasts and neutrophils.

Specific binding sites remain open from endothelial cells and non-cellular negatively charged surfaces such as kaolin, which have the general name "contact phase" and allow the localization of the collection of the main components. The transformation of the zymogen, E XII, into an active enzyme, E XIIa, activates the system itself from the contact phase of ISoitap eii ai., 1986, (sg. Chem. OpsoiI. Netayoi. 5: 57-85). As a result of the mutual activation (3) of surface-bound ХІІІ and РК, fixed on the surface through NK, ІІІІІ and РКа are formed, thus amplifying the initial signal. After that, factor ІІІІ can activate Factor ІІ, and therefore, the natural coagulation pathway is initiated. It is known that RK also hydrolyzes NC to form an effective nonapeptide, bradykinin (Cariap apa Ziimegrego, 1987, Viosa 70:1-15).Kinins are considered the main mediators of the inflammatory process, which cause pain, lead to dilation and impaired vascular permeability due to their direct action on endothelial cells, forcing them to contract and allow both the migration of neutrophils and the transudation of plasma components. Local production of prostaglandins and nitric oxide is also possible. Tag. 56:131-190). Therefore, activation of the contact phase system can lead to side effects, for example, inflammation, septic shock, respiratory distress syndrome in adults, disseminated intravascular coagulation, postoperative bleeding in cardiovascular operations.

In particular, the contact phase system can be activated during the period when neutrophils bind to endothelial cells in the presence of endotoxins and by bacterial infection (see Soitap et al., 1997, Biood 90:3819-3843). For example, it was discovered that during sepsis, the activation of factor Hi! and C of prekallikrein leads to cleavages that activate them to enzymes that quickly react with inhibitor C1 to form complexes inhibitor of factor HPa-C1 and inhibitor of kallikrein-C1. Significant increase in formation

From" the kallikrein-C1 inhibitor complex is observed in simulated cardiopulmonary bypass.

Aprotinin, an inhibitor of plasma plasmin and kallikrein, has been found to reduce blood loss after cardiac surgery and shorten the duration of severe postoperative bleeding. In particular, it was found that aprotinin reduces 49 complexes of kallikrein-C1 inhibitor and C1-C1 inhibitor in a bypass model of artificial blood circulation (M/asypiodei!ei and AI., 1993, U. Tpogas. Sagaiomazs Zygo. 106:1). When aprotinin is added to cardiopulmonary bypass systems that are used with whole blood treated with anticoagulation with heparin, aprotinin actually complements the action of heparin. U. Naet. 101:455-461). So, aprotinin has another effect, in addition to the one that was found in heparin-bound systems, - a hemostatic effect. o 20 Aprotinin also increases the viability of endothelial cells under hypoxic conditions of storage in a refrigerator, when such cells are contained in solutions for preserving organs, and improves the preservation of lung and myocardial cells in models of whole organs. Tpog Zygu 52:971-978 and Kobegiv et al., 1998, App. Extinguished Zygo. 66:225-2301|. In addition, if bradykinin caused greater vascular permeability, then aprotinin decreased this vascular permeability and the number of neutrophils (O'Wgiep et al.,, 1997, Sap. 9. Ruzio|. 22 Rnagtasoi. 75:741-749 and Omzhepdegega!., 1995, Eig. U). Siip. Snet Siip. Viospet. 34:207-214).

GF) Heparin-bound protein

Recently, the covalent structure of two related proteins isolated from human and pig peripheral neutrophil leukocytes was determined (cf. N. Rioddaaga ei ai., 1991, Eig. U. Viospet. 197:535-547; U.

Ropi eyi aYi.,, 1990, REV5 Yey. 272: 200131. Both proteins show high affinity to elastase 60 of neutrophils, but due to selective mutation of active serine 195 and histidine 57 (chymotrypsin numbering |B. 5.

Nashpeu, "Notoiodiez ip Zegipe Rgoieipazev", RA. Thapv Coe. boss. Zegpez 257, 1970, p. 77I.| proteins do not have protease activity. The proteins were respectively named human heparin-bound protein (HBP) and porcine heparin-bound protein (pHBP) due to their high affinity for heparin.

Thank you for your help. |(Zspatheg ei aiYi., 1984, Ipiesi. Ittyp. 53:651| called the protein cationic antimicrobial protein bo (SARZ7) due to its antimicrobial activity. HVR strongly binds to the lipid component AL P5 and endotoxin (Cave-0.8x109M 7 It was pointed out that the bactericidal action of HVR is due to the binding of lipid A (Re(exep ei ai.,, 1993, Eig. 9. Viospet. 8214: 271-279., Rioddaaga ei ai.,, 1994, 9. Seii. Viospet. Zirri. 78AArzig. E5BO5; Regeiga ei aiYi., 1993, Rgos. Mai. Asai. Zsi. BA 90:4733-4737) According to assumptions, the site

The binding of lipid A//P5 of natural HBP is located as an uncharged insert between the basic and acidic inserts on HBP and includes residues 20-26 and 41-43. In the lipid binding site of AL RZ Rpe25, Suza2b,

Suz42 and PNe43 form a hydrophobic niche capable of binding fatty acid chains or the glycosaminoglycan sugar ring of lipid A. Behind this niche is an ionic and hydrophilic niche (Avp20,

SbiIp21 and Agd23), which has a high ability to bind the glycosaminyl-connected phosphate group of lipid A 70 Tsmegvep eyi a!., 1997, Maishge Zighisi. Viiodu 4:265-268).

In addition, the results of the treatment of fecal peritonitis with the use of NVR in animals showed that, for example, mice with the help of this treatment can avoid fatal damage. Hogyt, pp. 105-108 and UMiskKeyi! ей ай., 1997, Ip DI |piegpaiopa)! Sopdgezv op (She Ittipe Sopzedcepsev oyi

Tgashta, ZnosK apa Zerziz, Mypisy, Sepgtapu, pp. 413-416). It was proposed that heparin-bound protein or 75 of it | Re-bound parts were used to treat septic shock (MUO95/28949, US patents

MoMo 5,458,874, 5,607,916 and 5,650,392I.

At first, studies of the antibiotic and I Rho-binding properties of NVR were carried out (|Sabauega)!., 1989,

Rgos. May Asai. All together. O.5.A. 86:5610-5614 and Regeiga et al., 1993, Rgos. May Asai. All together. BOTH 90: 4733-7|.

However, recently collected data support the concept that NVR, in addition to its bactericidal effect, causes the progression of inflammation due to its ability to influence the recruitment and activation of monocytes (Regeiiga ei ai.,, 1990, 9. Sp. Ipmevi. 85:1468-1476 and Kaztivzzep ei aiYi.,, 1996, REV5 Gen. 390:109-1121, involvement of T-cells (Spegiom ei ai!., 1996, U). Vioi. Spet. 271: 2935-2940).

It was found that HVR causes contraction in endothelial cells and fibroblasts | Oziegdaaga apa

Rioddaaga, 1992, 9. ek. Vioi 51:316-323) In this connection |in UUO 93/05396| describes a method of selecting HBP inhibitors by incubating HBP or HBP-producing cells with a substance considered to be an inhibitor of

HBP, and with tissue or cells capable of interacting with HBP; reduced interaction (for example, reduction of endothelial cells) indicates that the substance is an inhibitor of HBP.

IU applications UUO99/26647| describes the use of a heparin-bound protein to modulate or prevent apoptosis of mammalian cells. The application also describes that HVR frees rat insuloma cells from induced | -1 IS o) apoptosis.

It was also found that only human heparin-bound protein, and not pig heparin-bound protein, binds to aprotinin (PRTI). U. Viospet. 214:271-279). In particular, VRTI is still able to connect with NVR with K ah0.1x109M IResegzep ei ai., 1993, Eig. U. Viospet. B214:271-279). It was determined that

P1 specificity of NVR is primarily I uz or Ii ei IKislak ei ai!., 1999, Vioi. Spent 380:101-105). It was noted that the most prominent residues in HBP, which influence the binding of VRTI, are SIu169, (Iu175, Zeg192 and Azr201, which correspond to Avzr 189, beg195, SIu216 and Avzr226 in trypsin (Re(egvzep ei aiYi.,, 1993, Eisg. U. Viospet. B214: 271-279. Kislak et al., 1999, Vioi. Spec. 380: 101-106 | collected a library of mutants in the P1 side chain of aprotinin, using phage display methods. They found that in NVR a high degree affinity with Р1 ГГ ув, « as well as with uncharged Р1-amino acids, Гем, TAг, Мей, Сип.

A new fact was revealed that heparin-bound protein (HBP) serves as a signaling link in neutrophil-induced vascular permeability dysfunction and activation of the contact phase system with parallel h formation of bradykinin, and that it plays a role in HC-mediated cleavage of NC to produce » sequences of bradykinin. In addition, it was found that NVR antagonists reduce the permeability of endothelial cells. As already defined, "HVR antagonist" is a substance that interacts with heparin-bound protein and inhibits the action of heparin-bound protein. - The invention relates to a method of treatment or prevention of diseases that arise as a result of the release of bradykinin in mammals, in particular humans, especially mammals that produce NVR that binds to an antagonist

A method which involves administering to said mammal in need thereof an appropriate dose of a human heparin-bound protein antagonist to modulate or reduce the release of bradykinin in said mammal. o 20 These diseases include, among others, systemic inflammatory response syndrome, ischemic reperfusion, anaphylaxis, and allograft rejection. These diseases can also include respiratory distress syndrome in adults - a side effect of the systemic inflammatory response syndrome. Anaphylaxis can occur as a result of inappropriate activation of the RMM during cardiopulmonary bypass, lung surgery, head trauma, and extensive body trauma. Modulation or reduction in the release of bradykinin is achieved by preventing the contact of HVR with endothelial cells and/or with the contact phase system. In a specific embodiment of the invention

Gee! an HVR antagonist modulates or reduces kallikrein-mediated cleavage of H-kininogen to produce the bradykinin sequence. The invention also relates to the use of an NVR antagonist for the purpose of obtaining a medicament for the treatment of systemic inflammatory reaction syndrome, ischemic reperfusion, anaphylaxis and allograft rejection in a patient with an NVR that binds to an NVR antagonist. Antagonists of NVR 60 can also be used in the treatment or in the preparation of medication for the treatment of complications of the systemic inflammatory reaction syndrome - the respiratory disorder syndrome in adults.

In a specific embodiment, the invention relates to a method for the prevention of diseases resulting from the release of bradykinin in mammals, in particular humans, especially mammals that produce HVR, which binds to an HVR antagonist, which involves the administration to the above-mentioned mammal in need thereof, a domain or analogue of 65 a Kipii2-type serine protease inhibitor, or a derivative thereof, which binds to HVR, in an amount effective to modulate or reduce the release of bradykinin in the aforementioned mammal.

In another specific embodiment, the invention relates to a method of treating or preventing diseases resulting from the release of bradykinin in mammals, particularly humans, particularly mammals that produce

A HVR that binds to a monoclonal antibody that binds to at least one epitope on HVR, said epitope binding to the prekallikrein-H-kininogen complex and activating the release of bradykinin, and the method comprises administering to said mammal in need thereof , a monoclonal antibody that binds to at least one epitope on HVR, wherein said epitope binds to the prekallikrein-H-kininogen complex and activates the release of an appropriate amount of bradykinin to modulate or reduce the release of bradykinin in said mammal. 70 The invention also relates to methods of detecting an NVR antagonist. In one embodiment of the invention, the above-mentioned method includes: (a) culturing endothelial cells in the presence of HVR and in the presence and absence of a substance considered to be the above-mentioned antagonist, and (b) detecting any effect of the above-mentioned substance on the permeability function of endothelial cells, and a reduced permeability function of the above-mentioned endothelial cells compared to the permeability function of the above-mentioned cells when incubated in the presence of HVR without the above-mentioned substance indicates that the above-mentioned substance is an antagonist. In another embodiment of the invention, the aforementioned method provides for (a) incubating HBP with a first substance) that interacts with HBP and a second substance that is considered to be an antagonist of HBP, and (b) detecting any effect of the aforementioned second substance, which is considered to be an antagonist, on the interaction of HBP with the aforementioned first substance.

In a specific embodiment, the invention relates to methods of recognizing a monoclonal antibody that binds to at least one epitope of HVR, in particular human HVR, in which the aforementioned epitope binds to the prekallikrein-H-kininogen complex and activates the release of bradykinin. In one of the embodiments of the invention, the method includes: (a) culturing endothelial cells in the presence of HVR and in the presence and absence of a monoclonal antibody that is believed to bind to at least one epitope of HVR, in particular human HVR, and the aforementioned epitope of binds to the prekallikrein-H-kininogen complex and activates bradykinin release, and (b) detection of any effect of the aforementioned substance on the permeability function of the endothelial cells, and the permeability function of the aforementioned endothelial cells is reduced, compared to i) the permeability function of the aforementioned cells upon incubation in the presence of HBP without the above antibody indicates that the above antibody is a monoclonal antibody that binds to at least one epitope on HBP, and the above epitope binds to the prekallikrein-H-kininogen complex and activates the release of zo bradykinin. in another embodiment of the invention, the method provides for (a) incubation of the prekallikrein-H-kininogen complex in the presence of HBP and in the presence and absence of a monoclonal antibody, which is considered to bind to at least one epitope on HBP, in particular human HBP, and the aforementioned the epitope binds to the prekallikrein-H-kininogen complex and activates the release of bradykinin, and (b) detecting any effect of the above antibody on the release of bradykinin, a reduction in the release of bradykinin, indicating that the above antibody binds to the above epitope on NVR. uh-

The invention also relates to methods and kits for determining whether a mammal produces an NVR that binds to an NVR antagonist. This method provides for the following stages: (a) separation of HVR or cells or tissue producing HVR from the body of a mammal, in particular a human; (b) incubating the aforementioned HVR or HVR-producing cells or tissue with a substance, tissue, cells, or component thereof that interacts with the HVR and the aforementioned HVR antagonist and (c) detecting the effect of the aforementioned heparin-bound antagonist on with the interaction of HBP with the above-mentioned substance, tissue, cells or their components, the decrease in interaction indicates that the above-mentioned HBP binds to the above-mentioned HBP antagonist. Test set ;" includes (a) NVR antagonist; (b) a natural HBP and (c) a substance, tissue, cell, or component thereof that interacts with the HBP.

In a specific embodiment, the invention also relates to methods and kits for determining whether a mammal, in particular a human, produces an HVR that binds to a monoclonal antibody that binds to at least one epitope on a native HVR, in particular a human HVR, wherein the aforementioned epitope binds to the complex

Ш-prekallikrein-H-kininogen and activates the release of bradykinin, and also involves (a) separation of HVR or 2) cells or tissue producing HVR from the body of the aforementioned mammal; (b) cultivation of the above

HBP or HBP-producing cells or tissue with endothelial cells in the presence or absence of the above-mentioned antibody and (c) detecting any effect of the above-mentioned antibody on the permeability function of the endothelial cells, the reduced permeability function indicating that the above-mentioned HBP is binds to the above-mentioned antibody. In another embodiment, the method provides for (a) separation of HVR or cells or tissue producing HVR from the body of the aforementioned mammal; (b) incubating the aforementioned HVR or HVR-producing cells or tissue with a prekallikrein-H-kininogen complex in the presence and absence of the aforementioned antibody, and (c) detecting any effect of the aforementioned HVR on the release of bradykinin from

F) prekallikrein-H-kininogen complex, the decrease in the release of bradykinin indicates that the above-mentioned NVR binds to the above-mentioned antibody. The kit according to the present invention includes (a) a monoclonal antibody that binds to at least one epitope on natural HVR, in particular human HVR, and the aforementioned epitope binds to the prekallikrein-H-kininogen complex and activates the release of bradykinin, ( 6) natural human NVR and (c) prekallikrein-H-kininogen complex attached to a solid base.

Figure 1 shows the effect of cross-linking CO18 with the secondary goat anti-mouse E(ab) antibody.

Figure 2 shows the dose-dependent effect of NVR (25-75mg/ml) on the permeability function of a monolayer of endothelial cells. 65 Figure 3 shows the inhibition of HBP-induced increase in EC permeability by polyclonal antiserum to HBP.

Figure 4 shows the effect of anti-NVR antibodies on the effect of PMM secretion on endothelial barrier function.

Figure 5 shows the inhibition of bradykinin- and HVR-induced EC permeability increase by the monoclonal antibody MVKZ to bradykinin. Bradykinin (T00nNM; Fig. ba) or HVR (75μg/ml; Fig. 5b6) is injected at the beginning of the time count from the laminal side of ES monolayers pre-incubated with tAbB MVKZ (4Omkg/ml).

Figure 6 shows the inhibition of the NBP-induced increase in EC permeability by the monoclonal antibody RKNA to plasma kallikrein. HVR (75μg/ml; shaded symbols) or bradykinin (100nM; unshaded symbols).

Figure 7 shows the inhibition of the NBP-induced increase in EC permeability due to peptide treatment with NKH. 70 Figure 8 shows the inhibition of the HBP-induced increase in EC permeability due to peptide treatment of 5OOZ1.

Figure 9 shows the inhibition of the NBP-induced increase in EC permeability due to aprotinin treatment.

Fig. 10: extraction of 1I-6 from human monocytes. Monocytes are cultured in 1 ml of medium without serum for 24 hours together with I RZ and/or natural NVR/LK235,E25EINVRLO175O0NVR in the amounts indicated in Fig.

Fig. 11: saturation of natural NVR, IB235, E25EINVR and |C175O|NVR-(A) ZNA RU and (B) competition for 725I1-VRTI-binding with constant, increasing concentrations of unlabeled VRTI. (IN)

The concentration of 722I-VRTI is shown at 95 OnM of unlabeled VRTI. An obvious difference in binding between NVR and

IC235,P25E)NVR is discussed in the "Results" section. Bars represent standard deviation.

Heparin-bound protein

In the proper context, the term "heparin-bound protein" ("HBP") means a protein that is proteolytically inactive; (iii) stored in azurophilic granules of polymorphonuclear leukocytes; and (ii) is a chemoattractant for monocytes and/or activates monocytes. NVR can be a mammal, in particular a human. In particular, the HVR can be developed by a human HVR that has at least about 8095 identity with the amino acid sequence presented in ZEO 0 MO:1, preferably at least about 9095, more preferably at least EM about 9595, most preferably at least about 9795 (hereinafter "" homologous polypeptides"), which qualitatively preserve d) the activity of the above-mentioned HVR. zl SHE V Na I v

Amino acid sequences of homologous polypeptides differ from the amino acid sequence presented in ZEO I MO: 1 by the insertion or deletion of one or more amino acid residues and/or by the replacement of one or more amino acid residues by other amino acid residues. Amino acid changes are mostly minor, that is, conservative amino acid substitutions that do not have a significant impact on the composition and/or activity of the protein; small deletions, typically from one to about 30 amino acids; small extensions at the amino- or carboxyl terminus, such as an amino-terminal methionine residue; a small linker peptide of approximately 20-25 residues; or a small extension that facilitates purification by changing the resulting charge or other function, such as a polyhistidine tract, antigen-75 epitope, or binding domain.

Examples of conservative substitutions include basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine) and small amino acids (such as glycine, alanine, (ee)50 serine, threonine and methionine). Amino acid substitutions, which in general do not change the specific activity, are known and described by specialists (for example, in the works of N. Mega apa Kk... NiIIII, 1979, Te Rgoivipz, Asadetis

Rgezv, Mem/ MogKk)|. The most common changes are: Aia/Zeg, MaiLie, Azr/sISH, Tig/Zeg, AiIa/zIu, Aia/TNg, Zeg/Avzp,

Aia//ai, Zeg/SiIu, Tu/Rpe, AiIa/Rho, I uv/Agd, Avr/Avp, IeshIe, Ieshy/Mai, Aia/SiIi, Avzr/bIuU, as well as these changes in the reverse direction.

This term, in particular, covers peptide fragments of HVR, and especially fragments with a chemotactic effect,

HF) similar to natural NVR. In addition, HVR, which are used in the methods according to the present invention, 7 bind to HVR antagonists, such as aprotinin and/or a monoclonal antibody that is produced against natural human HVR. Natural human HVR in its developed form has the amino acid sequence shown in ZEO IO MO:1 and, in addition, () is proteolytically inactive; (iii) stored in azurophilic 60 granules of polymorphonuclear leukocytes; and (ii) is a chemoattractant for monocytes and/or activates monocytes.

HVR is isolated from platelets obtained from human blood using the procedures described in US Patent No. 5,814,6021. More precisely, the protein is produced by fractionating the platelet extract.

For this purpose, it is advisable to use column chromatography using heparin-sepharose. Such a chromatographic method, which includes a gradient elution with Masi from 0.5M to 3M column, through which 65 was initially passed the extract of blood platelets, as a result leads to the elution of two peaks. The first peak

- approximately 1.2M Mass - can be measured at 280nm as a large protein peak, which is platelet-derived growth factor (PGF) and is known in the art. The amount of protein of approximately 1.8M Mass is less than the limit of detection in the system used, but the fractions in this area have angiogenic activity. The active fractions are additionally purified by microcolumn antiphase NRIS on a C 4 column, at 214NM a completely purified protein can be detected, this protein is identically represented in the HVR of both porcine and human types, depending on the type of plates used.

If HBP is to be used to detect HBP antagonists, it is best obtained through the recombinant DNA methods presented below. The nucleic acid sequence encoding HVR can be synthetically produced by generally accepted standard methods, for example, the phosphoamidite method (described in 5... Veaisade apa MN SagtsiNegz, 1981, Teigapedgop I ebege 22:1859-1869, or by the method , described by Maipez et al., 1984, EMVO doigpa! 3:801-8051. According to the phosphoamidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, annealed, cross-linked and cloned into appropriate vectors.

Technologies for isolation or cloning of a nucleic acid sequence encoding a heparin-bound protein, used in the method according to the present invention, are known to specialists and include isolation from genomic

DNA, preparation from cDNA, or a combination of these methods. Cloning of nucleic acid sequences from genomic DNA according to the present invention is carried out, for example, by using the well-known polymerase chain reaction (PCR) or selection using antibodies of expression libraries to identify cloned DNA fragments with common structural fragments. See, |for example, Ippis ei a!., 1990, A

Scide o Meijodz apa Arriisayop, Asadetis Rgez5, Mem Mogk). Other nucleic acid amplification procedures are also used, such as ligase chain reaction (LCR), cross-linked activated transcription (GAT) and amplification based on nucleic acid sequence (NAS).

The nucleic acid sequence is then inserted into a recombinant expression vector, which can be any vector amenable to the recombinant DNA procedure. The choice of vector often depends on the host cell into which it is introduced. Thus, the vector can be an autonomous replication vector, i.e. i) a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. In an alternative embodiment, the vector may be one that, when introduced into a host cell, integrates into the genome of the host cell and replicates along with the chromosome(s) into which it has been integrated. The nucleic acid sequence that encodes ZEO AND MO:1, ZEO OO MO:2 can be functionally linked to a nucleic acid that encodes a heterologous gene and/or signal sequence. In this alternative embodiment, the nucleic acid that encodes ZEO IU MO5:4 (signal sequence - developed with

NVR) and 6 (signal sequence yrgo-sequence - developed NVR), ZEO IU MOB5:Z and 5, respectively, can be introduced into the recombinant vector. - shi ay 5 i Z -i -i (95) What (ee) 60 b5

AI

Iii ik ti sten - pole ki skit y mns s

POD t Kt eye KS IN Otyt VIT sn vd ZV pe nn nn an im

The nucleic acid sequence that encodes the HBP can also be operably linked to a suitable terminator, such as the human growth hormone terminator (Raiti(eg ei ai., or. siyi) The vector may also contain other elements. These include polyadenylation signals (for example, from ZM 40 or the region of adenovirus 5 EIyb), iv . vector can too

Contain a DNA sequence that allows the vector to replicate in a given host cell. This vector may also include a selectable marker, for example, a gene whose product complements a deficiency in the host cell, such as a gene encoding dihydrofolate reductase (ONEK), or one that confers drug resistance,

Sh- for example, neomycin, geneticin, ampicillin or hygromycin. 2) In a specific embodiment, HVR can be produced by culturing host cells containing a DNA sequence that encodes an evolved HVR located after the M-terminal extension in an appropriate co-culture medium under conditions that permit expression of HVR, and recovering the resulting HVR sp from the cultural environment as elongated at the M-end of the NVR.

The M-terminal extension can be a sequence that contains from about 5 to about 25 amino acid residues, in particular from about 8 to about 15 amino acid residues. The origin of the amino acid residues in the M-terminal sequence is considered insignificant.

To increase the production of the developed NVR, as a rule, the coding DNA sequence is chosen

F) extended at the M-end of the HBP, which contains the DNA sequence encoding the protease cleavage site, placed between the DNA sequence encoding the M-terminal extension and the DNA sequence encoding the developed HBP.

Examples of a suitable protease cleavage site are the enterokinase cleavage site with the amino acid sequence bo, the factor Xa cleavage site with the amino acid sequence.

In an alternative embodiment, host cells that contain DNA encoding an evolved HVR preceded by a signal sequence are cultured in an appropriate culture medium under conditions that permit expression of the HVR, and the resulting HVR is removed from the culture medium as an evolved HVR.

The procedures for splicing the nucleic acid sequences that encode HBP or M-terminally extended HBP, 65 drgo-HBP (signal e- extended HBP), promoter and terminator, respectively, and inserting them into appropriate vectors containing the information necessary for replication are well-known specialists (cf., for example,

zatrgook ei ai., op. 9U.

The host cell(s) into which the expression vector is introduced can be any cell(s) capable of producing HVR, and in an optimal embodiment is a eukaryotic cell(s). , for example, invertebrate (insect) cell(s) or vertebrate cell(s), for example, mammalian cell(s), in particular vertebrate and mammalian cell(s). In an optimal embodiment, the mammalian cell is a cell that can be cultured under anaerobic conditions after transfection with a nucleic acid encoding a mammalian HVR. A particularly preferred embodiment is where the mammalian cell is an adenovirus-transformed cell(s) or is derived from an embryonic cell(s). As defined by the authors, a cell derived from an embryonic cell 70 is a cell derived from a primary culture of embryonic cells or a cell(s) from a cell line originally derived from a primary culture of embryonic cells. An example of such an adenovirus-transformed cell or embryo-derived cell is a human embryonic kidney (HEK) cell(s), particularly HEK 293 cells. An insect cell may be, for example, a Lepidoptera or Drosophila cell.

Methods of transfecting mammalian cells and expressing DNA sequences introduced into cells are described,

For example, in the works of Katsitap apa Zpagr, 1982, 9. My. Vioi 159:601-621; bBotsiyegp apa Vego, 1982, 9. Mo).

Arri. Sepoys 1:327-341; I ouieg eyi aYi.,, 1982, Ros. May, Asad. All together. BOTH 79:422-426; MUidieg ei ai., 1978, Seii 14:725; Sogzago apa Reagzop, 1981, Botaiiis SeiYi Sepeiiisv 7:603, bSgapat apa map deg EB, 1973, Migoiyudu 52:456;

EgaIeu ei ai., 1980, UVS 225:10431; Saresspi, 1980, SeiI 22:479; M/iregod ei aiI., 1983, MAK 11:7287; and Mashtapp ei aI,, 1982, EMVO )). 1:841-845). Insect cells are transfected with a baculovirus vector as described in US Pat

Me4,745,0511.

The cell culture medium can be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a medium suitable for growing mammalian cells. All media that can be purchased commercially or obtained from published recipes (eg, American Type Culture Collection catalogs) are suitable. Cells are selected for resistance to antibiotics. After that, the selected clones are tested for HVR activity, using methods known to those skilled in the art, such as chemotactic i) analysis and analysis of cytokine release from monocytes | see, for example, Kaztivzzep ei and I., 1996, REV5Z and ei. 390:109-112).

The HVR produced by the cells is then removed from the culture medium by conventional methods, including separation of the host cells from the medium by centrifugation or filtration, precipitation of the protein components of the supernatant or filtrate with a salt such as ammonium sulfate, purification using various chromatographic procedures, for example, ion exchange chromatography, affinity chromatography, etc.

If the NVR is elongated at the M-end of the NVR, after removal from the culture medium it is extended at the

The M-ends of NBP are best cleaved by a suitable protease to form the developed (and active) NBP. Examples of such enzymes are, among others, enterokinase and factor Xa.

Antagonists of NVR

Anti-NVR polyclonal and monoclonal antibodies can be an antagonist of NVR. The following procedure is used to obtain anti-HVR polyclonal antibodies. To produce antibodies by injection of HVR, in the "optimal embodiment" - human HVR, various host animals are immunized, including, but not limited to, rabbits, mice, rats, goats, etc. In one of the embodiments, HVR can be conjugated with an immunogenic carrier, for example, bovine serum albumin (B5A) or hemocyanin of molluscs of the Astaea species (KIN). For ;" strengthening the immunological reaction, various immunostimulants are used, depending on the type of hosts, namely, among others, Freund's adjuvant (complete and incomplete), mineral gels, such as aluminum hydroxide, surface-active niacin, such as lysolecithin, surface-active polyols /polyanions, peptides, oil emulsions, hemocyanins -I of Astaea molluscs, dinitrophenol and potentially useful human immunostimulants, such as BSO (bacillus

Saitece-Sshiegip) and Soguperasiegisht ragmite. - Various procedures are used to obtain a monoclonal antibody that binds to at least one epitope of HVR, in particular human HVR, and the aforementioned epitope binds to the 5p prekallikrein-H-kininogen complex and activates the release of bradykinin. In an optimal embodiment, the monoclonal antibody is a human monoclonal antibody. Any technology that allows the production of sp antibody molecules by a continuous cell line in culture can be used. These include, among others, the hybridoma method, first developed by Kopieg apa Miisiyip (1975, Mayte 256:495-497)), as well as the triomic method, the human B-cell hybridoma method by Kogboghei a/!., 1983, IttypoYodu Todau 4:721, and EVMU - a hybridoma method for the formation of two human monoclonal antibodies |Soie eyi a), 1985, Moposiopa! Apirodiez apa Sapseg TNegaru, AiYap K. II ivv,

Ips., pp. 77-96Y1. In an additional embodiment of the invention, monoclonal antibodies can be produced in transgenics

F) animals using modern technologies |PCT/I590/02545; Sgeep, 1999, in). And so on. Meipodz 231:11-23 and ka pat. US MoMo 5,625,126 and 5,633,425). According to the invention, human antibodies can be used and obtained by using a human hybrid |Soie ei a). 1983, Rhos. May Asad All together. O.5.A. 80:2026-2030| or because the transformation of B-cells of a person by the virus EVM ip mygo |Soie ei ai., 1985, Moposiopa! Apiirodiev apa Sapseg

Tnegaru, Aiap K I izv, ss.7 7-96).

According to the invention, technologies for the production of single-chain antibodies are described (US Pat. No. 4,946,778)| can be adapted for the formation of HVR-specific single-chain antibodies. In an additional embodiment of the invention, the technologies described for the construction of Rab expression libraries are used (Nize ei ai!., 1989, Zsiepse 65 246:1275-1281), which allow quick and easy recognition of monoclonal PHar fragments with specificity to

NVR

Antibody fragments that contain an idiotype of the antibody molecule can be obtained by known methods.

For example, such fragments, among others, include: fragment E(abr').zyr.2, which can be obtained by splitting the antibody molecule with pepsin; Bar fragments, which can be obtained by reducing the disulfide bridges of the K(ab').zy0.2 fragment and Rab fragments, which can be obtained by treating the antibody molecule with papain and a reducing agent.

When obtaining antibodies, the selection of the necessary antibodies is carried out by methods known to specialists, for example, by the method of radioimmunoassay, EII5A (solid-phase immunosorbent assay), multilayer immunological analysis, immunoradiometric analyses, gel-diffusion reactions of precipitation, immunodiffusion 70 analyses, immunological analyzes ip zysh (using, for example , colloidal gold, enzyme or radioisotope labels), Western blotting, precipitation reactions, agglutination assays (eg, gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of the secondary antibody or reagent to the primary antibody. In another embodiment, the secondary antibody is labeled. Many methods of detecting binding in an immunoassay are known to those skilled in the art and are within the scope of this invention.

In an alternative embodiment, the anti-NVR antibody library is completed by the phage detection method (see, for example, KozsieivKa et al., 1998, Asia Viospitis Roiipisa 45:705-720). The strongest binding components are selected from the library and used as NVR antagonists.

An HVR antagonist can also be a polypeptide that contains a Cypiig-type domain of a serine protease inhibitor that binds to HVR. Peptides that contain Kipi:-type serine protease inhibitor domains typically have approximately 60 residues and six spaced cysteines present in disulfide bonds. In a specific embodiment, if the NVR is a human NVR, then the Kipii2-type inhibitor can be aprotinin, which is also known as a VRTI, or an analog thereof that binds to the NVR. In an optimal embodiment, the aprotinin analog has mutations at positions 12-19 and 34-39, with mutations at positions 15-19 being preferred. Інші і) аналоги, які можуть бути застосовані, описано (у патентах США МоМо5,162,498, 5,316,923, 5,395,922, 5,514,585, 5,510,249, 5,591,603, 5,618,915, 5,621,074, 5,673,090, 5,618,696 та 5,576,294|. У найхарактернішому втіленні мутаціями є А16Н, К15А, 118М , 1195, K17T

Large libraries (107-109) of polypeptides, which include Kipii-type domains, are assembled using various approaches. For example, the error-prone ROK (Zaiki et al., 1988, Science 293:487-491) can be applied to each individual Kipiig-type domain. This procedure involves the random inclusion of an incorrect nucleotide into the gene of each corresponding Kipii2-type domain by carrying out the PCR reaction under conditions where Tad polymerase incorporates incorrect nucleotides at the amplification stage. The library can be detected on phage particles to select the best binding component for HBP. -

In an alternative embodiment, certain loops can be subjected to mutagenesis by using degenerate oligonucleotides (Zspieg et al., 1996, Sepe 169:147-153). Synthetic random oligonucleotides are injected into

Kipii2-type domain during RSK, which completes the library with variants in a certain loop. "

According to another approach, libraries can be assembled through DNA shuffling (Zietteg et al., 70 1994, Maitsge 370:389-391). This method is used for recombination between homologous Kipii2-type domains. - c That is, the DNA encoding each variant is mixed and fragmented into small parts. These parts are then reassembled by a conventional ROK reaction. During this reaction, the small parts join together because "» they are complementary, which causes the recombination of the corresponding genes.

Detection methods

To identify antagonists of heparin-bound protein (HBP), it is necessary to determine whether a substance, -I, which is considered an antagonist of HBP, binds to HBP, for this purpose, (a) incubation of HBP with the first substance that interacts with HBP, and a second substance believed to be an antagonist of HBP, and (b) detecting any effect of said second substance thought to be an antagonist on the interaction of HBP with said first substance, wherein a decrease in the interaction of HBP with the first substance indicates that the second substance is antagonist of NVR. with NVR or the first substance can be labeled. The label is preferably selected from the group of colored enzymes or fluorescent substances, radioactive isotopes and complexing agents.

Examples of enzymes used as labels are peroxidases (e.g., horseradish peroxidase), phosphatases (e.g., acid or alkaline phosphatase), β-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, lysozyme, malate dehydrogenase, glucose-6- phosphate dehydrogenase, p-glucosidase, proteases, pyruvate decarboxylase, esterases, luciferase, etc.

F, Enzymes themselves cannot be detected, they must be in combination with a substrate to catalyze a reaction whose end product can be detected. Examples of substrates used according to the method of the present invention are hydrogen peroxide/tetramethylbenzidine or chloronaphthol or o-phenylenediamine or 3-(p-hydroxyphenyl) propionic acid or luminol, indoxyl phosphate, p-nitrophenyl phosphate, nitophenyl galactose, 4-methyl umbelliferyl-O-galactopyranoside or luciferin.

In an alternative embodiment, the labeled substance may contain colored or fluorescent substances, including gold particles, colored or fluorescent latex particles, dye particles, fluorescein, phycoerythrin or phycocyanin. 65 Radioactive isotopes used for this purpose are selected from 7291, 1911111), ЗН, 82Р, С and 358,

The radioactivity emitted by these isotopes is measured by a gamma counter or a scintillation counter in a manner known to those skilled in the art.

Complexing agents used for this purpose are selected from biotin (which forms a complex with avidin or streptavidin), avidin (which forms a complex with biotin), protein A (which forms

Complex with immunoglobulins) and lectins (which form a complex with carbohydrate receptors). Since the complex cannot be directly detected, it is necessary to label the substance with which this agent forms a complex.

Labeling is successful with any of the above labeled enzyme labeling agents.

In a specific embodiment, the binding of the NVR antagonist to the NVR is detected by using, for example, the ZRA technology from Ategepat Ragtasia Vioyesi. That is, biotinylated plasma membranes from endothelial 7/0 Umbilical vein cells (NOMES) bind to streptavidin-conjugated RUT ZRA granules. These granules are obtained from Ategepat Riagptasia Vioyesi. Labeled human kininogen (for example, ZN, 125|) is added to the membranes until the kininogen binding sites are saturated. HVR is added at increasing concentrations to displace labeled kininogen and the decrease in label incorporation is measured. A standard displacement curve is constructed. The HVR antagonist will be tested for its ability to inhibit HVR-mediated displacement of 75 radioactive kininogen for plasma membranes. In particular, HBPs are incubated with different concentrations, up to a molar excess of HBP antagonists, and their ability to inhibit HBP-mediated displacement of labeled kininogen from membranes is determined.

In an alternative embodiment, HVR antagonists are detected by culturing endothelial cells in the presence of HVR and in the presence and absence of a substance believed to be an antagonist. In a specific embodiment, the antagonist is a monoclonal antibody that binds to at least one epitope of HVR, preferably human HVR, and the aforementioned epitope binds to the prekallikrein-H-kininogen complex and activates the release of bradykinin. In an optimal embodiment, the monoclonal antibody is a human monoclonal antibody. In one embodiment, the antibody and HBP are pre-incubated together prior to incubation with endothelial cells for approximately 10 minutes at 372C. The effect of the substance on the permeability function of SM endothelial cells is determined by measuring the transendothelial electrical resistance or (5) determining the albumin gap, as described below in the Examples. A reduced permeability function should indicate that the substance does not act as an antagonist of NVR.

In a specific embodiment, the binding of a monoclonal antibody to at least one epitope of HVR, when the aforementioned epitope binds to the prekallikrein-H-kininogen complex and activates the release of bradykinin, IF) is determined by incubating the antibody with HVR and the prekallikrein-H-kininogen complex. As described in more detail in the Examples, HVR binds to the prekallikrein-H-kininogen complex, thus stimulating the release of bradykinin. A monoclonal antibody that binds to at least one HVR epitope should reduce or prevent the release of bradykinin. Analysis of the release of bradykinin is carried out by procedures known to specialists, for example, immunological analysis.

In one of the embodiments, the covering buffer contains 0.0595 Tmeep, 15.9 mM disodium carbonate, 35 mM sodium bicarbonate, pHO,b. Add H-kininogen and incubate at 492C overnight. The covering buffer is removed and the plate is washed with Tgiz buffer, pH 7.4, containing approximately 50 OM zinc. After that, prekallikrein is added and the mixture is incubated for an hour. at room temperature. HBP is then added to Ttgiz buffer and samples are removed at approximately 10 minute intervals for approximately 1 hour starting approximately 5 minutes after HBP addition and assayed for bradykinin release. In one of the embodiments, the release of bradykinin is analyzed using a kit for EI IZA analysis of mass production "" (MAKIT-M VgadukKkip, Oaipirrop RNaptaseshchiisa! So., (4). In this analysis, a competitive reaction is carried out " of bradykinin in the sample and labeled with peroxidase of bradykinin from an antibody against VK (rabbit) captured by the antibody

Id anti-rabbit (goat) covering the microstrip hole. The concentration of bradykinin is determined by the enzyme activity of peroxidase-labeled bradykinin bound to an anti-bradykinin antibody. - In an alternative embodiment, prekallikrein and H-kininogen are added in equimolar amounts to a monolayer of endothelial cells and incubated for an hour. at 372C. NBP is then added and samples are removed at approximately 10-minute intervals for approximately 1 hour, beginning approximately 5 minutes after the addition of NBP, and assayed for bradykinin release using the procedures described above. o 20 In another embodiment, prekallikrein and H-kininogen are added in equimolar amounts to a plasma solution containing an ACE (angiotensin-converting enzyme) inhibitor and incubated for an hour. at 3720. After this, HBP is added and samples are removed at approximately 10 minute intervals for approximately 1 hour starting approximately 5 minutes after the addition of HBP and assayed for bradykinin release using the procedures described above. 52 Methods of treatment

F! The composition according to the present invention contains part of an aqueous solution. The solution in the optimal version should be physiologically acceptable, so that after the introduction into the patient's body of the required sequence, the solution does not lead to a side effect on the electrolytic and volume balance of the patient. The aqueous medium for the composition, thus, may contain, for example, normal saline (0.995 Mass, 0.15M), riH7-7 4 or 60 other pharmaceutically acceptable salts thereof. Solutions for use are prepared in any way, well-known among pharmacists, described (for example, in the work "Ketipdiopye Rpagtaseciisa! Zsiepsev", (Seppago, A., ed.),

Musk Ryb., 19901.

The concentration of the HVR antagonist can vary, namely from about less than 0.595, for example, from 195 to 15-2095 by weight. A unit dose of the composition, as a rule, contains from about 100 mg to 1 g of the NVR antagonist. because Therapeutically effective doses are determined by ip mio or ip mimo.

The NVR antagonist is administered locally, subcutaneously, or by intravenous injection. The dosage is prescribed by a doctor according to the specific condition of a particular patient. The dosage and frequency of administration are carefully selected and adjusted according to the parameters determined by the doctor. The optimal route of administration can be, for example, intra-abdominal injection. Intravenous intra-abdominal injections of the NVR antagonist are carried out on a 24-hour basis, the dose is 0.1-100 mg, better - 0.5-50 mg, even better - 1-25 mg per 1 kg of body weight. The dose is administered 1-4 times per 24 hours or administered continuously through a catheter. In an alternative embodiment, the treatment is carried out by continuous intravenous infusion during surgery, followed by postoperative treatment for 1-4 days. 70 An NVR antagonist is administered only to patients who produce NVR that binds to the NVR antagonist. To determine whether a patient produces such HVR, HVR is isolated from the patient's platelets using the procedures described above. Binding of HBP with an antagonist of HBP (for example, a monoclonal antibody) or the effect of HBP on the permeability function of endothelial cells is detected using the procedures described above and compared with the effect of natural HBP. In an alternative embodiment, the effect of HVR on the contraction of endothelial /5 cells or fibroblasts or monocyte aggregation in the presence or absence of an HVR antagonist can also be determined using procedures (described in UUO93/05396). A reduced ability to contract or aggregate cells indicates the presence of an interaction between HBP and the HBP antagonist. A test kit used to determine whether a patient produces such an NVR includes (a) an NVR antagonist; (b) an HBP or a cell that produces a HBP, and (c) a substance, tissue, cell, or component that interacts with the HBP.

In a specific embodiment, the NVR or cells or tissue that produce the NVR are isolated from the patient's body.

HBP or cells or tissue producing the aforementioned HBP are incubated with endothelial cells and the effect on permeability function is determined in the presence and absence of a monoclonal antibody that binds to at least one epitope of the HBP, said epitope binding to the prekallikrein-H- complex kininogen and activates the release of bradykinin. This effect is compared with the effect of the natural NVR of the human body. In a specific embodiment, the natural HVR of a person can be a recombinant HVR that binds to the same monoclonal antibody. In an alternative embodiment of HVR, or cells or tissue that i) produce HVR, are incubated in the presence of a prekallikrein-H-kininogen complex and in the presence of a monoclonal antibody. Determine the effect of a monoclonal antibody on the release of bradykinin. A decrease in bradykinin release in the presence of antibody indicates that the patient is producing HVR that binds to the antibody. Under these conditions, the test kit should include (a) a monoclonal antibody that binds to at least one epitope on HVR, and the aforementioned epitope binds to the prekallikrein-H-kininogen complex and activates the release of bradykinin; (b) natural human NVR and (c) prekallikrein-H-kininogen complex attached to a solid base.

Examples in

Example 1: Activated neutrophils initiate changes in endothelial barrier function by signaling through

B2 integrins

Materials and methods

Reagents

Medium-199, KRMI-1640 (which contains I-glutamine), fetal bovine serum (EB5), trypsin-EOTA, phosphate buffered saline (PB5) and Hanks' balanced salt solution (HB55) are obtained from pl") with Zhe Thesppoiodiosis (Saipegerigo, MO, OA). Gelatin, collagenase, penicillin, streptomycin, catalase, bovine serum albumin (BSA), M-formyl-methionyl-leucyl-phenylalanine (MI R), blue dye;" Evans, hexadecyltrimethylammonium bromide and tetramethylbenzidine are obtained from bZidt Spetis! Co. (51. And fathers,

MO, OA). NhO" is received from E. MegskK (JuOagtviadi Segtapu). Viotaih And receive from Vioteaisai! Thesppoiodyesis Ips. (Ziotsdpiop, MA, OA) and Oehigap 70 (Masgodeh) and Risoi-Radze receive from Rnaptasia -I Viogesp AV (rrzaia, Zm'edep). The main culture medium contains a 1:11 mixture of C-199 and KRMI-1640 with the addition of heat-inactivated EB5 20905, penicillin (100 U/ml) and streptomycin (100 μg/ml).

Ш- Monoclonal antibody IBA4 against the usual B-chain (СО18) of Во-integrins was provided by Og. Siaez I ipabego, 2) RPpagtasia 8 Oriopp (Orrzaiia, Zul"edep) with kind permission of YuOg. Zatie! Yu. UMgidni, KosKetePeg Opimegeiu, tab 601, recognizing o,)chain of integrin (CO11B) provided by Og. Mapie! Raiagtouo, KagoiipzKa Ipziyshe, and OKES bo 200 (anti-I -selectin) were provided by Og. Eidepe Wiispeg, Iaptoga Opimegeiu, tAB 5K11 (anti-I -selectin) was obtained from 4 Vesiop OisKipzop, (Zap dose, SA) and tAB 0544- 26 (anti-CO44) was obtained from Riagm-ipdep (Zap Oiedo, CA). K(ab)" fragment of goat anti-mouse dO antibody was provided by DaskKvop IttypoKezeagsi I Arogayogiev, Ips. (M/evi Ogome, RA),

EITO-conjugated K(ar)" fragment of the rabbit anti-mouse Id antibody was obtained from Oaco A/5 (SiIovigir, Oeptagk).

Endothelial cells

Human umbilical vein endothelial cells (NOMES) and bovine aortic endothelial cells (IF) (BAES) are isolated and cultured as described above (Sashat et al., 1998, Vy. U. Rpagt. 125:1109-1114і4. NOMES ko or BAES of the first-fifth pass are separated by a short (2 min.) treatment with trypsin-EOTA (0.2590 trypsin / 0.0195 EOTA) and applied either to an inorganic Aporoge membrane with a pore size of 0.2 μm, or to polycarbonate filters with with a pore size of 3,000 mm (inclusion of tissue culture, 100 mm; MOMS, Kozkiide, Oeptagk).

To promote cell differentiation and strengthen the connection of endothelial cells (ES), the filters are pre-treated with 50mMA Viotaih I (167mg/ml) and air-dried. ECs are seeded at a density of 2 x 109 cells per filter and incubated in a culture medium at 372С in a humidified atmosphere of 595 СО" in air. ECs are grown to confluent monolayers, which can be detected by daily observation under a microscope and by measuring the electrical resistance across the monolayer (Sashat et al., 1998, Vii. U. Rpagt, 125:1109-1114).

Measurement of the EC barrier function

Two different approaches are used to determine stimulation-induced changes in EC permeability function. To measure the transendothelial electrical resistance (TEEC), the filter inserts from the ES are transferred to the resistance measurement chamber | for details, see in the work of Sashciat ei aiYi., 1998, Vol. U. Rpagt. 125:1109-1114). A chamber (lower/luminal compartment) with filter inserts (upper/luminal compartment), filled with 2ml and 40O0μl of culture medium, respectively, is placed in a cell culture incubator. The electrical resistance across the EC monolayer is measured at 372 using electrodes in each compartment positioned precisely relative to each other and to the monolayer. The results of the electrical resistance of a single EC monolayer are obtained by subtracting the resistance of the corresponding filter covered with Viotaigih, measured before seeding 7/0 endothelial cells.

In separate experiments, albumin (EVA) conjugated with Evans blue dye is used as a marker of macromolecular permeability of EC monolayers. Before EC stimulation, the culture medium in the upper compartment is replaced with a medium containing EVA (culture medium containing 4956 VBA mixed with Evans blue dye at a final concentration of 0.b7mg/ml). The concentration of EVA in samples of 75 liquids of the upper and lower compartment is determined spectrophotometrically by measuring the optical density at 620 nm (Tiepek MiyizKap MOSS; Riomzh/ I arogayugiez, ZoIpa, Zm"edep). The albumin gap is calculated according to the ratio: M1-A2 xM2x1/A1, where M1 is the clearance volume (ie, the theoretical volume of the luminal medium cleared of albumin by diffusion with abluminal compartment), M2 is the abluminal volume, and AT and A2 are the optical densities of the luminal and abluminal media, respectively. of any stimulus, an average of 0.08 and 0.0Zmcl/min., is subtracted from the volume of the gap obtained in response to the corresponding specific stimulus.

Obtaining and determining the number of RMM

Human PMM is isolated from plasma rich in leukocytes, as (described in the work of Satsiat ei Ai., 1998, Vgii...

Rpagt. 125:1109-1114), and resuspended in the culture medium at a concentration of 2-5x10 cells/ml. se 25 The purity of PMMs is 29895, and their viability when displacing trypan blue is 29595. Before adding to EC monolayers, PMMs are incubated for ZOhv. with monoclonal antibodies against cell surface molecules for subsequent cross-linking with secondary tAB (see Experimental Procedures).

In some experiments, the cell surface expression of SOT 16/СОО 18 is regulated by an increase in temperature

RMM before use. RMM in the presence of anti-CO 18 tAB IV4 is incubated at 42C for 10 min., transferred to Iz) 30 to 372 for 10 min., and then returned to 42 for another 10 min., after which it is washed twice. PMMs that cannot be treated with the antibody are obtained according to the same protocol, using the carrier instead of the antibody.

The adhesion of RMM to EC and transendothelial migration are quantified by analysis of me)

PMM-specific enzyme about myeloperoxidase (MPO). That is, PMM is dissolved in 0.595 parts of hexadecyltrimmonium bromide and centrifuged, the enzyme activity in the supernatant is determined

It is spectrophotometrically determined by the change in optical density at 65ОНМ, which occurs in the redox reaction of

H2Oo-tetramethylbenzidine, catalyzed by MPO |(|ByhykKi ei ai., 1983, ApaiY. Viospet. 132:345-352) MPO activity of linked and transferred PMMs, respectively, is correlated with the indicator of the total number of PMMs added to the EC monolayer. "

Experimental Procedures 40 Before transferring the filter inclusions to the resistance measurement chamber (which is kept at 37 2s), the media in the inclusions and in the chamber are replaced with fresh culture medium (372C) with the addition of 10MM "zNerez. In some experiments, the EC monolayer is incubated before stimulation with 1 Ωkg /ml of Negritusip A for 15 min at 372 C and washed twice.PMM (2x109) with or without monoclonal antibodies bound to cell surface molecules is added to the upper compartment (RPMI:EC-10:1 ratio) and left for deposition at -I 10 min on EC monolayer. Activation of RMM is caused either by MI R (10-7M), which is added to the lower compartment, or by cross-linking of cell surface molecules by secondary fragments of IdsS P(abB)2 goat-anti-mouse. - Transendothelial electrical resistance is measured before and every minute after the start of stimulation until reaching the c phase of the plateau in the resistance change, and then at 5-minute intervals.In experiments investigating albumin permeability and PMM migration through е monolayer EC medium in the inclusion, EVA was present. After checking (ee) immunity, filter inserts are transferred at 5-10 minute intervals to new wells containing fresh medium. Incubation in all cases is carried out at 372. At the end of the experiment, the lower wells are centrifuged at 425 g and 49 for 2 hours. and the supernatant is analyzed for EVA content. The amount of PMM that migrated through the monolayer is determined by analyzing the MPO activity of the granules remaining in the well. From

The medium in the filter inclusion sucks the liquid to determine the amount of unbound PMM and the filter, the EC membrane is removed from the insert to determine the amount of the bound PMM fraction. i) In separate experiments, purified PMM, pretreated with anti-CO 18 and IV4 (bmkg per 2x109 cells) for 30 minutes, are incubated in a culture medium with or without E(ab)" goat-anti-mouse for 10 minutes. at 372C. PMM is precipitated by centrifugation at 300 xg for 15 minutes. at room temperature and 60 the cell-free supernatant containing PMM secretion products is decanted for use in the ip mimo experiment (see below) or with EC monolayers. In the latter conditions, the medium in the filter/EC inclusion is replaced with medium containing PMM secretion, and data on the effect on TEEK and albumin permeability are recorded as described above. In some cases, the medium containing PMM secretion is incubated at elevated temperature (802C for 15 minutes) before use. for Determination of the formation of intracellular |(Са?LES and g-actin

Sensitive to Ca?" fluorescent probe Yaisho-Z/AM (Moiesciag Rgorez Eshgore VU, I eidep, Tpe MeiShiepapos3v) is used to determine changes in free intracellular Ca?" EC. Confluent BAES monolayers on filters are incubated for ЗОхв. at 372 with Yasho-3/AM (ZmkM in HB55 containing 296 ES and 10MM NEREZ), which is added to both the apical and basolateral surfaces. Monolayers are washed three times and incubated with fresh HBV5 in the dark for 20 min. at room temperature for complete hydrolysis of the ester-dye. Treated anti-CO is added to ES! 8 or anti-I-selectin tAb PMM and receptor cross-linking is stimulated with secondary tAbB as described above. In separate experiments, PMM secretion containing medium (see above) is used to stimulate ECs in the absence of PMM. Changes in Ca 27) ES in response to these stimuli 70 are measured through continuous registration of fluorescence intensity using a confocal imaging system with laser scanning (Ipzidni Riiv; Megidiap Ipzigitepiv Ips., OKetop, Mispidap).

To analyze the formation of β-actin in ECs in response to the activation of PMM, confluent monolayers of ECs, which were grown on covered Viotai glass, are incubated for 15 minutes. at 372C or from PMM cross-linked with CO18 antibody, or from PMM secretion containing a medium identical to that used for the above-described 75 procedures. Incubation of ES with IVA-treated RMMs serves as a control. Monolayers are fixed in 3.795 formaldehyde in RVZ for 1 hour. at room temperature, washed twice and permeabilized with 0.296 Top X-100 in RVZ5 (Ikhv. at 42С). The cells are washed twice and stained to detect the act of new elementary fibers

EITOS-conjugated phalloidin for 20 hours. at 37 2С. After three additional washes, endothelial cells are observed in a laser scanning confocal imaging system (Ipzidnpi Riiv; Megiaiap

Ipsigitepiv Ips., OKetop, Mispidap).

Ip past the experiments

Intravital microscopy of the microcirculation of the hamster's buccal sac was used to study the influence of the secretion products of the RMM on the microvascular permeability of the IP mimo. As described in detail above (Katsa 8. I iparot, 1994, Ittypoiodu oi (She Misgosigstiaiop, Asadetis Rgezz, 1994, ch. 7|, the left buccal sac of Syrian s 29 golden hamsters under anesthesia is turned out and prepared for microscopic observations under conditions of continuous Ge ) superfusion of bicarbonate buffer with maintenance of the physiological level of temperature, pH and gas tension.

EITS-conjugated dextran (Mol. mass: 150,000), introduced by intravenous injection (25O0mg/kg of body weight) is used as a plasma tracer, which provides visualization of changes in vascular permeability for macromolecules.

Microvessels in the open buccal sac are observed at low magnification in fluorescent light using a microscope and recorded on video. After the control recordings, the superfusion is stopped and a ml of the medium, which contains the RMM secretion caused by the crosslinking of CO18t, is applied locally to the buccal sac at 37 °C. In parallel experiments, treated anti-5O018 PMM medium, not subjected to cross-linking, was used. -

The results of

RMM (2x109), pretreated with anti-Z018 TAB (84), is added to EC/filter inclusions (luminal side of EC/upper compartment of the resistance chamber) and allowed to settle for 10 min. Cross-linking of CO18 with secondary antibody goat anti-mouse K(ab)" revealed a marked decrease in TEE, which is detected within 1 min., reaches its maximum (36-13 control) after 15 min. and remains at its level during the entire observation period (Fig. 1). Neither treatment of RMM with IV4 alone nor treatment with secondary tAbB without IV4 induced any 7 s changes in TEEK.

The effectiveness of CO18 cross-linking to increase the macromolecular flow and migration of PMM through the EC monolayer is investigated in additional experiments. PMM, pre-treated with a saturating concentration of IV4, is added to the EC/filter inclusions and allowed to settle for 1 hour. in an environment containing EVA.

Crosslinking of CO18 also occurs due to the effect of goat anti-mouse antibody E(ar)", and PMM/EVA containing filter -I inserts, which are transferred at equal intervals to new wells with fresh medium. In the absence of a secondary antibody, there is a slight constant gap of albumin, 0.08-40.03μclhv" (ser.-50, n-13), which did not change with time and exactly corresponded to the spontaneous range detected in filter inserts containing about untreated PMMs. Conversely, activation of PMMs by CO18 cross-linking resulted in an increase in the albumin gap that continued to increase over time (Fig. 1). The net increase in albumin gap increased from 0.5 -0.1 μl under (ee) I and o, time of the first 5-minute period to a total of 20.0-4.5 μl (ser.-50, n-10) per minute. In contrast to the increase in the albumin gap, CO18 crosslinking did not lead to appreciable adhesion of RMM to EC or transendothelial migration ( Fig. 1) Using the analysis of the activity of MRO in different compartments after cross-linking of CO18, it was found that 91 16905 (ser.--50, n-9) of the added PMM are restored in the medium collected from the upper compartment, while in ES/filters or in the environment of the lower compartment is significant

Gee! MRO activity was not detected. The data of direct microscopic observation of transparent filters, which are washed at the end of the experiment, confirmed the absence of PMMs attached to the EC monolayer. where endothelial cell monolayers are labeled with the Ca27-sensitive fluorophore Pio-3, and stimulus-induced changes in EC Ca") were observed by laser-scanning confocal microscopic imaging. 60 Addition of anti-CO18-treated PMMs to the EC monolayer did not induce any changes in ( Sa?) EC However, stitching

CO18 with secondary tABb showed a rapid increase in cytosolic free (Ca 7") EC, which peaked after about 100s and then slowly decreased. Cross-linking of the I-selectin antibody in an equivalent manner did not cause any change (Ca 211 EC, whereas the reaction of Ca 25. similar to that caused by CO18 crosslinking, 65 is observed after stimulation of untreated RMM YAMI R | cf. Sashciat eyi ai., 1998, Vy. 9. RVagtaso). 125:1109-1114). On the causal relationship between changes in intracellular activity Ca 7" and the functional response of EC to the activation of PMM are evidenced by experiments in which endothelial cells are pre-treated with VARTA AM calcium complexon. This treatment completely inhibited both the increase in free intracellular Ca?'EC and changes in TEEK in response to CO18 cross-linking.

In other experiments, ES monolayers are incubated with PMM, and then stained to detect actin elementary fibers with EITS-conjugated phalloidin. Confocal laser scanning microscopy imaging revealed that endothelial cells incubated with IV4-treated PMM in the absence of secondary antibody displayed multiple stress fibers and a thin band of actin along the edges of the cells in direct contact with the adjacent cell. On the other hand, endothelial cells incubated with PMM and cross-linked by CO18 showed a marked increase in the content of g-actin, as well as the number and density of stress fibers.

To examine whether factor(s) secreted by PMMs regulate the response that ultimately leads to increased EC permeability, PMMs (2x109) pretreated with anti-CO18 and IBA and washed to remove unbound antibody were incubated with goat anti-mouse antibody B(ab)" during ZOhv., and then subjected to light centrifugation. Addition of a cell-free pop-up layer to an EC monolayer showed a decrease in TEEK 75 and an increase in the albumin gap by the same amount at the same time interval as after crosslinking

СО8 in RMM added to EC. The pop-up layer also caused changes in ESIS?, and content and distribution

T-actin. In addition, local application of a floating layer containing PMM secretion to the buccal sac of a hamster i.p. mimo revealed an immediate outflow of plasma from postcapillary and small venules. Places of leakage appear after 1.5 minutes. after application, and the maximum reaction was detected after about 5 min. After washing the tissues with buffer, the leakage slowly decreased. This effect on the permeability function of microvessels disappeared after heat treatment of PMM-derived secretion, suggesting that the increase in permeability is due to a thermosensitive factor(s). Applying a pop-up layer of IV4-treated PMMs, not subjected to crosslinking, to the buccal bag did not lead to a noticeable leakage of plasma.

Discussion of p

Adhesion of polymorphocellular leukocytes to the endothelial layer and involvement in extravascular tissue during acute inflammation largely depends on the function of B-2 integrins. An increase in the permeability of blood vessels for macromolecules is stimulated in connection with this cellular reaction, which leads to plasma leakage and the formation of edema. The results of the above-described experiments indicate a causal relationship between the transmembrane (from outside to inside) signal from B2 integrins in activated PMMs and the ability of these cells to cause barrier M) EC dysfunction. It was found that antibody-induced crosslinking and formation of B2 integrin groups leads to an increase in EC permeability similar to that observed after stimulation of PMM with a chemoattractant. This cellular cross-linking occurs in the absence of physical binding of the PMM to the EC, which indicates that the coupling of the PMM and the structural connection itself do not directly transmit signals to the EC that mediate their permeability. Rather, ligand binding of the B2 integrin triggers intracellular PMM signaling cascades to trigger secretory events and the release of a chemical mediator that modulates PMM-dependent changes in EC permeability. Furthermore, the change in vascular permeability thus induced was demonstrated to be the consequence of an active tyrosine kinase-dependent EC response, including cytoskeletal reorganization and reversible intercellular gap formation, suggesting a nonlytic function in this regard of the PMM-derived factor(s). ).

For a more detailed study of the mechanisms by which activated PMMs can transmit signals to - with ECs, in this process, the interaction of PMMs/ECs and the binding-dependent participation of B" integrins through cross-linking of the CO11/2018 antibody are modeled. The possibility of cross-linking of B5 integrin antibodies with shunting of chemoattractant-induced cell activation demonstrates that the involvement and formation of B5 integrin receptor groups as such through intracellular signals leads to an increase in EC permeability. Since this reaction occurred independently of the binding of PMM to EC, the transmission of signals between PMM and EC cannot be explained by the interaction of СО11/2О018 with counterreceptors on the surface of EC, but rather should occur due to the release of a mediator molecule derived from PMM. This mechanism is also confirmed by the ability to cause EC hyperpermeability of the cell-free floating layer, which is achieved after cross-linking CO18 in the PMM suspension. o Example 2: Recognition of neutrophil-derived HBP (HBP) as a signaling link in o 20 neutrophil-induced vascular plasma leakage

In Example 1, PMM engagement via B" integrin is a prerequisite for PMM-induced effects on endothelial cell permeability and that Ca"-dependent transmembrane signaling through engagement of activated B0 integrin induces the secretion of soluble factor(s) that directly directly affect changes in the EC barrier function. 59 As described by us in Example 2, the soluble factor is defined as the human NVR. Example 2 also characterizes

GF) interaction of the NVR with the contact phase system.

Materials and methods o Reagents

Medium-199, KRMI-1640 (containing I-glutamine), fetal bovine serum (EB5), 60 trypsin-EOTA, phosphate buffered saline (PB5), and Hanks' balanced salt solution (HB55) are obtained from

Thesppoiodiese ((zaipegzrygo, MO, USA). Gelatin, collagenase, penicillin, streptomycin, bovine serum albumin (BSA), Evans blue dye, and RITO-conjugated phalloidin were obtained from Ziedt

Spetis! Co. (5. And otsivb, MO, PERSON). Risho-3/AM was obtained from Moscow, Russia (Endepe, OK, OA). Viotaigih received from Viotedis! Tesppoiodiev Ips. (Ziotsdpiop, MA, BA). JuOehigap 70 (Masgodeh) and RisoiI-Radce bo received from Rpagtasia Vioyesp AV (Orrzaia, Zm'edep). The main culture medium contains a 1:1 mixture of C-199 and KRMI-1640 with the addition of 2095 heat-inactivated EB5, penicillin (100U/ml) and streptomycin (100μg/ml). Monoclonal antibody IBA4 against the usual B-chain (СО18) of B»-integrins was provided by Og. Siaez I ipdrego, Rpagtasia 5 Oriopp (Orrzaiia, Zm'edep) with the kind permission of JuOg. Zatie! O. Uugdni,

KoskegeiPeg Opimegeiu. E(ar)" fragment of goat anti-mouse food was obtained from daskwop IttipokKezeagsi I arogafgiev,

Ips. (MUevi Sgome, RA).

Polyclonal antibodies to HVR are obtained according to the following procedure. New Zealand white rabbits received subcutaneous injections of 5 µg of human heparin-bound protein (HBP) emulsified in complete Freund's immunostimulant (ESA), followed by three booster injections of 10 µg of human HBP emulsified in 7/0 incomplete adjuvant Freund (RIA), every two weeks. Ten days after the last injection, blood is taken from the animals and the serum is separated.

Monoclonal antibodies to HVR are obtained according to the following procedure. CVE mice are immunized by subcutaneous injection of 200 μg of purified human HVR into the ECA, followed by two injections of 2 μg of human HVR into

ON. Mice with a high degree of reaction are given an enhanced intravenous injection of 20 mcg of NVR and after three days /5 the spleen is removed. Splenic cells are fused with the Roch myeloma cell line. The supernatants are selected for the production of antibodies against HVR by HVR-specific EHIZA analysis. Positive lines are selected for binding to the intracellular part of the macrophage line using standard GASZ5 methods.

Positive lines are cloned and retested in both assays. Monoclonal antibodies are purified by protein A-affinity chromatography.

Endothelial cells

Human umbilical vein endothelial cells (HOES) were isolated from fresh umbilical cords as described in

Uapye eyi a, 1973, 9. Sp. Ipmevi 52:2745-2756), and endothelial cells of the aorta of cattle (BAES) are isolated according to the method described | In the work of Vosuze ei aiYi., 1975, Tygot. Oh my Here you are! 24:825-839|, with some changes. That is, the lumen of the umbilical vein or aorta of cattle is incubated with 0.2596 s of collagenase solution for 1 hour. and detached endothelial cells are washed from PB5. Collected cells are pelleted and resuspended in a culture medium, seeded into tissue culture flasks and incubated under i) standard conditions. The medium is changed every 48 hours until the cultures reach confluency (3-5 days). In the experiments, NOMES or VAES of the first-fifth pass are used. The recognition and determination of the purity of ES is based on the morphology of the cobblestone and the characteristics according to the MI factor! L//t-antigen. Oh my Naettoggty 24:825-839). Staining for MISH/L/LLYA-factor in cells is homogeneous.

Seeding on permeable membranes

Cultured ECs are separated by a short (2 min.) treatment with trypsin-EOTA (0.2595 trypsin/0.0190 s

EOTA) and applied either to an inorganic Aporoge membrane with a pore size of 0.2 μm, or to polycarbonate filters with a pore size of Ж, Омм (tissue culture inclusion, уОмм; MOM, KozkKiide, Yueptagk). In order to promote cell differentiation and strengthen EC adhesion, filters are treated with 50 μl of Viotaih | (167μg/ml) and it is left to air dry. ECs are seeded at a density of 2 x 109 cells per filter and incubated in a culture medium at 372C in a humidified atmosphere of 596 СО" in air. ECs are grown to confluent monolayers, which can be detected by daily microscopic observations and measurements of the electrical resistance across the monolayer.

Measurement of transendothelial electrical resistance (TEE) - c To measure the electrical resistance across the EC monolayer, the filter inserts are transferred to the resistance measurement chamber (EMOONM-12; UUogpiy Rgesiviop Ipzigitepov, bZagavoya, RII, O5A), modified for the purpose of "" ensuring the exact location of the electrodes relative to to each other and filter inclusions during repeated measurements. The chamber (lower compartment) and filter inserts (upper compartment) are filled with 2 ml and 400 μl of culture medium, respectively. All measurements are performed at 37 C using - and equipment located in the cell culture incubator. Direct electrical resistance indicators - obtained from an ohmmeter (EMOM; UMogid Rhesiviop Ipvigitepiv, Zagazoi, RII, OBA) connected to the electrodes of the resistance chamber. The electrical resistance of individual EC monolayers is obtained by subtracting the resistance of the corresponding (95) filter covered with Viotaiikh, measured before seeding endothelial cells p. 50 Determination of albumin concentration ru

Albumin (EVA) conjugated with Evans blue dye is used as a marker of macromolecular permeability of EC monolayers. The dye in a final concentration of 0.b7mg/ml is mixed with a culture medium containing 495 B5A. In experiments in which the permeability to albumin is analyzed, the culture medium in the upper compartment is changed to a medium containing EVA. The concentration of EVA in the liquid samples of the upper and lower compartments is determined spectrophotometrically by measuring the optical density at about 620 nm (Tiepek Miyizkap MOSS; Riom/I aroghaigiez, ZoIpa, Zmedep). The albumin gap is calculated according to the ratio: M1-A2xM2x1/A1, where M1 is the volume of the gap (that is, the theoretical volume of the apical medium, ie) purified from albumin by diffusion with basolateral separation), M2 is the basolateral volume, and A1 and A? are the optical density of the apical and basolateral media, respectively. The base gap for 60 in the absence of any stimulus, on average 0.08 -0.01μl/min., is subtracted from the volume of the gap obtained in response to the corresponding specific stimulus.

Isolation and activation of RMM

Plasma rich in leukocytes, extracted from human whole blood by dextran precipitation, is carefully placed in layers on GrisoI-Radtse and centrifuged at 40000g for 30 minutes. Granules containing PMM are resuspended and washed twice (400 g 7 min.) in ice-cold RV5. PMM are resuspended in the culture medium at a final concentration of 2-5x109 cells/ml. The RMM purity is 29896 and the trypan blue displacement viability is 29590,

Antibody crosslinking of CO18 molecules on the surface of neutrophils is stimulated as described above. That is, cleaned

PMM (B5X105/ml) is incubated with anti-CO18 TAB IV4, 10 μg/ml in HB55 for 30 minutes. During the incubation period, the cells are subjected to a sharp change in temperature from 42 to 372 C to increase the surface expression of СО11/С018. Cells are washed twice and CO18 crosslinking is carried out by adding fragments (ab) of goat anti-mouse Id (1:20 dilution). PMM is precipitated by centrifugation at 300 g for 15 minutes. at room temperature and the cell-free supernatant is collected. In separate experiments, cross-linking of CO18 antibodies is stimulated 70 after addition to EC PMM monolayers pre-incubated with tAB IVA (see below).

Experimental procedures

Before transferring the filter inserts to the resistance measurement chamber (which is kept at 372C), the medium in the insert and in the chamber is replaced with fresh culture medium (3723) with the addition of 10mM Nerez. HVR (25-100μg/ml) or a secretory product of RMM is added to either the upper or lower compartment and changes in 72 transendothelial electrical resistance are measured every minute until the plateau phase is reached, and thereafter at 5-minute intervals. In separate experiments, RMM (2x109) pre-incubated with tAb IVA (1Okg/ml), added to the upper compartment (ratio RMM:ES-10:1) and left to settle for 1 Ohv.

The activation of RMM is stimulated through the crosslinking of leukocyte CO18 antibodies by adding goat anti-mouse ido K(ab)" diluted in a ratio of 1:20. In experiments investigating permeability to albumin across an EC monolayer, the media in the insert contained EVA. During the experiment, filter inserts are moved at 5-10 minute intervals to new wells containing fresh medium. Incubation in all cases is carried out at 372C. After the experiment, the medium in the lower wells was suctioned and analyzed for EVA content. rya Determination of intracellular Ca" EC and distribution of E-actin p

To determine changes in intracellular free Ca 2" EC, sensitive to Ca?" o) fluorescent probe Psio-3/"AM. Confluent NOMES or BAES monolayers cultured on covered Vioptaig I filters are incubated with RiIsho-Z3/AM solution (µm in HB55 containing 296 EC5 and 10 mm NEREZ), which is added to the apical and the basolateral surface, during incubation at 37 °C. Before use, the monolayers are washed three times and incubated with fresh NVZZ in a dark place for 20 min at room temperature for complete hydrolysis of the ester-dye to its calcium-sensitive free acid form. ECs stimulate NVR , which is added to the apical surface. Changes (Ca?) of ES in response to stimulation of the NVR are measured by continuous registration of fluorescence intensity using a confocal imaging system with laser scanning (Ipzidni Riv; Megiaiap Ipvigitepiv Ips., OKketop, Mispidap). -

To analyze the distribution of B-actin in ECs in response to HVR stimulation, confluent monolayers of ECs, which were grown on covered Viotaig glass, are incubated with HVR or the carrier itself at 372C for 15 minutes. Monolayers are washed twice in the culture medium and fixed in 3.790 formaldehyde in PB5 for 1 hour. at room temperature, ECs are permeabilized with 0.295 Tiyop X-100 in RVB5 (equivalent to 42С), washed twice and stained with RITO-conjugated phalloidin for detection of actin elementary fibers for 20 min. at 372C. Endothelial cells are again washed three times in RVZ and visualized using a confocal microscope with laser scanning. h Western blotting i A sample of purified HBP or PMM secretion is added to ZO5 sample buffer and boiled for 5 min.

Electrophoresis is performed by applying samples to 505 polyacrylamide gradient gels (3-1095). Proteins by electrophoresis are transferred to a nitrocellulose membrane (Vio-Kai Iabrogaigiez, Kispytopa, CA) for - immunoblot analysis. Additional binding sites are blocked by incubating the nitrocellulose membrane for -1 2 hours. in milk NVR is detected using Western blotting.

Results o Activated PMMs release HVR so 50 PMMs in suspension are activated due to cross-linking of CO18 antibodies. RMM is centrifuged, the supernatant is collected and applied to BO5-RACE. Western blotting with the use of antibodies directed against HVR allows to recognize a very noticeable band of 27KO in the secretion of RMM. No special bands were detected in the control rabbit's ido.

NVR increases the permeability of EC monolayers

Addition of HBP (75μg/ml) to the upper compartment caused a marked decrease in transendothelial resistance, which manifests itself within one minute, reaching its maximum (3095 -395 from control) after 15 minutes. and

GF! remains unchanged throughout the observation period (Figure 2). Testing of HVR immunity also caused an increase in the macromolecular permeability of the EC, which is reflected in a gradual increase in the albumin gap, which gives a net volume of the gap of 20.4-34 μl during the period of bOhv. (Fig. 2). The response of the EC to the stimulation of HBP is clearly dependent on the dose, which is confirmed by a not so clearly detected decrease in TEE in 60 steady state with HBP of 25 and 5 oOmkg/ml (Fig. 2). However, the rate of change at which the reaction occurs is the same for all doses of HVR.

Stimulation of EC HBP from the basolateral side of the monolayer (HBP was added to the lower compartment) caused the same changes in EC permeability as apical stimulation, but at slightly higher concentrations.

Neutralization of HBP attenuates PMMA4-induced increase in EC permeability because Purified rabbit antiserum against HBP is used to neutralize protein activity. Addition of HBP antibodies (5 Ωkg/ml) to the EC monolayer before HBP stimulation (75 μg/ml) markedly attenuated the induced changes in TEEK and macromolecular permeability. Changes in TEEK occurred with a certain delay and reached 7590-5965 from the control (Fig. 3), indicating an inhibition of the HVR-induced response by about 6595. Similarly, albumin efflux was inhibited by about 6095 to a net gap volume of 8.64 141 ,74 μl per box. (Fig. 3). Treatment with nonspecific rabbit IDS (5 Ωkg/ml) has no effect on the HVR-induced decrease in TEEK, which reaches 3195-5900 from the control in 15 min.

It is determined whether antibodies against HVR can also have an inhibitory effect on changes in barrier function

ES caused by activated PMMs. PMM in suspension is activated through crosslinking of CO18 leukocyte antibodies and 70 centrifugation. The floating layer, which contains PMM secretion products (see Materials and methods), is transferred to the EC monolayer. In the presence of anti-NVR antibodies, the decrease in TEEK and the increase in macromolecular permeability were markedly inhibited compared to the response to stimulation by PMM secretion in the absence of an anti-NVR antibody (Fig. 4). An almost identical effect is observed when cross-linking of the CO18 antibody is stimulated after the addition of PMM to the EC monolayer, and therefore, the achievement of the PMM secretion product of the EC monolayer.

Stimulation of NVR increases intracellular free Ca 7 EC

In order to determine whether EC responds to HBP stimulation by increasing cytosolic Ca 2", confocal microscopic visualization with laser scanning is used. Addition of HBP (75 μg/ml) to confluent NOMES or BAEC monolayers charged with the Ca 7"-sensitive fluorophore yШho-Z/AM, caused a rapid increase in the fluorescence intensity, which reached a peak during iv. and then gradually decreased to the baseline level. This mobilization of intracellular Ca 2" clearly indicates an active response of EC to stimulation

NVR

Stimulation of the NVR causes the reorganization of EC microfilaments

Confluent EC monolayers, incubated with HBP (75 μg/ml) or culture medium alone for 15 min, are stained with REITS-conjugated phalloidin to detect actin elementary fibers and visualized with laser scanning confocal microscopy. Compared with untreated ECs, which (U reveals low-density E-actin stress fibers and distinct compact peripheral bands along the cell edges, there are significant changes in E-actin content and distribution in stimulated NVR ECs. Increase in density

E-actin tension fibers, which tighten the crack and rupture of peripheral bands, indicates the reorganization of the cytoskeleton of the EC as a result of the stimulation of the NVR. at

NVR increases the permeability of blood vessels ip mimo (ee)

Local application of NVR (75μg/ml) to the microvessels of the buccal sac of a hamster in 2 minutes. leads to the formation of visible sites of leakage of the fluorescent label from both post-capillary and large venules. The maximum outflow is observed after 7-10 minutes. after checking immunity. After that, the outflow slowly decreases and completely disappears after ЗОхв.

Inhibition of bradykinin- and HVR-induced EC permeability increase by monoclonal antibody MVKZ - to bradykinin

Bradykinin (100OnM; Fig. 5a) or HVR (75μg/ml; Fig. 56b) is injected at the beginning of the time count from the luminal side of EC monolayers pre-incubated with a monoclonal antibody to bradykinin, MVKZ (4Omkg/ml) "

INaazetapp ei ai., 1991, 9. Ittipoi. 147:3882-3892). МВКЗ completely prevents the increase in EC permeability by 70 caused by bradykinin or HVR. The specificity of the effect of tAbB is confirmed by the lack of inhibition of histamine-induced changes in permeability (histamine was administered 15 minutes after stimulation with bradykinin or with NVR), M-6.

Inhibition of the NVR-induced increase in EC permeability by the RKNA monoclonal antibody to plasma kallikrein 395 NVR (75 μg/ml; shaded symbols) or bradykinin (100 nM; unshaded symbols) is injected at the beginning of the time countdown from the luminal side of EC monolayers pre-incubated with tAB RKNA (4 Ωg/ml) . The results are shown in Fig. b. RKH4 prevented the increase in EC permeability caused by HVR, but not caused by bradykinin. RKNA treatment did not affect the change in permeability caused by histamine (histamine is administered 15 min after stimulation of the NVR), M-6. (ee) 50 Inhibition of HBP-induced EC permeability increase by treatment with NKH2O-peptide sp Monolayers of EC are incubated with NKH2O (3 µg/ml) for 30 h, thereby removing surface-bound

H-kininogen. After washing, at the beginning of the time count, NVR (75 μg/ml) is injected from the luminal side of the EC, after which 15 min. bradykinin (T0ONM) is administered. NC is attached to the EC surface through domains 5 and 6, with domain 5 being the most important. The minimum amino acid sequence required for binding of human NK in 99 domain 5 to EC is represented by a peptide of 20 amino acids (NKH 20) with the following sequence:

GF) NKeKNONONNKKMKOKKMOKN (ZEO IO MO:7) (human H-kininogen 479-498). This peptide inhibits the attachment of NK to 7 EC (Nazap sei ai., 1995, y. Bioi Spec. 270:19256-19261 and Nepmuai sei ai., 1996, y. Bioi. Spect. 271:13040-13047) The results are shown in Fig.7. NKH2O treatment of the EC prevented the NVR-induced increase in EC permeability, but not the change in permeability caused by the subsequent administration of bradykinin, M-6. for Inhibition of HBP-induced increase in EC permeability by treatment with Z0OOZ31-peptide

ES monolayers are incubated with 5BOOYUZ1 (Zmkg/ml) for 30 minutes. 5HY0OZ1, also called NKZ1, is a peptide derived from domain 6 of human H-kininogen NK that blocks the binding of NK to plasma kallikrein and factor

XII (Tai ek ai., 1986, y. Vioi Spet. 261:15396-15401 and Mode! ei aiYi, 1990, y. Vioi Spet. 265:12494-12502). It has the sequence bo ZOOBMLROIYUTORMOI ZEMRIBOERROTTZRK (ZEO IYUO MO:8) (human H-kininogen 565-595). After washing, at the beginning of the time count, NVR (75 μg/ml) is injected from the luminal side of the EC, after which 15 min. bradykinin (10 ONM) is administered. The results are shown in Fig.8. ZOYUZ1-treatment of the EC allowed to significantly prevent the NVR-induced increase in EC permeability. EC response to direct bradykinin stimulation was maintained after ZHOYUZ1 treatment,

M-6.

Inhibition of HVR-induced EC permeability increase by aprotinin treatment

HVR (75 μg/ml) is injected at the beginning of the time count from the luminal side of EC monolayers pre-incubated with aprotinin (100 μg/ml; ZOkhv.). The results are shown in Fig.9. Treatment with aprotinin prevented to a large extent the NVR-induced increase in EC permeability, but not the change in permeability caused by the subsequent /o administration of bradykinin (TOONM), M-6.

The HVR mutant (0 175M) is unable to bind to aprotinin due to the mutation, but the three-dimensional structure and surface charge distribution remain unchanged. When tested in a leakage model, this mutant causes bradykinin release and increased EC permeability. However, the addition of aprotinin has no inhibitory effect. A similar result is obtained from pig NVR, which is highly homologous to human NVR, but /5 cannot bind to aprotinin due to the presence of K at the bottom of the degenerated catalytic cleft.

Example 3: Two mutants of heparin-bound protein

Two human NVR mutants, IK235,E25E|NVR and |(517591|NVR, were produced to investigate the structure-function relationship of residues in the possible binding site of AL RZ lipid and the binding site of VRTI (bovine pancreatic trypsin inhibitor (cattle) mutation 51750) does not change the structure of the protein, but the ability to bind to VRTI disappears. Its mutant mediates only a very limited stimulation of lipopolysaccharide (I RB)-induced cytokine release from human monocytes. The binding property of lipid AL P5 can be compared with this property of natural HVR. Mutation K235,E25E does not affect the binding of lipid AL P and

VRTI or release of lipopolysaccharide (I P5) release of cytokines from human monocytes.

Materials and methods of sch

Construction of a mutant protein

Rzho DNA polymerase was obtained from Voeghipdeg Mappeit, zatnNi, T4 DNA ligase was obtained from Mem Epdiap and)

Vioyar5, Zrodorieg Igidiregda cells (519), and pMI/1393 transfer plasmid were obtained from Ipmygodep, TC100, and

ЖЕ900-І produced at Momo Mogaizk A/5, bovine embryo serum (EC5) obtained from sirsovna All other chemicals are of the highest grade. yu zo IC2Z35,P25E)NVR mutant

ИК235,Р25Е)НВР mutant is constructed using the technology of ROK with overlap of INo, 5. M., Nypi, N. 0., Nogiop, co.

K. M., RyPep, 9. K., apa Rease, |! K. (1989) Sepe (Atvi.) 77: 51-59). Two mutated c fragments with partial overlap are obtained by PCR using oligonucleotides 1-2 and 3-6, and the resulting fragments are connected using oligonucleotide 1-6 (Table 1). cDNA of natural HNR of human food is used as a template for RSC-reaction. The resulting cDNA encodes 19 amino acid propeptides, 222 amino acid extended parts and three amino acid C-terminal extensions. WatH1t sites are included on each side of the mutated cDNA for cloning. The mutated fragment is inserted into pMmiI 1393 transfer plasmid (Ipmygodep). DNA for transfection is obtained using Oiadep tiadirger kits. After purification, the insert is sequenced. « shi mon BiKe ey wen - s Na Chen eimni CAN. shroud in VYN TIIIOVUVV Ж LV KU ЯОЕЯЯЯ d) ШО kavy ion na i nu і z

This mutant is generally constructed as described above. Two mutated fragments with a partial overlap are obtained by PCR using oligonucleotides 14 and 5-6 and the resulting fragments are connected using oligonucleotides 1146 (Table 1). ime) Expression and purification 8/9-insect cells are maintained in TS 100 medium supported by 1095 EC5. Before infection, the medium is changed to serum-free ZE9O00-II. The expression of both mutants in a medium without serum and the purification of mutants from the culture supernatant are carried out as described above for natural human HVR (Kaztyvzep, R.V., Viogp, 5., Kavigyr, 5., Mieivep, R. RG., Moitiv, K., Tit, Y., UMiregd, R. S., and Rioddaaga, N. (1996) REVZ I ecK 390:109-1121.

Analysis of I P5-induced release of II-6 from monocytes 65 Human monocytes are isolated and processed as described (Kaztizzep, R.V., Vishgp, 5., Kazighir, 5., MieiIzep, R.

E., Moitiv, K., Tit, Y., UMiregod, R. S, and Riosddaaga, N. (1996) REV5 ey. 390:109-112), except that 195 non-essential amino acids were excluded from the medium.

Analysis of ZN-I P5 binding to NVR

Affinity for lipopolysaccharide (LPO) is measured by scintillation affinity analysis (RA) as described (Ipde, M., Viogp, 5., Kavigir, 5. and Rioddaaga, N., 2000, Violations 28:218-2221) . All samples are presented in triplicate.

Analysis of binding of 722I-VRTI with NVR

Kinship according to VRTI is determined by applying RA analysis, almost as described for the analysis of I RB | ipae

M., Viotp, 5., Kavigir, 5. and Rioddaaga, N., 2000, VioFesppidnez 28:218-2221 with some modifications. ZN-I RU is replaced with 125|-VRTI (obtained by Momo MogaizkK A/5), and the buffer is replaced with РВ5 (20 mM phosphate, pH7.4 and 150 mM mass), including 0.0590 VBA and 0.0590 Ttyop X-100. The relative affinity of HBP and mutants is determined by carrying out competition studies with fixed, increasing concentrations of unlabeled BRTI.

The results

Biological Characterization 19 The biological activity of the two mutants is compared with natural HVR in terms of their ability to increase, in a dose-dependent manner, the I P5-induced release of 1II-6E from human monocytes and mice. | The re-affinity of two mutants and HVR is measured by scintillation proximity analysis (SPA) (I ipde, M., Vishgp, 5., Kavighir, 5. and

Eiododaaga, N., 2000, Vioiesppidtsev 28:218-2221. Both mutants bind to ZN-I RZ with the possibility of saturation with a similar visible Ku (approximately 0.7 g/ml), as can be observed for natural HVR (Fig. 11 a). In addition, in a similar scintillation affinity assay, affinity for BRTI is measured for both mutants and for natural HBP, with 722I-BRTI used as a ligand for HBP. (K235,E25E|NVR mutant, as well as natural

HVR binds to VRTI, while no binding is observed for (517502|HVR mutant (Fig. 116). Competition experiments, conducted by adding fixed, increasing concentrations of unlabeled VRTI, showed that 125|- VRTI binds to NVR and (K235,E25E|NVR) with similar apparent IC50 values of about 7 nm. As can be seen in Fig. 11c, (K235,B25E|ІНVR visually has a greater binding capacity compared to NVR. This the difference is possibly caused by the ability of (K235,E25E|HBP to bind to different forms of the applied iodinated BRTI. However, greater binding ability does not affect the affinity of 125|-BRTI. In addition, the ability of ZN-I RU to displace 122I -VRTI bound to the natural NVR of Imise megva was investigated by competition experiments: competition for binding to 722I-VRTI by adding to bmkg/ml of unlabeled PO and competition for ZN-Y Rb-binding by adding to 50MM of unlabeled VRTI No inhibitor was observed in either situation influence, this indicates that the binding sites of I RZ5 and VRTI are located in different parts of the NVR molecule. Cha

Data from the analysis of the biological activity of ip mio show that (51750О|НВР, in contrast to |(К2З35,Е25ЕЙ|НВР and

From the natural NVR, it mediates only a very limited stimulation of lipopolysaccharide (IRB)-induced cytokine release from human monocytes (Fig. 10).

The scope of the invention described and formulated by the authors is not limited to the specific embodiments described, as these embodiments illustrate several aspects of the invention. Any equivalent embodiments may be included within the scope of this invention. Therefore, various modifications of the invention, i.e. additional to those shown and described by the authors, become obvious to specialists based on the above description. Such changes are also covered by the scope of the attached claims. from" The links we provide are incorporated by reference in their entirety.

Claims (18)

45 Formula of the invention -I - 1. Use of a mammalian anti-HVR antibody for the preparation of a medicament for the prevention or treatment of a disease resulting from the release of bradykinin in a mammal, wherein said mammal produces an HVR that binds to a mammalian anti-HVR antibody, wherein the disease is selected from group, which at 20 includes systemic inflammatory response syndrome, ischemic reperfusion, anaphylaxis and allograft rejection. sl 2. Application according to claim 1, in which the amount of anti-HVR antibody administered to the mammal is 0.1-100 mg/kg of body weight. C. The use according to claim 1, in which the amount of anti-HVR antibody administered to the mammal is 0.5-50 mg/kg of body weight. Gee! 4. Application according to claim 1, in which the amount of anti-HVR antibody administered to the mammal is 1-25 mg/kg of body weight. co 5. Application according to claim 1, in which the antibody is a monoclonal antibody, preferably a human monoclonal antibody. 60 6. The application according to claim 5, in which the mammal is a human and the NVR is a human NVR. 7. A method for determining a monoclonal antibody that binds to at least one epitope on natural HBP, in which (a) culturing endothelial cells in the presence of HBP and in the presence and absence of a monoclonal antibody that is believed to bind to at least one epitope on HBP, and (b) reveal any effect of the substance on endothelial cell permeability, with a decrease in endothelial cell permeability compared with cell permeability when incubated in the presence of HBP without the substance, indicating that the monoclonal antibody binds to at least by one epitope on NVR. 8. The method according to claim 7, in which the natural NVR is a human NVR. 9. The method according to claim 7, in which the release of bradykinin is determined by immunological analysis. 10. A test kit that includes (a) an HBP antibody, (b) an HBP or a cell that produces HBP, and (c) a substance, tissue, cell, or component that interacts with the HBP. 11. Test kit according to claim 10, in which the NVR is labeled. 12. The test kit according to claim 10, wherein the test kit includes a substance that interacts with the HBP. 13. Test kit according to claim 12, in which the indicated substance is H-kininogen. 14. Test kit according to claim 12, in which the specified substance is labeled. 70 15. Test kit according to claim 12, which additionally includes granules. 16. A method for determining the production by a mammalian cell or tissue of HBP that binds to a monoclonal antibody that binds to at least one epitope on natural HBP, in which (a) the cell or tissue is cultured with endothelial cells in the presence or absence of the antibody, and (b) detect any effect of the antibody on the permeability of endothelial cells, whereby a decrease in permeability indicates that HBP binds to the antibody. 17. The method according to claim 16, in which the mammal is a human. 18. The method according to claim 16, in which the natural NVR is a human NVR. s schi 6) IF) (ee) (ze) cha i - - with . and? -I -I (95) (ee) sl ime) 60 b5