RU2528249C2 - Method for angiogenesis inhibition with recombinant forms of urokinase - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to molecular medicine, and can be used in medical practice for treating pathologies related to excessive angiogenesis, including malignant tumour growth, rheumatoid arthritis, psoriasis and diabetic retinopathy. The method involves administering an effective amount of proteolytically inactive recombinant forms of a urokinase-type plasminogen activator (urokinase) into excessive angiogenesis in a combination with a recombinant kringle domain of the urokinase-type plasminogen activator inhibiting the proteolytic action of the native urokinase-type plasminogen activator competitively and disconnecting receptor proteins spatially mediating regulatory effects of the urokinase system.

EFFECT: method enables inhibiting the vascular growth locally.

4 cl, 9 dwg, 5 ex

Description

The invention relates to the field of biotechnology, in particular to genetic engineering, and can be used in medicine for the treatment of pathologies associated with excessive angiogenesis, including the growth of malignant tumors, rheumatoid arthritis, psoriasis and diabetic retinopathy. A method is proposed that locally suppresses the growth of blood vessels by introducing in the field of excessive angiogenesis an effective amount of proteolytically inactive recombinant forms of the urokinase type plasminogen activator (uPA) that competitively suppress the proteolytic effect of the native urokinase type plasminogen activator and spatially uncoupling receptor proteins involved in its implementation .

Introduction

In the process of initiation of angiogenesis, the necessary moment is the destabilization of existing vessels, including the destruction of intercellular contacts of endothelial cells, the degradation of proteins of the basement membrane and extracellular matrix, the subsequent migration of endothelial cells and the formation of new vessels. Urokinase plays a key role in the initial stages of angiogenesis, providing both pericellular proteolysis and stimulation of vascular wall cell migration. Urokinase and the plasmin activated by it are directly involved in the degradation of basement membrane proteins such as fibronectin and laminin, and also cause an increase in expression and subsequent activation of matrix metalloproteinases. Urokinase stimulates the migration of vascular wall cells, as well as the proteolytic activation of growth factors such as VEGF and HGF, which stimulate the proliferation and migration of endothelial cells [1-5]. Thus, an increase in the expression of urokinase in endothelial cells increases their invasive potential. Urokinase is involved in the induction of angiogenesis in the retina under the action of HGF [6], as well as in stimulating the growth of blood vessels in the ischemic myocardium caused by the action of VEGF [7].

On the contrary, blocking the interaction of urokinase with its receptor suppresses its angiogenic effects. In nature, this can occur due to the formation of the so-called “binding peptide”, which is formed as a result of proteolytic cleavage of urokinase with plasmin or metalloproteinases at the Lys135-Lys136 or Glu143-Leu144 positions. The “binding peptide” blocks the interaction of urokinase with its receptor, which causes inhibition of endothelial cell migration and in vivo angiogenesis stimulated by angiogenic growth factors [8]. The same peptide is able to block the invasive properties of tumors and slow down their growth and metastasis due to the suppression of angiogenesis [9].

In vitro, it was shown that the addition of antibodies blocking the urokinase receptor to the culture medium inhibits the formation of capillary-like structures [10, 11]. Disruption of the uPA-uPAR complex inhibits the migration and proliferation of retinal endothelial cells that have been induced by HGF. Moreover, the urokinase gene knockout is manifested in a decrease in blood vessel growth in the ischemic hind limbs of mice [12], significantly worse myocardial recovery after a heart attack, and also a decrease in the effect in response to VEGF treatment.

Thus, based on a theoretical analysis of the issue, we can conclude that the process of angiogenesis can be suppressed by acting on the proteolytic activity of urokinase, as well as on its interaction with the receptor.

State of the art

According to literature analysis, the first evidence confirming the possibility of suppressing tumor progression by inhibiting urokinase activity was obtained using antibodies to urokinase [13, 14]. Subsequently, efforts in this area were directed to the development of low molecular weight synthetic uPA inhibitors with high efficacy, selectivity and pharmacokinetic properties, allowing them to be used as drugs for antitumor therapy. The design of trypsin-like enzyme inhibitors was based on a structural analysis and was based on the trypsin active center, the binding specificity of which is largely determined by the Asp 189 residue, located at the base of the primary anchor site for the substrates, called the specific pocket S1 [15]. The crystal structure of the uPA catalytic domain has a trypsin-like topology in which Asp189 is preserved, giving the S1 region affinity for positively charged Arg and Lys residues [16]. Therefore, most synthetic uPA inhibitors created to date have a common structural feature, which is contained in the mono- or biaromatic part, which has the function of amidine or guanidine and acts as an arginine mimetic. However, there is a strict restriction in the choice of components, due to the need to inhibit urokinase, without affecting the activity of other trypsin-like serine proteases, and especially tPA and plasmin, key enzymes of fibrinolysis.

In the earliest studies, parasubstituted benzamidine derivatives, as well as 4-chloro- and 4-trifluoromethylphenylguanidines and amiloride were considered selective urokinase inhibitors, but none of them was highly effective, since all of them had Ki within the micromolar range [17]. When the amiloride structure was taken as the starting point, the search for more potent inhibitors led to the synthesis of two benzo [b] thiophene-2-carboxamidines with 4 substitutions, B428 and B623 with Ki 0.53 and 0.16 microM, respectively [18]. The antitumor properties of B428 and B623 were tested in several studies that demonstrated the ability of these inhibitors to suppress urokinase-mediated processes in cell culture. In vivo, the mentioned inhibitors exhibited antiproliferative and anti-invasive effects when tested on tumor xenoplant models [19-22]. A study was conducted for the B428 inhibitor to determine the structural determinants of its affinity and selectivity for the urokinase molecule and other trypsin-like serine proteases [23, 24]. Interestingly, despite the crystallographic data indicating a high similarity of S1 to the uPA and tPA sites, significant specificity to the uPA molecule can be achieved through the interaction of inhibitors already at the S1 site alone. In fact, position 190 in tPA thrombin and factor Xa is occupied by alanine, while in uPA and trypsin it is occupied by serine; it is the latter that is able to create an additional hydrogen bond with amidine inhibitors, giving them a higher degree of affinity for the uPA molecule compared to the tPA molecule. Other elements of the amino acid sequence in the S1 region and in the flank regions introduce additional structural differences that more and more differentiate the S1 regions of trypsin-like serine proteases, giving a unique structure to each of the enzymes, and thus provide the opportunity to create new specific inhibitors. In addition, the accuracy of the interaction of uPA with inhibitors depends on small pockets and / or subsites of the enzyme, which can be used as additional targets for optimizing the structure of designed inhibitors. This is exactly the case for S1 beta and S1 ′ subsites, which were used to construct 8- and 6-substituted 2-naphthamidines due to the availability of the 8th and 6th positions of 2-naphthamidine, respectively [25-27]. In the past few years, many new low molecular weight urokinase inhibitors have been proposed, including derivatives of 4-aminoarylguanidine and 4-aminobenzamidine, 2-pyridinylguanidines, 1-isoquinolinylguanidines and 1- (7-sulfonamidoisoquinolinyl) -guanidino derivatives of 4-methoxymethyl 3-alkoxysocoumarins, 4-oxazolidinone analone UK122, 5-thiomethylthiophenamidine derivatives, N-1-adamantyl) -N ′ - (4-guanidinobenzyl) urea (WX-293T) and WX-UK1 (3-amidinophenylalanine derivative) [27] ]. However, among these inhibitors, only WX-UK1 (WILEX, Munich, Germany) reached the stage of clinical trials. WX-UK1, having a Ki to human urokinase of 0.6 microM, significantly reduced the invasion and spread of cancer cells, as well as the growth of the primary tumor in in vivo tumorigenesis models [36–38]. After these preclinical results, in the first phase of the studies, the maximum permissible doses were determined for the administration of WX-UK1: alone or in combination with capecitabine (XelodaTM), a 5-fluorouracil cytostatic drug for oral administration. The results of the study showed that WX-UK1 (alone) is safe and non-toxic in the range of doses tested. In addition, when WX-UK1 was administered in combination with capecitabine, no significant changes in the frequency or intensity of side effects typical of capecitabine were observed. This phase I trial included 25 patients with progressive solid tumors. As a result of treatment, the course of the disease stabilized in several of them, and a partial response was observed in three patients (two of which had metastatic breast cancer). These results are especially noteworthy because in these three patients, at the time of inclusion in the study, the disease had a progressive course. Since WX-UK1 was not absorbed in the gastrointestinal tract, Wilex has developed an oral drug, WX-671, for the systemic delivery of active WX-UK1. Following oral administration, WX-671 is cleaved to an active inhibitor of WX-UK1 by constitutive intracellular reductases. The effectiveness of this drug in combination with classic cytostatics is currently being evaluated in phase II of two clinical trials. One study included patients with locally advanced non-metastatic inoperable pancreatic cancer. The time of onset of the first metastasis and survival without disease progression were the main endpoints. Patients are treated with the cytostatic Gemcitabine: only him or in combination with WX-671. The second study is a double-blind, multicenter clinical trial of phase II, which included patients with metastatic breast cancer. This study will evaluate the efficacy of combination therapy with WX-671 and capecitabine compared with capecitabine monotherapy.

Another class of selective urokinase inhibitors is represented by peptide molecules identified using phage libraries. Ecotin, a dimeric broad-specific serine proteinase inhibitor found in the periplasm of E. coli, was used to construct phage libraries expressing ecotin variants, and those with high affinity for urokinase were selected; then, using a mixed combined design of the ecotin structure and screening of phage libraries of ecotin, new peptide derivatives of ecotin with higher activity and selectivity for uPA were obtained [39–41]. In a similar study using random phage peptide sequences and urokinase as a target, the disulfide-limited sequence CSWRGLENHRMC was isolated. This peptide, called upain-1 (upain-1), binds to both the active site and the surface loops of uPA and has a high specificity for inhibiting the urokinase molecule [42]. Moreover, peptidomimetic inhibitors were created based on minimal (from di- to tetrapeptide) sequences. At the binding site, they contain arginine, benzamidine, or phenylguanidine groups and form a short antiparallel beta structure with an enzyme segment from Ser214 to Gly216, which reproduces the method of binding PAIs inhibitors [43–46]. One of these inhibitors, benzylsulfonyl-D-Ser-Ser-amidinobenzialamide (with Ki 20 nm), showed significant antimetastatic activity and increased survival in the xenoplant model of fibrosarcoma [46].

Various natural and synthetic compounds are also known that have the ability to inhibit the formation of new vessels. Their action is usually directed either to inhibition of angiogenic growth factors (antibodies against VEGF, soluble forms of VEGF and FGF receptors), or to suppress the sensitivity of endothelial cells to growth factors (suramin, pentosan), or to block the conduct of intracellular signals or to suppress proliferation and migration endothelial cells. However, the range of these compounds is very limited today.

To date, most of the created uPA inhibitors act by a competitive and reversible mechanism; however, irreversible diphenylphosphonate-based inhibitors have recently been created. The diphenylphosphonate group reacts with the serine residue of the active center and forms a stable phosphonylated and irreversibly inhibited enzyme. Such a group was included in the structure of arginyl tripeptide or in aromatic analogues of arginine, in addition, additional structural modifications were made to optimize the inhibitory properties [47; 48].

Despite the large number of drug candidates aimed at inhibiting urokinase and its receptors, so far none of the compounds have successfully overcome preclinical and clinical trials. Mentioned candidates exhibit either insufficient selectivity or unacceptable toxicity.

In this regard, we attempted to develop a method for suppressing angiogenesis, devoid of the disadvantages characteristic of the use of known inhibitor compounds of the pro-angiogenic action of the urokinase-type plasminogen activator system.

Disclosure of invention

In developing the present invention, the following facts have been taken into account.

The first one. To suppress vascular growth, it is necessary to suppress cell migration and proliferation, as well as the formation of capillaries. Urokinase stimulates cell migration, proliferation, and capillary formation.

The second one. Urokinase stimulates vascular growth more effectively than the generally recognized stimulator of angiogenesis, vascular endothelial growth factor, which has a wider spectrum of action than previously thought and, therefore, undesirable side effects when used to stimulate the growth of blood vessels.

The third. In addition to stimulating endothelial cells, extracellular matrix remodeling is required for the formation of blood vessels. Urokinase, by activating plasmin and matrix metalloproteinases, also provides the necessary remodeling of the matrix.

Fourth. Different domains of urokinase mediate its different pro-angiogenic effects. The condition for the realization of the pro-angiogenic effects of urokinase is jointly localized activity of the urokinase domains.

With this in mind, the solution of the task involved:

a) the creation of genetic engineering constructs, including modified gene sequences of urokinase (uPA); production and purification of recombinant proteins based on the structure of urokinase;

b) the development of in vitro and in vivo models to assess the effect of recombinant proteins based on urokinase on the processes of migration, the formation of capillaries and vascular growth;

c) selection of optimal concentrations and time for recording the effects of recombinant proteins based on the urokinase structure to identify their influence on the process of angiogenesis;

d) an assessment of the efficiency of suppression by recombinant proteins based on the urokinase structure of endothelial cell migration, capillary formation and vascular growth in in vitro and in vivo models.

When carrying out the invention, recombinant plasmids were used for the first time containing cDNA sequences of recombinant proteins based on the urokinase structure (amino acid sequence of proteolytically inactive urokinase and the amino acid sequence of a recombinant protein based on the kringle domain of urokinase.

Recombinant forms of urokinase were obtained: full-sized proteolytically inactive urokinase and the kringle domain of urokinase. Using antibodies to native fragments of the urokinase structure and the recombinant urokinase receptor (uPAR), it was confirmed that the structure of the recombinant forms of urokinase and their binding to receptors is identical to the structure and binding to the native urokinase receptor (Example 1).

Based on the data obtained in further experiments to study the ability of recombinant forms of urokinase to suppress excessive angiogenesis, we used recombinant forms of urokinase, similar in structure to the native urokinase molecule.

In adequate in vitro and in vivo animal models, experimental evidence has been obtained that recombinant urokinase molecules are able to significantly inhibit endothelial cell migration, microtubule formation (an analogue of in vitro capillaries), and the growth of medium-sized capillaries and blood vessels in a Matrigel ™ implant. Migration of endotheliocytes was suppressed to a greater extent by the kringle domain of urokinase (Example 3), while full-sized proteolytically inactive urokinase suppressed the formation of microtubules in vitro and the growth of medium-sized capillaries and blood vessels in an in vivo tumor angiogenesis model (examples 4 and 5) .

For the first time, in order to suppress excessive angiogenesis, recombinant forms of urokinase were used: full-sized proteolytically inactive urokinase and the kringle domain of urokinase. It was found that the combined use of the kringle domain and proteolytically inactive urokinase more effectively inhibits the growth of medium-sized capillaries and blood vessels in the Matrigel ™ implant than using only one of them.

Thus, as a result of the creation of the present invention, a method for suppressing unwanted (excessive) angiogenesis, the essence of which is the local use of therapeutically effective amounts:

(a) from 50 nM to 0.5 μM recombinant full-length proteolytically inactive urokinase molecule with the amino acid sequence of SEQ ID NO: 1;

(b) from 50 nM to 0.5 μM recombinant kringle domain of urokinase molecule with the amino acid sequence of SEQ ID NO: 2;

(c) an equimolar (1: 1) combination of recombinant forms of urokinase, characterized in (a) and (b).

The main technical result is to increase the efficiency of the method: when using in vitro and in vivo models, it was possible to significantly reduce the degree of migration of endotheliocytes, reduce the formation of microtubules in vitro, as well as reduce the number of forming capillaries and vessels of medium caliber in comparison with control experiments in which an equivalent amount of wild-type full-length urokinase was used. An additional result of this method should be considered the rejection of the use to suppress excessive angiogenesis of unsafe synthetic serine protease inhibitors.

Brief Description of the Drawings

Figure 1. Electrophoresis of protein preparations in SDS - 14% PAAG. The gel was stained with Coomassie R-250 solution. 1-3 - in non-denaturing conditions: 1. kringle domain; 2. inactive urokinase; 3. full-sized urokinase; 4-6 - in denaturing conditions: 4. full-sized urokinase; 5. inactive urokinase; 6. kringle domain.

Figure 2. Immunochemiluminescent staining of the obtained preparations of recombinant forms of urokinase, separated by electrophoresis in SDS page under non-denaturing conditions. 1 - full-sized urokinase; 2 - proteolytically inactive urokinase; 3 - kringle domain; 4 - full-sized urokinase; 5 - proteolytically inactive urokinase; 6 - kringle domain.

Figure 3. Proteolytic activity of recombinant forms of urokinase. Proteolytic activity was evaluated using pyro-Glu-Gly-Arg-pNA synthetic peptide (S-2444, Sigma) as a substrate.

Figure 4. The binding of recombinant forms of urokinase with a specific urokinase receptor.

Figure 5. Migration of endothelial cells in the presence of recombinant proteins based on urokinase.

Figure 6. The formation of capillary-like structures in the presence of recombinant forms of urokinase. A) The figure on the left shows the formed capillary-like structures stimulated by: A. 5% fetal bovine serum; B. 15% fetal bovine serum; B. VEGF 1 ng / ml; G. VEGF 2 ng / ml; D. VEGF 5 ng / ml; E. VEGF 2 ng / ml in the presence of 150 nM kringle domain; G. VEGF 2 ng / ml in the presence of 150 nM proteolytically inactive urokinase; Z. VEGF 2 ng / ml in the presence of 150 nM native form of urokinase.

B) The figure shows the formed capillary-like structures stimulated by: A. 5% fetal bovine serum; B. 15% fetal bovine serum; B. bFGF 10 ng / ml in the presence of 150 nM kringle domain; G. bFGF 10 ng / ml in the presence of 150 nM proteolytically inactive urokinase; DE. bFGF 10 ng / ml in the presence of 150 nM full-length proteolytically active urokinase.

C) Formation of capillary-like structures by endothelial cells in the presence of 2 ng / ml vascular endothelial growth factor and 150 nM urokinase and recombinant proteins based on urokinase.

D) The formation of capillary-like structures by endothelial cells in the presence of 10 ng / ml of the main fibroblast growth factor and 150 nM urokinase and recombinant proteins based on urokinase.

Figure 7

A) Assessment of vascular formation in Matrigel ™ implants, in vivo model. The nuclei are colored blue, the vessels forming are red: A - without additional stimuli; B - vascular endothelial growth factor, 20 ng / ml; B - vascular endothelial growth factor, 50 ng / ml; G - the main growth factor of fibroblasts, 10 ng / ml; D - the main growth factor of fibroblasts, 20 ng / ml; E - vascular endothelial growth factor, 20 ng / ml + proteolite. inact. urokinase, 50 nM; W - vascular endothelial growth factor, 20 ng / ml + kringle-domain of urokinase, 50 nM; Z - the main growth factor for fibroblasts, 10 ng / ml + proteolite. inact. urokinase, 50 nM; And - the main fibroblast growth factor, 10 ng / ml + kringle domain of urokinase, 50 nM; K - vascular endothelial growth factor, 20 ng / ml + 50 nM proteolite. inact. urokinase and kringle domain; L is the main growth factor for fibroblasts, 10 ng / ml + 50 nM proteolite. inact. urokinase and kringle domain; M - 50 nM proteolite. inact. urokinase; H - 50 nM kringle domain of urokinase.

B) Quantitative processing of immunohistochemical analysis data. The number of vessels formed is normalized to the number of vessels formed in Matrigel ™ without the use of additional stimuli (column 1).

Stimuli and recombinant forms of urokinase were introduced into the subcutaneous Matrigel ™: 1 - without additional stimuli; 2 - vascular endothelial growth factor, 20 ng / ml; 3 - vascular endothelial growth factor, 50 ng / ml; 4 - the main fibroblast growth factor, 10 ng / ml; 5 - the main fibroblast growth factor, 20 ng / ml; 6 - vascular endothelial growth factor, 20 ng / ml + proteolite. inact. urokinase, 50 nM; 7 - vascular endothelial growth factor, 20 ng / ml + kringle domain of urokinase, 50 nM; 8 - the main growth factor for fibroblasts, 10 ng / ml + proteolite. inact. urokinase, 50 nM; 9 - the main fibroblast growth factor, 10 ng / ml + kringle domain of urokinase, 50 nM; 10 - vascular endothelial growth factor, 20 ng / ml + 50 nM proteolite. inact. urokinase and kringle domain; 11 - the main growth factor for fibroblasts, 10 ng / ml + 50 nM proteolite. inact. urokinase and kringle domain; 12-50 nM proteolite. inact. urokinase; 13-50 nM kringle domain of urokinase.

SEQ ID NO: 1

- триада аминокислот активного центра; жирным строчным - аминокислоты, отличные от опубликованной последовательности изоформы 1 (Swissprot Р00749).Fonts: bold uppercase (II) - the beginning of the catalytic domain; bold capitalized italics (PRF) - plasmin site; bold uppercase with underline (

- triad of amino acids of the active center; in bold lower case — amino acids other than the published isoform 1 sequence (Swissprot P00749).

SEQ ID NO: 2

Fonts: bold capital italics (PRF) - plasmin site; in bold lower case — amino acids other than the published isoform 1 sequence (Swissprot P00749).

The implementation of the invention

In the implementation of the invention, in addition to the methods described in detail in the following examples, standard genetic engineering technologies were used, as well as the previously published methods for culturing endothelial cells and models for studying angiogenesis in vitro and in vivo.

Example 1. Obtaining recombinant forms of urokinase

Recombinant forms of urokinase were obtained using recombinant DNA technology. For the production of recombinant forms of urokinase, producer strains were used that were created by the genetic engineering laboratory of the Research Institute of Experimental Cardiology of the Republic of Kazakhstan NPK MSZ RF (strain SC2404 to obtain proteolytically inactive urokinase; strain SC1923 to obtain the kringle domain).

Growth of producer strains

Expressing recombinant forms of urokinase cells were cultured using LB medium [Bertani, G., 1952] containing: 1% peptone, 0.5% yeast extract and 1% NaCl. The medium was autoclaved in a steam autoclave at a temperature of 121 ° C for 60 minutes. A standard commercially available LB medium supplied in the form of a dry powder was used and prepared according to the manufacturer's recommendations [Sigma Aldrich L-3022].

To produce recombinant proteins, bacterial producer strains based on E. coli cells of the BL21 line (DE3) were used (strain SC2404 to obtain proteolytically inactive urokinase; strain SC1923 to obtain the kringle domain). After thawing samples of the indicated producer strains, small cultures were obtained that were grown overnight at 37 ° C with intensive aeration (260 rpm). The main biomass was grown in a volume of at least 2 L for 12 hours at 37 ° C and intensive aeration (260 rpm).

Cell wall destruction

To isolate the proteins expressed in these producer strains, we used the enzymatic treatment of the bacterial cell walls with lysozyme, as well as the ultrasound treatment of the cells. Bacterial cells were treated with lysozyme for 30 minutes at + 4 ° C, and sonicated 3-4 times (40 seconds) at a power of 80-100 W while cooling in an ice bath. The resulting solution was centrifuged for 10 min at 20,000 g at a temperature of + 4 ° C.

Isolation and purification of proteolytically inactive urokinase and kringle domain from refolding mixture

Recombinant forms of urokinase were produced in the cells of the producer strain in the form of an insoluble fraction - inclusion bodies requiring three times washing with 50 mM Tris / HCl buffer, pH = 7.5, containing 1 M NaCl, and subsequent three washing with 50 mM Tris / HCl buffer, pH = 7.5 . The washed precipitate was dissolved in 6 M guanidine / HCl pH 9.0 containing 10 mM β-mercaptoethanol (50 ml of solution per 0.5 L of cell culture); if necessary, the solution was heated to 50-60 ° C. To the resulting solution, a 10-fold volume of 1 M guanidine / HCl pH 9.0 containing Red / Ox pair of 1 mM glutathione reduced / 0.1 mM glutathione oxidized was slowly added with stirring. The resulting protein solution was left for refolding for 14 hours at a temperature of + 4 ° C. The refolding mixture was centrifuged for 10 min at 20,000 g. The pH of the protein solution was adjusted with 1 M Tris-HCl pH = 2.5 to pH 7.4.

For purification of proteolytically inactive urokinase and kringle domain, two-stage chromatographic purification was used. The first step was purification using metal chelate affinity chromatography. The second stage of purification was immunoaffinity chromatography with antibodies immobilized on a sorbent.

Samples of the obtained recombinant proteins were separated by SDS electrophoresis in 14% SDS page according to the Laemmli method. The gel was stained with Coomassie R-250 solution. The gel was photographed on a transilluminator using a digital camera. As a result, it was found that the molecular weight of the obtained proteins corresponds to the proteolytically inactive urokinase and the kringle domain of urokinase (Fig. 1).

Example 2. Properties of recombinant forms of urokinase

The analysis of the functional characteristics of proteolytically inactive urokinase and the kringle domain obtained in Example 1 was carried out using immunoblotting and enzyme immunoassay.

The conformity of purified proteins

To determine the nativeness of the conformation of recombinant proteins, antibodies were used that recognized antigenic determinants formed by amino acid residues of the protein.

Proteins were separated by SDS-PAGE, and then transferred from the gel to the PVDF membrane, where they were stained using specific monoclonal antibodies against significant determinants of native human urokinase, at a concentration of 0.2 μg / ml (UNG - to the kringle domain , UIG - to the proteolytic domain). Goat antibodies to mouse immunoglobulins conjugated to AffiniPure horseradish peroxidase (H + L) at a 1: 2000 dilution were used as secondary antibodies. For protein detection, a membrane with a chemiluminescent substrate was incubated. Luminescent signals were recorded using a sensitive digital video camera. Proteins were separated by SDS electrophoresis in 14% PAG and electro-transfer to the membrane was performed.

Native urokinase and the resulting recombinant proteins bound UNG antibodies (lanes 1-3 in FIG. 2). Native urokinase and the resulting proteolytically inactive urokinase also bound UIG antibodies in contrast to the kringle domain (compare lanes 5 and 6 in FIG. 2). The binding of specific antibodies indicates the preservation of significant determinants in the structure of proteins, and hence the native conformation of the resulting recombinant proteins.

Proteolytic activity of recombinant proteins based on the urokinase structure

Urokinase activity was determined by proteolytic activity using pyro-Glu-Gly-Arg-pNA synthetic peptide (S-2444, Sigma) as a substrate. The enzymatic activity of urokinase in this approach is determined by comparing the activity of the studied urokinase preparation with the activity of the standard attached to the kit.

For this, the recombinant urokinase protein preparation at a concentration of 2 μM was preincubated with plasmin (final concentration 0.005 mg / ml) for 1 hour at 37 ° C in PBS buffer containing 0.1% BSA and 0.05% Tween-20. To determine the proportion of the double-stranded form of urokinase contained in the preparation, part of the sample was not activated with plasmin. A plasmin-activated sample was diluted to a final urokinase concentration of 5-30 nM with PBS / 0.1% BSA / 0.05% Tween-20 buffer containing a plasmin inhibitor aprotinin at a concentration of 100 kallikrein units / ml (940 μl buffer + 10 μl sample). An inactive sample was diluted with the same buffer to a final urokinase concentration of 50-300 nM. To the samples, the volume of which was 950 μl each, 50 μl of a solution of the specific substrate of urokinase S-2444 in PBS / 0.1% BSA / 0.05% Tween-20 buffer (1.92 mg / ml) was added. Catalytic activity was determined by the release of paranitroanilide by measuring absorbance at a wavelength of 405 nm every minute. The catalytic activity was calculated using the formula Mccat / L = 1.74 * Sample volume (1 ml) * A (average change in absorption over 1 min in a linear section) / Sample volume (10 μl).

We found that the obtained recombinant proteins (proteolytically inactive urokinase and kringle domain) did not have proteolytic activity, in contrast to the control urokinase (compare the graphs in figure 3).

The binding of recombinant proteins based on the structure of urokinase receptor urokinase

To test the ability of recombinant proteins based on the urokinase structure to bind to the urokinase receptor, we used the purified urokinase receptor property to retain the ability to bind ligands.

For this, the recombinant receptor was immobilized on the bottom of the wells of a 96-well ELISA plastic plate by incubation for 1 hour with the urokinase receptor (0.11 nmol in 0.1 M NaHCO ₃ pH 9.5 buffer) at 37 ° C with constant stirring. In parallel with the receptor wells, ligand proteins were applied to clean wells to control the quality of the titration system. The washing and blocking of non-specific binding sites hereinafter was carried out with a blocking buffer of 50 mM Tris / HCl pH 7.5 containing 10 mg / ml BSA, 20 mg / ml gelatin, 0.02% Tween-20. Recombinant forms of urokinase were added in an amount of 0-0.014 nmol and incubated for 1 hour. Washed 4 times for 5 minutes at 37 ° C with stirring in blocking buffer. Biotinylated polyclonal goat anti-urokinase antibodies (GAHUK) were added to the wells at a concentration of 2 μg / ml in blocking buffer. Washed 4 times for 5 minutes at 37 ° C with stirring in blocking buffer. Neutravidin, conjugated to horseradish polyperoxidase, specifically recognizing biotin residues, was introduced. Washed 4 times for 5 minutes at 37 ° C with stirring in blocking buffer. Orthophenylenediamine (0.1 mg / ml) was incubated with the substrate until color development. The reaction was stopped by adding 0.5 volume of 10% H ₂ SO ₄ . The color intensity was measured on a plate spectrofluorimeter at a wavelength of 492 nm.

It was found that the binding pattern of the recombinant proteolytically inactive form of urokinase containing the growth domain did not differ from the binding of the native standard full-length urokinase and the binding of the recombinant full-length urokinase (compare the curves in Fig. 4).

Thus, the functional activity of the obtained recombinant forms of urokinase corresponds to the proteolytically inactive urokinase and the kringle domain of urokinase.

Example 3. Inhibition of migration of endothelial cells under the action of recombinant forms of urokinase

The effect of the obtained proteolytically inactive urokinase and Kringle domain on the migration of endothelial cells was studied using a Boyden chamber manufactured by Neuroprobe Inc., USA. To this end, human umbilical cord vein endothelial cells that reached a confluent state were cultured before experiment in serum-free DMEM / 0.1% BSA for 24 hours.

As chemoattractants, various forms of human urokinase were used, which were placed in the lower cells of the Boyden chamber. The chemoattractant and endothelial cells were separated by a polycarbonate porous filter with a pore diameter of 8 μm (Nucleopore Co., USA) coated with type I collagen (100 ng / ml) (Vitrogen 100, Celtrix Pharmaceuticals Inc., USA).

50 μl of a suspension of endothelial cells obtained by treating a cell monolayer with a solution of trypsin and EDTA (0.05 / 0.02%) was added to the upper cells of the chamber in serum-free DMEM / 0.1% BSA. The number of endothelial cells introduced into the well was 75,000 cells. A chamber with endothelial cells and added control stimuli and test substances was incubated in a CO ₂ incubator for 3 hours. Emigrated endothelial cells were removed with a special scraper from the upper surface of the filter, and endothelial cells on the lower surface were fixed in methanol and stained with DiffQuick stain (Baxter, USA). A membrane with stained endothelial cells was scanned on a ScanJet II CX scanner using Deskscan and NIH Image software. The migration intensity was estimated by the peak area obtained by scanning the field of stained endothelial cells. The results are presented as the ratio of the obtained areas to the control area obtained for cells that passed through the filter in the absence of a chemoattractant.

When analyzing the ability of recombinant forms of urokinase to suppress endothelial cell migration, it was found that wild-type urokinase does not affect endothelial cell migration in response to 10 ng / ml bFGF, and recombinant proteolytically inactive urokinase and its recombinant kringle domain cause a decrease in cell migration below the initial level (compare the uPAwt column with the uPA HQ and kringle columns in FIG. 5). The kringle domain is very active in this regard (compare the uPA HQ and kringle columns in FIG. 5).

Thus, recombinant proteolytically inactive urokinase and the recombinant kringle domain of urokinase inhibit endothelial cell migration, while full-sized proteolytically active urokinase does not affect endothelial cell migration. Based on the results of the negative effect of proteolytically inactive urokinase and the recombinant kringle domain of urokinase on the migration of endothelial cells, it can be concluded that these recombinant proteins can be used to suppress angiogenesis.

Example 4. Inhibition of the formation of capillary-like structures in vitro under the action of recombinant forms of urokinase

To assess the formation of capillary-like structures, a 24-well plate was used, the bottom of which was coated with Matrigel ™ (BD Biosciences). 5 × 10 ⁴ HUVEC (endothelial cells from human umbilical cord) were seeded into each well. To determine the effect of immobilized forms of urokinase on the formation of capillaries, the studied stimulants or inhibitors were introduced into Matrigel ™. Cells were incubated in a culture medium for endothelial cells in a CO ₂ incubator at 37 ° C for 24 hours. The formed capillary-like structures were photographed. The total length of the forming capillary-like structures was evaluated in 5 random fields of view in each well using the MetaMorph 5.0 program (Universal Imaging). To ensure the reliability of the results, the experiments were repeated three times in 3 parallels using 9 wells per experimental point.

In the presented figures it is seen (Fig. 6) that the recombinant kringle domain and proteolytically inactive urokinase influence the formation of endothelial microtubules stimulated by the main growth factor for fibroblasts. At the same time, proteolytically inactive urokinase inhibits the formation of endothelial microtubules.

The graphs (Fig.6) presents the results of mathematical processing of the obtained images. Mathematical processing allows us to take into account the length of the formed endothelial microtubules per unit area.

A similar effect was demonstrated when the main fibroblast growth factor was used as a stimulator for the formation of microtubules. The graph shows a slight decrease in the length of endothelial microtubules when using proteolytically inactive urokinase as an inhibitor.

The results of mathematical processing demonstrate a slight, but significant reduction in the length of endothelial microtubules when using proteolytically inactive urokinase as an inhibitor and vascular endothelial growth factor as a stimulant.

Nevertheless, a visual assessment of the formation of endothelial microtubules shows that this process is affected not only by proteolytically inactive urokinase, but also by the recombinant kringle domain of urokinase.

As a result of the study of the ability of recombinant proteolytically inactive urokinase and the recombinant kringle domain of urokinase to inhibit the formation of microtubules, it was demonstrated that proteolytically inactive urokinase slightly, but significantly reduces the length of the formed endothelial microtubules. The effect of recombinant proteolytically inactive urokinase and its recombinant kringle domain on the morphology of the resulting microtubules has been demonstrated.

Based on the results of a study of the ability of recombinant proteolytically inactive urokinase and the recombinant kringle domain of urokinase to influence the formation of microtubules, it can be concluded that these recombinant constructs can be used to suppress angiogenesis.

Example 5. Inhibition of angiogenesis in vivo under the action of recombinant forms of urokinase

To test the hypothesis that recombinant proteolytically inactive urokinase and its recombinant kringle domain can inhibit the formation of blood vessels in the body, a study was made of the effect of recombinant forms of urokinase on angiogenesis in an in vivo model.

To evaluate the effect of recombinant forms of urokinase on blood vessel growth in vivo, a model of angiogenesis in a subcutaneous Matrigel ™ implant was chosen.

Matrigel ™ is a secretion product of the tumor cell line (mouse sarcoma Engelbreth-Holm-Svarm) and therefore can be considered as a model of tumor angiogenesis. Matrigel ™, which contains a complex of growth factors and matrix proteins, stimulates vascular germination. By adding the studied factors to its composition, we can evaluate their effect on angiogenesis.

The mouse was anesthetized by intraperitoneal administration of a 2.5% solution of avertin;

Matrigel ™ was thawed on ice, without heating above + 4 ° C (at + 8 ° C the gel polymerizes). The tested additives were introduced into Matrigel ™, preventing the temperature from rising. Each mouse was injected subcutaneously in each flank with 500 μl of Matrigel ™. Injections were performed with an insulin syringe with a needle 23 G thick so that Matrigel ™ had time to polymerize under the skin and form an irregularly shaped jelly implant. The experimental groups included 5 mice. 10 days after subcutaneous administration of Matrigel ™ with the studied additives, mice were sacrificed by cervical dislocation. The implants were then removed and frozen in liquid nitrogen in a Tissue-Tek tissue freezing medium (Sakura, Japan). The assessment of the density of blood vessels on sections of Matrigel ™ was performed using immunofluorescence staining. To do this, using a cryomicrotome, sections were prepared with a thickness of 6 μm; which were fixed in a 4% formalin solution (Sigma, USA) at room temperature for 10 min; washed the fixed sections with phosphate-buffered saline (PBS); incubated in a 0.05 M solution of ammonium acetate in PBS (10 minutes). Then, the PBS sections were washed and the sections were incubated for 30 minutes in a 1% BSA solution (bovine serum albumin) on PBS containing 10% serum. Then the sections were incubated for 1 hour at room temperature with monoclonal antibodies to CD31; washed PBS three times and placed in a solution of secondary antibodies for 1 hour. As a negative control, some sections were incubated with non-specific isotypic immunoglobulins instead of the first antibodies. Cell nuclei were stained with DAPI fluorescent dye. After washing three times in PBS, the sections were enclosed in Mounting Medium Vectashield ™ (Vector Laboratories Inc.). To prevent "burnout" of fluorescent labels, the finished samples were stored at 4 ° C in the dark. The resulting preparations were analyzed using a ZeissAxiovert200M fluorescence microscope. Image documentation was performed using an Axiocam HRc digital video camera (Zeiss, Germany). Images were processed in the Axiovision program. Vascular size and density were evaluated using MetaMorph 5.0 (Universal Imaging) and ClickCounter software. Vascular counting was performed in 5 fields of view per section (12 sections per implant) at a 20-fold increase, separately identifying capillaries, medium-diameter vessels and large vessels. The resulting number of vessels in the field of view was normalized per unit area of the slice.

We found that the recombinant proteolytically inactive form of urokinase and the recombinant kringle domain of urokinase inhibit the formation of blood vessels in the Matrigel ™ implant both against the background of stimulation of angiogenesis with vascular endothelial growth factor and the main fibroblast growth factor, and the basal level of vascular formation in Matrigel ™ without the introduction of additional stimulants angiogenesis (Fig.7). According to the results of mathematical processing, the observed effects of recombinant forms of urokinase on angiogenesis were significant.

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Claims (4)

1. A method for locally suppressing blood vessel growth by co-administering to the site of excessive angiogenesis an effective amount of a proteolytically inactive recombinant form of an urokinase type plasminogen activator and a recombinant urokinase type plasminogen activator domain, wherein a recombinant non-molecular oresin molecule is used as a recombinant form of plasminogen activator the amino acid sequence of SEQ ID NO: 1., inhibiting proteolyte activity of endogenous urokinase, and the recombinant kringle domain is the recombinant kringle urokinase with the amino acid sequence of SEQ ID NO: 2, spatially uncoupling receptor proteins that mediate the effects of urokinase, and also displacing the proteolytic activity of endogenous urokinase from the binding sites of the kringle domain urokinase. 2. The method according to claim 1, characterized in that a combination of (1: 1) full-length proteolytically inactive urokinase with the amino acid sequence of SEQ ID NO: 1 and the kringle domain of urokinase with the amino acid sequence of SEQ ID NO: 2 is used. 3. Recombinant full-sized proteolytically inactive urokinase having the amino acid sequence of SEQ ID NO: 1, for use in the method according to claim 1. 4. Recombinant proteolytically inactive kringle domain of urokinase having the amino acid sequence of SEQ ID NO: 2, for use in the method according to claim 1.

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