WO2011011841A1

WO2011011841A1 - Human blood coagulation factor ix recombinant protein, composition, use of a factor ix recombinant protein, use of a composition, method of obtaining human blood coagulation factor ix recombinant protein and use of the factor ix recombinant protein

Info

Publication number: WO2011011841A1
Application number: PCT/BR2009/000240
Authority: WO
Inventors: Aparecida Maria Fontes; Dimas Tadeu Covas
Original assignee: Fundacao Hemocentro de Ribeirao Preto; Universidade de Sao Paulo USP
Current assignee: Fundacao Hemocentro de Ribeirao Preto; Universidade de Sao Paulo USP
Priority date: 2009-07-31
Filing date: 2009-07-31
Publication date: 2011-02-03
Anticipated expiration: 2012-01-31

Abstract

The present invention refers to a human blood coagulation factor IX recombinant protein and a composition containing it. The present invention also deals with the use of the protein or composition of the invention for the manufacture of a medicine to treat hemophilia B. Additionally, the present invention refers to the method of obtaining a human blood coagulation factor IX mutated recombinant protein. Another object of the present invention is the mutated recombinant protein obtained by the method described herein, and its use in order to prepare a medicine for the treatment of hemophilia B.

Description

"HUMAN BLOOD COAGULATION FACTOR IX RECOMBINANT PROTEIN, COMPOSITION, USE OF A FACTOR IX RECOMBINANT PROTEIN, USE OF

A COMPOSITION, METHOD OF OBTAINING HUMAN BLOOD

COAGULATION FACTOR IX RECOMBINANT PROTEIN AND USE OF THE FACTOR IX RECOMBINANT PROTEIN"

FIELD OF THE INVENTION

The present invention refers to a human blood coagulation factor IX (FIX) recombinant protein.

Additionally, the present invention refers to the method of obtaining the aforementioned recombinant protein and its use in order to prepare a medicine for the treatment of hemophilia B.

BACKGROUND OF THE INVENTION

Hemophilia B is a hereditary illness caused by the deficiency of blood coagulation factor IX in the blood plasma. Clinically, this illness has many similarities with hemophilia A, whereby the patient has frequent episodes of bleeding, in most cases in the mucocutaneous, skeletal muscle and soft tissue regions. The bleeding can also occur in other critical spaces such as, for example, in the intracranial or retroperitoneal space, which can be fatal.

The blood coagulation factor IX, or anti-hemophilia B globulin, is one of the factors of blood coagulation. It is a soluble protein that forms the fibrin matrix of a blood coagulum.

FIX products derived from human plasma were routinely used in the technique to treat individuals with hemophilia B. FIX concentrations with low specific activity and low purity were developed for over 30 years and the use of these concentrations increased the duration and quality of life of individuals with hemophilia B. However, these products infected the patients with the hepatitis B virus (HBV), the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV). The FIX concentrations used also contained prothrombin, factor VII, and factor X, and its use to treat hemophilia B resulted in thrombosis in some patients, generally attributed to the accumulation or delay in the release of these active and non-active vitamin K dependent proteins.

Accordingly, years later, new concentrations of FIX derived from plasma were developed. These were highly purified and substituted the first concentrations, without presenting the problem related to thrombosis. However, the new concentrations, despite being highly purified, still presented the inconvenience of contaminating the patients with several viruses, such as the hepatitis B virus (HBV), the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV).

The handicaps related to the concentrations derived from plasma, purified or not, stimulated the attempts to develop the recombinant products of FIX for use patients with hemophilia B.

The gene that codifies the FIX protein is located in the region 27.1 of the long arm of the chromosome X (Xq27.1) and has an extension of 33.5 Kb (Camerino, G., Grzeschik, K.H., Jaye, M., De La SaIIe, H., Tolstoshev, P., Lecocq, J. P., Heilig, R., and Mandel, J. L. (1984). Regional localization on the human X chromosome and polymorphism of the coagulation factor IX gene (hemophilia B locus). Proc Natl Acad Sci USA 81, 498-502). The RNA relative to the FIX is composed of 2802 nucleotides, having a small 5' untranslated region (29 nucleotides), the region between the start codon and the end codon (1383 nucleotides), and a long 3' untranslated region (1390 nucleotides) (Kurachi, K., and Davie, E.W. (1982). Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci USA 79, 6461-6464.) and (Yoshitake, S., Schach, B.G., Foster, D.C., Davie, E.W., and Kurachi, K. (1985). Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 24, 3736-3750).

The protein d educed from the sequence of nucleotides o f the cDNA of the FIX contains 461 amino acids, where the first twenty seven amino acids represent the signal peptide sequence. After the removal of the signal peptide sequence, the protein is submitted to a second cleavage, which results in the removal of the cysteine -19 to arginine -1 residues, and the other 404 amino acids constitute the mature form of the human blood coagulation FIX recombinant protein. As well as the removal of the pre and pro-peptide sequences, the factor IX, before its secretion, has 05 other post-translational modifications, among them: γ-carboxylation; β-hydroxylation; glycosylation; sulfatation and phosphorylation. These modifications have the purpose to ensure that the protein attains the adequate folding, in order that the secretion and its function are appropriate, making it biologically active if necessary (Kaufman, R.J. (1998). Post-translational modifications required for coagulation factor secretion and function. Thromb Haemost 79, 1068-1079.); ( White, G. C₁ 2nd, Pickens, E. M., Liles, D.K., and Roberts, H. R. (1998). Mammalian recombinant coagulation proteins: structure and function. Transfus Sci 19, 177- 189).

Therefore, the complexity of the post-translational modifications of the factor IX resulted in researchers focusing their initial studies on the investigation of the most appropriate cell line for the production of the biologically active factor IX.

Accordingly, recombinant FIX was developed in the state of the art, representing a free source of pathogens of the factor IX. The recombinant FIX of the state of the art is produced from the cell lines of non-human mammals, particularly the cell line of the Chinese hamster ovary, free from these infectious agents.

In the production process of the FIX recombinant, the non-human cell line is transfected with complementary DNA (cDNA) that codifies human FIX. The first studies used conventional plasmid vectors and the vaccinia virus system. In these studies, the FIX was produced in different cell lines, including rat hepatoma (H4-11-E-C3) and the fibroblast (LM-TK), kidney (BHK) and ovary (CHO) of mice. The analysis of the recombinant FIX present in the supernatant of these cell cultures indicated that it was not possible to produce biologically active factor IX in the line of the mouse fibroblast, which resulted in the authors suggesting the absence of an efficient γ-carboxylase system in this cell line (Anson, D.S.; Austen, D. E.; Brownlee, G.G. (1985). Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells. Nature, 315, 683-5). On the other hand, in the other lines, the presence of the biologically active factor IX was diagnosed, and also an increase of at least twofold in the level of the active factor IX after the addition of vitamin K to the culture medium. However, in all the cases, the levels of rFIX were inferior to the quantity of the factor IX present in the plasma (1 IU/mL = 5 ug/m!_) (de Ia SaIIe, H., Altenburger, W., Elkaim, R., Dott, K., Dieterle, A., Drillien, R., Cazenave, JP. , Tolstoshev, P., and Lecocq, JP. (1985). Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques. Nature 316, 268-270), (Busby, S., Kumar, A., Joseph, M., Halfpap, L., Insley, M., Berkner, K., Kurachi, K., and Woodbury, R. (1985). Expression of active human factor IX in transfected cells. Nature 316, 271-273).

The same did not occur with the use of the cell line CHO-dhfr-, which allowed the amplification of the gene of interest after the selection with the drug methotrexate and resulted in a level of rFIX approximately 20 times superior to the level of factor IX present in the plasma (100 μg/mL), in the presence of vitamin K, although only 2% of the protein is functional (1.5 μg/mL/24 h) (Kaufman, RJ. , Wasley, LC., Furie, B.C., Furie, B., and Shoemaker, CB. (1986). Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. J Biol Chem 261, 9622-9628).

In conjunction, these studies demonstrate the importance of the vitamin K as a co-factor of the factor IX, as well as the importance of the development of selection strategies that allow the stable and enduring expression of the recombinant FIX (rFIX) in the culture medium.

Additionally, other expression systems developed in the state of the art, in particular an adenoviral and retroviral expression system, which resulted in the generation of new productive recombinant cell lines of FIX

(Gordon, E.M., Tang, H., Salazar, R.L., and Kohn, D. B. (1993). Expression of coagulation factor IX (Christmas factor) in human hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer. Am J Pediatr Hematol Oncol

15, 196-203), ( Miao, C.H., Ohashi, K., Patijn, GA, Meuse, L., Ye, X.,

Thompson, A.R., and Kay, M.A. (2000). Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. MoI Ther 1, 522-532).

These studies indicate that the retroviral system is an appropriate system for the production of the recombinant FIX and allows the generation of cell lines with the stable and enduring expression of the rFIX.

An example of a commercially available recombinant molecule is BeneFIX, manufactured by the Genetics Institute, Inc., USA, developed for the treatment of hemophilia B. This molecule was licensed in 1997 and it is estimated that the number of individual carriers of hemophilia B that have access to the recombinant form of the factor IX is approximately 6500 persons.

Hemophilia B is a genetic illness that affects 1 in every 20,000 male births in the world. It is estimated that there are 1000 registered hemophiliacs B in Brazil and data from the Ministry of Health indicates that the treatment of these is with the factor IX concentration derived from plasma.

The recombinant product has been manufactured in only some regions by a single company and this has resulted in a shortage in the supply of the product.

Additionally, it is observed that the recombinant factor IX produced by non-human lines, can result in the patient developing activity neutralizing antibodies (inhibitors).

Accordingly, the high cost of the recombinant factor IX, the limited quantity available in the market and the possibility of developing inhibitor antibodies, are factors that encourage researchers to develop new formulations of rFIX, making it more accessible to the hemophilic B population and more clinically effective and viable.

BRIEF DESCRIPTION OF THE INVENTION

From a profound study of the molecular mechanisms that govern the gene expression that codifies the FIX protein, as well as the detailed biochemical characterization of the FIX protein, the Applicant succeeded in developing a new recombinant molecule of the FIX, produced in high levels from human cell lines.

The molecule of the present invention, as well as being produced in high levels, presents increased biological activity and does not present the handicap of inducing the development of inhibitor antibodies because it is produced from human cell line.

Furthermore, the FIX protein of the invention offers advantages in relation to the proteins obtained by murine cell lines, by dealing with complex proteins that present many post-translational modifications during their synthesis in the human organism.

Therefore, the present invention refers to a human blood coagulation factor IX recombinant protein, which presents a mutation in the position 359, where the aspartic acid amino acid (D) is substituted by valine (V), and the recombinant protein is produced by human lines. Another object of the present invention is a composition that comprises the factor IX recombinant protein, which is mutated from the present invention.

The present invention also refers to the use of the mutated recombinant protein herein described, or the composition that contains it, in the manufacture of a medicine to treat hemophilia B.

The present invention also has the object of a method of obtainment of a mutated human blood coagulation factor IX recombinant protein, in which the method comprises:

a) isolation and purification of the human blood coagulation factor

IX;

b) insertion of restriction sites and substitution of the aspartic acid amino acid in position 359 by valine;

c) introduction of the factor IX molecule obtained by step (b) in a vector;

d) transfection of human cells with the vector obtained by step (c); e) treatment of the human cell culture with increasing antibiotic concentrations;

f) culture of the rescued cultures; and

g) recuperation of the recombinant factor IX obtained by the aforementioned cultures.

Additionally, the present invention has the object of the mutated human blood coagulation factor IX recombinant protein, obtained by the above method, and the use of this protein in the manufacture of a medicine for the treatment of hemophilia B.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates the cloning of the cDNA relative to the factor IX in the pLXIN retroviral plasmid vector by 1 % agarose gel stained with ethidium bromide, after the electrophoresis. Band 1 : recombinant clone before the digestion. Band 2: DNA pLXIN-FIX after the digestion with the restriction enzymes Hpal + BamHI resulting in two fragments: 6.1 kb referring to the vector and 1.3 kb referring to the cDNA of factor IX. Band M1 : 1 kb molecular weight marker (fragments: 10.0; 8.0; 6.0; 5.0; 4.0; 3.0; 2.0; 1.5; 1.0; 0.5) and Band M2: ΦX-174 (fragments 1.3; 1.0; 0.8; 0.6; 0.2 kb).

Figure 2 illustrates the cDNA sequencing relative to the factor IX of the present invention, cloned in the vector pLXIN, demonstrating the presence of the mutation in the position 1243 A-»T of the cDNA relative to the factor IX conveying the substitution of an aspartic acid in the position 359 for a valine

(D359V).

Figure 3 schematically illustrates the human blood coagulation recombinant protein FIX_D359vLXIN of the present invention.

Figure 4 illustrates the pLXIN retroviral vector that is a carrier of the cDNA relative to the factor IX of the present invention between the restriction sites Hpal and BamHI.

Figure 5 illustrates the strategy for the production of the recombinant factor IX of the present invention in the HepG2 and Hek293 human cell lines using the retroviral system.

Figure 6 illustrates the analysis of the biological activity of the human blood coagulation factor IX of the present invention in 2 human cell lines HepG2 and Hek293. These cell lines indicate FIX expression levels in the order of 9 to 11 IU/mL as evaluated by the prothrombin partial activation test.

Figure 7 illustrates the expression of the mRNA relative to the recombinant factor IX of the invention in the cell lines Hep-G2/FIXo₃5₉vLXIN and

Hek293- FIXDSSWLXIN. Left: Band M1 : DNA of the phage ΦX-174 (fragments

1.3; 1.0; 0.8; 0.6; 0.2 kb). Band C: positive control of the PCR reaction. FIX primers were used in the bands 1 to 4 and the bands 5 to 8 represent the same samples of 1 to 4 using the β-actin primers. The bands 1 and 3 represent the virgin Hep-G2 and Hep-G2/FIX_D359vLXIN cell lines, respectively, indicating the absence of the mRNA of the FIXD35ΘVLXIN in the virgin cell and the presence of the same in the recombinant cell. The bands 2 and 4 represent the negative controls of the reactions 1 and 3 (absence of the reverse transcriptase enzyme). The bands 5 and 7 indicate the presence of the endogenous mRNA of the β- actin both in the virgin cell line and the recombinant. The negative controls of the respective reactions are in the bands 6 and 8 (absence of the reverse transcriptase enzyme). Band M2: 1 kb molecular weight marker (fragments: 10.0; 8.0; 6.0; 5.0; 4.0; 3.0; 2.0; 1.5; 1.0; 0.5). Right: Band M1 : DNA of the phage ΦX-174. FIX primers were used in the bands 1 to 4 and the bands 5 to 8 represent the same samples of 1 to 4 using the β-actin primers. The bands 1 and 3 represent the virgin Hek-293 and He/f-293/FIX_D359vLXIN cell lines, respectively, indicating the absence of the mRNA of the rFIX in the virgin cell and the presence of the same in the recombinant cell. The bands 2 and 4 represent the negative controls of the reactions 1 and 3 (absence of the reverse transcriptase enzyme). The bands 5 and 7 indicate the presence of the endogenous mRNA of the β-actin both in the virgin cell line and the recombinant. The negative controls of the respective reactions are in the bands 6 and 8 (absence of the reverse transcriptase enzyme). The band C- represents the negative control of the PCR reaction.

Figure 8 illustrates the rFIX expression by the Cell Lines Hep- G2/FIX_D3δ9VLXIN and Hek293- FIX_D359VLXIN after the selection with geneticin, indicating the analysis by cytometry of the percentage flow of cells expressing the protein FIXD359VLXIN (which represents the mutated recombinant protein of the present invention). (A) SSCXFSC dot plot graph indicating the size and complexity of the virgin HepG2 cells; (B) %GFP X SSC dot plot graph indicating the absence of FIX in the virgin HepG2 cell; (C) dot plot graph of the HepG2/FIX_D359vLXIN cells marked with anti-FIX-FITC indicating that after the selection with the antibiotic a recombinant cell population is obtained in which 46% of the cells express the protein FIXDSSQVLXIN. (D) SSCXFSC dot plot graph indicating the size and complexity of the virgin Hek-293 cells; (E) %GFP X SSC dot plot graph indicating the absence of rFIX in the virgin Hek-293 cell; (F) dot plot graph of the Hek-293/FIX_D359VLXIN cells marked with anti-FIX-FITC indicating that after the selection with the antibiotic a recombinant cell population is obtained in which 21 % of the cells express the protein rFIX.

Figure 9 illustrates the analysis of the FIXD359VLXIN protein expression in the supernatant of the Hep-G2/FIXo₃₅₉\_/LXIN and Hek293/ FIXD359VLXIN recombinant cells and the absence of the same in the virgin cells. Left: analysis of the HepG2/F IX-LXI N cell line. Band 1 : Positive control - FIX derived from the plasma (1.5 μg/μL - Octanyne^®). Band 2: virgin Hep-G2 (15 μg) and Band 3: Hep-G2/FIX_D359vLXIN+ (15 μg). First - gel stained with Coomassie and alongside - autoradiogram of the western blot after the immunodetection using the anti-FIX antibody. Right: analysis of the Hek- 293/FIX-LXIN cell line. Band 1 : Positive control - FIX derived from the plasma (1.5 μg/μL - Octanyne^®). Band 2: virgin Hek-293 (15 μg) and Band 3: Hek- 293/FIX_D359vLXIN+ (15 μg). First - gel stained with Coomassie and alongside - autoradiogram of the western blot after the immunodetection using the anti-FIX antibody. An immunoreactive band of the expected size of 57 kDa is observed in the rFIX producer cell and in the positive control and the absence of the same in the virgin cell. The LMVV molecular weight marker (fragments 97, 66, 45, 30 and 20 kDa.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the present invention refers to a human blood coagulation factor IX recombinant protein, which presents a mutation in the position 359, where the aspartic acid amino acid (D) is substituted by valine (V), and the recombinant protein is produced by human lines.

Within a particular embodiment of the present invention, the human cell lines are selected from hepatic and renal lines. Preferentially, the human lines used by the present invention for the obtainment of a mutated human blood coagulation factor IX recombinant protein are HepG2 and Hek293.

The mutated human blood coagulation factor IX recombinant protein (D359V) of the present invention is produced in elevated levels and has increased biological activity. It is appropriate for use in the treatment of hemophilia B.

The present invention also deals with a composition that comprises the factor IX recombinant protein of the present invention and pharmaceutically acceptable vehicles, excipients or stabilizers. The pharmaceutical composition in accordance with the present invention can be liquid, semi-solid or solid and can be adapted for any enteral or parenteral administration route, whether for immediate or modified release. In a particular embodiment, the aforesaid pharmaceutical composition is adapted for oral administration, more particularly in the form of tablets, capsules, tinctures, emulsions, liposomes, micro-capsules or nanoparticles.

Vehicles, excipients or stabilizers appropriate to the invention are, for example, and without any limitation, those cited in the reference work

Remington's Pharmaceutical Sciences, by the American publisher Mack

Publishing, and also in the European Pharmacopeia and the Brazilian

Pharmacopeia.

In another embodiment, the present invention deals with the use of the mutated recombinant FIX protein of the invention, or the compositions that contain it, for the preparation of medicines to treat hemophilia B.

In another embodiment, the present invention refers to a method of obtainment of a mutated human blood coagulation factor IX recombinant protein, in which the method comprises:

a) isolation and purification of the human blood coagulation factor IX;

b) insertion of restriction sites and substitution of the aspartic acid amino acid in position 359 by valine (D359V);

c) introduction of the factor IX molecule obtained by step (b) in a vector;

T) culture of the rescued cultures; and

In accordance with a preferential embodiment of the present invention, step (b) of the above method is performed by PCR using specific primers that are carriers of restriction sites. Preferentially, the inserted restriction sites are Hpa I and Bam HI.

Step (c) is performed particularly by the digestion of the molecule of step (b) with specific endonucleases, preferentially Hpa I and Bam HI. The bond of the molecule is then treated to the DNA of the linearized vector with the same restriction enzymes.

In a preferential embodiment, the vector used by the present invention is a retroviral vector, particularly, plasmid retroviral. In accordance with a preferential embodiment, the vector used is pLXIN.

In accordance with another embodiment of the present invention, the transfection of the human cells with the vector obtained by step (c) can be performed by any method known in the state of the art. Particularly, the transfection is performed by the method of electroporation. Particularly, the method of the present invention presents two retrovirus production systems in order to increase the viral title. Primarily, the FIX is transfected in ecotropic retrovirus productive cells. Next, the retrovirus produced by the abovementioned cells is transduced into amphotropic cell lines. Finally, the human cell is then transduced with the retrovirus produced by the amphotropic line.

Furthermore, the antibiotic used by the present invention for the treatment of the transfected cultures can be any one that is appropriate in the state of the art, as long as the vector used contains a resistance gene for the same. In a particular embodiment, the antibiotic used is geneticin, without excluding any other appropriately selected system.

As described above, the selection of the transduced cultures is performed by growth treatment with an antibiotic. In accordance with the present invention, the doses of antibiotic used in the first selection (low doses) are between 400 and 600 μg/mL, and the doses of antibiotic used in the second selection (high doses) are between 1 ,000 and 2,000 μg/mL.

The culture of the cells is performed under standard conditions, which can be determined by a person skilled in the art depending on the type of cell line used. Particularly, the culture is performed at a temperature of 37⁰C and 5% of CO2.

Furthermore, the recuperation of the mutated recombinant factor IX obtained by the method of the present invention can be performed by any known method in the state of the art.

The method of the present invention allows the obtainment of mutated human recombinant FIX proteins in high levels. The proteins have a high biological activity and do not present the handicaps presented by the recombinant proteins of the state of the art.

In accordance with an additional embodiment, the present invention refers to the mutated factor IX recombinant protein obtained by the abovementioned method, presenting a substitution of aspartic acid amino acid of position 359 by valine.

A further object of the present invention refers to the use of the protein obtained by the method of the present invention in the preparation of a medicine to treat hemophilia B.

The present invention can be understood more clearly and precisely by a reading of the following examples, which illustrate the present invention without presenting any limiting character.

EXAMPLES

EXAMPLE 1

OBTAINMENT OF A VECTOR CONTAINING THE MUTANT FACTOR IX MOLECULE

The recombinant clone that is a carrier of cDNA sequences relative to the factor IX named 7-5A2 was isolated and purified after four consecutive traces of a human liver cDNA library, using the pMac-FIX plasmid clone as a probe, kindly granted by Prof. Dr. Marcelo Brigido of the UnB. Next, this clone was submitted to the PCR reaction using specific primers that are carriers of the sites for the restriction enzymes Hpa I + Bam HI in their extremities, and a fragment of the expected size of 1.4 kb was obtained. After the purification of this DNA fragment, the digestion with specific endonucleases (Hpa I + Bam Hf) was performed and this was submitted to bond to the pLXIN retroviral plasmid DNA linearized with the same restriction enzymes, as demonstrated by figure 1.

Next, the cDNA sequence relative to the factor IX cloned in the pLXIN vector was submitted to the sequencing, which indicated the presence of the D359V mutation in the catalytic domain of the protein (heavy chain), illustrated by figure 2. The schematic design of the human blood coagulation FIX_D359vLXIN recombinant protein of the present invention can be ascertained in figure 3. EXAMPLE 2

PRODUCTION OF THE MUTANT FIX FROM HUMAN LINES

The HepG2 and Hek293 human cell lines were transduced with the pLXIN-FIX retroviral vector obtained by example 1. After the treatment with growth concentrations of geneticin, the analysis of the biological activity of the factor IX present in the supernatant of these cell lines was performed. Figure 5 indicates the strategy used for the generation of human cell lines with a stable expression of human blood coagulation factor IX and elevated levels of these. After the selection of the HepG2-FIX_D359vLXIN and Hek293- FIX_D359vLXIN recombinant cell lines, the test of activated partial thromboplastin time (aPTT) was performed. This indicated that both cell lines expressed levels of the human blood coagulation factor IX of the present invention, from 9 to 11 times superior to the level of factor IX present in the blood plasma (figure 6), and to the recombinant factor IX produced by the murine lines described in the state of the art.

EXAMPLE 3

EVALUATION OF THE PRODUCTION LEVEL OF THE RECOMBINANT MOLECULE OF THE INVENTION BY THE HEPG2 AND HEK293 HUMAN CELL LINES The evaluation of the production level of the FIX-LXIN recombinant molecule by the HepG2 and Hek293 human cell lines can be performed by conventional RT-PCR; flow cytometry, test of activated partial thromboplastin time (aPTT) and western blot.

CONVENTIONAL RT-PCR

The evaluation by conventional RT-PCR, illustrated by figure 7, diagnoses the presence of mRNA relative to the coagulation factor IX of the present invention in the HepG2-FIX_D359vLXIN and Hek293-FIX_D35₉vLXIN recombinant cell lines, using the following procedure: the extraction of the total RNA is performed using the RNAsy Mini Kit (Qiagen), in accordance with the manufacturer's instructions. Next, 1 to 3 μg of the total RNA is converted into cDNA, using the Superscript Il (Invitrogen) kit, in accordance with the manufacturer's instructions, and 50 pmoles of the specific oligonucleotides (P5 e P3). Afterwards, the DNA fragment relative to the factor IX of the invention is amplified in a reactional mixture that contains: 2 μL of the cDNA, 0.2 M of dNTPs; 1 U of the Taq DNA polymerase enzyme {Amersham Bioscience), 2.5 μL of the 10x buffer of the respective enzyme (Amersham Bioscience) and 10 pmoles of each P3FIX5seq and P5FIX3seq oligonucleotide. This reaction was performed in a thermocycler with the program: 95⁰C for 2 minutes; 35 cycles of 95⁰C for 40 seconds, 55⁰C for 40 seconds and 72⁰C for 1 minute; and 72⁰C for 10 minutes.

FLOW CYTOMETRY

For the intracellular marking of the mutated recombinant factor IX protein of the present invention, approximately 1x10⁶ of HepG2/FIX-LXIN and Hek-293/F7X-LX//V cells were fixed with 1% (w/v) paraformaldehyde during 20 minutes at 4°C and next permeabilized with 0.5% Tween for 15 minutes at 37°C. After the washing step with 2% PBS-BSA, the cells are incubated with a blocking solution (of 10% goat serum) for 1 hour at 37°C. Next, the anti-factor IX monoclonal primary antibody is placed diluted in a 2% (1 :100) PBS-BSA solution, for approximately 24 hours at an ambient temperature. Next, the washing is performed with 2% PBS-BSA and the cells are incubated with the secondary antibody diluted in 2% (1:400) PBS-BSA solution during 45 minutes in an ambient temperature. After this period, a second washing is performed with PBS 1X, and the cells are fractionated in 100μL of PBS 1X, for the subsequent analysis of the emission of fluorescence by the recombinant cells by the flow cytometry. The quantification of the emission of fluorescence was performed with a cellular suspension using 10,000 events (cells) in laminar flow cytometry {FACSort, Beckton Dickinson, San Jose, CA, USA).

The evaluation by flow cytometry indicated that the recombinant cell populations, HepG2-FIX_D359vLXIN and Hek-293-FIX_D359vLXIN, present a stable expression of the FIX in the order of 46% and 21%, respectively, as illustrated by figure 8.

TEST OF ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

The evaluation of the biological activity is performed by the test of activated partial thromboplastin time (aPTT), illustrated by table 1.

TABLE 1

The test of the aPTT coagulation, measures the velocity in vitro that a sample of plasma containing the FIX takes to coagulate a sample of plasma deficient in FIX. The measured parameter is the time necessary for the prothrombinic activity. The time taken for the coagulation varies in accordance with the concentration of the FIX present in the sample to be analyzed. In the present invention, the sample to be tested was diluted 1:10 in a buffer (Owren^'s Veronal - Biomerieux) and after 20 seconds mixed with the plasma deficient in human FIX (Biomerieux, Durham), phospholipids and a contact activator (Platelin® LS - Biomerieux). After the incubation for approximately 240 seconds at 37°C, 100 μl_ calcium chlorate (CaCI₂ 0.25M) was added, and the time for the formation of the coagulation was marked using the automatic coagulometer (COAG-A-MATE® XM - Organon Teknika), in accordance with the manufacturer's instructions. Before initiating the dosage, a standard calibration curve was constructed using FIX derived from plasma (Verify - Reference plasma - (Organon Teknika, Durham), reconstituted with 1mL of distilled water and diluted for a concentration of (1 U ml_ FIX). Six dilutions (1 :5; 1 :10; 1 :20; 1 :40; 1 :80; 1 :160) were performed in a buffer (Owren^*s Veronal - Biomerieux), and used for the construction of the curve for each sample. A linear relation was traced in a graph, with FIX activity in a logarithm scale (abscissa) and the time of the partially active thromboplastin (se conds), and the percentage of the calculated FIX activity was obtained with the average of the curve inclination.

Table 1 indicates the analysis of the biological activity of the FIX present in the supernatant of the two HepG2/FIX_D3δ9vLXIN and Hek- 293/FIXD₃59VLXIN cell populations by the aPTT test. It can be observed that the recombinant cell populations that are carriers of the FIX, after the treatment with geneticin, present levels of biological activity of FIX in the order of 9 to 11 times the level of factor IX present in the blood plasma.

WESTERN BLOT

The characterization of the FIXD35WLXIN protein, produced by the HepG2/FIX_D359vLXIN and Hek-293/FIX_D359vLXIN cell lines, was performed by western blot.

For the preparation of the gel, 7 mL of supernatant are collected of

1x10⁷ HepG2/FIX-LXIN and Hek-293/F IX-LXI N cells, and submitted to concentration in columns Centricon® Amicon® (Millipore), by centrifugation at 7500 rpm during 2 to 4 hours. Next, a washing is performed, using 3 ml of autoclaved MiIIiQ H₂O, by centrifugation at 6000 rpm. Subsequently, the columns were inverted for the fractionation of the samples in approximately 150 μl of autoclaved MiIIiQ H₂O, and centrifuged at 6000 rpm during 15 minutes.

After the fractionation of the protein, the samples are quantified in a spectrophotometer by the Bradford Method.

For the performance of the Western Blot, the samples were submitted to electrophoresis using the Phast System. Approximately 10 μL of the sample (30 μg) were mixed with 10 μl of the sample buffer (10 mM Tris-HCI, pH 8.0; 2.5% SDS (w/v); 1 mM EDTA; 0.01% (v/v) of bromophenol blue), and 1.5 μl of β-mercaptoethanol (Sigma). The mixture is submitted to heating and applied in the gel. The electrophoresis is performed in a fast buffer for the PhastGel (SDS Buffer - Amersham Biosciences) at 100 V, during approximately 40 minutes. Two gels, exactly identical, are submitted to electrophoresis, one to be stained and the other for the transfer. After the electrophoresis, the gel to be stained is incubated for 1 hour in a fixing solution (40% ethanol (v/v), 10% acetic acid (v/v), sterile water qsp 0.125L) and subsequently stained with Coomassie brilliant blue solution (1.3% phosphoric acid (v/v), 0.75 M (NH₄)₂ SO₄, 0.1% Coomassie B-Blue G250 (v/v)), during 18 hours. Next, the gel is washed from 3 to 5 times with MiIIiQ water, and mounted between two sheets of cellophane power in order to complete the drying.

After the electrophoresis, the proteins contained in the polyacrylamide gel are transferred to a nitrocellulose membrane, by the semi- dry electro-transfer method with the Phast System device - Amersham Biosciences, in accordance with the manufacturer's instructions. The electrophoresis is performed in a transfer buffer (0.25 M of Tris-Base; 0.96 M of glycine; 0.5% SDS (sodium dodecyl sulfate); sterile water qsp 1 L) at 90 V, during approximately 2 hours and 30 minutes. When the transfer is concluded, the membrane is prepared for the immunodetection.

The nitrocellulose membrane is submitted to the blockage of the unspecified sites by the addition of the blockage solution TBS-T (10 mM Tris pH 7.5, 150 mM NaCI, 0.1% Tween-20), in 5% of powdered milk (w/v), for approximately 16 hours. After the blockage, the membrane is rapidly washed twice with TBS 1X, 0.1% Tween-20, and incubated with the monoclonal primary antibody (QED), diluted in solution TBS-T (1 :800) during 2 hours at an ambient temperature, and constantly agitated. Next, the washing of the membrane is performed with TBS 1X, 0.1% Tween-20 (2X / 15 minutes; 3X / 5 minutes), and the membrane is incubated with the secondary antibody diluted in solution TBS-T (1:3000), during 1 hour. After the incubation with the second antibody, the membrane is washed again and the detection procedure begins. For the detection, the ECL Western Blotting Kit {Amersham Biosciences) is used, in accordance with the manufacturer's instructions. Finally, the membrane is exposed to the X-ray film for 15 seconds and the development is performed in automatic KODAK machines.

As illustrated by figure 9, an immunoreactive band of the expected size of 57 kDa can be observed, referring to the human blood coagulation factor IX protein, present in the supernatants of the FIXD359V recombinant cells and the absence of the same in the virgin HepG2 and Hek-293 cells.

As persons skilled in the art understand well, numerous modifications and variations of the present invention are possible in the light of the above teachings, without removing the scope of the protection, as delimited by the attached claims.

Claims

1. HUMAN BLOOD COAGULATION FACTOR IX RECOMBINANT PROTEIN, characterized by the fact that it comprises a substitution of the aspartic acid amino acid in the position 359, by valine.

2. PROTEIN, of claim 1 , characterized by the fact that it is obtained by human cell lines.

3. PROTEIN, of claim 2, characterized by the fact that the cell lines are human renal or hepatic lines.

4. PROTEIN, of claim 3, characterized by the fact that the human cell lines are HepG2 and Hek293.

5. COMPOSITION, characterized by the fact that it comprises the factor IX recombinant protein, as described in any one of claims 1 to 4, and pharmaceutically acceptable carriers, excipients or stabilizers.

6. USE OF THE FACTOR IX RECOMBINANT PROTEIN, as described in any one of claims 1 to 4, characterized by the fact that it is in the preparation of a medicine to treat hemophilia B.

7. USE OF A COMPOSITION, as described in claim 5, characterized by the fact that it is in the preparation of a medicine to treat hemophilia B.

8. METHOD OF OBTAINING A HUMAN BLOOD

COAGULATION FACTOR IX RECOMBINANT PROTEIN, characterized by the fact that it comprises:

a) isolation and purification of the human blood coagulation factor

IX;

c) introduction of the factor IX molecule obtained by step (b) in a vector; d) transfection of human cells with the vector obtained by step (c); e) treatment of the human cell culture with increasing antibiotic concentrations;

f) culture of the rescued cultures; and

9. METHOD, of claim 8, characterized by the fact that the step (b) is performed by PCR using specific primers that are carriers of restriction sites.

10. METHOD, of claim 9, characterized by the fact that the restriction sites are Hpa I and Bam HI.

11. METHOD, of claim 8, characterized by the fact that the vector is retroviral.

12. METHOD, of claim 11 , characterized by the fact that the vector is pLXIN.

13. METHOD, of claim 8, characterized by the fact that the transfection of the human cells with the vector obtained by step (c) is performed by electroporation.

14. METHOD, of claim 8, characterized by the fact that it presents two retrovirus production systems: (1) the FIX is transfected in ecotropic retrovirus productive cells and (2) the retrovirus produced by the ecotropic cells is transduced in amphotropic cell lines.

15. METHOD, of claim 8, characterized by the fact that the growth doses of antibiotic of the step (e) are comprised between 400 to 600 μg/mL in the first selection (low doses) and between 1000 to 2000 μg/mL in the second selection (high doses).

16. HUMAN BLOOD COAGULATION FACTOR IX RECOMBINANT PROTEIN, characterized by the fact that it is obtained from the method as described in any one of claims 8 to 15 and contains a substitution of the aspartic acid amino acid in the position 359 by valine.

17. USE OF THE FACTOR IX RECOMBINANT PROTEIN, as described in claim 16, characterized by the fact that it is in the preparation of a medicine to treat hemophilia B.