BRPI0802690A2 - recombinant human blood coagulation factor ix protein, composition, use of a recombinant ix factor protein, use of a composition, method of obtaining recombinant human blood coagulation factor ix protein and use thereof - Google Patents
recombinant human blood coagulation factor ix protein, composition, use of a recombinant ix factor protein, use of a composition, method of obtaining recombinant human blood coagulation factor ix protein and use thereof Download PDFInfo
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- BRPI0802690A2 BRPI0802690A2 BRPI0802690A BRPI0802690A2 BR PI0802690 A2 BRPI0802690 A2 BR PI0802690A2 BR PI0802690 A BRPI0802690 A BR PI0802690A BR PI0802690 A2 BRPI0802690 A2 BR PI0802690A2
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- protein
- factor
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- human blood
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Abstract
PROTEÍNA RECOMBINANTE DO FATOR IX DE COAGULAÇçO SANGUÍNEA HUMANA, COMPOSIÇçO, USO DE UMA PROTEÍNA RECOMBINANTE DO FATOR IX, USO DE UMA COMPOSIÇçO, MÉTODO DE OBTENÇçO DE PROTEÍNA RECOMBINANTE DO FATOR IX DE COAGULAÇçO SANGUÍNEA HUMANA E USO DA MESMA. A presente invenção refere-se a uma proteína recombinante mutada do fator IX de coagulação sanguínea humana e a uma composição que a contém. A presente invenção trata ainda do uso da proteína ou composição da invenção para a fabricação de um medicamento para tratar hemofilia B. Adicionalmente, a presente invenção refere-se ao método de obtenção de uma proteína recombinante mutada do fator IX de coagulação sanguínea humana. Ainda outro objeto da presente invenção é a proteína recombinante mutada obtida pelo método aqui descrito, e seu uso para preparar um medicamento para o tratamento de hemofilia B.HUMAN BLOOD COAGULATION FACTOR IX RECOMBINANT PROTEIN, COMPOSITION, USE OF A FACTOR RECOMBINANT PROTEIN, USE OF A COMPOSITION, HUMAN BLOOD FACTORY RECOMBINANT PROTEIN METHOD AND METHOD The present invention relates to a mutated recombinant human blood coagulation factor IX protein and a composition containing it. The present invention further relates to the use of the protein or composition of the invention for the manufacture of a medicament for treating hemophilia B. In addition, the present invention relates to the method of obtaining a mutated recombinant human blood coagulation factor IX protein. Still another object of the present invention is the mutated recombinant protein obtained by the method described herein, and its use to prepare a medicament for treating hemophilia B.
Description
"PROTEÍNA RECOMBINANTE DO FATOR IX DE COAGULAÇÃOSANGÜÍNEA HUMANA, COMPOSIÇÃO, USO DE UMA PROTEÍNARECOMBINANTE DO FATOR IX, USO DE UMA COMPOSIÇÃO, MÉTODODE OBTENÇÃO DE PROTEÍNA RECOMBINANTE DO FATOR IX DECOAGULAÇÃO SANGÜÍNEA HUMANA E USO DA MESMA""RECOMBINATING PROTEIN OF HUMAN BLOOD COAGULATION FACTOR IX, COMPOSITION, USE OF A COMPACTING FACTOR IX PROTEIN, USE OF A COMPOSITION, METHOD OF OBTAINING RECOMBINING FACTOR PROTEIN OF BLOOD SUMMARY USE"
Campo da InvençãoField of the Invention
A presente invenção refere-se a uma proteína recombinante dofator IX (FIX) de coagulação sangüínea humana.The present invention relates to a recombinant human blood coagulation protein IX (FIX) protein.
Adicionalmente, a presente invenção refere-se ao método deobtenção da mencionada proteína recombinante e seu uso para preparar ummedicamento para o tratamento de hemofilia B.Additionally, the present invention relates to the method of obtaining said recombinant protein and its use to prepare a medicament for the treatment of hemophilia B.
Antecedentes da InvençãoA hemofilia B é uma doença hereditária causada pela deficiênciado fator IX de coagulação sangüínea no plasma sangüíneo. Clinicamente, estadoença apresenta muitas similaridades com a hemofilia A, isto é, o pacienteapresenta freqüentes episódios de sangramento, na maioria das vezes emregiões muco-cutâneas, músculo-esquelético e em tecidos moles. Osangramento pode ocorrer também em outros espaços críticos como, porexemplo, no espaço intracranial ou retroperitoneal, o qual pode ser fatal.BACKGROUND OF THE INVENTION Hemophilia B is an inherited disease caused by blood coagulation factor IX deficiency in blood plasma. Clinically, the condition has many similarities with hemophilia A, that is, the patient has frequent episodes of bleeding, most often in mucocutaneous, musculoskeletal and soft tissue regions. Bleeding may also occur in other critical spaces such as intracranial or retroperitoneal space, which can be fatal.
O fator IX de coagulação sangüínea, ou globulina anti-hemofílicaB, é um dos fatores de coagulação do sangue. É uma proteína solúvel queforma a matriz de fibrina de um coágulo de sangue.Blood coagulation factor IX, or antihemophilic globulin B, is one of the blood clotting factors. It is a soluble protein that forms the fibrin matrix of a blood clot.
Produtos de FIX derivado de plasma humano foramrotineiramente utilizados na técnica para tratar indivíduos com hemofilia B.Human plasma derived FIX products have been routinely used in the art to treat individuals with hemophilia B.
Concentrados de FIX com baixa atividade específica e baixa pureza foramdesenvolvidos há mais de 30 anos, e o uso destes concentrados aumentou aduração e qualidade de vida das pessoas com hemofilia B. No entanto, estesprodutos infectaram os pacientes com vírus da hepatite B (HBV), vírus dahepatite C (HCV) e vírus da imunodeficiência humana (HIV). Os concentradosde fator IX utilizados continham ainda protrombina, fator VII, e fator, X, e seuuso para tratar hemofilia B gerou trombose em alguns pacientes, geralmenteatribuída ao acúmulo ou demora de liberação destas proteínas dependentes devitamina K, ativadas e não-ativadas.Low specific activity low purity FIX concentrates have been developed for over 30 years, and the use of these concentrates has increased the height and quality of life of people with hemophilia B. However, these products have infected patients with hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The factor IX concentrates used also contained prothrombin, factor VII, and factor X, and their use to treat hemophilia B generated thrombosis in some patients, generally attributed to the accumulation or delayed release of these activated and inactivated vitamin D-dependent proteins.
Desta forma, anos depois, foram desenvolvidos novosconcentrados de FIX derivados de plasma, altamente purificados, quesubstituíram os primeiros concentrados, sem apresentar o problemarelacionado à trombose. No entanto, os novos concentrados, mesmo altamentepurificados, ainda apresentavam o inconveniente de contaminar os pacientescom diversos vírus, como o vírus da hepatite B (HBV), vírus da hepatite C(HCV) e vírus da imunodeficiência humano (HIV).Thus, years later, new highly purified plasma-derived FIX concentrates were developed that replaced the first concentrates without presenting the thrombosis-related problem. However, the new concentrates, even highly purified, still had the drawback of contaminating patients with various viruses, such as hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV).
Os inconvenientes relacionados aos concentrados derivados deplasma, purificados ou não, estimularam as tentativas no desenvolvimento deprodutos recombinantes de FIX para uso em pacientes com hemofilia B.The drawbacks of purified or non-purified plasma derived concentrates have spurred attempts to develop recombinant FIX products for use in patients with hemophilia B.
O gene que codifica a proteína do FIX situa-se na região 27.1 dobraço longo do cromossomo X (Xq27.1) e apresenta uma extensão de 33.5 Kb(Camerino, G., Grzeschik, K.H., Jaye, M., De La Salle, H., Tolstoshev, P.,Lecocq, J.P., Heilig, R., and Mandei, J.L. (1984). Regional localization on thehuman X chromosome and polymorphism of the coagulation factor IX gene(hemophilia B locus). Proc Natl Acad Sei U S A 81, 498-502). O RNA relativo aoFIX é composto por 2802 nucleotídeos, possuindo uma pequena região 5' não-traduzida (29 nucleotídeos), a região entre o códon iniciador e o códon detérmino (1383 nucleotídeos), e uma longa região 3' não-traduzida (1390nucleotídeos) (Kurachi, K., and Davie, E.W. (1982). Isolation andcharacterization of a cDNA coding for human factor IX. Proc Natl Acad Sei U SA 79, 6461-6464.) e (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 gene encoding the FIX protein is located in the X-chromosome 27.1 long-fold region (Xq27.1) and has a length of 33.5 Kb (Camerino, G., Grzeschik, KH, Jaye, M., De La Salle, H., Tolstoshev, P., Lecocq, JP, Heilig, R., and Mandei, JL (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). FIX-related RNA is composed of 2802 nucleotides, having a small untranslated 5 'region (29 nucleotides), the region between the primer codon and the terminus codon (1383 nucleotides), and a long 3' untranslated region (1390nucleotides). ) (Kurachi, K., and Davie, EW (1982). Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci SA 79, 6461-6464.) And (Yoshitake, S., Schach, BG, Foster, DC, Davie, EW, and Kurachi, K. (1985) .Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 24, 3736-3750).
A proteína deduzida da seqüência de nucleotídeos do cDNA doFIX contém 461 aminoácidos, onde os primeiros vinte e sete aminoácidosrepresentam a seqüência peptídica sinal. Após a remoção da seqüênciapeptídica sinal, a proteína é submetida a uma segunda clivagem, o que resultana remoção dos resíduos cisteína -19 a arginina -1, e os outros 404aminoácidos constituem a forma madura da proteína do FIX de coagulaçãosangüínea humana. Além da remoção das seqüências pré e pró-peptídica, ofator IX, antes de sua secreção, apresenta 05 outras modificações pós-traducionais, entre eles: v-carboxilação; (3-hidroxilação; glicosilação; sulfataçãoe fosforilação. Estas modificações têm a finalidade de que a proteína atinja odobramento adequado, para que sua secreção e o seu funcionamento sejamapropriados, tornando-a biologicamente ativa se necessário (Kaufman, RJ.(1998). Post-translational modifications required for coagulation factor secretionand function. Thromb Haemost 79, 1068-1079.); ( White, G.C., 2nd, Pickens,E.M., Liles, D.K., and Roberts, H.R. (1998). Mammalian recombinantcoagulation proteins: structure and function. Transfus Sei 19, 177-189).The protein deduced from the doFIX cDNA nucleotide sequence contains 461 amino acids, where the first twenty-seven amino acids represent the signal peptide sequence. After removal of the signal peptide sequence, the protein is subjected to a second cleavage resulting in the removal of cysteine -19 to arginine -1 residues, and the other 404 amino acids constitute the mature form of the human blood coagulation FIX protein. In addition to the removal of the pre and propeptide sequences, factor IX, prior to secretion, has 05 other post-translational modifications, including: v-carboxylation; (3-hydroxylation; glycosylation; sulfation and phosphorylation. These modifications are intended for the protein to achieve adequate folding so that its secretion and functioning are appropriate, making it biologically active if necessary (Kaufman, RJ. (1998). Post -translational modifications required for coagulation factor secreting function Thromb Haemost 79, 1068-1079.) (White, GC, 2nd, Pickens, EM, Liles, DK, and Roberts, HR (1998) Mammalian recombinantcoagulation proteins: structure and function Transfus Sci 19, 177-189).
Assim, a complexidade das modificações pós-traducionais dofator IX conduziram os pesquisadores a focarem seus estudos iniciais nainvestigação da linhagem celular mais apropriada para a produção do fator IXbiologicamente ativo.Thus, the complexity of post-translational modifications of factor IX led the researchers to focus their initial studies on the investigation of the most appropriate cell line for the production of biologically active factor IX.
Desta forma, FIX recombinante foi desenvolvido na técnica,representando uma fonte livre de patógenos do fator IX. O FIX recombinante datécnica é produzido a partir de linhagens de células de mamífero não-humanas,particularmente, linhagem de célula de ovário de hamster chinês, livre destesagentes infecciosos.Thus, recombinant FIX was developed in the art, representing a source free of factor IX pathogens. Recombinant FIX technique is produced from non-human mammalian cell lines, particularly Chinese hamster ovary cell line, free of these infectious agents.
No processo de produção de FIX recombinante, a linhagemcelular não-humana é transfectada com DNA complementar (cDNA) quecodifica FIX humano.In the process of producing recombinant FIX, the non-human cell line is transfected with complementary DNA (cDNA) which encodes human FIX.
Os primeiros estudos utilizaram vetores plasmidiais convencionaise o sistema viral vaccinia. Nesses estudos, o FIX foi produzido em diferenteslinhagens celulares, entre elas, de hepatoma de rato (H4-11-E-C3) e fibroblasto(LM-TK), rim (BHK) e ovário (CHO) de camundongo. A análise do FIXrecombinante presente no sobrenadante destas culturas celulares mostrou quenão foi possível a produção do fator IX biologicamente ativo na linhagem defibroblasto de camundongo, o que levou aos autores sugerirem a ausência deum sistema v-carboxilase eficiente nesta linhagem celular (Anson, D.S.; Austen, D.E.; Brownlee, G.G. (1985). Expression of active human clotting factorIX from recombinant DNA clones in mammalian cells. Nature, 315, 683-5). Poroutro lado, nas demais linhagens, a presença de fator IX biologicamente ativofoi diagnosticada, e ainda um aumento de pelo menos 2 vezes no nível de fatorIX ativo após a adição de vitamina K ao meio de cultura. Entretanto, em todosos casos, os níveis de rFIX foram inferiores à quantidade de fator IX presenteno plasma (1 lU/mL = 5 ug/mL) (de Ia Salle, H., Altenburger, W., Elkaim, R.,Dott, K., Dieterle, A., Drillien, R., Cazenave, J.P., Tolstoshev, P., and Lecocq,J.P. (1985). Active gamma-carboxylated human factor IX expressed usingrecombinant 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).Early studies used conventional plasmid vectors and the vaccinia viral system. In these studies, FIX was produced in different cell lines, including mouse hepatoma (H4-11-E-C3) and mouse fibroblast (LM-TK), kidney (BHK) and ovary (CHO). The analysis of recombinant FIX present in the supernatant of these cell cultures showed that it was not possible to produce biologically active factor IX in the mouse defibroblast line, leading the authors to suggest the absence of an efficient v-carboxylase system in this cell line (Anson, DS; Austen Brownlee, GG (1985) Expression of active human clotting factorIX from recombinant DNA clones in mammalian cells., Nature, 315, 683-5). On the other hand, in the other strains, the presence of biologically active factor IX was diagnosed, and at least a 2-fold increase in the active factorIX level after the addition of vitamin K to the culture medium. However, in all cases, rFIX levels were lower than the amount of plasma factor IX present in plasma (1 lU / mL = 5 µg / mL) (from Ia Salle, 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).
O mesmo não ocorreu com o uso da linhagem celular CHO-dhfr-,que permitiu a amplificação do gene de interesse após a seleção com a drogametotrexato e resultou em um nível de rFIX de cerca de 20 vezes superior aonível de fator IX presente no plasma (100 ug/mL), na presença de vitamina K,embora somente 2% da proteína seja funcional (1,5 ug/mL/24 h) (Kaufman,R.J., Wasley, L.C., Furie, B.C., Furie, B., and Shoemaker, C.B. (1986).Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. J Biol Chem261, 9622-9628).The same did not occur with the use of the CHO-dhfr- cell line, which allowed the amplification of the gene of interest after selection with drogamethotrexate and resulted in an approximately 20-fold higher rFIX level of plasma factor IX ( 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, BC, Furie, B., and Shoemaker, CB (1986) .Expression, purification, and characterization of gamma-carboxylated factor IX recombinant synthesized in Chinese hamster ovary cells (J Biol Chem261, 9622-9628).
Em conjunto, estes estudos demonstram a importância davitamina K como um co-fator do fator IX, bem como a importância dodesenvolvimento de estratégias de seleção que permitam a expressão estávele duradoura do FIX recombinante (rFIX) no meio de cultura.Taken together, these studies demonstrate the importance of davitamin K as a factor IX cofactor, as well as the importance of developing selection strategies that allow stable and lasting expression of recombinant FIX (rFIX) in the culture medium.
Adicionalmente, desenvolveram-se no estado da técnica, outrossistemas de expressão, em particular, sistema de expressão adenoviral eretroviral, o que resultou na geração de novas linhagens celularesrecombinantes produtoras de FIX (Gordon, E.M., Tang, H., Salazar, R.L., andKohn, D.B. (1993). Expression of coagulation factor IX (Christmas factor) inhuman hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer.Am J Pediatr Hematol Oncol 15, 196-203), ( Miao, C.H., Ohashi, K., Patijn, G.A., Meuse, L, Ye, X., Thompson, A.R., and Kay, M.A. (2000). Inclusion of thehepatic locus control region, an intron, and untranslated region increases andstabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol Ther 1,522-532).In addition, other expression systems have been developed in the prior art, in particular the eretroviral adenoviral expression system, resulting in the generation of new FIX-producing recombinant cell lines (Gordon, EM, Tang, H., Salazar, RL, andKohn). , DB (1993) Expression of coagulation factor IX (Christmas factor) inhuman hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer.Am J Pediatr Hematol Oncol 15, 196-203), (Miao, CH, Ohashi, K. , Patijn, GA, Meuse, L., Ye, X., Thompson, AR, and Kay, MA (2000) Inclusion of the hepatic locus control region, intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro, Mol Ther 1.522-532).
Esses estudos mostram que o sistema retroviral é um sistema apropriado para a produção do FIX recombinante e permite a geração delinhagens celulares com expressão estável e duradoura de rFIX.These studies show that the retroviral system is an appropriate system for the production of recombinant FIX and allows the generation of stable and enduring expression of rFIX cell lines.
Um exemplo de molécula recombinante comercialmentedisponível é a BeneFIX, fabricada por Genetics Institute, Inc., USA,desenvolvida para o tratamento de hemofilia B. Essa molécula foi licenciada em1997 e estima-se que o número de indivíduos portadores de hemofilia B quepuderam ter acesso à forma recombinante do fator IX é cerca de 6500pessoas.An example of a commercially available recombinant molecule is BeneFIX, manufactured by 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 individuals with hemophilia B who could have access to The recombinant form of factor IX is about 6500 people.
A hemofilia B é uma doença genética que afeta 1 em cada 20.000nascimentos masculinos em todo o mundo. No Brasil, estima-se a existência de1000 hemofílicos B cadastrados e dados do Ministério da Saúde mostram queo tratamento dos mesmos é com o fator IX concentrado derivado do plasma.Haemophilia B is a genetic disease that affects 1 in 20,000 male births worldwide. In Brazil, it is estimated that there are 1000 registered B hemophiliacs and data from the Ministry of Health show that their treatment is with concentrated factor IX derived from plasma.
O produto recombinante tem sido fabricado em apenas algumasregiões por uma única empresa e isso leva a uma carência no fornecimento doproduto.The recombinant product has been manufactured in just a few regions by a single company and this leads to a shortage in product supply.
Adicionalmente, observou-se que o fator IX recombinanteproduzido por linhagens não-humanas, pode levar o paciente a desenvolveranticorpos neutralizadores de atividade (inibidores).Additionally, it was observed that recombinant factor IX produced by non-human strains may lead the patient to develop activity neutralizing antibodies (inhibitors).
Desta forma, o alto custo do fator IX recombinante, sua limitadaquantidade disponível no mercado e a possibilidade de desenvolver anticorposinibidores, são fatores que têm incentivado os pesquisadores aodesenvolvimento de novas formulações de rFIX, tornando-o mais acessível apopulação de hemofílicos B e mais clinicamente eficazes e viáveis.Thus, the high cost of recombinant factor IX, its limited amount available on the market and the possibility of developing inhibitor antibodies are factors that have encouraged researchers to develop new rFIX formulations, making it more accessible and more clinically effective. and viable.
Descrição Resumida da InvençãoBrief Description of the Invention
A partir de um aprofundado estudo dos mecanismos molecularesque governam a expressão do gene que codifica a proteína do FIX, bem comoa caracterização bioquímica minuciosa da proteína do FIX, a Depositanteconseguiu desenvolver uma nova molécula recombinante do FIX, produzida emelevados níveis a partir de linhagens celulares humanas.From an in-depth study of the molecular mechanisms that govern the expression of the FIX protein-encoding gene as well as the detailed biochemical characterization of the FIX protein, Depositant was able to develop a new recombinant FIX molecule produced at high levels from human cell lines. .
A molécula da presente invenção, além de ser produzida emníveis elevados, apresenta atividade biológica aumentada e não apresenta oinconveniente de induzir o desenvolvimento de anticorpos inibidores, uma vezque é produzida a partir de linhagem celular humana.The molecule of the present invention, in addition to being produced at high levels, exhibits increased biological activity and has no inconvenience to induce the development of inhibitory antibodies as it is produced from human cell line.
Ainda, a proteína FIX da invenção oferece vantagens em relaçãoàs proteínas obtidas por linhagens celulares murinas, em se tratando deproteínas complexas que apresentam muitas modificações pós-traducionaisdurante sua síntese no organismo humano.Assim, a presente invenção refere-se a uma proteínarecombinante do fator IX de coagulação sangüínea humana, que apresentauma mutação na posição 359, onde o aminoácido ácido aspártico (D) ésubstituído por valina (V), sendo que a proteína recombinante é produzida porlinhagens humanas.In addition, the FIX protein of the invention offers advantages over proteins obtained from murine cell lines as complex deproteins that exhibit many post-translational modifications during their synthesis in the human organism. Thus, the present invention relates to a recombinant factor IX protein. human blood coagulation, which has a mutation at position 359, where the amino acid aspartic acid (D) is replaced by valine (V), and the recombinant protein is produced by human lines.
Outro objeto da presente invenção é uma composição quecompreende a proteína recombinante do fator IX mutada da presente invenção.Another object of the present invention is a composition comprising the mutant factor IX recombinant protein of the present invention.
A presente invenção refere-se ainda ao uso da proteínarecombinante mutada aqui descrita, ou da composição que a contém, para a fabricação de um medicamento para tratar hemofilia B.The present invention further relates to the use of the mutated recombinant protein described herein, or the composition containing it, for the manufacture of a medicament for treating hemophilia B.
A presente invenção tem também por objeto um método deobtenção de uma proteína recombinante do fator IX de coagulação sangüíneahumana mutada, em que o método compreende:The present invention is also directed to a method of obtaining a recombinant mutated human blood coagulation factor IX protein, wherein the method comprises:
a) isolamento e purificação do fator IX de coagulação sangüíneahumano;a) isolation and purification of human blood coagulation factor IX;
b) inserção de sítios de restrição e substituição do aminoácidoácido aspártico na posição 359 por valina;b) insertion of restriction sites and replacement of the aspartic amino acid at position 359 by valine;
c) introdução da molécula de fator IX obtido pela etapa (b) em umvetor;c) introducing the factor IX molecule obtained by step (b) into a vector;
d) transfecção de células humanas com o vetor obtido pela etapa (c);d) transfecting human cells with the vector obtained by step (c);
e) tratamento da cultura de células humanas com concentraçõescrescentes de antibiótico;e) treating human cell culture with increasing antibiotic concentrations;
f) cultivo das culturas recuperadas; ef) cultivation of recovered crops; and
g) recuperação do fator IX recombinante obtido pelasmencionadas culturas.g) recovery of recombinant factor IX obtained by said cultures.
Adicionalmente, a presente invenção tem por objeto a proteínarecombinante do fator IX de coagulação sangüínea humana mutada, obtidapelo método acima, e ao uso desta proteína para a fabricação de ummedicamento para o tratamento de hemofilia B.Additionally, the present invention relates to the recombinant mutant human blood coagulation factor IX protein obtained by the above method and to the use of this protein for the manufacture of a drug for the treatment of hemophilia B.
Breve Descrição das FigurasBrief Description of the Figures
A figura 1 ilustra a clonagem do cDNA relativo ao fator IX no vetorplasmidial retroviral pLXIN através de gel de agarose 1% corado com brometode etídeo, após a eletroforese. Raia 1: clone recombinante antes da digestão.Raia 2: DNA pLXIN-FIX após a digestão com as enzimas de restrição Hpal +BamHI resultando em dois fragmentos: 6,1kb referente ao vetor e 1,3 kbreferente ao cDNA do fator IX. Raia M1: marcador de peso molecular 1 kb(fragmentos: 10.0; 8.0; 6.0; 5.0; 4.0; 3.0; 2.0; 1.5; 1.0; 0.5) e Raia M2: OX-174(fragmentos 1.3; 1.0; 0.8; 0.6; 0.2 kb).Figure 1 illustrates the cloning of factor IX cDNA in the pLXIN retroviral plasmid vector by ethidium brometode-stained 1% agarose gel after electrophoresis. Lane 1: recombinant clone prior to digestion. Lane 2: DNA pLXIN-FIX after digestion with restriction enzymes Hpal + BamHI resulting in two fragments: 6.1 kb relative to the vector and 1.3 kb relative to factor IX cDNA. Lane 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 Lane M2: OX-174 (fragments 1.3; 1.0; 0.8; 0.6; 0.2 kb).
A figura 2 ilustra o seqüenciamento do cDNA relativo ao fator IXda presente invenção, clonado no vetor pLXIN, demonstrando a presença damutação mutação na posição 1243 A->T do cDNA relativo ao Fator IXconduzindo a substituição de um ácido aspártico na posição 359 por umavalina (D359V).Figure 2 illustrates the factor IX cDNA sequencing of the present invention cloned into the pLXIN vector, demonstrating the presence of a mutation at position 1243 A-> T of Factor IX cDNA leading to the replacement of an aspartic acid at position 359 by umavaline ( D359V).
A figura 3 ilustra esquematicamente a proteína recombinanteFIXD359vLXIN de coagulação sangüínea humana da presente invenção.Figure 3 schematically illustrates the recombinant human blood coagulation protein FIXD359vLXIN of the present invention.
A figura 4 ilustra o vetor retroviral pLXIN portador do cDNArelativo ao fator IX da presente invenção entre os sítios de restrição Hpal eBamHI.Figure 4 illustrates the factor IX cDNA-bearing retroviral vector pLXIN of the present invention between the Hpal eBamHI restriction sites.
A figura 5 ilustra a estratégia para a produção do fator IXrecombinante da presente invenção nas linhagens celulares humanas HepG2 eHek293 utilizando o sistema retroviral.Figure 5 illustrates the strategy for the production of recombinant factor IX of the present invention in HepG2 and Hek293 human cell lines using the retroviral system.
A figura 6 ilustra a análise da atividade biológica do fator IX decoagulação sangüínea humana da presente invenção em 2 linhagens celulareshumanas HepG2 e Hek293. Essas linhagens celulares mostraram níveis deexpressão do FIX da ordem de 9 a 11 lU/mL conforme avaliado pelo teste deativação parcial de protrombina.Figure 6 illustrates the analysis of the biological activity of human blood coagulation factor IX of the present invention in 2 human and HepG2 and Hek293 cell lines. These cell lines showed FIX expression levels in the range of 9 to 11 lU / mL as assessed by the partial prothrombin activation test.
A figura 7 ilustra a expressão do mRNA relativo ao fator IXrecombinante da invenção nas linhagens celulares Hep-G2/FIXD359vLXIN eHek293- FIXD359vLXIN. Á esquerda: Raia M1: DNA do fago OX-174 (fragmentos1.3; 1.0; 0.8; 0.6; 0.2 kb). Raia C: controle positivo da reação de PCR. Nasraias 1 a 4 foram utilizados os "primers" do FIX, e as raias 5 a 8 representam asmesmas amostras de 1 a 4 utilizando os "primers" da B-actina. As Raias 1 e 3representam as linhagens celulares Hep-G2 virgem e /-/ep-G2/FIXD359vLXIN,respectivamente, mostrando a ausência do mRNA do FIXD359vLXIN na célulavirgem e a presença do mesmo na célula recombinante. As raias 2 e 4representam os controles negativos das reações 1 e 3 (ausência da enzimatranscriptase reversa). As raias 5 e 7 mostram a presença do mRNA endógenoda B-actina tanto na linhagem celular virgem como recombinante. Os controlesnegativos das respectivas reações estão nas raias 6 e 8 (ausência da enzimatranscriptase reversa). Raia M2: marcador de peso molecular 1 kb (fragmentos:10.0; 8.0; 6.0; 5.0; 4.0; 3.0; 2.0; 1.5; 1.0; 0.5). À direita: Raia M1: DNA do fagoOX-174. Nas raias 1 a 4 foram utilizados os primers do FIX, e as raias 5 a 8representam as mesmas amostras de 1 a 4 utilizando os primers da B-actina.As Raias 1 e 3 representam as linhagens celulares Hek-293 virgem e Hek-293/FIXd359vLXIN, respectivamente, mostrando a ausência do mRNA do rFIXna célula virgem e a presença do mesmo na célula recombinante. As raias 2 e4 representam os controles negativos das reações 1 e 3 (ausência da enzimatranscriptase reversa). As raias 5 e 7 mostram a presença do mRNA endógenoda p-actina tanto na linhagem celular virgem como recombinante. Os controlesnegativos das respectivas reações estão nas raias 6 e 8 (ausência da enzimatranscriptase reversa). A raia C- representa o controle negativo da reação dePCR.Figure 7 illustrates the expression of the recombinant factor IX mRNA of the invention in the Hep-G2 / FIXD359vLXIN and Hek293-FIXD359vLXIN cell lines. Left: Lane M1: OX-174 phage DNA (fragments 1.3; 1.0; 0.8; 0.6; 0.2 kb). Lane C: Positive control of PCR reaction. In lanes 1 through 4, FIX primers were used, and lanes 5 through 8 represent the same samples as 1 through 4 using B-actin primers. Lanes 1 and 3 represent the virgin Hep-G2 and / - / ep-G2 / FIXD359vLXIN cell lines, respectively, showing the absence of FIXD359vLXIN mRNA in the cell cell and its presence in the recombinant cell. Lanes 2 and 4 represent negative controls for reactions 1 and 3 (absence of reverse enzyme transcriptase). Lanes 5 and 7 show the presence of endogenous B-actin mRNA in both virgin and recombinant cell lines. Negative controls of the respective reactions are in lanes 6 and 8 (absence of reverse transcriptase enzyme). Lane 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: Lane M1: phageOX-174 DNA. Lanes 1 through 4 used FIX primers, and lanes 5 through 8 represent the same samples 1 through 4 using B-actin primers. Lanes 1 and 3 represent virgin Hek-293 and Hek-293 cell lines. / FIXd359vLXIN, respectively, showing the absence of rFIX mRNA in the virgin cell and its presence in the recombinant cell. Lanes 2 and 4 represent negative controls for reactions 1 and 3 (absence of reverse enzyme transcriptase). Lanes 5 and 7 show the presence of endogenous p-actin mRNA in both virgin and recombinant cell lines. Negative controls of the respective reactions are in lanes 6 and 8 (absence of reverse transcriptase enzyme). Lane C- represents the negative control of the dePCR reaction.
A figura 8 ilustra a expressão de rFIX pelas Linhagens CelularesHep-G2/FIXD359vLXIN e Hek293- FIXD359vl-XIN após a seleção com geneticina,mostrando a análise por citometria de fluxo da percentagem de célulasexpressando a proteína FIXD359vLXIN (que representa a proteína recombinantemutada da presente invenção). (A) gráfico "dot plot SSCXFSC mostrando otamanho e complexidade das células HepG2 virgens; (B) gráfico "dor plof%GFP X SSC mostrando a ausência de FIX na célula HepG2 virgem; (C)gráfico dot plot das células HepG2/FIXD359vLXIN marcadas com anti-FIX-FITCmostrando que após a seleção com o antibiótico obteve-se uma populaçãocelular recombinante em que 46% das células expressam a proteínaFIXD359VLXIN. (D) gráfico "dot plot' SSCXFSC mostrando o tamanho ecomplexidade das células Hek-293 virgens; (E) gráfico "dor plot' %GFP X SSCmostrando a ausência de rFIX na célula Hek-293 virgem; (F) gráfico dot plotdas células Hek-293/FIXD35gvl-XIN marcadas com anti-FIX-FITC mostrando queapós a seleção com o antibiótico obteve-se uma população celularrecombinante em que 21% das células expressam a proteína rFIX.Figure 8 illustrates the expression of rFIX by the Hep-G2 / FIXD359vLXIN and Hek293-FIXD359vl-XIN Cell Lines after geneticin selection, showing flow cytometric analysis of the percentage of cells expressing the FIXD359vLXIN protein (representing the recombinant protein of the present invention). ). (A) plot plot SSCXFSC showing the size and complexity of virgin HepG2 cells; (B) plot plof% GFP X SSC pain showing the absence of FIX in the virgin HepG2 cell; (C) dot plot plot of anti-FIX-FIT-labeled HepG2 / FIXD359vLXIN cells showing that after selection with the antibiotic a recombinant cell population was obtained in which 46% of the cells express the FIXD359VLXIN protein. (D) SSCXFSC dot plot graph showing size and complexity of virgin Hek-293 cells (E) pain plot% GFP X SSC graph showing absence of rFIX in virgin Hek-293 cell; (F) dot plot plotted anti-FIX-FITC-labeled Hek-293 / FIXD35gvl-XIN cells showing that after antibiotic selection, a recombinant cell population was obtained in which 21% of the cells express the rFIX protein.
A figura 9 ilustra a análise da expressão da proteína doFIXD359vLXIN no sobrenadante das células recombinantes Hep-G2/FIXD359vLXIN e Hek293/ FIXd359vLXIN e ausência do mesmo nas célulasvirgens. À esquerda: análise da linhagem celular HepG2/FIX-LXIN. Raia 1:Figure 9 illustrates analysis of the expression of doFIXD359vLXIN protein in Hep-G2 / FIXD359vLXIN and Hek293 / FIXd359vLXIN recombinant cells supernatant and absence thereof in virgin cells. Left: HepG2 / FIX-LXIN cell line analysis. Lane 1:
Controle positivo - FIX derivado do plasma (1,5 ug/uL - Octanyne®). Raia 2:Hep-G2 virgem (15 ug) e Raia 3: Hep-G2/FIXD3s9vLXIN+ (15 ug). Primeiro - gelcorado com Coomassie e ao lado - autoradiograma do western blot após aimunodetecção utilizando o anticorpo anti-FIX. À direita análise da linhagemcelular Hek-293/FIX-LXIN. Raia 1: Controle positivo - FIX derivado do plasma(1,5 ug/uL- Octanyne®). Raia 2: Hek-293 virgem (15 ug) e Raia 3: Hek-293/FIXD359vLXIN+ (15 ug). Primeiro - gel corado com Coomassie e ao lado -autoradiograma do western blot após a imunodetecção utilizando o anticorpoanti-FIX. Observa-se uma banda imunoreativa do tamanho esperado de 57 kDana célula produtora de rFIX e no controle positivo e a ausência da mesma nacélula virgem. O marcador peso molecular LMW (fragmentos 97, 66, 45, 30 e20 kDa.Positive control - plasma derived FIX (1.5 µg / µL - Octanyne®). Lane 2: Virgin Hep-G2 (15 µg) and Lane 3: Hep-G2 / FIXD3s9vLXIN + (15 µg). First - Coomassie gelcorated and next - western blot autoradiogram after aim detection using anti-FIX antibody. Right analysis of Hek-293 / FIX-LXIN cell line. Lane 1: Positive Control - Plasma derived FIX (1.5 µg / µL-Octanyne®). Lane 2: virgin Hek-293 (15 µg) and Lane 3: Hek-293 / FIXD359vLXIN + (15 µg). First - Coomassie stained gel and next to the western blot autoradiogram after immunodetection using the anti-FIX antibody. An immunoreactive band of the expected size of 57 kDana from the rFIX-producing cell is observed and in the positive control and the absence of the same virgin cell. The molecular weight marker LMW (fragments 97, 66, 45, 30 and 20 kDa.
Descrição Detalhada da InvençãoDetailed Description of the Invention
Em uma primeira realização, a presente invenção refere-se a umaproteína recombinante do fator IX de coagulação sangüínea humana, queapresenta uma mutação na posição 359, onde o aminoácido ácido aspártico(D) é substituído por valina (V), sendo que a proteína recombinante é produzidapor linhagens humanas.In a first embodiment, the present invention relates to a recombinant human blood coagulation factor IX protein, which has a mutation at position 359, where the amino acid aspartic acid (D) is replaced by valine (V), the recombinant protein being It is produced by human lineages.
Dentro de uma realização particular da presente invenção, aslinhagens celulares humanas são selecionadas entre linhagens hepáticas erenais. De preferência, as linhagens humanas utilizadas pelo presenteinvenção para a obtenção de uma proteína recombinante do fator IX decoagulação sangüínea humana mutada são HepG2 e Hek293.Within a particular embodiment of the present invention, human cell lines are selected from erenal liver lines. Preferably, the human strains used by the present invention to obtain a mutated human blood coagulation factor IX recombinant protein are HepG2 and Hek293.
A proteína recombinante do fator IX de coagulação sangüíneahumana mutada (D359V) da presente invenção é produzida em níveis elevadose apresenta atividade biológica aumentada, sendo apropriada para uso notratamento de hemofilia B.The recombinant mutated human blood coagulation factor IX protein (D359V) of the present invention is produced at high levels and has increased biological activity and is suitable for use in hemophilia B treatment.
A presente invenção trata ainda de uma composição quecompreende a proteína recombinante do fator IX da presente invenção eveículos, excipientes ou estabilizantes farmaceuticamente aceitáveis. Acomposição farmacêutica de acordo com a presente invenção pode ser líquida,semi-sólida ou sólida e podem ser adaptadas para qualquer rota deadministração enteral ou parenteral, seja de liberação imediata ou modificada.The present invention further relates to a composition comprising the recombinant factor IX protein of the present invention and pharmaceutically acceptable carriers, excipients or stabilizers. The pharmaceutical composition according to the present invention may be liquid, semi-solid or solid and may be adapted to any enteral or parenteral route of administration, whether immediate or modified release.
Em realização particular, dita composição farmacêutica é adaptada paraadministração oral, mais particularmente na forma de comprimidos, cápsulas,tinturas, emulsões, lipossomas, microcápsulas ou nanoparticulas.In particular embodiment, said pharmaceutical composition is adapted for oral administration, more particularly in the form of tablets, capsules, tinctures, emulsions, liposomes, microcapsules or nanoparticles.
Veículos, excipientes ou estabilizantes adequados à invençãosão, por exemplo, e sem qualquer limitação, aqueles citados na obraRemington's Pharmaceutical Sciences, da editora americana Mack Publishing,ou ainda na Farmacopéia Européia ou Farmacopéia Brasileira.Suitable vehicles, excipients or stabilizers for the invention are, for example, and without limitation, those cited in Remington's Pharmaceutical Sciences, by the American publisher Mack Publishing, or in the European Pharmacopoeia or Brazilian Pharmacopoeia.
Em outro aspecto, a presente invenção trata do uso da proteínaFIX recombinante mutada da invenção, ou de composições que a contêm, paraa preparação de medicamentos para tratar hemofilia B.In another aspect, the present invention addresses the use of the mutant recombinant FIX protein of the invention, or compositions containing it, for the preparation of medicaments for treating hemophilia B.
Em outra realização, a presente invenção refere-se a um métodode obtenção de uma proteína recombinante do fator IX de coagulaçãosangüínea humana mutada, em que o método compreende:a) isolamento e purificação do fator IX de coagulação sangüíneahumano;In another embodiment, the present invention relates to a method of obtaining a mutated human blood coagulation factor IX recombinant protein, wherein the method comprises: a) isolating and purifying human blood coagulation factor IX;
b) inserção de sítios de restrição e substituição do aminoácidoácido aspártico na posição 359 por valina (D359V);b) insertion of restriction sites and replacement of the aspartic amino acid at position 359 by valine (D359V);
c) introdução da molécula de fator IX obtido pela etapa (b) em um vetor;c) introducing the factor IX molecule obtained by step (b) into a vector;
d) transfecção de células humanas com o vetor obtido pela etapa (c);d) transfecting human cells with the vector obtained by step (c);
e) tratamento da cultura de células humanas com concentraçõescrescentes de antibiótico;e) treating human cell culture with increasing antibiotic concentrations;
f) cultivo das culturas recuperadas; ef) cultivation of recovered crops; and
g) recuperação do fator IX recombinante obtido pelasmencionadas culturas.g) recovery of recombinant factor IX obtained by said cultures.
De acordo com um aspecto preferencial da presente invenção, aetapa (b) do método acima é realizada através de PCR utilizando primers específicos portadores de sítios de restrição. De preferência, os sítios derestrição inseridos são Hpa I e Bam Hl.In accordance with a preferred aspect of the present invention, step (b) of the above method is performed by PCR using specific restriction site-bearing primers. Preferably, the restriction sites inserted are Hpa I and Bam Hl.
A etapa (c) é realizada particularmente a partir da digestão damolécula da etapa (b) com endonucleases específicas, preferencialmente Hpa Ie Bam Hl, e ligação da molécula assim tratada ao DNA do vetor linearizadocom as mesmas enzimas de restrição.Step (c) is performed particularly from the molecule digestion of step (b) with specific endonucleases, preferably Hpa Ie Bam H1, and ligation of the thus treated molecule to the linearized vector DNA with the same restriction enzymes.
Em uma realização preferencial, o vetor utilizado pela presenteinvenção é um vetor retroviral, particularmente, plasmidial retroviral. De acordocom uma realização preferencial, o vetor utilizado é pLXIN.In a preferred embodiment, the vector used by the present invention is a retroviral vector, particularly a retroviral plasmid. According to a preferred embodiment, the vector used is pLXIN.
De acordo com outro aspecto da presente invenção, a transfecçãodas células humanas com o vetor obtido pela etapa (c)vpode ser realizada porqualquer método conhecido na técnica. Particularmente, a transfecção érealizada através do método de eletroporaçãoParticularmente, o método da presente invenção apresenta doissistemas de produção de retrovírus para aumentar o título viral. Primeiramente,o FIX é transfectado em células produtoras de retrovírus ecotrópicas. Naseqüência, o retrovírus produzido pelas células mencionadas acima étransduzido em linhagens de células anfotrópicas. Por fim, a célula humana é então transduzida com o retrovírus produzido pela linhagem anfotrópica.According to another aspect of the present invention, transfection of human cells with the vector obtained by step (c) can be performed by any method known in the art. Particularly, the transfection is performed by the electroporation method. In particular, the method of the present invention has two retrovirus production systems to increase the viral titer. First, FIX is transfected into ecotropic retrovirus producing cells. Accordingly, the retrovirus produced by the cells mentioned above is translated into amphotropic cell lines. Finally, the human cell is then transduced with the retrovirus produced by the amphotropic strain.
Ainda, o antibiótico utilizado pela presente invenção para otratamento das culturas transfectadas pode ser qualquer um apropriado noestado da técnica, desde que o vetor utilizado contenha um gene de resistênciapara o mesmo. De forma particular o antibiótico utilizado é geneticina, semexcluir qualquer outro sistema de seleção adequadoFurther, the antibiotic used by the present invention for the treatment of transfected cultures may be any suitable in the art provided that the vector used contains a resistance gene for the same. In particular, the antibiotic used is geneticin, without excluding any other appropriate selection system.
Conforme descrito acima, a seleção das culturas transduzidas, érealizada a partir de tratamento crescente com antibiótico. De acordo com apresente invenção, as doses de antibiótico utilizadas na primeira seleção(baixas doses) estão compreendidas entre 400 e 600 ug/mL, e as doses deantibiótico utilizadas na segunda seleção (altas doses) estão compreendidasentre 1.000 e 2.000 ug/mL.As described above, selection of transduced cultures is performed from increasing antibiotic treatment. According to the present invention, the antibiotic doses used in the first selection (low doses) are between 400 and 600 µg / mL, and the antibiotic doses used in the second selection (high doses) are between 1,000 and 2,000 µg / mL.
O cultivo das células é realizado sob condição padrão, podendoser determinado pelo técnico no assunto dependendo do tipo de linhagemcelular utilizada. Particularmente, o cultivo é realizado a uma temperatura de37°C e 5% de C02.Cultivation of cells is performed under standard condition, and may be determined by the subject matter expert depending on the type of cell lineage used. Particularly, cultivation is carried out at a temperature of 37 ° C and 5% CO2.
Ainda, a recuperação do fator IX recombinante mutado obtido pelométodo da presente invenção pode ser realizada por qualquer métodoconhecido na técnica.Further, recovery of the mutant recombinant factor IX obtained by the method of the present invention may be accomplished by any method known in the art.
O método da presente invenção permite obter proteínas FIXrecombinantes humanas mutadas em altos níveis, sendo que as proteínasapresentam alta atividade biológica e não apresentam os inconvenientesapresentados pelas proteínas recombinantes da técnica.The method of the present invention makes it possible to obtain high-level mutated human recombinant FIX proteins, the proteins having high biological activity and not having the drawbacks of recombinant proteins in the art.
De acordo com uma realização adicional, a presente invençãorefere-se à proteína recombinante do fator IX mutada obtida pelo métododescrito acima, apresentando uma substituição de aminoácido ácido asparticoda posição 359 por valina.According to a further embodiment, the present invention relates to the mutated factor IX recombinant protein obtained by the method described above, showing an amino acid substitution of aspartic acid 359 for valine.
Ainda outro objeto da presente invenção refere-se ao uso da proteína obtida pelo método da presente invenção na preparação de ummedicamento para tratar hemofilia B.Still another object of the present invention relates to the use of the protein obtained by the method of the present invention in the preparation of a medicament for treating hemophilia B.
A presente invenção pode ser compreendida de forma mais clarae precisa através da leitura dos exemplos a seguir, que ilustram a presenteinvenção sem apresentar qualquer caráter limitativo.The present invention may be more clearly and precisely understood by reading the following examples which illustrate the present invention without any limiting character.
ExemplosExamples
Exemplo 1Example 1
Obtenção de vetor contendo a molécula do Fator IX mutanteObtaining vector containing the mutant Factor IX molecule
O clone recombinante portador de seqüências de cDNA relativasao fator IX nomeado 7-5A2 foi isolado e purificado após quatro rastreamentosconsecutivos de uma biblioteca de cDNA de fígado humana, utilizando comosonda o clone plasmidial pMac-FIX, gentilmente cedido pelo Prof. Dr. MarceloBrígido da UnB. Em seguida, esse clone foi submetido à reação de PCRutilizando primers específicos portadores dos sítios para as enzimas derestrição Hpa I + Bam Hl em suas extremidades, e um fragmento de tamanhoesperado de 1,4 kb foi obtido. Após a purificação desse fragmento de DNA, foirealizada a digestão com endonucleases específicas (Hpa I + Bam Hl) e omesmo submetido à ligação ao DNA plasmidial retroviral pLXIN linearizadocom as mesmas enzimas de restrição, conforme demonstrado pela figura 1.The recombinant clone carrying factor IX cDNA sequences named 7-5A2 was isolated and purified after four consecutive screenings of a human liver cDNA library using as the plasmid clone pMac-FIX, kindly provided by Prof. Dr. MarceloBrigido from UnB. This clone was then subjected to the PCR reaction using site-specific primers for Hpa I + Bam Hl restriction enzymes at their ends, and an expected 1.4 kb fragment size was obtained. After purification of this DNA fragment, digestion with specific endonucleases (Hpa I + Bam Hl) was performed and the same was ligated to the pLXIN retroviral plasmid DNA linearized with the same restriction enzymes, as shown in Figure 1.
Em seguida, a seqüência do cDNA relativa ao fator IX clonado novetor pLXIN foi submetida ao seqüenciamento, o que mostrou a presença damutação D359V no domínio catalítico da proteína (cadeia pesada), ilustrado pelafigura 2. O desenho esquemático da proteína recombinante FIXd359vLXIN decoagulação sangüínea humana da presente invenção pode ser verificado na figura 3.Subsequently, the cloning factor IX cDNA sequence from novel pLXIN cloned factor was sequenced, which showed the presence of D359V mutation in the protein (heavy chain) catalytic domain, illustrated by Figure 2. Schematic drawing of recombinant human FIXd359vLXIN protein and blood coagulation of the present invention can be seen in figure 3.
Exemplo 2Example 2
Produção de FIX mutante a partir de Linhagens HumanasMutant FIX Production from Human Lineages
As linhagens celulares humanas HepG2 e Hek293 foram transduzidas com o vetor retroviral pLXIN-FIX o btido pelo exemplo! Após otratamento com concentrações crescentes de geneticina, foi realizada a análise daatividade biológica do fator IX presente no sobrenadante dessas linhagenscelulares. A figura 5 mostra a estratégia utilizada para a geração de linhagenscelulares humanas com expressão estável do fator IX de coagulação sangüínea humana, e elevados níveis do mesmo. Após a seleção das linhagens celularesrecombinantes HepG2-FIXD359vLXIN e Hek293- FIXD359vLXIN, foi realizado oensaio do tempo de tromboplastina parcial ativada (aPTT), que permitiu mostrarque ambas linhagens celulares expressam níveis do fator IX de coagulaçãosangüínea humana da presente invenção, de 9 a 11 vezes superiores ao nível defator IX presente no plasma sangüíneo (figura 6), e ao fator IX recombinanteproduzido pelas linhagens murinas descritas no estado da técnica.Human cell lines HepG2 and Hek293 were transduced with the retroviral vector pLXIN-FIX as shown in the example! After treatment with increasing concentrations of geneticin, the biological activity of factor IX present in the supernatant of these cell lines was analyzed. Figure 5 shows the strategy used to generate human cell lines with stable expression of human blood coagulation factor IX, and high levels of it. After selection of the recombinant HepG2-FIXD359vLXIN and Hek293-FIXD359vLXIN cell lines, the activated partial thromboplastin time (aPTT) assay was performed, which showed that both cell lines express human blood coagulation factor IX levels of 9 to 11 times. higher than the defactor level IX present in blood plasma (Figure 6), and the recombinant factor IX produced by the murine strains described in the state of the art.
Exemplo 3Example 3
Avaliação do nível de produção da molécula recombinante da invençãopelas linhagens celulares humanas HEPG2 e HEK293Evaluation of the level of production of the recombinant molecule of the invention by HEPG2 and HEK293 human cell lines
A avaliação do nível de produção da molécula recombinante FIX-LXIN pelas linhagens celulares humanas HepG2 e Hek293 pode ser realizadapor RT-PCR convencional; citometria de fluxo, ensaio de tempo detromboplastina parcial ativada (aPTT) e western blot.Evaluation of the production level of the recombinant FIX-LXIN molecule by HepG2 and Hek293 human cell lines can be performed by standard RT-PCR; flow cytometry, activated partial detromboplastin time assay (aPTT) and western blot.
RT-PCR convencionalConventional RT-PCR
A avaliação por RT-PCR convencional, ilustrada pela figura 7,permitiu diagnosticar a presença do mRNA relativo ao fator IX de coagulaçãoda presente invenção nas linhagens celulares recombinantes HepG2-FIXD359vLXIN e Hek293-FIXD359vLXIN, utilizando o seguinte procedimento: érealizada a extração de RNA total utilizando o kit RNAsy Mini kit (Quiagen), deacordo com as instruções do fabricante. Em seguida, 1 a 3 ug de RNA total éconvertido em cDNA, utilizando o kit Superscript II (Invitrogen), conforme asinstruções do fabricante, e 50 pmoles dos oligonucleotídeos específicos (P5 eP3) Posteriormente, o fragmento de DNA relativo ao fator IX da invenção éamplificado em uma mistura reacional que contém: 2 uL do cDNA, 0,2 M dedNTPs; 1 U da enzima Taq DNA polimerase (Amershan Bioscience), 2,5 uL dotampão 10x da respectiva enzima (Amershan Bioscience) e 10 pmoles de cadaoligonucleotídeo P3FIX5seq e P5FIX3seq. Essa reação foi realizada em termociclador com o programa: 95°C por 2min; 35 ciclos de 95°C por 40s, 55°Cpor 40s e 72°C por 1 min; 72°C por 10min.Conventional RT-PCR evaluation, illustrated in Figure 7, allowed the diagnosis of the presence of coagulation factor IX mRNA of the present invention in the HepG2-FIXD359vLXIN and Hek293-FIXD359vLXIN recombinant cell lines using the following procedure: Total RNA extraction is performed. RNAsy Mini kit (Quiagen) according to the manufacturer's instructions. Then 1 to 3 µg of total RNA is converted to cDNA using the Superscript II kit (Invitrogen) as per manufacturer's instructions and 50 pmoles of specific oligonucleotides (P5 eP3). Next, the factor IX DNA fragment of the invention It is amplified in a reaction mixture containing: 2 µl cDNA, 0.2 M dedNTPs; 1 U of Taq DNA polymerase enzyme (Amershan Bioscience), 2.5 µl with 10x buffer of respective enzyme (Amershan Bioscience) and 10 pmoles of each P3FIX5seq and P5FIX3seq oligonucleotide. This reaction was performed in a thermal cycler with the program: 95 ° C for 2min; 35 cycles of 95 ° C for 40s, 55 ° C for 40s and 72 ° C for 1 min; 72 ° C for 10min.
Citometria de fluxoPara marcação intracelular da proteína do fator IX recombinantemutado da presente invenção, cerca de 1x106 de células HepG2/F/X-LX7/V eHek-293/F/X-LX7A/ são fixadas com paraformaldeído 1% (m/v) durante 20minutos a 4°C e, em seguida, permeabilizadas com Tween 0,5% por 15minutos a 37°C. Após a etapa de lavagem com PBS-BSA 2%, as células sãoincubadas com solução de bloqueio (soro de cabra 10%) por 1 hora a 37°C. Aseguir, é colocado o anticorpo primário monoclonal anti-fator IX diluído emsolução PBS-BSA 2% (1:100), por aproximadamente 24 horas a temperaturaambiente. Em seguida, é realizada a lavagem com PBS-BSA 2% e as célulassão incubadas com o anticorpo secundário diluído em solução PBS-BSA 2%(1:400), durante 45 minutos em temperatura ambiente. Após este período, érealizada uma segunda lavagem com PBS 1X, e as células são eluídas emIOOjxL de PBS 1X, para posterior análise de emissão de fluorescência pelascélulas recombinantes por de citometria de fluxo. A quantificação da emissãode fluorescência foi realizada com uma suspensão celular utilizando 10.000 eventos (células) em citômetro de fluxo laminar (FACSort, Beckton Dickinson,San Jose, CA,EUA).Flow cytometry For intracellular labeling of the recombinant factor IX protein of the present invention, about 1x10 6 HepG2 / F / X-LX7 / V and Hek-293 / F / X-LX7A / cells are fixed with 1% (m / v) paraformaldehyde for 20 minutes at 4 ° C and then permeated with 0.5% Tween for 15 minutes at 37 ° C. After the 2% PBS-BSA wash step, cells are incubated with blocking solution (10% goat serum) for 1 hour at 37 ° C. Next, diluted anti-factor IX monoclonal primary antibody in 2% PBS-BSA (1: 100) solution is placed for approximately 24 hours at room temperature. Washing with 2% PBS-BSA is then performed and the cells are incubated with secondary antibody diluted in 2% PBS-BSA solution (1: 400) for 45 minutes at room temperature. After this time, a second wash with 1X PBS is performed, and the cells are eluted in 10x1 PBS 1X for further fluorescence emission analysis by recombinant cells by flow cytometry. Quantitation of fluorescence emission was performed with a cell suspension using 10,000 events (cells) in laminar flow cytometer (FACSort, Beckton Dickinson, San Jose, CA, USA).
A avaliação por citometria de fluxo mostrou que as populaçõescelulares recombinantes, HepG2-FIXD359vLXIN e Hek-293-FIXD359vLXIN,apresentam expressão estável do FIX da ordem de 46% e 21%,respectivamente, conforme ilustrado pela figura 8.Flow cytometric evaluation showed that the recombinant cell populations, HepG2-FIXD359vLXIN and Hek-293-FIXD359vLXIN, showed stable expression of FIX of 46% and 21%, respectively, as shown in Figure 8.
Ensaio de tempo de tromboplastina parcial ativada f aPTT)Activated Partial Thromboplastin Time Assay (aPTT)
A avaliação da atividade biológica é realizada pelo teste de tempode tromboplastina parcial ativada (aPTT), ilustrado pela tabela 1.The evaluation of biological activity is performed by the activated partial thromboplastin time test (aPTT), illustrated by table 1.
Tabela 1Table 1
<table>table see original document page 18</column></row><table><table> table see original document page 18 </column> </row> <table>
O teste da coagulação aPTT, mede a velocidade in vitro que umaamostra de plasma contendo o FIX leva para coagular uma amostra de plasmadeficiente em FIX. O parâmetro medido é o tempo necessário para a atividadeprotrombínica, sendo assim, o tempo levado para coagulação varia de acordocom a concentração de FIX presente na amostra a ser analisada. Na presenteinvenção, a amostra a ser testada foi diluída 1:10 em tampão (Owren's Veronal- Biomérieux) e, depois de 20 segundos, misturada com o plasma deficiente emFIX humano (Biomérieux, Durham), fosfolipídeos e um ativador de contato(Platelin® LS - Biomérieux). Depois da incubação por aproximadamente 240segundos a 37°C, 100 uL de cloreto de cálcio (CaCI2 0,25M) foi adicionado, e otempo para a formação do coágulo foi marcado utilizando o coagulômetroautomático (COAG-A-MATE® XM - Organon Teknika), de acordo com asinstruções do fabricante. Antes de iniciar a dosagem, uma curva de calibraçãopadrão foi construída utilizando FIX derivado do plasma (Verify - Referenceplasma - (Organon Teknika, Durham), reconstituído com 1ml_ de águadestilada e diluído para concentração de (1 U ml_ FIX). Seis diluições (1:5;1:10; 1:20; 1:40; 1:80; 1:160) são realizadas em tampão (Owrens Veronal -Biomérieux), e usadas para construção da curva para cada amostra. Umarelação linear foi traçada em um gráfico, com atividade de FIX em escalalogarítima (abscissa) e o tempo de tromboplastina parcialmente ativa(segundos), e a porcentagem de atividade de FIX calculada foi obtida com amédia da inclinação da curva.The aPTT coagulation test measures the in vitro velocity that a plasma sample containing FIX leads to coagulate a sample of FIX-deficient plasmid. The measured parameter is the time required for prothrombin activity, so the time taken for coagulation varies according to the FIX concentration present in the sample to be analyzed. In the present invention, the sample to be tested was diluted 1:10 in buffer (Owren's Veronal-Biomérieux) and, after 20 seconds, mixed with human FIX deficient plasma (Biomérieux, Durham), phospholipids and a contact activator (Platelin®). LS - Biomérieux). After incubation for approximately 240 seconds at 37 ° C, 100 µl of calcium chloride (0.25M CaCl2) was added, and the time for clot formation was labeled using the automated coagulometer (COAG-A-MATE® XM - Organon Teknika). in accordance with the manufacturer's instructions. Prior to initiating dosing, a standard calibration curve was constructed using plasma derived FIX (Verify - Referenceplasma - (Organon Teknika, Durham), reconstituted with 1 ml of distilled water and diluted to a concentration of 1 U ml FIX. : 5; 1: 10; 1:20; 1:40; 1:80; 1: 160) are made in buffer (Owrens Veronal -Biomérieux), and used to construct the curve for each sample. graph, with scalalogarithmic FIX activity (abscissa) and partially active thromboplastin time (seconds), and the percentage of calculated FIX activity was obtained by averaging the slope of the curve.
A tabela 1 mostra a análise da atividade biológica do FIX presenteno sobrenadante das duas populações celulares HepG2/FIXD35gVLXIN e Hek-293/FIXd359vLXIN através do teste aPTT. Pode-se observar que as populaçõescelulares recombinantes portadoras do FIX após o tratamento com geneticina,apresentam níveis de atividade biológica de FIX da ordem de 9 a 11 vezes aonível de fator IX presente no plasma sangüíneo.Table 1 shows the analysis of the biological activity of the present supernatant FIX of the two cell populations HepG2 / FIXD35gVLXIN and Hek-293 / FIXd359vLXIN by the aPTT test. It can be observed that FIX-bearing recombinant cell populations after geneticin treatment have levels of FIX biological activity of 9 to 11 times the level of factor IX present in blood plasma.
Western BlotWestern blot
A caracterização da proteína FIXD359VLXIN, produzida pelaslinhagens celulares HepG2/FIXd359vLXIN e Hek-293/FIXD359vLXIN, foi realizadapor western blot.The characterization of the FIXD359VLXIN protein produced by HepG2 / FIXd359vLXIN and Hek-293 / FIXD359vLXIN cell lines was performed by western blot.
Para o preparo do gel, 7 ml_ de sobrenadante são coletados de1x107 células HepG2/FIX-LXIN e Hek-293/FIX-LXIN, e submetidos aconcentração em colunas Centrícon® Amicon® (Millipore), por centrifugação à7500 rpm durante 2 a 4 horas. Em seguida, é realizada uma lavagem,utilizando 3 ml de H20 MHHQ autoclavada, por centrifugação à 6000 rpm.Posteriormente, as colunas foram invertidas para eluição das amostras emaproximadamente 150 ul de H20 MHHQ autoclavada, e centrifugadas à 6000rpm durante 15 minutos. Após a eluição da proteína, as amostras sãoquantificadas em um espectrofotômetro pelo Método de Bradford.For the preparation of the gel, 7 ml of supernatant is collected from 1x10 7 HepG2 / FIX-LXIN and Hek-293 / FIX-LXIN cells and subjected to Centrícon® Amicon® columns (Millipore) by centrifugation at 7500 rpm for 2 to 4 hours. . A wash is then performed using 3 ml of autoclaved H20 MHHQ by centrifugation at 6000 rpm. Subsequently, the columns were inverted to elute the samples to approximately 150 µl of autoclaved H20 MHHQ, and centrifuged at 6000rpm for 15 minutes. After protein elution, samples are quantified on a spectrophotometer by the Bradford Method.
Para a realização do Western Blot, as amostras foram submetidasà eletroforese utilizando o sistema Phast System. Cerca de 10 uL da amostra(30 ug) foram misturadas com 10 pi do tampão de amostra (10 mM Tris-HCI,pH 8,0; 2,5% SDS (m/v); 1 mM EDTA; 0,01% (v/v) de azul de bromofenol), e1,5 pi de p-mercaptoetanol (Sigma). A mistura é submetida ao aquecimento eaplicada no gel. A eletroforese é realizada em tampão de corrida para oPhastGel (SDS Buffer - Amersham Biociences) a 100 V, duranteaproximadamente 40 minutos. Dois géis, exatamente idênticos, são submetidosà eletroforese, um para ser corado e outro para a transferência. Após aeletroforese, o gel a ser corado é incubado por 1 hora em solução fixadora(etanol 40% (v/v), ácido acético 10% (v/v), água estéril qsp 0,125L) e,posteriormente, corado com solução de azul brilhante de Coomassie (1,3%ácido fosfórico (v/v), 0,75 M (NH4)2 S04, 0,1% Coomassie B-Blue G250 (v/v)),durante 18 horas. Em seguida, o gel é lavado de 3 a 5 vezes com água MilliQ,e montado entre duas folhas de papel celofane para a completa secagem.For Western blotting, the samples were electrophoresed using the Phast System. About 10 µl of the sample (30 µg) was mixed with 10 µl of the sample buffer (10 mM Tris-HCl, pH 8.0; 2.5% SDS (m / v); 1 mM EDTA; 0.01% (v / v) bromophenol blue), and 1.5 µg p-mercaptoethanol (Sigma). The mixture is subjected to heating and applied to the gel. Electrophoresis is performed in PhastGel running buffer (SDS Buffer - Amersham Biociences) at 100 V for approximately 40 minutes. Two exactly identical gels are electrophoresed, one for staining and one for transfer. After electrophoresis, the gel to be stained is incubated for 1 hour in a fixative solution (40% (v / v) ethanol, 10% (v / v) acetic acid, 0.25L qsp sterile water) and then stained with Coomassie bright blue (1.3% phosphoric acid (v / v), 0.75 M (NH4) 2 SO4, 0.1% Coomassie B-Blue G250 (v / v)) for 18 hours. The gel is then washed 3 to 5 times with MilliQ water, and mounted between two sheets of cellophane paper for complete drying.
Após a eletroforese, as proteínas contidas no gel de poliacrilamidasão transferidas para uma membrana de nitrocelulose, pelo método de eletro-transferência semi-seca com o aparelho Phast System - Amersham Biociences,conforme as instruções do fabricante. A eletroforese é realizada em tampão detransferência (0,25 M de Tris-Base; 0,96 M de glicina; 0,5% SDS (sódioduodecil sulfato); água estéril qsp 1L) a 90 V, durante aproximadamente 2horas e 30 minutos. Concluída a transferência a membrana é preparada paraimunodetecção.After 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 - Amersham Biociences apparatus according to the manufacturer's instructions. Electrophoresis is performed in a transfer buffer (0.25 M Tris-Base; 0.96 M Glycine; 0.5% SDS (Sodium Dioduodecyl Sulphate); sterile water qsp 1L) at 90 V for approximately 2 hours and 30 minutes. After the transfer is complete the membrane is prepared for immunodetection.
A membrana de nitrocelulose é submetida ao bloqueio de sítiosinespecíficos pela adição da solução de bloqueio TBS-T (10 mM Tris pH 7,5, 150mM NaCI, 0,1% Tween-20), em 5% de leite em pó (m/v), por cerca de 16 horas.Após o bloqueio, a membrana é lavada rapidamente 2 vezes com TBS 1X, 0,1%Tween-20, e incubada com anticorpo primário monoclonal (QED), diluído emsolução TBS-T (1:800) durante 2 horas à temperatura ambiente, e sob agitaçãoconstante. Em seguida, é realizada a lavagem da membrana com TBS 1X, 0,1%Tween-20 (2X / 15 minutos; 3X / 5 minutos), e a membrana é incubada com oanticorpo secundário diluído em solução TBS-T (1:3000), durante 1 hora. Após aincubação com o segundo anticorpo, a membrana é lavada novamente e inicia-se o procedimento de detecção. Para a detecção, é utilizado o "kit ECL Westernblotting" (Amersham Biosciences), conforme instruções do fabricante.Finalmente, a membrana é exposta ao filme de raio-X por 15 segundos e arevelação é realizada em máquinas automáticas da KODAK.The nitrocellulose membrane is subjected to nonspecific site blockade by the addition of the TBS-T blocking solution (10 mM Tris pH 7.5, 150mM NaCl, 0.1% Tween-20) in 5% milk powder (m / w v) for about 16 hours. After blockage, the membrane is rapidly washed 2 times with 1X TBS, 0.1% Tween-20, and incubated with primary monoclonal antibody (QED), diluted with TBS-T solution (1: 800) for 2 hours at room temperature and under constant stirring. The membrane is then washed with 1X TBS, 0.1% Tween-20 (2X / 15 minutes; 3X / 5 minutes), and the membrane is incubated with the secondary antibody diluted in TBS-T solution (1: 3000 ) for 1 hour. After incubation with the second antibody, the membrane is washed again and the detection procedure is initiated. For detection, the "Western Blotting ECL Kit" (Amersham Biosciences) is used per manufacturer's instructions. Finally, the membrane is exposed to the X-ray film for 15 seconds and development is performed on KODAK vending machines.
Conforme ilustrado pela figura 9, pode-se observar uma bandaimuno-reativa do tamanho esperado de 57 kDa, referente à proteína do fator IXde coagulação sangüínea humana, presente nos sobrenadantes das célulasrecombinantes FIXD359V e a ausência da mesma nas células HepG2 e Hek-293 virgens.As shown in Figure 9, an expected 57 kDa bandimmuno-reactive antibody can be observed for the human blood coagulation factor IX protein present in the supernatants of the FIXD359V recombinant cells and in the virgin HepG2 and Hek-293 cells. .
Como bem compreendem os técnicos no assunto, são possíveisnumerosas modificações e variações da presente invenção à luz dosensinamentos acima, sem se afastar do seu escopo de proteção, conformedelimitado pelas reivindicações anexas.As is well understood by those skilled in the art, numerous modifications and variations of the present invention are possible in light of the above teachings, without departing from their scope of protection as limited by the appended claims.
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