ES2170815T5 - Use of a PM-1 antibody or an MH166 antibody to enhance the anti-tumor effect of cisplatin or carboplatin - Google Patents
Use of a PM-1 antibody or an MH166 antibody to enhance the anti-tumor effect of cisplatin or carboplatin Download PDFInfo
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- ES2170815T5 ES2170815T5 ES95942318T ES95942318T ES2170815T5 ES 2170815 T5 ES2170815 T5 ES 2170815T5 ES 95942318 T ES95942318 T ES 95942318T ES 95942318 T ES95942318 T ES 95942318T ES 2170815 T5 ES2170815 T5 ES 2170815T5
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
POTENCIADOR PARA AGENTES ANTITUMORALES COMO POR EJEMPLO, COMPUESTOS DE PLATINO TALES COMO CISPLATINO Y CARBOPLATINO, MITOMICINA C, ETC., QUE INCLUYE UN ANTAGONISTA DE INTERLEUCINA 6 (IL - 6) TALES COMO UN ANTICUERPO FRENTE A IL - 6, UN ANTICUERPO FRENTE AL RECEPTOR DE IL - 6, UN ANTICUERPO FRENTE A LA PROTEINA GP130, ETC.POTENTIATOR FOR ANTITUMOR AGENTS AS FOR EXAMPLE, PLATINUM COMPOUNDS SUCH AS CISPLATINO AND CARBOPLATINO, MITOMYCIN C, ETC., WHICH INCLUDES AN INTERLEUCINE ANTAGONIST 6 (IL - 6) AS AN ANTIBODY AGAINST ILL-6 FROM IL - 6, AN ANTIBODY AGAINST PROTEIN GP130, ETC.
Description
Uso de un anticuerpo PM-1 o de un anticuerpo MH166 para potenciar el efecto antitumoral de cisplatino o carboplatino. Use of a PM-1 antibody or an MH166 antibody to enhance the antitumor effect of cisplatin or carboplatin.
Campo técnico Technical field
Esta invención se refiere a un potenciador de los efectos de un agente antitumoral que comprende un antagonista deinterleucina-6 que ayuda y potencia los efectos de agentes antitumorales en el tratamiento de tumores. This invention relates to an enhancer of the effects of an antitumor agent comprising an interleukin-6 antagonist that aids and enhances the effects of antitumor agents in the treatment of tumors.
En el pasado se han usado para quimioterapia sobre tumores humanos diversos fármacos como agentesalquilantes, antagonistas del metabolismo, antibióticos antitumorales y compuestos de platino. En el caso de que no se observen efectos terapéuticos notables cuando se usan agentes antitumorales solos, se han ideado procedimientos terapéuticos en los que se usan una pluralidad de agentes antitumorales de forma concomitante(Frei, E. III, Cancer Res., 32, 2593-2607, 1992). Las células tumorales presentan diversas sensibilidades a losagentes antitumorales y se sabe que ciertas células presentan resistencia a la terapia por los agentes antitumorales(Magrath, I., New Directions in Cancer Treatment, 1989, Springer-Verlag). Se dice que la adquisición de resistencia a la terapia por las células tumorales está causada, por ejemplo, por la aparición de resistencia multifármaco (MDR) (Tsuruo, T. et al., Cancer Res., 42, 4730-4733, 1982), por reducción en la recaptación de agente antitumoral(Sherman, S.E. et al., Science, 230, 412, 1985), por una mayor actividad reparadora del DNA (Borch, R.F., Metabolism and Action of Anticancer Drugs, Powis, G. & Prough, R. ed., Taylor & Francis, Londres, 1987, 163-193),In the past, various drugs such as alkylating agents, metabolism antagonists, antitumor antibiotics and platinum compounds have been used for chemotherapy on human tumors. In the event that no notable therapeutic effects are observed when antitumor agents are used alone, therapeutic procedures have been devised in which a plurality of antitumor agents are used concomitantly (Frei, E. III, Cancer Res., 32, 2593 -2607, 1992). Tumor cells have various sensitivities to antitumor agents and it is known that certain cells have resistance to therapy by antitumor agents (Magrath, I., New Directions in Cancer Treatment, 1989, Springer-Verlag). It is said that the acquisition of resistance to therapy by tumor cells is caused, for example, by the appearance of multi-drug resistance (MDR) (Tsuruo, T. et al., Cancer Res., 42, 4730-4733, 1982) , by reduction in the reuptake of antitumor agent (Sherman, SE et al., Science, 230, 412, 1985), by a greater DNA repair activity (Borch, RF, Metabolism and Action of Anticancer Drugs, Powis, G. & Prough, R. ed., Taylor & Francis, London, 1987, 163-193),
o promover la inactivación del agente antitumoral en las células (Teicher, B.A. et al., Cancer Res., 46, 4379, 1986).En tales casos, existen muchas ocasiones en las que los efectos terapéuticos esperados no se observan sencillamente administrando un agente antitumoral. or promote the inactivation of the antitumor agent in cells (Teicher, BA et al., Cancer Res., 46, 4379, 1986) .In such cases, there are many occasions when the expected therapeutic effects are not simply observed by administering an agent. antitumor
El carcinoma de células renales presenta resistencia a los agentes antitumorales como el cisplatino, adriamicina y vinblastina (Kakehi, Y. et al., J. Urol., 139, 862-864, 1988; Kanamaru, H. et al., J. Natl. Cancer Inst., 81, 844-847, 1989; Teicher, B.A. et al., Cancer Res., 47, 388-393, 1987). Los compuestos de platino como cisplatino que poseen efectos antitumorales se unen al DNA e inhiben la síntesis del DNA y la división celular (Pinto, A.L. et al., Biochica etBiophysica Acta, 780, 167-180, 1985). Renal cell carcinoma exhibits resistance to antitumor agents such as cisplatin, adriamycin and vinblastine (Kakehi, Y. et al., J. Urol., 139, 862-864, 1988; Kanamaru, H. et al., J. Natl. Cancer Inst., 81, 844-847, 1989; Teicher, BA et al., Cancer Res., 47, 388-393, 1987). Platinum compounds such as cisplatin that possess antitumor effects bind to DNA and inhibit DNA synthesis and cell division (Pinto, A.L. et al., Biochica etBiophysica Acta, 780, 167-180, 1985).
La expresión de glutatión-S-transferasa-n (GST-n), la inhibición de los efectos de cisplastino provocados por unaumento en los niveles intracelulares de sustancias que contienen grupos sulfhidrilo, el incremento de actividadesreparadoras de DNA o la activación de oncogenes como c-myc se considera que están implicados en la resistencia ala terapia del carcinoma de células renales al cisplatino (Sklar, M.D. et al, Cancer Res., 51, 2118-2123, 1991;Mizutani, Y. et al., Cancer in press, 1994; Nakagawa, K. et al., Japan J. Cancer Res., 79, 301-305, 1988). The expression of glutathione-S-transferase-n (GST-n), the inhibition of cisplastine effects caused by an increase in the intracellular levels of substances containing sulfhydryl groups, the increase in DNA repair activities or the activation of oncogenes such as c -myc are considered to be involved in resistance to renal cell carcinoma therapy to cisplatin (Sklar, MD et al, Cancer Res., 51, 2118-2123, 1991; Mizutani, Y. et al., Cancer in press, 1994; Nakagawa, K. et al., Japan J. Cancer Res., 79, 301-305, 1988).
Además, se dice que los cambios en la permeabilidad de la membrana y las capacidades de transporte en célulastumorales causan una reducción de la recaptación de cisplatino en las células, aumentando así la resistencia a laterapia del cisplatino (Richon, V., et al., Cancer Res., 47, 2056-2061, 1987); Waud, W.R. et al., Cancer Res., 47, 6549-6555, 1987). Glutatión, un ejemplo de una sustancia que contiene grupos sulfhidrilo que está presente engrandes cantidades en las células de mamíferos, se dice que inactiva el cisplatino en las células y ciertos tipos detumores han demostrado tener niveles más elevados de glutatión y de metalotioneína intracelulares (Hromas, R.A. etal., Cancer LETT., 34, 9-13, 1987; Taylor, D.M. et al., Eur. J. Cancer, 12, 249-254, 1976). In addition, it is said that changes in membrane permeability and transport capacities in tumoural cells cause a reduction in cisplatin reuptake in cells, thereby increasing resistance to cisplatin laterapy (Richon, V., et al., Cancer Res., 47, 2056-2061, 1987); Waud, W.R. et al., Cancer Res., 47, 6549-6555, 1987). Glutathione, an example of a substance containing sulfhydryl groups that is present in large quantities in mammalian cells, is said to inactivate cisplatin in cells and certain types of tumors have been shown to have higher levels of intracellular glutathione and metallothionein (Hromas, RA etal., Cancer LETT., 34, 9-13, 1987; Taylor, DM et al., Eur. J. Cancer, 12, 249-254, 1976).
Glutatión es un tiol tripéptido. Desempeña una función importante en la inactivación de sustancias que se unen alDNA como los agentes alquilantes y cisplatino y en la reparación de las lesiones celulares originadas por estos. Unode los efectos de GST-n es que promueve la inactivación de agentes antitumorales haciendo que los agentesantitumorales del tipo descrito antes se unan a glutatión. Glutathione is a tripeptide thiol. It plays an important role in the inactivation of substances that bind alDNA such as alkylating agents and cisplatin and in the repair of cellular lesions caused by them. One of the effects of GST-n is that it promotes the inactivation of antitumor agents by causing antitumor agents of the type described above to bind glutathione.
Puesto que el carcinoma de células renales produce interleucina-6 (IL-6) y expresa el receptor de IL-6 (IL-6R), se hasugerido que la IL-6 desempeña una función en la actividad del crecimiento del carcinoma de células renales (Miki, S., et al. FEBS Lett., 250, 607-610, 1989; Takenawa, J. et al., J. Natl. Cancer Inst., 83, 1668-1672, 1991). Además,se ha descrito que el nivel de IL-6 en el suero aumenta cuando el pronóstico para el tratamiento de pacientes concarcinoma de células renales es malo (Blay, J. et al., Cancer Res., 52, 3317-3322, 1992; Tsukamoto, T. et al., J. Urol., 148, 1778-1782, 1992). Sin embargo, todavía no se ha encontrado y sigue siendo desconocida una clararelación entre IL-6 y la resistencia a la terapia del carcinoma de células renales a los agentes antitumorales. Since renal cell carcinoma produces interleukin-6 (IL-6) and expresses the IL-6 receptor (IL-6R), it has been suggested that IL-6 plays a role in the growth activity of renal cell carcinoma. (Miki, S., et al. FEBS Lett., 250, 607-610, 1989; Takenawa, J. et al., J. Natl. Cancer Inst., 83, 1668-1672, 1991). In addition, it has been reported that the level of serum IL-6 increases when the prognosis for the treatment of patients with renal cell carcinoma is poor (Blay, J. et al., Cancer Res., 52, 3317-3322, 1992 ; Tsukamoto, T. et al., J. Urol., 148, 1778-1782, 1992). However, a clear relationship between IL-6 and resistance to renal cell carcinoma therapy to antitumor agents remains unknown and remains unknown.
IL-6 es una citocina multifuncional denominada célula B que estimula el factor 2 o interferón 12. IL-6 se descubrió como un factor de diferenciación implicado en la activación de las células linfoides B (Hirano, T. et al., Nature, 324,73-76, 1986). Posteriormente, se demostró claramente que era una citocina multifuncional que afectaba a lasfunciones de diversas células (Akira, S. et al., Adv. in Immunology, 54, 1-78, 1993). IL-6 transmite su actividad biológica por medio de dos proteínas presentes en las células. IL-6 is a multifunctional cytokine called B cell that stimulates factor 2 or interferon 12. IL-6 was discovered as a differentiation factor involved in the activation of lymphoid B cells (Hirano, T. et al., Nature, 324 , 73-76, 1986). Subsequently, it was clearly demonstrated that it was a multifunctional cytokine that affected the functions of various cells (Akira, S. et al., Adv. In Immunology, 54, 1-78, 1993). IL-6 transmits its biological activity through two proteins present in the cells.
Una de estas proteínas es IL-6R, una proteína de unión a ligando que tiene un peso molecular de aproximadamente80 KD y a la que se une IL-6. Además de la forma de unión celular que se expresa en la membrana celular pasandoa través de la membrana celular, también está presente en forma IL-6R soluble (sIL-6R), que está constituidafundamentalmente de su región extracelular. Otra proteína es gp130, que tiene un peso molecular de aproximadamente 130KD y que está implicada en la transmisión de señal de la unión a no ligandos. IL-6 e IL-6Rforman un complejo IL-6/IL-6R. Como resultado su unión con la otra proteína de membrana, gp130, se transmite laactividad biológica de IL-6 a la célula (Taga, et al., J. Exp. Med., 196, 967, 1987). One of these proteins is IL-6R, a ligand binding protein that has a molecular weight of approximately 80 KD and to which IL-6 binds. In addition to the cell binding form that is expressed in the cell membrane passing through the cell membrane, it is also present in soluble IL-6R form (sIL-6R), which is essentially constituted of its extracellular region. Another protein is gp130, which has a molecular weight of approximately 130KD and is involved in signal transmission of non-ligand binding. IL-6 and IL-6Rform an IL-6 / IL-6R complex. As a result its binding with the other membrane protein, gp130, the biological activity of IL-6 is transmitted to the cell (Taga, et al., J. Exp. Med., 196, 967, 1987).
Aunque los compuestos de platino como cisplatino y agentes antitumorales como mitomicina C inducen la apóptosis en las células tumorales, se ha descrito que IL-6 inhibe la apóptosis inducida por agentes antitumorales (Kerr, J. etal., Cancer, 73, 2013-2026, 1994; Sachs, L. et al., Blood, 82, 15-21, 1993). Además, agentes antitumorales como cisplatino y mitomicina C tienen efectos citotóxicos sobre las células tumorales como resultado de la producción deradicales libres (Oyanagi, Y. et al., Biochem. Pharmacol., 26, 473-476, 1997; Nakano, H. et al., Biochem. Biophys.Acta., 796, 285-293, 1984). Sin embargo, IL-6 es conocida por promover la expresión de la manganeso superóxidodismutasa (MnSOD), que tiene el efecto de descomponer los radicales libres y el anticuerpo frente a IL-6 inhibe laexpresión de MnSOD promovida (Ono, M. et al., Biochem. Biophys. Res. Commun., 182, 1100-1107, 1992; Dougall,W.C., et al., Endocrinology, 129, 2376-2384, 1991). Although platinum compounds such as cisplatin and antitumor agents such as mitomycin C induce apoptosis in tumor cells, IL-6 has been described to inhibit apoptosis induced by antitumor agents (Kerr, J. etal., Cancer, 73, 2013-2026 , 1994; Sachs, L. et al., Blood, 82, 15-21, 1993). In addition, antitumor agents such as cisplatin and mitomycin C have cytotoxic effects on tumor cells as a result of the production of free radicals (Oyanagi, Y. et al., Biochem. Pharmacol., 26, 473-476, 1997; Nakano, H. et al., Biochem. Biophys. Act., 796, 285-293, 1984). However, IL-6 is known to promote the expression of manganese superoxide dismutase (MnSOD), which has the effect of breaking down free radicals and the antibody against IL-6 inhibits the expression of promoted MnSOD (Ono, M. et al. , Biochem. Biophys. Res. Commun., 182, 1100-1107, 1992; Dougall, WC, et al., Endocrinology, 129, 2376-2384, 1991).
Sin embargo, ninguno de estos estudios establece que los efectos de los agentes antitumorales se ven potenciadosbloqueando la actividad biológica de IL-6. Además, no existen estudios del intento actual de usar un antagonista deIL-6 como potenciador de los efectos de agentes antitumorales. However, none of these studies establish that the effects of antitumor agents are enhanced by blocking the biological activity of IL-6. In addition, there are no studies of the current attempt to use an IL-6 antagonist as an enhancer of the effects of antitumor agents.
Aunque con anterioridad se han usado agentes antitumorales en el tratamiento de tumores, puesto que grandesdosis de estos fármacos producen efectos secundarios adversos como nauseas, vómitos, trastornos funcionalesrenales y hepáticos y la inhibición de la función de la médula ósea, han habido casos en los que resultaba peligrosoadministrar la dosis requerida de agente antitumoral para que demostrara de forma adecuada los efectos antitumorales. Además, puesto que existen tumores que tienen resistencia a la terapia, en los que la quimioterapiaque usa agentes antitumorales convencionales no es eficaz, existe la necesidad de un potenciador de los efectosque aumente la sensibilidad de estos tumores a los agentes antitumorales. Although previously antitumor agents have been used in the treatment of tumors, since large doses of these drugs produce adverse side effects such as nausea, vomiting, renal and hepatic functional disorders and inhibition of bone marrow function, there have been cases in which it was dangerous to administer the required dose of antitumor agent to adequately demonstrate the antitumor effects. In addition, since there are tumors that have resistance to therapy, in which chemotherapy using conventional antitumor agents is not effective, there is a need for an effect enhancer that increases the sensitivity of these tumors to antitumor agents.
El objeto de la presente invención es proporcionar un nuevo potenciador de los efectos de un agente antitumoral queayuda y potencia los efectos de los agentes antitumorales y aumenta la sensibilidad de las células tumoralesresistentes al tratamiento con agentes antitumorales. Así, la presente invención se refiere al uso de un antagonista de IL-6 para la preparación de un medicamento para potenciar el efecto de cisplatino o carboplatino en el tratamientode un tumor, en donde el antagonista de IL-6 es el anticuerpo PM-1 o MH166 y dicho tumor es un carcinoma de células renales. The object of the present invention is to provide a new enhancer of the effects of an antitumor agent that helps and enhances the effects of antitumor agents and increases the sensitivity of tumor cells resistant to treatment with antitumor agents. Thus, the present invention relates to the use of an IL-6 antagonist for the preparation of a medicament to enhance the effect of cisplatin or carboplatin in the treatment of a tumor, wherein the IL-6 antagonist is the PM-1 antibody. or MH166 and said tumor is a renal cell carcinoma.
Como resultado de diversos estudios sobre los efectos de antagonistas de IL-6 sobre los cambios en la sensibilidadde las células tumorales hacia agentes antitumorales, los autores de la presente invención encontraron que losantagonistas de IL-6 como el anticuerpo frente a IL-6 o anticuerpo frente a IL-6R incrementaban la sensibilidad delas células tumorales hacia los agentes antitumorales, que los efectos terapéuticos se observan con dosis menoresde agentes antitumorales y que los efectos terapéuticos aparecen mediante el uso concomitante de agentesantitumorales convencionales con un potenciador de sus efectos que comprende un antagonista de IL-6 frente atumores que presentan resistencia a la terapia. As a result of various studies on the effects of IL-6 antagonists on changes in the sensitivity of tumor cells towards antitumor agents, the authors of the present invention found that IL-6 antagonists as the antibody against IL-6 or antibody against IL-6R they increased the sensitivity of tumor cells towards antitumor agents, that the therapeutic effects are observed with lower doses of antitumor agents and that the therapeutic effects appear through the concomitant use of conventional antitumor agents with a potentiator of their effects comprising an antagonist of IL-6 versus tumors presenting resistance to therapy.
A saber, la presente invención se refiere a un potenciador de los efectos de un agente antitumoral que contiene un antagonista de IL-6. Más específicamente, la presente invención se refiere al uso de un antagonista de IL-6 para lapreparación de un medicamento para potenciar los efectos de un agente antitumoral. Namely, the present invention relates to an enhancer of the effects of an antitumor agent containing an IL-6 antagonist. More specifically, the present invention relates to the use of an IL-6 antagonist for the preparation of a medicament to enhance the effects of an antitumor agent.
Ejemplos específicos de anticuerpo PM-1 son anticuerpo PM-1 humanizado. Agentes antitumorales usados encombinación con antagonistas IL-6 son cisplatino y carboplatino. Specific examples of PM-1 antibody are humanized PM-1 antibody. Antitumor agents used in combination with IL-6 antagonists are cisplatin and carboplatin.
Breve descripción de los dibujos Brief description of the drawings
Las Figuras 1A y 1B indican la citotoxicidad frente a la línea de carcinoma de células renales Caki-1 en presencia decisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 1A) o anticuerpo frente a IL-6R(Fig. 1B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino, los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, lostriángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R,y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 1A and 1B indicate cytotoxicity against the Caki-1 renal cell carcinoma line in the presence of decisplatin in a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 1A) or antibody against IL-6R (Fig. 1B). The diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml of antibody against IL-6 or antibody against IL-6R, and the cytotoxicity circles in the presence of cisplatin and 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
Las Figuras 2A y 2B indican la citotoxicidad frente a la línea de carcinoma de células renales Caki-1 en presencia demitomicina C en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 2A) o anticuerpo frente a IL-6R (Fig. 2B). Los rombos indican la citotoxicidad (%) en presencia únicamente de mitomicina, los cuadrados lacitotoxicidad en presencia de mitomicina y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, lostriángulos la citotoxicidad en presencia de mitomicina y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL6R, y los círculos la citotoxicidad en presencia de mitomicina y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 2A and 2B indicate cytotoxicity against the Caki-1 renal cell carcinoma line in the presence of demitomycin C at a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig 2A) or antibody against IL-6R (Fig. 2B). The rhombuses indicate cytotoxicity (%) in the presence of mitomycin only, the square lacitotoxicity in the presence of mitomycin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles cytotoxicity in the presence of mitomycin and 1 µg / ml antibody against IL-6 or antibody against IL6R, and cytotoxicity circles in the presence of mitomycin and 10 µg / ml antibody against IL-6 or antibody against IL-6R .
Las Figuras 3A y 3B indican la citotoxicidad frente a la línea de carcinoma de células renales Caki-1/DDP enpresencia de cisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 3A) o anticuerpo frente a IL-6R (Fig. 3B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino, los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL6R, los triángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 3A and 3B indicate cytotoxicity against the Caki-1 / DDP renal cell carcinoma line in cisplatin presence at a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 ( Fig. 3A) or antibody against IL-6R (Fig. 3B). The rhombuses indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml of antibody against IL-6 or antibody against IL6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml antibody against IL-6 or antibody against IL-6R, and cytotoxicity circles in the presence of cisplatin and 10 µg / ml antibody against IL-6 or antibody against IL -6R.
Las Figuras 4A y 4B indican la citotoxicidad frente a la línea de carcinoma de células renales ACHN en presencia decisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 4A) o anticuerpo frente a IL-6R(Fig. 4B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino, los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, lostriángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R,y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 4A and 4B indicate cytotoxicity against the ACHN renal cell carcinoma line in the presence of decisplatin in a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 4A) or antibody against IL-6R (Fig. 4B). The diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml of antibody against IL-6 or antibody against IL-6R, and the cytotoxicity circles in the presence of cisplatin and 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
Las Figuras 5A y 5B indican la citotoxicidad frente a la línea de carcinoma de células renales A704 en presencia decisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 5A) o anticuerpo frente a IL-6R(Fig. 5B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino, los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, lostriángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R,y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 5A and 5B indicate cytotoxicity against the A704 renal cell carcinoma line in the presence of decisplatin in a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 5A) or antibody against IL-6R (Fig. 5B). The diamonds indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml of antibody against IL-6 or antibody against IL-6R, and the cytotoxicity circles in the presence of cisplatin and 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
Las Figuras 6A y 6B indican la citotoxicidad frente a carcinoma de células renales recientes obtenidas del paciente 1en presencia de cisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 6A) o anticuerpo frente a IL-6R (Fig. 6B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino,los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente aIL-6R, los triángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 6A and 6B indicate cytotoxicity against recent renal cell carcinoma obtained from patient 1 in the presence of cisplatin in a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 6A ) or antibody against IL-6R (Fig. 6B). The rhombuses indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml of antibody against IL-6 or antibody against IL-6R, and the cytotoxicity circles in the presence of cisplatin and 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
Las Figuras 7A y 7B indican la citotoxicidad frente a carcinoma de células renales recientes obtenidas del paciente 2en presencia de cisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 7A) o anticuerpo frente a IL-6R (Fig. 7B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino,los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente aIL-6R, los triángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 7A and 7B indicate cytotoxicity against recent renal cell carcinoma obtained from patient 2 in the presence of cisplatin in a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 7A ) or antibody against IL-6R (Fig. 7B). The rhombuses indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml of antibody against IL-6 or antibody against IL-6R, and the cytotoxicity circles in the presence of cisplatin and 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
Las Figuras 8A y 8B indican la citotoxicidad frente a carcinoma de células renales recientes obtenidas del paciente 3en presencia de cisplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 8A) o anticuerpo frente a IL-6R (Fig. 8B). Los rombos indican la citotoxicidad (%) en presencia únicamente de cisplatino,los cuadrados la citotoxicidad en presencia de cisplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente aIL-6R, los triángulos la citotoxicidad en presencia de cisplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, y los círculos la citotoxicidad en presencia de cisplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 8A and 8B indicate cytotoxicity against recent renal cell carcinoma obtained from patient 3 in the presence of cisplatin in a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 8A ) or antibody against IL-6R (Fig. 8B). The rhombuses indicate the cytotoxicity (%) in the presence of cisplatin only, the cytotoxicity squares in the presence of cisplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of cisplatin and 1 µg / ml of antibody against IL-6 or antibody against IL-6R, and the cytotoxicity circles in the presence of cisplatin and 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
Las Figuras 9A y 9B indican la citotoxicidad frente a la línea de carcinoma de células renales Caki-1 en presencia decarboplatino en una concentración de 0, 0,1, 1 ó 10 !g/ml y anticuerpo frente a IL-6 (Fig. 9A) o anticuerpo frente a IL6R (Fig. 9B). Los rombos indican la citotoxicidad (%) en presencia únicamente de carboplatino, los cuadrados lacitotoxicidad en presencia de carboplatino y 0,1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, lostriángulos la citotoxicidad en presencia de carboplatino y 1 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL6R, y los círculos la citotoxicidad en presencia de carboplatino y 10 !g/ml de anticuerpo frente a IL-6 o de anticuerpo frente a IL-6R. Figures 9A and 9B indicate cytotoxicity against the Caki-1 renal cell carcinoma line in the presence of carboplatin at a concentration of 0, 0.1, 1 or 10 µg / ml and antibody against IL-6 (Fig. 9A) or antibody against IL6R (Fig. 9B). The rhombuses indicate the cytotoxicity (%) in the presence of carboplatin only, the square lacitotoxicity in the presence of carboplatin and 0.1 µg / ml antibody against IL-6 or antibody against IL-6R, the triangles the cytotoxicity in the presence of carboplatin and 1 µg / ml antibody against IL-6 or antibody against IL6R, and cytotoxicity circles in the presence of carboplatin and 10 µg / ml antibody against IL-6 or antibody against IL-6R .
En la Figura 10, A es un diagrama que muestra los resultados de la transferencia Northern del RNA total de Caki-1usando una sonda de cDNA de GST-n con el fin de investigar la expresión de mRNA de GST-n en la línea de carcinoma de células renales Caki-1 tratada con un medio de cultivo (control), cisplatino en 10 !g/ml o anticuerpo frente a IL-6 o anticuerpo frente a IL-6R en 10 !g/ml. La banda 1 muestra Caki-1 tratada con medio de cultivoúnicamente, la banda 2 Caki-1 tratada con cisplatino, la banda 3 Caki-1 tratada con anticuerpo frente a IL-6 y labanda 4 Caki-1 tratada con anticuerpo frente a IL-6R. En la Figura 10, B es un diagrama de un gel usado entransferencia Northern, teñido con bromuro de etidinio. El diagrama muestra que el RNA está presente en cadabanda. In Figure 10, A is a diagram showing the results of Northern blotting of the total Caki-1 RNA using a GST-n cDNA probe in order to investigate the expression of GST-n mRNA in the carcinoma line of Caki-1 renal cells treated with a culture medium (control), cisplatin in 10 µg / ml or antibody against IL-6 or antibody against IL-6R in 10 µg / ml. Band 1 shows Caki-1 treated with culture medium only, band 2 Caki-1 treated with cisplatin, band 3 Caki-1 treated with antibody against IL-6 and band 4 Caki-1 treated with antibody against IL- 6R. In Figure 10, B is a diagram of a gel used in Northern interference, stained with ethidinium bromide. The diagram shows that RNA is present in each band.
El potenciador del efecto del agente antitumoral que comprende un antagonista de IL-6 de la presente invenciónpotencia los efectos antitumorales al ser usado en combinación con los agentes antitumorales durante el tratamientode tumores. Además, permite reducir la dosis requerida de agente antitumoral puesto que tiene el efecto de aumentar la sensibilidad hacia los agentes antitumorales de tumores resistentes a tratamiento para los cuales no seobservan los efectos terapéuticos con tratamiento convencional de quimioterapia. The anti-tumor agent effect enhancer comprising an IL-6 antagonist of the present invention potentiates the anti-tumor effects when used in combination with the anti-tumor agents during the treatment of tumors. In addition, it allows the required dose of antitumor agent to be reduced since it has the effect of increasing the sensitivity towards antitumor agents of treatment resistant tumors for which the therapeutic effects are not observed with conventional chemotherapy treatment.
Los agentes antitumorales para los cuales se potencian los efectos antitumorales por medio del potenciador de losefectos de la presente invención son fármacos para quimioterapia que tienen efectos terapéuticos frente a tumoresque inhiben el desarrollo y el crecimiento de células tumorales actuando sobre las citadas células tumorales. Estos fármacos para quimioterapia son compuestos de platino seleccionados de cisplatino y carboplatino. El antibióticoantitumoral mitomicina C y los compuestos de platino que tienen efectos antitumorales son agentes antitumoralesespecialmente preferidos. Los compuestos de platino tienen un átomo de platino que forma complejos con otrosátomos. Estos compuestos presentan efectos antitumorales al unirse al DNA, inhibiendo la síntesis de DNA de lascélulas tumorales e inhibiendo la división de las células tumorales. Ejemplos conocidos de compuestos de platinoque tienen efectos antitumorales usados hasta ahora incluyen cisplatino (cis-diamina dicloroplatino(II), que tiene laformula estructural mostrada a continuación): The antitumor agents for which the antitumor effects are enhanced by means of the lymphatic enhancer of the present invention are chemotherapy drugs that have therapeutic effects against tumors that inhibit the development and growth of tumor cells by acting on said tumor cells. These chemotherapy drugs are platinum compounds selected from cisplatin and carboplatin. The antibiotic antitumor mitomycin C and platinum compounds that have antitumor effects are especially preferred antitumor agents. Platinum compounds have a platinum atom that complexes with other atoms. These compounds exhibit antitumor effects by binding to DNA, inhibiting DNA synthesis of tumor cells and inhibiting the division of tumor cells. Known examples of platinum compounds that have antitumor effects used so far include cisplatin (cis-diamine dichloroplatin (II), which has the structural formula shown below):
carboplatino (cis-diamina (1,1-ciclobutanodicarboxilato)platino(II), que tiene la fórmula estructural mostrada a continuación): carboplatin (cis-diamine (1,1-cyclobutanedicarboxylate) platinum (II), which has the structural formula shown below):
5 Estos agentes antitumorales se pueden preparar usando procedimientos de rutina. Por ejemplo, los compuestos de platino se pueden usar por vía oral o parenteral, y con preferencia en forma de formulación en inyección, mezclandocon un vehículo y excipiente farmacéutico, si fuera necesario. Para preparar una formulación para inyección, éste sepuede mezclar con agua destilada, una solución salina como cloruro sódico o cloruro potásico, solución glucosada osolución salina fisiológica. La cantidad de agente antitumoral en estas formulaciones es preferiblemente una unidad5 These antitumor agents can be prepared using routine procedures. For example, platinum compounds can be used orally or parenterally, and preferably in the form of an injection formulation, mixing with a pharmaceutical carrier and excipient, if necessary. To prepare a formulation for injection, it can be mixed with distilled water, a saline solution such as sodium chloride or potassium chloride, a greased solution or physiological saline solution. The amount of antitumor agent in these formulations is preferably a unit.
10 de dosis que sea conveniente para usar conforme a la edad del paciente, síntomas y factores similares. Por ejemplo, al usar para el tratamiento de un tumor en un adulto, se administra a diario de 10 a 2000 mg/m2 (superficie corporal), administrado de forma continua durante 5 días conforme a la dosis, o se puede disponer un período de recuperación de 1 a 4 semanas entre las administraciones. 10 doses that are convenient for use according to the patient's age, symptoms and similar factors. For example, when used for the treatment of a tumor in an adult, it is administered daily from 10 to 2000 mg / m2 (body surface), administered continuously for 5 days according to the dose, or a period of 1 to 4 week recovery between administrations.
Las células tumorales en las que se observan efectos antitumorales debidos al potenciador de los efectos de laTumor cells in which antitumor effects due to the enhancer of the effects of
15 presente invención son tumores que tienen IL-6R y presentan crecimiento y/o resistencia al tratamiento proporcionado por IL-6 como una de sus sustancias fisiológicamente activas. De acuerdo con la presente invenciónel tumor es carcinoma de células renales (Miki, S. et al., FEBS Letter, 250, 607-610, 1989). The present invention are tumors that have IL-6R and exhibit growth and / or resistance to the treatment provided by IL-6 as one of its physiologically active substances. In accordance with the present invention the tumor is renal cell carcinoma (Miki, S. et al., FEBS Letter, 250, 607-610, 1989).
El antagonista de IL-6 usado en la presente invención es un anticuerpo frente a IL-6 o un anticuerpo frente a IL-6R. The IL-6 antagonist used in the present invention is an antibody against IL-6 or an antibody against IL-6R.
No existe una limitación particular sobre la especie animal de las células productoras de anticuerpos monoclonales,There is no particular limitation on the animal species of monoclonal antibody producing cells,
20 con tal que sea de mamífero y puedan originarse en un anticuerpo humano o en un anticuerpo de mamífero no humano. Los anticuerpos monoclonales de origen de conejo o de roedor son anticuerpos monoclonales preferidosde origen mamífero no humano debido a su facilidad de preparación. Aunque no existen limitaciones particularesrespecto a los anticuerpos de roedores, los ejemplos preferidos incluyen anticuerpos de ratón, de rata y de hámster. 20 provided it is mammalian and can originate from a human antibody or a non-human mammalian antibody. Monoclonal antibodies of rabbit or rodent origin are preferred monoclonal antibodies of non-human mammalian origin due to their ease of preparation. Although there are no particular limitations regarding rodent antibodies, preferred examples include mouse, rat and hamster antibodies.
El anticuerpo IL-6 de acuerdo con la presente invención es el anticuerpo MH166 (Matsuda, et al., Eur. J. Immunol.,25 18, 951-956, 1988). El anticuerpo IL-6R de acuerdo con la presente invención es el anticuerpo PM-1 (Hirata, et al., J. Immunol., 143, 2900-2906, 1989). The IL-6 antibody according to the present invention is the MH166 antibody (Matsuda, et al., Eur. J. Immunol., 18, 951-956, 1988). The IL-6R antibody according to the present invention is the PM-1 antibody (Hirata, et al., J. Immunol., 143, 2900-2906, 1989).
De estos anticuerpos, son particularmente preferidos los anticuerpos IL-6R, es decir el anticuerpo PM-1 anteriormente citado. Of these antibodies, IL-6R antibodies, ie the aforementioned PM-1 antibody, are particularly preferred.
Los anticuerpos monoclonales se pueden preparar de la siguiente forma, usando básicamente tecnología conocida.Monoclonal antibodies can be prepared as follows, basically using known technology.
30 A saber, usando IL-6 o IL-6R para el antígeno sensibilizador, se inmuniza una célula hospedadora conforme a un procedimiento de inmunización convencional, se fusionan las células inmunizadas resultantes con células parentalesconocidas por un método de fusión de rutina y se selecciona entonces una célula productora de un anticuerpomonoclonal por procedimientos de cribado de rutina. Namely, using IL-6 or IL-6R for the sensitizing antigen, a host cell is immunized according to a conventional immunization procedure, the resulting immunized cells are fused with parental cells known by a routine fusion method and then selected a cell producing an monoclonal antibody by routine screening procedures.
De forma más específica, se puede llevar a cabo el siguiente procedimiento para preparar anticuerposMore specifically, the following procedure can be carried out to prepare antibodies
35 monoclonales. Por ejemplo, para el antígeno sensibilizador anteriormente citado es preferible un antígeno de origen humano y, en el caso de IL-6 humana, se obtiene el antígeno usando la secuencia génica de IL-6 humana descritaen Hirano, et al., Nature, 324, 73, 1986. Después de insertar la secuencia génica de IL-6 humana en un sistemavector de expresión conocido y transformar las células hospedadoras adecuadas, se purifica la proteína IL-6 diana apartir de las células hospedadoras o el líquido sobrenadante del cultivo y se puede usar entonces la proteína IL-6 purificada como antígeno sensibilizador. 35 monoclonal. For example, for the aforementioned sensitizing antigen, an antigen of human origin is preferable and, in the case of human IL-6, the antigen is obtained using the human IL-6 gene sequence described in Hirano, et al., Nature, 324 , 73, 1986. After inserting the human IL-6 gene sequence into a known expression system and transforming the appropriate host cells, the target IL-6 protein is purified from the host cells or the culture supernatant and You can then use the purified IL-6 protein as a sensitizing antigen.
En el caso de IL-6R humana, la proteína IL-6R se puede obtener siguiendo un procedimiento similar al de IL-6humana descrito anteriormente usando la secuencia génica descrita en la publicación de patente europea nºEP325474. Existen dos tipos de IL-6R, a saber, la que se expresa en la membrana celular y un tipo soluble que selibera de la membrana celular (sIL-6R) (Yasukawa, et al., J. Biochem., 108, 673-676, 1990). sIL-6R está compuestafundamentalmente de la región extracelular de IL-6R unida a la membrana celular y se diferencia de IL-6R unida a lamembrana porque carece de una región de permeación en la membrana celular o de región de permeación en lamembrana celular y de región intracelular. In the case of human IL-6R, the IL-6R protein can be obtained following a procedure similar to that of human IL-6 described above using the gene sequence described in European Patent Publication No. EP325474. There are two types of IL-6R, namely, the one expressed in the cell membrane and a soluble type that selects the cell membrane (sIL-6R) (Yasukawa, et al., J. Biochem., 108, 673- 676, 1990). sIL-6R is primarily composed of the extracellular region of cell membrane bound IL-6R and differs from membrane-bound IL-6R because it lacks a permeation region in the cell membrane or permeation region in the cell membrane and region intracellular
Aunque no existen limitaciones particulares, los mamíferos que se inmunizan con antígeno sensibilizador se seleccionan preferiblemente considerando la compatibilidad con las células parentales usadas en la fusión celular y se usan corrientemente ratones, ratas, hámsteres o conejos. Although there are no particular limitations, mammals that are immunized with sensitizing antigen are preferably selected considering compatibility with parental cells used in cell fusion and mice, rats, hamsters or rabbits are commonly used.
La inmunización de un animal con antígeno sensibilizador se lleva a cabo de acuerdo con procedimientos conocidos. Como ejemplo típico, la inmunización se lleva a cabo por inyección intraperitoneal o subcutánea de un antígenosensibilizador en un animal. De forma más específica, después de diluir y suspender el antígeno sensibilizador enuna cantidad adecuada de solución salina tamponada con fosfato (PBS) o solución salina fisiológica, se mezcla lasuspensión resultante con una cantidad adecuada de un adyuvante convencional, tal como adyuvante completo deFreund, como se desee, seguido por emulsificación. La emulsión se administra entonces preferiblemente al mamífero en varias dosis cada 4 a 21 días. Además, se puede usar un vehículo adecuado durante la inmunizacióncon antígeno sensibilizador. Immunization of an animal with sensitizing antigen is carried out according to known procedures. As a typical example, immunization is carried out by intraperitoneal or subcutaneous injection of an antigen sensitizer in an animal. More specifically, after diluting and suspending the sensitizing antigen in a suitable amount of phosphate buffered saline (PBS) or physiological saline, the resulting suspension is mixed with a suitable amount of a conventional adjuvant, such as complete adjuvant of Freund, as desired, followed by emulsification. The emulsion is then preferably administered to the mammal in several doses every 4 to 21 days. In addition, a suitable vehicle can be used during immunization with sensitizing antigen.
Así, después de inmunizar el mamífero y confirmar que se ha alcanzado en el suero el nivel deseado de antígeno,se extraen células inmunizadas del mamífero y se usan en la fusión celular. Un ejemplo particularmente preferido decélulas inmunizadas son las células de bazo. Thus, after immunizing the mammal and confirming that the desired level of antigen has been reached in the serum, immunized cells are extracted from the mammal and used in cell fusion. A particularly preferred example immunized cells are spleen cells.
Se usan preferiblemente diversos tipos de líneas de células conocidas para las células de mieloma de mamífero quesirven como células parentales que se fusionan con las células inmunizadas anteriormente citadas, ejemplos de lascuales incluyen P3 (P3x63Ag8.653) (J. Immunol., 123, 1548, 1978), p3-U1 (Current Topics in Microbiology andImmunology, 81, 1-7, 1978), NS-1 (Eur. J. Immunol., 6, 511-519, 1976), MPC-11 (Cell, 8, 405-415, 1976), SP2/0 (Nature, 276, 269-270, 1978), F0 (J. Immunol. Meth., 35, 1-21, 1980), S194 (J. Exp. Med., 148, 313-323, 1978) y R210 (Nature, 277, 131-133, 1979). Various types of cell lines known for mammalian myeloma cells are preferably used as parental cells that fuse with the aforementioned immunized cells, examples of which include P3 (P3x63Ag8.653) (J. Immunol., 123, 1548 , 1978), p3-U1 (Current Topics in Microbiology and Immunology, 81, 1-7, 1978), NS-1 (Eur. J. Immunol., 6, 511-519, 1976), MPC-11 (Cell, 8 , 405-415, 1976), SP2 / 0 (Nature, 276, 269-270, 1978), F0 (J. Immunol. Meth., 35, 1-21, 1980), S194 (J. Exp. Med., 148, 313-323, 1978) and R210 (Nature, 277, 131-133, 1979).
La fusión celular de las células inmunizadas anteriormente citadas y células de mieloma se puede llevar a cabo deacuerdo con procedimientos conocidos, un ejemplo de los cuales es el procedimiento de Milstein, et al., (Milstein, etal., Methods Enzymol., 73, 3-46, 1981). Cell fusion of the aforementioned immunized cells and myeloma cells can be carried out according to known procedures, an example of which is the procedure of Milstein, et al., (Milstein, et al., Methods Enzymol., 73, 3-46, 1981).
De forma más específica, la fusión celular anteriormente citada, por ejemplo, se lleva a cabo en un líquido de cultivonutriente convencional en presencia de un promotor de fusión celular. Ejemplos de promotores de fusión que seusan incluyen polietilenglicol (PEG) y virus de Sendai (HVJ). Por otro lado, se desea se puede añadir también unagente adyuvante de la fusión como dimetil sulfóxido para aumentar la eficacia de la fusión. More specifically, the aforementioned cell fusion, for example, is carried out in a conventional culture fluid in the presence of a cell fusion promoter. Examples of fusion promoters that are used include polyethylene glycol (PEG) and Sendai virus (HVJ). On the other hand, it is also desirable to add a fusion adjuvant such as dimethyl sulfoxide to increase the efficiency of the fusion.
La relación de células inmunizadas y células de mieloma usadas varía preferiblemente, por ejemplo, de 1 a 10 vecesmás células inmunizadas que células de mieloma. Como líquido de cultivo usado para la fusión celular anteriormentecitada se puede usar el medio de cultivo RPMI 1640, medio de cultivo MEM u otro medio de cultivo convencionalusado en este tipo de cultivos celulares. Por otro lado, estos medios de cultivo se pueden usar combinados con unsuplemento sérico como suero bovino fetal (FCS) The ratio of immunized cells and myeloma cells used preferably varies, for example, from 1 to 10 times more immunized cells than myeloma cells. As the culture liquid used for the aforementioned cell fusion, RPMI 1640 culture medium, MEM culture medium or other conventional culture medium used in this type of cell culture can be used. On the other hand, these culture media can be used in combination with a serum supplement such as fetal bovine serum (FCS)
Para la fusión celular, se mezclan perfectamente las cantidades prescritas de las células inmunizadas anteriormentecitadas y las células de mieloma en el medio de cultivo anteriormente citad, seguido por la adición de la una soluciónde PEG, tal como una solución de PEG que tiene un peso molecular medio de 1000 a 6000, calentada conantelación hasta aproximadamente 37ºC, normalmente en una concentración de 30 a 60% (p/v) y se mezclanformando las células fusionadas diana (hibridoma). A continuación, repitiendo un procedimiento que consiste enañadir de forma secuencial un medio de cultivo adecuado, centrifugar y eliminar el líquido sobrenadante, se puedeneliminar los agentes de fusión celular y elementos no deseados para el crecimiento del hibridoma. For cell fusion, the prescribed amounts of the aforementioned immunized cells and myeloma cells in the aforementioned culture medium are perfectly mixed, followed by the addition of a PEG solution, such as a PEG solution having a molecular weight medium from 1000 to 6000, heated with constellation to about 37 ° C, usually in a concentration of 30 to 60% (w / v) and mixed forming the target fused cells (hybridoma). Then, by repeating a procedure consisting in sequentially adding a suitable culture medium, centrifuging and eliminating the supernatant liquid, the cell fusion agents and unwanted elements for hybridoma growth can be eliminated.
Dicho hibridoma se selecciona cultivando en un medio de cultivo selectivo convencional como medio de cultivo HAT (medio de cultivo que contiene hipoxantina, aminopterina y timidina). El cultivo en dicho medio de cultivo HAT seprolonga normalmente durante varios días a varias semanas dejando el tiempo adecuado para que las célulasdistintas a las células del hibridoma diana (células no fusionadas) mueran. A continuación, se lleva a cabo elprocedimiento de dilución limitada convencional seguido por el cribado y clonación simple del hibridoma que produceel anticuerpo diana. Said hybridoma is selected by culturing in a conventional selective culture medium as HAT culture medium (culture medium containing hypoxanthine, aminopterin and thymidine). The culture in said HAT culture medium is normally prolonged for several days to several weeks leaving adequate time for the different cells to the target hybridoma cells (non-fused cells) to die. Next, the conventional limited dilution procedure is carried out followed by simple screening and cloning of the hybridoma that produces the target antibody.
El hibridoma así preparado que produce anticuerpo monoclonal se puede subcultivar en un medio de cultivo convencional y puede almacenarse durante un largo período de tiempo en nitrógeno líquido. The hybridoma so prepared that produces monoclonal antibody can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.
Con el fin de obtener el anticuerpo monoclonal de dicho hibridoma, se cultiva el mismo conforme a procedimientosconvencionales y se obtiene en forma de líquido sobrenadante del cultivo. Como alternativa, el hibridoma se desarrolla administrándolo a un mamífero con el cual tenga compatibilidad y obteniendo el anticuerpo monoclonal ensu líquido ascítico. El procedimiento primero es adecuado para la obtención de anticuerpo muy puro, mientras que elprocedimiento siguiente es adecuado para la producción de un gran volumen de anticuerpos. In order to obtain the monoclonal antibody of said hybridoma, it is cultured according to conventional procedures and is obtained as a culture supernatant. As an alternative, the hybridoma develops by administering it to a mammal with which it has compatibility and obtaining the monoclonal antibody in its ascites fluid. The first procedure is suitable for obtaining very pure antibody, while the following procedure is suitable for the production of a large volume of antibodies.
Además, el anticuerpo monoclonal no solo se puede obtener a partir del hibridoma formado por fusión de célulasproductoras de anticuerpos obtenidos inmunizando con antígeno, sino que también se puede usar el anticuerpomonoclonal que se produce usando tecnología génica recombinante clonando el gen del anticuerpo, incorporando elmismo en un vector adecuado e introduciendo el vector en una línea de células conocida como COS o CHO (véase, por ejemplo, Vandamme, A-M. et al., Eur. J. Biochem., 192, 767-775, 1990). In addition, the monoclonal antibody can not only be obtained from the hybridoma formed by fusion of antibody-producing cells obtained by immunizing with antigen, but also the monoclonal antibody that is produced using recombinant gene technology can be used by cloning the antibody gene, incorporating the same in a suitable vector and introducing the vector into a cell line known as COS or CHO (see, for example, Vandamme, AM. et al., Eur. J. Biochem., 192, 767-775, 1990).
Por otro lado, el anticuerpo monoclonal obtenido por el procedimiento anteriormente citado se puede purificar hastauna elevada pureza usando medios de purificación convencionales como la precipitación de sales, la filtración en gel On the other hand, the monoclonal antibody obtained by the aforementioned procedure can be purified to high purity using conventional purification means such as salt precipitation, gel filtration.
o la cromatografía de afinidad. or affinity chromatography.
El anticuerpo monoclonal preparado de este modo puede reconocer el antígeno con una elevada sensibilidad y altaprecisión por medios inmunológicos convencionales tales como radioinmunoensayo (RIA), inmunoensayo ligado aenzima (EIA, ELISA) y análisis de inmunofluorescencia. The monoclonal antibody thus prepared can recognize the antigen with high sensitivity and high accuracy by conventional immunological means such as radioimmunoassay (RIA), aenzyme-linked immunoassay (EIA, ELISA) and immunofluorescence analysis.
El anticuerpo monoclonal usado en la presente invención no queda limitado a anticuerpos monoclonales producidospor hibridoma. Es más preferido el anticuerpo monoclonal que ha sido alterado artificialmente con el objeto dereducir la heteroantigenicidad a seres humanos y similares. Por ejemplo, se puede usar el anticuerpo quiméricoformado por la región variable del anticuerpo monoclonal de mamífero diferente del anticuerpo monoclonal humano,por ejemplo anticuerpo monoclonal de ratón, y la región constante del anticuerpo monoclonal humano. Este tipo de anticuerpo quimérico puede producirse usando procedimientos conocidos de producción de anticuerpos quiméricos y, en particular, tecnología génica recombinante. The monoclonal antibody used in the present invention is not limited to monoclonal antibodies produced by hybridoma. More preferred is the monoclonal antibody that has been artificially altered in order to reduce heteroantigenicity to humans and the like. For example, the chimeric antibody formed by the variable region of the mammalian monoclonal antibody other than the human monoclonal antibody, for example mouse monoclonal antibody, and the constant region of the human monoclonal antibody can be used. This type of chimeric antibody can be produced using known methods of producing chimeric antibodies and, in particular, recombinant gene technology.
Además, también se pueden usar en la presente invención anticuerpos humanos remodeladas. En este tipo deanticuerpos, se injerta la región determinante de la complementariedad del anticuerpo de mamífero no humano, conla región determinante de la complementariedad del anticuerpo humano, por una técnica de recombinación génicaconocida general. Se puede obtener un anticuerpo remodelado humano que es de utilidad en la presente invenciónusando esta técnica conocida, un ejemplo preferido del cual es el anticuerpo PM-1 remodelado (véase, por ejemplo,la publicación de patente internacional nº WO92-19759). In addition, remodeled human antibodies can also be used in the present invention. In this type of antibodies, the region determining the complementarity of the non-human mammalian antibody is grafted, with the region determining the complementarity of the human antibody, by a generally known gene recombination technique. A human remodeled antibody that is useful in the present invention can be obtained using this known technique, a preferred example of which is the remodeled PM-1 antibody (see, for example, International Patent Publication No. WO92-19759).
Por otro lado, los aminoácidos de la región marco conservada (FR) de la región variable de un anticuerpo se puedensustituir para que la región determinante de la complementariedad del anticuerpo humano remodelado forme un sitiode unión a anticuerpo adecuado (Sato, et al., Cancer Res., 53, 1-6, 1993). Además, se puede construir un gen quecodifique el fragmento de anticuerpo tal como F(ab’)2, Fab, Fv, Fv de cadena sencilla (scFv) que se une a Fv de lacadena H y la cadena L con un engarce adecuado, y se puede expresar entonces el fragmento de anticuerpo en unacélula hospedadora adecuada y puede usarse con el fin anteriormente citado con tal que se una con el antígeno einhiba la actividad de IL-6 (véase, por ejemplo, Bird, et al., TIBTECH, 9, 132-137, 1991). On the other hand, the amino acids of the conserved framework region (FR) of the variable region of an antibody can be substituted so that the region determining the complementarity of the remodeled human antibody forms a suitable antibody binding site (Sato, et al., Cancer Res., 53, 1-6, 1993). In addition, a gene can be constructed that encodes the antibody fragment such as F (ab ') 2, Fab, Fv, single chain Fv (scFv) that binds Fv of chain H and chain L with a suitable linker, and the antibody fragment can then be expressed in a suitable host cell and can be used for the purpose mentioned above as long as the activity of IL-6 is linked with the einhiba antigen (see, for example, Bird, et al., TIBTECH, 9 , 132-137, 1991).
Un scFv está formado uniendo la región V de la cadena H y la región V de la cadena L de un anticuerpo. En estescFv, la región V de la cadena H y la región V de la cadena L se unen por medio de un engarce, y preferiblemente,por un engarce peptídico (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883, 1988). La región V de la cadena H y la región V de la cadena L en scFv pueden derivarse de cualquiera de los anticuerpos anteriormentecitados. Estas regiones V se engarzan preferiblemente por medio de un engarce peptídico. Ejemplos de engarces peptídicos que se usan incluyen cualquiera de los péptidos de cadena sencilla que comprenden 12 a 19 restos aminoácidos. An scFv is formed by joining the V region of the H chain and the V region of the L chain of an antibody. In estescFv, the V region of the H chain and the V region of the L chain are joined by a linker, and preferably, by a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA , 85, 5879-5883, 1988). The V region of the H chain and the V region of the L chain in scFv can be derived from any of the aforementioned antibodies. These V regions are preferably crimped by means of a peptide linker. Examples of peptide linkers that are used include any of the single chain peptides comprising 12 to 19 amino acid residues.
El DNA que codifica scFv se obtiene usando DNA que codifica la cadena H o la región V de la cadena H del anticuerpo anteriormente citado y DNA que codifica la cadena L o la región V de la cadena L como moldes, amplificando la porción de DNA de sus secuencias que codifican la secuencia de aminoácidos deseada usando unapareja de cebadores que definen sus dos extremos, y combinando con una pareja de cebadores que definen el DNAque codifica la porción del engarce peptídico, así como ambos extremos para unirse con la cadena H y con lacadena L. The DNA encoding scFv is obtained using DNA encoding the H chain or the V region of the H chain of the aforementioned antibody and DNA encoding the L chain or the V region of the L chain as templates, amplifying the DNA portion of its sequences that encode the desired amino acid sequence using a pair of primers that define its two ends, and combining with a pair of primers that define the DNA that encodes the peptide linker portion, as well as both ends to bind with the H chain and chain L.
Además, una vez que se ha preparado el DNA que codifica el scFv, se puede obtener conforme a procedimientosconvencionales un vector de expresión que lo contenga y un hospedador transformado por dicho vector de expresión. Además, scFv se puede obtener de acuerdo con procedimientos convencionales usando dichohospedador. Puesto que scFv tiene una mayor capacidad para migrar en el tejido que la molécula de anticuerpo,cabe esperar que se use como sustancia que tiene funciones similares que el anticuerpo humano remodelado. Furthermore, once the DNA encoding the scFv has been prepared, an expression vector containing it and a host transformed by said expression vector can be obtained according to conventional procedures. In addition, scFv can be obtained in accordance with conventional procedures using such a viewer. Since scFv has a greater ability to migrate in tissue than the antibody molecule, it can be expected to be used as a substance that has similar functions as the remodeled human antibody.
El potenciador del efecto de un agente antitumoral que comprende un agonista de IL-6 de la presente invención sepuede usar de forma eficaz en el tratamiento de cualquier tumor que tenga IL-6R, se desarrolle usando IL-6 comouna de sus sustancias fisiológicamente activas y/o presente resistencia al tratamiento, con tal que bloquee latransmisión de señal de IL-6 y ayude y potencie el efecto del agente antitumoral. The effect enhancer of an antitumor agent comprising an IL-6 agonist of the present invention can be used effectively in the treatment of any tumor having IL-6R, is developed using IL-6 as a physiologically active substance and / or present resistance to treatment, as long as it blocks the signal transmission of IL-6 and helps and enhances the effect of the antitumor agent.
El potenciador del efecto de un agente antitumoral que comprende un antagonista de IL-6 de la presente invenciónse puede administrar de forma sistémica o local, y preferiblemente de forma parenteral, por ejemplo, por inyecciónintravenosa, inyección intramuscular, inyección intraperitoneal o inyección subcutánea. Además, se puede usar enforma de una composición farmacéutica o estuche con al menos un vehículo o diluyente farmacéutico. The effect enhancer of an antitumor agent comprising an IL-6 antagonist of the present invention can be administered systemically or locally, and preferably parenterally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection. In addition, it can be used in the form of a pharmaceutical composition or case with at least one pharmaceutical carrier or diluent.
La dosis en seres humanos del potenciador del efecto de un agente antitumoral que comprende un antagonista deIL-6 de la presente invención varía conforme al estado y edad del paciente o conforme al procedimiento deadministración. Sin embargo, es necesario seleccionar una dosis adecuada. Por ejemplo, en el caso de anticuerpofrente a IL-6R, se puede seleccionar una dosis dividida de cuatro administraciones o menos en un intervalo deaproximadamente 1 a 1.000 mg/paciente. Además, puede administrarse también en una dosis de 1 a 10 mg/kg/semana. No obstante, el potenciador del efecto de un agente antitumoral que comprende un antagonista deIL-6 de la presente invención no está limitado a estas dosis. The dose in humans of the effect enhancer of an antitumor agent comprising an IL-6 antagonist of the present invention varies according to the condition and age of the patient or according to the administration procedure. However, it is necessary to select an appropriate dose. For example, in the case of antibody to IL-6R, a divided dose of four administrations or less can be selected in a range of approximately 1 to 1,000 mg / patient. In addition, it can also be administered in a dose of 1 to 10 mg / kg / week. However, the effect enhancer of an antitumor agent comprising an IL-6 antagonist of the present invention is not limited to these doses.
El potenciador del efecto de un agente antitumoral que comprende un antagonista de IL-6 de la presente invenciónse puede preparar conforme a procedimientos convencionales (Remington’s Pharmaceutical Science, última edición,Mark Publishing Company, Easton, Estados Unidos de América). Por ejemplo, se pueden preparar preparacionespara inyección disolviendo el antagonista de IL-6 purificado en un disolvente como solución salina fisiológica, tampón The effect enhancer of an antitumor agent comprising an IL-6 antagonist of the present invention can be prepared according to conventional procedures (Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, United States of America). For example, preparations for injection can be prepared by dissolving the purified IL-6 antagonist in a solvent such as physiological saline, buffer
o solución glucosada y añadiendo un agente de prevención de la adsorción como Tween 80, gelatina o albúmina sérica humana (HSA). Como alternativa, también se puede preparar por liofilización para la reconstitución antes deusar. Ejemplos de vehículos que se pueden usar para la liofilización incluyen alcoholes azúcares y azúcares comomanitol y glucosa. or glucose solution and adding an adsorption prevention agent such as Tween 80, gelatin or human serum albumin (HSA). Alternatively, it can also be prepared by lyophilization for reconstitution before use. Examples of vehicles that can be used for lyophilization include sugar alcohols and comomanitol sugars and glucose.
Ejemplos Examples
5 Aunque a continuación se proporciona una explicación detallada de la presente invención usando ejemplos, ejemplos de referencia y experimentos, la presente invención no queda limitada a los mismos. 5 Although a detailed explanation of the present invention is provided below using examples, reference examples and experiments, the present invention is not limited thereto.
Ejemplo 1. Efecto de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R sobre la sensibilidad de células tumoraleshacia agentes antitumorales Example 1. Effect of antibody against IL-6 or antibody against IL-6R on the sensitivity of tumor cells to antitumor agents
Se investigó el efecto del anticuerpo frente a IL-6 o el anticuerpo frente a IL-6R sobre la sensibilidad de célulastumorales hacia diversos agentes antitumorales. The effect of the antibody against IL-6 or the antibody against IL-6R on the sensitivity of cellular cells to various antitumor agents was investigated.
(1) Preparación de carcinoma de células renales humanas (1) Preparation of human renal cell carcinoma
Se cultivaron la línea de células renales humanas Caki-1, la cepa resistente a cisplatino Caki-1/DDP, una sublineade Caki-1, carcinoma de células renales ACHN y carcinoma de células renales A704 (Giard, D.J. et al., J. Natl. Cancer Inst., 51, 1417-1423, 1973) para formar una monocapa en placas de plástico en medio de cultivo RPMI 1640 The Caki-1 human renal cell line, the Caki-1 / DDP cisplatin resistant strain, a Caki-1 sublineade, ACHN renal cell carcinoma and A704 renal cell carcinoma (Giard, DJ et al., J. were cultured). Natl. Cancer Inst., 51, 1417-1423, 1973) to form a monolayer in plastic plates in RPMI 1640 culture medium
15 que contenía HEPES 25 mM, L-glutamina 2 mM, aminoácidos no esenciales al 1%, 100 U/ml de penicilina, 100 !g/ml de estreptomicina y suero bovino fetal (FBS) térmicamente inactivado al 10% (todos fabricados por Gibco) (sedenomina como medio de cultivo completo). 15 containing 25 mM HEPES, 2 mM L-glutamine, 1% non-essential amino acids, 100 U / ml penicillin, 100 µg / ml streptomycin and 10% thermally inactivated fetal bovine serum (FBS) (all manufactured by Gibco) (sedenomine as a complete culture medium).
Por otro lado, se obtuvieron células tumorales recientes de pacientes con carcinoma de células renales conforme alprocedimiento de Mizutani, Y. et al., (Cancer, 69, 537-545, 1992). El tejido tumoral del carcinoma de células renalesse obtuvo durante una operación quirúrgica en tres pacientes con carcinoma de células renales. Después deconfirmar que el tejido era carcinoma de células renales por clasificación histológica, se descompuso finamente eltejido tumoral por 3 mg/ml de colagenasa (Sigma Chemical Co.) para preparar una suspensión de células tumorales.Después de lavar tres veces con medio de cultivo RPMI 1640, se depositó una capa de suspensión celular sobre ungradiente no continuo, cada uno de 2 ml de Ficol-Hypaque al 100%, al 80% y al 50% en tubos de plástico de 15 ml,On the other hand, recent tumor cells were obtained from patients with renal cell carcinoma according to the procedure of Mizutani, Y. et al., (Cancer, 69, 537-545, 1992). The tumor tissue of renal cell carcinoma was obtained during a surgical operation in three patients with renal cell carcinoma. After confirming that the tissue was renal cell carcinoma by histological classification, the tumor tissue was broken down finely by 3 mg / ml collagenase (Sigma Chemical Co.) to prepare a tumor cell suspension.After washing three times with RPMI culture medium 1640, a layer of cell suspension was deposited on a non-continuous gradient, each 2 ml of 100% Ficol-Hypaque, 80% and 50% in 15 ml plastic tubes,
25 seguido por centrifugación durante 30 minutos a 400 xg. La capa de monocitos rica en linfocitos contenida en la capa al 100% citada antes se retiró y se obtuvieron células mesoteliales y tumorales de la capa al 80%. 25 followed by centrifugation for 30 minutes at 400 xg. The lymphocyte-rich monocyte layer contained in the 100% layer cited above was removed and 80% mesothelial and tumor cells were obtained from the layer.
Para evitar la contaminación por otras células, se depositó una capa de la suspensión celular rica en célulastumorales sobre un gradiente no continuo formado por 3 ml de Percol al 25%, al 15% y al 10% en medio de cultivocompleto contenido en tubos de plástico de 15 ml, seguido por centrifugación durante 7 minutos a 25 xg y a temperatura ambiente. Después de lavar las células tumorales resultantes y suspender en medio de cultivo completo, se confirmó la presencia de células tumorales por tinción con azul de triptano. Las células tumoralespreparadas de este modo se usaron en el siguiente experimento. In order to avoid contamination by other cells, a layer of the cell suspension rich in cellular cells was deposited on a non-continuous gradient formed by 3 ml of 25%, 15% and 10% Percol in complete culture medium contained in plastic tubes 15 ml, followed by centrifugation for 7 minutes at 25 xg and at room temperature. After washing the resulting tumor cells and suspending in complete culture medium, the presence of tumor cells was confirmed by staining with triptane blue. Tumor cells prepared in this way were used in the following experiment.
(2) Confirmación por ELISA de la producción de IL-6 del carcinoma de células renales (2) Confirmation by ELISA of the production of IL-6 renal cell carcinoma
La presencia de IL-6 en el sobrenadante del cultivo de la línea de carcinoma de células renales Caki-1, Caki-1/DDP,35 ACHN, A704 y células de tumor recientes de pacientes con carcinoma de células renales (Números 1 a 3) se investigó por ELISA (ensayo de inmunoabsorbente ligado a enzima). The presence of IL-6 in the culture supernatant of the Caki-1, Caki-1 / DDP renal cell carcinoma line, 35 ACHN, A704 and recent tumor cells of patients with renal cell carcinoma (Numbers 1 to 3 ) was investigated by ELISA (enzyme-linked immunoabsorbent assay).
Se añadieron 100 !l de anticuerpo frente a IL-6 a placas ELISA de 96 pocillos para revestir las placas ELISA con anticuerpo frente a IL-6 dejando reposar al menos durante toda la noche. Estas placas se almacenaron durante unmáximo de 4 semanas a 4ºC hasta el momento de uso. Las placas revestidas con anticuerpo frente a IL-6 se lavarontres veces y bloquearon durante una hora con PBS para ELISA que contenía PBS (albúmina sérica bovina) al 1%.Después de lavar dos veces, se añadieron a cada pocillo 100 !l de líquido sobrenadante del cultivo de células tumorales o IL-6 recombinante de E. coli como control (Yasukawa, et al., Biotechnol. Lett., 12, 419, 1990). 100 µl of antibody against IL-6 was added to 96-well ELISA plates to coat the ELISA plates with antibody against IL-6 allowing to stand at least overnight. These plates were stored for a maximum of 4 weeks at 4 ° C until the time of use. The plates coated with antibody against IL-6 were washed three times and blocked for 1 hour with PBS for ELISA containing 1% PBS (bovine serum albumin). After washing twice, 100 µl of liquid was added to each well Supernatant of tumor cell culture or recombinant E. coli IL-6 as a control (Yasukawa, et al., Biotechnol. Lett., 12, 419, 1990).
Las placas se incubaron durante 1 hora y se lavaron tres veces, seguido por la adición a cada pocillo de 100 !l de anticuerpo policlonal frente a IL-6 (Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988). Las placas se incubaron The plates were incubated for 1 hour and washed three times, followed by the addition to each well of 100 µl of polyclonal antibody against IL-6 (Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988). The plates were incubated
45 entonces durante 1 hora, seguido por la adición de IgG de cabra anticonejo unida a fosfato alcalino a cada pocillo y se incubó durante una hora más. Las placas se lavaron e incubaron con sustrato de fosfato alcalino (Sigma 104,Sigma Chemical Co.). Dos horas más tarde, se midió la absorbancia a 405 nm con el lector de ELISA (Immunoreader, Japan Intermed Co., Ltd.). Conforme a estos resultados, se demostró claramente que todos loscarcinomas de células renales producían IL-6 (véase la Tabla 1). Then for 1 hour, followed by the addition of alkaline phosphate-bound goat IgG to each well and incubated for another hour. The plates were washed and incubated with alkaline phosphate substrate (Sigma 104, Sigma Chemical Co.). Two hours later, absorbance at 405 nm was measured with the ELISA reader (Immunoreader, Japan Intermed Co., Ltd.). Based on these results, it was clearly demonstrated that all renal cell carcinomas produced IL-6 (see Table 1).
Tabla 1 Table 1
Concentración de IL-6 en el líquido sobrenadante Concentration of IL-6 in the supernatant liquid
Células de CCR Concentración de IL-6 (pg/ml; media+desviación típica) RCC cells IL-6 concentration (pg / ml; mean + standard deviation)
- Caki-1 Caki-1
- 1337+35 1337 + 35
- Caki-1/DDP Caki-1 / DDP
- 3900+325 3900 + 325
- A704 A704
- 1290+141 1290 + 141
- ACHN ACHN
- 1282+106 1282 + 106
- Células recientes de CCR (Paciente 1) Recent RCC cells (Patient 1)
- 1236+71 1236 + 71
- Células recientes de CCR (Paciente 2) Recent RCC cells (Patient 2)
- 42+4 42 + 4
- Células recientes de CCR (Paciente 3) Recent RCC cells (Patient 3)
- 2579+219 2579 + 219
(3) Efecto del anticuerpo frente a IL-6 o el anticuerpo frente a IL-6R sobre la citotoxicidad de agentes antitumorales (3) Effect of the antibody against IL-6 or the antibody against IL-6R on the cytotoxicity of antitumor agents
Se usó el procedimiento MMT (Mizutani, Y. et al., Cancer, 73, 730-737, 1994) para investigar el efecto del anticuerpofrente a IL-6 o el anticuerpo frente a IL-6R sobre la sensibilidad de diversos carcinomas de células renales, a saberThe MMT procedure (Mizutani, Y. et al., Cancer, 73, 730-737, 1994) was used to investigate the effect of the antibody against IL-6 or the antibody against IL-6R on the sensitivity of various cell carcinomas renal, namely
5 Caki-1, Caki-1/DDP, A704, ACHN y células tumorales recientes derivadas de pacientes con carcinoma de células renales (Pacientes números 1 a 3) a diversas concentraciones de agentes antitumorales, a saber, cisplatino (cisdiaminadicloroplatino (II)), mitomicina C (MMC), adriamicina (ADR), vinblastina (VBL) y 5-fluorouracilo (5-FU). 5 Caki-1, Caki-1 / DDP, A704, ACHN and recent tumor cells derived from patients with renal cell carcinoma (Patients numbers 1 to 3) at various concentrations of antitumor agents, namely cisplatin (cisdiaminadichloroplatin (II)) , mitomycin C (MMC), adriamycin (ADR), vinblastine (VBL) and 5-fluorouracil (5-FU).
Se añadieron 100 !l de las suspensiones de células (2 x 104 células) tumorales de carcinoma de células renalesanteriormente citados a una placa de microvaloración de superficie inferior de 96 pocillos (Corning Glass Works, 10 Corning). La placa se incubó en un ambiente húmedo a 37ºC en presencia de CO2 al 5% y las células tumorales se cultivaron durante 24 horas. El líquido sobrenadante del cultivo celular se aspiró y se lavaron las células tumoralestres veces con medio de cultivo RPMI 1640. Se añadieron a cada pocillo 200 !l de una solución que contenía cadaagente antitumoral o control en forma de una solución de cultivo completa, en presencia de anticuerpo frente a IL-6(Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988) o anticuerpo frente a IL-6R (Hirata, Y. et al., J. Immunol., 100 µl of the cell suspensions (2 x 104 cells) of renal cell carcinoma tumor cited above were added to a 96-well bottom surface microtiter plate (Corning Glass Works, 10 Corning). The plate was incubated in a humid environment at 37 ° C in the presence of 5% CO2 and the tumor cells were cultured for 24 hours. The cell culture supernatant was aspirated and the tumor cells were washed three times with RPMI 1640 culture medium. 200 µl of a solution containing each antitumor or control agent was added to each well in the form of a complete culture solution, in the presence of antibody against IL-6 (Matsuda, T. et al., Eur. J. Immunol., 18, 951-956, 1988) or antibody against IL-6R (Hirata, Y. et al., J. Immunol .,
15 143, 2900-2906, 1989), seguido por el cultivo durante 24 horas a 37ºC. Se añadieron a cada pocillo 20 !l de solución de MTT (5 mg/ml; Sigma Chemical Co.), después de lo cual se continuó el cultivo durante 4 horas en un ambientehúmedo a 37ºC y en presencia de CO2 al 5%. El medio de cultivo se eliminó de cada pocillo y se reemplazó porisopropanol que contenía HCl 0,05N (Sigma Chemical Co.). 15 143, 2900-2906, 1989), followed by cultivation for 24 hours at 37 ° C. 20 µl of MTT solution (5 mg / ml; Sigma Chemical Co.) was added to each well, after which the culture was continued for 4 hours in a humid environment at 37 ° C and in the presence of 5% CO2. The culture medium was removed from each well and replaced by isopropanol containing 0.05N HCl (Sigma Chemical Co.).
Se midió la absorbancia de la solución en cada pocillo a 540 nm con un lector de placas de microcultivo 20 (Immunoreader, Japan Intermed Co., Ltd.). Se calculó el porcentaje de citotoxicidad con la siguiente fórmula. The absorbance of the solution in each well at 540 nm was measured with a microculture plate reader 20 (Immunoreader, Japan Intermed Co., Ltd.). The percentage of cytotoxicity was calculated with the following formula.
Citotoxicidad (%) = [1-(absorbancia del grupo experimental/absorbancia del grupo control] x 100 Cytotoxicity (%) = [1- (absorbance of the experimental group / absorbance of the control group] x 100
Como resultado, la citotoxicidad provocada por cisplatino (véanse las Figuras 1A y 1B) o MMC (véanse lasFiguras 2A y 2B) en células Caki-1 aumentó claramente en presencia de anticuerpo. En el grupo experimental al quese añadió anticuerpo MOPC3 como control (J. Natl. Cancer Inst. (Bethesda), 41, 1083, 1986) no se observaron As a result, cytotoxicity caused by cisplatin (see Figures 1A and 1B) or MMC (see Figures 2A and 2B) in Caki-1 cells clearly increased in the presence of antibody. In the experimental group to which MOPC3 antibody was added as a control (J. Natl. Cancer Inst. (Bethesda), 41, 1083, 1986) no observed
25 efectos sobre la sensibilidad hacia cisplatino o MMC. En comparación con el caso de añadir un agente antitumoral solo, la cantidad de agente antitumoral requerida para obtener efectos citotóxicos equivalentes a los obtenidos enpresencia de agente antitumoral y anticuerpo frente a IL-6 o anticuerpo frente a IL-6R fue de 1/10 a 1/100. Por otro lado, no se produjo cambio en la sensibilidad de las células tumorales hacia agentes antitumorales en presencia deanticuerpo frente a IL-6 o anticuerpo frente a IL-6R y ADR, VBL o 5-FU. 25 effects on sensitivity to cisplatin or MMC. In comparison with the case of adding an antitumor agent alone, the amount of antitumor agent required to obtain cytotoxic effects equivalent to those obtained in the presence of antitumor agent and antibody against IL-6 or antibody against IL-6R was 1/10 a 1/100. On the other hand, there was no change in the sensitivity of the tumor cells towards antitumor agents in the presence of antibody against IL-6 or antibody against IL-6R and ADR, VBL or 5-FU.
30 La resistencia de células Caki-1/DDP al cisplatino se superó estando presentes anticuerpo frente a IL-6 o anticuerpo frente a IL-6R con el cisplatino (véanse las Figuras 3A y 3B). Los efectos combinados de anticuerpo frente a IL-6 oanticuerpo frente a IL-6R y cisplatino se observaron de igual forma para las otras líneas de carcinoma de célulasrenales ACHN (véanse las Figuras 4A y 4B) y A704 (véanse las Figuras 5A y 5B), así como para las células detumor recientes obtenidas de pacientes con carcinoma de células renales (véanse las Figuras 6A, 6B, 7A, 7B, 8A y 30 The resistance of Caki-1 / DDP cells to cisplatin was overcome by the presence of antibody against IL-6 or antibody against IL-6R with cisplatin (see Figures 3A and 3B). The combined effects of antibody to IL-6 or antibody to IL-6R and cisplatin were observed in the same way for the other ACHN renal cell carcinoma lines (see Figures 4A and 4B) and A704 (see Figures 5A and 5B) , as well as for recent tumor cells obtained from patients with renal cell carcinoma (see Figures 6A, 6B, 7A, 7B, 8A and
35 8B). 35 8B).
Ejemplo 2. Efecto del anticuerpo frente a IL-6 o el anticuerpo frente a IL-6R sobre la sensibilidad del carcinoma decélulas renales hacia carboplatino Example 2. Effect of antibody against IL-6 or antibody against IL-6R on the sensitivity of renal cell carcinoma towards carboplatin
Se examinó la citotoxicidad cuando estaba presente anticuerpo frente a IL-6 o anticuerpo frente a IL-6R concarboplatino en diversas concentraciones de la misma forma que en el Ejemplo 1 usando la línea de carcinoma deCytotoxicity was examined when antibody to IL-6 or antibody to IL-6R concarboplatin was present in various concentrations in the same manner as in Example 1 using the carcinoma line of
40 células renales Caki-1. Como resultado, se potenció la sensibilidad de las células Caki-1 hacia carboplatino (véanse las Figuras 9A y 9B). 40 renal cells Caki-1. As a result, the sensitivity of Caki-1 cells to carboplatin was potentiated (see Figures 9A and 9B).
Ejemplo 3. Efecto del anticuerpo frente a IL-6 o el anticuerpo frente a IL-6R sobre la acumulación intracelular deagentes antitumorales Example 3. Effect of the antibody against IL-6 or the antibody against IL-6R on the intracellular accumulation of antitumor agents
Se cultivaron células Caki-1 durante 24 horas en presencia de una combinación de 10 !g/ml de cisplatino o 100 Caki-1 cells were cultured for 24 hours in the presence of a 10 µg / ml combination of cisplatin or 100
45 !g/ml de 5-FU o medio de cultivo como control, y 10 !g/ml de anticuerpo MOPC3/C como control o 10 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R. 45 µg / ml of 5-FU or culture medium as control, and 10 µg / ml of MOPC3 / C antibody as control or 10 µg / ml of antibody against IL-6 or antibody against IL-6R.
A continuación, se retiró el medio de cultivo y se lavaron las células tres veces con medio de cultivo RPMI 1640. Semidió la acumulación intracelular de cisplatino por espectrometría de absorción atómica sin llama (Daley-Tates, P.T. et al., Biochem. Pharmacol., 34, 2263-2369; Riley, C.M. et al., Analytical Biochem, 124, 167-179, 1982). Como50 instrumento de medida se usó el Zeeman Z-8000 (Espectrofotómetro Zeeman Z-8000, Hitachi Co., Ltd.). Además, la acumulación intracelular de 5-FU se midió por cromatografía de gases y por espectrometría de masas (Marunaka, T.et al., J. Pharm. Sci., 69, 1296-1300, 1980). Como instrumento de medida se usó el espectrómetro de masas JMS-D300 equipado con el cromatógrafo de gases JGC-20KP (JOEL). Los resultados se muestran en la Tabla 2. Losresultados muestran claramente que el anticuerpo frente a IL-6 o el anticuerpo frente a IL-6R no tienen efecto algunoNext, the culture medium was removed and the cells were washed three times with RPMI 1640 culture medium. The intracellular accumulation of cisplatin was semi-determined by flameless atomic absorption spectrometry (Daley-Tates, PT et al., Biochem. Pharmacol. , 34, 2263-2369; Riley, CM et al., Analytical Biochem, 124, 167-179, 1982). As a measuring instrument, the Zeeman Z-8000 (Zeeman Z-8000 Spectrophotometer, Hitachi Co., Ltd.) was used. In addition, intracellular accumulation of 5-FU was measured by gas chromatography and mass spectrometry (Marunaka, T. et al., J. Pharm. Sci., 69, 1296-1300, 1980). The JMS-D300 mass spectrometer equipped with the JGC-20KP gas chromatograph (JOEL) was used as a measuring instrument. The results are shown in Table 2. The results clearly show that the antibody against IL-6 or the antibody against IL-6R has no effect.
55 sobre la acumulación de cisplatino y 5-FU en las células tumorales. 55 on the accumulation of cisplatin and 5-FU in tumor cells.
Tabla 2 Table 2
Acumulación intracelular de CDDP o 5-FU (ng/107 células) Tratamiento Intracellular accumulation of CDDP or 5-FU (ng / 107 cells) Treatment
- Medio de cultivo control Control culture medium
- mAb mAb mAb mAb
- Fármaco Drug
- Ab control Anti-IL-6 anti-IL-6R Ab control Anti-IL-6 anti-IL-6R
- CDDP CDDP
- 0,28+0,05 0,27+0,02 0,26+0,05 0,27+0,02 0.28 + 0.05 0.27 + 0.02 0.26 + 0.05 0.27 + 0.02
- 5-FU 5-FU
- 1,48+0,32 1,57+0,45 1,50+0,19 1,63+0,31 1.48 + 0.32 1.57 + 0.45 1.50 + 0.19 1.63 + 0.31
Los datos de la tabla se calcularon a partir de los datos obtenidos de tres experimentos (media + desviación típica). The data in the table were calculated from the data obtained from three experiments (mean + standard deviation).
Ejemplo 4. Efecto de cisplatino, anticuerpo frente a IL-6 o anticuerpo frente a IL-6R sobre la expresión de glutatión S5 transferasa-n (GST-n) Example 4. Effect of cisplatin, antibody against IL-6 or antibody against IL-6R on the expression of glutathione S5 transferase-n (GST-n)
Se cultivaron células Caki-1 durante 4 horas con medio de cultivo control, 100 !g/ml de cisplatino o 10 !g/ml de anticuerpo frente a IL-6 o anticuerpo frente a IL-6R. A continuación, se preparó el RNA total de las células conformela procedimiento de Mizutani, Y. et al. (Cancer, 73, 730-737, 1994), seguido por electroforesis en gel de agarosa al1,2%-HCHO 2,2M en 1 x tampón MOPS que contenía MOPS (3-[N-morfolino]propano-sulfonato) 200 mM, acetato 10 sódico 50 mM y EDTA sódico 10 mM, dando lugar a 10 !g de RNA por banda. A continuación, se realizó la transcripción del RNA a una membrana Biodyne A (Poll) en 20 x solución SSC que contenía NaCl 3M y citrato sódico0,3M (pH 7,0). Se marcó una sonda de cDNA de GST-n de 50 a 100 ng (Nakagawa, K. et al., J. Biol. Chem., 265, 4296-4301, 1990) por elongación de oligocebador al azar usando a32P-dCTP (NEN). La membrana de nylon anteriormente citada a la que se transcribe el RNA se reticuló con radiación ultravioleta y se hibridó con la sondaCaki-1 cells were cultured for 4 hours with control culture medium, 100 µg / ml cisplatin or 10 µg / ml antibody against IL-6 or antibody against IL-6R. Next, the total RNA of the cells was prepared according to the procedure of Mizutani, Y. et al. (Cancer, 73, 730-737, 1994), followed by 1.2% agarose gel electrophoresis - 2.2M HCHO in 1 x MOPS buffer containing MOPS (3- [N-morpholino] propane sulphonate) 200 mM , 50 mM sodium acetate 10 and 10 mM sodium EDTA, resulting in 10 µg of RNA per band. Next, RNA transcription was performed on a Biodyne A (Poll) membrane in 20 x SSC solution containing 3M NaCl and 0.3M sodium citrate (pH 7.0). A GST-n cDNA probe of 50 to 100 ng (Nakagawa, K. et al., J. Biol. Chem., 265, 4296-4301, 1990) was labeled by random oligoceator elongation using a32P-dCTP ( NEN) The above-mentioned nylon membrane to which the RNA is transcribed was crosslinked with ultraviolet radiation and hybridized with the probe
15 anteriormente citada. Los resultados se muestran en la Figura 10. 15 cited above. The results are shown in Figure 10.
La expresión de mRNA de GST-n de células Caki-1 no se vio afectada en absoluto por cisplatino. Sin embargo,cuando se añadió anticuerpo frente a IL-6 o anticuerpo frente a IL-6R, se redujo la expresión de mRNA de GST-n. En base a estos hallazgos, se sugirió que la disminución del nivel de expresión de mRNA de GST-n está implicada en el aumento en la sensibilidad del carcinoma de células renales hacia cisplatino provocada por el anticuerpo frenteGST-n mRNA expression of Caki-1 cells was not affected at all by cisplatin. However, when antibody to IL-6 or antibody to IL-6R was added, the expression of GST-n mRNA was reduced. Based on these findings, it was suggested that the decrease in the level of GST-n mRNA expression is implicated in the increased sensitivity of renal cell carcinoma towards cisplatin caused by the antibody against
20 a IL-6 o el anticuerpo frente a IL-6R. 20 to IL-6 or the antibody against IL-6R.
Aplicabilidad industrial Industrial applicability
Se observó la sensibilidad de células tumorales a agentes antitumorales a menores dosis como resultado decombinar antagonistas de IL-6 como el anticuerpo frente a IL-6 o el anticuerpo frente a IL-6R con agentes antitumorales, confirmando de este modo que se demuestran efectos combinados por antagonistas de IL-6 y The sensitivity of tumor cells to antitumor agents at lower doses was observed as a result of combining IL-6 antagonists such as the antibody against IL-6 or the antibody against IL-6R with antitumor agents, thereby confirming that combined effects are demonstrated. by IL-6 antagonists and
25 agentes antitumorales. Además, se demostró que las células tumorales que presentan resistencia al tratamiento con agentes antitumorales podían tratarse como resultado de que los antagonistas de IL-6 potenciaban su sensibilidad aagentes antitumorales. 25 antitumor agents. In addition, it was shown that tumor cells that exhibit resistance to treatment with antitumor agents could be treated as a result of IL-6 antagonists enhancing their sensitivity to antitumor agents.
Además, el potenciador del efecto de un agente antitumoral que comprende un antagonista de IL-6 de la presente invención puede reducir los efectos citotóxicos de agentes antitumorales sobre los tejidos permitiendo reducir laIn addition, the potentiator of the effect of an antitumor agent comprising an IL-6 antagonist of the present invention can reduce the cytotoxic effects of antitumor agents on the tissues allowing reducing the
30 dosis requerida de agente antitumoral, dándole así un considerable valor en uso como potenciador de los efectos de agentes antitumorales. 30 required dose of antitumor agent, thus giving it considerable value in use as an enhancer of the effects of antitumor agents.
Claims (3)
- 3.3.
- El uso según la reivindicación 1, en el que dicho anticuerpo es un anticuerpo PM-1 humanizado. The use according to claim 1, wherein said antibody is a humanized PM-1 antibody.
- 4.Four.
- Uso de un antagonista de IL-6 y un agente antitumoral seleccionado de cisplatino y carboplatino para la preparación de una composición para el tratamiento de un tumor, en donde la composición tiene un efecto antitumoral potenciado con respecto al efecto del agente antitumoral en solitario y en donde dicho antagonista de IL Use of an IL-6 antagonist and an antitumor agent selected from cisplatin and carboplatin for the preparation of a composition for the treatment of a tumor, wherein the composition has an enhanced antitumor effect with respect to the effect of the antitumor agent alone and in where said IL antagonist
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WO1996020728A1 (en) | 1996-07-11 |
DE69525971D1 (en) | 2002-04-25 |
CN1174507A (en) | 1998-02-25 |
AU704723B2 (en) | 1999-04-29 |
DE69525971T3 (en) | 2013-01-10 |
CA2209124A1 (en) | 1996-07-11 |
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