Method for controlling the activity of immunologically functional molecule
Abstract
IgG antibody that exhibits a cell cytotoxicity activity dependent on promoted antibody, the antibody can be obtained by introducing a gene encoding said antibody into a host cell, non-human animal or plant, in which said host cell, non-human animal or plant, does not has α1,6-fucosyltransferase activity due to the elimination of the gene encoding said enzyme or the addition of a mutation to said gene to eliminate said enzymatic activity, and being bound to said antibody a chain of N-glycoside-linked sugar of the double-stranded complex type, and in which it is not fucose-bound to the N-acetylglucosamine of the reducing end of said sugar chain, in which said linked sugar chain a N-glycoside of the double-stranded complex type has mainly the following structure ** Formula ** in which the antibody has a heavy chain constant region and a light chain constant region of a human IgG antibody.
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10 claims: 2 independent, 8 dependent
- 1ES 2 568 898 T3 REIVINDICACIONES 1. Anticuerpo IgG que presenta una actividad de citotoxicidad celular dependiente de anticuerpo promovida, pudiendo obtenerse el anticuerpo introduciendo un gen que codifica dicho anticuerpo en una célula anfitriona, animal no humano o planta, en el que dicha célula anfitriona, animal no humano o planta, no presenta actividad de a1,6-fucosiltransferasa debido a la eliminación del gen que codifica dicha enzima o la adición de una mutación a dicho gen para eliminar dicha actividad de enzimática, y estando unida a dicho anticuerpo una cadena de azúcar unida a N-glucósido de tipo complejo bicatenaria, y en el que no está unida fucosa a la N-acetilglucosamina del extremo reductor de dicha cadena de azúcar, en el que dicha cadena de azúcar unida a N-glucósido de tipo complejo bicatenaria presenta principalmente la estructura siguiente ±Gslpl—4GlcNAcfH —* 2Manal iGJcNAcpJ —» 4GlcNAcpi“* 4GlcNAc ±Galpl—4GlcNAcpl —2Manal en el que el anticuerpo presenta una región constante de cadena pesada y una región constante de cadena ligera de un anticuerpo IgG humano.
- 2Anticuerpo según la reivindicación 1, en el que el anticuerpo reconoce un antígeno relacionado con tumores, siendo opcionalmente el antígeno relacionado con tumores el gangliósido GD3.
- 3Anticuerpo según la reivindicación 1, en el que el anticuerpo reconoce un antígeno relacionado a una alergia o inflamación, siendo opcionalmente el antígeno relacionado a una alergia o inflamación la cadena α de receptor de interleucina 5 humana.
- 4Anticuerpo según la reivindicación 1, en el que el anticuerpo reconoce (i) un antígeno relacionado a una enfermedad cardiovascular, o (ii) un antígeno relacionado a una infección vírica o bacteriana.
- 5Medicamento que comprende el anticuerpo según cualquiera de las reivindicaciones 1 a 4.
- 6Medicamento según la reivindicación 5, en el que dicho medicamento (a) contiene dicho anticuerpo y está destinado a ser administrado como un fármaco terapéutico solo;o (b) comprende además uno o más vehículos farmacéuticamente aceptables.
- 7Agente para la utilización en el diagnóstico, el tratamiento o la prevención de un cáncer, que comprende el anticuerpo según la reivindicación 2 como un principio activo.
- 8Agente para la utilización en el diagnóstico, el tratamiento o la prevención de una alergia o inflamación, que comprende el anticuerpo según la reivindicación 3 como un principio activo.
- 9Agente para la utilización en el diagnóstico, el tratamiento o la prevención de una enfermedad cardiovascular, que comprende el anticuerpo según la reivindicación 4 (i) como un principio activo.
- 10Agente para la utilización en el diagnóstico, el tratamiento o la prevención de una infección vírica o bacteriana, que comprende el anticuerpo según la reivindicación 4 (ii) como un principio activo.
Independent claims10
504 paragraphs in 16 sections, as filed
ES 2 568 898 T3
DESCRIPTION
Procedure to control the activity of an immunofunctional molecule.
Technical field
The present disclosure relates to a method of monitoring the activity of an immunofunctional molecule, such as an antibody, a protein or a peptide, an agent for stimulating the activity of an immunofunctional molecule, and an immunofunctional molecule having the stimulated activity.
Previous technique
Since antibodies have high binding activity, binding specificity, and high stability in blood, their applications to the diagnosis, prevention, and treatment of various human diseases have been tried (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1 (1995)). However, an antibody from a non-human animal, such as a mouse antibody, is recognized as foreign material when administered to a human, thereby producing a human antibody against the mouse antibody (human anti-mouse antibody : hereinafter referred to as HAMA) in the human body, and HAMA is known to cause side effects by reaction with administered mouse antibody (J. Clin. Oncol., 2, 881 (1984); Blood, 65, 1349 (1985 ); J. Natl. Cancer Inst., 80, 932 (1988); Proc. Natl. Acad. Sci. USA, 82, 1242 (1985)), causes the disappearance of the mouse antibody administered in the blood (J. Nuc. Med., 26, 1011 (1985); Blood, 65, 1349 (1985); J. Natl Cancer Inst., 80, 937 (1988)) and reduces the diagnostic, preventive and therapeutic effects of the mouse antibody (J. Immunol., 135, 1530 (1985); Cancer Res., 46, 6489 (1986)) .
In order to solve these problems, an attempt has been made to convert an antibody from a non-human animal into a humanized antibody, such as a human hybrid antibody or an antibody with grafted human complementarity determining region (hereinafter referred to as RDC), using techniques of gene recombination. The human hybrid antibody is an antibody in which its antibody variable region (hereinafter referred to as the V region) is from an antibody from a non-human animal and its constant region (hereinafter referred to as the C region) is from a human antibody (Proc. Natl. Acad. Sci. USA, 81, 6851 (1984)). It has been reported that the administration of such hybrid antibodies to humans eliminates serious side effects and the half-life in the blood was approximately 6-fold prolonged compared to a mouse antibody (Proc. Natl. Acad. Sci. USA, 86, 4220 (1989)). RDC grafted human antibody is an antibody in which RDC of a human antibody is replaced by RDC of a non-human antibody (Nature, 321, 522 (1986)). It has been reported that, in an experiment using monkey, the immunogenicity of a human antibody grafted to the RDC was reduced and its half-life in the blood was prolonged 4 to 5 times compared to a mouse antibody (J. Immunol., 147 , 1352 (1991)). These publications demonstrate that a humanized antibody is expected to have sufficient effects, as an antibody to be applied to the diagnosis, prevention and treatment of various human diseases, even though it is not a fully human antibody. In fact, clinical trials have been performed with antitumor antibodies, such as a human anti-CD20 hybrid antibody, Rituxan (IDEC, Inc.), and an anti-HER2 / neu DRC-grafted human antibody, Herceptin (Genentech, Inc. ). The safety and therapeutic effects of human anti-CD20 hybrid antibody and anti-HER2 / neu RDC-grafted human antibody, to some extent, have been confirmed in B lymphoma and breast cancer, respectively (J. Clin. Oncol., 16, 2825 (1998); J. National Cancer Institute, 90, 882 (1998)). On the other hand, a fragment (Fab ') of a hybrid human anti-GPIIb / IIIa antibody, ReoPro (Centocor, Inc.), is marketed in Europe and America as a drug for the prevention of secondary disease after percutaneous transluminal coronary angioplasty. . Currently, a large number of clinical trials are being conducted with other humanized antibodies. Most of these humanized antibodies have been prepared using gene recombination techniques and have been produced using appropriate animal cells.
Five classes of antibodies, ie, IgM, IgD, IgG, IgA, and IgE, have been shown to be present in mammals. Human IgG class antibodies are mainly used in the diagnosis, prevention and treatment of various human diseases, due to their long half-life in the blood and functional characteristics, such as various effector functions and the like (Monoclonal Antibodies: Principles and Applications, Wiley -Liss, Inc., Chapter 2.1 (1995)). The human IgG class antibody is classified into the following 4 subclasses: IgG1, IgG2, IgG3 and IgG4. A large number of studies have been carried out so far for antibody-dependent cellular cytotoxicity activity (hereinafter referred to as ADCC activity) and complement-dependent cytotoxicity activity (hereinafter referred to as CDC) as effector functions of the class antibody. IgG, and it has been reported that antibodies of the IgG1 subclass have the highest ADCC activity and CDC activity among human antibodies of the IgG class (Chemical Immunology, 65, 88 (1997)). Therefore, most of the humanized anti-tumor antibodies that require high effector function are antibodies of the human IgG1 subclass, including earlier Rituxan and Herceptin.
The expression of the ADCC activity and CDC activity of antibodies of the human IgG1 subclass requires the binding of the Fc region of the antibody to an existing antibody receptor on the surface of an effector cell, such as a killer cell, a lymphocyte natural cytolytic, an activated macrophage or similar (hereinafter referred to as
ES 2 568 898 T3
FcyR) and different complement components. Several amino acid residues in the second domain of the antibody hinge region and the C region (hereinafter referred to as the Cy2 domain) have been suggested (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 ( 1995), Chemical Immunology, 65, 88 (1997)) and a sugar chain linked to the Cy2 domain are also important for this binding reaction (Chemical Immunology, 65, 88 (1997)). Regarding the sugar chain, Boyd et al. have examined the effects of a sugar chain on ADCC activity and CDC activity, by treating an antibody with human grafted RDC, CAMPATH-1H (human IgG1 subclass), produced using Chinese hamster ovary cells (CHO cell) or mouse myeloma NS0 cells with various sugar hydrolyzing enzymes, and reported that removal of sialic acid from the non-reducing terminal has no influence on both activities. In addition, the elimination of the remaining galactose, however, was published to influence only the CDC activity, decreasing approximately 50% of its activity. It was reported that the complete removal of the sugar chain causes the disappearance of both activities (Molecular Immunol., 32, 1311 (1995)). On the other hand, Lifely et al. have analyzed the sugar chain of an antibody with human grafted RDC, CAMPATH-1H (subclass human IgG1) that was produced using CHO cells, NS0 cells or YO cells of rat myeloma, its ADCC activity was determined and it was published that the CAMPATH-1H from YO cells exhibits the highest ADCC activity, suggesting that Nacetylglucosamine at the bisector position is important for activity (Glycobiology, 5, 813 (1995): WO 99/54342). Rothman et al (Mol Immunol, 1989, 26: 1113-1123) report that glycosylation inhibitors impact antibody activity under certain conditions. These publications demonstrate that the structure of the sugar chain plays an important role in the effector function of antibodies of the human IgG1 subclass, and that it may be possible to prepare an antibody with a higher effector function by changing the structure of the sugar chain. In reality, however, the structures of the sugar chains are complex and vary greatly. It is therefore necessary to study the structure more in depth to obtain a greater effector function.
Exhibition exhibition
An objective of the present invention is to specify a sugar chain that increases the activity of ADCC, analyzing the sugar chains of the antibodies of the human IgG1 subclass produced by various animal cells, and therefore also to provide a method to monitor the activity. of an immunofunctional molecule. Since ADCC activity is enhanced in such antibodies, increased therapeutic effect of various human diseases can be expected by using not only anti-tumor antibodies, but also other antibodies against diseases, as well as proteins or peptides against various diseases. In particular, in the clinical application of antitumor antibodies, the antitumor effect of an antibody alone is insufficient in many of the current cases. Known antibody deficiencies have required the simultaneous use of chemotherapy (Science, 280, 1197, 1998). The dependence on chemotherapy however will be reduced, with a reduction in side effects, if a stronger antitumor effect of an antibody is provided by the enhancement of ADCC activity alone. The in vitro activity of various humanized antibodies of the human IgG1 subclass produced by two types of Chinese hamster ovary cells, CHO / dhFr cells (ATCC CRL 9096) and CHO / DG44 cells (Somatic Cell and Molecular Genetics, 12 , 555 (1986)), mouse myeloma NS0 cells (RcB 0213, BIO / TECHNOLOGY, 10, 169 (1992)), mouse myeloma SP2 / 0-Ag14 cells (hereinafter referred to as SP2 / 0 cells; ATCC CRL 1581) and YB2 / 3HL.P2.G11.16Ag.20 rat myeloma cells (hereinafter referred to as YB2 / 0 cells; ATCC CRL 1662) and have found, as a result, that the ADCC activity of a humanized antibody produced by rat myeloma YB2 / 0 cells is considerably higher than that of humanized antibodies produced by other cells. Furthermore, as a result of an in vivo activity evaluation using Macaca phaseicularis, it has been found that the humanized antibody produced by YB2 / 0 cells exhibits the greatest effect, suggesting the utility of an antibody having elevated ADCC activity in a human clinical application. Furthermore, a sugar chain with the ability to increase ADCC activity has been identified by analyzing and comparing the sugar chain structures of humanized antibodies produced by various animal cells in detail, and the present invention has been achieved. .
More specifically, the present invention relates to items (1) to (10) below.
[1]. IgG antibody exhibiting a promoted antibody-dependent cellular cytotoxicity activity, the antibody being able to be obtained by introducing a gene encoding said antibody into a host cell, non-human animal or plant, wherein said host cell, non-human animal or plant, does not has a1,6-fucosyltransferase activity due to the deletion of the gene encoding said enzyme or the addition of a mutation to said gene to eliminate said enzyme activity, and a sugar chain linked to N-glucoside of the double-stranded complex type being linked to said antibody, and in which fucose is not linked to the N-acetylglucosamine of the reducing end of said sugar chain, wherein said sugar chain linked to Double-stranded complex type N-glucoside mainly has the following structure
ES 2 568 898 T3 ± Galpl — ► 4GlcNAc £ l + Ga) pJ - · - 4GicNAcftl * - 2Manal.
± G) cNAcpl —4Μ «ηβ1 — ► 4GJcNAcpj - *« GícNAc * 2Manal 'in which the antibody has a heavy chain constant region and a light chain constant region of a human IgG antibody.
[two] . The antibody of claim 1, wherein the antibody recognizes a tumor-related antigen, optionally the tumor-related antigen being ganglioside GD3.
[3] . The antibody of claim 1, wherein the antibody recognizes an antigen related to an allergy or inflammation, optionally the antigen related to an allergy or inflammation being the human interleukin-5 receptor α chain.
[4] . The antibody according to claim 1, wherein the antibody recognizes (i) an antigen related to a cardiovascular disease, or (ii) an antigen related to a viral or bacterial infection.
[5] . Drug comprising the antibody according to any of claims 1 to 4.
[6]. Drug according to claim 5, wherein said drug (a) contains said antibody and is intended to be administered as a therapeutic drug alone; or (b) further comprises one or more pharmaceutically acceptable carriers.
[7]. Agent for use in the diagnosis, treatment or prevention of cancer, comprising the antibody according to claim 2 as an active principle.
[8]. Agent for use in the diagnosis, treatment or prevention of an allergy or inflammation, comprising the antibody according to claim 3 as an active principle.
[9]. Agent for use in the diagnosis, treatment or prevention of cardiovascular disease, comprising the antibody according to claim 4 (i) as an active principle.
[10]. Agent for use in the diagnosis, treatment or prevention of a viral or bacterial infection, comprising the antibody according to claim 4 (ii) as an active principle.
This disclosure also provides the following items (1) to (62):
(1) Procedure to control the activity of an immunofunctional molecule, comprising the regulation of the presence or absence of fucose binding to M-acetylglucosamine of the reducing terminal of a sugar chain attached to the M-glucoside that binds to the immunofunctional molecule .
(2) Method according to item (1), in which the sugar chain attached to the M-glucoside that binds to the immunofunctional molecule comprises:
<img file="ES2568898T3_D0001.tif" />
(3) Process to improve the activity of an immunofunctional molecule, which comprises joining a sugar chain in which fucose is not present in M-acetylglucosamine of the reducing terminal of a sugar chain linked to M-glucoside to the immunofunctional molecule.
(4) Process according to point (3), in which the sugar chain comprises:
ES 2 568 898 T3
Well.
®Manp1
Manal4GlcNAc01
4GlcNAc (5) Process according to point (3), in which the sugar chain is synthesized in a cell that has a low enzymatic activity of the addition of fucose to M-acetylglucosamine of the reducing terminal or does not have said enzymatic activity.
(6) Method according to item (5), in which the enzyme that adds fucose to M-acetylglucosamine of the reducing terminal is a fucosyltransferase.
(7) Process according to item (6), wherein the fucosyltransferase is α-1,6-fucosyltransferase.
(8) Procedure according to item (3), wherein the sugar chain is synthesized in a rat myeloma cell.
(9) Procedure according to item (8), wherein the rat myeloma cell is YB2 / 3HL.P2.G11.16Ag.20 (ATCC CRL 1662).
(10) Process for inhibiting the activity of an immunofunctional molecule, which comprises joining a sugar chain in which fucose is present in N-acetylglucosamine of the reducing terminal of a sugar chain linked to the N-glucoside to an immunofunctional molecule.
(11) Process according to point (10), in which the sugar chain comprises:
<img file="ES2568898T3_D0002.tif" />
(12) Procedure according to point (10), in which the sugar chain is synthesized in a cell that has a high enzymatic activity of addition of fucose to M-acetylglucosamine of the reducing terminal.
(13) Method according to item (12), in which the enzyme that adds fucose to M-acetylglucosamine of the reducing terminal is a fucosyltransferase.
(14) Process according to item (13), wherein the fucosyltransferase is α-1,6-fucosyltransferase.
(15) Method according to item (1) to (14), in which the immunofunctional molecule is an antibody, a protein or a peptide.
(16) Agent stimulating the activity of an immunofunctional molecule, comprising a sugar chain in which fucose is not present in M-acetylglucosamine at the reducing terminal of a sugar chain attached to the N-glucoside.
(17) Agent stimulating the activity of an immunofunctional molecule according to item (16), in which the sugar chain comprises:
<img file="ES2568898T3_D0003.tif" />
(18) Agent stimulating the activity of an immunofunctional molecule according to point (16), in which the sugar chain is synthesized in a cell that has a low enzymatic activity of addition of fucose to M-acetyl-glucosamine of the reducing terminal or it does not have such enzymatic activity.
(19) Agent stimulating the activity of an immunofunctional molecule according to item (18), in which the enzyme that adds fucose to M-acetylglucosamine of the reducing terminal is a fucosyltransferase.
(20) Agent stimulating the activity of an immunofunctional molecule according to item (19), wherein the fucosyltransferase is α-1,6-fucosyltransferase.
ES 2 568 898 T3 (21) Agent stimulating the activity of an immunofunctional molecule according to item (16), in which the sugar chain is synthesized in a rat myeloma cell.
(22) Agent stimulating the activity of an immunofunctional molecule according to item (21), wherein the rat myeloma cell is YB2 / 3HL.P2.G11.16Ag.20 (ATCC CRL 1662).
(23) Agent stimulating the activity of an immunofunctional molecule according to any one of (16) to (22), in which the immunofunctional molecule is an antibody, a protein or a peptide.
(24) Immunofunctional molecule having an immunofunctional stimulated activity, to which the molecule of a sugar chain in which fucose is not present in M-acetylglucosamine of the reducing terminal of a sugar chain attached to the M-glucoside is attached.
(25) Immunofunctional molecule having an inhibited immunofunctional activity to which the molecule of a sugar chain in which fucose is present in M-acetylglucosamine of the reducing terminal of a sugar chain attached to the M-glucoside is attached.
(26) Immunofunctional molecule according to item (24), wherein the immunofunctional molecule is an antibody, a protein or a peptide.
(27) Immunofunctional molecule according to item (25), wherein the immunofunctional molecule is an antibody, a protein or a peptide.
(28) Process to produce the immunofunctional molecule according to point (24), which comprises the use of a cell that has a low enzymatic activity of the addition of fucose to M-acetylglucosamine of the reducing terminal or does not have said enzymatic activity.
(29) Process according to item (28), in which the enzyme that adds fucose to M-acetylglucosamine of the reducing terminal is a fucosyltransferase.
(30) Process according to item (29), wherein the fucosyltransferase is α-1,6-fucosyltransferase.
(31) Method for producing the immunofunctional molecule according to item (24), wherein a rat myeloma cell is used in the method for producing an immunofunctional molecule having an immunofunctional inhibited activity.
(32) Method according to item (31), wherein the rat myeloma cell is YB2 / 3HL.P2.G11.16Ag.20.
(33) Procedure to produce the immunofunctional molecule according to point (25), in which a cell having a high enzymatic activity of fucose addition to M-acetylglucosamine of the reducing terminal is used.
(34) Process according to item (33), in which the enzyme that adds fucose to M-acetylglucosamine of the reducing terminal is a fucosyltransferase.
(35) Process according to item (34), wherein the fucosyltransferase is α-1,6-fucosyltransferase.
(36) Immunofunctional molecule according to item (26), in which the antibody recognizes a tumor antigen.
A tumor antigen used according to the present invention is an antigen that is expressed in a tumor cell in greater quantity compared to normal cells. Examples include ganglioside GD2, GD3, and GM2 (Cancer Immunol. Immunother., 43, 152 (1996)), HER2 (J. Surgical Research, 77, 85 (1998)), CD52 (Leukemia Research, 22, 185 (1998) ) and MAGE (APMiS, 106, 665 (1998)). Furthermore, a factor that causes the growth of a tumor cell and its receptor are also tumor antigens. Examples include a basic fibroblast growth factor and its receptor (Pancreas, 17, 169 (1998)) and a vascular endothelial cell growth factor and its receptor (Pathology International, 48, 499 (1998)).
(37) Immunofunctional molecule according to item (36), in which the tumor antigen is ganglioside GD3.
(38) Immunofunctional molecule according to item (36), in which 09/07/51 (FERM BP-6691) produces the antibody.
(39) Immunofunctional molecule according to item (26), in which the antibody recognizes an antigen related to an allergy or inflammation.
An allergy or inflammation-related antigen used according to the present invention is an antigen that causes an allergy or inflammation and an antigen that is produced accompanied by an allergy or inflammation. The
Examples ES 2 568 898 T3 include interleukin 5 and its receptor (International Archives. Allergy. Immunol., 117, 11 (1998)), a tumor necrosis factor and its receptor (Citokine, 8, 651 (1996)).
(40) Immunofunctional molecule according to item (39), in which the antigen related to an allergy or inflammation is the α chain of the human interleukin-5 receptor.
(41) Immunofunctional molecule according to item (39), in which number 3 (FERM BP-6690) produces the antibody.
(42) Immunofunctional molecule according to item (26), in which the antibody recognizes an antigen related to cardiovascular disease.
A heart disease-related antigen used according to the present invention is an antigen that is involved in heart disease caused by thrombus or vascular restenosis. Examples include platelet GPIIb / IIIa (Thrombosis Research, 89, 129 (1998)), a platelet-derived growth factor and its receptor (American J. Physiology, 269, 1641 (1995)), and a blood clotting factor. blood (Thrombosis. Haemostasis, 79, 14 (1998)).
(43) Immunofunctional molecule according to item (27), in which the antibody recognizes an antigen related to an autoimmune disease.
An autoimmune disease-related antigen used according to the present invention is an autoantigen that elicits an immune response as the cause of a disease and an antigen that increases the response. Examples include auto-DNA (Rheumatology International, 17, 223 (1998)) and CD4 ((Rheumatic Diseases Clinics. North America, 24, 567 (1998)).
(44) Immunofunctional molecule according to item (26), in which the antibody recognizes an antigen related to a viral or bacterial infection.
A viral or bacterial infection-related antigen used according to the present invention is an antigen related to its infection and growth on a viral or bacterial target cell and also includes a viral or bacterial product. Examples include gp120 (Virology, 248, 394 (1998)), CXCR4 (J. Virology, 72, 8453 (1998)), and Vero toxin (J. Clinical Microbiology, 34, 2053 (1996)).
(45) Agent for the diagnosis of cancer, comprising the immunofunctional molecule according to point (36) as an active principle.
(46) Agent for the treatment of cancer, comprising the immunofunctional molecule according to point (36) as an active principle.
(47) Agent for the prevention of cancer, which comprises the immunofunctional molecule according to point (36) as an active principle.
(48) Agent for the diagnosis of an allergy or inflammation, which comprises the antibody according to point (39) as an active principle.
(49) Agent for the treatment of an allergy or inflammation, which comprises the antibody according to point (39) as an active principle.
(50) Agent for the prevention of an allergy or inflammation, which comprises the antibody according to point (39) as an active principle.
(51) Agent for the diagnosis of cardiovascular disease, which comprises the antibody according to point (42) as an active principle.
(52) Agent for the treatment of cardiovascular disease, which comprises the antibody according to point (42) as an active principle.
(53) Agent for the prevention of cardiovascular disease, which comprises the antibody according to point (42) as an active principle.
(54) Agent for the diagnosis of an autoimmune disease, comprising the antibody according to point (43) as an active principle.
(55) Agent for the treatment of an autoimmune disease, comprising the antibody according to point (43) as an active principle.
ES 2 568 898 T3 (56) Agent for the prevention of an autoimmune disease, which comprises the antibody according to point (43) as an active principle.
(57) Agent for the diagnosis of a viral or bacterial infection, which comprises the antibody according to point (44) as an active principle.
(58) Agent for the treatment of a viral or bacterial infection, comprising the antibody according to point (44) as an active principle.
(59) Agent for the prevention of a viral or bacterial infection, which comprises the antibody according to point (44) as an active principle.
(60) Agent for the diagnosis of various diseases, comprising the peptide or protein according to point (26) or (27) as an active principle.
Examples of the various diseases according to the present invention include a cancer, an allergic disease, an inflammatory disease, a cardiovascular disease, an autoimmune disease, a viral or bacterial infection, and the like.
(61) Agent for the treatment of various diseases, comprising the peptide or protein according to point (60) as an active principle.
(62) Agent for the prevention of various diseases, comprising the peptide or protein according to point (60) as an active principle.
Based on the way immunofunctional molecules are attached, the sugar chain is roughly classified into two types, namely a sugar chain that binds to asparagine (called an N-glucoside-linked sugar chain) and a sugar chain which binds to serine, threonine and the like (called O-glucoside linked sugar chain).
The sugar chain linked to N-glucoside according to the present invention has several structures (Biochemical Experimentation Method 23 - Method for Studying Glycoprotein Sugar Chains (Gakkai Shuppan Center), edited by Reiko Takahashi (1989)), but each case has the following structure common core.
<img file="ES2568898T3_D0004.tif" />
In the above structure, the terminal of the sugar chain that is attached to asparagine is called the reducing terminal, and the opposite side is called the non-reducing terminal. Fucose can be attached to reducing terminal N-acetylglucosamine, for example, via an α-1,3 bond or an α1,6 bond.
Examples of N-glucoside-linked sugar chains include a high-mannose type, in which only mannose binds to the non-reducing end of the core structure; a complex type, in which the non-reducing terminal side of the core structure has one or more galactose-N-acetylglucosamine branches (hereinafter referred to as Gal-GlcNAc) and the non-reducing terminal side of Gal-GlcNAc also has a structure such as for example a sialic acid or bisecting N-acetylglucosamine; a hybrid type, in which the non-reducing terminal side of the core structure has both branches of the sugar chain attached to the N-glucoside rich in mannose and the complex sugar chain attached to the N-glucoside.
Included in all of these types is a sugar chain in which fucose binds to N-acetylglucosamine on the reducing terminal side, and the sugar chain of the present invention includes not only the above sugar chains but also any another sugar chain, as long as the fucose is not bound to Nacetylglucosamine.
An immunofunctional molecule is a molecule that originally originates from the living body and is involved in various immune responses. Specifically, it includes for example antibodies, proteins and peptides.
An antibody is a protein that is produced in vivo by an immune response as a result of stimulation by a foreign antigen and has an activity to specifically bind to the antigen. Examples of the antibody include an antibody secreted by a hybridoma cell prepared from spleen cells of an immunized animal after immunization of the animal with an antigen, as well as an antibody prepared by gene recombination techniques, namely an antibody obtained introducing an antibody encoding the antibody expression vector inserted into the gene into a host cell. Examples include an antibody
ES 2 568 898 T3 produced by a hybridoma, a humanized antibody and a human antibody.
A hybridoma is a cell that produces a monoclonal antibody that has a desired antigen specificity and is obtained by cell fusion of a B lymphocyte prepared by immunizing a mammal other than human with an antigen, with a myeloma cell derived, for example, from a mouse.
Humanized antibodies include, for example, a human hybrid antibody, an antibody grafted onto a human complementarity determining region (hereinafter referred to as RDC).
A human hybrid antibody is an antibody that comprises a variable region of the antibody heavy chain (hereinafter also called HV or VH, in which the heavy chain is an H chain and the variable region is a V region) and a region variable of the antibody light chain (hereinafter also called LV or VL, where the light chain is an L chain) from a non-human animal, a heavy chain constant region (hereinafter also called CH, wherein the constant region is a C region) of a human antibody and a light chain constant region (hereinafter also referred to as CL) of a human antibody. Non-human animals can be, for example, any of mouse, rat, hamster and rabbit, as long as a hybridoma can be prepared from them.
The human hybrid antibody can be produced by obtaining the cDNAs encoding VH and VL from a hybridoma producing a monoclonal antibody, inserting each of the cDNAs into an expression vector for a host cell that has a gene encoding the CH of the human antibody, and the human antibody CL to construct a human hybrid antibody expression vector, and then introducing the vector into a host cell to express the antibody.
Any CH of the human hybrid antibody can be used, as long as it belongs to a human immunoglobulin (hereinafter referred to as hIg), but those of the hIgG class are preferred and any of the subclasses belonging to the hIgG class can be used, such as hIgG1, hIgG2, hIgG3 and hIgG4. On the other hand, any CL of the human hybrid antibody can be used, as long as it belongs to hIg, and those of the κ class or the λ class can be used.
A human CDR-grafted antibody is an antibody in which the amino acid sequences of the VH and VL CDRs of an antibody from a non-human animal are grafted to the appropriate positions of the VH and VL of a human antibody.
The human RDC-grafted antibody can be produced by constructing the cDNAs encoding the V regions in which the VH and VL RDC sequences of an antibody from a non-human animal are grafted to the VH and VL RDC sequences of an antibody from a non-human animal each of the cDNAs are grafted into an expression vector for a host cell that has a gene encoding the CH of a human antibody and the CL of a human antibody to constructing a human CDR-grafted antibody expression vector, and introducing the expression vector into a host cell to express the human CDR-grafted antibody.
The CH of the human CDR-grafted antibody can be any region belonging to hIg, but those of the hIgG class are preferred. Any of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3, and hIgG4 can be used. Furthermore, the CL of the human CDR-grafted antibody can be any region belonging to hIg, and those of the κ class or the λ class can be used.
A human antibody has to originally be an antibody naturally occurring in the human body, but also includes antibodies obtained from a human antibody phage library and from a transgenic animal that produces human antibodies or from a transgenic plant that produces human antibodies, which are prepared based on recent advances in genetic engineering, cell engineering, and developmental engineering techniques.
Antibody existing in the human body can be obtained, for example, by isolating a lymphocyte from human peripheral blood, immortalizing it by infection with the EB virus, followed by cloning, cultivating a lymphocyte capable of producing the antibody, and purifying the antibody from the culture mix.
The human antibody phage library is a library in which an antibody fragment, such as for example Fab or a single chain antibody, is expressed on the surface of the phage by inserting an antibody gene prepared from human B lymphocytes into a gene of the phage. A phage expressing an antibody fragment with the desired antigen-binding activity can be retrieved from this library, using its activity to bind an immobilized antigen substrate as a marker. The antibody fragment can be further converted into a human antibody molecule comprising two full H chains and two full L chains by genetic engineering techniques.
A non-human transgenic animal that produces human antibodies is an animal in which a gene encoding
ES 2 568 898 T3 human antibodies are integrated into cells. Specifically, a transgenic animal that produces human antibodies can be prepared by introducing a gene encoding human antibodies into a mouse ES cell, transplanting the ES cell into an early-stage embryo from another mouse, and growing an animal. The human antibody can be prepared and accumulated in a culture mixture of the transgenic animal that produces human antibodies by obtaining a hybridoma that produces human antibodies according to a hybridoma preparation method generally carried out in non-human mammals and then culturing the hybridoma.
An activity of the antibodies of the present invention includes the activity of ADCC.
ADCC activity, as used herein, refers to an activity to damage, for example, a tumor cell by activating an effector cell by binding the Fc region of an antibody to an existing Fc receptor. on the surface of an effector cell such as a killer cell, a natural killer cell or an activated macrophage (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., chapter 2.1 (1995)).
Any protein and peptide can be used, as long as multiple immune responses can be activated. Examples include interferon molecules, such as interleukin-2 (IL-2) (Science, 193, 1007 (1976)) and interleukin-12 (IL-12) (J. Leuc. Biol., 55, 280 ( 1994)), colony stimulating factors, such as granulocyte colony stimulating factor (G-CSF) (J. Biol .. Chem., 258, 9017 (1983)), macrophage colony stimulating factor (M-CSF ) (J. Exp. Med., 173, 269 (1992)) and granulocyte macrophage colony stimulating factor (GM-CSF) (J. Biol .. Chem., 252, 1998 (1977)); growth factors, such as erythropoietin (EPO) (J. Biol..Chem., 252, 5558 (1977)) and thrombopoietin (TPO) (Nature, 369, 533 (1994)).
The activities of the antibodies of the present invention are the activities of various immunocompetent cells, including lymphocytes (eg, T lymphocytes and B lymphocytes) and macrophages, or various immune response reactions, when the protein and peptide containing the chain sugar are administered into the living body.
The stimulation of the activities of the antibodies of the present invention includes the activation of natural killer cells and T lymphocytes by IL-2 and IL-12, the stimulating activities of erythrocyte production by EPO, which are further increased.
1. Procedure for the analysis of the sugar chain of an immunofunctional molecule (1) Analysis of the composition of neutral sugar and amino sugar
As described above, the sugar chain of IgG comprises a neutral sugar, such as for example galactose, mannose or fucose, an amino sugar, such as N-acetylglucosamine, and an acidic sugar, such as for example sialic acid.
Regarding analysis of the composition of the sugar chain of an antibody, the proportion in the composition can be analyzed by the release of neutral sugars or amino sugars by acid hydrolysis of the sugar chain.
Specific methods include a method using a sugar composition analyzer (BioLC) produced by Dionex. The BioLC is an apparatus for the analysis of the sugar composition by the HPAEC-PAD method (high performance anion exchange chromatography with pulsed amperometric detection) (J. Liq. Chromatogr., 6, 1577 (1983)).
The proportion in the composition can also be analyzed by a fluorescence labeling method using 2-aminopyridine. Specifically, the proportion in the composition can be calculated by fluorescence labeling of an acid hydrolyzed sample with 2-aminopyridine according to a known method (Agric. Biol .. Chem., 55 (1), 283284 (1991)) and leading to carry out HPLC analysis.
(2) Analysis of the structure of the sugar chain
The structure of the sugar chain of an antibody can be analyzed by a two-dimensional sugar chain mapping method (Anal. Biochem., 171, 73 (1988), Biochemical Experimentaron Method 23 - Method for Studying Glycoprotein Sugar Chains ( Gakkai Shuppan Center (Gakkai Shuppan Center), edited by Reiko Takahashi (1989)). The two-dimensional sugar chain mapping method is a method in which the structure of the sugar chain is estimated, for example, by plotting the retention time or elution position of the sugar chain by phase chromatography. inverse on the X axis and the retention time or elution position of the sugar chain by normal phase chromatography on the Y axis, and comparing the results with those of known sugar chains.
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Specifically, the sugar chain is released from the antibody by hydrazinolysis of the antibody, fluorescence labeling of the sugar chain is carried out with 2-aminopyridine (hereinafter referred to as PA) (J. Biochem., 95, 197 (1984)) , and then the sugar chain is separated from excess PA reagent by gel filtration and subjected to reverse phase chromatography. Subsequently, each peak of the fractionated sugar chain is analyzed by normal phase chromatography. Based on these results, the structure of the sugar chain can be estimated by plotting the points on a two-dimensional map of the sugar chain and comparing them with those of the patterns of the sugar chain (prepared by Takara Shuzo) or a reference (Anal. Biochem., 171, 73 (1988)).
Furthermore, the structure estimated by the two-dimensional sugar chain mapping method can be confirmed by mass spectrometry, such as for example MALDI-TOF-MS, of each sugar chain.
two. Method to monitor the activity of the immunofunctional molecule
The method of the present disclosure for monitoring the activity of an immunofunctional molecule is described below using immunoglobulin G (hereinafter referred to as IgG) as an example.
The sugar chain attached to the M-glucoside that binds to IgG is a complex biantennial sugar chain mainly composed of the following structure (hereinafter referred to as biantennial).
+ Fucal + Gaipi — 4GlcNAc01 —► 2Manal | iGlcNAcfJl ^ Manpi — v- 4GlcNAcpl —4G1cNAe ± Galfl — ►- 4GtcNAcpl —2Manal
The present invention also includes similar sugar chains in which an acid sugar, sialic acid, is further added to Gal of the non-reducing terminal of the sugar chain attached to the M-glucoside or to a bisecting Macetylglucosamine is added to the sugar chain. attached to the M-glucoside.
In an IgG type, a sugar chain attached to the M-glucoside is attached to a position in the Fc region. Since an IgG type comprises two H chains, the Fc moiety is present at two positions on an antibody molecule. Consequently, the sugar chain binding region is also present in two positions.
IgG activity changes as a function of the number of M-glucoside-bound sugar chains in which fucose is not bound to M-acetylglucosamine, to add to the two sugar chain-binding regions above. That is, when the sugar chain linked to the M-glucoside in which the fucose is not linked to N-acetyl-glucosamine is added to at least one of the binding regions of the sugar chain, the activity of the immunofunctional molecule. As an example, the degree of activity of IgG will be as follows: antibody F0> antibody F1> antibody F2, where antibody F0 designates an antibody in which the sugar chain attached to M-glucoside in which fucose is not present. linked to M-acetylglucosamine it is added to both two regions of binding to the sugar chains; the F1 antibody designates an antibody in which the M-glucoside-bound sugar chain in which fucose is not bound to M-acetylglucosamine is added to one of the sugar chain-binding regions, and the F2 antibody designates an antibody in which the sugar chain linked to the M-glucoside in which the fucose is linked to M-acetylglucosamine is added to both binding regions of the sugar chain.
The antibody produced may not always have a single sugar chain structure, and the F0 antibody, the F1 antibody, and the F2 antibody may be present as a mixture when the presence or absence of fucose is taken into account. To monitor the ADCC activity of the produced antibody, the antibody-bound sugar chain is analyzed using the above method for analyzing the sugar chain of an immunofunctional molecule, and using the analyzed result as an index.
The ADCC activity of the produced antibody can be stimulated by increasing the ratio between the F1 antibody and the F0 antibody. Specifically, the F1 antibody and the F0 antibody can be purified, or expression in a host cell can be regulated such that the M-glucoside-bound sugar chain in which fucose is not bound to M-acetylglucosamine is added to the immunofunctional molecule.
The ADCC activity of the produced antibody can be inhibited by increasing the existing proportion of the F2 antibody. Specifically, the F2 antibody can be purified, or expression in a host cell can be regulated such that the M-glucoside-linked sugar chain in which fucose is linked to M-acetylglucosamine is added to the immunofunctional molecule.
As described above, the strength of the desired activity can be controlled by regulating the existing ratio of F0 antibody, F1 antibody, and F2 antibody.
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3. Procedure for the production of an immunofunctional molecule
A procedure for producing an immunofunctional molecule having an N-glucoside-linked sugar chain in which fucose is not linked to N-acetylglucosamine or an immunofunctional molecule having an N-glucoside-linked sugar chain is described below. which fucose is bound to Nacetylglucosamine.
To link a desired sugar chain to an antibody, peptide, or protein, it can be produced by introducing a gene encoding the antibody, peptide, or protein of interest into a host cell and culturing the resulting cell. Alternatively, it can also be produced by introducing a gene encoding the antibody, peptide or protein of interest into an animal or plant and cultivating the resulting animal or plant.
The host cell, animal or plant useful in the production of an immunofunctional molecule that has a sugar chain attached to the N-glucoside in which the fucose is not attached to N-acetylglucosamine can be any cell, animal or plant, as long as , for example, have low enzymatic activity of adding fucose to Nacetylglucosamine that binds to the Fc region of an antibody or has no enzymatic activity. Examples of the cell that has low enzymatic activity from the addition of fucose to N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzymatic activity include, for example, a rat myeloma cell, YB2 / 3HL cell .P2.G11.16Ag. 20 (ATCC CRL 1662 (hereinafter called YB2 / 0 cell)).
Also, a cell, animal or plant, that has low or no enzymatic activity associated with an α1,6 bond can be constructed, for example, by deleting a gene encoding the enzyme that carries the α1,6 bond in the host cell, animal. or plant or adding a mutation to the gene to reduce or eliminate enzyme activity, and can be used as a host cell, animal or plant. The enzyme bearing the α1,6 bond includes fucosyltransferases, and is preferably α1,6-fucosyltransferase (hereinafter referred to as FUT8).
The host cell, animal or plant that is used in the production of an immunofunctional molecule that has a sugar chain linked to N-glucoside in which fucose is linked to N-acetylglucosamine can be any cell, animal or plant, as long as when, for example, it has a high enzymatic activity of fucose addition to N-acetylglucosamine that binds to the Fc region of an antibody.
Furthermore, a cell, animal or plant that has a high enzymatic activity associated with an α1,6 bond can be prepared by introducing a gene encoding the enzyme that carries the α1,6 bond into the host cell, animal or plant or by adding a mutation. to the gene to increase enzyme activity, and can be used as a host cell, animal or plant. The enzyme bearing the α1,6 bond includes fucosyltransferases, and is preferably FUT8 for example.
The host cells can be, for example, any of bacteria, yeast, animal cells, insect cells, plant cells and the like, as long as they can express the gene of interest.
Examples of bacterial host cells include microorganisms belonging to the genus Escherichia, the genus Serratia, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, the genus Microbacterium, and the genus Pseudomonas, such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue. , Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli GI698, Escherichia coli TB1, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium CCC, Brevibacterium flamagenesium, Brevibacterium 140CC, Brevibacterium flamariovibacterium 140CC68, Brevibacterium flamariovibacterium 140CC, Brevibacterium Brevibacterium 140CC, Brevibacterium flagiosumCC, Brevibacterium 140CC Brevibacterium, Brevibacterium, Brevibacterium 140CC, Brevibacterium flamarioc 14067, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas putida and Pseudomonas sp. D-0110.
Examples of yeast host cells include microorganisms belonging to the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces, the genus Pichia, the genus Candida, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis lactomyces , Trichosporon pullulans, Schwanniomyces alluvius and Candida utilis.
Examples of animal host cells include mouse myeloma cells, such as NS0 cells and SP2 / 0 cells, Chinese hamster ovary cells, such as CHO / dhfr cells.<sup>-</sup> and CHO / DG44 cells; rat myeloma cells, such as YB2 / 0 cells and IR983F cells; monkey cells, such as COS cells, human myeloma cells, such as Namalwa cells. Preferably, Chinese hamster ovary cells, such as CHO / DG44 cells, can be used.
Examples of insect host cells include Spodoptera frugiperda ovary cells, such as Sf9 and Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)); a
ES 2 568 898 T3 Trichoplusia ni ovarian cell, such as High 5 (prepared by Invitrogen).
Examples of plant host cells include plant cells from tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, and barley.
An immunofunctional molecule can be produced by culturing the obtained transformant in a medium to form and accumulate the immunofunctional molecule in the resulting culture, and then recovering it from the culture.
Furthermore, an immunofunctional molecule can also be produced by constructing a transgenic animal or plant and raising the resulting animal or plant.
The animal or plant for the production of an immunofunctional molecule having a sugar chain linked to N-glucoside in which fucose is not linked to N-acetylglucosamine can be any animal or plant, as long as, for example, it has a low activity fucose addition to N-acetylglucosamine that binds to the Fc region of an antibody or has no enzymatic activity.
Also, a transgenic non-human animal or a transgenic plant that has low or no enzymatic activity relative to an α1,6 bond can be prepared and used by deleting a gene encoding the enzyme that carries the α1,6 bond in the animal or plant. or adding a mutation to the gene to reduce or eliminate enzyme activity. The enzyme bearing the α1,6 bond includes fucosyltransferases and is preferably FUT8.
Any animal or plant can be used as an animal or plant for use in the production of an immunofunctional molecule having a sugar chain attached to the N-glucoside in which the fucose is attached to Nacetylglucosamine, as long as, for example, with respect to to an antigen, it has a high enzymatic activity of fucose addition to N-acetylglucosamine that binds to the Fc region of the antibody.
In addition, a non-human transgenic animal or transgenic plant having a high enzyme activity carrying an α1,6 linkage can be prepared and used by introducing a gene encoding the enzyme carrying the α1,6 linkage into the animal or plant or by adding a mutation. to the gene to increase enzyme activity. The enzyme bearing the α1,6 bond includes fucosyltransferases and is preferably FUT8.
The transgenic non-human animal can be obtained by directly injecting a desired gene into a fertilized egg (Proc. Natl. Acad. Sci. USA, 77, 7380 (1980)).
Genetically modified non-human animals include mouse, rat, rabbit, poultry, goat, or cattle.
Furthermore, a transgenic non-human animal or a genetically modified non-human animal having a desired gene can be achieved by introducing the desired gene into an embryonic stem cell and preparing the animal by a hybrid method of aggregation or a hybrid method of injection (Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, IRL Press in Oxford University Press (1993); Biomaterial Series 8, Gene Targeting, Gene Targeting, Preparation of mutation mouse using ES cell, Yodo-sha (1995)).
Examples of the embryonic stem cell include mouse (Nature, 292, 154 (1981)), rat, poultry, pig, monkey, goat, or cattle embryonic stem cells.
Furthermore, the non-human transgenic animal or the genetically modified non-human animal can also be prepared using a clonal technique in which a nucleus into which a desired gene is introduced is transplanted into a denuclear egg (Science, 280, 1256, (1998) ; Science, 278, 824, (1997)).
An immunofunctional molecule can be produced by introducing DNA encoding the immunofunctional molecule into an animal prepared by the above procedure to thereby form and accumulate the immunofunctional molecule in the animal, and then harvesting the immunofunctional molecule from the animal. The immunofunctional molecule can be prepared, for example, to form and accumulate in milk (Japanese Unexamined Published Patent Application No. 309192/88), or egg of the animal.
The procedure for producing a transgenic plant is described, for example, in a reference (Biol. Chem., 380, 825 (1999)). The procedure for producing a genetically modified plant is described, for example, in a reference (Plant Journal, 11, 1195 (1997)).
Regarding the process for the production of an immunofunctional molecule using a plant, the immunofunctional molecule can be produced, for example, by cultivating a transgenic plant in which it introduces DNA encoding the immunofunctional molecule, according to a known procedure (Tissue Culture, 20, (1994); Tissue Culture, 21, (1995); Trends in Biotechnology, 15, 45 (1997)) to thereby form and accumulate the immunofunctional molecule in the plant, and then collect the immunofunctional molecule from the plant.
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In addition, a genetically modified animal gene that can produce an immunofunctional molecule that has a sugar chain attached to N-glucoside in which fucose is not attached to N-acetylglucosamine or an immunofunctional molecule that has a sugar chain attached to N- glucoside in which fucose is bound to Nacetylglucosamine can be obtained by crossing a transgenic non-human animal or genetically modified non-human animal of a fucosyltransferase, preferably FUT8, with a homologous but different lineage from the transgenic animal of a desired immunofunctional molecule. The crossing procedure includes, for example, natural crossing, in vitro fertilization.
Furthermore, it is possible to carry out the mass production of the sugar chain by introducing a group of genes encoding the isolated enzymes, for example, in yeast, E. coli (Nature Biotechnology, 16, 847 (1998)). Furthermore, the enzyme produced can be used in the modification of an antibody, peptide or protein with the sugar chain or in the production thereof.
Furthermore, a sugar chain that stimulates the activity of an immunofunctional molecule, according to the present invention, can be substituted with a peptide (J. Immunol., 160, 293 (1998)). Said peptide has utility in the above process for the use of sugar chains and is also excellent in view of convenience, since it can be easily fused with an immunofunctional molecule.
A method for producing an immunofunctional molecule having a stimulated immunofunctional activity is described below. While a method for producing a humanized antibody is described as an example herein, other immunofunctional molecules may be prepared by the aforementioned procedure or a procedure similar thereto.
Four. Procedure for the production of humanized antibody (1) Construction of the vector for the expression of the humanized antibody
The vector for the expression of the humanized antibody is an expression vector for use in an animal cell into which the genes encoding C regions of the heavy chain (hereinafter referred to as the H chain) and the light chain (referred to as the H chain) are inserted. hereinafter L chain) of a human antibody, and can be constructed by cloning each of the genes encoding the C regions of the H chain and the L chain of a human antibody into an expression vector for animal cells.
The C regions of a human antibody may be the C regions of the H chain and the L chain of a suitable human antibody, and examples include the C region of the IgG1 subclass of an H chain of a human antibody (hereinafter referred to as hCy1 ) and the C region of a κ class of a human antibody L chain (hereinafter referred to as hCK).
The genes encoding the C regions of the H chain and the L chain of a human antibody can be a chromosomal DNA comprising the exon and intron or a cDNA.
The expression vector for animal cells can be any vector, as long as a gene encoding the C region of a human antibody can be inserted and expressed. Examples include pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223 (1984)), pKCR (Proc. Natl. Acad. Sci. USA, 78, 1527 (1981), pSG1β d2-4 (Cytotechnology, 4, 173 (1990)). The activator and enhancer to be used in the expression vector for animal cells includes the SV40 early activator and enhancer (J. Biochem., 101, 1307 (1987)), the mouse Moloney leukemia virus LTR ( Biochem. Biophys. Res. Comun., 149, 960 (1987)), the immunoglobulin H chain activator (Cell, 41, 479 (1985)) and the enhancer (Cell, 33, 717 (1983)).
The vector for the expression of the humanized antibody can be any vector in which the H chain and the L chain of the antibody are present in separate vectors or a vector in which they are present in the same vector (hereinafter referred to as a tandem vector); however, a tandem vector for the expression of the humanized antibody is preferable because such tandem humanized antibody expression vectors are easily constructed and introduced into an animal cell and the expression amounts of the H chain and the L chain of the antibody in the animal cell it can be equilibrated (J. Immunol. Methods, 167, 271 (1994)).
The vector constructed for the expression of the humanized antibody can be used for the expression of a human hybrid antibody and a human CDR-grafted antibody in animal cells.
(2) Preparation of the cDNA encoding the V region of the antibody from non-human animals
The cDNA encoding the H chain and L chain V regions of an antibody from a non-human animal, such as a mouse antibody, can be obtained as described below.
The cDNA is synthesized by extracting mRNA from a hybridoma cell capable of producing the mouse antibody of
ES 2 568 898 T3 interest. The synthesized cDNA is cloned into a vector, such as a phage or plasmid, to prepare a cDNA library. A recombinant phage or a recombinant plasmid containing a cDNA encoding the H chain V region and a recombinant phage or a recombinant plasmid containing a cDNA encoding the L chain V region are respectively isolated from the library using a residue of the C region or a residue of the V region of a known mouse antibody as a probe. The complete nucleotide sequences of the regions are determined
V of the H chain and L chain of the mouse antibody of interest in the recombinant phage or recombinant plasmid, and the complete amino acid sequences of the H chain and L chain V regions are deduced from nucleotide sequences.
The non-human animal can be any animal, such as, for example, mouse, rat, hamster, or rabbit, as long as a hybridoma cell can be produced therefrom.
The process for the preparation of complete RNA from a hybridoma cell includes a guanidine thiocyanate-cesium trifluoroacetate method (Methods in Enzymol., 154, 3 (1987)). The procedure for the preparation of mRNA from complete RNA includes, for example, an oligo (dT) column method immobilized on cellulose (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab .. Press, New York, 1989) .Also, the Fast Track mRNA isolation kit (produced by Invitrogen), the Quick Prep mRNA purification kit (produced by Pharmacia) can be given as examples of a kit for the preparation of mRNA a starting from a hybridoma cell.
Examples of the procedure for cDNA synthesis and preparation of a cDNA library include standard procedures (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab .. Press, New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34), a procedure using a commercially available kit such as the Super Script ™ Plasmid System for cDNA synthesis and plasmid cloning (prepared by GIBCO BRL) or the ZAP kit -DNA (produced by Stratagene).
The vector in which the cDNA synthesized using mRNA extracted from a hybridoma cell is inserted into the preparation of a cDNA library can be any vector, as long as the cDNA can be inserted. Examples include ZAP Express (Strategies, 5, 58 (1992)), PBluescript II SK (+) (Nucleic Acids Research, 17, 9494 (1989)), AzapII (produced by Stratagene), Agt10 and Agt11 (DNA Cloning: A Practical Approach, I, 49 (1985)), Lambda BlueMid (produced by Clontech), AExCell and pT7T3 18U (produced by Pharmacia), pcD2 (Mol. Cell. Biol., 3, 280 (1983)) and PUC18 (Gene, 33, 103 (1985)).
The E. coli to be used for the introduction of the cDNA library constructed by a phage or plasmid vector can be any strain, as long as the cDNA library can be introduced, expressed and maintained. Examples include XL1-Blue MRF '(Strategies, 5, 81 (1992)), C600 (Genetics, 39, 440 (1954)), Y1088 and Y1090 (Science, 222, 778 (1983)), NM522 (J. Mol Biol., 166, 1 (1983)), K802 (J. Mol. Biol., 16, 118 (1966)) and JM105 (Gene, 38, 275 (1985)).
A colony hybridization or plaque hybridization method using an isotope or fluorescently labeled probe can be used to select a cDNA clone that encodes the H chain and L chain V regions of an antibody from a non-human animal of the cDNA library (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab .. Press, New York, 1989). Furthermore, the cDNA encoding the regions
V of the H chain and the L chain can be prepared by polymerase chain reaction (hereinafter referred to as PCR; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab .. Press, New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34) by preparing primers and using cDNA prepared from mRNA or a cDNA library as a template.
The nucleotide sequence of the DNA selected by the above procedure can be determined, for example, by digesting the cDNA with appropriate restriction enzymes, cloning the fragments into a plasmid, such as pBluescript SK (-) (produced by Stratagene), carrying out the reaction by a commonly used nucleotide analysis method, such as the dideoxy method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)), and then analyzing the sequence using an automatic nucleotide sequence analyzer such as the ALF DNA sequencer (produced by Pharmacia).
Whether the obtained cDNA encodes the entire amino acid sequence of the V regions of the H chain and of the L chain of the antibody that contains a secretion signal sequence can be confirmed by estimating the complete amino acid sequence of the V regions of the H chain and of the L chain of the determined nucleotide sequence and comparing it with the complete amino acid sequences of the V regions of the H chain and the L chain of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991).
(3) Analysis of the V region of the amino acid sequence of the antibody from non-human animals
Regarding the complete amino acid sequence of the V regions of the H chain and the L chain of the antibody comprising a secretion signal sequence, the length and the terminal amino acid sequence
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N of the secretion signal sequence can be estimated and the subgroups to which they belong can be known by comparing it with complete amino acid sequences of the V regions of the H chain and the L chain of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. . Health and Human Services, 1991). Each RDC amino acid sequence of the H chain and L chain V regions can also be identified by comparing it with amino acid sequences of the H chain and L chain V regions of known antibodies (Sequences of Proteins of Immunological Interest , US Dept. Health and Human Services, 1991).
(4) Construction of the human hybrid antibody expression vector
A human hybrid antibody expression vector can be constructed by cloning cDNA encoding the V regions of the H chain and the L chain of an antibody from a non-human animal upstream of a gene encoding the C regions of the H chain and of the L chain of a human antibody in the humanized antibody expression vector described in point 4 (1). For example, a human hybrid antibody expression vector can be produced by connecting the cDNAs encoding the V regions of the H chain and the L chain from an antibody from a non-human animal, respectively, with a synthetic cDNA comprising sequences nucleotides from the 3 'terminal side of the V regions of the H chain and the L chain of an antibody from a non-human animal, a nucleotide sequence on the 5 'terminal side of the C regions of the H chain and the L chain from a human antibody and sequences that recognize the appropriate restriction enzyme at both terminal ends, and cloning them upstream of a gene encoding the C regions of the H chain and L of the chain of a human antibody in the humanized antibody expression vector described in point 4 (1) in such a way as to expressed in an appropriate way.
(5) Construction of cDNA encoding human CDR-grafted antibody V region
The cDNA encoding the H chain and L chain V regions from a human CDR-grafted antibody can be obtained as described below. First, the framework amino acid sequence (hereinafter referred to as FR) of the H chain and L chain V regions of a human antibody is selected to graft CDR of the H chain and L chain V regions. of an antibody from a non-human animal. Any sequence such as the FR amino acid sequence of the H chain and L chain V regions of a human antibody can be used, as long as it is derived from a human antibody. For example, the FR amino acid sequences of the H chain and L chain V regions of human antibodies recorded in a database, such as the Protein Data Bank, can be used, a common amino acid sequence in each subgroup of FR of V regions of the H chain and L chain of human antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991) and the like, but to prepare a human RDC-grafted antibody having sufficient activity, it is desirable to select an amino acid sequence that has high homology (at least 60% or more) to the sequence of Target amino acids of the V regions of the H chain and the L chain of an antibody from a non-human animal.
Next, the RDC target amino acid sequence of the H chain and L chain V regions of an antibody from a non-human animal is grafted to the FR selected amino acid sequence of the H chain and V regions. of the L chain of a human antibody, and the amino acid sequences of the V regions of the H chain and of the L chain of the human CDR-grafted antibody are designed. Taking into account the codon utilization found in the nucleotide sequence of the antibody gene (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), the designed amino acid sequences are converted into DNA sequences and designed DNA sequences encoding the amino acid sequences of the H chain and L chain V regions of the human CDR-grafted antibody. Based on the designed DNA sequences, several synthetic DNA fragments that are approximately 100 bases in length are synthesized, and PCR is carried out using these fragments. In this case, based on the efficiency of the PCR reaction and the length of the DNA that can be synthesized, it is desirable to design 6 synthetic DNA fragments for each H chain and L chain.
Furthermore, cloning into the vector for the expression of the humanized antibody constructed at point 4 (1) can be easily carried out by introducing the appropriate restriction enzyme that recognizes sequences at the 5 'terminals of the synthetic DNA located at both ends. After PCR, a plasmid with a DNA sequence encoding the amino acid sequence of the V regions of the H chain and the L chain of the desired human CDR-grafted antibody is obtained by cloning the expanded product into the plasmid, such as pBluescript SK (-) (produced by Stratagene), and determination of the nucleotide sequence by the method described in point 4 (2).
(6) Modification of the amino acid sequence of the V region of the antibody with human RDC grafted
It is known that when only the RDC of the V regions of the H chain and the L chain of an antibody from a non-human animal of interest is simply grafted to the FR of the V regions of the H chain and the L chain of a human antibody, the antigen-binding activity of the human-grafted RDC antibody is reduced
ES 2 568 898 T3 compared to the activity of the original antibody from a non-human animal (BIO / TECHNOLOGY, 9, 266 (1991)). As the cause of this, not only the RDC amino acid sequences, but also various FR amino acid sequences of the V regions of the H chain and the L chain of the original antibody from a non-human animal are considered to be direct. or indirectly related to antigen-binding activity, and these amino acid residues are transformed into different FR amino acid residues of the V regions of the H chain and of the L chain of the human antibody accompanied by the RDC graft. To solve this problem, in RDC-grafted human antibodies, an attempt has been made to identify, between the FR amino acid sequences of the V regions of the H chain and the L chain of a human antibody, an amino acid residue directly related to the binding. to the antibody, an amino acid residue that interacts with an amino acid residue of RDC and / or an amino acid residue that maintains the three-dimensional structure of the antibody and is directly related to its binding to the antigen, and to increase the reduced antigen-binding activity by changing these residues of amino acids in amino acid residues found in the original antibody from a non-human animal (BIO / TECHNOLOGY, 9, 266 (1991)).
In preparing a human RDC-grafted antibody, it is preferable to efficiently identify these amino acid residues of FR related to antigen-binding activity, so that the construction and analysis of the three-dimensional structure of antibodies is carried out. preferably using crystal X-ray analysis (J. Mol. Biol., 112, 535 (1977)) and computer simulation (Protein Engineering, 7, 1501 (1994)). Although information on these three-dimensional antibody structures has provided useful information for the preparation of human CDR-grafted antibodies, a procedure for producing a human CDR-grafted antibody applicable to all antibodies has not yet been established. It is therefore preferable to carry out several trial and error experiments on individual antibodies, for example, preparing various modified products thereof and examining their correlation with the respective antigen-binding activities.
Modification of the FR amino acid residues of the H chain and L chain V regions of a human antibody can be achieved by the PCR described in item 4 (5) using synthetic DNA for further modification. The achievement of the target modification is confirmed by determining the nucleotide sequence of the expanded fragment after PCR, by the method described in point 4 (2).
(7) Construction of human RDC grafted antibody expression vector
A human CDR grafted antibody expression vector can be constructed by cloning the cDNA encoding the V regions of the H chain and L chain of the human CDR grafted antibody constructed at current points 4 (5) and 4 (6). above the gene encoding the C regions of the H chain and the L chain of a human antibody in the humanized antibody expression vector described in point 4 (1). For example, among the synthetic DNA fragments used in the construction of the V regions of the H chain and the L chain of the antibody with human CDR grafted in (5) and (6) of point 4, the sequences that recognize the enzyme appropriate restriction are introduced into the 5 'terminals of a synthetic DNA fragment located at both ends, and they were cloned upstream of the gene encoding the C regions of the H chain and the L chain of a human antibody in the vector for the expression of the humanized antibody described in point 4 (1), in such a way that they can be expressed suitably to thereby construct a human CDR-grafted antibody expression vector.
(8) Stable production of humanized antibody
A transformant that can produce a humanized antibody stably can be obtained by introducing the humanized antibody expression vector described in items 4 (4) and 4 (7) into an appropriate animal cell.
The method of introducing an expression vector into an animal cell includes, for example, an electroporation method (Japanese Patent Application Published Unexamined No. 257891/90, Cytotechnology, 3, 133 (1990)).
The animal cell into which a humanized antibody expression vector is introduced can be any cell, as long as it is an animal cell that can produce the humanized antibody. Preferred examples include a cell that has a low enzymatic activity of adding fucose to N-acetylglucosamine to bind to the Fc region of the produced antibody and a cell that does not have such enzymatic activity.
The cell that has a low enzymatic activity of adding fucose to N-acetylglucosamine to bind to the Fc region of the antibody or does not have such enzymatic activity is a cell that has fewer or no enzymes related to the α1,6- bond. Examples include a cell that has low fucosyltransferase activity, preferably FUT8 activity, and a cell that does not have such activity.
Examples of cells that have low enzymatic activity of adding fucose to N-acetylglucosamine to bind to the Fc region of the antibody or have no enzymatic activity include, for example, a rat myeloma cell and a YB2 / 0 cell. A cell in which a gene involved in the enzyme that carries the α1,6 bond is
ES 2 568 898 T3 eliminates or the enzymatic activity is reduced or eliminated by adding a mutation to the gene can also be used as an antibody-producing cell.
Specific examples include mouse myeloma cells, such as NS0 cell and SP2 / 0 cell, Chinese hamster ovary cells, such as CHO / dhfr cell and CHO / DG44 cell; rat myeloma cells, such as the YB2 / 0 cell and the IR983F cell; human myeloma cells, such as Namalwa cells. Preferably, Chinese hamster ovary cells, such as CHO / DG44 cells, can be used.
After introduction of the expression vector, the transformant capable of stably producing the humanized antibody can be selected using an animal cell culture medium containing a drug, such as G418 sulfate (hereinafter referred to as G418; produced by SIGMA) by the method described in Japanese Published Unexamined Patent Application No. 257891/90. Animal cell culture medium includes, for example, RPMI 1640 medium (produced by Nissui Pharmaceutical), GIT medium (produced by Nippon Pharmaceutical), EX-CELL 302 medium (produced by JRH), IMDM medium (produced by GIBCO BRL) , Hybridoma-SFM medium (produced by GIBCO BRL), or a medium prepared by adding various additives, such as fetal bovine serum (hereinafter referred to as SBF), to each of these media. The humanized antibody can be produced by culturing the obtained transformant in a medium, and accumulated in a culture supernatant. The amount produced and the antigen-binding activity of the humanized antibody in the culture supernatant can be measured by the enzyme-linked immunosorbent assay (hereinafter referred to as ELISA; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1998; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996). In addition, the production of the humanized antibody by the transformant can be increased by using a DHFR gene amplification system by the procedure described in Japanese Unexamined Published Patent Application No. 257891/90.
The humanized antibody can be purified from a culture supernatant containing transformant using a protein A column (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, nineteen ninety six). Also, other generally used purification procedures for protein purification can be used. For example, it can be purified by a combination of gel filtration, ion exchange chromatography, and ultrafiltration. The molecular weight of the H chain, the L chain or the entire antibody molecule of the purified humanized antibody can be measured by polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE; Nature, 227, 680 (1970)), Western immunoblot method (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 12, 1988; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996).
An antibody production method has been demonstrated above using an animal cell as a host, and as described in point 3 above, it can also be produced by a bacterium, a yeast, an insect cell, a plant cell, an animal or a vegetable.
(9) Evaluation of humanized antibody activity
The activity of the purified humanized antibody to bind an antigen or a cultured cell line to the antigen can be measured by the ELISA method and fluorescence antibodies (Cancer Immunol. Immunother., 36, 373 (1993)). Cytotoxic activity for antigen positive cultured cell lines can be evaluated by determining their CDC activity, ADCC activity (Cancer Immunol. Immunother., 36, 373 (1993)). Furthermore, the safety and therapeutic effects of the humanized antibody in humans can be evaluated using an appropriate model of an animal species relatively close to humans, such as Macaca phaseicularis.
5. Immunofunctional molecule application procedure
As shown in the humanized antibody described in item 4 above, an antibody having high ADCC activity is useful in the prevention and treatment of various diseases, including cancer, allergy, cardiovascular disease, and viral or bacterial infection. .
In cancer, that is, a malignant tumor, cancer cells proliferate. Conventional anticancer agents have a characteristic of inhibiting the proliferation of cancer cells. On the other hand, since an antibody having a high ADCC activity can treat cancers by affecting the proliferation of cancer cells by its cytotoxic effect, it is more effective as a therapeutic drug than conventional anticancer agents.
Since the release of a mediator molecule from the immune cells causes the allergic reaction, it can be inhibited by the elimination of the immune cells using an antibody that has a high ADCC activity.
Cardiovascular disease includes, for example, arteriosclerosis. Atherosclerosis is currently treated with a balloon catheter, but cardiovascular disease can be prevented and treated by inhibiting the proliferation of cells.
ES 2 568 898 T3 in restenosis after treatment, using an antibody having a high ADCC activity.
Various diseases, including viral or bacterial infections can be prevented and treated by inhibiting the proliferation of cells infected by viruses or bacteria using an antibody having high ADCC activity.
Furthermore, an antibody having inhibited ADCC activity is useful in the prevention and treatment of autoimmune diseases. The antibody having inhibited ADCC activity is also useful in the prevention and treatment of autoimmune diseases from the viewpoint of suppressing the stimulated immune response in autoimmune diseases.
The drug containing the antibody according to the present invention can be administered as a therapeutic drug alone, but in general, it is desirable to provide it in the form of a pharmaceutical preparation produced by an appropriate method well known in the field of the art of pharmaceutical preparation, mixing it with one or more pharmaceutically acceptable carriers.
It is desirable to select a route of administration that is the most effective in carrying out a treatment. Examples include oral administration and parenteral administration, such as, for example, buccal, respiratory, rectal, subcutaneous, intramuscular, or intravenous. In an antibody preparation, intravenous administration is preferred.
The dosage form includes, for example, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, and strips.
Liquid preparations, such as emulsions and syrups, can be produced using, as additives, water; saccharides, such as sucrose, sorbitol, fructose, etc .; glycols, such as polyethylene glycol, propylene glycol, etc .; oils, such as sesame oil, olive oil, soybean oil, etc .; antiseptics, such as p-hydroxybenzoic acid esters, etc .; flavors, such as strawberry, mint flavor, etc.
Capsules, tablets, powders and granules can be produced using, as additives, fillers, such as lactose, glucose, sucrose, mannitol, etc .; disintegrating agents, such as starch, sodium alginate, etc .; lubricants, such as magnesium stearate, talc, etc .; binders, such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc .; surfactants, such as fatty acid ester, etc .; plasticizers, such as glycerol, etc.
Examples of the pharmaceutical preparation suitable for parenteral administration include injections, suppositories, and sprays.
Injections can be prepared using a vehicle, such as a saline solution, a glucose solution, or a mixture of both. Alternatively, powdered injections can be prepared by lyophilizing the humanized antibody in the usual way and adding sodium chloride thereto.
Suppositories can be prepared using a carrier such as cocoa butter, a hydrogenated fat, or carboxylic acid.
Furthermore, sprays can be prepared using the compound as such or using a vehicle that does not stimulate the oral mucous membrane or airways of the patient and can facilitate absorption of the compound by dispersing it as fine particles.
Examples of the vehicle include lactose and glycerol. Depending on the properties of the compound and the vehicle to be used, it is possible to produce pharmaceutical preparations such as sprays and dry powders. Furthermore, the components exemplified as additive agents for oral preparations can also be added to these parenteral preparations.
Although the clinical dose or frequency of administration varies depending, for example, on the target therapeutic effect, the method of administration, the period of treatment, age and body weight, it is generally 10 pg / kg to 20 mg / kg. kg per day and per adult.
Also, in relation to the method for examining the antitumor effect of antibodies on various tumor cells, in vitro assays include, for example, the CDC activity measurement method, the ADCC activity measurement method, and In vivo assays include, for example, an antitumor experiment using a tumor system in an experimental animal such as a mouse.
CDC activity and ADCC activity measurements and antitumor experiments can be carried out according to the methods described in the references (Cancer Immunology Immunotherapy, 36, 373 (1993); Cancer Research, 54, 1511 (1994)).
ES 2 568 898 T3
6. Procedure to stimulate or inhibit the activity of immunofunctional molecules
The activity of an immunofunctional molecule can be stimulated by the production of an antibody, peptide, or protein to which a fucose-free sugar chain is attached by the above procedure.
When the immunofunctional molecule with stimulated activity is administered to the living body, various immunocytes, including cells such as killer lymphocytes, natural killer lymphocytes, and macrophages activated as effector cells in relation to ADCC activity are activated in the living body, accordingly. so that it becomes possible to control various immune responses.
Furthermore, the activity of an immunofunctional molecule can be inhibited by producing an antibody, a peptide, or a protein to which an existing fucose sugar chain is attached by the above procedure.
When the immunofunctional molecule with inhibited activity is administered to the living body, the activities of various immunocytes involved in ADCC activity are weakened in the living body, so that control of various immune responses becomes possible.
Examples in the present disclosure are shown below, but the scope of the present disclosure is not limited thereto.
Brief explanation of the drawings
Fig. 1 is a graph showing SDS-PAGE electrophoresis patterns of five purified anti-GD3 hybrid antibodies (using 4-15% gradient gel). The upper drawing and the lower drawing show a result of electrophoresis under non-reducing conditions and under reducing conditions, respectively. Lanes 1 to 7 show a high molecular weight marker electrophoresis pattern, a YB2 / 0-GD3 hybrid antibody electrophoresis pattern, a CHO / DG44-GD3 hybrid antibody electrophoresis pattern, a hybrid antibody electrophoresis pattern SP2 / 0-GD3, an NSO-GD3 (302) hybrid antibody electrophoresis standard, a NSO-GD3 hybrid antibody electrophoresis (GIT) standard, and a low molecular weight marker electrophoresis standard, respectively.
Fig. 2 is a graph showing the activity of five purified anti-GD3 hybrid antibodies to bind GD3, as measured by the change in antibody concentration. The ordinate axis and the abscissa axis show GD3 binding activity and antibody concentration, respectively. Open circles, solid circles, open squares, black squares and open triangles show YB2 / 0-GD3 hybrid antibody activity, CHO / DG44-GD3 hybrid antibody activity, SP2 / 0 hybrid antibody activity -GD3, the activity of hybrid antibodies NS0-GD3 (302) and the activity of hybrid antibodies NS0-GD3 (GIT), respectively.
Fig. 3 is a graph showing the ADCC activity of five purified anti-GD3 hybrid antibodies to a human melanoma G-361 cell line. The ordinate axis and the abscissa axis show cytotoxic activity and antibody concentration, respectively. Open circles, solid circles, open squares, black squares and open triangles show YB2 / 0-GD3 hybrid antibody activity, CHO / DG44-GD3 hybrid antibody activity, SP2 / 0 hybrid antibody activity -GD3, the activity of hybrid antibodies NS0-GD3 (302) and the activity of hybrid antibodies NS0-GD3 (GIT), respectively.
Fig. 4 is a graph showing SDS-PAGE electrophoresis patterns of three RDC grafted purified anti-hIL5Ra antibodies (using 4-15% gradient gel). The upper drawing and the lower drawing show the results of electrophoresis carried out under non-reducing conditions and n reducing conditions, respectively. Lanes 1 to 5 show a high molecular weight marker electrophoresis pattern, a YB2 / 0-hIL-5RCDR antibody electrophoresis pattern, a CHO / d-hIL-5RCDR antibody electrophoresis pattern, a NSO-hIL-5RCDR antibodies and a low molecular weight marker electrophoresis pattern, respectively.
Fig. 5 is a graph showing the activity of three purified anti-hIL-5Ra antibodies grafted on CDR to bind hIL-5Ra, as measured by the change in the concentration of the antibodies. The ordinate axis and the abscissa axis show hIL-5Ra binding activity and antibody concentration, respectively. Open circles, solid circles, and open squares show YB2 / 0-hIL5RaRDC antibody activity, CHO / d-hIL-5RCDR antibody activity, and NS0-hIL-5RCDR antibody activity, respectively.
Fig. 6 is a graph showing the ADCC activity of three purified anti-hIL-5Ra grafted onto the RDC antibodies for a cell line expressing mouse T CTLL hIL-5R-2 (H5R). The ordinate axis and the abscissa axis show the cytotoxic activity and the antibody concentration, respectively. Open circles, black circles and open squares show the activity of the YB2 / 0-hIL-5RaCDR antibody, the
ES 2 568 898 T3 activity of the CHO / d-hIL-5RCDR antibody, and the activity of the NS0-hIL-5RCDR antibody, respectively.
Fig. 7 is a graph showing the inhibition activity of three purified anti-hIL-5Ra antibodies grafted onto CDR in a Macaca phaseicularis model that increases eosinophils produced on hIL-5. The ordinate axis and the abscissa axis show the number of eosinophils in the peripheral blood and the number of days (the day of the start of the administration of antibodies and hIL-5 was defined as day 0). The results in the group without antibody administration are shown in 101 and 102, the results in the group administered with YB2 / 0-hIL-5RCDR antibodies are shown in 301, 302 and 303, the results in the group administered with CHO / d-hIL-5RCDR are shown at 401, 402, and 403 and results in the group administered with NS0-hIL-5RDC antibodies are shown at 501, 502, and 503.
Fig. 8 is a graph showing an elution pattern of the reverse phase HPLC elution of a Pa-treated sugar chain (left side), and an elution pattern obtained by treating the PA-treated sugar chain with α-L -fucosidase and then analyzed by reverse phase HPLC (right side), of the YB2 / 0 grafted purified anti-hIL-5Ra antibody produced by YB2 / 0 (upper side) and the NS0-grafted purified anti-hIL-5Ra antibody produced by NS0 (lower side). The ordinate axis and the abscissa axis show the relative fluorescence intensity and elution time, respectively.
Fig. 9 is a graph showing an elution pattern obtained by preparing a PA-treated sugar chain of the purified anti-hIL-5Ra antibody grafted onto the CDR produced by CHO / d cells and analyzing it by reverse phase HPLC. The ordinate axis and the abscissa axis show the relative fluorescence intensity and elution time, respectively.
Fig. 10 is a graph showing the GD3 binding activity of the non-adsorbed fraction and a part of the adsorbed fraction, measured by changing the antibody concentration. The ordinate axis and the abscissa axis show GD3 binding activity and antibody concentration, respectively. Black circles and open circles show the non-adsorbed fraction and a part of the adsorbed fraction, respectively. The lower graph shows the ADCC activity of the non-adsorbed fraction and a part of the adsorbed fraction for a human melanoma line G-361. The ordinate axis and the abscissa axis show the cytotoxic activity and the antibody concentration, respectively. Black circles and open circles show the non-adsorbed fraction and a part of the adsorbed fraction, respectively.
Fig. 11 is a graph showing elution patterns obtained by analyzing PA-treated sugar chains prepared from the non-adsorbed fraction and a part of the adsorbed fraction by reverse HPLC. The left side drawing and the right side drawing show an elution pattern of the non-adsorbed fraction and an elution pattern of a part of the adsorbed fraction, respectively. The ordinate axis and the abscissa axis show the relative fluorescence intensity and elution time, respectively.
Fig. 12 is a graph showing the amount of FUT8 transcription product by respective host cell lines when a rat FUT8 sequence is used as an internal standard reference. Black circles and open circles show the result when the CHO cell line was used and the result when the YB2 / 0 cell line was used as the host cell, respectively.
Best way to put the invention into practice
Example 1
Production of human anti-ganglioside GD3 hybrid antibody:
1. Construction of the tandem expression vector, pChiLHGM4, for the human anti-ganglioside GD3 hybrid antibody
A plasmid, pChi641LGM40, was constructed by ligating an approximately 4.03 kb fragment containing an L chain cDNA, obtained by digesting an L chain expression vector, pChi641LGM4 (J. Immunol. Methods, 167, 271 (1994)) for human anti-ganglioside GD3 hybrid antibody (hereinafter referred to as anti-GD3 hybrid antibody) with restriction enzymes, Mul I (produced by Takara Shuzo) and Sal I (produced by Takara Shuzo) , with a fragment of around 3.40 kb containing a G418 resistant gene and a splicing signal, obtained by digestion of an expression vector pAGE107 (Cytotechnology, 3, 133 (1990)) for animal cells with enzymes restriction, Mul I (produced by Takara Shuzo) and Sal I (produced by Takara Shuzo), using the DNA ligation kit (produced by Takara Shuzo), followed by transformation of E. coli hB101 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab .. Press, New York, 1989) with the product ligated using the DNA ligation kit (produced by Takara Shuzo).
Next, a fragment of approximately 5.68 kb containing an L-chain cDNA, obtained by digestion of the plasmid pChi641LGM40 constructed with a restriction enzyme, ClaI (produced by Takara Shuzo),
ES 2 568 898 T3 by truncating the ends using DNA Blunting Kit (produced by Takara Shuzo) and digesting it further with MluI (produced by Takara Shuzo), it was ligated with a fragment of approximately 8.40 kb containing an H-chain cDNA, obtained by digestion of an anti-GD3 hybrid antibody H chain expression vector, pChi641HGM4 (J. Immunol. Methods, 167, 271 (1994)) with a restriction enzyme, Xho I (produced by Takara Shuzo), by truncating the ends using DNA Blunting Kit (produced by Takara Shuzo) and digesting it further with MluI (produced by Takara Shuzo), using DNA Ligation Kit (produced by Takara Shuzo), then HB101 from E. coli (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 1989) was transformed with the ligated product to thereby construct a tandem expression vector, pChi641LHGM4, for the hybrid anti-GD3 antibody.
two. Production of cells stably producing hybrid anti-GD3 antibody
Using the tandem expression vector, pChi641LHGM4, for the anti-GD3 hybrid antibody constructed in point 1 of Example 1, cells capable of stably producing an anti-GD3 hybrid antibody were prepared as described below.
(1) Production of producer cells using rat myeloma YB2 / 0 cells
After introducing 5 pg of the hybrid anti-GD3 antibody expression vector, pChi641LHGM4, at 4 × 10<sup>6 </sup>rat YB2 / 0 myeloma cells by electroporation (Cytotechnology, 3, 133 (1990)), cells were suspended in 40 ml of RPMI1640-SBF (10) (RpM medium | 1640 containing 10% SBF (produced by GIBCO BRL)) and distributed at 200 µl / well in a 96-well culture plate (manufactured by Sumitomo Bakelite). Twenty-four hours after culturing at 37 ° C in a 5% CO2 incubator, G418 was added at a concentration of 0.5 mg / ml, followed by cultivation for 1 to 2 weeks. The culture supernatant was recovered from the respective wells in which colonies of transformants exhibiting G418 resistance were formed and colony growth was observed, and the antigen-binding activity of the hybrid anti-GD3 antibody in the supernatant was determined by the ELISA assay is shown in point 3 of Example 1.
Regarding the transformants in the wells in which production of the hybrid anti-GD3 antibody was observed in culture supernatants, to increase the amount of antibody production using a DHFR gene amplification system, each of them was put into suspension in RPMI1640-SBF medium (10) containing 0.5 mg / ml of G418 and 50 nM DHFR inhibitor, methotrexate (hereinafter referred to as MTX; produced by SIGMA) to provide a density of 1 to 2x10<sup>5</sup> cells / ml, and the suspension was distributed over 2 ml into the wells of a 24-well plate (manufactured by Greiner). Transformants showing resistance to 50 nM MTX were produced by culturing at 37 ° C for 1 to 2 weeks in a 5% CO2 incubator. The antigen-binding activity of the hybrid anti-GD3 antibody in culture supernatants in the wells in which growth of the transformants was observed was determined by the ELISA assay as shown in point 3 of example 1. Regarding the transformants in the wells in which production of the hybrid anti-GD3 antibody was observed in culture supernatants, the concentration of MTX increased to 100 nM and then to 200 nM, and a transformant capable of growing in the RPMI1640 medium- SBF (10) containing 0.5 mg / ml of G418 and 200 nM MTX and producing the anti-GD3 hybrid antibody in large quantities, was finally obtained by the same procedure described above. The transformant obtained was made in a single cell (cloning) limiting the dilution twice.
The clone 7.9.51 of transformed cells producing hybrid anti-GD3 antibodies obtained has been deposited on April 5, 1999, as FERM BP-6691 at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (Higashi 1 -1-3, Tsukuba, Ibaraki, Japan).
(2) Production of producer cells using CHO / DG44 cells
After introduction of 4 pg of the anti-GD3 hybrid antibody expression vector, pChi641LHGM4, at 1.6χ10<sup>6</sup> CHO / DG44 cells by electroporation (Cytotechnology, 3, 133 (1990)), cells were suspended in 10 ml of IMDM-SBf (10) (IMDM medium containing 10% SBF and 1 χ concentration of supplement HT (produced by GIBCO BRL)) and distributed at 200 µl / well in a 96-well culture plate (manufactured by Iwaki Glass). Twenty-four hours after culturing at 37 ° C in a 5% CO2 incubator, G418 was added at a concentration of 0.5 mg / ml, followed by cultivation for 1 to 2 weeks. The culture supernatant was recovered from the respective well in which colonies of transformants showing resistance to G418 were formed and growth of the colonies was observed, and the antigen-binding activity of the hybrid anti-GD3 antibody in the supernatant was determined by the ELISA assay is shown in point 3 of Example 1.
Regarding the transformants in the wells in which the production of anti-GD3 hybrid antibodies was observed in culture supernatants, to increase the amount of antibody production using a DHFR gene amplification system, each of them was put into suspension in iMDM-dSBF medium (10) (IMDM medium containing 10% dialyzed fetal bovine serum (hereinafter referred to as dSBF; produced by GIBCO BRL)) containing 0.5 mg / ml G418 and 10 nM MTX to provide a density of 1 to 2x10<sup>5</sup> cells / ml, and the suspension was partitioned 0.5 ml into the wells of a 24-well plate (manufactured by Iwaki Glass). The transformants that
ES 2 568 898 T3 exhibits resistance to MTX 10 nM were produced by culturing at 37 ° C for 1 to 2 weeks in an incubator with 5% CO2. Regarding the transformants in the wells in which their growth was observed, the concentration of MTX was increased to 100 nM, and a transformant capable of growing in IMDM-dSBF medium (10) containing 0.5 mg / ml of G418 and 100 nM MTX and producing the anti-GD3 hybrid antibody in large quantity was finally obtained by the same method described above. The obtained transformant was prepared in a single cell (cloning) by limiting the dilution twice.
(3) Production of producer cells using mouse myeloma NS0 cells
After introducing 5 pg of the anti-GD3 hybrid antibody expression vector, pChi641LHGM4, at 4 * 10<sup>6 </sup>mouse myeloma NS0 cells by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 40 ml of EX-CELL302-SBF (10) (EX-CELL302 medium containing 10% SBF and L 2 mM -glutamine (hereinafter L-Gln; produced by GIBCO BRL)) and distributed at 200 µl / well in a 96-well culture plate (manufactured by Sumitomo Bakelite). Twenty-four hours after culturing at 37 ° C in a 5% CO2 incubator, G418 was added at a concentration of 0.5 mg / ml, followed by cultivation for 1 to 2 weeks. The culture supernatant was recovered from the respective wells in which colonies of, the transformants exhibiting G418 resistance were formed and colony growth was observed, and the antigen-binding activity of the hybrid anti-GD3 antibody in the supernatant was determined by the ELISA assay as shown in point 3 of example 1.
Regarding the transformants in the wells in which the production of the hybrid anti-GD3 antibody was observed in culture supernatants, to increase the amount of antibody production using a DHFR gene amplification system, each of them was put suspended in EX-CELL302-dSBF (10) medium (EX-CELL302 medium containing 10% dSBF and 2 mM L-Gln) containing 0.5 mg / ml G418 and 50 nM MTX to provide a density of 1 to 2x10<sup>5</sup> cells / ml, and the suspension was distributed over 2 ml into the wells of a 24-well plate (manufactured by Greiner). Transformants showing resistance to 50 nM MTX were produced by culturing at 37 ° C for 1 to 2 weeks in a 5% CO2 incubator. The antigen-binding activity of the hybrid anti-GD3 antibody in culture supernatants in the wells in which growth of the transformants was observed was determined by the ELISA test shown in point 3 of example 1. Regarding the transformants in the wells in which the production of the hybrid anti-GD3 antibody was observed in culture supernatants, the concentration of MTX was increased to 100 nM and then to 200 nM, and a transformant capable of growing in the medium EX-CELL302-dSBF (10) containing 0.5 mg / ml of G418 and 200 nM MTX and to produce the anti-GD3 hybrid antibody in large quantity was finally obtained by the same procedure described above. The obtained transformant was prepared in a single cell (cloning) by limiting the dilution twice.
3. Measurement of antibody binding activity to GD3 (ELISA)
The binding activity of the antibody to GD3 was determined as described below.
In 2 ml of ethanol solution containing 10 pg of dipalmitoylphosphatidylcholine (produced by SIGMA) and 5 pg of cholesterol (produced by SIGMA), 4 nmoles of GD3 were dissolved. In each well of a 96-well ELISA plate (manufactured by Greiner), 20 µl of the solution (40 pmol / well in final concentration) was dispensed, followed by air drying, PBS containing 1% serum albumin bovine (hereinafter referred to as ASB, produced by SIGMA) (hereinafter referred to as PBS-1% BSA) was partitioned in 100 µl / well, and then the reaction was carried out at room temperature for 1 hour to block the remaining active groups. After discarding PBS1% BSA, a culture supernatant of a transformant or a diluted solution of a human hybrid antibody was partitioned into 50 µl / well to carry out the reaction at room temperature for 1 hour. After the reaction, each well was washed with PBS containing 0.05% Tween 20 (produced by Wako Pure Chemical Industries) (hereinafter referred to as Tween-PBS), a solution of anti-human IgG antibodies (H&L). In peroxidase-labeled goat (produced by American Qualex) diluted 3000 times with PBS-1% BSA was partitioned 50 µl / well as a secondary antibody solution, and then the reaction was carried out at room temperature for 1 hour. After the reaction and subsequent washing with Tween-PBS, the ABTS substrate solution (solution prepared by dissolving 0.55 g of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2) and addition of 1 µl / ml hydrogen peroxide to the solution just before use) was distributed over 50 µl / well for color development, and then measured the absorbance at 415 nm (hereinafter referred to as OD415).
Four. Anti-GD3 hybrid antibody purification (1) Producer cell culture from YB2 / 0 cells and antibody purification
The hybrid anti-GD3 antibody producing transformed cell clone obtained in point 2 (1) above of example 1 was suspended in the Hybridoma-SFM medium containing 0.2% BSA, 200 nM MTX and 100 nM triiodothyronine (hereinafter referred to as T3, produced by SIGMA) to provide a density of
3x10<sup>5</sup> cells / ml and cultured using a 2.0 liter capacity spinner flask (manufactured by Iwaki
ES 2 568 898 T3
Glass) while stirring at a speed of 50 rpm. Ten days after culturing at 37 ° C in a temperature control room, the culture supernatant was recovered. Hybrid anti-GD3 antibody was purified from the culture supernatant using a Prosep-A column (manufactured by Bioprocessing) according to the manufacturer's instructions. The purified anti-GD3 hybrid antibody was named YB2 / 0-GD3 hybrid antibody.
(2) Culture of producer cells from CHO / DG44 cells and purification of antibodies
The hybrid anti-GD3 antibody producing transformed cell clone obtained in point 2 (2) above of Example 1 was suspended in EX-CELL302 medium containing 3 mM L-Gln, concentrated 0 fatty acid solution, 5% (hereinafter referred to as CDLC; produced by GIBCO BRL) and 0.3% Pluronic F68 (hereinafter referred to as PF68; produced by GIBCO BRL) to provide a density of 1x10<sup>6</sup> cells / ml, and the suspension was distributed over 50 ml in 175 mm flasks<sup>2</sup> (made by Greiner). Four days after cultivation at 37 ° C in a 5% CO 2 incubator, the culture supernatant was recovered. The hybrid antiGD3 antibody was purified from the culture supernatant using a Prosep-A column (manufactured by Biopreocessing) according to the manufacturer's instructions. The purified anti-GD3 hybrid antibody was named CHO / DG44-GD3 hybrid antibody.
(3) Culture of producer cells from NS0 cells and purification of antibodies
The clone of hybrid anti-GD3 antibody-producing transformed cells obtained in point 2 (3) above of example 1 was suspended in the EX-CELL302 medium containing 2 mM L-Gln, 0.5 mg / ml of G418 , MTX 200 nM and 1% SBF, to provide a density of 1x10<sup>6</sup> cells / ml, and the suspension was distributed over 200 ml in 175 mm flasks<sup>2</sup> (made by Greiner). Four days of culture at 37 ° C in an incubator with 5% CO 2, the culture supernatant was recovered. The hybrid anti-GD3 antibody was purified from the culture supernatant using a Prosep-A column (manufactured by Biopreocessing) according to the manufacturer's instructions. The purified hybrid anti-GD3 antibody was named hybrid NS0-GD3 antibody (302). In addition, the transformed cell clone was suspended in GIT medium containing 0.5 mg / ml G418 and 200 nM MTX to provide a density of 3 * 10<sup>5</sup> cells / ml, and the suspension was distributed over 200 ml in 175 mm flasks<sup>2 </sup>(made by Greiner). Ten days after cultivation at 37 ° C in a 5% CO 2 incubator, the culture supernatant was recovered. The hybrid anti-GD3 antibody was purified from the culture supernatant using a Prosep-A column (manufactured by Biopreocessing) according to the manufacturer's instructions. The purified hybrid anti-GD3 antibody was named hybrid NS0-GD3 antibody (GIT).
(4) Culture of producer cells from SP2 / 0 cells and purification of antibodies
The hybrid anti-GD3 antibody-producing transformed cell clone described in Japanese Unexamined Published Patent Application No. 304989/93 was suspended in GIT medium containing 0.5 mg / ml G418 and 200 nM MTX for provide a density of 3x10<sup>5</sup> cells / ml, and the suspension was distributed over 200 ml in 175 mm flasks<sup>2</sup> (made by Greiner). Eight days after culturing at 37 ° C in a 5% CO2 incubator, the culture supernatant was recovered. The hybrid anti-GD3 antibody was purified from the culture supernatant using a Prosep-A column (manufactured by Biopreocessing) according to the manufacturer's instructions. The purified anti-GD3 hybrid antibody was named SP2 / 0-GD3 hybrid antibody.
5. Analysis of Purified Hybrid Anti-GD3 Antibodies
According to a known method (Mature, 227, 680, 1970), 4 pg of each of the five anti-GD3 hybrid antibodies produced by and purified from animal cells, obtained in point 4 of example 1 above, were subjected to SDS-PAGE to analyze molecular weight and degree of purification. The results are shown in Fig. 1. As shown in Fig. 1, a single band of approximately 150 kilodaltons (hereinafter referred to as Kd) in molecular weight was found under non-reducing conditions, and two bands of approximately 50 Kd and approximately 25 Kd under reducing conditions, in each of the purified hybrid antiGD3 antibodies. These molecular weights almost matched the deduced molecular weights of the antibody H chain and L chain cDNA nucleotide sequences (H chain: approximately 49 Kd, L chain: approximately 23 Kd, whole molecule: about 144 Kd), and they also concurred with reports indicating that the IgG antibody has a molecular weight of about 150 Kd under non-reducing conditions and degrades in H chains that have a molecular weight of about 50 Kd and in L chains that have a molecular weight of approximately 25 Kd under reducing conditions due to the cleavage of the disulfide bond (hereinafter SS bond) in the molecule (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1998; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996), so that it was confirmed that each hybrid anti-GD3 antibody was expressed and purified as an antibody molecule having the true structure.
Example 2
Evaluation of the activity of the hybrid anti-GD3 antibody:
ES 2 568 898 T3
1. GD3 Hybrid Anti-GD3 Antibodies Binding Activity (ELISA)
The activity of the five purified hybrid anti-GD3 antibodies obtained in point 4 above of Example 1 to bind GD3 (produced by Snow Brand Milk Products) was determined by the ELISA assay shown in point 3 of Example 1. Fig. 2 shows a result of the examination of the binding activity measured by changing the concentration of the hybrid anti-GD3 antibody to be added. As shown in Fig. 2, the five hybrid anti-GD3 antibodies showed almost the same GD3 binding activity. This result demonstrates that the antigen-binding activities of these antibodies are constant regardless of the antibody-producing cells of animal origin and their culture methods. Furthermore, it was suggested from the comparison of the hybrid NS0-GD3 antibody (302) with the hybrid NS0-GD3 antibody (GIT) that the antigen-binding activities are constant regardless of the media used in culture.
two. In Vitro Cytotoxic Activity (ADCC Activity) of Hybrid Anti-GD3 Antibody
In order to evaluate the in vitro cytotoxic activity of the five purified hybrid anti-GD3 antibodies obtained in point 4 above of Example 1, the ADCC activity was determined according to the following procedure.
(1) Preparation of the target cell solution
A human melanoma cultured G-361 cell line (ATCC CRL 1424) was cultured using RPMI1640-SBF medium (10) to prepare 1> <10<sup>6</sup> cells, and the cells were labeled with radioisotopes by reacting them with 3.7 MBq equivalents of a radioactive substance Na2<sup>51</sup>CrO4 at 37Ό for 1 hour. After the reaction, the cells were washed three times with their suspension in the RPMI1640-SBF medium (10) and centrifugation, they were resuspended in the medium and then left to stand at 4<sup>r</sup>C for 30 minutes on ice for spontaneous dissolution of the radioactive substance. After centrifugation, the precipitate was adjusted to 2 * 10<sup>5</sup> cells / ml by adding 5 ml of RPMI1640-SBF (10) and used as the target cell solution.
(2) Preparation of effector cell solution
50 ml of venous blood was collected from a healthy person, and mixed gently with 0.5 ml of sodium heparin (produced by Takeda Pharmaceutical). The mixture was centrifuged to isolate a mononuclear cell layer using Lymphoprep (produced by Nycomed Pharma AS) according to the manufacturer's instructions. After washing with RPMI1640-SBF (10) centrifugation medium three times, the resulting precipitate was resuspended to provide a density of 2 * 10<sup>6</sup> cells / ml using the medium and was used as the effector cell solution.
(2) Measurement of ADCC activity
In each well of a 96-well plate with a U-shaped bottom (manufactured by Falcon), 50 µl of the target cell solution prepared in point (1) above (1 χ 10<sup>4</sup> cells / well). Then 100 μΙ of the effector cell solution prepared in point (2) above (2 χ 10<sup>5</sup> cells / well, the ratio of effector cells to target cells becomes 20:01). Subsequently, each of the hybrid anti-GD3 antibodies was added to provide a final concentration of 0.0025 to 2.5 pg / ml, followed by reaction at 37Ό for 4 hours. After the reaction, the plate was centrifuged, and the amount of<sup>51</sup>Cr in the supernatant using a y counter. The amount of<sup>51</sup>Spontaneously released Cr was calculated by the same operation using only the medium instead of the effector cell solution and the antibody solution and measuring the amount of <sup>51</sup>Cr in the supernatant. The amount of<sup>51</sup>Total Cr released was calculated by the same operation using only the medium instead of the antibody solution and adding 1 N hydrochloric acid instead of the effector cell solution, and measuring the amount of <sup>51</sup>Cr in the supernatant. The ADCC activity was calculated from the following equation.
51
Cr in the supernatant sample - Spontaneously released Cr
ADCC% activity = £ ¡~ x 100
Total released Cr - Spontaneously released Cr
The results are shown in Fig. 3. As shown in Fig. 3, among the five hybrid anti-GD3 antibodies, the hybrid YB2 / 0-GD3 antibody showed the highest ADCC activity, followed by the SP2 / 0 antibody. -GD3 hybrid, the hybrid NSO-GD3 antibody and the hybrid CHO-GD3 antibody in that order. No difference in ADCC activity was found between hybrid NS0-GD3 antibody (302) and hybrid NS0-GD3 antibody (GIT) prepared using different media in culture. The above results demonstrate that the ADCC activity of antibodies varies greatly depending on the animal cells to be used in their production. As its mechanism, since its antigen-binding activities were identical, it is considered that it was caused by a difference in the structure of the antibody Fe region.
ES 2 568 898 T3
Example 3
Production of anti-human interleukin-5 receptor α-chain human grafted RDC antibody:
1. Production of cells that stably produce anti-human interleukin-5 receptor α-chain human-grafted RDC antibody (1) Producer cell production using rat myeloma YB2 / 0 cells
Using the anti-human interleukin-5 receptor α-chain human grafted RDC antibody expression vector (hereinafter referred to as anti-hIL-5R antibody grafted into RDC α), pKANTEX1259HV3LVO, described in WO 97/10354 Cells capable of stably producing RDC α grafted anti-hIL-5R α antibody were prepared as described below.
After the introduction of 5 pg of the expression vector of the anti-hIL-5R antibody grafted in the RDC α, pKANTEX1259HV3LV0, in 4 * 10<sup>6</sup> YB2 / 0 rat myeloma cells by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 40 ml of RPMI 1640-SBF (10) and distributed in 200 µl / well in a plate of 96-well culture (manufactured by Sumitomo Bakelite). Twenty-four hours after culturing at 37 ° C in a 5% CO 2 incubator, G418 was added to provide a concentration of 0.5 mg / ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from the respective wells in which colonies of transformants showing resistance to G418 were formed and colony growth was observed, and the antigen-binding activity of anti-hIL-5R antibodies grafted onto the α-DRC in the supernatant it was determined by the ELISA test as shown in point 2 of example 3.
Regarding the transformants in wells in which the production of anti-hIL-5R antibodies grafted onto the RDC α was observed in culture supernatants, in order to increase the amount of antibody production using a DHFR gene extension system , each of them was suspended in the RPMI1640-SBF medium (10) containing 0.5 mg / ml of G418 and 50 nM MTX to provide a density of 1 to 2 x 10<sup>5</sup> cells / ml, and the suspension was distributed over 2 ml into wells of a 24-well plate (manufactured by Greiner). Transformants exhibiting resistance to 50 nM MTX were produced by culturing at 37 ° C for 1 to 2 weeks in a 5% CO2 incubator. The antigen-binding activity of the anti-hIL-5R antibodies grafted onto the RDC α in the culture supernatants in the wells in which growth of transformants was observed was determined by the ELISA test shown in point 2 of example 3. Regarding the transformants in wells in which production of the anti-hIL-5R antibody grafted in the RDC α was observed in culture supernatants, the concentration of mTx increased to 100 nm and then to 200 nM, and a transformant capable of growing in the RPMI1640-SBF medium (10) containing 0.5 mg / ml of G418 and 200 nM MTX and to produce the anti-hIL-5R antibody grafted on the RDC α in a large quantity was finally obtained in the same way as described above. The obtained transformant was prepared in a single cell (cloning) by limiting the dilution twice. Clone No. 3 of transformed cell producing anti-hIL-5R antibodies grafted to RDC α has been deposited on April 5, 1999, as FERM BP-6690 at the National Institute of Human Bioscience and Technology, Agency for Science and Technology Industrial (Higashi 1-1-3, Tsukuba, Ibaraki, Japan).
(2) Production of producer cells using CHO / dhfr cells<sup>-</sup>
After introducing 4 pg of the expression vector of the anti-hIL-5R antibody grafted in the RDC α, pKANTEX1259HV3LV0, described in document WO 97/10354 in 1.6 x 10<sup>6</sup> CHO / dhfr 'cells by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 10 ml of IMDM-SBF (10) and distributed in 200 µl / well in a 96-cell culture plate. wells (manufactured by Iwaki Glass). Twenty-four hours after culturing at 37 ° C in a 5% CO2 incubator, G418 was added to provide a concentration of 0.5 mg / ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from the respective wells in which colonies of transformants showing resistance to G418 were formed and colony growth was observed, and the antigen-binding activity of anti-hIL-5R antibodies grafted onto the α-DRC in the supernatant it was determined by the ELISA assay as shown in point 2 of example 3.
Regarding the transformants in the wells in which the production of anti-hIL-5R antibodies grafted onto the RDC α was observed in culture supernatants, in order to increase the amount of antibody production using a DHFR gene extension system, each of the transformants was suspended in IMDM-dSBF medium (10) containing 0.5 mg / ml G418 and 10 nM MTX to provide a density of 1 to 2 x 10<sup>5</sup> cells / ml, and the suspension was partitioned 0.5 ml into the wells of a 24-well plate (manufactured by Iwaki Glass). Transformants showing resistance to 10 nM mTx were produced by culturing at 37 ° C for 1 to 2 weeks in a 5% CO2 incubator. Regarding the transformants in the wells in which their growth was observed, the concentration of MTX increased to 100 nM and then to 500 nM, and a transformant capable of growing in IMDM-dSBF medium (10) containing 0.5 mg / ml of G418 and 500 nM of MTX and to produce anti-hIL-5R antibody grafted on the RDC α in large quantity was finally obtained in the same way as previously described. The obtained transformant was produced in a single cell (cloning) by limiting the dilution twice.
ES 2 568 898 T3 (3) Producer cell production using NS0 mouse myeloma cells
An expression vector of the anti-hIL-5R antibody grafted to the α-DRC was prepared according to the method of Yarranton et al. (BIO / TECHNOLOGY, 10, 169 (1992)) and using cDNA of the H chain and the L chain of the antibody in the expression vector of the anti-hIL-5R antibody grafted in the α-DRC, pKANTEX1259HV3LV0, described in WO 97/10354, and NS0 cells were transformed to obtain a transformant capable of producing the antihIL-5Rα antibody grafted on the RDC in a large quantity. The transformant obtained was made in a single cell (cloning) limiting the dilution twice.
two. Measurement of hIL-5R α antibody binding activity (ELISA)
The hIL-5Rα binding activity of the antibody was determined as described below.
A solution was prepared by diluting the anti-hIL-5R antibody, mouse KM1257α, described in WO 97/10354 with PBS to provide a concentration of 10 pg / ml, and 50 µl of the resulting solution was partitioned into each well of a 96-well plate for ELISA (produced by Greiner), followed by reaction at 4 ° C for 20 hours. After the reaction, 1% BSA-PBS was partitioned into 100 µl / well, and then the reaction was carried out at room temperature for 1 hour to block the remaining active groups. After discarding PBS-1% BSA, a solution prepared by diluting the soluble hIL-5R α described in WO 97/10354 with 1% BSA-PBS to give a concentration of 0.5 pg / ml was partitioned into 50 pl / well, followed by reaction at 4 ° C for 20 hours. After the reaction, each well was washed with Tween-PBS, the culture supernatants of transformants or the diluted solutions of a purified human antibodies grafted in RDC were distributed in 50 g / well to carry out the reaction at room temperature for 2 hours. After the reaction, each well was washed with Tween-PBS, a solution of peroxidase-labeled goat anti-human IgG (H&L) antibodies (produced by American Qualex) diluted 3000 times with 1% BSA-PBS was partitioned in 50 µl / as well as a secondary antibody solution, followed by reaction at room temperature for 1 hour. After reaction and subsequent washing with Tween-PBS, the ABTS substrate solution (a solution prepared by dissolving 0.55 g of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2) and the addition of 1 µl / ml of hydrogen peroxide to the solution just before use) were distributed over 50 µl / well for color development, and then measured the absorbance at OD415.
3. Purification of anti-hIL-5R α antibodies grafted on the RDC (1) Culture of producer cells from YB2 / 0 cells and purification of antibodies
The anti-hIL-5R α antibody-producing transformed cell clone obtained in point 1 (1) above of Example 3 was suspended in GIT medium containing 0.5 mg / ml of G418 and 200 nM of MTX to provide a density of 3x10<sup>5</sup> cells / ml and distributed over 200 ml into 175 mm flasks<sup>2</sup> (made by Greiner). The culture supernatant was recovered eight days after cultivation at 37 ° C in an incubator with 5% CO2. The RDC grafted anti-hIL-5Rα antibody was purified from the culture supernatant using ion exchange chromatography and a gel filtration method. The purified anti-hIL-5R α antibody grafted onto the RDC was named YB2 / 0-hIL-5RCDR antibody.
(2) Culture of producer cells from sCHO / dhfr cells<sup>-</sup> and antibody purification
The anti-hIL-5R α antibody-producing transformed cell clone obtained in point 1 (2) above of Example 3 was suspended in EX-CELL302 medium containing 3 mM L-Gln, 0.3% CDlC and 0.5% PF68 to provide a density of 3x10<sup>5</sup> cells / ml and cultured using a 4.0 liter capacity spinner flask (produced by Iwaki Glass) shaking at a speed of 100 rpm. Ten days after culturing at 37 ° C in a temperature controlled room, the culture supernatant was recovered. The RDC grafted anti-hIL-5Rα antibody was purified from the culture supernatant using ion exchange chromatography and a gel filtration method. The purified anti-hIL-5R α antibody grafted onto the RDC was called the CHO / d-hIL-5RCDR antibody.
(3) Culture of producer cells from NS0 cells and purification of antibodies
The anti-hIL-5R α antibody producing transformed cell clone obtained in item 1 (3) above of Example 3 was cultured according to the method of Yarranton et al. (BIO / TECHNOLOGY, 10, 169 (1992)) and then a culture supernatant was recovered. RDC grafted anti-hIL-5Rα antibody was purified from culture supernatant using ion exchange chromatography and gel filtration method. The purified anti-hIL-5R α antibody grafted onto the RDC was designated as the NS0-LIS-5RCDR antibody.
Four. Analysis of anti-hIL-5R α antibodies grafted onto purified CDR
According to a known method (Nature, 227, 680, (1970)), 4 pg of each of the three anti-hIL-5R α antibodies
ES 2 568 898 T3 grafted in RDC produced by the respective animal cells and purified therein, obtained in point 3 above of example 3, were subjected to SDS-PAGE to analyze the molecular weight and the degree of purification. The results are shown in Fig. 4. As shown in Fig. 4, a single band of approximately 150 Kd in molecular weight was observed under non-reducing conditions, and two bands of approximately 50 Kd and approximately 25 Kd under reducing conditions, in each of the anti-hIL antibodies. Purified -5Rα grafted onto RDC. These molecular weights nearly matched the deduced molecular weights of the antibody H chain and L chain cDNA nucleotide sequences (H chain: approximately 49 Kd, L chain: approximately 23 Kd, whole molecule: approximately 144 Kd), and were also in agreement with reports indicating that the IgG antibody has a molecular weight of approximately 150 Kd under non-reducing conditions and degrades into H chains that have a molecular weight of approximately 50 Kd and L chains that have a molecular weight of approximately 25 Kd under reducing conditions due to the cleavage of the disulfide bond (hereinafter referred to as the SS bond) in the molecule (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1998; Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996), so it was confirmed that each anti-hIL-5Rα antibody grafted to the RDC was expressed and purified as an antibody molecule having the true structure.
Example 4
Evaluation of the activity of the anti-hIL-5R α antibody grafted in the RDC:
1. Binding activity of anti-hIL-5R α antibodies grafted on the CDR to hIL-5R α (ELISA)
The activity of the three RDC grafted purified anti-hIL-5R α antibodies obtained in point 2 above of example 3 to bind to hIL-5R α was determined by the ELISA assay shown in point 2 of example 3. Fig. 5 shows a result of examination of the binding activity measured by changing the concentration of the anti-hIL-5R antibodies grafted on the α-DRC to be added. As shown in Fig. 5, the three anti-hIL5R α antibodies grafted onto the RDC exhibited almost the same binding activity to α hIL-5R. This result demonstrates that the antigen-binding activities of these antibodies are constant regardless of the antibody-producing cells of animal origin and their culture methods, similar to the result of point 1 of example 2.
two. In vitro cytotoxic activity (ADCC activity) of anti-hIL-5R α antibodies grafted on the RDC
In order to evaluate the in vitro cytotoxic activity of the three RDC grafted purified anti-hIL-5R α antibodies obtained in point 3 above of Example 3, the ADCC activity was determined according to the following method.
(1) Preparation of the target cell solution
A line of mouse T lymphocytes CTLL-2 (h5R) expressing the α chain and β chain of hIL-5R described in WO 97/10354 was cultured using RPMI1640-SBF medium (10) to prepare a suspension of 1 * 10<sup>6 </sup>cells / 0.5 ml, and the cells were labeled with radioisotopes by reacting them with 3.7 MBq equivalents of a radioactive substance Na2<sup>51</sup>CrO4 at 37 ° C for 1.5 hours. After the reaction, the cells were washed three times through their suspension in RPMI1640-SBF medium (10) and centrifugation, resuspended in the medium and then allowed to stand at 4 ° C for 30 minutes in ice for the spontaneous dissolution of the radioactive substance. After centrifugation, the precipitate was adjusted to 2 * 10<sup>5</sup> cells / ml by adding 5 ml of RPMI1640-SBF medium (10) and used as target cell solution.
(2) Preparation of effector cell solution
50 ml of venous blood was collected from a healthy person and mixed gently with 0.5 ml of sodium heparin (produced by Takeda Pharmaceutical). The mixture was centrifuged to separate a mononuclear cell layer using Polymorphprep (produced by Nycomed Pharma AS) and according to the manufacturer's instructions. After washing with RPMI1640-SBF medium (10) by centrifugation three times, the resulting cells were resuspended to provide a density of 9 * 10<sup>6</sup> cells / ml using the medium and were used as the effector cell solution.
(3) Measurement of ADCC activity
In each well of a 96-well U-bottom plate (manufactured by Falcon), 50 µl of the target cell solution prepared in point (1) above (1 * 10<sup>4</sup> cells / well). Next, 100 μl of the effector cell solution prepared in point (2) above (9 * 10<sup>5</sup> cells / well, the ratio of effector cells to target cells becomes 90: 1). Subsequently, each of the RDC grafted anti-hIL-5Rα antibodies was added to provide a final concentration of 0.001 to 0.1 pg / ml, followed by reaction at 37 ° C for 4 hours. After the reaction, the plate was centrifuged, and the amount of<sup>51</sup>Cr in the supernatant. The amount of spontaneously released<sup>51</sup> Cr was calculated by the same operation using only the medium instead of the effector cell solution and the antibody solution and determining the amount of <sup>51</sup> Cr in the supernatant. The amount of<sup>51</sup>Total Cr released was calculated by the same operation using only the
ES 2 568 898 T3 medium instead of the antibody solution and the addition of 1 N hydrochloric acid, instead of the effector cell solution, and determining the amount of <sup>5l</sup>Cr in the supernatant.
The ADCC activity was calculated from the following equation.
<sup>51</sup> Cr in the supernatant sample -<sup>51</sup> Spontaneously released Cr
ADCC activity% = ~ "x 100
Total released Cr - Spontaneously released Cr
The results are shown in Fig. 6. As shown in Fig. 6, among the three anti-hlL-5R a antibodies grafted on RDC, the YB2 / 0-hlL-5RCDR antibody showed the highest ADCC activity, followed CHO / d-hlL-5RCDR antibody and NS0-hlL-5RCDR antibody in this order. Like the result of point 2 of example 2, the above results demonstrate that the ADCC activity of the antibodies varies greatly depending on the animal cells to be used in their production. Furthermore, since the antibodies produced by the YB2 / 0 cell showed the highest ADCC activity in both cases of the two humanized antibodies, it was revealed that an antibody having high ADCC activity can be produced by using the cell YB2 / 0.
3. Evaluation of the in vivo activity of antl-h! L-5R α antibodies grafted in the RDC
In order to evaluate the in vivo activity of the three RDC-grafted purified anti-hIL-5R antibodies obtained in point 3 above of Example 3, the inhibition activity was examined in a model of Macaca phaseicularis that increases eosinophilia produced by hlL-5 according to the following procedure.
HIL-5 (preparation procedure described in WO 97/10354) was administered to Macaca phaseicularis under the dorsal skin at a dose of 1 pg / kg, starting on the first day and once a day for a total of 14 times. Each RDC-grafted anti-hIL-5R a antibody was administered intravenously at a dose of 0.3 mg / kg one hour before hIL-5 administration on day zero. A group with no added antibodies was used as a reference. In the groups administered with antibodies, three Macaca phaseicularis animals were used in each group (No. 301, No. 302, No. 303, No. 401, No. 402, No. 403, No. 501, No. 502 and No. 503), and two animals (# 101 and # 102) were used in the group without antibodies. From 7 days before the start of the administration and up to 42 days after the administration, about 1 ml of blood was collected periodically from the saphenous vein or from the femoral vein, and the number of eosinophils in 1 µl of peripheral blood was determined. . The results are shown in Fig. 7. As shown in Fig. 7, the increase of eosinophils in the blood was completely inhibited in the group to which the YB2 / 0-hIL-5RCDR antibody was administered. On the other hand, complete inhibition activity was found in one animal in the group to which the CHO / d-hIL-5RCDR antibody was administered, but no complete inhibition activity was found and its effect was not sufficient. In the group to which NSO-hIL-5RCDR antibody was administered, complete inhibitory activity was not found and its effect was not sufficient. The above results demonstrate that the in vivo activity of the antibodies varies greatly depending on the animal cells to be used in their production. Furthermore, since a positive correlation was found between the degree of in vivo activity of the anti-hIL-5R antibody grafted onto the RDC and the degree of its ADCC activity described in point 2 of the example
4, it was indicated that the degree of activity of ADCC is very important for its expression of activity.
Based on the above results, it is expected that an antibody having high ADCC activity is also useful in the clinical field for various diseases in humans.
Example 5
Sugar chain analysis that increases ADCC activity:
1. Preparation of 2-aminopyridine-labeled sugar chain (particle-treated sugar chain)
The humanized antibody of the present invention was acid hydrolyzed with hydrochloric acid to remove sialic acid. After hydrochloric acid was completely removed, the sugar chain was cut from the protein by hydrazinolysis (Method of Enzymology, 83, 263, 1982). The hydrazine was removed, and N-acetylation was carried out by adding an aqueous solution of ammonium acetate and acetic anhydride. After lyophilization, fluorescence labeling was carried out with 2-aminopyridine (J. Biochem., 95, 197 (1984)). The fluorescence-labeled sugar chain (PA-treated sugar chain) was removed as an impurity using the Surperdex Peptide HR 10/30 column (manufactured by Pharmacia). The sugar chain fraction was dried using a centrifugal concentrator and used as the purified PA-treated sugar chain.
two. Reverse Phase HPLC Analysis of the PA-Treated Sugar Chain of Purified Anti-h! L-5Rα Antibody Grafted on the RDC
ES 2 568 898 T3
Using the respective PA-treated sugar chains of anti-hIL-5R α antibody grafted to the RDC prepared in item 1 above of Example 5, reverse phase HPLC analysis was carried out by the CLC-ODS column (manufactured by Shimadzu). An excess amount of α-L-fucosidase (from bovine kidney, produced by SIGMA) was added to the PA-treated sugar chain for digestion (37 ° C, 15 hours), and the products were then analyzed by HPLC in reverse phase (Fig. 8). Using PA-treated sugar chain standards prepared by Takara Shuzo it was confirmed that the asparagine-bound sugar chain elutes over a period of 30-80 minutes. The ratio of sugar chains whose reversed phase HPLC elution positions shifted (sugar chains eluted over a period of 48 minutes to 78 minutes) was calculated by digestion with α-L-fucosidase. The results are shown in Table 1.
Table 1
<td>Antibody-producing cells</td><td>Sugar chain attached to α-1,6-fucose (%)</td>
<td>YB2 / 0</td><td> 47</td>
<td>NS0</td><td> 73</td>
Approximately 47% of the anti-hIL-5RCDR-grafted antibodies produced by the YB2 / 0 cell and approximately 73% of the anti-hIL-5RCDR-grafted antibodies produced by the NS0 cell were sugar chains having α-1, 6-fucose. Therefore, sugar chains lacking α-1,6-fucose were more frequent in the antibody produced by the YB2 / 0 cell than the antibody produced by the NS0 cell.
3. Analysis of the monosaccharide composition of purified anti-hIL-5R α antibody grafted on the DRC
RDC-grafted anti-hIL-5Rα antibody sugar chains produced by YB2 / 0 cells, NS0 cells, and CHO / d cells were hydrolyzed into monosaccharides by acid hydrolysis with trifluoroacetic acid, and monosaccharide composition analysis was carried out using BioLC (produced by Dionex).
Among the N-glucoside-linked sugar chains, there are 3 mannose units in a sugar chain in the complex-type N-glucoside-linked sugar chain. A relative proportion of each monosaccharide obtained by calculating the sum of mannoses as 3 is shown in Table 2.
Table 2
<td>Antibody-producing cell</td><td>Fuc</td><td>GlcNAc</td><td>Gal</td><td>Man</td><td>ADCC activity (%) *</td>
<td>YB2 / 0</td><td> 0,60</td><td> 4,98</td><td> 0,30</td><td> 3,00</td><td> 42,27</td>
<td>NS0</td><td> 1,06</td><td> 3,94</td><td> 0,66</td><td> 3,00</td><td> 16,22</td>
<td>CHO / DHFr<sup>-</sup></td><td> 0,85</td><td> 3,59</td><td> 0,49</td><td> 3,00</td><td> 25,73</td>
<td></td><td> 0,91</td><td> 3,80</td><td> 0,27</td><td> 3,00</td><td></td>
*: Antibody concentration: 0.01 pg / ml
Since the relative fucose proportions were in the order YB2 / 0 <CHO / d <NS0, the sugar chain produced in the antibody produced by the YB2 / 0 cells had the lowest fucose content as also shown in the present results.
Example 6
Analysis of the sugar chain of the antibody produced by CHO / dhfr cells<sup>-</sup>:
PA-treated sugar chains were prepared from RDC-grafted purified anti-hIL-5Rα antibodies produced by CHO / dhfr cells.<sup>-</sup>, and reverse phase HPLC analysis was carried out using a CLC-ODS column (manufactured by Shimadzu) (Fig. 9). In Fig. 9, an elution time of 35 to 45 minutes corresponded to sugar chains having no fucose and an elution time of 45 to 60 minutes corresponded to sugar chains having fucose. As in the case of the antibody produced by mouse myeloma NS0 cells, the RDC-grafted anti-hIL-5R α antibody produced by CHO / dhfr<sup>-</sup> Cell had less sugar chain content without fucose than the antibody produced by rat myeloma YB2 / 0 cells.
Example 7
Separation of antibodies with high ADCC activity:
RDC-grafted anti-hIL-5Rα antibody produced by YB2 / 0 rat myeloma cells was separated using a lectin column that binds to fucose-bearing sugar chains. HPLC was carried out using LC6A produced by Shimadzu at a flow rate of 1 ml / min and at room temperature as the column temperature. After equilibration with 50 mM Tris-sulfate buffer (pH 7.3), the purified anti-hIL-5R α antibody was injected
ES 2 568 898 T3 grafted onto the DRC and then eluted by a linear density gradient (60 minutes) of 0.2M α-methylmannoside (produced by Nakalai Tesque). The anti-hIL-5R α antibody grafted to the RDC separated into the non-adsorbed fraction and the adsorbed fraction. When the non-adsorbed fraction and a part of the adsorbed fraction were sampled and their binding activity to hIL-5R α was determined, they exhibited similar binding activity (Fig. 10, top graph). When ADCC activity was determined, the non-adsorbed fraction exhibited higher ADCC activity than the portion of the adsorbed fraction (Fig. 10, bottom graph). Furthermore, PA-treated sugar chains were prepared from the non-adsorbed fraction and a part of the adsorbed fraction, and reverse HPLC analysis was carried out using a CLC-ODS column (manufactured by Shimadzu) (Fig. eleven). The non-adsorbed fraction was an antibody having mainly fucose-free sugar chains, and the portion of the adsorbed fraction was an antibody having mainly fucose-containing sugar chains.
Example 8
Determination of the transcription product of the a1,6-fucosyltransferase (FUT8) gene in the host cell line:
(1) Preparation of single-stranded cDNA from various cell lines
CHO / DG44 cells from Chinese hamster ovary were suspended in IMDM medium (produced by Life Technologies) enriched with 10% SBF (produced by Life Technologies) and 1χ concentration of HT supplement (produced by Life Technologies) and inoculated into a T75 cell adhesion culture flask (produced by Greiner) at a density of 2 × 10<sup>5</sup> cells / ml. In addition, the YB2 / 0 cells from rat myeloma were suspended in RPMI1640 medium (produced by Life Technologies) enriched with 10% SBF (produced by Life Technologies) and 4 mM glutamine (produced by Life Technologies) and inoculated in a T75 cell suspension flask (manufactured by Greiner) at a density of 2 × 10<sup>5</sup> cells / ml. These cells were cultured at 37 ° C in an incubator with 5% CO2, and 1 χ 10<sup>7</sup> cells from each host cell were recovered on the first, second, third, fourth, and fifth days to extract full-length RNA using RNAeasy (produced by QUIAGEN).
The complete RNA was dissolved in 45 µl of sterile water, mixed with 0.5 U / µl of RQ1 DNase without RNase (produced by Promega) and 5 µl of 10 χ DNase coupled buffer and 0.5 µl of RNasin ribonuclease inhibitor (produced by Promega), followed by reaction at 37 ° C for 30 minutes. After the reaction, the complete RNA was purified again using RNAeasy (produced by QUIAGEN) and dissolved in 50 µl of sterile water.
According to the SUPERSCRIPT ™ Preampling System for First Strand cDNA Synthesis (produced by Life Technology), 3 pg of each obtained complete RNA was subjected to a reverse transcription reaction in a 20 pl system using oligo (dT) as primer to thereby synthesize cDNA. A 1 concentration solution of the solution after the reverse transcription reaction was used for the cloning of FUT8 and β-actin from each host cell, and a solution after the reverse transcription reaction further diluted 50 times with water was used. used for the determination of the amount of transcription of each gene using competitive PCR, and each of the solutions were stored at -80 ° C until use.
(2) Preparation of the respective partial cDNA fragments of Chinese hamster FUT8 and rat FUT8
The respective partial fragments of Chinese hamster FUT8 and rat FUT8 cDNA were obtained as described below. First, primers (shown in SEQ ID No. 1 and SEQ ID No. 2) specific for common nucleotide sequences in a human FUT8 cDNA were designed (Journal of Biochemistry, 121, 626 ( 1997)) and a porcine FUT8 cDNA (Journal of Biological Chemistry, 271, 27810 (1996)).
Next, using an ExTaq DNA polymerase (produced by Takara Shuzo), 25 µl of a reaction solution consisting of ExTaq buffer (produced by Takara Shuzo), 0.2 mM dNTP, 0.5 mM of each of the specific primers above (SEQ ID No. 1 and SEQ ID. n ° 2), and 1 pl of each of the cDNAs from CHO cells and the cDNAs from YB2 / 0 cells, each one obtained on the 2nd day of culture in (1), was prepared, and carried out perform the polymerase chain reaction (PCR). The PCR was carried out under conditions where, after heating to 94 ° C for 1 minute, a cycle consisting of reactions at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 seconds. minutes is repeated 30 cycles and then the reaction solution is heated at 72 ° C for 10 minutes. Each 979 bp specific expanded fragment obtained by PCR was connected to a plasmid pCR2.1 using TOPO TA Cloning Kit (produced by Invitrogen) to obtain a respective partial cDNA fragment containing either Chinese hamster FUT8 or rat FUT8 plasmid (CHFT8 -pCR2.1 or YBFT8pCR2.1).
The nucleotide sequence of each obtained cDNA was determined using DNA Sequencer 377 (produced by Perkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (produced by Parkin Elmer) to confirm that the obtained cDNAs encode the partial sequences of the open reading frame. (ORF) of Chinese hamster FUT8 and rat FUT8 (shown in SEQ. ID. Nos. 3 and 4).
ES 2 568 898 T3 (3) Preparation of cDNA with Chinese hamster β-actin and rat β-actin
Since the β-actin gene is considered to be constantly transcribed in each cell and its amount of transcription is almost the same between cells, the amount of transcription of the β-actin gene is determined as a standard for the efficiency of the reaction. synthesis of cDNA from the respective cells.
Chinese hamster β-actin and rat β-actin were obtained by the following procedure. First, a forward primer (shown in SEQ ID NO 5) specific for a common sequence containing a translation initiation codon and reverse primers (shown in SEQ ID NO 6 and SEQ ID NO. n ° 7) specific for the respective sequence containing a translation termination codon were designed from a genomic sequence of Chinese hamster β-actin (GenBank, U20114) and from a genomic sequence of rat β-actin ( Nucleic Acid Research, 11, 1759 (1983)).
Then, using a DNA polymerase, KOD (produced by TOYOBO), 25 µl of a reaction solution constituted by KoD buffer n ° 1 (produced by TOyObO), 0.2 mM dNTP, 1 mM MgCl2, 0, was prepared. 4 μΜ of each of the above gene specific primers (SEQ ID NO 5 and SEQ ID NO 6, or SEQ ID NO 5 and SEQ ID NO. n ° 7), 5% DMSO, and 1 μl of each of the cDNAs from CHO cells and the cDNAs from YB2 / 0 cells, each one obtained on the 2nd day of culture in (1), and carried out the polymerase chain reaction (PCR). The PCR was carried out under conditions in which, after heating to 94 ° C for 4 minutes, a cycle consisting of reactions at 98 ° C for 15 seconds, 65 ° C for 2 seconds, and 74 ° C for 30 seconds, 25 cycles are repeated. The 5 'terminus of each 1128 bp specific expanded fragment obtained by PCR was phosphorylated using MEGALABEL (produced by Takara Shuzo) and then digested with a restriction enzyme, Eco RV, and the resulting fragment (2.9 Kb) was connected to pBluescript II (KS (+) (produced by Stratagene) using Ligation High (produced by Toyobo) to obtain a plasmid containing a complete ORF of the respective β-actin cDNAs Chinese hamster or rat β-actin (CHAc-pBS or YBAc-pBS).
The nucleotide sequence obtained from the respective cDNAs was determined using DNA Sequencer 377 (produced by Perkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (produced by Perkin Elmer) to confirm that they respectively encode complete β-cDNA ORF sequences. Chinese hamster actin and rat β-actin.
(4) Preparation of the standard and internal reference sequence
In order to measure the amount of transcription of the FUT8 gene mRNA in producer cells, a calibration curve was first prepared.
As the FUT8 standard to be used in the calibration curve, the plasmids, CHFT8-pCR2.1 and YBFT8pCR2.1, obtained in (2) by inserting partial fragments of the respective cDNA, Chinese hamster FUT8 or rat FUT8 in pCR2.1 were digested with a restriction enzyme, EcoRI, and the resulting DNA fragments were used after being converted to straight chains.
As an internal standard to be used in the determination of FUT8, between CHFT8-pCR2.1 and YBFTS-pCR2.1, pCR2.1-CHFT8d and YBFTBd-pCR2.1 obtained by deleting 203 bp between ScaI-HindIII of internal nucleotide sequences Chinese hamster FUT8 or rat FUT8 were digested with the restriction enzyme, EcoRI, and the resulting DNA fragments were used after being converted to straight chains.
As a standard for the amount of mRNA transcribed from the β-actin gene in producer cells, the plasmids CHAc-pBS and YBAc-PBS obtained in (3) by integration of the complete ORF of the respective Chinese hamster β-actin cDNA and Rat β-actin in pBluescript II KS (+) were digested respectively, the first with HindIII and Pst I and the last with HindIII and KpnI, and the resulting DNA fragments were used by converting them into linear chains.
As internal standard for the determination of β-actin, between CHAc-pBS and YBAc-pBS, CHAcd-pBS and YBAcd-pBS obtained by deleting 180 bp between DraIII-DraIII of the internal nucleotide sequences of Chinese hamster β-actin and β Rat actin were digested, the former with HindIII and PstI and the latter with HindIII and KpnI, and the resulting DNA fragments were used after converting them to straight chains.
(5) Determination of the amount of transcription by competitive RT-PCR
In the first place, a set of primers (shown in SEQ ID No. 8 and No. 9) was designed common to the specific sequence for the internal sequences of the partial ORF sequences of Chinese hamster FUT8 and rat FUT8. obtained in (2).
Next, PCR was performed using an ExTaq DNA polymerase (produced by Takara Shuzo) in 20 μl in the total volume of a reaction solution consisting of ExTaq buffer (produced by Takara Shuzo), 0.2 mM dNTP, 0.5 mM of each of the previous gene-specific primers (SEQ. ID. No. 8 and SEQ. ID. No. 9), 5% DMSO and 5 μl of a 50-fold diluted solution of each of the cDNAs from respective lines of
ES 2 568 898 T3 host cells obtained in (1) and 5 pl (10 fg) of the plasmid for internal standard. PCR was carried out by heating at 94 ° C for 3 minutes and then repeating 30 cycles using reactions at 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute as one cycle.
The β-actin transcription product was determined as described below. The specific genes of the primer sets were designed respectively for the internal sequences of the complete ORFs of Chinese hamster β-actin and rat β-actin obtained in (3) (the former are shown in SEQ ID NO. 10 and SEQ ID No. 11, and the second in SEQ ID No. 12 and SEQ ID No. 13).
Next, PCR was performed using an ExTaq DNA polymerase (produced by Takara Shuzo) in 20 µl in the total volume of a reaction solution consisting of ExTaq buffer (produced by Takara Shuzo), 0.2 mM dNTP, 0.5 mM of the above gene specific primers (SEQ ID NO 10 and SEQ ID NO 11, or SEQ ID NO 12 and SEQ ID NO. n ° 13), 5% DMSO, and 5 µl of a 50-fold diluted solution of each of the cDNAs from the respective host cell lines obtained in (1) and 5 µl (1 µg) of plasmid for the standard internal. PCR was carried out by heating at 94 ° C for 3 minutes and then repeating 17 cycles using reactions at 94 ° C for 30 seconds, 65 ° C for 1 minute, and 72 ° C for 2 minutes as one cycle.
Table 3
<td>Target gene</td><td>* Set of primers</td><td colspan="2">Product size (bp) expanded by RCP</td>
<td></td><td></td><td>Diana</td><td>Competitor</td>
<td>FUT8</td><td>F: 5'-GTCCATGGTGATCCTGCAGTGTGG-3 'R: 5'-CACCAATGATATCTCCAGGTTCC-3'</td><td> 638</td><td> 431</td>
<td>β-actin (Chinese hamster)</td><td>F: 5'-GATATCGCTGCGCTCGTTGTCGAC-3 'R: 5'-CAGGAAGGAAGGCTGGAAAAGAGC-3'</td><td> 789</td><td> 609</td>
<td>β-actin (rat)</td><td>F: 5'-GATATCGCTGCGCTCGTCGTCGAC-3 'R: 5'-CAGGAAGGAAGGCTGGAAGAGAGC-3'</td><td> 789</td><td> 609</td>
* F: forward primer, R: reverse primer
Determinative PCR was carried out using the primer sets shown in Table 3. As a result, the DNA fragment having the size shown in the target column of Table 3 was amplified from the respective transcription product of the gene and the corresponding standard, and the DNA fragment having the size shown in the competitor column of Table 3 was amplified from the corresponding internal standard.
After 7 µl of the solution after PCR was subjected to 1.75% agarose gel electrophoresis, the gel was stained with SYBR Green I Nucleic Acid Gel Stain (produced by Molecular Probes). The amount of enlarged DNA fragments was determined by calculating the luminescence intensity of each of the amplified DNA fragments using FluorImager SI (produced by Molecular Dynamics).
In addition, PCR was performed by changing the amount of the standard plasmid prepared in (4) to 0.1 fg, 1 fg, 5 fg, 10 fg, 50 fg, 100 fg and 500 fg, instead of the cell-derived cDNA, and the number of expanded products was determined. A calibration curve was prepared by plotting the measured values against the amounts of standard plasmid.
Using this calibration curve, the amount of gene of interest cDNA in each cell was calculated from the amount of the expanded product when the complete cDNA from each cell was used as a template, and the amount was defined as the amount of transcription. of mRNA in each cell.
The amounts of the FUT8 transcription product in each host cell line using rat FUT8 sequences as the standard and internal standard are shown in Fig. 12. The CHO cell line showed a 10-fold or greater amount of transcription. to that of the YB2 / 0 cell line throughout the culture period. This trend was also found when Chinese hamster FUT8 sequences were used as the standard and internal standard.
Furthermore, the amount of FUT8 transcript is shown in Table 4 as a value relative to the amount of β-actin transcript.
Table 4
<td rowspan="2">Cell line</td><td colspan="5">Cultivation days</td>
<td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 4</td><td>Day 5</td>
<td>CHO</td><td> 2,0</td><td> 0,90</td><td> 0,57</td><td> 0,52</td><td> 0,54</td>
<td>YB2 / 0</td><td> 0,07</td><td> 0,13</td><td> 0,13</td><td> 0,05</td><td> 0,02</td>
ES 2 568 898 T3
While the amount of FUT8 transcription from the YB2 / 0 cell line was approximately 0.1% β-actin, that from the CHO cell line was 0.5 to 2%.
Based on the above results, the amount of FUT8 transcription product in the YB2 / 0 cell line was shown to be significantly smaller than that in the CHO cell line.
Industrial applicability
The present disclosure refers to a sugar chain that controls the activity of an immunofunctional molecule, such as an antibody, a protein or a peptide, as well as an antibody, a protein or a peptide having the sugar chain. The present disclosure further relates to processes for the production of the sugar chain and an antibody, a protein or a peptide having the sugar chain, as well as a diagnostic agent, a preventive agent, and a therapeutic agent containing these. products as an active ingredient.
The following numbered clauses are also explicitly disclosed herein:
1. Method for controlling the activity of an immunofunctional molecule, which comprises regulating the presence or absence of fucose binding to the N-acetylglucosamine of the reducing terminal of a sugar chain linked to Nglucoside that binds to the immunofunctional molecule.
two. Procedure according to clause 1, in which the N-glucoside-linked sugar chain that binds to the immunofunctional molecule comprises:
Manal -
Man31 ----- ► 4GlcNAcpi ----- ► 4GlcNAc.
Manal
<img file="ES2568898T3_D0005.tif" />
3. Procedure to increase the activity of an immunofunctional molecule, which comprises the union of a sugar chain in which fucose is not present in the N-acetylglucosamine of the reducing terminal of a sugar chain linked to N-glucoside to an immunofunctional molecule .
Four. Procedure according to clause 3, in which the sugar chain comprises:
<img file="ES2568898T3_D0006.tif" />
5. Procedure according to clause 3, in which the sugar chain is synthesized in a cell that has a low enzymatic activity of addition of fucose to the N-acetylglucosamine of the reducing terminal or does not present said enzymatic activity.
6. Procedure according to clause 5, in which the enzyme that adds fucose to the reducing terminal N-acetylglucosamine is a fucosyltransferase.
7. Procedure according to clause 6, in which the fucosyltransferase is α1,6-fucosyltransferase.
8. Procedure according to clause 3, in which the sugar chain is synthesized in a rat myeloma cell.
9. Procedure according to clause 8, wherein the rat myeloma cell is the YB2 / 3HL.P2.G11.16Ag.20 cell (ATCC CRL 1662).
10. Process for inhibiting the activity of an immunofunctional molecule, which comprises joining a sugar chain in which fucose is present in the N-acetylglucosamine of the reducing terminal of a sugar chain linked to N-glucoside to an immunofunctional molecule.
eleven. Procedure according to clause 10, in which the sugar chain comprises:
ES 2 568 898 T3
<img file="ES2568898T3_D0007.tif" />
12. Procedure according to clause 10, in which the sugar chain is synthesized in a cell that shows a high enzymatic activity of addition of fucose to the N-acetylglucosamine of the reducing terminal.
13. Procedure according to clause 12, in which the enzyme that adds fucose to the reducing terminal N-acetylglucosamine is a fucosyltransferase.
14. Procedure according to clause 13, wherein the fucosyltransferase is α1,6-fucosyltransferase.
fifteen. Method according to any one of clauses 1 to 14, in which the immunofunctional molecule is an antibody, a protein or a peptide.
16. Agent for stimulating the activity of an immunofunctional molecule, which comprises a sugar chain in which fucose is not present in the N-acetylglucosamine of the reducing terminal of a sugar chain linked to N-glucoside.
17. Agent for stimulating the activity of an immunofunctional molecule according to clause 16, in which the sugar chain comprises:
<img file="ES2568898T3_D0008.tif" />
18. Agent for stimulating the activity of an immunofunctional molecule according to clause 16, in which the sugar chain is synthesized in a cell that has a low enzymatic activity of addition of fucose to the N-acetylglucosamine of the reducing terminal or does not present said enzymatic activity.
19. Agent for stimulating the activity of an immunofunctional molecule according to clause 18, in which the enzyme that adds fucose to the reducing terminal N-acetylglucosamine is a fucosyltransferase.
twenty. Agent for stimulating the activity of an immunofunctional molecule according to clause 19, in which the fucosyltransferase is α1,6-fucosyltransferase.
twenty-one. Agent for stimulating the activity of an immunofunctional molecule according to clause 16, in which the sugar chain is synthesized in a rat myeloma cell.
22. Agent for stimulating the activity of an immunofunctional molecule according to clause 21, wherein the rat myeloma cell is the YB2 / 3HL.P2.G11: 16Ag.20 cell (ATCC CRL 1662).
2. 3. Agent for stimulating the activity of an immunofunctional molecule according to any one of clauses 16 to 22, wherein the immunofunctional molecule is an antibody, a protein or a peptide.
24. Immunofunctional molecule exhibiting stimulated immunofunctional activity, said molecule being linked to a sugar chain in which fucose is not present in the N-acetylglucosamine of the reducing terminal of a sugar chain linked to N-glucoside.
25. Immunofunctional molecule that exhibits inhibited immunofunctional activity, a sugar chain in which fucose is present in the N-acetylglucosamine of the reducing terminal of a sugar chain linked to N-glucoside being attached to said molecule.
26. Immunofunctional molecule according to clause 24, wherein the immunofunctional molecule is an antibody, a protein or a peptide.
27. Immunofunctional molecule according to clause 25, wherein the immunofunctional molecule is an antibody, a protein or a peptide.
28. Process for producing the immunofunctional molecule according to clause 24, which comprises using a cell that has a low enzymatic activity of adding fucose to the N-acetylglucosamine of the reducing terminal or does not present said enzymatic activity.
ES 2 568 898 T3
29. Procedure according to clause 28, in which the enzyme that adds fucose to the reducing terminal M-acetylglucosamine is a fucosyltransferase.
30. Procedure according to clause 29, wherein the fucosyltransferase is α1,6-fucosyltransferase.
31. A method for producing the immunofunctional molecule according to clause 24, wherein a rat myeloma cell is used in the method for producing an immunofunctional molecule exhibiting inhibited immunofunctional activity.
32. Procedure according to clause 31, where the rat myeloma cell is the YB2 / 3HL.P2.G11.16Ag.20 cell.
33. Process for producing the immunofunctional molecule according to clause 25, in which a cell that exhibits high enzymatic activity is used by adding fucose to the reducing terminal M-acetylglucosamine.
3. 4. Procedure according to clause 33, in which the enzyme that adds fucose to the reducing terminal M-acetylglucosamine is a fucosyltransferase.
35. Procedure according to clause 34, wherein the fucosyltransferase is α1,6-fucosyltransferase.
36. Immunofunctional molecule according to clause 26, in which the antibody recognizes a tumor-related antigen.
37. Immunofunctional molecule according to clause 36, in which the tumor-related antigen is ganglioside GD3.
38. Immunofunctional molecule according to clause 36, in which the antibody is produced by 7-9-51 (FERM BP6691).
39. Immunofunctional molecule according to clause 26, in which the antibody recognizes an antigen related to an allergy or inflammation.
40. Immunofunctional molecule according to clause 39, wherein the allergy or inflammation related antigen is a human interleukin 5 receptor a chain.
41. Immunofunctional molecule according to clause 39, in which the antibody is produced by n ° 3 (FERM BP-6690).
42. Immunofunctional molecule according to clause 26, in which the antibody recognizes an antigen related to a cardiovascular disease.
43. Immunofunctional molecule according to clause 27, in which the antibody recognizes an antigen related to an autoimmune disease.
44. Immunofunctional molecule according to clause 26, in which the antibody recognizes an antigen related to a bacterial or viral infection.
Four. Five. Agent for diagnosing cancer, comprising the immunofunctional molecule according to clause 36 as an active principle.
46. Agent for treating cancer, comprising the immunofunctional molecule according to clause 36 as an active principle.
47. Agent for preventing cancer, comprising the immunofunctional molecule according to clause 36 as an active principle.
48. Agent for diagnosing an allergy or inflammation, comprising the antibody according to clause 36 as an active principle.
49. Agent for treating an allergy or inflammation, comprising the antibody according to clause 39 as an active principle.
fifty. Agent for preventing an allergy or inflammation, comprising the antibody according to clause 39 as an active principle.
Contents16
102 members in 13 offices
Priority claims2
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| EP2275541B1 | European Patent Office (EPO) | B1 | |
| EP2278003B1 | European Patent Office (EPO) | B1 | |
| EP2270150B1 | European Patent Office (EPO) | B1 | |
| ES2568898T3This record | Spain | T3 | |
| ES2568899T3 | Spain | T3 | |
| DK2270149T3 | Denmark | T3 | |
| DK2275540T3 | Denmark | T3 | |
| DK2275541T3 | Denmark | T3 | |
| ES2569919T3 | Spain | T3 | |
| DK2278003T3 | Denmark | T3 | |
| ES2571230T3 | Spain | T3 | |
| ES2572623T3 | Spain | T3 | |
| EP3031917A1 | European Patent Office (EPO) | A1 | |
| DK2270150T3 | Denmark | T3 | |
| ES2574826T3 | Spain | T3 | |
| CY1114164T1 | Cyprus | T1 | |
| CY1114197T1 | Cyprus | T1 | |
| EP2270147B1 | European Patent Office (EPO) | B1 | |
| CA2704600C | Canada | C | |
| DK2270147T3 | Denmark | T3 | |
| ES2601882T3 | Spain | T3 | |
| CY1117551T1 | Cyprus | T1 | |
| US2017240647A1 | United States of America | A1 | |
| LUC00074I2 | Luxembourg | I2 | |
| BE2018C020I2 | Belgium | I2 | |
| US10233247B2 | United States of America | B2 | |
| EP2270150B2 | European Patent Office (EPO) | B2 | |
| DK2270150T4 | Denmark | T4 | |
| US2020017593A1 | United States of America | A1 | |
| ES2574826T5 | Spain | T5 | |
| EP2270147B2 | European Patent Office (EPO) | B2 | |
| EP2278003B2 | European Patent Office (EPO) | B2 | |
| DK2270147T4 | Denmark | T4 | |
| DK2278003T4 | Denmark | T4 | |
| NL300939I2 | Netherlands (Kingdom of the) | I2 |
Numbers
- Publication
- 2568898
- Application
- 10180045
Titles2
- Spanish
- Procedimiento para controlar la actividad de una molécula inmunofuncional
- English
- Procedure to control the activity of an immunofunctional molecule
Classification
- CPC, 21
- C07K16/2866
- A61K2039/505
- C07K16/30
- C07K2317/24
- C07K2317/41
- C07K2317/73
- C07K2317/732
- C07K2319/00
- G01N33/53
- C07K2317/52
- C07K16/3084
- A61P29/00
- A61P31/00
- A61P35/00
- A61P37/00
- A61P37/06
- A61P37/08
- A61P9/00
- C07K2317/21
- C07K2317/565
- C12P21/005
- IPC, 9
- C12N15 09
- C12P21 00
- C12P21 08
- A61K39 00
- G01N33 53
- A61K39 395
- C07K16 28
- C07K16 30
- C07K16 46