WO2001023573A1 - Human type complementation-determining domain transplanted antibody against ganglioside gd2 and derivative of this antibody - Google Patents

Abstract

A monoclonal antibody against ganglioside GD2 having a novel complementation-determining domain to ganglioside GD2; a fused protein of this antibody with a cytokine; and therapeutic and diagnostic uses of the above-described antibody and fused protein.

Description

 Description Antibody transplanted with human-type complementarity determining region against ganglioside GD2 and derivatives of said antibody

 The present invention relates to a human complementarity-determining region-grafted antibody against ganglioside GD2 (hereinafter referred to as GD2), CDR1, CDR2, and CDR3 of the heavy chain variable region are represented by SEQ ID NOS: 3, 4, and 5, respectively, and CDR1 of the light chain variable region. The present invention also relates to a monoclonal antibody against ganglioside GD2, wherein CDR2 and CDR3 each contain an amino acid sequence represented by SEQ ID NOs: 6, 7, and 8, and derivatives of the antibody and the antibody fragment. The present invention further relates to DNA sequences encoding the above antibodies and derivatives. The present invention relates to a vector comprising the DNA sequence and a cell transformed with the vector. The present invention further relates to a method for producing the above-described antibody and derivative using the transformed cell, and a therapeutic and diagnostic agent for cancer using the antibody and the derivative. Background art

 When an antibody from a non-human animal, such as a mouse antibody, is administered to a human, it is recognized as a foreign substance, and a human antibody against the mouse antibody (Human Anti Mouse Antibody: hereinafter, referred to as HAMA) is induced in the human body. It is known. HAMA reacts with the administered mouse antibody and causes side effects [J. Clin. Oncol., 2, 881 (1984), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80, 932. Natl. Acad. Sci. USA, 82, 1242 (1985)], accelerates the disappearance of administered mouse antibodies from the body [J. Nucl. Med., 26, 1011 (1985), Blood , 65, 1349 (1985), J. Natl. Cancer Inst., 80, 937 (1988)], and is known to reduce the therapeutic effect of mouse antibodies [J. Immunol., 135, 1530 (1988). 1985), Cancer Res., 46, 6489 (1986)].

In order to solve these problems, using a gene recombination technique, an antibody from a non-human animal was transplanted into a human chimeric antibody or a human complementarity determining region (Complementarity Determining Region: hereinafter referred to as CDR). For humanized antibodies such as Has been attempted. A human chimeric antibody is an antibody in which the antibody variable region (hereinafter, referred to as V region) is a non-human animal antibody and the constant region (hereinafter, referred to as C region) is a human antibody [Pnx Natl. Acad. Sci. USA, 81, 6851 (1984)], and a human CDR-grafted antibody refers to the amino acid sequence of the CDR in the V region of an antibody of a non-human animal. It is an antibody that has been implanted at the site [Nature, 522 (1986)]. These humanized antibodies have various advantages in human clinical application compared to non-human animal antibodies such as mouse antibodies. For example, with regard to immunogenicity and stability in blood, it has been reported that human chimeric antibodies have a half-life increased by about 6 times in blood when administered to humans compared to mouse antibodies. Natl. Acad. Sci. USA, 86, 4220 (1989)]. It has been reported that in human monkey CDR-grafted antibodies, immunogenicity was reduced and half-life in blood was extended 4 to 5 times compared to mouse antibodies in experiments using monkeys (J.

 Immunol., 147, 1352 (1991)]. That is, humanized antibodies are expected to have fewer side effects and to maintain their therapeutic effects for a long period of time as compared to antibodies of non-human animals. In particular, when considering application as an antitumor antibody, complement-dependent cytotoxicity (hereinafter referred to as CDC activity) via the Fc region of the antibody (the region following the hinge region of the antibody heavy chain) and The degree of cytotoxic activity such as antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC activity) is important for its therapeutic effect. The Fc region of a human antibody more efficiently activates human complement components and human Efecta cells having Fc receptors such as mononuclear cells, macrophages, and NK cells on the cell surface than the Fc region of an antibody It has been reported that it is better because it can For example, a human chimeric antibody obtained by converting the Fc region of a mouse antibody to GD2 (hereinafter, a mouse antibody to GD2 is referred to as an anti-GD2 mouse antibody) to the Fc region of a human antibody (hereinafter, referred to as an anti-GD2 chimeric antibody) Has been reported to increase tumor cell damage activity by human effector cells [J. Immunol., 144, 1382 (1990)]. The same applies to human CDR-grafted antibodies against CAMPATH-1 antigen. Results have been reported [Nature, 332, 323 (1988)].

 The above results clearly show that humanized antibodies are more preferable than non-human animal antibodies such as mouse antibodies for use in human clinical applications.

Furthermore, with recent advances in protein engineering and genetic engineering, Fab, Fab ', F (ab') ₂ , single-chain antibodies [Science, 242, 423 (1988)], disulfide-stabilized V region fragments (hereinafter dsFv) [Molecular Immunol., 32, 249 (1995)]. These fragments have a smaller molecular weight than the complete antibody molecule, and therefore have excellent transferability to target tissues [Cancer Res., 52, 3402 (1992)]. Also in the case of clinical application to humans, it is considered that these fragments are more preferably derived from humanized antibodies than non-human animal antibodies such as mouse antibodies.

 Ganglioside, a kind of glycolipid containing sialic acid, constitutes the cell membrane of animals and is a molecule composed of sugar chains, which are hydrophilic side chains, and sphingosine, which is a hydrophobic side chain, and fatty acids. is there. It is known that the type and expression level of gangliosides vary depending on the cell type, organ type, animal type and the like. It is also known that ganglioside expression changes quantitatively and qualitatively in the process of canceration of cells [Cancer Res., 45, 2405 (1985)]. GD2 is present in very small amounts in normal cells, but is present in large amounts in cancer cells such as small cell lung cancer, malignant melanoma, and neuroblastoma.An antibody against GD2 (hereinafter referred to as anti-GD2 antibody) ) Is considered to be useful in the treatment of these cancers [Proc. Natl. Acad. Sci. USA, 79, 7629 (1982), Cancer Res., 44, 5914 (1984), Cancer Res. 45, 2642 (1985), Cancer Res., 47, 1098 (1987)]. To date, two types of anti-GD2 chimeric antibodies have been produced [J. Immunol., 144, 1382 (1990), Biotechnology, 10, 1121 (1992)], and their therapeutic effects have been confirmed in human clinical trials. [Eur. J. Cancer, 31A, 261 (1995)]. However, on the other hand, it has also been shown that a human antibody against the V region derived from the mouse antibody in the administered anti-GD2 chimeric antibody is induced and an allergic reaction is induced [Cancer J. From Scientific American , 3, S121 (1997)]. In order to solve this problem, production of a human antibody or humanized antibody against GD2, which is considered to be less immunogenic in humans, is desired. For antibody fragments against GD2, single-chain antibodies derived from mouse antibodies have been produced [Hybridoma, 16, 335 (1997)], but reports on the production of antibody fragments derived from human or humanized antibodies have been reported. There is no. Therefore, if an antibody fragment derived from a humanized antibody against GD2 (hereinafter, referred to as an anti-GD2 humanized antibody) can be prepared, it has excellent target tissue transportability in humans and immunogenicity is reduced. It is expected.

As described above, the humanized antibody and its fragment can be used alone. Is expected to have the effect of diagnosis and treatment, but it is being studied to further enhance the effect by using it in combination with other molecules. For example, cytokines are used as one of these molecules. Cytokine is a general term for various humoral factors that control cell-cell interactions in the immune response. CDC activity and ADCC activity are known as the cytotoxic activity of antibodies, but ADCC activity is carried by effector cells having Fc receptors of monocytes, macrophages, and NK cells on the cell surface [J. Immunol., 138, 1992 (1987)]. Since various cytokines activate these effector cells, they have been administered in combination with antibodies for the purpose of enhancing ADCC activity and the like of the antibodies. For example, with respect to the anti-GD2 mouse antibody 14.G2a and the anti-GD2 chimeric antibody chl4.18, the site of human interleukin 2 (hereinafter referred to as hIL-2) or human granulocyte-macrophage colony Administration to humans in combination with a stimulating factor (hereinafter referred to as hGM-CSF) has been performed [Cancer, 80, 317 (1997), Cancer J. From Scientific American, 3, S121 (1997)]. Anti-ganglioside GD3 mouse antibody R24 was also used in combination therapy with various cytokine molecules [Cancer Res., 50, 7490 (1990), Proc. Am. Soc. Clin. Oncol., 1186, 345 (1992) , J. Biol. Response Mod., 9, 319 (1990), Proc. Am. Soc. Clin. Oncol., 1182, 344 (1992), Proc. Am. Soc. Clin. Oncol., 1188, 346 (1992). )]. However, these combination therapies have not been as effective as expected due to the antigenicity of mouse antibodies or side effects of cytokines. Therefore, derivatives are produced by chemically or genetically fusing radioisotopes, proteins (cytokines, toxins, enzymes, etc.), low-molecular-weight drugs, etc. to antibodies and their fragments. Has been studied [New Eng. J. Med., 329, 459 (1993), Anticancer Res., 17, 1735 (1997), Blood, 78, 1173 (1991), J. Clin. Oncol. , 15, 723 (1997), Biocon jugate Chem., 7, 606 (1997), Cancer, 61, 881 (1988), Jpn. J. Cancer Res., 85, 167 (1994), Antibody Immunoconjugates and Radiopharmaceuticals, 3 , 60 (1990), Surgery, 106, 533 (1989)]. These derivatives are more effective and have fewer side effects by accumulating radioisotopes, proteins (cytokines, toxins, enzymes, etc.), low-molecular-weight drugs, etc. around the target tissue according to the binding specificity of the antibody. It is expected to enable diagnosis and treatment. For example, among the cytokines used in the combination therapy described above, ML-2, which showed some antitumor effect, was fused with the anti-GD2 chimeric antibody chl4.18. It has been reported that the synthetic protein was engineered and its antitumor effect was superior to that of co-administration of the anti-GD2 chimeric antibodies chl4.18 and hIL-2 in experiments using mice [Proc. Natl Acad. Sci. USA, 89, 1428 (1992), Proc. Natl. Acad. Sci. USA, 91, 9626 (1994), Cancer Immunol. I maraud nother., 42, 88 (1996), Blood, 91, 1706 (1998)]. Therefore, if a humanized anti-GD2 antibody and its fragment can be obtained by fusing radioisotopes, proteins (toxins, enzymes, etc.), low-molecular-weight drugs, etc., including fusion proteins with various cytokines, human When administered to the body, immunogenicity is reduced, side effects are reduced, and a stronger antitumor effect at the tumor site is expected. Disclosure of the invention

 The present inventors have obtained antibody H chain cDNA and L chain cDNA from a hybridoma KM666 (FERMBP-6786) that produces a mouse monoclonal antibody against GD2 belonging to the IgG3 class, and CDRs of their V regions have novel amino acids. It was found to have an acid sequence. The cDNA encoding the H chain V region and the L chain V region having the novel CDR is cloned into an animal cell expression vector having the cDNA encoding the human antibody H chain C region and the human antibody L chain C region, and humanized. An antibody expression vector was constructed. The expression vector was introduced into animal cells to express and purify anti-GD2 chimeric antibody KM1138 and anti-GD2 human CDR-grafted antibody (hereinafter referred to as anti-GD2CDR. Transplanted antibody) KM8138. KM1138 and KM8138 were found to react specifically with GD2 and show strong cytotoxic activity against antigen-positive human cancer cell lines. Furthermore, a cDNA encoding hIL-2 is ligated to the 3 'end of the cDNA encoding the H chain of the anti-GD2 CDR-grafted antibody KM8138 to construct a cDNA, and the cDNA encoding the L chain of KM8138 Was cloned into an expression vector for animal cells to construct an expression vector for a fusion protein of anti-GD2CDR-grafted antibody KM8138 and hIL-2 (hereinafter referred to as KM8138-hIL-2). The KM8138-hIL-2 expression vector was introduced into animal cells to express and purify KM8138-hIL-2. The present inventors have found that KM8138-hIL-2 specifically reacts with GD2 and shows a biological activity equivalent to that of hIL-2, thereby completing the present invention.

 The present invention relates to the following (1) to (51).

(1) A human-type complementarity determining region (CDR) -grafted antibody or an antibody fragment thereof that specifically reacts with ganglioside GD2. (2) The human type according to (1) above, wherein the human CDR-grafted antibody comprises CDRs of a heavy chain (H chain) variable region (V region) and a light chain (L chain) V region of a monoclonal antibody against ganglioside GD2. CDR-grafted antibody or said antibody fragment.

 (3) Human CDR-grafted antibody contains CDR of H chain V region and L chain V region of monoclonal antibody against ganglioside GD2 and framework region (FR) of H chain V region and L chain V region of human antibody The human CDR-grafted antibody or the antibody fragment according to the above (1).

 (4) The human CDR-grafted antibody is composed of the CDRs of the H chain V region and L chain V region of the monoclonal antibody against ganglioside GD2, the FRs of the H chain V region and L chain V region of the human antibody, and the H antibody of the human antibody. The antibody or the antibody fragment thereof according to the above (1), which is a human CDR ^ -planted antibody comprising a chain constant region (C region) and an L chain C region.

 (5) The CDR according to any one of (1) to (4), wherein CDR1, CDR2, and CDR3 of the V region of the H chain of the antibody include the amino acid sequences represented by SEQ ID NOS: 3, 4, and 5, respectively. Or the human CDR-grafted antibody or the antibody fragment thereof.

 (6) The human according to any one of (1) to (4), wherein CDR1, CDR2, and CDR3 of the V region of the L chain of the antibody include the amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively. Type CDR-grafted antibody or said antibody fragment.

 (7) CDR1, CDR2, and CDR3 of the H chain V region of the antibody are SEQ ID NOs: 3, 4, and 5, respectively, and CDR1, CDR2, and CDR3 of the L chain V region are the amino acids represented by SEQ ID NOs: 6, 7, and 8, respectively. The human CDR-grafted antibody or the antibody fragment thereof according to any one of the above (1) to (4), comprising an acid sequence.

 (8) The human CDR-grafted antibody or the antibody fragment thereof according to any one of (1) to (4) above, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 13.

 (9) The human CDR-grafted antibody or the antibody fragment thereof according to any one of the above (1) to (4), wherein the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 14.

 (10) The human CDR graft according to any one of the above (1) to (4), wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 13, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 14. An antibody or an antibody fragment thereof.

(11) The human CDR according to any one of (1) to (4) above, wherein the H chain V region of the antibody comprises an amino acid sequence represented by SEQ ID NO: 13, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 14. Transplant Antibody KM8138 or an antibody fragment thereof.

(12) The antibody fragment is an antibody fragment selected from Fab, Fab \ F (ab,) ₂ , a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv) and a peptide containing CDR. The antibody fragment according to any one of the above (1) to (11).

 (13) A DNA encoding the human CDR-grafted antibody according to any one of the above (1) to (12).

 (14) A recombinant vector containing the DNA according to (13).

 (15) A transformant obtained by introducing the recombinant vector according to (14) into a host cell

 (16) A transformant KM8138 (FER BP-6788) that produces the antibody of (12) above

(17) The transformant described in (15) or (16) above is cultured in a medium, and the human CDR-grafted antibody or antibody fragment described in (1) to (12) is produced and accumulated in the culture. And recovering the antibody from the culture.

 (18) CDR1, CDR2, and CDR3 of the H chain V region of the monoclonal antibody are SEQ ID NOs: 3, 4, and 5, respectively, and CDR1, CDR2, and CDR3 of the L chain V region are the amino acids represented by SEQ ID NOs: 6, 7, and 8, respectively. A monoclonal antibody against ganglioside GD2 or a fragment thereof comprising the sequence.

 (19) The antibody according to the above (18), wherein the monoclonal antibody is selected from an antibody produced by a hybridoma, a humanized antibody and a human antibody.

 (20) The antibody described in (19) above, wherein the monoclonal antibody class belongs to the human antibody IgG type.

 (21) The antibody KM666 according to the above (19), which is produced by a mouse hybridoma KM666 (FERMBP-6786).

 (22) The humanized antibody according to (19), wherein the humanized antibody is a human chimeric antibody.

 (23) The human chimeric antibody or the antibody fragment thereof according to the above (22), wherein the human chimeric antibody comprises the H chain V region and the L chain V region of a monoclonal antibody against ganglioside GD2.

(24) A human chimeric antibody is a monoclonal antibody against ganglioside GD2 The human chimeric antibody or the antibody fragment thereof according to the above (22), which is a human chimeric antibody comprising the H chain V region and L chain V region of the above, and the H chain C region and L chain C region of a human antibody.

 (25) The human type according to the above (23) or (24), wherein the amino acid sequences of the H chain V region and the L chain V region include the amino acid sequences of the H chain V region and the L chain V region of the mouse monoclonal antibody KM666. Chimeric antibodies or antibody fragments thereof.

 (26) the antibody H chain V region comprises the amino acid sequence represented by SEQ ID NO: 32,

The human chimeric antibody or the antibody fragment thereof according to (23) or (24).

 (27) the antibody (L) wherein the V region comprising the amino acid sequence represented by SEQ ID NO: 33;

23. The human chimeric antibody or the antibody fragment thereof according to (24).

 (28) The human chimeric antibody according to (23) or (24), wherein the H chain V region comprises the amino acid sequence represented by SEQ ID NO: 32, and the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 33. The antibody fragment.

 (29) The human chimeric antibody KM1138 or the antibody according to the above (23) or (24), wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 32 and the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 33 fragment.

 (30) The human chimeric antibody according to any one of the above (23) to (29), or a DNA encoding the antibody.

 (31) A recombinant vector containing the MA described in (30) above.

 (32) A transformant obtained by introducing the recombinant vector according to (31) into a host cell.

 (33) a transformant KM1138 (FERM BP-6787) that produces the antibody of (29) above;

) o

 (34) The transformant according to (32) or (33) is cultured in a medium, and the human chimeric antibody according to (23) to (29) is produced and accumulated in the culture. A method for producing an antibody, comprising collecting the antibody.

(35) The antibody fragment is an antibody fragment selected from Fab, Fab, F (ab,) ₂ , single-chain antibody (scFv), disulfide-stabilized V region fragment (dsFv), and a peptide containing CDR. The antibody fragment according to the above (19).

(36) The antibody fragment has CDR1, CDR2 and CDR3 of the H chain V region of the antibody, respectively. The antibody fragment according to (35) above, wherein the CDR1, CDR2, and CDR3 of the L chain V region have the amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively.

 (37) The antibody fragment according to (35), wherein the antibody fragment comprises an amino acid sequence represented by SEQ ID NO: 32 in the H chain V region of the antibody and SEQ ID NO: 33 in the L chain V region of the antibody.

 (38) The antibody or the antibody fragment according to any one of (1) to (12), (18) to (29), (35), (36) and (37) above, and a radioisotope An antibody derivative conjugated with a protein or a small molecule drug.

 (39) The antibody derivative according to the above (38), wherein the antibody derivative is obtained by binding a radioisotope, a protein or a low-molecular-weight drug to the C-terminal side of the H chain of the antibody.

 (40) The derivative of the antibody according to (38) or (39), wherein the protein is a cytokine.

 (41) The derivative of the antibody according to (40) above, wherein the cytokine is human inulin leukin 2 (hIL-2).

 (42) The antibody derivative according to any of (38) to (41), wherein the antibody derivative comprises a human CDR-grafted antibody KM8138 and ML-2.

 (43) The antibody derivative KM8138-hIL-2 according to the above (38) to (41), wherein the antibody derivative comprises the human CDR-grafted antibody KM8138 and hIL-2.

 (44) A DNA encoding the derivative of the antibody according to any one of (38) to (43).

 (45) A recombinant vector containing the DNA according to (44).

 (46) A transformant obtained by introducing the recombinant vector according to (45) into a host cell.

 (47) A transformant producing the derivative according to (43) KM8138hIL2 (FERM BP-6789) o

 (48) The transformant described in (46) or (47) above is cultured in a medium, and the derivative of the antibody described in (38) to (43) is produced and accumulated in the culture. A method for producing a derivative of an antibody, which comprises collecting the derivative.

(49) The antibody and the antibody fragment according to (1) to (12), (18) to (29), (35), (36) and (37), and the above (38) to (43) A medicament comprising at least one selected from the derivatives of the antibodies described therein. (50) The antibody and the antibody fragment described in (1) to (12), (18) to (29), (35), (36) and (37), and the above (38) to (43) For the treatment of cancers containing as an active ingredient at least one selected from derivatives of the listed antibodies

(5 1) The antibody and the antibody fragment according to (1) to (12), (18) to (29), (35), (36) and (37), and the above (38) to (43) A diagnostic drug for cancer, comprising as an active ingredient at least one selected from the derivatives of the antibodies described above.

 Examples of the monoclonal antibody of the present invention include antibodies produced by hybridomas, humanized antibodies, human antibodies, and antibody fragments thereof.

 A hybridoma is a monoclonal antibody having a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing a mammal other than human with an antigen and myeloma cells derived from a mouse or the like. Means a producing cell.

 Examples of the humanized antibody include a human chimeric antibody and a human CDR-grafted antibody. The human chimeric antibody is a non-human animal heavy chain variable region (hereinafter, the heavy chain is a heavy chain and the variable region Is the HV or VH as the V region, the variable region of the antibody light chain (hereinafter the light chain is described as LV or VL as the L chain) and the heavy chain constant region of the human antibody (the constant region is the C region). CH) and the light chain constant region of a human antibody (hereinafter, referred to as CL). As animals other than humans, any animal can be used as long as hybridomas can be produced, such as mice, rats, hamsters, and rabbits.

 The human quinula antibody of the present invention obtains cDNAs encoding VH and VL from a hybridoma producing a monoclonal antibody that specifically reacts with GD2, and encodes human antibody CH and human antibody CL. A human-type chimeric antibody expression vector is constructed by inserting each into an expression vector for animal cells having the gene to be expressed, and then introduced into animal cells for expression and production.

As the CH of the humanized chimeric antibody, any CH may be used as long as it belongs to human immunoglobulin (hereinafter, referred to as hlg), but those of the hlgG class are suitable, and hIgGl, hIgG2, hIgG3, Use any of the subclasses such as hIgG4 Can be The CL of the human chimeric antibody may be any CL as long as it belongs to hlg, and a class or human class CL can be used.

 As specific examples of the anti-GD2 quinula antibody, VH of the antibody has the amino acid sequence of SEQ ID NO: 32, CH has the amino acid sequence of the hlgGl subclass, VL of the antibody has the amino acid sequence of SEQ ID NO: 33, and CL has the amino acid sequence of SEQ ID NO: 33. Antibody KM1138 having an amino acid sequence of the human antibody class is exemplified.

 The human CDR-grafted antibody refers to an antibody obtained by grafting the amino acid sequence of the CDRs of VH and VL of a non-human animal antibody to an appropriate position of VH and VL of a human antibody.

 The human CDR-grafted antibody of the present invention comprises a V region obtained by grafting the VH and VL CDR sequences of an antibody of an animal other than the seventeenth animal specifically reacting with GD2 into the VH and VL CDR sequences of any human antibody. A cDNA encoding the human-type CDR and a human-type CDR-grafted antibody expression vector are constructed by inserting cDNAs into the expression vectors for animal cells having genes encoding human antibody CH and human antibody CL. By introducing the expression vector into animal cells, a human CDR-grafted antibody can be expressed and produced.

 The CH of the human CDR-grafted antibody may be any CH as long as it belongs to hlg, but is preferably of the hlgG class, and any subclass such as hIgGl, hIgG2, hIgG3, hIgG4 belonging to the hlgG class may be used. it can. As the CL of the human CDR-grafted antibody, any CL belonging to hlg may be used, and a class or human class CL may be used.

 As specific examples of the anti-GD2 CDR-grafted antibody, VH of the antibody has the amino acid sequence of SEQ ID NO: 13, CL has the amino acid sequence of the human antibody IgGl subclass, and VL of the antibody has the amino acid sequence of SEQ ID NO: 14, CL Is an antibody KM8138 having an amino acid sequence of the human antibody class.

 Originally, human antibodies refer to antibodies naturally occurring in the human body. However, human antibody phage libraries and libraries produced by recent advances in genetic engineering, cell engineering, and developmental engineering have been developed. Antibodies obtained from human antibody-producing transgenic animals are also included.

Antibodies present in the human body can be obtained, for example, by isolating human peripheral blood lymphocytes, infecting EB virus and the like, immortalizing them, and cloning the cells to produce the antibody-producing lymphocytes. Antibodies can be purified. The human antibody phage library 1 is a library in which antibody fragments such as Fab and single-chain antibodies are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. Phage expressing an antibody fragment having a desired antigen-binding activity can be recovered from the library using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted to a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.

 Human antibody-producing transgene: A nick animal refers to an animal in which a human antibody gene has been integrated into cells. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into an early embryo of another mouse, and then developing the embryo. The method for producing human antibodies from human antibody-producing transgenic animals is performed by obtaining and culturing human antibody-producing hybridomas by the usual method for producing hybridomas in mammals other than humans. Human antibodies can be produced and accumulated in the culture.

Examples of the antibody fragment include Fab, Fab \ F (ab ') ₂ , scFv, dsFv, a peptide containing CDR, and the like.

 Fab is a fragment obtained by treating IgG with proteolytic enzyme papain (which is cleaved at the 224th amino acid residue of H chain). It is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is linked by a sulfide bond. The Fab of the present invention can be obtained by treating an antibody specifically reacting with GD2 with proteolytic enzyme papain. Alternatively, a DNA encoding the Fab of the antibody is inserted into one of prokaryotic expression vectors or eukaryotic expression vectors, and the vector is expressed by introducing the prokaryotic cell into a eukaryote. Can be manufactured.

F (ab ') ₂ is a fragment obtained by treating IgG with the protease pepsin (which is cleaved at the 234th amino acid residue in the H chain), and Fab is a disulfide bond in the hinge region. This is an antibody fragment having a molecular weight of about 100,000 and having an antigen-binding activity, which is slightly larger than that bound through the DNA.

The F (ab ') ₂ of the present invention can be obtained by treating an antibody that specifically reacts with GD2 with the protease pepsin. Alternatively, the following Fab ′ can be prepared by making it a chain bond or a disulfide bond. Fab is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity in which the disulfide bond in the hinge region of F (ab ') ₂ is cleaved.

Fab ′ of the present invention can be obtained by treating F (ab ′) ₂ that specifically reacts with GD2 with a reducing agent dithiothreitol. Alternatively, DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or an eukaryotic expression vector, and the vector is introduced into a prokaryotic expression vector eukaryote. Fab 'can be expressed and produced.

 scFv refers to a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter, referred to as P). VH and VU contained in the scFv of the present invention, and any of the antibodies, humanized antibodies, and human antibodies produced by the hybridoma of the present invention can be used.

 The scFv of the present invention obtains cDNAs encoding VH and VL of an antibody that specifically reacts with GD2, constructs a DNA encoding the scFv, and uses the DNA for prokaryotic expression vectors or eukaryotic organisms. The scFv can be produced by inserting it into an expression vector and introducing the expression vector into a prokaryote or eukaryote for expression.

 dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL has been substituted with a cysteine residue, which is linked via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. [Protein Engineering, 7, 697 (1994)]. As the VH and VL contained in the dsFv of the present invention, any of the antibodies, humanized antibodies, and human antibodies produced by the hybridoma of the present invention can be used.

 The dsFv of the present invention obtains cDNA encoding VH and VL of an antibody that specifically reacts with GD2, constructs a DNA encoding dsFv, and uses the DNA for prokaryotic expression vector or DsFv can be produced by inserting it into a eukaryotic expression vector and introducing the expression vector into a prokaryote or eukaryote to express it.

 The peptide containing the CDR comprises at least one region of the H chain or L chain CDR. A plurality of CDRs can be linked directly or via an appropriate peptide linker.

The peptide containing the CDR of the present invention can cope with VH and VL of an antibody that specifically reacts with GD2. After obtaining the cDNA to be encoded, a DNA encoding the CDR is constructed, the DNA is inserted into a prokaryotic expression vector or an eukaryotic expression vector, and the expression vector is converted to a prokaryotic expression vector. It can be expressed by introduction into a product or a eukaryote to produce a peptide containing CDR.

 In addition, peptides containing CDR can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

 The derivative of the antibody of the present invention relates to an antibody produced by the hybridoma of the present invention, a humanized antibody, a human antibody or an antibody fragment thereof, to which a radioisotope, a protein or a low-molecular drug is bound.

 The derivative of the antibody of the present invention may be prepared by reacting an antibody or antibody fragment specifically reacting with GD2 with the H chain or V, the N-terminal side or the C-terminal side of the L chain, an appropriate substituent in the antibody or antibody fragment or Radioisotope, protein, or low-molecular-weight drugs, etc., are applied to the side chains, and also to the sugar chains in the antibody or antibody fragment, using chemical techniques [Introduction to Antibody Engineering (Osamu Kanemitsu, 1994, Jichijinshokan Co., Ltd.)] It can be manufactured by bonding.

 Alternatively, DNA encoding an antibody or antibody fragment that specifically reacts with GD2 and DNA encoding the protein to be bound are ligated and inserted into an expression vector, and the expression vector is introduced into host cells. I do. It can also be produced by the genetic engineering techniques described above.

Radioisotopes, ¹³¹ 1, ¹²⁵ 1, and the like, for example, more chloramine T method or the like, can be attached to the antibody.

Low-molecular-weight drugs include alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexate, daunomycin, bleomycin, mitomycin (, daunorubicin, doxorubicin, etc. Antibiotics such as antibiotics, plant alkaloids such as vincristine, vinblastine, and vindesine, and hormonal drugs such as evening moxifen and dexamethasone [Clinical Oncology (Japanese Society of Clinical Oncology, 1996, Cancer and Chemotherapy)], or Steroids such as hydrocortisone and prednisone, non-steroids such as aspirin and indomethacin, immunological modulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine; Murray Anti-inflammatory agents such as antihistamines such as chlorpheniramine dichloramine and clemacitin [Inflammation and anti-inflammatory therapy 1977, Ichiyokuto Shuppan Co., Ltd.]. For example, as a method for binding daunomycin to an antibody, a method for binding between daunomycin and the amino group of the antibody via glutaraldehyde, and a method for binding the amino group of daunomycin to the carboxyl group of the antibody via water-soluble carbodiimide And the like.

 Suitable proteins are cytokines that activate immunocompetent cells, for example, hIL-2, hGM-CSF, seven tomacrophagic colony-stimulating factor (hereinafter referred to as hM-CSF), One leukin 12 (hereinafter referred to as hlL-12) and the like. Also, toxins such as ricin and diphtheria toxin can be used to directly damage cancer cells. For example, for a fusion antibody with a protein, a cDNA encoding the protein is linked to a cDNA encoding the antibody or antibody fragment, a DNA encoding the fusion antibody is constructed, and the DNA is converted to a prokaryotic or eukaryotic organism. Thus, a fusion antibody can be produced by inserting the expression vector into a prokaryote or eukaryote and expressing the expression vector.

 Examples of the derivative of the antibody of the present invention include a fusion protein of a humanized anti-GD2 antibody and cytokine. Specifically, the fusion protein of the H chain and hlL-2 of the antibody has the amino acid sequence of SEQ ID NO: 31, the V region of the L chain of the antibody is the amino acid sequence of SEQ ID NO: 14, and the C region of the L chain is human. KM8138-hlL-2, which is a fusion protein of anti-GD2 CDR-grafted antibody KM8138 and hlL-2, which has an amino acid sequence of the human antibody class.

 The human CDR-grafted antibody that specifically reacts with GD2, the monoclonal antibody that specifically reacts with GD2 and has a novel amino acid sequence in the V region of the H chain and L chain, and A method for producing a human chimeric antibody and a fusion protein of these anti-GD2 antibodies and cytokines will be described.

 1. Production of anti-GD2 monoclonal antibody produced by Hypri-doma

 (1) Preparation of antigen

 Antigens required for producing anti-GD2 monoclonal antibodies include tissues or cell lines that highly express GD2, or GD2 extracted and purified from such tissues or cell lines. [Anticancer Res., 13, 331 (1993)].

(2) Animal immunization and preparation of antibody-producing cells The animal used for immunization may be any animal, such as a mouse, a rat, a hamster, or a rabbit, as long as a hybridoma can be produced. The following describes an example using a mouse and a rat.

 A mouse or rat aged 3 to 20 weeks is immunized with the antigen prepared in 1 (1) above, and antibody-producing cells are collected from the spleen, lymph nodes, and peripheral blood of the animal. Immunization is performed by subcutaneously, intravenously or intraperitoneally administering the antigen several times with an appropriate adjuvant to the animal. Adjuvants include Complete Freund's Adjuvant, or aluminum hydroxide gel and B. pertussis vaccine. On days 3 to 7 after each administration, blood is collected from the venous plexus or tail vein of the immunized animal, and the reactivity to GD2 used as an antigen is confirmed by enzyme immunoassay or the like. ELISA method): published by Medical Shoin (1976)], using mice or rats whose serum shows a sufficient antibody titer as the source of antibody-producing cells. Three to seven days after the final administration of the antigenic substance, a method known from the immunized mouse or rat [Antibodies: Laboratory Manual, Cold Springing Laboratory Laboratory] — (Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, 1988), the spleen is excised and fused with spleen cells and myeloma cells according to Antibody's: “Laboratory I” manual. Let it.

 (3) Preparation of myeloma cells

Myeloma cells include 8-azaguanine-resistant mice (derived from BALB / c) myeloma cell line P3-X63Ag8-Ul (P3-Ul), a cell line obtained from mice [Euro. J. Immunol. , 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol., 123, 1548 (1979) ], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)], and any other myeloma cells that can grow in vitro. Culture and passage of these cell lines should be performed according to a known method (Antibodies: Laboratory Manual) to secure a cell number of 2 × 10 ⁷ or more by the time of cell fusion.

 (4) Cell fusion

After washing the antibody-producing cells and myeloma cells obtained above, a cell-aggregating medium such as polyethylene glycol 1000 (PEG-1000) is added, and the cells are fused and suspended in a medium. For washing cells, use MEM medium or PBS (Ninator phosphate). 1.83 g of water, 0.21 g of phosphoric acid monophosphate A, 7.65 g of salt, 1 liter of distilled water, pH 7.2). As a medium for suspending the fused cells, a HAT medium (normal medium [glumin (1.5 mM) in RPMI-1640 medium, 2-mercaptoethanol) so that only the desired fused cells can be selectively obtained. (5 x l0-), Jen evening puromycin (10〃G / ml) and fetal calf serum (FCS) (CSL Ltd., 10) was added medium] hypoxanthine (10- ⁴ M), thymidine (1.5 xl (T ⁵ M) and after culturing using a culture medium} plus aminopterin (4 x l0- ⁷ M), a portion of the culture supernatant by enzyme immunoassay, in response to an antigen protein, non-antigen Select a sample that does not react with the protein, then perform cloning by the selective dilution method, and select a monoclonal antibody-producing hybridoma strain that has a stable and high antibody titer by enzyme immunoassay.

 (5) Selection of anti-GD2 monoclonal antibody produced by Hypri-doma

 The selection of a hybridoma producing an anti-GD2 monoclonal antibody is carried out according to the method described in the Antibody's: a laboratory 'manual and by the measurement method described below. By these methods, the binding activity of the anti-GD2 humanized antibody described below, the anti-GD2 antibody contained in the culture supernatant of the transformant producing the antibody fragment, or all the purified anti-GD2 antibodies can be measured.

Enzyme immunoassay

 The antigen or cells expressing the antigen are coated on a 96-well plate, and the primary antibody is reacted with the hybridoma culture supernatant or the purified antibody obtained by the above method.

 After the first antibody reaction, the plate is washed and the second antibody is added.

 The second antibody is an antibody obtained by labeling an antibody capable of recognizing the immunoglobulin of the first antibody with biotin, an enzyme, a chemiluminescent substance, a radiation compound, or the like. Specifically, if a mouse was used for preparing the hybridoma, an antibody capable of recognizing mouse simnoglobulin would be used as the second antibody.

 After the reaction, a reaction according to the substance labeled with the second antibody is performed, and the antibody is selected as a hybridoma that produces a monoclonal antibody that specifically reacts with the antigen.

A specific example of the hybridoma strain is the hybridoma strain KM666 (FERM BP-6786). (6) Purification of monoclonal antibody

Pristane treatment [intraperitoneal administration of 0.5 ml of 2,6,10,14-tetramethylpentene decane (Pristane) and breeding for 2 weeks] 8 to; 10-week-old mice or nude mice received 1 (4 ) obtained in the anti-GD2 monoclonal antibody producing High Priestess dormer cells 2 x: injected into l0 ⁷ ~5 x 10 ⁶ cells / mouse intraperitoneally. Hypridoma develops ascites cancer in 10 to 21 days. Ascites is collected from the mouse or nude mouse, centrifuged, salted out with 40-50% saturated ammonium sulfate, force prillic acid precipitation, DEAE-Sepharose column, Protein A-column or Cellulofine GSL2000 (raw) The IgG or IgM fraction is collected using a column (manufactured by Chemical Industry Co., Ltd.) and used as a purified monoclonal antibody. The subclass of the purified monoclonal antibody can be determined using a mouse monoclonal antibody evening kit, a rat monoclonal antibody typing kit, or the like. The protein content can be calculated by the Lowry method or the absorbance at 280 nm.

 The antibody subclass refers to an isotype within the class, and includes IgGl, IgG2a, IgG2b, and IgG3 in mice and IgGK IgG2, IgG3, and IgG4 in humans.

 Mouse IgG2a, IgG2b, IgG3 and human IgGl, IgG3 types have relatively strong complement-dependent cytotoxicity (hereinafter referred to as CDC activity) and antibody-dependent cytotoxicity (hereinafter referred to as ADCC activity). It is useful in the application of.

 2. Preparation of humanized antibody

 (1) Construction of humanized antibody expression vector

 A humanized antibody expression vector is an expression vector for animal cells into which genes encoding human antibody CH and CL have been incorporated. Can be constructed by cloning each gene to be cloned.

 The C region of the human antibody can be CH and CL of any human antibody. For example, the C region of the IgG1 subclass of the H chain of the human antibody (hereinafter referred to as hCα1) and the L chain of the human antibody Class C region (hereinafter referred to as hC). As a gene encoding human antibody CH and CL, chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used.

As an expression vector for animal cells, a gene encoding the C region of a human antibody is incorporated. Any substance can be used as long as it can be expressed only. For example, 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)], pSGlld2-4 [Cytotechnology, A, 173 (1990)] and the like. Promoters and enhancers used for expression vectors for animal cells include the early promoter and enhancer of SV40 [J. Biochem., 101, 1307 (1987)], and the LTR promoter of Moroni murine leukemia virus. Ito and Enhancer [Biochem. Biophys. Res. Co. un., 149, 960 (1987)], Immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and Enhancer [Cell, 33, 717 (1983)] ] And the like.

 The humanized antibody expression vector can be used in either the type in which the antibody H chain and the L chain are present on separate vectors, or the type in which the antibody is present on the same vector (hereinafter referred to as tandem type). However, the ease of construction of a humanized antibody expression vector, the ease of introduction into animal cells, and the balance of antibody H chain and L chain expression levels in animal cells are balanced. Therefore, a vector for expressing a tandem humanized antibody is preferred [J. Immunol. Methods, 167, 271 (1994)]. Vectors for expressing tandem humanized antibodies include PKANTEX93 (W097 / 10354), pEE18 [HYBRID0MA, 17, 559 (1998)] and the like.

 The constructed humanized antibody expression vector can be used for expression of a human chimeric antibody and a human CDR-grafted antibody in animal cells.

 (2) Acquisition of cDNA encoding V region of antibody derived from non-human animal and analysis of amino acid sequence

 CMA encoding VH and VL of a non-human animal antibody, for example, a mouse antibody, is obtained as follows.

Extracts mRNA from hybridoma cells producing mouse antibodies and synthesizes cMA. The synthesized cDNA is cloned into a vector such as phage or plasmid to prepare a cDNA library. Using the C region or V region of the mouse antibody as a probe from the library, a recombinant phage or recombinant plasmid having a cDNA encoding VH and a cDNA encoding VL The recombinant phage or the recombinant plasmid having the following are respectively isolated. Determine the entire nucleotide sequence of VH and VL of the target mouse antibody on the recombinant phage or recombinant plasmid. And deduce the entire amino acid sequence of VL.

 As animals other than humans, any mouse, rat, hamster, rabbit and the like can be used as long as hybridoma cells can be produced.

 Methods for preparing total RNA from hybridoma cells include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], and methods for preparing mRNA from total RNA using oligo ( dT) Immobilized cellulose column method [Molecular Cloning: A Laboratory Manual], Cold Spring Harbor Lab. Press New York, 1989, below. Molecular Cloning: A Laboratory Manual. Notation] and so on. Examples of kits for preparing mRNA from hybridoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen) and Quick Prep mRNA Purification Kit (manufactured by Pharmacia).

 For the method of cDNA synthesis and cDNA library production, use the usual method [Molecular 'cloning: a · laboratory · manual; current' protocols 'in' molecule · · ·, etc. (Current Protocols in Molecular Biology ),

Supplement 34, hereinafter referred to as the current 'protocols' in 'molecular' biology, or commercially available kits such as the Super Script ™ Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) or ZAP- Examples include a method using a cDNA Synthesis Kit (manufactured by Stratagene).

 In preparing a cDNA library, any vector can be used as a vector for incorporating cDNA synthesized by converting mRNA extracted from hybridoma cells into type II as long as it can incorporate the cDNA. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], et zap II (Stratagene), et gtlO, Agtll [DNA Cloning : A Practical Approach, I, 49 (1985)], Lambda BlueMid (Clontech), AExCel K pT7T3 18U (Pharmacia), pcD2 [Mol. Cell. Biol., 280 U983)] and pUC18 [Gene , 103 (1985)].

Escherichia coli to which a cDNA library constructed from a phage or plasmid vector can be introduced should be able to introduce, express, and maintain the cDNA library. Any material can be used. For example, XLl-Blue MRF '[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science ₅ 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1, 1983], concealed [J. Mol. Biol., 16, 118 (1966)] and JM105 [Gene, 38, 275 (1985)].

 As a method for selecting cDNA clones encoding VH and VL of non-human animal antibodies from cDNA libraries, colony hybridization or plaque hybridization (PBS) using an isotope or a fluorescently labeled probe can be used. Molecular Cloning: Can be selected by the lab. In addition, primers are prepared, and cDNA or cDNA library synthesized from mRNA is used as type III for polymerase chain reaction (hereinafter referred to as PCR method; molecular 'cloning: a' laboratory 1 'manual; current' CDNA encoding VH and VL can also be prepared by Luzin 'Molecular' biology.

 The cDNA selected by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and a commonly used nucleotide sequence analysis method, for example, Sanger, F.) et al. (Proc, Natl. Acad. Sci., USA, 74, 5463 (1977)) and the like, and a base sequence automatic analyzer, for example, ALF DNA Sequencer (Pharmacia) The nucleotide sequence of the cDNA can be determined by analysis using such methods.

From the determined nucleotide sequence, the entire amino acid sequence of VH and VL was deduced, and the entire amino acid sequence of VH and VL of a known antibody [Sequences 'ob', 'proteins', 'obn' of Proteins of Immunological Interest), US Dept. Health and Human Services, 1991, hereinafter referred to as “sequences” of “proteins”, “of” immunological, and “in-rest”. It can be confirmed that the cDNA encodes the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence. For the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence, refer to the entire amino acid sequence of VH and VL of the known antibody (sequences of proteins, proteins and proteins). By comparing with (rest), the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and the subgroup to which they belong can be known. The amino acid sequence of each CDR of VH and VL The sequence can also be found by comparing the amino acid sequence of the VH and VL of the known antibody (Sequences of Proteins' ob 'Immunological').

 Furthermore, the BLAST method [J. Mol. Biol., 215, 403, 1990] for any database using the complete amino acid sequence of VH and VL, for example, SWISS-PROT, PIR-Protein, etc.

] And the like, and the novelty of the sequence can be examined.

 (3) Construction of human-type chimeric antibody expression vector

 A cDNA encoding VH and VL of a non-human animal antibody was cloned upstream of the gene encoding the CH and CL of the human antibody in the humanized antibody expression vector described in 2 (1) of this section. Thus, a human chimeric antibody expression vector can be constructed. For example, the cDNA encoding the VH and VL of the antibody of a non-human animal can be obtained by combining the nucleotide sequence of the 3 'end of the VH and VL of the non-human animal with the 5' end of CH and CL of the human antibody. And ligation with a synthetic DNA having a recognition sequence for an appropriate restriction enzyme at both ends, and linking each of them to the human antibody CH of the vector for expressing a humanized antibody described in (1) of this section 2. And CL upstream of the genes encoding CL and CL so that they can be expressed in an appropriate form to construct a human chimeric antibody expression vector.

 (4) Construction of cDNA encoding V region of human CDR-grafted antibody

CDNA encoding VH and VL of the human CDR-grafted antibody can be constructed as follows. First, the amino acid sequences of the VH and VL framework regions (hereinafter referred to as FR) of the human antibody to which the VH and VL CDR amino acid sequences of the desired non-human animal antibody are transplanted are selected. As the amino acid sequence of FRs of VH and VL of a human antibody, any amino acid sequence can be used as long as it is derived from a human antibody. For example, the amino acid sequences of the FRs of VH and VL of human antibodies and the common amino acid sequences of each subgroup of FR of VH and VL of human antibodies (Sequences' Among them, in order to produce a human CDR-grafted antibody having sufficient activity, the antibody of the target non-human animal can be used. It is desirable to select an amino acid sequence having as high a homology as possible (at least 60% or more) to the amino acid sequence of FR of VH and VL. Next, the amino acid sequences of the VH and VL CDRs of the antibody of the target non-human animal are transplanted into the VH and VL FR amino acid sequences of the selected human antibody, and the VH and VL of the human CM-grafted antibody are transplanted. Design the amino acid sequence of VL. The designed amino acid sequence is converted into a DNA sequence in consideration of the frequency of codon usage (sequences, protein, protein, immunological, and protein rest) found in the nucleotide sequence of the antibody gene. A DNA sequence encoding the amino acid sequence of VH and VL of the CDR-grafted antibody is designed. Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR is performed using them. In this case, it is preferable to design six synthetic DNAs for both the H chain and the L chain in view of the reaction efficiency in PCR and the length of DNA that can be synthesized.

 In addition, by introducing an appropriate restriction enzyme recognition sequence at the 5 'end of the synthetic DNA located at both ends, it can be easily cloned into the humanized antibody expression plasmid constructed in (1) of this section. You can ping. After the PCR reaction, the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the nucleotide sequence is determined by the method described in (2) of this section 2, and the desired human CDR graft is obtained. A plasmid having a DNA sequence encoding the amino acid sequence of VH and VL of the antibody is obtained.

 (5) Amino acid sequence modification of V region of human CDR-grafted antibody

A human CDR-grafted antibody has the antigen-binding activity of the original non-human animal by only grafting the VH and VL CDRs of the target non-human animal antibody to the human antibody VH and VL FR. It is known to be lower than antibodies [BI0 / TECHN0L0GY, 9, 266 (1991)]. This is because, in the original non-human animal antibodies VH and VL, not only CDRs but also some amino acid residues of FRs are directly or indirectly involved in the antigen binding activity. It is believed that the grafting of CDRs to the FRs of a human antibody changes those amino acid residues to different amino acid residues of the FRs of the human antibody, thereby reducing antigen-binding activity. In order to solve this problem, the human CDR-grafted antibody uses the amino acid residue of CDR and the amino acid residue of CDR in the amino acid sequence of FR of human antibody VH and VL that are directly involved in antigen binding. Identify amino acid residues that interact with amino acid residues and amino acid residues that maintain the antibody's tertiary structure and that indirectly participate in antigen binding, and substitute them for the amino acid residues of the original non-human animal antibody. To reduce the antigen-binding activity [BI0 / TECHN0L0GY, 9, 266 (1991)]. In the production of human CDR-grafted antibodies, the most important point is how to efficiently identify the amino acid residues of FR involved in these antigen-binding activities. For this reason, X-ray crystallography [J · Mol Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)]. Although information on the three-dimensional structure of these antibodies has provided much useful information for the production of human CDR-grafted antibodies, on the other hand, a method for producing human CDR-grafted antibodies that can be applied to all types of antibodies has not yet been established. At present, various trials and errors are necessary, such as preparing several variants for each antibody and examining the correlation with each antigen binding activity. The modification of the FR amino acid residues of VH and VL of a human antibody can be achieved by performing the PCR method described in (4) of this section 2 using synthetic DNA for modification. Determine the nucleotide sequence of the amplified product after PCR by the method described in (2) of this section 2 and confirm that the target modification has been performed.

 (6) Construction of human-type CDR-grafted antibody expression vector

 In the vector for humanized antibody expression described in (1) of this section 2, the human antibody constructed in (4) and (5) of this section should be located upstream of the genes encoding CH and CL of the human antibody. The cDNA encoding the VH and VL of the CDR-grafted antibody can be cloned to construct a human CDR-grafted antibody expression vector.

 For example, of the synthetic MA used for constructing the human CDR-grafted antibody VH and VL in (4) and (5) of this section 2, an appropriate restriction enzyme at the 5 'end of the synthetic DNA located at both ends. By introducing the recognition sequence, they are expressed in an appropriate form upstream of the genes encoding CH and CL of the human antibody of the humanized antibody expression vector described in (1) of this section 2. Can be cloned as follows.

 (7) Transient expression of humanized antibody

 In order to efficiently evaluate the antigen-binding activity of the various types of humanized antibodies prepared, the humanized antibody expression vectors described in (3) and (6) of this section 2 or an expression vector modified from them are used. One can be used for transient expression of a humanized antibody. As the host cell into which the expression vector is introduced, any host cell that can express the humanized antibody can be used.However, due to its high expression level, COS-7 cells (ATCC CRL1651) Is commonly used [Methods in Nucleic Acids Res., CRC press, 283 (1991)]. Methods for introducing an expression vector into COS-7 cells include the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press, 283 (1991)] and the lipofection method [Proc. Natl. Acad. Sci., USA, 84 , 7413 (1987)].

After introduction of the expression vector, the expression level and antigen-binding activity of the humanized antibody in the culture supernatant Enzyme-linked immunosorbent assay (hereinafter referred to as ELISA method); Antibodies: A 'Laboratory' Manual, Monoclonal 'Antibodies: Principles' and 'Practice and Practice', Academic Press Limited, 1996 In the following, it can be measured by a monoclonal antibody “antibody: principals” and “practice”.

 (8) Stable expression of humanized antibody

 By introducing the humanized antibody expression vector described in (3) and (6) of this section 2 into an appropriate host cell, a transformant that stably expresses the humanized antibody can be obtained.

 Examples of a method for introducing an expression vector into a host cell include an electroporation method [Japanese Unexamined Patent Publication (Kokai) No. 2-257891, Cytotechnology, 3, 133 (1990)] and the like.

 As the host cell into which the humanized antibody expression vector is introduced, any cell can be used as long as it can express the humanized antibody. For example, mouse SP2 / 0-Agl4 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CH0 cells deficient in the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Pro Natl. Acad USA, USA, 4216 (1980)], rat

YB2 / 3HL.P2. G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as YB2 / 0 cells) and the like. Humanized antibodies expressed in YB2 / 0 cells are preferred because they increase ADCC activity.

After the introduction of the expression vector, a transformant that stably expresses the humanized antibody can be treated with a drug such as G418 sulfate (hereinafter referred to as G418: manufactured by SIGMA) according to the method disclosed in JP-A-2-2577891. By culturing in a medium for culturing animal cells. The medium for animal cell culture includes RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL302 medium (manufactured by JRH), I bandage medium (manufactured by GIBCO BRL), Hybridoma- An SFM medium (GIBC0 BRL) or a medium to which various additives such as fetal bovine serum (hereinafter referred to as FBS) are added to these mediums can be used. By culturing the obtained transformant in a medium, the humanized antibody can be expressed and accumulated in the culture supernatant. The expression level and antigen-binding activity of the humanized antibody in the culture supernatant can be measured by ELISA or the like. In addition, the transformed strain can increase the expression level of the humanized antibody by using a dhfr amplification system or the like according to the method disclosed in Japanese Patent Application Laid-Open No. 2577891/1990. The humanized antibody can be purified from the culture supernatant of the transformant using Protein A force column (Antibodies: A Laboratory I Manual, monoclonal • Antibodies: Principles 'and' practice) . In addition, a purification method usually used for protein purification can be used. For example, purification can be performed by a combination of gel filtration, ion exchange chromatography, and ultrafiltration. The molecular weight of the purified humanized antibody H-chain, L-chain or whole antibody molecule can be determined by polyacrylamide gel electrophoresis [SDS-PAGE: Nature, ^, 680 (1970)] or Western blotting ( Antibodies: can be measured using 'Laboratory One' manual, monoclonal 'Antibodies: Principles' and 'Practice', etc.

 (9) Evaluation of humanized antibody activity

 The binding activity of the purified humanized antibody to an antigen and the binding activity to a cultured cancer cell line can be measured by an ELISA method and a fluorescent antibody method [Cancer Immunol. I. unothe, 36, 373 (1993)] and the like. The cytotoxic activity against an antigen-positive cultured cancer cell line can be evaluated by measuring CDC activity, ADCC activity and the like [Cancer Immunol. Immunother., 36, 373 (1993)].

 (10) How to use humanized antibodies

 The humanized antibody of the present invention specifically binds to GD2 expressed in a human-derived cultured cancer cell line, and exhibits cytotoxic activities such as CDC activity and ADCC activity. It is considered useful in the diagnosis and treatment of human cancers such as tumors and neuroblastomas. In addition, since most of the amino acid sequence is derived from the amino acid sequence of human antibodies compared to non-human animal antibodies, it exhibits a strong antitumor effect in the human body, does not show immunogenicity, and its effect is prolonged. It is expected to last for a long time.

 Although the humanized antibody of the present invention can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers and is well known in the technical field of pharmacology. It is desirable to provide it as a pharmaceutical preparation manufactured by any method.

It is desirable to use the most effective route for treatment, including oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous administration. In the case of antibody or peptide preparations, preferably intravenous administration I can give it.

 Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

 Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

 Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, P -Can be manufactured using preservatives such as hydroxybenzoic acid esters and flavors such as strawberry flavor and peppermint as additives.

 Capsules, tablets, powders, granules, etc. are excipients such as lactose, pudose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl It can be produced using a binder such as alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.

 Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. An injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.

 Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Sprays are also prepared using the antibody or peptide itself, or a carrier that does not irritate the oral and respiratory mucosa of the recipient and disperses the antibody or peptide as fine particles to facilitate absorption. You.

 Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the antibody or peptide and the carrier used. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

 The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is generally 10 mg / kg to 8 mg / kg per day for an adult.

3. Preparation of fusion protein between humanized antibody and site

(1) Construction of gene encoding fusion protein between humanized antibody and cytokine By linking the gene encoding the cytokine to the 5 'end or 3' end of the gene encoding the H chain or L chain of the humanized antibody via an appropriate synthetic DNA, the humanized antibody can be linked to the cytokine. A gene encoding the fusion protein can be constructed. In addition, when amplifying the gene encoding cytokine by PCR, an appropriate restriction enzyme recognition sequence is introduced into the 5 'end of the amplification primer, and the H-chain or L-chain of the humanized antibody is cloned. By linking the gene to the 5 'end or the 3' end of the gene, a gene encoding a fusion protein of a humanized antibody and cytokine can be constructed. As the gene encoding a cytokine, either chromosomal DNA or cDNA can be used. The nucleotide sequence of the constructed gene encoding the fusion protein of the humanized antibody and cytokine is determined by the method described in (2) of this section 2 to confirm that it is the target sequence.

 (2) Construction of expression vector for fusion protein of humanized antibody and cytokines H chain of humanized antibody on humanized antibody expression vector described in (3) and (6) of this section 2 or By replacing a part or all of the gene encoding the L chain with the gene encoding the fusion protein of the humanized antibody and cytokine described in (1) of this section 3, humanization is achieved. An expression vector for a fusion protein of an antibody and a cytokine can be constructed. For example, when producing a fusion protein in which a cytokine is fused to the C-terminus of the H chain of a humanized antibody, in (1) of section 3 of this section, a site is added to the 3 ′ end of the gene encoding CH of the humanized antibody. A gene encoding a fusion protein of humanized antibody CH and cytokine is constructed by ligating the gene encoding tokine, and the gene and the gene described in Section 2 of this section are constructed.

An expression vector can be prepared by replacing the gene encoding CH of the humanized antibody on the humanized antibody expression vector described in (3) and (6).

 (3) Stable expression of fusion protein between humanized antibody and site

 Using the expression vector for the fusion protein of the humanized antibody and cytokine described in (2) of Section 3 of this section, the humanized antibody and cytokinin were synthesized in accordance with the method described in (8) of Section 2 of this section. By performing stable expression of the fusion protein with human, a transformant that stably expresses the fusion protein between the humanized antibody and cytokine is obtained, and from the culture supernatant, the fusion protein between the humanized antibody and cytokine is obtained. It can be purified and its molecular weight can be analyzed.

 (4) Activity evaluation of fusion protein between humanized antibody and site force protein

Among the activities of the fusion protein between the purified humanized antibody and the cytokinin, The activity of the portion, that is, the binding activity to an antigen and the binding activity to a cultured cancer cell line can be measured by an ELISA method, a fluorescent antibody method, or the like. The cytotoxic activity against the antigen-positive cultured cancer cell line can be evaluated by measuring CDC activity, ADCC activity and the like. On the other hand, the activity of the cytokine moiety can be evaluated, for example, by using the growth of a cultured cell line showing a concentration-dependent growth for the cytokine as an index [Proc. Natl. Acad. Sci., USA, 91, 9626 (1994)].

 The antitumor effect of a fusion protein of a humanized antibody and cytokine can be evaluated, for example, by administering it to a wild-type mouse transplanted with a cultured mouse cancer cell line expressing GD2, and By comparing with humanized antibody alone, cytoforce alone or co-administration of humanized antibody and cytokine, a stronger antitumor effect in vivo can be evaluated [Cancer Immunol. , 42, 88 (1996)].

 (5) Method of using fusion protein between humanized antibody and site force protein

 The fusion protein of the humanized antibody of the present invention and cytokine specifically binds to GD2 expressed in a human-derived cultured cancer cell line, and exhibits cytotoxic activities such as CDC activity and ADCC activity. It is considered to be useful in the diagnosis and treatment of human cancers such as small cell lung cancer, malignant melanoma, and neuroblastoma. Humanized antibodies have a strong antitumor effect in the human body and do not show immunogenicity because most of the portion derived from the amino acid sequence of the human antibody is less than that of non-human animal antibodies. It is expected to last for a long period of time, and since the activity of the fused cytokine can activate immunocompetent cells in the vicinity of the cancer, it is possible to use humanized antibodies alone, cytokines alone or humanized antibodies. It is expected to have a stronger antitumor effect as compared to simultaneous administration of cysteine and cytokine, etc., and to reduce side effects as compared to systemic administration of cytokine.

 Although the fusion protein of the humanized antibody of the present invention and cytokines can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers to prepare a preparation. It is desirable to provide it as a pharmaceutical preparation manufactured by any method well known in the technical field of science.

It is desirable to use the most effective route for treatment, including oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous administration. In the case of protein preparations, preferably intravenous administration it can.

 Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

 Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

 Liquid preparations such as emulsions and syrups are prepared from water, sucrose, sorbitol, saccharides such as fructose, glycols such as polyethylene glycol and propylene glycol.

It can be manufactured using oils such as sesame oil, olive oil and soybean oil, preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.

 Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol , Hydroxypropylcellulose, gelatin and the like, surfactants such as fatty acid esters, and plasticizers such as glycerin as additives.

 Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. An injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.

 Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. A spray is prepared using the fusion protein itself or a carrier which does not irritate the oral and respiratory tract mucosa of the recipient and disperses the fusion protein as fine particles to facilitate absorption.

 Specific examples of the carrier include lactose and glycerin. Depending on the properties of the fusion protein and the carrier used, preparations such as aerosols and dry powders can be made. Also, in these parenteral preparations, the components exemplified as additives in the oral preparation can be added.

The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 mg / kg to 8 mg / kg per adult per day. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a diagram showing the construction process of plasmid PKANTEX666H.

 Fig. 2 is a diagram showing the construction process of plasmid PKANTEX666.

 FIG. 3 is a view showing an electrophoresis pattern of purified anti-GD2 chimeric antibody KM1138 by SDS-PAGE (4 to; using a 15% gradient gel). The left side shows the results of electrophoresis under non-reducing conditions, and the right side shows the results of electrophoresis under reducing conditions. Lane 1 is high molecular weight, 2 is KM1138

, 3 indicates the low molecular weight marker, and 4 indicates the migration pattern of KM1138.

 FIG. 4 is a diagram showing the binding activity of purified anti-GD2 mouse antibody KM666 and purified anti-GD2 chimeric antibody KM1138 to GD2 measured by changing the antibody concentration. The vertical axis shows the binding activity to GD2

The horizontal axis shows the antibody concentration. 〇 indicates the activity of KM666 and mushroom KM1138, respectively.FIG. 5 shows the binding activity of purified anti-GD2 mouse antibody KM666 and purified anti-GD2 chimeric antibody KM1138 to GD2 measured by varying the amount of GD2 adsorbed on the plate. FIG. The vertical axis indicates the binding activity to GD2, and the horizontal axis indicates the amount of GD2 adsorbed on the plate. 〇 indicates the activity of KM666 and mushroom KM1138, respectively.

FIG. 6 shows the reactivity of the purified anti-GD2 mouse antibody KM666 and the purified anti-GD2 chimeric antibody KM1138 with various gangliosides. The vertical axis indicates the type of ganglioside, and the horizontal axis indicates the binding activity. AcGM2 refers to N-acetyl GM2, GcGM2 refers to N-glycolyl GM2, AcGM3 refers to N-acetyl GM3, and GcGM3 refers to N-glycolyl GM3. □ indicates the reactivity of KM666 and cheat indicates the reactivity of KM1138.

FIG. 7 shows the reactivity of the purified anti-GD2 mouse antibody KM666 and the purified anti-GD2 chimeric antibody KM1138 with various human cancer cell lines. The vertical axis shows the number of cells, and the horizontal axis shows the fluorescence intensity. Each figure shows the reactivity of the control, KM666, and KM1138, respectively, from the bottom.

FIG. 8 shows the CDC activities of the purified anti-GD2 mouse antibody KM666 and the purified anti-GD2 chimeric antibody KM1138 against the human neuroblastoma cell line IMR32 and the human brain tumor cell line T98G. The vertical axis indicates cytotoxic activity, and the horizontal axis indicates antibody concentration. The mouth shows the activity of KM666, and the picture shows the activity of KM1138.

FIG. 9 shows ADCC activities of purified anti-GD2 mouse antibody KM666 and purified anti-GD2 chimeric antibody KM1138 on human neuroblastoma IMR32 and human brain tumor T98G. The vertical axis shows the cytotoxic activity, and the horizontal axis shows the antibody concentration. The mouth is KM666 and the garden is KM1138 Shows the activity of each.

 FIG. 10 is a diagram showing a process of constructing plasmid phKM666H.

 FIG. 11 is a diagram showing a process for constructing plasmid phKM666L.

 Fig. 12 is a diagram showing the process of forming plasmid PT666.

 FIG. 13 is a diagram showing a process for forming a plasmid PT666LCDR.

 FIG. 14 is a diagram showing a process of forming a plasmid PT666HLCDR.

 FIG. 15 is a diagram showing the activity evaluation by transient expression of anti-GD2 chimeric antibody and anti-GD2 CDR-grafted antibody using plasmids pT666 and pT666HLCDR. The vertical axis shows the antibody name, and the horizontal axis shows the relative activity value when the activity of the anti-GD2 chimeric antibody is set to 100.

FIG. 16 is a diagram showing a construction process of a plasmid PKA TEX666HLCDR.

FIG. 17 is a diagram showing an electrophoresis pattern of the purified anti-GD2 quinula antibody KM1138 and the purified anti-GD2 CDR-grafted antibody KM8138 by SDS-PAGE (4 to; using a 15% gradient gel). The left side shows the results of electrophoresis under non-reducing conditions, and the right side shows electrophoresis under reducing conditions. Lane 1 shows the high molecular weight marker, 2 shows ΚΜ1138, 3 shows ΚΜ8138, 4 shows the low molecular weight marker, 5 shows KM1138, and 6 shows the migration pattern of KM8138, respectively.

FIG. 18 is a diagram showing the binding activity of purified anti-GD2 chimeric antibody KM1138 and purified anti-GD2 CDR-grafted antibody # 8138 to GD2 as measured by changing the antibody concentration. The vertical axis shows the binding activity to GD2, and the horizontal axis shows the antibody concentration. 〇 shows activity of ΚΜ1138, Okina shows activity of KM8138, respectively.

FIG. 19 is a graph showing the binding activity of the purified anti-GD2 quinula antibody KM1138 and the purified anti-GD2 CDR-grafted antibody KM8138 to GD2 measured by changing the amount of GD2 adsorbed on the plate. The vertical axis indicates the binding activity to GD2, and the horizontal axis indicates the amount of GD2 adsorbed on the plate. 〇 indicates the activity of KM1138, · indicates the activity of KM8138.

FIG. 20 shows the reactivity of the purified anti-GD2 quinula antibody KM1138 and the purified anti-GD2 CDR-grafted antibody KM8138 with various gangliosides. The vertical axis indicates the type of ganglioside, and the horizontal axis indicates the binding activity. AcGM2 refers to Ν-acetyl GM2, GcGM2 refers to N-glycolyl GM2, AcGM3 refers to N-acetyl GM3, and GcGM3 refers to N-glycolyl GM3. The mouth shows the reactivity of KM8138.

FIG. 21 shows the reactivity of the purified anti-GD2 chimeric antibody KM1138 and the purified anti-GD2 CDR-grafted antibody KM8138 with the human glioblastoma cell line IMR32 and the human brain tumor cell line T98G. The vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity. Each figure shows the reactivity of the control, KM1138s and KM8138, from the bottom.

 FIG. 22 shows the CDC activities of the purified anti-GD2 quinula antibody KM1138 and the purified anti-GD2 CDR-grafted antibody KM8138 on human neuroblastoma cell line IME32 and human brain tumor cell line T98G. The vertical axis indicates cytotoxic activity, and the horizontal axis indicates antibody concentration. The mouth shows the activity of KM1138, and the garden shows the activity of KM8138.

 FIG. 23 shows ADCC activities of purified anti-GD2 chimeric antibody KM1138 and purified anti-GD2 CDR-grafted antibody KM8138 against human neuroblastoma cell line IMR32 and human brain tumor cell line T98G. The vertical axis shows cytotoxic activity, and the horizontal axis shows antibody concentration. The mouth shows the activity of KM1138, and the picture shows the activity of KM8138.

 FIG. 24 is a diagram showing a process for constructing plasmid pBSA-B.

 FIG. 25 is a diagram showing a construction process of plasmid pBSAhCat IL-2.

FIG. 26 is a diagram showing a construction process of plasmid pBShC; I-IL-2.

FIG. 27 is a diagram showing a construction process of plasmid PKA TEX8138-hIL2.

Fig. 28 shows the purified anti-GD2 CDR-grafted antibody KM8138 and the purified fusion protein KM8138-hlL-

FIG. 2 is a view showing an electrophoresis pattern of SDS-PAGE 2 (using a 4 to 15% gradient gel). The left side shows the results of electrophoresis under non-reducing conditions, and the right side shows the results of electrophoresis under reducing conditions. Lane 1 is low molecular weight marker, 2 is KM8138—hIL-2, 3 is KM8138, 4 is low molecular weight marker

-, 5 show the migration pattern of KM8138-hIL-2 and 6, respectively.

Figure 29 shows the purified anti-GD2 CDR-grafted antibody KM8138 and the purified fusion protein KM8138—hIL-

FIG. 2 is a diagram showing the binding activity of No. 2 to GD2 measured by changing the antibody concentration. The vertical axis indicates the binding activity to GD2, and the horizontal axis indicates the antibody concentration. 〇 indicates the activity of KM8138, and 拿 indicates the activity of KM8138-hi2.

FIG. 30 shows the reactivity of the purified anti-GD2 CDR-grafted antibody KM8138 and the purified fusion protein KM8138-hIL-2 with various gangliosides. The vertical axis indicates the type of ganglioside, and the horizontal axis indicates the binding activity. AcGM2 refers to N-acetyl GM2, GcGM2 refers to N-glycolyl GM2, AcGM3 refers to N-acetyl GM3, and GcGM3 refers to N-glycolyl GM3. □ indicates the reactivity of 、 8138 and 鱺 indicates the reactivity of KM8138-hIL-2.

FIG. 31 is a graph showing the growth supporting activity of hIL-2 and the purified fusion protein KM8138-hIL-2 on hIL-2-dependent cell CTLL-2, measured by varying the concentration of each protein. The vertical axis is The growth supporting activity and the abscissa indicate the protein concentration, respectively. 〇 indicates hIL-2 activity, and Hata indicates KM8138-hIL-2 activity. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited thereto.

 Example 1 Production of Hybridoma Cells Producing Mouse Anti-GD2 Monoclonal Antibody

 (1) Preparation of antigen solution

 GD2 was purified from a human neuroblastoma cell line IMR32 (ATCC CCL127) according to a known method [J. Biol. Chem., 263, 10915, 1988]. 5 g of GD2 is 0.5 mol of dipalmitoylphosphatidylcholine (manufactured by SIGMA), 0.5 / mol of cholesterol (manufactured by Nakarai Tesque), 0.05 mol of dipalmitoylphosphatidic acid (manufactured by SIGMA) and lipid The solution was dissolved in 30 ml of a form / methanol (2/1) solution containing 0.5 / mol A (manufactured by Funakoshi) and heated to 45 ° C. to remove the solvent, thereby forming a uniform lipid thin film. Furthermore, the solvent was completely removed by suction with a vacuum pump for 1 hour, 0.5 ml of PBS was added, and the mixture was dissolved by stirring at 45 ° C to obtain an antigen solution.

 (2) Immunization of mice and preparation of splenocytes

 0.5 ml of the antigen solution prepared in (1) of Example 1 was administered to the tail vein of Balb / c mice (manufactured by Japan SLC) once a week for a total of seven times to immunize. The spleen was removed from the mouse 3 days after the final administration, cut in a MEM medium (manufactured by Nissui Pharmaceutical), dissociated using forceps, and centrifuged (1200 rpm, 5 minutes) to remove the supernatant. Then, with 3 ml of Tris-ammonium chloride buffer (PH7.65); Treated for ~ 2 minutes to remove red blood cells. After washing three times with MEM medium, the cells were subjected to cell fusion.

 (3) Preparation of mouse myeloma cells

 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.l (ATCC CRL1597, P

3-）) were cultured and used as a parent strain in cell fusion.

 (4) Preparation of Hypri-doma cells

The spleen cells and myeloma cells obtained in (2) and (3) of Example 1 were mixed in a ratio of 10: 1, centrifuged (1200 rpm, 5 minutes), and the supernatant was removed. Cell group 37 ° C under conditions in polyethylene glycol Ichiru solution to - a (2g polyethylene glycol Ichiru of 1000, 2 ml of MEM medium and 0.7ml solution consisting DMS0) of 10 was added ⁸ splenocytes per 0.5 ml, well suspended It became cloudy. Further, every 1 to 2 minutes, 1 to 2 ml of MEM medium was added several times, and the total amount was finally made up to 50 ml with MEM medium. After removing the supernatant by centrifugation (900 rpm, 5 minutes), suspend the cells in 100 ml of HAT medium, and place them in a 96-well mic-mouthed Thai Yuichi plate (manufactured by Sumitomo Bei-Client); They were cultured for 14 days; 10 at 37 ° C for at dispensed with 5 0 ₂ Inkyube evening within one. The binding activity to GD2 in the culture supernatant of the gel in which the proliferation of the fused cells was observed was measured by the ELISA method described in Example 2, paragraph (3). For the wells in which activity was observed, clone the cells twice by limiting dilution, once with HT medium and once with RPMI1640-FBS (IO) medium. Was. In this way, a hybridoma cell KM666 producing a mouse antibody KM666 specifically reacting with GD2 was obtained. Hypri-doma cell KM666 was deposited as FERM BP-6786 on July 22, 2001 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-3-3 Tsukuba East, Ibaraki, Japan). I have.

Example 2 Preparation of anti-GD2 chimeric antibody

 1. Isolation and analysis of cDNA encoding V region of anti-GD2 mouse antibody

 (1) Preparation of mRNA from anti-GD2 mouse antibody-producing hybridoma cells

MRNA was recovered from the hybridoma cells KM666 described in Example 1. Using a Fast Track mRNA Isolation Kit (manufactured by Invitrogen), an mRNA preparation kit, about 30 zg of mRNA was prepared from 1 × 10 ⁸ cells of the hybridoma cell KM666 according to the attached instruction manual.

 (2) Preparation of H-chain and L-chain cDNA libraries of anti-GD2 mouse antibody

 From 5 〃g of KM666 mRNA obtained in Example 1, paragraph 1 (1), cDNA Synthesis Kit

(Manufactured by Pharmacia), and cDNA having EcoRI-Notl Adabu Yuichi at both ends was synthesized according to the attached instruction manual. After dissolving about 6 g of the prepared cDNA in 10 滅菌 of sterile water, fractionation by agarose gel electrophoresis, a cDNA fragment of about 1.5 kb corresponding to the H chain of IgG type antibody and A: type L chain Approximately 0.1 µg of each corresponding approximately 1. Okb cDNA fragment was recovered. Next, 0.1 g of each about 1.5 kb cDNA fragment and 0.1 g of about l. Okb cDNA fragment were digested with the restriction enzyme EcoRI, and the ends were dephosphorylated with Calf Intestine Alkaline Phosphatase. Use the ZAPI I Cloning Kit (Stratagene) to attach one lg of ZAPI I vector and attach Were connected according to the instruction manual. After ligation, 4〃1 of each reaction solution was packaged into the phage using Gigapackll Packaging Extracts Gold (manufactured by Stratagene) according to the attached instruction manual, and an appropriate amount of E. coli strain XU-Blue [ Biotechniques, 5, 376 (1987)], and about 4 × 10 ³ phage clones were obtained as one H chain cDNA library and one L chain cDNA library of KM666. Next, each phage was fixed on a nitrocellulose filter according to a conventional method (Molecular Cloning: Manual of Laboratory 1).

 (3) Cloning of H chain and L chain cDNA of anti-GD2 mouse antibody

 Using the ECL Direct Nucleic Acid Labeling and Detection Systems (manufactured by Amersham), attach the two-nitrocellulose filter of the KM666 H chain cDNA library and L chain cDNA library prepared in section 1 (2) of Example 2. According to the manufacturer's instructions, cDNA for the mouse antibody C region [H chain is a BamHI-Xhol fragment of mouse C3 cDNA [EMBO J., 3, 2041 (1984)], L chain is Hpal-Xhol of mouse Cc cDNA. The fragment [Cell, 22, 197 (1980)] was detected as a probe, and phage clones that strongly bound to the probe were cloned in 10 clones each for H and L chains. Next, each phage clone was converted into a plasmid by the in vivo excision method according to the instruction manual of the human ZAP I Cloning Kit (Stratagene). The nucleotide sequence of cMA contained in each of the thus obtained plasmids was determined by the dideoxy method (Molecular Cloning: “Laboratory” manual) using Sequenase Version 2.0 DNA Sequencing Kit (manufactured by United States Biochemical Corporation). As a result, a plasmid PKM666H4 containing a full-length functional H chain cDNA having an ATG sequence presumed to be an initiation codon at the 5 ′ end of the cDNA and a plasmid pKM666L4 containing an L chain cDNA were obtained.

 (4) Analysis of amino acid sequence of V region of anti-GD2 mouse antibody

SEQ ID NO: 1 contains the entire nucleotide sequence of VH contained in plasmid PKM666H4, SEQ ID NO: 32 contains the entire amino acid sequence of secreted VH deduced therefrom, and SEQ ID NO: 2 contains all of the VL contained in plasmid PKM666L4 The nucleotide sequence of SEQ ID NO: 33 shows the entire amino acid sequence of secreted VL deduced therefrom. Comparison with the known mouse antibody sequence data (Sequences, Proteins, Proteins, Immunology, Inrest Rest) and purification of the N-terminal amino acid sequences of the H and L chains of the purified anti-GD2 mouse antibody KM666. Automated Edman degradation using a Protein Sequencer 470A (Applied Biosystems) From the comparison with the results of the analysis, each isolated cDNA was a full-length cDNA encoding the anti-GD2 mouse antibody KM666 including a secretory signal sequence, and the H chain was derived from 1 of the amino acid sequence described in SEQ ID NO: 1. It was revealed that the 19th amino acid sequence of the L chain was a secretory signal sequence from the 1st to the 22nd amino acid sequence described in SEQ ID NO: 2.

 Next, the novelty of the amino acid sequence (sequence excluding the secretory signal sequence) of the V region of the H chain (SEQ ID NO: 32) and the V region of the L chain (SEQ ID NO: 33) of the anti-GD2 mouse antibody KM666 investigated. Using the GCG Package (version 9.1, Genetics Computer Group) as a sequence analysis system, the amino acid sequence database of existing proteins was used.

[SWISS-PROT (Release 35.0), PIR-Protein (Release 56.0)] was searched by the BLAST method [J. Mol. Biol., 215, 403 (1990)]. As a result, no completely identical sequence was found in both the H chain and the L chain, confirming that VH and VL of the anti-GD2 mouse antibody KM666 are novel amino acid sequences.

 Further, the VH and VL CDRs of the anti-GD2 mouse antibody KM666 were identified by comparing with the amino acid sequences of known antibodies. The amino acid sequence of CDR1, CDR2 and CDR3 of the V region of the H chain of the anti-GD2 mouse antibody KM666 is shown in SEQ ID NOS: 3, 4 and 5, and the amino acids of CDR1, CDR2 and CDR3 of the V region of the L chain. The sequences are shown in SEQ ID NOs: 6, 7, and 8, respectively.

 2. Stable expression of anti-GD2 chimeric antibody in animal cells

 (1) Construction of anti-GD2 chimeric antibody expression vector pKANTEX666

 Using the humanized antibody expression vector PKA TEX93 described in W097 / 10354 and the plasmids PKM666H4 and PKM666L4 obtained in Example 1, paragraph 1 (3), the anti-GD2 chimeric antibody expression vector Yuichi pKANTEX666 is as follows. Was built.

 3 zg of the plasmid PKM666H4 obtained in item (3) of Example 2 was added to a buffer solution consisting of 10 〃 1 of 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride, and ImM DTT. In addition, 10 units of restriction enzyme Pstl (Takara Shuzo) was further added and reacted at 37 ° C for 1 hour. The reaction mixture was precipitated with ethanol and buffered with 10/1 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride, lmM DTT, 100 µg / ml BSA and 0.01% Triton X-100. In addition to the solution, 10 units of a restriction enzyme Notl (Takara Shuzo) was further added and reacted at 37 ° C. for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 0.33 g of a Notl-Pstl fragment of about 0.43 kb was recovered.

Next, 3 µg of the humanized antibody expression vector PKANTEX93 was added to 10 µl of lOmM Tris-HCl ( pH 7.5), 10 mM restriction enzyme Apal (Takara Shuzo) in addition to a buffer solution consisting of 10 mM magnesium chloride and ImM DTT, and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol, and a buffer consisting of 10: 1 50 mM Tris-hydrochloric acid (pH 7.5), 100 mM sodium chloride, ΙΟηιΜ magnesium chloride, ImM DTT, 100 mg / ml BSA and 0.01% Triton X-100 was used. In addition to the solution, 10 units of a restriction enzyme Notl (Takara Shuzo) was further added and reacted at 37 ° C. for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 g of an Apa I-Not I fragment of about 12.75 kb was recovered.

 Next, synthetic DNAs having the nucleotide sequences of SEQ ID NOS: 9 and 10 were synthesized using an automatic DNA synthesizer (380A, manufactured by Applied Biosystems). Each 0.3 μg of the obtained synthetic DNA was added to 15/1 sterilized water and heated at 65 ° C. for 5 minutes. After allowing the reaction solution to stand at room temperature for 30 minutes, 2〃10 buffer solution [500 mM Tris-HCl (pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2/1 lOmM ATP were added. Further, 10 units of T4 Polynucleotide Kinase (manufactured by Takara Shuzo) was added and reacted at 37 ° C for 30 minutes to phosphorylate the 5 'end.

 Next, 0.1 μg of the Notl-Pstl fragment derived from the above-obtained plasmid PKM666H4, 0.1 μg of the plasmid 1 (^ 1; 1 & 1 fragment derived from £ 93), and 0.05 zg of phosphorylated synthetic MA加 え Ligation was performed using Ready-To-Go T4 DNA Ligase (manufactured by Pharmacia) in addition to the sterilized water of 1. Escherichia coli HB101 strain was transformed using the recombinant plasmid DNA solution thus obtained. Then, the plasmid PKANTEX666H shown in Fig. 1 was obtained, and 10 µg of the obtained plasmid was reacted with the AutoRead Sequencing Kit (Pharmacia) according to the instructions attached thereto, followed by ALF DNA Electrophoresis was performed with a Sequencer (Pharmacia), and the nucleotide sequence was determined. As a result, it was confirmed that a plasmid containing the target DNA was obtained.

 Next, 3 μg of the plasmid PKM666L4 obtained in the item (3) of Example 2 above was obtained from 10 μl of 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and ImM DTT. In addition to the buffer solution, 10 units of restriction enzyme EcoRI (Takara Shuzo) and restriction enzyme EcoT14I (Takara Shuzo) were further added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 0.31 g of an EcoRI-EcoT14I fragment of about 0.41 kb was recovered.

Next, 3 μg of the plasmid PKANTEX666H obtained above was added with 10 μl of 50 mM Tris-HCl (pH 7.5), lOOmM sodium chloride, lOmM magnesium chloride, lmM DTT and 100 μg / ml. In addition to the buffer consisting of BSA, 10 units of restriction enzyme EcoRI (Takara Shuzo) and restriction enzyme SplI (Takara Shuzo) were further added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 μg of an EcoRI-SplI fragment of about 13.20 kb was recovered.

 Next, synthetic DNAs having the nucleotide sequences of SEQ ID NOS: 11 and 12 were synthesized using an automatic MA synthesizer (380A, manufactured by Applied Biosystems). 0.3 g each of the obtained synthetic DNA was added to 15 il of sterilized water, and heated at 65 ° C for 5 minutes. After allowing the reaction solution to stand at room temperature for 30 minutes, 2〃10 buffer solution [500 mM Tris-HCl (pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2〃1 OmM ATP were added. Further: 10 units of T4 Polynucleotide Kinase (Takara Shuzo) was added and reacted at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end.

 Next, 0.1 μg of the EcoRI-EcoT14I fragment derived from the plasmid PKM666L4 obtained above, 0.1 μg of the EcoRI-SplI fragment derived from the plasmid pKANTEX666H, and 0.05 μg of phosphorylated synthetic DNA were sterilized in a total amount of 201. In addition to water, ligation was performed using Ready-To-Go T4 DNA Ligase (Pharmacia). Escherichia coli HB101 strain was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid PKA TEX666 shown in FIG. Using 10 zg of the obtained plasmid, a reaction was performed according to the instructions attached to the AutoRead Sequencing Kit (Pharmacia), followed by electrophoresis with an ALF DNA Sequencer (Pharmacia), and the nucleotide sequence was determined. It was confirmed that a plasmid in which the target DNA was cloned was obtained.

 (2) Stable expression of anti-GD2 chimeric antibody using animal cells

 Using the anti-GD2 chimeric antibody expression vector PKANTEX666 obtained in Example 2, paragraph 2 (1), expression of the anti-GD2 quinula antibody in animal cells was performed as follows.

Plasmid and 4 g of de pKNATEX666 4x l0 ⁶ cells Rattomiero Ichima cell line YB2 / 0 cells (ATCC CRL 1581) to electroporation Chillon method after introduction by [Cytotechnology, 3, 133 (1990 )], of 40 ml RPMI640- The cells were suspended in FBS (10) [RPI1640 medium containing 10% fetal bovine serum (FBS)] and dispensed into a 96-well microtiter plate (manufactured by Sumitomo Bei-Client) in 200 l / l wells. In 5% C0 ₂ incubator base Isseki within one 37 ° C, after 24 hours of incubation, was added so as to be a G418 to 0.5mg / inl;! Were cultured for 2 weeks to. G418-resistant transformant colonies appeared, and the culture supernatant was recovered from the confluent gel. The antigen-binding activity of the anti-GD2 chimeric antibody in the supernatant is shown in Example 2, item 2 (3). Measured by ELISA (peroxidase-labeled goat anti-human IgG (a) antibody is used as the secondary antibody) did.

For the transformed strain of Pelles in which the expression of the anti-GD2 chimeric antibody was observed in the culture supernatant, 0.5 mg / ml of G418 and dhfr gene were used to increase the amount of antibody expression using the dhfr gene amplification system. 1-2x10 ⁵ medium containing RPMO 1640-FBSU0 containing 50 nM of methotrexet (hereinafter referred to as MTX: SIGMA), an inhibitor of the product dihydrofolate reductase (hereinafter referred to as DHFR) The cells were suspended to give cells / ml, and 2 ml was dispensed into a 24-well plate (manufactured by Greiner). After culturing at 37 ° C. for 1 to 2 weeks in a 5% CO ₂ incubator, a transformant showing 50 nM MTX resistance was induced. When the transformant became confluent in the well, the antigen-binding activity of the anti-GD2 chimeric antibody in the culture supernatant was measured by the ELISA method described in Example 2, paragraph (3). For the transformed strain of Pellet in which the expression of the anti-GD2 chimeric antibody was observed in the culture supernatant, the MTX concentration was sequentially increased to 100 nM and 200 nM by the same method as above, and finally G418 was 0.5 mg / ml. In addition, a transformant capable of growing on an RPMI 1640-FBS (IO) medium containing MTX at a concentration of 200 nM and highly expressing an anti-GD2 chimeric antibody was obtained. The resulting transformant was transformed into a single cell (cloning) by the limiting dilution method twice, and the clone with the highest expression of the anti-GD2 chimeric antibody was named KM1138. The expression level of KM1138 anti-GD2 quinula antibody was about 5 μg / 10 ⁶ cells / 24 hours. KM1138 has been deposited as FERM BP-6787 on July 22, 1999 with the Institute of Life Science and Industrial Technology, the Institute of Industrial Science and Technology (1-3 Tsukuba East, Ibaraki, Japan).

 (3) Measurement of antibody binding activity to various gangliosides (ELISA)

 2 nmol of various gangliosides were dissolved in 2 ml of an ethanol solution containing 10 μg of dipalmitoylphosphatidylcholine (SIGMA) and 5 μg of cholesterol (SIGMA). 20〃1 of the solution (20 pmol / ゥ: per well) or 20〃1 of a solution obtained by diluting the solution with ethanol is distributed to each well of a 96-well ELISA plate (manufactured by Greiner). After the mixture was air-dried, PBS containing BSA (hereinafter referred to as 1% BSA-PBS) was added at 100/1 / mL, and reacted at room temperature for 1 hour to block the remaining active groups.

The 1% BSA-PBS was discarded, and the culture supernatant of the transformant, various diluted solutions of purified mouse antibody or purified humanized antibody were added at 50〃1 / ゥ ヱ, and reacted at room temperature for 1 hour. After the reaction, each gel was washed with PBS containing 0.05% Tween 20 (hereinafter referred to as Tween-PBS), and the mouse antibody-added gel was diluted with IPSA-PBS to 400-fold peroxidase. A labeled heron anti-mouse Ig antibody solution (manufactured by DAKO) and a humanized antibody-added well were diluted 1000 times with 1% BSA-PBS in a peroxidase-labeled goat anti-human IgG (a) antibody solution (Kirkegaard & (Perry Laboratories) as secondary antibody solutions were added at 50〃 / ゥ, respectively, and reacted at room temperature for 1 hour. After the reaction, wash with Tween-PBS and add 0.55 g of ABTS substrate solution [2,2, -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium] to 1 L of 0.1 M citrate buffer. (PH4.2), and immediately before use, add a solution of hydrogen peroxide (1 l / ml) at 50/1 / well to develop color, and measure the absorbance at 415 nm (hereinafter referred to as 0D415). It was measured. Alternatively, a biotinylated protein A solution (VECTOR, diluted 200-fold with BSA-PBS) is used at 50〃1 / 抗体 in place of the secondary antibody solution, and reacted at room temperature for 1 hour. After washing with Tween-PBS, peroxidase-labeled Avidin D solution (VECTOR, diluted 1: 4000 with 1 BSA-PBS) was added at 50〃1 / well, and reacted at room temperature for 1 hour. -After washing with PBS, the color was developed in the same manner with the ABTS substrate solution, and the binding activity of the mouse antibody and the humanized antibody was measured.

 (4) Purification of anti-GD2 chimeric antibody from culture supernatant

GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.) containing the transformed cell clone KM1138 expressing the anti-GD2 chimeric antibody obtained in Section 2 (2) of Example 2 at a concentration of 0.5 mg / ml of G418 and 200 nM of MTX. the l~2 x: l0 ⁵ was suspended so as to be cells / ml, was dispensed One not a 200ml in 175cm ² flasks (Greiner Co.). In 5% C0 ₂ incubator and cultured at 37 ° C 5 to 7 days, the culture supernatant was recovered when they became Konfuruento. The anti-GD2 chimeric antibody KM1138 was purified from about 1 L of the culture supernatant using a Prosep-A (manufactured by Bioprocessing) column according to the attached instructions to obtain about 4 mg of the purified protein. About 2 g of the obtained anti-GD2 chimeric antibody KM1138 was subjected to electrophoresis according to a known method [Nature, 227, 680 (1970)], and the molecular weight and the degree of purification were determined. The results are shown in FIG. As shown in FIG. 3, the purified anti-GD2 chimeric antibody KM1138 has a molecular weight of about 150 kilodaltons (Kd) under non-reducing conditions, and about 50Kd and about 25Kd under reducing conditions. Two bands were observed. These molecular weights are almost the same as the molecular weights (H chain: about 49 Kd, L chain: about 23 Kd, whole molecule: about 144 Kd) deduced from the nucleotide sequences of the H chain and L chain cDNAs of KM1138. An IgG-type antibody has a molecular weight of about 150Kd under non-reducing conditions, and has a molecular weight of about 50Kd under reducing conditions by cutting intramolecular disulfide bonds (hereinafter referred to as SS bonds). Reported to be broken down into H chains and L chains with a molecular weight of about 25Kd (Antibodies: α's Laboratory. It was confirmed that the anti-GD2 chimeric antibody KM1138 was expressed as an antibody molecule with the correct structure, which was consistent with the manual, Monocchi's 'Antibody's: Principles' and Practice. In addition, the N-terminal amino acid sequences of the H-chain and L-chain of the purified anti-GD2 chimeric antibody KM1138 were analyzed by automatic Edman degradation using a protein sequencer (470A, manufactured by Applied Biosystems). It was confirmed that the sequence matched the N-terminal amino acid sequence of the L chain and the L chain.

 3. Evaluation of anti-GD2 chimeric antibody activity

 (1) Reactivity of anti-GD2 chimeric antibody to GD2 (ELISA)

The reactivity of the purified anti-GD2 quinula antibody KM1138 to GD2 was measured by the ELISA method described in Example 2, paragraph 2 (3). Detection was performed with a solution of a biotinylated protein A and peroxidase-labeled avidin! ) Solution. GD2 was purified from human neuroblastoma cell line 32 (ATCC CCL127) according to a known method [J. Biol. Chem., 263, 10915 (1988)]. Fig. 4 shows that the amount of GD2 adsorbed to each well of the ELISA plate was fixed at 20 pmol / well, and the reactivity was examined by changing the concentration of the added anti-GD2 chimeric antibody KM1138 and anti-GD2 mouse antibody KM666. The result. As shown in FIG. 4, the anti-GD2 chimeric antibody KM1138 was shown to have the same binding activity to GD2 as the fang GD2 mouse antibody KM666. Figure 5 shows the results of examining the reactivity of the anti-GD2 chimeric antibody KM1138 and the anti-GD2 mouse antibody KM666 at a constant concentration (20 / g / ml) by changing the amount of GD2 adsorbed to each well of the ELISA plate. It is. As shown in FIG. 5, the anti-GD2 kinase antibody KM1138 was shown to have the same binding activity to GD2 as the anti-GD2 mouse antibody KM666. Fig. 6 shows the results obtained by changing the type of ganglioside adsorbed to each well of the ELISA plate (adsorption amount: 20 pmol / ml) to obtain a constant concentration (10 µg / ml) of anti-GD2 chimeric antibody KM1138 and anti-GD2 mouse. It is a result of examining the reactivity of the antibody KM666. The gangliosides used were GM1, N-acetyl GM 2 (manufactured by Boehringer mannheim, hereinafter referred to as AcGM2), N-glycolyl GM 2 (hereinafter referred to as GcGM2), N-acetyl GM 3 (hereinafter AcGM3). ), N-glycolyl GM 3 (hereinafter referred to as GcGM3), GD la, GD Ib (DIA-IATR0N), GD 2, GD 3 (DIA-IATRON), GQ 1 b ( DIA-IATRON) and GTlb (Funakoshi). GM 1 and GD Ia are from mouse brain, N-glycolyl GM 2 and N-glycolyl GM 3 are from mouse liver, N-acetyl GM 3 is from canine erythrocytes, and GD 2 is human neuroblastoma culture Cell line IMR32 ( ATCC CCL127) according to a known method [J. Biol. Chem., 263, 10915 (1988)].

 As shown in FIG. 6, the anti-GD2 quinula antibody # 38 was shown to specifically bind to GD2 in the same manner as the anti-GD2 mouse antibody KM666.

 (2) Reactivity of anti-GD2 chimeric antibody with human cancer cells (fluorescent antibody method)

The reactivity of the purified anti-GD2 chimeric antibody KM1138 with human cancer cells was measured as follows. Human neuroblastoma cell line YT-nu [Acta Path. Jap., 27, 697 (1977)], NAGAI [Acta Path. Jap., 29, 289 (1979)], IMR32 (ATCC CCL127), human brain tumor cell line T98G (ATCC C L1690), respectively lx 10 ⁶ cells of human malignant melanoma cell line G361 (ATCC CRL1424) were suspended into PBS, Ri taken in a microtube (Treff Co.), centrifuged ( After washing the cells at 2000 rpm for 2 minutes), add 50 zl of anti-GD2 chimeric antibody KM1138 or anti-GD2 mouse antibody KM666 (solution adjusted to 50 μg / ml with BSA-PBS) and stir at 4 ° C. For 1 hour. After the reaction, the mixture was centrifuged three times with PBS and washed, and then a protein A solution (fluorescently labeled with fluorescein isocyanate (hereinafter referred to as FITC)) (30 times with BSA-PBS, manufactured by Boehringer Mannheim) (Diluted and used) and stirred, and reacted at 4 ° C for 1 hour. After the reaction, the mixture was centrifuged three times with PBS and washed, then suspended in PBS, and analyzed using a flow cytometer EPICS Elite (manufactured by COULTER). As a control, the same operation as described above was performed without addition of the antibody, and the analysis was performed. Figure 7 shows the results. As shown in FIG. 7, the anti-GD2 chimeric antibody KM1138 (upper) and the anti-GD2 mouse antibody KM666 (middle) showed three out of three neuroblastoma lines, one out of brain tumor lines, and one malignant black. One of the tumor cell lines responded. The reaction intensity was almost the same between the anti-GD2 chimeric antibody KM1138 and the anti-GD2 mouse antibody KM666. The above results indicate that the anti-GD2 chimeric antibody KM1138 is useful for the diagnosis and treatment of human neuroblastoma, brain tumor, and malignant melanoma.

 (3) In vitro cytotoxic activity (CDC activity) of anti-GD2 chimeric antibody

 To evaluate the in vitro cytotoxic activity of the anti-GD2 chimeric antibody KM1138, CDC activity was measured according to the method described below.

a. Preparation of target cell solution

5 x 10 ⁶ cells of human neuroblastoma cell line IMR32 (ATCC CCL127) and human brain tumor cell line T98G (ATCC CRL1690) cultured in RPMI 1640-FBS (10) medium It was prepared and the Na ₂ ⁵¹ Cr0 ₄ is a radioactive substance 3.7MBq eq addition reacted at 37 ° C, radiolabeled cells. After the reaction, the cells were washed with RPMI1640-FBS (10) medium three times by suspending and centrifuging, resuspended in the medium, and left on ice at 4 ° C for 30 minutes to dissociate the radioactive substances naturally. . After centrifugation, 5 ml of RPMI1640-FBS (10) medium was added to adjust to lx10 ⁶ cells / ml, and used as a target cell solution.

b. Preparation of complement solution

 Serum from three healthy individuals was mixed and used as a source of human complement. At the time of use, it was diluted to 15% vol./vol. With RPMI 1640-FBSU0) medium and used as a complement solution.

c Measurement of CDC activity

To each plate of a 96-well U-shaped bottom plate (manufactured by Falcon), add 50 溶 1 of the target cell solution prepared in a. (5 x 10 ⁴ cells / well), and then add the anti-GD2 chimeric antibody KM1138 or The anti-602 mouse antibody 1 »1666 was heated to a final concentration of 0.05 to 50〃 /] 01, and reacted at room temperature for 30 minutes. After the reaction, the plate was centrifuged, the supernatant was removed, the human complement solution prepared in b. Was added, and the mixture was reacted at 37 ° C for 1 hour. After centrifugation, the amount of ⁵¹ Cr released into the supernatant was measured with an a counter. The amount of spontaneously dissociated ⁵¹ Cr was determined by performing the same operation as above using only the medium instead of the antibody solution and the complement solution, and measuring the amount of ⁵¹ Cr in the supernatant. The total amount of dissociated ⁵¹ Cr was determined by adding the medium alone in place of the antibody solution and adding 5 N sodium hydroxide instead of the complement solution, and performing the same operation as above, and measuring the amount of ⁵¹ Cr in the supernatant. I asked. CDC activity was determined by the following equation.

CDC activity) = ¹ volume in the sample supernatant-51 volumes of isolation _χ

Total dissociation ⁵¹ Cr Amount of spontaneous dissociation ⁵¹ Cr The results are shown in FIG. As shown in FIG. 8, the anti-GD2 chimeric antibody KM1138 and the anti-GD2 mouse antibody KM666 show equivalent CDC activities in all cell lines, and in particular, the human neuroblastoma cell line IMR32 Showed a very strong cytotoxic activity.

 (4) In vitro cytotoxic activity of anti-GD2 chimeric antibody (ADCC activity)

To evaluate the in vitro cytotoxic activity of the anti-GD2 chimeric antibody KM1138, ADCC activity was measured according to the method described below. a. Preparation of target cell solution

RPMI1640-FBS (10) human neuroblastoma cell lines IMR32 were cultured in medium (ATCC CCL127) and respectively to prepare a I x lO ⁶ cells of human brain tumor cell lines T98G (ATCC CRL1690), a radioactive substance Na ₂ ⁵¹ Cr0 ₄ reacted at a 1.85MBq eq addition 37 ° C, radiolabeled cells. After the reaction, the cells were washed three times by suspending and centrifuging in RPMI1640-FBS (IO) medium, resuspended in the medium, and left on ice at 4 ° C for 30 minutes to spontaneously dissociate the radioactive substance. After centrifugation, RPMI1640-FBS (10) medium was added 5 ml, adjusted to 2 x l0 ⁵ cells / ml, and a target cell solution.

b. Preparation of effector-cell solution

50 ml of venous blood of a healthy person was collected, and 0.5 ml of heparin sodium (manufactured by Takeda Pharmaceutical Company Limited) was added and mixed gently. This was centrifuged (1500 to I800 xg, 30 minutes) using Polymorphprep (manufactured by Nycomed Pharma AS) according to the instruction manual to separate a mononuclear cell layer. RPMH640-FBS (10) 3 times centrifugation medium (1500~; 1800 xg, 5 minutes) washed, re at a concentration of 1 X 10 ⁷ cells / ml or 5 X 10 ⁶ cells / ml using a medium This was suspended to obtain an effect cell solution.

c Measurement of ADCC activity

To each well of a 96-well U-shaped bottom plate (manufactured by Falcon), 50〃1 (I × 10 ⁴ cells / well) of the target cell solution prepared in a. Was dispensed. For human neuroblastoma cell line IMR32 is then the ratio of b. 1 X 10 ⁷ cells / ml in effect evening single cell suspension prepared in the 100 UL \ (lx lO ⁶ cells / Ueru, effector and target cells 100:. 1 a), for the human brain tumor cell line T98G, b of 5 X 10 ⁶ cells / ml prepared in effector cell solution 100 zl ⁽⁵ X 10 5 cells / Ueru, and Efuwekuta single cell The ratio of target cells was 50: 1). Further, the anti-GD2 chimeric antibody KM1138 or the anti-GD2 mouse antibody KM666 was heated at 37 ° C. for 4 hours at a final concentration of 0.05 to 50〃 / 1111. After the reaction, the plate was centrifuged, and the amount of ⁵¹ Cr in the supernatant was measured at a county. The amount of spontaneously dissociated ⁵¹ Cr was determined by performing the same operation as above using only the medium instead of the effector cell solution and the antibody solution, and measuring the amount of ⁵¹ Cr in the supernatant. For the total amount of dissociated ⁵¹ Cr, add only the medium instead of the antibody solution, and add 5 N sodium hydroxide instead of the effector cell solution, perform the same operation as above, and measure the amount of the supernatant ⁵ Determined by ADCC activity was determined by the following equation. Amount of ⁵¹ Cr in sample supernatant - spontaneously dissociated ⁵¹ Cr amount

 ADCC activity (%) = X 100

Total dissociated ⁵¹ Crft "Amount of spontaneously dissociated ⁵¹ Cr The results are shown in Fig. 9. As shown in Fig. 9, the anti-GD2 chimeric antibody KM1138 showed ADCC activity against all cell lines, It was found that the activity was higher than that of the anti-GD2 mouse antibody KM666.The above results indicate that the anti-GD2 kinula antibody KM1138 can more efficiently transform the human effector cells than the anti-GD2 mouse antibody KM666. Since it can be activated, it has been shown to be useful in the treatment of human cancer.

Example 3 Preparation of anti-GD2 CDR-grafted antibody

 1. Construction of cDNA encoding VH and VL of anti-GD2 CDR-grafted antibody

 (1) Design of VH and VL amino acid sequences of anti-GD2 CDR-grafted antibody

 First, the VH amino acid sequence of the anti-GD2CDR-grafted antibody was designed as follows. The amino acid sequence of FR of human antibody VH for transplanting the amino acid sequence of CDR of VH of anti-GD2 mouse antibody KM666 identified in section (4) of Example 2 was selected. Liedbat et al. Classified the VHs of various known human antibodies into three types of human subgroups (HSG I-III) based on their amino acid sequence homology, and reported common sequences for each of these subgroups. (Sequences of Proteins of Immunology 'Interest'). Since there is a possibility that the immunogenicity of these consensus sequences is further reduced in humans, the VH amino acid sequence of the anti-GD2 CDR-grafted antibody was designed based on these consensus sequences. In order to produce a more active anti-GD2 CDR-grafted antibody, the amino acid sequence of the FR of the KM666 VH and the FR amino acid sequence of the KM666 VH An amino acid sequence of FR having high homology was selected. Table 1 shows the homology search results. As shown in Table 1, the amino acid sequence of the FR of KM666 VH had the highest homology with subgroup 11. Table 1

 Human antibody H chain V region © Common rooster of each subgroup 1 | FR Amino g wing i column and K 666 H chain \. 'Region FR amino translation

 HSGI HSGII HSG0I

51.2% 67.8% 63.2% From the above results, the VH CDR amino acid sequence of the anti-GD2 mouse antibody KM666 was grafted to the appropriate position of the FR amino acid sequence of the consensus sequence of the VH subgroup II of the human antibody. The amino acid sequence HV.0 was designed.

 Next, the amino acid sequence of VL of the anti-GD2CDR-grafted antibody was designed as follows. The amino acid sequence of the FR of VL of the human antibody to which the amino acid sequence of the CDR of the VL of the anti-GD2 mouse antibody KM666 identified in Section 2 (4) of Example 2 was selected. Liedbat et al. Classified the VLs of various known human antibodies into four types of human subgroups (HSG I-IV) based on their amino acid sequence homology, and reported common sequences for each of these subgroups. Yes (Synchronization / Problems / Ops / Imnomological 'In Evening Rest). Therefore, in the same manner as in the case of the H chain, the amino acid sequence of FR having the highest homology with the amino acid sequence of FR of KM666 VL among the amino acid sequences of 共通 of the common sequence of the four subgroups of human antibody VL Was selected. Table 2 shows the homology search results. As shown in Table 2, the amino acid sequence of FR of VL of KM666 had the highest homology with subgroup I.

 Table 2

の間の相同性 Each amino acid subunit of the L region V region of the human antibody FR amino acid translation 1 | of the ffiffi? L | and the amino acid FR of the 666 L chain V region

Homology between

 HSGI HSGII HSGIII HSGIV

 70.1% 61.2% 66.2% 65.0%

 From the above results, the VL CDR amino acid sequence of the anti-GD2 mouse antibody KM666 was grafted to the appropriate position of the FR amino acid sequence of the consensus sequence of the VL subgroup I of the human antibody, and the VL amino acid of the anti-GD2 CDR-grafted antibody was transplanted. Acid sequence LV.0 was designed.

The VH amino acid sequence HV.0 and VL amino acid sequence LV.0 of the anti-GD2 CDR-grafted antibody designed above was used to graft only the CDR amino acid sequence of the anti-GD2 mouse antibody KM666 into the FR amino acid sequence of the selected human antibody. It is the array which did. In general, the activity of a human CDR-grafted antibody is often reduced only by grafting the amino acid sequence of the mouse antibody CDR to the amino acid sequence of the human antibody RF. To avoid antibody activity, the amino acid residues of FR that differ between human and mouse antibodies, which are thought to affect the activity, may be transplanted together with the amino acid sequence of CDR. Is being done. Thus, in the present Example, it was examined to identify the amino acid residues of FR which are considered to affect the activity. First, the anti-GD2 CDR-grafted antibody VH The three-dimensional structure of the antibody V region (hereinafter, referred to as HVOLVO) consisting of the amino acid sequence HV.O and the amino acid sequence LV.O of VL was constructed using a computer-modeling technique. The three-dimensional structure coordinates were prepared using software AbM (manufactured by Oxford Molecular) and the three-dimensional structure was displayed using software Pro-Explor (manufactured by Oxford Molecular) according to the attached instruction manual. In addition, a computer model of the three-dimensional structure of the V region of the anti-GD2 mouse antibody KM666 was similarly constructed. Furthermore, in the amino acid sequence of FR of each of VH and VL of HVOLVO, amino acid residues different from anti-GD2 mouse antibody KM666 are sequentially changed to residues found at corresponding positions of anti-GD2 mouse antibody KM666. A three-dimensional structure model comprising the modified amino acid sequence was similarly constructed, and the three-dimensional structures of the V regions of the anti-GD2 mouse antibodies KM666, HVOLVO, and the modified product were compared. As a result, among the amino acid residues of FR of HVOLVO, those amino acid residues which change the three-dimensional structure of the antigen binding site and are thought to affect the activity of the antibody were selected. The FR amino acid residue of the selected HVOLVO was modified to a residue found in the mouse antibody KM666, and as a result, the amino acid sequence of the VH amino acid sequence hKM666H of the anti-GD2 CDR-grafted antibody shown in SEQ ID NO: 13 and the antibody shown in SEQ ID NO: 14 The amino acid sequence MM666L of the VL of the GD2CDR-grafted antibody was designed. In hKM666H, in the amino acid sequence of FR of HV.0, Leu at position 20, Ser at position 30, Ile at position 37, Ile at position 48, Val at position 67, Val at position 71, and position 73 Thr, 78th Phe, 79th Ser, 82th Leu, 85th Val, 97th Arg are the amino acid residues I found at the corresponding positions of the VH of the anti-GD2 mouse antibody KM666, respectively. le, Ala, Val, Leu, Leu, Lys, Asn, Val, Phe, Met ヽ Leu ヽ Lys. In the MM666L, in the amino acid sequence of FR of LV.O, 1st Asp, 2nd Ile, 21st Ile, 41st Pro, 47th Leu, 48th Leu Phe at position 72 was modified to Glu, Asn, Met, Pro, Leu, Leu, and Phe, which are amino acid residues found at the corresponding positions of VL of anti-GD2 mouse antibody KM666.

 (2) Construction of cDNA encoding anti-GD2 CDR-grafted antibody VH

 A cDNA encoding the VH amino acid sequence MM666H of the anti-GD2CDR-grafted antibody designed in section 1 (1) of Example 3 was constructed by PCR using the following method.

First, the complete amino acid sequence was obtained by connecting the designed amino acid sequence to the secretion signal sequence of the H chain of the anti-GD2 mouse antibody KM666 described in SEQ ID NO: 1. Next, the amino acid sequence was converted to a gene codon. Multiple gene copies for one amino acid residue When a don was present, the corresponding gene codon was determined in consideration of the frequency of use (Sequences of Proteins' Ob 'Immunological') in the nucleotide sequence of the antibody gene. By connecting the determined gene codons, the nucleotide sequence of the cDNA that encodes the complete amino acid sequence of the antibody V region is designed, and the 5'-end and the 3'-end are the binding bases of the primer for amplification during PCR. A sequence (including a restriction enzyme recognition sequence for cloning into a humanized antibody expression vector) was added. Divide the designed base sequence into a total of 6 base sequences, each of which is approximately 100 bases from the 5 'end (adjacent base sequences should have a duplicated sequence of 20 bases at their ends). Synthesis was performed using an automatic DNA synthesizer (380A, manufactured by Applied Biosystems) in the order of alternating sense and antisense strands. Actually, six synthetic DNAs each having the nucleotide sequence of SEQ ID NOS: 15 to 20 were synthesized. Each DNA was adjusted to 50 最終 1 lOmM Tris-HCl (pH 8.3), 50m potassium chloride, 1.5mM magnesium chloride, 0.001% gelatin, 200〃M dNTPs, 0.5l In addition to a buffer consisting of U¾Ml 3 primer RV (Takara Shuzo), 0.5〃M M13 primer M4 (Takara Shuzo) and 2 units of TaKaRa Taq DNA polymerase (Takara Shuzo), cover with 50〃1 mineral oil, The sample was set in a DNA thermal cycler (PJ480, manufactured by PERK IN ELMER) and subjected to 30 cycles of 2 minutes at 94 ° C, 2 minutes at 55 ° C, and 2 minutes at 72 ° C. After purifying the reaction mixture using QIAquick PCR Purification Kit (manufactured by QIAGEN) according to the attached instruction manual, a buffer consisting of 30〃1 lOmM Tris-HCl (ρΗ7.5), 10 mM magnesium chloride and ImM DTT The mixture was further added with 10 units of restriction enzyme Apal (Takara Shuzo) and reacted at 37 ° C for 1 hour. The reaction mixture was precipitated with ethanol, and buffered with 10 1 50 mM Tris-hydrochloric acid (PH7.5), 100 mM sodium chloride, 10 mM magnesium chloride, ImM DTT, 100 μg / ml BSA and 0.01% Triton X-100. In addition to the solution, 10 units of Notl (a product of Takara Shuzo Co., Ltd.) was further added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and an Apal-Notl fragment of about 0.44 kb was recovered at about 0.2 / g.

Next, 3 〃g of plasmid pBluescript SK (-) (manufactured by Stratagene) was added to a buffer solution consisting of 10〃1 lOmM tris-hydrochloride (pH 7.5), lOmM magnesium chloride and ImM DTT, followed by another 10 units. Was added, and the mixture was reacted at 37 ° C. for 1 hour. The reaction mixture was precipitated with ethanol and consisted of 10〃1 50 Tris-HCl (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride, lfflM DTT, 100〃g / ml BSA and 0.0ΠTriton X-100. Add 10 units of restriction enzyme Notl (Takara Shuzo) to the buffer at 37 ° C. For 1 hour. The reaction mixture was fractionated by agarose gel electrophoresis, and about 2.95 g of an Apal-Notl fragment of about 2.95 kb was recovered.

 Next, 0.1 μg of the Apal-Notl fragment of the VH PCR product of the anti-GD2 CDR-grafted antibody obtained above and 0.1 μg of the Apal-Notl fragment of plasmid pBluescript SK (-) were sterilized in a total volume of 20 μl. In addition to water, ligation was performed using Ready-To-Go T4 DNA Ligase (Pharmacia). Escherichia coli HB101 was transformed using the recombinant plasmid DNA solution thus obtained. Each plasmid DNA was prepared from 10 clones of the transformed strain, and AutoRead

After reaction with Sequencing Kit (Pharmacia) according to the instructions attached, electrophoresis was performed with ALF DNA Sequencer (Pharmacia), and the base sequence was determined. As a result, the plasmid shown in Fig. 10 having the target base sequence was obtained. phKM666H was obtained.

 (3) Construction of cDNA encoding VL of anti-GD2 CDR-grafted antibody

 The cMA encoding the VL amino acid sequence MM666L of the anti-GD2CDR-grafted antibody designed in section 1 (1) of Example 3 was subjected to PCR using the PCR method in the same manner as VH in section 1 (2) of Example 3 as follows. And built. However, as the secretory signal sequence, the sequence of the L chain of the anti-GD2 mouse antibody KM666 described in SEQ ID NO: 2 was used.

 First, six synthetic DNAs having the nucleotide sequences of SEQ ID NOS: 21 to 26 were synthesized using an automatic DNA synthesizer (380A, manufactured by Applied Biosystems). Each synthesized DNA was adjusted to a final concentration of 0.1 M with 50 mM 1 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 M dNTPs, 0.5 M χ Μ Μ13 rimer RV

(Takara Shuzo), OzM MlS primer MA (Takara Shuzo) and 2 units of TaKaRa Taq DNA polymerase (Takara Shuzo) in addition to a buffer solution, covered with 501 mineral oil. And PERK IN ELMER), and 30 cycles of 94 ° C for 2 minutes, 55 ° C for 2 minutes, and 72 ° C for 2 minutes were performed. The reaction solution was purified using a QIAquick PCR Purification Kit (manufactured by QIAGEN) according to the attached instruction manual, and then purified, 30〃1 of 50 mM Tris-hydrochloric acid (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride, ImM A buffer consisting of DTT and 100 g / ml BSA, plus 10 units of EcoM

(Takara Shuzo) and restriction enzyme SplI (Takara Shuzo) were added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 0.29 g of an EcoRI-SplI fragment of about 0.39 kb was recovered.

Next, 3 μg of the plasmid pBSL3 described in JP-A-10-257893 was added to 10 μl of 50 mM Tris- In addition to a buffer solution consisting of hydrochloric acid (pH 7.5), lOOmM sodium chloride, ΜπιΜ magnesium chloride, Im DTT and 100 zg / inl BSA, 10 units of restriction enzyme EcoRI (Takara Shuzo) and restriction enzyme Spl l (Takara Shuzo) Was added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 g of an EcoRI-SplI fragment of about 2.95 kb was recovered. Next, 0.1 〃g of the EcoRI-SplI fragment of the PCR product of the VL of the anti-GD2CDR-grafted antibody obtained above and 0.1 〃g of the EcoRI-Spll fragment of plasmid pBSL3 were added to a total volume of 20〃1 sterile water. Ligation was performed using Ready-To-Go T4 DNA Ligase (Pharmacia). Escherichia coli HB101 was transformed using the recombinant plasmid DNA solution thus obtained. Each plasmid DNA was prepared from 10 clones of the transformant, reacted with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, and electrophoresed with ALF DNA Sequencer (Pharmacia) to determine the nucleotide sequence. As a result, the plasmid phKM666L having the target nucleotide sequence shown in FIG. 11 was obtained.

 2. Activity evaluation of anti-GD2 CDR-grafted antibody by transient expression using animal cells

 To more quickly evaluate the activity of the anti-GD2 CDR-grafted antibody, transient expression of the anti-GD2 CDR-grafted antibody was performed using COS-7 cells (ATCC CRL1651) as follows. (1) Construction of transient expression vector for anti-GD2 chimeric antibody

 For use as a positive control in the activity evaluation by transient expression of the anti-GD2 CDR-grafted antibody, a transient expression vector of the anti-GD2 chimeric antibody was constructed as follows.

 In general, the efficiency of transient expression using animal cells depends on the number of copies of the expression vector introduced. Therefore, it is considered that the expression vector having a smaller size has higher expression efficiency. Therefore, the region which is considered to have no effect on the antibody expression of PKA TEX666 obtained in paragraph (2) of Example 2 was deleted, and the smaller anti-GD2 kinula antibody expression vector PT666 was deleted as follows. Built.

3 zg of plasmid pKANTEX666 was added to a buffer solution consisting of 10〃1 lOmM Tris-HCl (pH 7.5), 50 mM sodium chloride, lOmM magnesium chloride and ImM DTT, and an additional 10 units of restriction enzyme Hindl II (Takara Shuzo) ) Was added and reacted at 37 ° C for 1 hour. The reaction mixture was precipitated with ethanol, added to a buffer solution consisting of 10〃1 of 50 mM Tris-hydrochloric acid (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and ImM DTT, and further added 10 units of restriction enzyme Mlul (Takara Shuzo). Was added thereto and reacted at 37 ° C for 1 hour. The reaction mixture was precipitated with ethanol, and the 5'-protruding end generated by restriction enzyme digestion was digested with DNA Blunting Kit (Takara Shuzo). Changed to blunt ends. The reaction solution was fractionated by agarose gel electrophoresis, and about 2.60 g of a DNA fragment of about 9.60 kb was recovered. 0.1 µg of the recovered DNA fragment was added to a total of 20〃1 of sterile water, and ligated using Ready-To-Go T4 DNA Ligase (Pharmacia). Escherichia coli HB101 strain was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain plasmid PT666 shown in FIG.

 (2) Construction of transient expression vector for anti-GD2 CDR-grafted antibody

 The anti-GD2 chimeric antibody expression vector pT666 obtained in the item (2) (1) of Example 3 and the plasmids phKM666H and phKM666L obtained in the items (2) and (3) of Example 3 were used. Thus, a transient expression vector for the anti-GD2 CDR-grafted antibody was constructed as follows.

 3 / g of the plasmid PMM666L obtained in paragraph (3) of Example 3 was added to 10〃1 of 50 mM tris-hydrochloric acid (pH 7.5), lOOmM sodium chloride, lOmM magnesium chloride, ImM DTT and lOO ^ g / ml BSA, and 10 units of restriction enzyme EcoRI (Takara Shuzo) and restriction enzyme Sppl (Takara Shuzo) were further added thereto and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 0.29 g of an EcoRI-SplI fragment of about 0.39 kb was recovered.

 Next, 3 μg of the anti-GD2 chimeric antibody expression vector pT666 was obtained from 10 μl of 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride, ImM DTT and 100 μg / ml BSA. 10 units of restriction enzyme EcoRI (manufactured by Takara Shuzo) and restriction enzyme Spll (manufactured by Takara Shuzo) were added thereto, followed by reaction at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2.60 g of an EcoRI-SplI fragment of about 9.60 kb was recovered.

 Next, 0.1 μg of the EcoRI-Spll fragment derived from the plasmid phKM666L obtained above and the EcoRI-Spll fragment of the plasmid pT666 were added to the entire amount of sterilized water, and the Ready-To-Go T4 DNA Ligase (Pharmacia (Made by the company). Escherichia coli HB101 strain was transformed with the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid pT666LCDR shown in FIG.

Next, 3 μg of the plasmid PMM666H obtained in paragraph (1) (2) of Example 3 was added to a buffer consisting of 10〃1 lOmM tris-hydrochloride (PH7.5), 10 mM magnesium chloride and lmM DTT. Further, 10 units of restriction enzyme Apal (Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C. for 1 hour. The reaction mixture was precipitated with ethanol, and 10% of 50 mM Tris-hydrochloric acid (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride, lmM DTT, 100 μg / ml BSA and 0.01% triton X-100 And 10 units of a restriction enzyme Notl (Takara Shuzo) was added thereto, followed by reaction at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and an Apal-Notl fragment of about 0.44 kb was recovered at about 0.2 / g.

 Next, 3 zg of the plasmid PT666LCDR obtained above was added to 10 1 of a buffer solution containing 10 mM Tris-hydrochloric acid (PH7.5), lOm magnesium chloride and ImM DTT, and an additional 10 units of restriction enzyme Apal (Takara Shuzo) Was added and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol and consisted of 10〃1 of 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 100 mM magnesium chloride, ImM DTT, 100 zg / ml BSA and 0.0 Triton X-100. In addition to the buffer solution, another 10 units of Notl (Takara Shuzo Co., Ltd.) was added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and an Apal-NotI fragment of about 10.000 kb was recovered at about 2 / g.

 Next, 0.1 μg of the Apal-Notl fragment derived from the plasmid PMM666H obtained above and 0.1 zg of the Apal-Notl fragment of the plasmid pT666LCDR were added to a total volume of 20〃1 of sterile water. Ligation was carried out using -Go T4 DNA Ligase (Pharmacia). Escherichia coli HB101 strain was transformed with the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid pT666HLCDR shown in FIG.

 (3) Evaluation of activity of anti- & 02 chimeric antibody and anti-GD2 CDR-grafted antibody by transient expression using animal cells

 Transient expression of the antibody using the transient expression vector PT666 and the transient expression vector PT666HLCDR of the anti-GD2 CDR-grafted antibody obtained in Example 3, paragraph 2 (1) and (2) above. Was performed as follows.

COS-7 cells (ATCC CRL1651) were dispensed 2nd at 1 × 10 ⁵ cells / ml into 6-well plates (Falcon) and cultured overnight at 37 ° C. Add 2 μg of each expression vector to 100 （1 OPT I-MEM medium (GIBCO BRL), and add 10〃1 to 100 01 0PTI-MEM medium.

A solution to which LIPOFECTAMINE Reagent (manufactured by GIBCO BRL) was added was added, and the mixture was reacted at room temperature for 40 minutes to form a DNA-ribosome complex. After washing the COS-7 cells cultured overnight with 2 ml of 0PTI-MEM medium (GIBCO BRL) twice, add a solution containing 0.8 ml of 0PTI-MEM medium to the solution containing the complex, and add 37 ° C After culturing for 7 hours, the solution was removed, 2 ml of DME medium (manufactured by GIBCO BRL) containing 10% FBS was added, and the cells were cultured at 37 ° C. After 72 hours from the introduction of the expression vector, the culture supernatant is collected and concentrated if necessary. The binding of humanized anti-GD2 antibody to GD2 in the culture supernatant by the ELISA method described in Example 2, paragraph 2 (3) [peroxidase-labeled goat anti-human IgG (a) antibody was used as the secondary antibody] The total activity was measured by measuring the concentration of the anti-GD2 humanized antibody in the culture supernatant by the ELISA method described in (4) of this section, and the activity was determined from the values to determine the activity of the anti-GD2 chimeric antibody as a positive control. Was calculated as the relative activity value. The results are shown in FIG. As shown in FIG. 15, the anti-GD2 CDR-grafted antibody derived from the transient expression vector PT666HLCDR showed about 40% of the binding activity as compared to the anti-GD2 chimeric antibody.

 (4) Measurement of concentration of humanized antibody in culture supernatant of transient expression by ELISA

 50〃1 of a goat anti-human IgG (r-chain) antibody (manufactured by Medical Biology Laboratories) diluted 400 times with PBS was used for each plate of a 96 ゥ ELISA plate (Greiner). And reacted at 4 ° C. overnight. After removing the antibody solution, 1¾BSA-PBS was added at 100〃1 / ゥ ヱ and reacted at room temperature for 1 hour to block the remaining active groups. 1% BSA-PBS was discarded, and a transient expression culture supernatant or various dilutions of the purified anti-GD2 chimeric antibody were added at 50 1 / well, and reacted at room temperature for 1 hour. After the reaction, wash each well with Tween-PBS, add a solution of peroxidase-labeled mouse anti-human A: L chain antibody (Zymed) diluted 500 times with PBS at 50〃1 / ゥ ヱ, and add 1 hour at room temperature. Reacted. After the reaction, wash each well with Tween-PBS, and add 0.55 g of the substrate solution [2,2, azinobis (3-ethylbenzothiazoline-6-sulfonic acid) to 1 L of 0.1 M citrate buffer. The solution was dissolved in a solution (pH 4.2), and immediately before use, a solution I added with hydrogen peroxide at 11 / ml was added at 50〃1 / ゥ ヱ to develop color, and 0D415 was measured.

 3. Stable expression of anti-GD2 CDR-grafted antibody using animal cells

 The results described in item 2 of Example 3 above suggested that the anti-GD2 CDR-grafted antibody derived from the expression vector PT666HLCDR had a binding activity to GD2 of about 40% as compared to the anti-GD2 chimeric antibody. . Therefore, in order to evaluate the activity of the anti-GD2 CDR-grafted antibody in more detail, a transformed cell line stably expressing the anti-GD2 CDR-grafted antibody was obtained according to the method described below, and the anti-GD2 CDR-grafted antibody was purified from the culture supernatant of the transformed cell strain. The GD2CDR-grafted antibody was purified. (1) Construction of stable expression vector for anti-GD2 CDR-grafted antibody

The transient expression vector PT666HLCDR constructed in paragraph 2 (2) of Example 3 lacks the gene conferring resistance to the drug G418 and the dhfr gene. Selection by drug resistance and gene amplification by MTX in the method described in Cannot be used to obtain a transformed cell line using Therefore, PT666HLCDR was modified according to the method shown below to construct a stable expression vector PKANTEX666HLCDR for an anti-GD2CDR-grafted antibody.

 First, 3 g of the humanized antibody expression vector pKANTEX796 described in JP-A-10-257893 was added to a buffer consisting of 101/1 20 mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, ImM DTT and 100 mM chloride medium. In addition, 10 units of restriction enzyme BainHI (Takara Shuzo) and restriction enzyme Xhol (Takara Shuzo) were further added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 0.2 µg of a BamHI-Xhol fragment of about 8.67 kb was recovered. Next, 3 / g of plasmid pT666HLCDR was added to a buffer consisting of 10〃1 of 20 mM Tris-HCl (pH 8.5), lOmM magnesium chloride, ImM DTT and lOOmM potassium chloride, and further 10 units of restriction enzyme BamHI ( (Takara Shuzo), restriction enzyme Xhol (Takara Shuzo) and restriction enzyme Stul (Takara Shuzo) were added and reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 0.2 μg of a BamHI-Xhol fragment of about 4.90 kb was recovered.

 Next, the BajnHI-Xhol fragment O.l / zg derived from the plasmid PKANTEX796 obtained above and 0.1 μg of the BamHI-Xhol fragment derived from the plasmid pT666HLCDR were added to a total volume of sterilized water of 20〃1, and -Ligation was performed using To-Go T4 DNA Ligase (Pharmacia). Escherichia coli HB10 verm was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid PKANTEX666HLCDR shown in FIG.

 (2) Expression of anti-GD2 CDR-grafted antibody in animal cells

 The anti-GD2 CDR-grafted antibody expression vector obtained in Example 3, paragraph 3 (1)

YB2 / 0 cells (ATCC CRL1581) were transformed with 4 μg of PKANTEX666HLCDR according to the method described in paragraph 2 (2) of Example 2, and finally G418 (0.5 mg / ml) and MTX (200 nM ) To obtain transformed cell clone KM8138 showing an expression level of about 5 μg / 10 ⁶ cells / 24 hours. KM8138 was deposited as FERM BP-6788 on July 22, 1999 with the Institute of Biotechnology, Institute of Biotechnology, Industrial Science and Technology Institute (1-3 Tsukuba East, Ibaraki, Japan). .

 (3) Purification of anti-GD2 CDR-grafted antibody from culture supernatant

The transformed cell clone KM8138 expressing the anti-GD2 CDR-grafted antibody obtained in Example 3, paragraph (2) was cultured according to the method described in Example 2, paragraph (4), and about 1 L of the culture supernatant was used. About 5 m of the purified anti-GD2 CDR-grafted antibody KM8138 was obtained. Figure 17 shows the purified KM8138 The result of SDS-PAGE was shown. As shown in FIG. 17, it was confirmed that KM8138 was expressed as an antibody molecule having a correct structure consisting of an H chain of about 50 Kd and an L chain of about 25 Kd. In addition, their molecular weights are almost the same as the molecular weights estimated from the nucleotide sequences of KM8138 H and L chain cDNAs (H chain: about 49 Kd, L chain: about 23.5 Kd, whole molecule: about 145 Kd) did. Further, the N-terminal amino acid sequences of the purified H and L chains of KM8138 were analyzed by automatic Edman degradation using a protein sequencer (470A, manufactured by Applied Biosystems), and as a result, the protein was designed in Example 3, item 1 (1). It was confirmed that it matched the N-terminal amino acid sequence of MM666H and MM666L.

 4. Activity evaluation of anti-GD2 CDR-grafted antibody

 (1) Reactivity of anti-GD2 CDR-grafted antibody to GD2 (ELISA)

 The reactivity of the purified anti-GD2 CDR-grafted antibody KM8138 to GD2 was measured according to the method described in Example 2, section 3 (1). Figure 18 shows that the amount of GD2 adsorbed on each well of the EUSA plate was fixed at 20 pmol / well, and the reactivity was examined by changing the concentration of the added anti-GD2 chimeric antibody KM1138 and anti-GD2 CDR-grafted antibody KM8138. The result. As shown in FIG. 18, the anti-GD2 CDR-grafted antibody KM8138 was shown to have the same GD2 binding activity as the anti-GD2 chimeric antibody KM1138. Figure 19 shows the results of examining the reactivity of the anti-GD2 chimeric antibody KM1138 and the anti-GD2 CDR-grafted antibody KM8138 at a constant concentration (10 zg / ml) by changing the amount of GD2 adsorbed to each well of the ELISA plate. It is. As shown in FIG. 19, the anti-GD2 CDR-grafted antibody KM8138 was shown to have the same GD2 binding activity as the anti-GD2 chimeric antibody KM1138. Fig. 20 shows the constant concentration (li ^ g / ml) of anti-GD2 chimeric antibody KM1138 and anti-GD2CDR by changing the type of ganglioside adsorbed to each well of the ELISA plate (adsorption amount: 20 pmol / well). This is the result of examining the reactivity of the transplant antibody KM8138. As shown in FIG. 20, the anti-GD2 CDR-grafted antibody KM8138 was shown to specifically bind to GD2 in the same manner as the anti-GD2 chimeric antibody KM1138.

 (2) Reactivity of anti-GD2 CDR-grafted antibody with human cancer cells (fluorescent antibody method)

The reactivity of the purified anti-GD2CDR-grafted antibody KM8138 with human cancer cells was measured according to the method described in Example 2, section 3 (2). As human cancer cell lines, a human neuroblastoma cell line IMR32 (ATCC CCL127) and a human brain tumor cell line T98G (ATCC CRL1690) were used. The results are shown in FIG. As shown in Fig. 21, anti-GD2 CDR-grafted antibody KM8138 (upper) reacts with IMR32 and T98G with the same strength as anti-GD2 chimeric antibody KM1138 (middle). did.

 (3) In vitro cytotoxic activity (CDC activity) of anti-GD2 CDR-grafted antibody

 The CDC activity of the anti-GD2 CDR-grafted antibody KM8138 was measured according to the method described in Example 2, section 3 (3). The results are shown in FIG. As shown in FIG. 22, it was confirmed that the anti-GD2 CDR-grafted antibody KM8138 had a high CDC activity equivalent to that of the anti-GD2 chimeric antibody KM1138.

 (4) In vitro cytotoxic activity (ADCC activity) of anti-GD2 CDR-grafted antibody

 The ADCC activity of the anti-GD2 CDR-grafted antibody KM8138 was measured according to the method described in Example 2, section 3 (4). The ratio of effector cells to target cells was 100: 1. The results are shown in FIG. As shown in FIG. 23, it was confirmed that the anti-GD2 CDR-grafted antibody KM8138 had as high ADCC activity as the anti-GD2 chimeric antibody KM1138. The results of paragraph 4 (1) to (4) of Example 3 indicate that the anti-GD2 CDR-grafted antibody KM8138 is useful for the diagnosis and treatment of human cancer, as well as the anti-602 chimeric antibody KM1138.

Example 4. Preparation of fusion protein of anti-GD2 humanized antibody and human cytokine

 As a specific example of a fusion protein of anti-GD2 humanized antibody and human cytokinin, KM8138-hIL-2, a fusion protein of anti-GD2 CDR-grafted antibody KM8138 and human IL-2, was prepared as follows. The activity was evaluated.

 1. Construction of cDNA encoding fusion protein between hCα1 and hIL-2

 (1) Construction of plasmid pBSAhCα1-IL-2 having cDNA consisting of about 65 bases at the 3 'end of cMA of hCyl and full-length cMA of mature hIL-2

 Add 3 μg of plasmid pBluescript SK (-) (Stratagene) to a buffer consisting of 10〃1 lOmM Tris-HCl (PH7.5), lOmM magnesium chloride and ImM DTT, and add another 10 units of restriction enzyme. Apal (Takara Shuzo) was added and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol, added to a buffer solution consisting of 10〃1 of 20 mM Tris-hydrochloric acid (pH 8.5), 10 mM magnesium chloride, ImM DTT and 100 mM potassium chloride, and further added 10 units of restriction enzyme BamHI (Takara Shuzo Co., Ltd.). ) Was added and reacted at 30 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2.95 g of an Apal-BamHI fragment of about 2.95 kb was recovered.

Next, synthetic DNAs having the nucleotide sequences of SEQ ID NOs: 27 and 28 were synthesized using an automatic DNA synthesizer (380A, manufactured by Applied Biosystems). 0.3 g of each of the obtained synthetic DNAs was added to 15-1 sterile water, and heated at 65 C for 5 minutes. Bring the reaction to room temperature And left for 30 minutes, add 2〃10x buffer [500 mM Tris-HCl (pH7.6), 100 mM magnesium chloride, 50 mM DTT] and 2〃1 lOmM ATP, and add 10 units of T4 Polynucleotide Kinase (Takara Shuzo) was added and reacted at 37 ° C for 30 minutes to phosphorylate the 5 'end. Next, 0.1 μg of the Apal-BajnHI fragment derived from the plasmid HpBluescript SK (-) obtained above and phosphorylated synthetic MAO.05 / g were added to a total of 10〃1 sterile water, and the DNA ligation Kit Ver.2 ( (Takara Shuzo) according to the instruction manual. Escherichia coli DH5 strain (manufactured by Stratagene) was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain a plasmid pBSA-B shown in FIG. Using 10 g of the obtained plasmid, react with AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, and electrophoresed with ALF DNA Sequencer (Pharmacia) to determine the nucleotide sequence. It was confirmed that a plasmid in which the DNA was cloned was obtained. Next, 3 / g of the plasmid pBSA-B obtained above was added to a buffer solution consisting of 10/1 50 mM Tris-hydrochloric acid (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and lmM DTT. 10 units of restriction enzyme EcoRI (Takara Shuzo) was added and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol, added to a buffer solution consisting of 10〃1 of 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT and 100 ig / ml BSA. The restriction enzyme Smal (Takara Shuzo) was added and reacted at 30 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and an EcoRI-Smal fragment of about 3.00 kb was recovered.

Next, the following PCR was performed using Plasmid KpILL4 [Agric. Biol. Chem., 51, 1135 (1987)] containing the full-length cDNA of mature hIL-2 as type III. Add lng of plasmid pILL4 to 100〃1 reaction mixture [1x concentration Ex Taq buffer (Takara Shuzo), 200〃M dNTPs, 1.0 / M revl primer (SEQ ID NO: 29), 1.0〃 ^ 1 ^ 2 primer (SEQ ID NO: 30) and 2.5 units of TaKaRa Ex Taq DNA polymerase (manufactured by Takara Shuzo)], covered with 100] 1 mineral oil, and placed in a DNA thermocycler (PJ480, manufactured by PERKIN ELMER). After reacting at C for 3 minutes, 30 cycles of 96 ° C for 30 seconds, 55 ° C for 1 minute, 72 ° C for 1 minute are performed 30 times, and finally, reaction at 72 ° C for 7 minutes Was done. The reaction solution was precipitated with ethanol, added to a buffer containing 30% of 50 mM Tris-hydrochloric acid (PH7.5), 100 mM sodium chloride, 10 mM magnesium chloride and ImM DTT, and further 10 units of restriction enzyme EcoRI (Takara Shuzo Co., Ltd.). ) Was added and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol and 10〃1 of 33 mM In addition to a buffer consisting of squirt-acetic acid (pH 7.9), 66πιΜ potassium acetate, lOmM magnesium acetate, 0.5 mM DTT and 100 g / ml BSA, add 10 units of restriction enzyme Smal (Takara Shuzo) and add 30 ° The reaction was performed at C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about llg of an EcoRI-Smal fragment of about 0.41 kb was recovered.

 Next, 0.1 〃g of the EcoRI-Smal fragment of the plasmid pBSA-B obtained above and 0.1 〃g of the EcoRI-Smal fragment of the PCR product of the full-length cDNA of mature hIL-2 were used in a total amount of 10 〃1. In addition to sterile water, ligation was performed using DNA ligation Kit Ver.2 (Takara Shuzo) according to the instruction manual. Using the recombinant plasmid DNA solution thus obtained, Escherichia coli DH5 strain was transformed. Each plasmid DNA was prepared from 10 clones of the transformant, reacted with the AutoRead Sequencing Kit (Pharmacia) according to the attached instructions, and then electrophoresed with ALF DNA Sequencer (Pharmacia) and inserted. As a result of determining the nucleotide sequence of the obtained cDNA, the plasmid pBSAhCat IL-2 shown in FIG. 25 having the desired nucleotide sequence was obtained.

 (2) Construction of plasmid pBShCat IL-2 having cDNA consisting of full-length cDNA of hCα1 and full-length cDNA of mature hIL-2

First, 3 〃g of plasmid pBShCα1 described in Japanese Patent Application No. 10-257893 was added to a buffer solution consisting of 10〃1 lOmM tris-hydrochloride (PH7.5), lOmM magnesium chloride and ImM DTT. Was added with 10 units of restriction enzyme Apal (Takara Shuzo) and reacted at 37 ° C for 1 hour. The reaction solution was precipitated with ethanol, added to a buffer consisting of 10〃1 of 50 mM Tris-HCl (pH 7.5), lOm magnesium chloride, ImM DTT and lOOm sodium chloride, and further added 10 units of restriction enzyme EcoT22I (Takara Shuzo Co., Ltd.). Was added and the mixture was reacted at 37 ° C for 1 hour. The reaction mixture was fractionated by agarose gel electrophoresis, and about 1 g of an ApaI-EcoT22I fragment of about 0.92 kb was recovered. Next, 3 μg of the plasmid pBSAhCat IL-2 obtained in paragraph (1) of Example 4 above was combined with 10 μl of 10 mM Tris-HCl (pH 7.5), lOmM magnesium chloride and ImM DTT And 10 units of restriction enzyme Apal (Takara Shuzo Co., Ltd.) was added thereto, followed by reaction at 37 ° C. for 1 hour. The reaction solution was precipitated with ethanol, added to a buffer consisting of 10〃1 of 50 mM Tris-HCl (pH 7.5), lOmM magnesium chloride, ImM DTT and lOOmM sodium chloride, and further added 10 units of restriction enzyme EcoT22I (Takara Shuzo Co., Ltd.). Was added and the mixture was reacted at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and about 2 μg of an Apal-EcoT22I fragment of about 3.40 kb was recovered. Next, add 0.1 μg of the ApaI-EcoT22I fragment of the plasmid pBShCα1 obtained above and 0.1 μg of the Apal-EcoT22I fragment of the plasmid pBSAhCATAIL-2 to a total of 10〃1 sterile water, and use the DNA ligation Kit. Ver.2 (Takara Shuzo) was connected according to the instruction manual. Escherichia coli DH5 strain was transformed using the recombinant plasmid DNA solution obtained in this manner to obtain plasmid pBShCIL-2 shown in FIG. Using 10 / g of the obtained plasmid, react with AutoRead Sequencing Kit (Pharmacia) according to the instructions attached, electrophorese with ALF DNA Sequencer (Pharmacia), and determine the nucleotide sequence of the inserted cDNA. As a result of the determination, it was confirmed that a plasmid having the target nucleotide sequence was obtained.

 2. Stable expression of KM8138-hIL-2 in animal cells

 (1) Construction of KM8138—hIL-2 stable expression vector

 Fusion of PKANTEX666HLCDR, a stable expression vector of anti-GD2CDR-grafted antibody KM8138 obtained in section 3 (1) of Example 3 with hCy1 and hIL-2 obtained in section 1 (2) of Example 4 above A stable expression vector of KM8138-hIL-2 was constructed as follows using plasmid pBShCat IL-2 having cDNA encoding the protein. One

3 g of the plasmid PKANTEX666HLCDR obtained in Section 3 (1) of Example 3 was added to a buffer solution consisting of 10 〃 1 of 10 mM Tris-hydrochloric acid (PH7.5), 10 mM magnesium chloride and ImM DTT. A unit of restriction enzyme Apal (manufactured by Takara Shuzo) was added and reacted at 37 ° C. for 1 hour. The reaction mixture was precipitated with ethanol, added to a buffer solution consisting of 10 1 of 201 Tris-hydrochloric acid (pH 8.5), lOmM magnesium chloride, lmM DTT and lOOmM potassium chloride, and further added 10 units of restriction enzyme BajnHI (Takara Shuzo). Was added and reacted at 30 ° C. for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and an Apal-BamHI fragment of about 12.57 kb was recovered at about 2 / g. Next, the plasmid pBShC protein obtained in Example 4, paragraph 1 (2) was recovered. Add 3〃g of IL-2 to a buffer solution consisting of 10 101 of 10mM Tris-HCl (pH 7.5), lOmM magnesium chloride and lmM DTT, and further add 10 units of restriction enzyme Apal (Takara Shuzo). The reaction was performed at 37 ° C for 1 hour. The reaction mixture was precipitated with ethanol, added to a buffer consisting of 10〃1 of 20 mM Tris-HCl (pH 8.5), lOmM magnesium chloride, lmM DTT and lOOmM potassium chloride, and further added 10 units of restriction enzyme BamHI (Takara Shuzo Co., Ltd.). ) Was added and reacted at 30 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis, and the Apal-BamHI fragment of about 1.45 kb was Received.

 Next, 0.1 μg of the Apal-BamHI fragment derived from the plasmid PKANTEX666HLCDR obtained above and 0.1 μg of the Apal-BamHI fragment derived from the plasmid pBShCatIL-2 were added to the entire amount of sterilized water, and the DNA ligation Kit Ver. .2 (Takara Shuzo) according to the instruction manual. Using the recombinant plasmid DNA solution thus obtained, Escherichia coli DH5 strain was transformed to obtain a stable expression vector pKANTEX8138-hIL2 of KM8138-hIL-2 shown in FIG. After 10 µg of the obtained plasmid was reacted with the AutoRead Sequencing Kit (Pharmacia) according to the instructions attached thereto, it was subjected to electrophoresis using an ALF DNA Sequencer (Pharmacia), and the nucleotide sequence was determined. It was confirmed that a plasmid in which the DNA was cloned was obtained.

 (2) Expression of KM8138-hIL-2 in animal cells

Using 4 / g of the stable expression vector PKANTEX8138-hIL2 of KM8138-hIL-2 obtained in Section 2 (1) of Example 4, YB2 / 0 cells (ATCC CRL1581), and finally selected with G418 (0.5 mg / ml) and MTX (200 nM), and the transformed cells showing an expression level of about 4 μg / 10 ⁶ cells / 24 hours The clone KM8138hIL2 was obtained. KM8138hIL2 has been deposited as FERM BP-6789 on July 22, 1999 with the Institute of Biotechnology, Industrial Science and Technology, Tsukuba-Higashi 1-chome, Ibaraki Pref., Japan.

 (3) KM8138—purification of ML-2 from culture supernatant

 The transformed cell clone KM8138hIL2 that expresses KM8138-hIL-2 obtained in Example 4, paragraph 2 (2) is cultured according to the method described in Example 2, paragraph 2 (4), and the culture supernatant is removed. About 9.7 m of purified KM8138-hIL-2 was obtained from about 3 L. FIG. 28 shows the result of SDS-PAGE of the purified KM8138-hIL-2. As shown in FIG. 28, the purified KM8138-hIL-2 had a molecular weight of about 180 Kd under non-reducing conditions, and two bands of about 65 Kd and about 25 Kd were observed under reducing conditions. These molecular weights are based on the molecular weights estimated from the nucleotide sequences of the KM8138-hIL-2 H chain and hIL-2 and L chain cDNAs (H chain and ML-2: about 64 Kd, L chain: about 23.5 Kd, molecular weight Overall: approximately 177 Kd), confirming that the structure as an antibody molecule is maintained even after fusion of hIL-2.

3. KM8138—hIL-2 activity evaluation

(1) KM8138—Reactivity of hIL-2 to GD2 (EUSA method) The reactivity of purified KM8138-hIL-2 to GD2 was measured according to the method described in Example 2, section 3 (1). However, a peroxidase-labeled goat anti-human IgG (H & L) antibody (manufactured by American Qualex, diluted 1: 3000 with 1 BSA-PBS) was used as the secondary antibody solution. Fig. 29 shows that the amount of GD2 adsorbed to each well of the ELISA plate was fixed at 20 pmol / well, and the reactivity was examined by changing the concentration of the added anti-GD2 CDR-grafted antibody KM8138 and KM8138-hIL-2. The result. As shown in FIG. 29, KM8138-hIL-2 was shown to have the same binding activity to GD2 as the anti-GD2 CDR-grafted antibody KM8138. Figure 30 shows that the type of ganglioside to be adsorbed to each well of the ELISA plate was changed (adsorption amount: 20 pmol / ゥ ヱ), and a constant concentration (10 / g / ml) of anti-GD2CDR-grafted antibodies KM8138 and KM8138-h It is a result of examining the reactivity of IL-2. As shown in FIG. 30, KM8138-hIL-2 was shown to bind most strongly to GD2, similarly to the anti-GD2 CDR-grafted antibody KM8138. The above results indicate that the activity of KM8138-ML-2 as an anti-GD2CDR-grafted antibody KM8138 is maintained even after fusion with hIL-2.

 (2) Evaluation of hIL-2 activity of KM8138-hIL-2

The activity of purified KM8138-hIL-2 as hIL-2 was measured according to the method described below. HIL- 2 was suspended in RPMI1640- FBS (IO) medium mouse T cell line CTLL 2 a (ATCC TIB214) at a concentration of 2 x l0 ⁵ cells / ml showing the concentration-dependent growth relative, 96-well microphone port In the evening, 50 1 / ゥ l was dispensed into an Italian plate (manufactured by Sumitomo BeiClient). The 50 zl of the solution diluted to various concentrations added at each © E (manufactured by R & D SYSTEMS Inc.) yl the hi 2 or purified KM8138- ML-2 and RPMI1640- FBS (IO) medium, 5% (0 ₂ Inkyube Isseki The cells were cultured for 30 hours at 37. C. After the culture, the number of viable cells was measured using the Cell Counting Kit (manufactured by Dojindo Laboratories) according to the instruction manual, and the results are shown in Fig. 31. As shown in Fig. 31, KM8138-hIL-2 showed the same level of growth supporting activity of CTLL-2 cells as hIL-2.The above results indicate that KM8138-hIL-2 is expressed as hIL-2. This activity is maintained even after fusion with the anti-GD2 CDR-grafted antibody KM8138.

According to the present invention, a monoclonal antibody against GD2, including a novel CDR against GD2, and a fusion protein of the antibody and cytokine are provided. "Rooster System ¹ J Nori Ast"

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Claims

The scope of the claims 1 Human-type complementarity-determining region (CDR) -grafted antibody or antibody fragment that specifically reacts with ganglioside GD2.  2. The human according to claim 1, wherein the human CDR-grafted antibody comprises CDRs of a heavy chain (H chain) variable region (V region) and a light chain (L chain) V region of a monoclonal antibody against ganglioside GD2. Type CDR-grafted antibody or said antibody fragment.  3 The human CDR-grafted antibody contains CDRs of the H chain V region and L chain V region of the monoclonal antibody against ganglioside GD2 and the framework regions (FR) of the H chain V region and L chain V region of the human antibody. A human CDR-grafted antibody according to claim 1 or an antibody fragment thereof.  4 The human CDR-grafted antibody is a monoclonal antibody against ganglioside GD2, the CDRs of the H chain V region and L chain V region, the FRs of the H chain V region and L chain V region of the human antibody, and the H chain constant of the human antibody. The antibody or the antibody fragment thereof according to claim 1, which is a human CDR-grafted antibody comprising a region (C region) and an L chain C region.  The human-type CDR-grafted antibody according to any one of claims 1 to 4, wherein CDR1, CDR2, and CDR3 of the H chain V region of the antibody 5 include the amino acid sequences represented by SEQ ID NOS: 3, 4, and 5, respectively. Or said antibody fragment.  6 The human CDR-grafted antibody according to any one of claims 1 to 4, wherein CDIU of the L chain V region of the antibody, CDH2 and CDR3 each comprise an amino acid sequence represented by SEQ ID NOS: 6, 7 and 8. The antibody fragment.  7 CDR1, CDR2, and CDR3 of the H chain V region of the antibody correspond to SEQ ID NOs: 3, 4, and 5, respectively, and CDR1, CDR2, and CDR3 of the L chain V region correspond to the amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively. The human CDR-grafted antibody or the antibody fragment thereof according to any one of claims 1 to 4, which comprises the antibody.  8. The human CDR-grafted antibody or the antibody fragment thereof according to any one of claims 1 to 4, wherein the H chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 13.  9. The human CDR-grafted antibody or the antibody fragment thereof according to any one of claims 1 to 4, wherein the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 14. The human CDR graft according to any one of claims 1 to 4, wherein the H chain V region of the 10 antibody comprises the amino acid sequence represented by SEQ ID NO: 13 and the L chain V region by SEQ ID NO: 14. Antibodies or The antibody fragment.  The humanized CDR according to any one of claims 1 to 4, wherein the H chain V region of the 11 antibody comprises the amino acid sequence represented by SEQ ID NO: 13 and the L chain V region by SEQ ID NO: 14. The transplant antibody KM8138 or the antibody fragment. 12 Claim that the antibody fragment is an antibody fragment selected from Fab, Fab \ F (ab,) ₂ , single-chain antibody (scFv), disulfide-stabilized V region fragment (dsFv) and a peptide containing CDR. Item 14. The antibody fragment according to any one of Items 1 to 11.  13. A DNA encoding the human CDR-grafted antibody or the antibody fragment thereof according to any one of claims 1 to 12.  1. A recombinant vector containing the DNA according to claim 13.  15. A transformant obtained by introducing the recombinant vector according to claim 14 into a host cell.  16 A transformant KM8138 (FERM BP-6788) that produces the antibody of claim 12. (17) The transformant according to (15) or (16) is cultured in a medium, and the human CDR-grafted antibody or antibody fragment according to claims (1) to (12) is produced and accumulated in the culture. A method for producing an antibody, comprising collecting the antibody.  1 8 CDR1, CDR2, and CDR3 of the V region of the H chain of the monoclonal antibody are SEQ ID NOs: 3, 4, and 5, respectively, and CDR1, CDR2, and CDR3 of the V region of the L chain are the amino acid sequences represented by SEQ ID NOs, 6, 7, and 8, respectively. A monoclonal antibody or an antibody fragment thereof against ganglioside GD2, comprising:  19. The antibody according to claim 18, wherein the 19 monoclonal antibody is selected from an antibody produced by a hybridoma, a humanized antibody, and a human antibody.  The geometry according to claim 19, wherein the class of the 20 monoclonal antibody belongs to the human antibody IgG type.  21. The antibody KM666 or the antibody fragment thereof according to claim 19, wherein the antibody KM666 is produced by 21 mouse hybridoma KM666 (FERM BP-6786). 22. The humanized antibody according to claim 19, wherein the humanized antibody is a human chimeric antibody. 23. The human chimeric antibody or the antibody fragment thereof according to claim 22, wherein the human chimeric antibody comprises an H chain V region and an L chain V region of a monoclonal antibody against ganglioside GD2. 24. The human chimeric antibody according to claim 2, wherein the human-type chimeric antibody is a human chimeric antibody comprising the H chain V region and L chain V region of a monoclonal antibody against ganglioside GD2, and the H chain C region and L chain C region of a human antibody. 3. The human chimeric antibody or the antibody fragment thereof according to 2. 25. The human chimer according to claim 23 or 24, wherein the amino acid sequences of the H chain V region and L chain V region include the amino acid sequences of the H chain V region and L chain V region of mouse monoclonal antibody KM666. An antibody or an antibody fragment thereof.  26. The human chimeric antibody or the antibody fragment thereof according to claim 23, wherein the H chain V region of the antibody 26 comprises the amino acid sequence represented by SEQ ID NO: 32.  27. The human chimeric antibody or the antibody fragment thereof according to claim 23, wherein the L chain V region of the 27 antibody comprises the amino acid sequence represented by SEQ ID NO: 33.  The human chimeric antibody according to claim 23 or 24, wherein the 28 H chain V region comprises an amino acid sequence represented by SEQ ID NO: 32, and the L chain V region comprises an amino acid sequence represented by SEQ ID NO: 33. The antibody fragment.  The human chimeric antibody KM1138 or the human chimeric antibody according to claim 23 or 24, wherein the H chain V region of the 29 antibody comprises the amino acid sequence represented by SEQ ID NO: 32, and the L chain V region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 33. Antibody fragment.  30 A DNA encoding the human chimeric antibody or the antibody fragment thereof according to any one of claims 23 to 29.  31. A recombinant vector containing the DNA of claim 30.  32. A transformed strain obtained by introducing the recombinant vector according to claim 31 into a host cell.  33. A transformant KM1138 (FERM BP-6787) that produces the antibody of claim 29. 34 The transformed strain according to claim 32 or 33 is cultured in a medium, the human chimeric antibody according to claims 23 to 29 is produced and accumulated in the culture, and the antibody is collected from the culture. A method for producing an antibody. 35. Claim that the antibody fragment is an antibody fragment selected from Fab, Fab \ F (ab,) ₂ , a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv), and a peptide including CDR. Item 19. The antibody fragment according to Item 19. 36 The antibody fragment has CDR1, CDR2 and CDR3 of the H chain V region of SEQ ID NOS: 3, 4 and 5, respectively, and the CDR1, CDR2 and CDR3 of the L chain V region has SEQ ID NOs, 6, 7 and 8, respectively. 36. The antibody fragment according to claim 35, comprising the amino acid sequence represented by:  37 Antibody fragment, H chain V region of antibody is SEQ ID NO: 32, L chain V region of antibody is SEQ ID NO: 36. The antibody fragment according to claim 35, comprising the amino acid sequence represented by 33.  38 The antibody or the antibody fragment according to any one of claims 1 to 12, 18, 18 to 29, 35, 36, and 37, and a radioisotope, protein, or small molecule drug. A derivative of an antibody bound to.  39. The antibody derivative according to claim 38, wherein the derivative of the antibody is obtained by binding a radioisotope, a protein, or a low molecular drug to the C-terminal side of the H chain of the antibody.  30. The antibody derivative according to claim 38 or 39, wherein the 40 protein is a cytokine. 41. The antibody derivative according to claim 40, wherein the 41 cytokine is human interleukin 2 (hIL-2).  42. The antibody derivative according to any one of claims 38 to 41, wherein the derivative of the antibody 42 comprises the human CDR-grafted antibody KM8138 and hIL-2.  43. The antibody derivative KM8138-hIL-2 according to any one of claims 38 to 41, wherein the derivative of the 43 antibody comprises a human CDR-grafted antibody KM8138 and ML-2.  4. A DNA encoding a derivative of the antibody according to any one of claims 38 to 43. 45 A recombinant vector containing the DNA according to claim 44.  46.Transformation obtained by introducing the recombinant vector according to claim 45 into a host cell. 47. A transformant KM8138ML2 (FERM BP-6789) that produces the derivative according to claim 43.  48 The transformed strain according to claim 46 or 47 is cultured in a medium, and the derivative of the antibody according to claims 38 to 43 is produced and accumulated in the culture, and the derivative is collected from the culture. A method for producing a derivative of an antibody, comprising:  49 At least one selected from the antibody and the antibody fragment thereof according to claims 1-12, 18-29, 35, 36 and 37, and the derivative of the antibody according to claims 38-43. One kind of medicine. 50 At least one selected from the antibodies and antibody fragments according to claims 1-12, 18-29, 35, 36 and 37, and derivatives of the antibodies according to claims 38-43. A therapeutic agent for cancer containing one as an active ingredient. 51 As an active ingredient, at least one selected from the antibody and the antibody fragment according to claims 1 to 12, 18, 29, 35, 36, and 37 and the derivative of the antibody according to claims 38 to 43 is contained. Diagnostic agent for cancer.