JPH1192500A - Antibodies to human parathyroid hormone-related peptides - Google Patents

Abstract

(57) [Problem] To provide an antibody against a human parathyroid hormone-related peptide. An antibody against a human parathyroid hormone-related peptide, a DNA encoding the antibody, a recombinant vector containing the DNA, a transformant transformed with the recombinant vector, a method for producing the antibody, and the antibody Uses.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human / mouse chimeric antibody comprising a variable region (V region) of a mouse monoclonal antibody to a parathyroid hormone-related peptide and a constant region (C region) of a human antibody, and a parathyroid hormone-related peptide. Light chain of mouse monoclonal antibody against peptide (L
A humanized antibody in which the capture region of the V region and the heavy chain (H chain) V region are grafted to a human antibody; the L chain and the H chain of the antibody; and the L chain of the antibody Or H
The present invention relates to a polypeptide containing a V region constituting a chain.

[0002] The invention further relates to the antibodies described above, in particular their V
The present invention relates to a DNA containing a base sequence encoding a region and a DNA encoding an L chain or H chain containing a V region. The present invention further relates to a recombinant vector containing the DNA, and a host transformed with the vector.

[0003] The present invention further relates to methods for producing chimeric and humanized antibodies against parathyroid hormone-related peptides. The present invention further relates to a pharmaceutical composition containing an antibody against a parathyroid hormone-related peptide as an active ingredient, and a hypercalcemia inhibitor and a hypophosphatemic ameliorating agent.

[0004]

2. Description of the Related Art Hypercalcemia associated with a malignant tumor is a serious complication in 5 to 20% of all malignant tumor patients. It is believed that there is. Control of hypercalcemia has a clinically important role because it greatly affects the treatment prognosis and quality of life (QOL) of patients.

[0005] Hypercalcemia in patients with malignant tumors generally involves HHM due to tumor-producing humoral bone resorption factor.
(Humoral hypercalcemia of malignancy) and LOH (Local O
steolytic hypercalcemia). HHM is thought to increase calcium efflux due to enhanced bone resorption or bone destruction, resulting in hypercalcemia in combination with decreased renal calcium excretion (Seiki Wada and Naoichi Nagata, Internal Medicine 69, 644-648).

[0006] In hypercalcemia, serum calcium levels are increased.
When the dose exceeds 12 mg / dl, it is considered that the symptoms appear, and as the symptoms, anorexia, nausea, and vomiting are non-specifically recognized in malignant tumor patients at an early stage. When hypercalcemia worsens, polyuria occurs due to a decrease in the water concentrating power due to damage to the distal renal tubule, and dehydration occurs due to insufficient intake of water due to nausea and vomiting.

HH among hypercalcemia associated with malignant tumors
As a humoral factor causing M, PTH (parathyroid hormone; Pa
Parathyroid Hormone related Peptid, a substance similar to rathyroid Hormone
e, hereinafter referred to as "PTHrP") was found by Moseley, JM et al. (Proc. Natl. Acad. Sci. USA (1987), 84,
5048-5052).

[0008] Thereafter, the gene encoding PTHrP was isolated (Suva, LJ et al., Science (1987) 237, 893).
The analysis shows that human PTHrP has three types of 139, 141 and 173 amino acids based on the alternative splicing of the gene, and that PTHrP has the full structure in blood.
Various fragments based on limited digestion of P (1-139) were found to exist (Baba, H. Clinical Calc
ium (1995) 5, 229-223). PTHrP is the same as PTH in 8 out of 13 amino acids at the N-terminal 1st to 13th positions,
It is presumed that the amino acids at positions 14 to 34 also exhibit a steric structure similar to that of PTH. At least at the N-terminal side, they bind to the PTH / PTHrP receptor common to PTH (J
ueppner, H. et al., Science (1991) 254, 1024-102
6, Abou-Samra, AB. Et al., Proc. Natl. Acad. Sci.
USA (1992) 89, 2732-2736).

It has been reported that PTHrP is produced from various tumor tissues. Not only tumors but also skin, central nervous system, uterus, placenta, lactating mammary gland, thyroid gland, parathyroid gland, adrenal gland, liver, kidney It has been shown to be produced by various normal tissues from the fetus to the adult, including the bladder (Burtis, WJ Clin. Chem. (1992) 38, 217).
1-2183, Stewart, AF & Broadus, AEJ Clin. E
ndocrinol. (1991) 71, 1410-1414). Also, PTHrP is
It is thought to play an important role in regulating calcium metabolism, which is kept higher than the mother from fetal to neonatal.

The PTH / PTHrP receptor is mainly present in bone and kidney (Shino, Chohei, Clinical Calcium (1995) 5, 355-359),
It is known that binding of PTHrP to a receptor activates a plurality of intracellular signaling systems. One is adenyl cyclase and the other is phospholipase C. Activation of adenyl cyclase increases intracellular cAMP concentration and activates protein kinase A. Phospholipase C also degrades phosphatidylinositol 4,5-bisphosphonate to produce inositol 1,4,5-triphosphonate and diacylglycerol. G proteins are involved in these signaling systems (Coleman, DT et al.,
Biochemical mechanisms of parathyroid hormone acti
on. In: "Theparathyroids" (Bilezikian, JP et a
l.), Raven press, New York, (1994) page 239).

[0011] PTHrP is responsible for the hypercalcemia and hypophosphatemia observed in HHM via these signal transduction systems.
It causes decreased renal phosphorus reabsorption and increased excretion of renal cAMP. Thus, PTHrP has been shown to be closely related to hypercalcemia associated with malignant tumors. For the treatment of hypercalcemia associated with malignant tumors, in addition to rehydration, calcitonin, steroids, indomethacin, inorganic phosphate, bisphosphonate and the like are used. However, these drugs are expected to use drugs having a higher therapeutic effect and fewer side effects, because their effects are reduced by continuous use, strong side effects are exhibited, or the drug effect is lately expressed.

On the other hand, as a new trial for the treatment of hypercalcemia associated with malignant tumors, Kukreja, SC et al. Developed PTHrP into athymic mice in which hypercalcemia occurred by transplanting human lung cancer cells or human laryngeal cancer cells. It has been reported that administration of neutralizing antiserum to sera reduced blood calcium levels and urinary cAMP levels (J. Clin. Invest. (1988)
82, 1798-1802). Sato, M., et al. Reported that administration of an antibody against PTHrP (1-34) to nude mice transplanted with PTHrP-producing human tumors reduced hypercalcemia and greatly increased the survival time of mice. Yes (J. bon
e & Mine. Res. (1993) 8, 849-860). In addition, JP-A-4-24-2
No. 28089 discloses a mouse / human chimeric antibody against human PTHrP (1-34).

[0013] Mouse monoclonal antibodies are highly immunogenic (sometimes referred to as "antigenic") in humans, which limits the therapeutic value of mouse monoclonal antibodies in humans. For example, when a mouse antibody is administered to humans, it can be metabolized as a foreign substance, so that the half-life of mouse antibodies in humans is relatively short, and the expected effects cannot be sufficiently exerted. In addition, human anti-mouse antibodies (HAMA) raised against administered mouse antibodies elicit inconvenient and dangerous immune responses to patients, such as serum sickness or other allergic reactions. Therefore, mouse monoclonal antibodies cannot be frequently administered to humans.

In order to solve these problems, a method for reducing the immunogenicity of a non-human-derived antibody, for example, a mouse-derived monoclonal antibody, has been developed. One method is to prepare a chimeric antibody in which the variable region (V region) of the antibody is derived from the original mouse monoclonal antibody and the constant region (C region) is derived from an appropriate human antibody.

Since the obtained chimeric antibody contains the complete variable region of the original mouse antibody, it can be expected to bind to the antigen with the same specificity as the original mouse antibody. Furthermore, the ratio of amino acid sequences derived from non-human is substantially reduced in the chimeric antibody, and therefore, it is expected that the immunogenicity is lower than that of the original mouse antibody. Chimeric antibodies bind antigen similarly to the original mouse monoclonal antibody and are less immunogenic, but may still generate an immune response against the mouse variable region (LoBu
glio, AFet al., Proc. Natl. Acad. Sci. USA, 86, 4220-42
24,1989).

The second method for reducing the immunogenicity of a mouse antibody is more complex, but is expected to further reduce the potential immunogenicity of the mouse antibody. In this method, a complementarity determining region (complementarity determining region) is determined from the variable region of a mouse antibody.
n; CDR) alone into a human variable region and "reconstituted"
Generate (reshaped) human variable regions. However, if necessary, in order to bring the CDR structure of the reshaped human variable region closer to the structure of the original mouse antibody, the amino acid sequence of a part of the framework region (FR) supporting the CDR may be replaced with a mouse. In some cases, the variable region of the antibody is transplanted to a human variable region.

Next, these humanized reshaped human variable regions are ligated to human constant regions. The portion derived from the non-human amino acid sequence of the finally reconstructed humanized antibody is only the CDRs and only a part of the FRs. CDR
Are composed of hypervariable amino acid sequences, which do not show species-specific sequences. For this reason, humanized antibodies carrying mouse CDRs should no longer have stronger immunogenicity than native human antibodies containing human CDRs.

For humanized antibodies, Riechm
ann, L. et al., Nature, 332, 323-327, 1988; Verhoeye, Me
t al, Science, 239,1534-1536,1988; Kettleborough,
CAet al., Protein Engng., 4,773-783, 1991; Maeda, H.
et al., Human Antibodies and Hybridoma, 2,124-134,199
1; Gorman, SD et al., Proc. Natl. Acad. Sci. USA, 8
8,4181-4185,1991; Tempest, PRet al., Bio / Technolo
gy, 9, 266-271, 1991; Co, MSet al., Proc. Natl. A
cad.Sci. USA, 88, 2869-2873, 1991; Carter, P. et al.,
Proc. Natl. Acad. Sci. USA, 89,4285-4289,1992; C
o, MS et al., J. Immunol., 148, 1149-1154, 1992; and Sato, K. et al., Cancer Res., 53, 851-856, 1993.

As described above, humanized antibodies are expected to be useful for therapeutic purposes, but humanized antibodies against PTHrP are not known, and the literature does not suggest them. In addition, there is no uniform method that can be universally applied to any antibody in the method for producing a humanized antibody, and a humanized antibody showing sufficient binding activity and neutralizing activity for a specific antigen Requires various contrivances (for example, Sato, K. et al., Cancer Res., 53, 851-8).
56, 1993).

[0020]

The present invention relates to a human / mouse chimeric antibody comprising a variable region (V region) of a mouse monoclonal antibody to PTHrP and a constant region (C region) of a human antibody, and a mouse monoclonal antibody to PTHrP. The humanization (humani) in which the light-chain (L-chain) V region and heavy-chain (H-chain) V-region capture-determining regions are transplanted into a human antibody
zed) antibody, light chain and heavy chain of the antibody, and light chain of the antibody
It is an object of the present invention to provide a polypeptide comprising a V region constituting a chain or an H chain.

The present invention further relates to the antibodies described above, in particular
DNA encoding a base sequence encoding a region, and DN encoding an L chain or H chain including a polypeptide including a V region
A is intended to be provided. The present invention further provides the D
It is an object to provide a recombinant vector containing NA and a host transformed with the vector. Another object of the present invention is to provide a method for producing a chimeric antibody and a humanized antibody against PTHrP. Another object of the present invention is to provide an antibody against PTHrP having a high neutralizing activity. Another object of the present invention is to provide a pharmaceutical composition containing an antibody to PTHrP or a humanized antibody as an active ingredient, and a hypercalcemia inhibitor, hypophosphatemia improver and alkalosis improver.

[0022]

Means for Solving the Problems The present inventors have conducted intensive studies based on the above-mentioned problems, and as a result, have succeeded in obtaining an antibody in which the immunogenicity of a mouse monoclonal antibody against PTHrP in humans has been reduced. Thus, the present invention has been completed. That is, the present invention provides an L chain C region of a human antibody,
Chain V of mouse monoclonal antibody against PTHrP and PTHrP
It is a chimeric L chain containing a region. Examples of the L chain V region include those containing the amino acid sequence represented by SEQ ID NO: 45, and examples of the L chain C region include a Cλ region.

Further, the present invention relates to the C region of the H chain of a human antibody and the H region of a mouse monoclonal antibody against PTHrP.
It is a chimeric H chain containing a chain V region. Examples of the H chain V region include those containing the amino acid sequence represented by SEQ ID NO: 46, and examples of the C region include those of the Cγ1 region. Furthermore, the present invention is a chimeric monoclonal antibody against PTHrP, comprising the chimeric L chain and the chimeric H chain.

Further, the present invention provides a framework region 1 to 4 of the L chain V region of a human antibody and a complementarity determining region 1 of the L chain V region of a mouse monoclonal antibody to PTHrP.
A polypeptide comprising an L chain V region of a humanized antibody, comprising: Examples of the complementarity determining regions 1 to 3 include those containing the amino acid sequences represented by SEQ ID NOS: 59 to 61, respectively. The framework regions 1 to 3 are derived from the framework regions 1 to 3 of the human antibody HSU03868, respectively, and the framework region 4 is the human antibody S257
55 framework 4 origin. Alternatively, the framework regions 1 to 3 are substantially the same as the framework regions 1 to 3 of the human antibody HSU03868, respectively, and the framework region 4 is a human antibody
Substantially the same as the framework region 4 of S25755.

Here, “substantially the same” means that the mouse monoclonal antibody has the same activity as the mouse monoclonal antibody in the framework region of the human antibody used in the humanized antibody. It means that deletions, substitutions, additions, and the like of amino acids necessary for forming the complementarity determining region of the antibody may occur.

Furthermore, the present invention relates to the definition of Kabat in the framework region (Kabat, EA et al., US Dept. Health and
Human Services, US Government Printing Offices, 199
The 36th amino acid according to 1) is tyrosine, and
A polypeptide comprising an L chain V region of a humanized antibody, wherein the 49th amino acid is aspartic acid.

Further, the present invention relates to a humanized antibody L comprising any one of the amino acid sequences represented by SEQ ID NOs: 48 to 51.
A polypeptide comprising a chain V region. Further, the present invention provides a polypeptide comprising the L chain V region of a humanized antibody, wherein the amino acid at position 45 according to Kabat's definition in the framework region is lysine and the amino acid at position 87 is isoleucine. Is a peptide. Furthermore, the present invention is a polypeptide comprising the L chain V region of a humanized antibody, comprising any of the amino acid sequences represented by SEQ ID NOS: 52 to 55.

Furthermore, the present invention provides a humanized humanized antibody comprising framework regions 1 to 4 of the H chain V region of a human antibody and complementarity determining regions 1 to 3 of the H chain V region of a mouse monoclonal antibody to human PTHrP. It is a polypeptide containing the V region of the H chain of the antibody. Examples of the complementarity determining regions 1 to 3 include those containing the amino acid sequences represented by SEQ ID NOs: 62 to 64, and the framework regions 1 to 4 include human subgroup III (Human Subgroup III (HSG III)). , Kaba
t, EAet al., US Dept.Health and Human Services, US
(Government Printing Offices, 1991), derived from framework regions 1 to 4 of human antibodies, more specifically, those derived from framework regions 1 to 4 of human antibody S31679, respectively, or framework regions of human antibody S31679. Substantially the same as 1-4.

Furthermore, the present invention relates to a polypeptide comprising the H chain V region of a humanized antibody, comprising the amino acid sequence represented by SEQ ID NO: 56. Further, the present invention provides a polypeptide comprising the L chain V region of the humanized antibody and the L
The light chain of a humanized antibody against human PTHrP containing a polypeptide comprising a chain C region. Here, the C region is a Cλ region, the framework regions 1 to 3 are substantially the same as the framework regions 1 to 3 of the human antibody HSU03868, respectively, and the framework region 4 is a human antibody
Substantially the same as framework region 4 of S25755,
Examples of the amino acid sequences of the complementarity determining regions 1 to 3 include those represented by SEQ ID NOS: 59 to 61, respectively.

Furthermore, the present invention relates to the human antibody C
The heavy chain of a humanized antibody against human PTHrP, including a polypeptide comprising a region and a polypeptide comprising a heavy chain V region. The C region is the Cγ1 region, the framework regions 1 to 4 are those derived from framework regions 1 to 4 derived from a human antibody belonging to HSGIII, and the complementarity determining region 1
Nos. To 3 include those containing the amino acid sequences represented by SEQ ID NOs: 62 to 64, respectively.

Furthermore, the present invention relates to an anti-PTHrP antibody having weak antigenicity and high neutralizing activity. The PTHrP antibody includes a human antibody, a humanized antibody, a chimeric antibody, a primed antibody, and the like, which can be used for treatment of human diseases.
In addition, the antibody has a low dissociation constant. Furthermore, since the antibody of the present invention has a low dissociation constant and a high neutralizing activity, it can be used for treating human diseases.

The antibody of the present invention has a dissociation constant of 1.86 × 10 ⁻⁷ [M] or less, a dissociation rate constant of 1.22 × 10 ⁻¹ [1 / Sec] or less, and 6.55 × 10 ⁴ [1 / M.Sec]. It has the above binding rate constant. Further, these constants can be measured by Scatchard analysis using a RI-labeled ligand, a surface plasmon resonance sensor, or the like.

Furthermore, the present invention relates to a DNA containing a nucleotide sequence encoding the L chain V region or a DNA containing a nucleotide sequence encoding the H chain V region of a mouse monoclonal antibody against human PTHrP. Examples of the L chain V region and the H chain V region include those containing the amino acid sequences represented by SEQ ID NOS: 45 and 46, and examples of the DNA containing the base sequence encoding the L chain V region include SEQ ID NO: 65. D containing a nucleotide sequence encoding the H chain V region
Examples of NA include those represented by SEQ ID NO: 57.

Furthermore, the present invention is a DNA encoding the chimeric L chain or the chimeric H chain. Examples of the DNA encoding the L chain include those containing the nucleotide sequence represented by SEQ ID NO: 65, and examples of the DNA encoding the H chain include those containing the nucleotide sequence represented by SEQ ID NO: 57. .

Further, the present invention relates to the humanized antibody L
DNA containing a base sequence encoding the V region of the H chain or DNA containing the base sequence encoding the V region of the H chain. The DNA containing the nucleotide sequence encoding the L chain V region includes SEQ ID NO:
Examples include those containing any of the nucleotide sequences represented by SEQ ID NOs: 66 to 74, and examples of the DNA containing the nucleotide sequence encoding the H chain V region include those represented by SEQ ID NO: 58.

Further, the present invention relates to a DNA of the L chain V region of a humanized antibody, comprising a base sequence encoding any of the amino acid sequences represented by SEQ ID NOs: 47 to 55. The DN
Examples of A include those containing any of the base sequences represented by SEQ ID NOs: 66 to 74. Furthermore, the present invention relates to a DNA for the H chain V region of a humanized antibody, which encodes the amino acid sequence represented by SEQ ID NO: 56. Examples of the DNA include a DNA containing the base sequence represented by SEQ ID NO: 58.

Further, the present invention provides any one of the above DNAs
It is a recombinant vector containing: Furthermore, the present invention is a transformant transformed by the above-mentioned recombinant vector. Furthermore, the present invention provides a method for culturing the transformant and collecting a chimeric antibody or a humanized antibody against a human parathyroid gland-related peptide from the obtained culture. This is a method for producing a shaped antibody.

Further, the present invention is a pharmaceutical composition containing the antibody as an active ingredient, and a hypercalcemia inhibitor and a hypophosphatemia ameliorating agent. The calciumemia is caused by a malignant tumor, and hypophosphatemia is often observed in patients with hypercalcemia associated with a malignant tumor. Therefore, the antibody of the present invention can be used for treating the above malignant tumors or reducing hypercalcemia or hypophosphatemia. In addition, as a malignant tumor, for example, pancreatic cancer, lung cancer, pharyngeal cancer, laryngeal cancer, tongue cancer,
Gingival, esophageal, gastric, bile duct, breast, kidney, bladder,
At least one selected from the group consisting of uterine cancer, prostate cancer, and malignant lymphoma is included, but is not limited to these cancers, and all malignant tumors that cause hypercalcemia are those of the present invention. It can be the subject of application of the agent. Hereinafter, the present invention will be described in detail.

[0039]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Preparation of a mouse monoclonal antibody against human PTHrP A mouse monoclonal antibody against PTHrP is prepared by preparing a hybridoma by cell fusion of an antibody-producing cell obtained from an animal immunized with an antigen with myeloma cells, and specifically measuring PTHrP activity from the obtained hybridoma. It can be prepared by selecting a clone that produces an inhibitory antibody.

(1) Preparation of Antigen The PTHrP used for immunization of animals includes all or part of the amino acid sequence of PTHrP prepared by a recombinant DNA method or chemical synthesis, or cancer cells that cause hypercalcemia. And PTHrP derived from the culture supernatant. For example, the known PTHrP (Kemp, BE et al., Science
(1987) 238, 1568-1570), the peptide consisting of the 1st to 34th amino acids (PTHrP (1-34)) can be used as the antigen. Note that human PTHrP (1-34) has the amino acid sequence represented by SEQ ID NO: 75.

After binding the obtained PTHrP to a carrier protein (eg, thyroglobulin), an adjuvant is added. Examples of the adjuvant include Freund's complete adjuvant and Freund's incomplete adjuvant, and any of these may be mixed.

(2) Immunization and collection of antibody-producing cells The antigen obtained as described above is administered to mammals, for example, mammals such as mice, rats, horses, monkeys, rabbits, goats and sheep. Immunization can be performed by any of the existing methods, but is mainly performed by intravenous injection, subcutaneous injection, intraperitoneal injection, or the like. The interval of immunization is not particularly limited, and immunization is performed at intervals of several days to several weeks, preferably at intervals of 4 to 21 days.

The antibody-producing cells are collected 2-3 days after the last immunization. Antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, and spleen cells are generally used. The amount of antigen immunization used is 100 μg per mouse at a time.

(3) Measurement of antibody titer In order to confirm the immune response level of the immunized animal and to select a desired hybridoma from the cells after the cell fusion treatment, the antibody titer in the blood of the immunized animal or the antibody was determined. The antibody titer in the culture supernatant of the producer cells is measured. Examples of the method for detecting the antibody include known techniques such as EIA (enzyme immunoassay), RIA (radioimmunoassay), and ELISA (enzyme linked immunosorbent assay).

(4) Cell fusion Myeloma (myeloma) cells to be fused with antibody-producing cells are derived from various animals such as mice, rats, and humans.
Use cell lines generally available to those of skill in the art. As a cell line to be used, a cell line which has drug resistance, cannot survive in a selection medium (for example, HAT medium) in an unfused state, and can survive only in a fused state is used. Generally, an 8-azaguanine resistant strain is used. This cell line lacks hypoxanthine-guanine-phosphoribosyltransferase and cannot grow on a hypoxanthine-aminopterin-thymidine (HAT) medium.

Myeloma cells include various cell lines already known, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 12
3: 1548-1550), P3x63Ag8U.1 (Current Topics in Micr
obiology and Immunology (1978) 81: 1-7), NS-1 (Kohle
r, G. and Milstein, C., Eur. J. Immunol. (1976) 6: 511-51
9), MPC-11 (Margulies, DH et al., Cell (1976) 8:40
5-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276:
269-270), FO (de St. Groth, SF et al., J. Immunol. M
ethods (1980) 35: 1-21), S194 (Trowbridge, IS, J.
Exp.Med. (1978) 148: 313-323), R210 (Galfre, G. et a
l., Nature (1979) 277: 131-133) and the like are preferably used.

Antibody-producing cells are obtained from spleen cells, lymph node cells, and the like. That is, spleen from the various animals,
Lymph nodes and the like are removed or collected, and these tissues are crushed.
The obtained crushed product is suspended in a medium or buffer such as PBS, DMEM, RPMI1640 or the like, filtered through a stainless steel mesh or the like, and then centrifuged to prepare a desired antibody-producing cell.

Next, the myeloma cells are fused with the antibody-producing cells. For cell fusion, MEM, DMEM, RPME-1
Contacting myeloma cells and antibody-producing cells in a medium for animal cell culture such as 640 medium at a mixing ratio of 1: 1 to 1:10 at 30 to 37 ° C for 1 to 15 minutes in the presence of a fusion promoter. Done by In order to promote cell fusion, a fusion promoter or a fusion virus such as polyethylene glycol, polyvinyl alcohol or Sendai virus having an average molecular weight of 1,000 to 6,000 can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (eg, electroporation).

(5) Selection and Cloning of Hybridoma A desired hybridoma is selected from the cells after the cell fusion treatment. Examples of the method include a method utilizing selective growth of cells in a selection medium. That is, the cell suspension is diluted with an appropriate medium, spread on a microtiter plate, a selection medium (such as a HAT medium) is added to each well, and the culture is performed after appropriately changing the selection medium. As a result, the cells that grow can be obtained as hybridomas. Hybridoma screening is performed by a limiting dilution method, a fluorescence excitation cell sorter method, or the like, and finally a monoclonal antibody-producing hybridoma is obtained.

(6) Collection of Monoclonal Antibody As a method for collecting a monoclonal antibody from the obtained hybridoma, a usual cell culture method, ascites formation method and the like can be mentioned. In cell culture methods, hybridomas
RPMI-1640 medium containing 10-20% fetal bovine serum, MEM medium,
Alternatively, the cells are cultured in an animal cell culture medium such as a serum-free medium under ordinary culture conditions (for example, 37 ° C., 5% CO ₂ concentration) for 2 to 14 days, and the antibody is obtained from the culture supernatant.

In the ascites formation method, a hybridoma is administered intraperitoneally to an animal of the same species as a mammal derived from a myeloma cell, and the hybridoma is grown in large quantities. And
One to four weeks later, ascites or serum is collected. In the above antibody collection method, if purification of the antibody is required, a known method such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, or the like is appropriately selected, or the purification is performed by combining these methods. I do.

2. Construction of Chimeric Antibody (1) Cloning of DNA Containing Base Sequence Encoding V Region of Mouse Monoclonal Antibody Against Human PTHrP (i) Preparation of mRNA V of Mouse Monoclonal Antibody Against Human PTHrP
To perform cloning of the DNA containing the nucleotide sequence encoding the region, known methods such as guanidine-ultracentrifugation (Chirgwin, JM et al.) Are used from the recovered hybridomas.
Biochemistry (1979), 18, 5294-5299), the AGPC method (Chomczynski, P et al., Analytical Biochemistry (198
7), 162, 156-159), etc., and prepare total RNA.
A Attached to Purification Kit (Pharmacia)
MRNA is prepared using an Oligo (dT) -cellulose span column or the like. Also, Quick Prep mRNA Purification Kit
By using (Pharmacia), mRNA can be prepared without performing a total RNA extraction operation.

(Ii) Preparation and Amplification of cDNA From the mRNA obtained in (i) above, cDNA in the V region of the L chain and H chain is synthesized using reverse transcriptase. cDNA is synthesized by using Oligo-dT primer or L chain C
An appropriate primer (for example, an MHC2 primer having the base sequence represented by SEQ ID NO: 1) that hybridizes with the region or the H chain C region can be used. cDN
In the A synthesis reaction, the mRNA and the primer are mixed,
The reaction is carried out, for example, at 52 ° C. for 30 minutes in the presence of reverse transcriptase.

The amplification of the cDNA was carried out for both the L and H chains.
5 'using 5'-Ampli FINDER RACE kit (CLONTECH)
-RACE method (Frohman, MA et al., Proc. Natl. Acad. S
ci. USA 85, 8998-9002, 1988; Belyavsky, A. et al., Nucle.
ic Acids Res. 17, 2919-2932, 1989)
(Polymerase chain reaction). That is, Ampli FIND was added to the 5 'end of the cDNA synthesized above.
ER Anchor (SEQ ID NO: 42) is ligated, and a DNA containing a base sequence encoding an L chain V region and an H chain V region (hereinafter, referred to as DNA
The DNA containing the nucleotide sequence encoding the L chain V region may be referred to as “DNA of the L chain V region” or “DN encoding the L chain V region.
PCR is also performed for some cases, which may be abbreviated as “A” (the same applies to the H chain V region, C region, etc.).

As a primer for amplifying the DNA of the L chain V region, for example, Anchor primer (SEQ ID NO: 2) and the L chain λ chain constant region (Cλ region) of a mouse antibody
Primers (for example, SEQ ID NO: 4)
(MLC primer having the base sequence represented by Formula (1)) can be used. Further, as primers for amplifying the DNA of the H chain V region, for example, Anchor primer (SEQ ID NO: 2) and MHC-G1 primer (SEQ ID NO: 3) (ST Jones et al., Biotechnology, 9, 88, 1991)
Can be used.

(Iii) Purification of DNA and Determination of Nucleotide Sequence The PCR product was subjected to agarose gel electrophoresis according to a known method to cut out a target DNA fragment, and then the DNA was recovered and purified, and ligated to vector DNA. I do. For purification of DNA, use a commercially available kit (for example, GENECL
EAN II; BIO101). Known vector DNAs for retaining DNA fragments (for example, pUC1
9, Bluescript, etc.).

The above DNA and the above vector DNA are ligated using a known ligation kit (Takara Shuzo) to obtain a recombinant vector. Next, the resulting recombinant vector is introduced into E. coli JM109 or the like, and ampicillin-resistant colonies are selected, and vector DNA is prepared according to a known method (J. Sambrook, et al., Molecular Cloning, Co., Ltd.).
ld Spring Harbor Laboratory Press, 1989). The base sequence of the target DNA is determined by a known method (eg, dideoxy method) after digesting the above vector DNA with a restriction enzyme (J. Sambrook, et al., Molecular Cloning,
Cold Spring Harbor Laboratory Press, 1989). In the present invention, an automatic base sequencer (DNA Sequencer 373A;
ABI) can be used.

(Iv) Complementarity determining region The V region of the H chain and the V region of the L chain form an antigen binding site,
Its general structure is similar to each other. That is, each of the four framework regions (FR) portions comprises three hypervariable regions, namely, the complementarity determining regions (CD).
R). The amino acid sequence of the FR is relatively well conserved, while the amino acid sequence of the CDR region is extremely variable (Kabat, EA et al., "Sequence
of Proteinsof Immunological Interest "US Dept. H
ealth and Human Services, 1983).

Most of the four FRs adopt a β-sheet structure, so that the three CDRs form a loop. CDRs may in some cases form part of a β-sheet structure. Thus, the three CDRs are held sterically very close to each other by the FR and
R together with the three CDRs of the paired region form an antigen binding site.

Based on these facts, human PTHrP
The amino acid sequence of the variable region of the mouse monoclonal antibody against the amino acid sequence database of the antibody prepared by Kabat et al. (“Sequence of Proteins of Immu
nological Interest "US Dept. Health and Human Ser
vices, 1983), CDR regions can be found by examining homology.

(2) Preparation of Expression Vector for Chimeric Antibody Mouse L chain of mouse monoclonal antibody (hereinafter referred to as “L chain or H chain of antibody”, “mouse L chain” for mouse and H chain of human antibody May be abbreviated as "human H chain".) And the DNA fragment encoding the H chain V region is cloned into these mouse V
A chimeric anti-human PTHrP antibody can be obtained by ligating the DNA encoding the region with the DNA encoding the human antibody constant region and expressing it.

The basic method for producing a chimeric antibody is to use the mouse leader sequence and V region sequence present in the cloned cDNA to encode the human antibody C region already present in the mammalian cell expression vector. Linking the sequence. Alternatively, the method comprises ligating the sequence of the mouse leader sequence and the V region present in the cloned cDNA to a sequence encoding the human antibody C region, and then ligating the sequence to a mammalian cell expression vector.

The polypeptide containing the human antibody C region can be the H chain C region of any human antibody and the L chain C region of a human antibody. For example, for human H chain, Cγ1, Cγ2, Cγ3 or Cγ4, and Cλ or Cκ for the L chain, respectively.

For production of a chimeric antibody, first, mouse L-L under the control of two expression vectors, ie, an expression control region such as an enhancer / promoter system, is used.
An expression vector containing a DNA encoding the chain V region and a DNA encoding the human L chain C region, and a DNA encoding the mouse H chain V region and a human H chain C under an expression control region such as an enhancer / promoter system. An expression vector containing a DNA encoding the region is prepared. next,
Host cells, such as mammalian cells, are co-transformed with these expression vectors, and the transformed cells are cultured in vitro or in vivo to produce chimeric antibodies (see, eg, WO 91/16928).

Alternatively, a mouse leader sequence present in the cloned cDNA, a DNA encoding the mouse L chain V region and a DNA encoding the human L chain C region
And a mouse leader sequence, a DNA encoding the mouse H chain V region and a DNA encoding the human H chain C region in a single expression vector (for example, WO94 / 11523).
And transforming the host cells with the vector, and then culturing the transformed host in vivo or in vitro to produce the chimeric antibody of interest.

(I) Construction of Chimeric Antibody H Chain An H chain expression vector for a chimeric antibody is a cDNA containing a nucleotide sequence encoding a mouse H chain V region (hereinafter referred to as “H chain V
Region cDNA) is also referred to as genomic DNA containing a base sequence encoding the H chain C region of a human antibody (hereinafter referred to as "H region").
It can also be obtained by introducing the gene into an appropriate expression vector containing a genomic DNA of the chain C region) or a cDNA encoding the region (hereinafter, also referred to as “cDNA of the H chain C region”). Examples of the H chain C region include Cγ
1, Cγ2, Cγ3 or Cγ4 regions.

(Ia) Genome DN encoding H chain C region
Construction of Chimeric H Chain Expression Vector Containing A As an expression vector having a genomic DNA encoding the H chain C region, for an expression vector encoding the Cγ1 region, for example, HEF-PMh-gγ1 (WO92 / 197)
59) or DHFR- △ E-RVh-PM1-f
(See WO92 / 19759).

Here, c coding for the mouse H chain V region
In inserting DNA into these expression vectors, P
An appropriate nucleotide sequence can be introduced into the cDNA by the CR method. For example, Kozak should have a recognition sequence for an appropriate restriction enzyme at the 5'-end of the cDNA, and Kozak immediately before the start codon of the cDNA to improve transcription efficiency.
A PCR primer designed to have a consensus sequence, and a splice to have an appropriate restriction enzyme recognition sequence at the 3'-end of the cDNA, and to ensure that the primary transcript of the genomic DNA is correctly spliced into mRNA. By performing PCR using PCR primers designed to have a donor site, these appropriate nucleotide sequences can be introduced into an expression vector.

The cDNA encoding the mouse H chain V region thus constructed is treated with an appropriate restriction enzyme, and inserted into the above expression vector to obtain a chimeric H containing genomic DNA encoding the H chain C region (Cγ1 region). Construct the chain expression vector.

(Ib) c containing the base sequence encoding the H chain
Construction of chimeric H chain expression vector containing DNA cDNA encoding H chain C region (eg, Cγ1 region)
Can be constructed as follows. That is, the genomic DNAs of the H chain V region of the humanized PM1 antibody and the C region Cγ1 of the human antibody H chain (N.T.
akahashi, et al., Cell 29, 671-679 1982) containing an expression vector DHFR-ΔE-RVh.
-PM1-f (see WO92 / 19759) and an expression vector RV1-PM1a (WO92 / 19) containing DNA encoding the genomic DNA of the humanized PM1 antibody L chain V region and the genomic DNA of the human antibody L chain κ chain C region. MRNA was prepared from CHO cells into which R
By T-PCR, cDNA encoding the humanized PM1 antibody H chain V region and cDNA encoding the human antibody H chain C region (Cγ1) were cloned, and the cDNAs were treated with appropriate restriction enzymes for expression in animal cells. The desired expression vector is constructed by ligating the expression vector.

Here, c coding for the mouse H chain V region
The DNA was converted to a cD encoding human antibody H chain C region Cγ1.
Upon direct ligation with NA, an appropriate base sequence can be introduced into the fragment containing the cDNA encoding the H chain V region by PCR. For example, 5′-
A PCR primer designed to have an appropriate restriction enzyme recognition sequence at its end and a Kozak consensus sequence immediately before the start codon of the cDNA to improve transcription efficiency, and a 3′-end of the cDNA PCR is performed using a PCR primer designed to have a recognition sequence for an appropriate restriction enzyme for directly linking to the DNA of the H chain C region Cγ1 to introduce these appropriate nucleotide sequences into the cDNA.

The thus constructed cDNA encoding the mouse H chain V region is treated with an appropriate restriction enzyme and ligated to the cDNA encoding the H chain C region Cγ1 to obtain pCO
By inserting it into an expression vector such as S1 or pCHO1, an expression vector containing a cDNA encoding the chimeric H chain can be constructed.

(Ii) Construction of Chimeric Antibody L Chain The chimeric antibody L chain expression vector is obtained by ligating a cDNA encoding the mouse L chain V region and a genomic DNA or cDNA encoding the human antibody L chain C region. By introducing it into an appropriate expression vector. Examples of the L chain C region include a κ chain and a λ chain.

(Ii-a) cD encoding chimeric L chain λ chain
Construction of Expression Vector Containing NA In constructing an expression vector containing cDNA encoding the mouse L chain V region, an appropriate nucleotide sequence can be introduced into the expression vector by PCR. For example,
Koz to have an appropriate restriction enzyme recognition sequence at the 5'-end of the cDNA and to improve transcription efficiency
using a PCR primer designed to have an ak consensus sequence and a PCR primer designed to have an appropriate restriction enzyme recognition sequence at the 3′-end.
By performing CR, these appropriate nucleotide sequences are
To be introduced.

CDNA encoding human L chain λ chain C region
Can be constructed by synthesizing the entire base sequence with a DNA synthesizer and using the PCR method. At least four types of human L chain λ chain C regions are known depending on the isotype, and any of these isotypes can be used for construction of an expression vector. For example, a search for homology with the cloned mouse monoclonal antibody L chain λ chain C region revealed that the isotype of the human L chain λ chain C region fragment was Mc
g + Ke + Oz- (accession No. X57819) (PD
ariavach et al., Proc. Natl. Acad. Sci. USA, 84, 9074-9.
078, 1987) can be used for constructing the expression vector. A known human light chain λ chain C region, such as M
In order to construct cDNA of cg + Ke + Oz-, for example, it is divided into the following four primers shown in SEQ ID NOS: 11 to 14. Primers MBC1HGP1 (SEQ ID NO: 11) and MBC1HGP3 (SEQ ID NO: 13) have a sense DNA sequence and MBC1HGP2 (SEQ ID NO: 1).
2) and MBC1HGP4 (SEQ ID NO: 14) have an antisense DNA sequence and are designed to have complementary sequences of 20 to 23 bp at both ends of each primer.

MBC1HGPS (SEQ ID NO: 15) and M
BC1HGPR (SEQ ID NO: 16) is called an external primer and has sequences homologous to MBC1HGP1 and MBC1HGP4, respectively, and includes recognition sequences for appropriate restriction enzymes. Four primers are assembled by PCR, a full-length cDNA is synthesized, and an external primer is added to amplify the cDNA. The assembly by the PCR method refers to MBC1HGP1
And MBC1HGP2, or MBC1HGP3 and MBC1
HGP4 anneals with its complementary sequence,
BC1HGP1-MBC1HGP2 fragment and MBC1HG
It indicates that a P3-MBC1HGP4 fragment is synthesized and further annealed with a complementary sequence of each fragment to synthesize a cDNA encoding a full-length human L chain λ chain C region.

The cDNA encoding the human L chain λ chain C region constructed in this way and the cDNA encoding the mouse L chain V region constructed as described above are ligated between appropriate restriction enzyme sites. And pCOS1 or pCHO
By inserting the cDNA into the expression vector as in Example 1, an expression vector containing the cDNA encoding the L chain λ chain of the chimeric antibody can be constructed.

(Ii-b) cDN encoding chimeric L chain κ chain
Construction of Expression Vector Containing A In constructing an expression vector containing a cDNA encoding the mouse L chain V region, the cDNA
An appropriate nucleotide sequence can be introduced into the DNA. For example, Koza to have an appropriate restriction enzyme recognition sequence at the 5'-end of the cDNA and to improve transcription efficiency.
PC using a PCR primer designed to have a k consensus sequence and a PCR primer designed to have a recognition sequence for an appropriate restriction enzyme at the 3′-end.
By performing R, these appropriate nucleotide sequences are introduced into the cDNA.

DNA coding for human L chain κ chain C region to be ligated to DNA coding for mouse L chain V region
Is, for example, HEF-PM1k-gk containing genomic DNA (WO9
2/19759). PC
By the R method, 5 ′ of the DNA encoding the L chain κ chain C region
A DNA encoding the mouse L chain V region and a DNA encoding the L chain κ chain C region were ligated by introducing appropriate restriction enzyme recognition sequences into the -terminal and the 3'-terminal. , PCOS1 or pCHO1 to construct an expression vector containing a cDNA encoding the L chain κ chain of the chimeric antibody.

3. Preparation of Humanized Antibody (1) Homology Search with Human Antibody To prepare a humanized antibody in which the CDRs of a mouse monoclonal antibody are transplanted to a human antibody, the FR of a mouse monoclonal antibody and the FR of a human antibody It is desirable that there be high homology between Therefore, the V regions of the H and L chains of the mouse anti-human PTHrP monoclonal antibody are compared to the V regions of all known antibodies whose structures have been elucidated using the Protein Data Bank. Also,
At the same time, Kabat et al. (HSG: Human subgroup) (Kabat, EA et al., US Dep.) Classified by antibody FR length, amino acid homology, etc.
p.Health and Human Services, US Government Printin
g Offices, 1991).

The human H chain V region can be classified into HSGI to III by the HSG classification according to Kabat et al., And the mouse anti-human PTHrP monoclonal antibody H
The chain V region has 82.7% homology with the HSGIII consensus sequence. On the other hand, the human L chain λ chain V region
can be classified into HSGI to VI by the HSG classification according to abat et al., and the mouse anti-human PTHrP monoclonal antibody L chain λ chain V region has a high homology with the consensus sequence of the human L chain λ chain V region belonging to any subgroup. Do not have.

Therefore, when humanizing a mouse anti-human PTHrP monoclonal antibody, it belongs to HSGIII as the human H chain V region and has the most homologous human H chain V region or canonical structure (Chothia C, et a).
l., J. Mol. Biol. 196, 901-917, 1987)
It is desirable to use a human H chain V region having the structure for construction of a humanized antibody. In addition, since there is no consensus sequence with high homology in the human L chain λ chain V region subgroup, the human antibody L chain λ chain V region having the highest homology registered in the Protein Data Bank was identified. It is desirable to use it for construction of a humanized antibody.

(2) D coding for humanized antibody V region
Design of NA The first step in designing DNA encoding a humanized antibody V region is to select a human antibody V region on which to base the design. In the present invention, a human antibody V region FR having 80% or more homology with a mouse antibody V region FR can be used as a humanized antibody. here,
As the FRs of the H chain V region, those belonging to subgroup III, for example, S31679 (NBRF-PDB, Cuisinier AM et al., Eu
rJImmunol. 23, 110-118, 1993) can be mentioned as fragments of substantially the same FR. Also, L
As the FR of the chain V region, for example, the human antibody HSU038
68 (GEN-BANK, Deftos M et al., Scand. J. Immunol. 39,
95-103, 1994) and FR4 from human antibody S25755 (NBRF-PDB).
And as substantially identical FR fragments. In addition, human antibody S31679 was obtained from human fetal liver c.
It is an antibody cloned from a DNA library, and the human antibody HSU03868 is an antibody cloned as a novel human L chain λ chain V region gene.

(3) Preparation of Polypeptide Containing Humanized Antibody V Region In the humanized antibody of the present invention, the C region of the antibody and the framework (FR) region of the V region are of human origin. , The complementarity determining region (CDR) of the V region is derived from a mouse (FIG. 1). The polypeptide containing the V region of the humanized antibody of the present invention can be prepared by a technique called CDR-grafting by PCR if a DNA fragment of a human antibody serving as a template is available. "C
"DR-grafting" refers to a technique of preparing a DNA fragment encoding a mouse-derived CDR and replacing it with the CDR of a human antibody serving as a template.

When a DNA fragment of a human antibody serving as a template is not available, a base sequence registered in a database is synthesized by a DNA synthesizer, and DNA of a humanized antibody V region is prepared by PCR. Can be. Further, when only the amino acid sequence is registered in the database, the data from Kabat, EA et al. (US Dep. Health and Human Services, US Government)
Based on the codon usage of the antibody (Printing Offices, 1991), the entire nucleotide sequence can be inferred. The target polypeptide can be prepared by synthesizing this base sequence with a DNA synthesizer, preparing DNA of the humanized antibody V region by PCR, introducing this into a suitable host, and expressing it. . In the following, when a DNA fragment of a human antibody serving as a template is available,
Shows a general overview of R-grafting.

(I) CDR-grafting As shown in FIG. 2, the DNA encoding the V region is FR
1, DNAs encoding CDR1, FR2, CDR2, FR3, CDR3 and FR4 are assumed to be linked in this order.

First, a mouse-derived DNA fragment corresponding to each CDR is synthesized. CDRs 1 to 3 are DNAs synthesized based on the nucleotide sequences of the mouse H chain V region and L chain V region previously cloned. The grafting primer B has a sequence that hybridizes to the mouse CDR1 in the sense direction and FR2 of the human antibody, and the grafting primer E has a sequence that hybridizes to the CDR1 in the antisense direction and FR1 of the human antibody. Synthesize (similar for grafting primers C and F and grafting primers D and G) (FIG. 2 (1)
). In addition, appropriate primers (referred to as external primers; A and H in FIG. 2A) capable of hybridizing to the region upstream of FR1 and the region downstream of FR4 are also synthesized. The separation and extraction of the grafting primer can be performed by a known method (Sambrook, et.
al., Molecular Cloning: A Laboratory Manual, Cold S
pringHarbor Laboratory Press, 1989).

Next, first PCR is performed using the grafting primer E and the external primer A, the grafting primers B and F, the grafting primers C and G, and the grafting primer D and the external primer H.
As a result, fragment A-E, fragment BF, fragment C-
G and fragment DH are obtained (FIG. 2 (2)).

As described above, grafting primer B
Are designed so that the upstream region of the primer and a part of the downstream region of the grafting primer E overlap (similarly for the grafting primers C and F, and D and G), these fragments are prepared under appropriate temperature conditions. By reacting with
By annealing to each complementary sequence and performing PCR, it is possible to assemble into a DNA having a length from A to H. Then, when one DNA fragment encoding the V region was obtained, external primers A and H were added, and a second PCR was performed to obtain FR1
DNAs encoding human V regions derived from mouse but CDRs 1-3 are mouse-derived DNAs
Get. Then, by introducing this into an appropriate host and expressing it, the desired polypeptide can be obtained (FIG. 2 (3)).

(Ii) DN encoding humanized H chain V region
A and Construction of Expression Vector In the present invention, since the DNA encoding the H chain V region of the human antibody as a template for the humanized antibody cannot be obtained from nature, the DNA is a DN encoding the H chain V region.
The entire base sequence of A can be synthesized by a DNA synthesizer and constructed by the PCR method.

The mouse anti-human PTHrP monoclonal antibody H chain V region was identified as S31 belonging to human subgroup III.
679 has high homology. In order to construct a DNA encoding the humanized H chain V region using this human antibody as a template, the DNA is used, for example, by dividing into four primers shown in SEQ ID NOS: 23 to 26. Primer MBC1HGP1
(SEQ ID NO: 23) and MBC1HGP3 (SEQ ID NO: 2)
4) has a sense DNA sequence, and MBC1HGP2 (SEQ ID NO: 25) and MBC1HGP4 (SEQ ID NO: 26) have an antisense DNA sequence and are designed to have a complementary sequence of 15 to 21 bp at both ends of each primer. I do.

The external primers MBC1HVS1 (SEQ ID NO: 27) and MBC1HVR1 (SEQ ID NO: 28)
It has a sequence homologous to each of 1HGP1 and MBC1HGP4, and contains a recognition sequence for an appropriate restriction enzyme, respectively. Using the PCR method, four primers are assembled to synthesize a full-length cDNA, and then an external primer is added to amplify the DNA. The assembly by the PCR method includes MBC1HGP1 and MBC1HGP.
2, or MBC1HGP3 and MBC1HGP4 anneal with their complementary sequences, and MBC1HGP1-
MBC1HGP3 fragment and MBC1HGP2-MBC1H
GP4 fragments are synthesized and further annealed with the complementary sequence of each fragment to synthesize a full-length humanized H chain V region DNA.

The human antibody H chain C region can be any human H chain C region, for example, human H chain Cγ1, Cγ2,
Cγ3 or Cγ4 can be mentioned. The DNA of the humanized antibody H chain V region constructed as described above can be ligated to any human antibody H chain C region, for example, DNA of the human H chain C region Cγ1 region. After treatment with an appropriate restriction enzyme as described in the construction of the chimeric antibody H chain,
An expression vector containing humanized H chain V region and human H chain C region DNA is prepared by ligating the DNA encoding the human H chain C region under an expression control region such as an enhancer / promoter system.

(Iii) D encoding humanized L chain V region
Construction of NA and Expression Vector In the present invention, as in the case of the DNA encoding the H chain V region, the DNA of the L chain V region of the human antibody used as a template cannot be obtained from nature. The entire base sequence of the DNA to be encoded is synthesized using a DNA synthesizer, and PCR is performed.
It can be constructed by the method.

Human antibody HSU03 having the highest homology with mouse anti-human PTHrP monoclonal antibody L chain V region
In order to construct a humanized L chain V region DNA using 868 as a template, for example, the primers are used by dividing into four primers shown in SEQ ID NOS: 29 to 32. Primer MBC1LG
P1 (SEQ ID NO: 29) and MBC1LGP3 (SEQ ID NO: 30) have a sense DNA sequence and MBC1LGP2
(SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 3)
2) has an antisense DNA sequence and is designed to have a complementary sequence of 15 to 21 bp at both ends of each primer.

The external primers MBC1LVS1 (SEQ ID NO: 33) and MBC1LVR1 (SEQ ID NO: 34)
It has a sequence homologous to each of 1LGP1 and MBC1LGP4, and each contains a recognition sequence for an appropriate restriction enzyme. Using the PCR method, four primers are assembled to synthesize a full-length DNA, and further, external primers are added to amplify the DNA. The assembly by the PCR method includes MBC1LGP1 and MBC1LGP3,
Alternatively, MBC1LGP2 and MBC1LGP4 anneal with their complementary sequences, and MBC1LGP1-MB
C1LGP3 fragment and MBC1LGP2-MBC1LGP
Four fragments are synthesized and further annealed with the complementary sequence of each fragment to synthesize DNA encoding the full-length humanized H chain V region.

The human antibody L chain C region can be any human L chain C region, for example, human L chain Cλ or Cκ. The DNA of the humanized antibody L chain V region constructed as described above may be any human antibody L chain C region,
For example, it can be linked to a human L chain Cλ region. After treatment with an appropriate restriction enzyme, it is ligated to DNA encoding the human L chain λ chain C region under an expression control region such as an enhancer / promoter system, and the humanized L chain V region and human L chain λ chain An expression vector containing a DNA encoding the C region is prepared.

As described above, even if a polypeptide containing the V region of a humanized antibody is prepared, whether or not the polypeptide has an antibody activity (antigen binding activity, neutralizing activity, etc.) Is not always clear. Especially L
In the case of the chain, since the mouse anti-human PTHrP monoclonal antibody L chain V region is derived from a very rare Vλx gene, it is expressed in an animal cell such as COS-7 in combination with a humanized H chain, and the activity of It is necessary to consider the existence.

As a method for clarifying which FRs of the humanized antibody V region contribute to the binding activity and neutralizing activity of the humanized antibody, a hybrid V region was constructed (Ohto
mo, T. et al. Molecular Immunology, 32,407-416,199
5) It is effective to confirm. Humanized antibody L of the present invention
In order to examine which amino acids in the chain V region can be mutated to obtain an active one, DNA encoding a humanized antibody FR region fragment recombined with a mouse-derived FR region fragment is used. Is constructed, and each region for humanization is evaluated.

As shown in FIG. 3, FR1 and FR2 are derived from a human antibody, but an antibody having a polypeptide containing a V region obtained by recombining FR3 and FR4 from a mouse antibody. "Hybrid antibody"), a hybrid antibody in which only FR1 is recombined with human, and a hybrid antibody in which only FR2 is recombined with human. Then, DNAs encoding these hybrid antibodies are incorporated into an expression vector, the humanized antibody is transiently expressed, and the presence or absence of the activity of the antibody is examined.

The present inventors have examined the antigen binding activity and the neutralizing activity of the polypeptide containing the L chain V region by using this method. As a result, it has been found that amino acids to be substituted exist in FR2 and FR3. . The present inventors have found that amino acids contributing to the activity exist in the FR2 and FR3 regions, and have found that Kabat, EA et al. (US Dep. Health and Human
Services, US Government Printing Offices, 1991), the amino acids 36, 45 and 49 (present in the FR2 region) and the 87th amino acid (present in the FR3 region) of the antibody determined by the antibody are active. Amino acids that contribute to

Therefore, in the present invention, a polypeptide containing a V region in which these amino acids are mutated (for example, substituted) is prepared. First, by the CDR-grafting,
A polypeptide containing a V region having a basic amino acid sequence for introducing an amino acid mutation is prepared. This basic polypeptide contains the amino acid sequence represented by SEQ ID NO: 47 and is referred to as “version a” (a in Table 1).

Next, based on this version a, F
Various mutant fragments in which several amino acids of R are mutated are prepared. Mutation can be introduced by designing an oligonucleotide primer (mutagenic primer) encoding an amino acid into which a desired mutation is to be introduced, and performing PCR using the primer. Thus, F
Polypeptides (versions bt) containing V regions in which specific amino acids of R2 and FR3 are mutated are produced (bt in Table 1).

[0104]

[Table 1]

The humanized antibody L constructed as described above
The DNA encoding each version of the chain V region can be ligated to any human antibody L chain C region, for example, DNA of the human L chain Cλ region. After treatment with appropriate restriction enzymes,
DNA encoding each version of humanized L chain V region linked to DNA encoding human L chain λ chain C region under an expression control region such as an enhancer / promoter system
An expression vector containing A and DNA encoding the humanized L chain λ chain C region is prepared.

The DNAs encoding the humanized antibody H chain V region and human H chain C region constructed as described above
And a DNA encoding a humanized L chain V region and a human L chain C region by a single expression vector (for example, WO94
/ 11523) and transforming host cells with the vector, then culturing the transformed host in vivo or in vitro to produce the desired humanized antibody Can be done.

4. Production of Chimeric Antibody and Humanized Antibody In order to produce a chimeric antibody or a humanized antibody, two types of expression vectors are prepared as described above. That is, for a chimeric antibody, an expression vector containing DNA encoding a mouse H chain V region and a human H chain C region under the control of an expression control region such as an enhancer / promoter system, and an expression vector such as an enhancer / promoter system. An expression vector containing DNAs encoding the mouse L chain V region and the human L chain C region under the control region is prepared. For the humanized antibody, the expression vector is controlled by an expression control region such as an enhancer / promoter system. Encoding a humanized H chain V region and a human H chain C region
NA-containing expression vector, and a humanized L chain V under an expression control region such as an enhancer / promoter system.
An expression vector containing the DNA encoding the region and the human L chain C region is prepared.

Next, host cells such as mammalian cells are co-transformed with these expression vectors, and the transformed cells are cultured in vitro or in vivo to produce chimeric or humanized antibodies. (For example, WO
91/16928).

Further, the DNA encoding the H chain V region and the H chain C region and the DNA encoding the L chain V region and the L chain C region are ligated into a single vector, and a suitable host cell is transformed. Antibodies can be produced. That is, for expression of the chimeric antibody, the cloned cDN
A single expression of the DNA encoding the mouse leader sequence, the mouse H chain V region and the human H chain C region, and the DNA encoding the mouse leader sequence and the mouse L chain V region and the human L chain C region present in A Vector (for example,
WO94 / 11523). For expression of a humanized antibody, a DNA encoding a humanized H chain V region and a human H chain C region and a DNA encoding a humanized L chain V region and a human L chain C region are expressed as a single unit. It is introduced into an expression vector (for example, see WO94 / 11523). Then, host cells are transformed with these vectors,
Next, the transformed host is cultured in vivo or in vitro to produce the desired chimeric or humanized antibody.

The transformant transformed with the DNA encoding the chimeric or humanized antibody of interest as described above is cultured, and the chimeric or humanized antibody produced is obtained from inside or outside the cell. It can be separated and purified to homogeneity. The chimeric antibody or humanized antibody as the target protein of the present invention can be separated and purified using a protein A agarose column. In addition, other separation / purification methods used for ordinary proteins may be used, and there is no particular limitation. For example, if various kinds of chromatography, ultrafiltration, salting, dialysis, etc. are appropriately selected and combined, a chimeric antibody or humanized antibody can be separated and
It can be purified.

To produce the chimeric or humanized antibody of the present invention against human PTHrP, any expression system can be used. For example, when eukaryotic cells are used, animal cells (for example, established mammalian cell lines) and eukaryotic fungal cells or yeast cells are used. When prokaryotic cells are used, bacterial cells (for example, E. coli cells and the like) are used. can do. Preferably, the chimeric or humanized antibody of the present invention is a mammalian cell, such as a COS cell or CHO.
It is expressed in cells.

In these cases, conventional promoters useful for expression in mammalian cells can be used. For example, human cytomegalovirus early (human cytome
It is preferred to use the galovirus immediate early (HCMV) promoter. Examples of expression vectors containing the HCMV promoter include HCMV-VH-HCγ1, HC
MV-VL-HCK and the like derived from pSV2neo (WO92-19759) are included.

In addition, promoters for gene expression in mammalian cells that can be used for the present invention include retroviruses, polyomaviruses,
Viral promoters such as adenovirus and simian virus 40 (SV40) and human polypeptide chain elongation factor 1α (HEF-1
A promoter derived from a mammalian cell such as α) may be used. For example, when using the SV40 promoter, the method of Mulligan et al. (Nature 277, 108, 1979)
When using the HEF-1α promoter, use Mizushim
a, S. et al.'s method (Nucleic Acids Research, 18, 5322,
1990).

The origin of replication includes SV40, polyoma virus, adenovirus, bovine papilloma virus (B
PV) or the like, and for amplification of the gene copy number in a host cell system, the expression vector is a phosphotransferase AP as a selection marker.
H (3 ′) II or I (neo) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene,
It may contain a dihydrofolate reductase (DHFR) gene and the like.

[0115] 5. Evaluation of Antigen Binding Activity and Neutralizing Activity of Chimeric Antibody and Human Antibody (1) Measurement of Antibody Concentration The concentration of the obtained purified antibody can be measured by ELISA. An ELISA plate for antibody concentration measurement is prepared as follows. Each well of a 96-well plate for ELISA (eg, Maxisorp, NUNC) was filled with 1 μg /
Immobilized with 100 μl of goat anti-human IgG antibody prepared to a concentration of ml. 200 μl of dilution buffer (eg 50 mM T
ris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween20,
0.02% NaN ₃ , 1% bovine serum albumin (BSA), pH 7.
After blocking in 2), the culture supernatant of COS-7 cells or CHO cells expressing the chimeric antibody, hybrid antibody or humanized antibody, or purified chimeric antibody, hybrid antibody or humanized antibody is serially diluted. 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody was added to each well, and a 1 mg / ml substrate solution (Si
gma 104, p-nitrophenyl phosphate, SIGMA) and then the absorbance at 405 nm is measured on a microplate reader (Bio Rad). Hu IgG1λ Purified (The Binding Site) can be used as a standard for concentration measurement.

(2) Measurement of antigen binding ability An ELISA plate for antigen binding measurement is prepared as follows. Human PTHrP (1-34) 10 was prepared at a concentration of 1 μg / ml in each well of a 96-well plate for ELISA.
Immobilize with 0 μl. After blocking with 200 μl of the dilution buffer, COS-7 cells expressing the chimeric antibody, hybrid antibody or humanized antibody or CH
O cell culture supernatant, or purified chimeric antibody, hybrid antibody or humanized antibody serially diluted and added to each well,
Next, 100 μl of an alkaline phosphatase-conjugated goat anti-human IgG antibody was added, and a 1 mg / ml substrate solution (Sigma 104,
p-Nitrophenylphosphoric acid, SIGMA), then add 405n
The absorbance at m is measured with a microplate reader (BioRad).

(3) Measurement of Neutralizing Activity The neutralizing activity of the mouse antibody, chimeric antibody and humanized antibody was measured by, for example, rat osteosarcoma cell line ROS17 / 2.8.
-5 cells (Sato, K. et al., Acta Endocrinology 116, 113-1)
20, 1987). That is, ROS1
7 / 2.8-5 cells are stimulated with 4 mM hydrocortisone to induce PTH / PTHrP receptors. 1m
M isobutyl-1-methylxanthine (IBMX, SI
GMA) inhibits cAMP-degrading enzyme, and measures neutralizing activity.
HrP (1-34) is mixed in an equal amount, and each antibody and PTHrP
Add the mixture of (1-34) to each well. Upon stimulation of PTHrP, the rat osteosarcoma cell line ROS17 / 2.8-
By measuring the amount of cAMP produced by the 5 cells,
The neutralizing ability of a mouse antibody, chimeric antibody or humanized antibody can be evaluated.

(4) Kinetic Analysis of the Interaction between PTHrP and Anti-PTHrP Antibody In the present invention, the kinetics of the interaction between PTHrP and anti-PTHrP antibody can be analyzed using various techniques. Specifically, Scatchard analysis and dissociation constant by surface plasmon resonance sensor called BIACORE (developed and commercialized by Pharmacia Biotech)
Although it is possible to measure the dissociation rate constant and the binding rate constant, in the present invention, as an example, the case of analyzing with a surface plasmon resonance sensor called BIACORE will be described.

The basic structure of BIACORE is a light source and a prism,
It consists of a detector and a microchannel. In practice, a ligand is immobilized on a cassette-type sensor chip, and an analyte is injected there. If both have an affinity, the amount of binding is optically detected.

The detection principle is a phenomenon called surface plasmon resonance. That is, of the light that is incident on the interface between the glass and the metal thin film so as to be totally reflected, the incident light at a certain angle is used for exciting surface plasmons and is attenuated. The angle varies depending on the change in the concentration of the solvent in contact with the metal thin film (sensor). BIACORE detects this change.

In BIACORE, this change is represented by a resonance signal (SPR
signal), and a change of 0.1 degree is 1000RU (resonance un
its). 1000 RU is the amount of change when about 1 ng of protein binds to a thin gold sensor having a surface area of 1 mm ^{2. For} proteins, a change of about 50 RU (50 pg) can be sufficiently detected. The computer attached to BIACORE converts the detected signal into a binding curve called a sensorgram, which is drawn on a computer display in real time (Toru Natsume et al., (1995) Experimental Medicine, 13, p563-569.) (Karlsson, R. et al., (1991)
J. Immunol. Methods 145, p229-240.).

The BIACORE can measure the kinetic parameters of the anti-PTHrP antibody of the present invention, namely, the dissociation constant (KD), dissociation rate constant (Kdiss) and binding rate constant (Kass). The anti-PTHrP antibody of the present invention is preferred because the smaller the dissociation constant (KD value), the more neutralizing activity it has. In the anti-PTHrP antibody of the present invention, the KD value is preferably 1.86 × 10 ⁻⁷ or less, and 1.
86 × 10 ^-8 or less, more preferably 3.58 × 10 ^-10
The following are most preferred.

The KD value is determined from two parameters, a dissociation rate constant (Kdiss) and a binding rate constant (Kass) (KD = Kdiss / Kass). Therefore, Kdi
It is clear that the smaller the value of ss and the larger the value of Kass, the smaller the KD value. Specifically, the anti-PT of the present invention
In the case of the HrP antibody, the value of Kdiss may be 1.22 × 10 ⁻¹ [1 / Sec] or less. Preferably, the value of Kdiss is 1.22 × 10 ⁻² or less, more preferably 3.16 × 10 ⁻⁴ or less, and most preferably 2.32 × 10 ⁻⁴ [1 / Sec] or less.

On the other hand, the value of Kass is 6.55 × 10 ⁴ [1 / M.Sec]
All that is required is the above. Preferably, the value of Kass is 6.55 × 10 ⁵
Or more, more preferably 0.883 × 10 ⁶ or more,
Most preferably, it is 1.03 × 10 ⁶ [1 / M.Sec] or more. Furthermore, anti-PTHrP with a Kdiss value of 1.22 × 10 ⁻¹ [1 / Sec] or less and a Kass value of 6.55 × 10 ⁴ [1 / M.Sec] or more
Antibodies are also preferred.

More specifically, the anti-PTHrP antibody of the present invention has a KD value in the range of 1.02 × 10 ^{-11 to} 1.86 × 10 ^-7 [M], and 1.02 × 10 ^{-10 to} 1.86 × M. 10 ^-8 [M] is preferred, 1.34 × 10 ^{-10 to} 3.58 × 10 ^-10 [M] is more preferred, and 2.25 × 10 ^{-10 to} 3.58 × 10 ^-10 [M] is most preferred. . The Kdiss value is 7.38 × 10 ^{−6 to} 1.22 × 10 ⁻¹ [1 / S
ec], preferably 7.38 × 10 ^{−5 to} 1.22 × 10 ⁻² [1 / Sec], more preferably 1.66 × 10 ^{−4 to} 3.16 × 10 ⁻⁴ [1 / Sec], Those having a ratio of 1.66 × 10 ^{−4 to} 2.32 × 10 ⁻⁴ [1 / Sec] are most preferable.

The Kass value is 6.55 × 10 ^{4 to} 1.24 × 10 ⁷
[1 / M.Sec], from 6.55 × 10 ^{5 to} 1.24 × 10 ⁶ [1 / MS
ec] is preferred, and 7.23 × 10 ^{5 to} 1.03 × 10 ⁶ [1 / MS
ec], more preferably 0.883 × 10 ^{6 to} 1.03 × 10 ⁶ [1 /
M.Sec] is most preferred. These KD values, Kdis
The s and Kass values are based on Scatchard analysis or B
It can be obtained using a surface plasmon resonance sensor such as IACORE, but is preferably obtained using BIACORE.

6. To confirm the therapeutic effect of an anti-PTHrP antibody or a humanized antibody as a pharmaceutical composition and a hypercalcemia inhibitor PTHrP or a humanized antibody as an active ingredient, an antibody or a humanized antibody to PTHrP is The therapeutic effect can be confirmed by administering to an animal exhibiting hypercalcemia and measuring an index of hypercalcemia. In addition, hypophosphatemia is often observed in animals exhibiting hypercalcemia and hypercalcemia patients, and the antibody of the present invention can also be used to improve this hypophosphatemia.

The antibodies used in the present invention are anti-PTHrP antibodies (including human antibodies, chimeric antibodies, and primed antibodies) having the above dissociation constants, dissociation rate constants and association rate constants, or humanized against PTHrP. Antibodies. This antibody neutralizes the activity of PTHrP by binding to PTHrP, and particularly preferably includes the humanized # 23-57-137-1 antibody. Humanization # 23-57
The method for producing the -137-1 antibody is described in Examples 1 to 3.

The antibody used in the present invention can be prepared by
Purification can be performed with high purity by combining ordinary purification means such as a gel filtration method using E.g. and the like, and an affinity chromatography method using a protein A column. Antibodies purified in this way can be used for radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), or immunofluorescence (Im
It is possible to confirm that PTHrP is recognized with high accuracy by ordinary immunological means such as munofluorescence analysis).

Animals presenting with hypercalcemia include PTHr
Model animals produced by transplanting P-producing tumor cells into experimental animals with reduced or deleted immune function can be used. The tumor cells to be transplanted are preferably human-derived tumor cells, for example, human pancreatic cancer PAN-7.
Is mentioned. In addition, examples of animals in which the immune function for transplanting tumor cells is reduced or deleted include nude mice and SCID mice. Evaluation of suppression of hypercalcemia is performed by observing blood calcium concentration, weight loss, or decrease in exercise amount over time and evaluating the degree of improvement.

The pharmaceutical composition of the present invention containing an antibody against PTHrP or a humanized antibody as an active ingredient and a hypercalcemia inhibitor can be parenterally administered systemically or locally. For example, intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection such as infusion can be selected, and the administration method can be appropriately selected according to the age and symptoms of the patient. The effective dose is selected in the range of 0.01 mg to 1000 mg per kg of body weight at a time. Alternatively, 5-1 per patient
A dose of 0000 mg / body, preferably 50-1000 mg / body, can be chosen.

The pharmaceutical composition of the present invention containing an antibody to PTHrP or a humanized antibody as an active ingredient and a hypercalcemia-suppressing agent contain both pharmaceutically acceptable carriers and additives depending on the administration route. There may be. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, Sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol,
Stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like. The additives to be used are appropriately selected from the above or in combination according to the dosage form of the present invention, but are not limited thereto.

The antibodies of the present invention can be widely used for hypercalcemia induced by various cancers (malignant tumors). These cancer types are not particularly limited, and include not only a single cancer but also a combination of a plurality of cancers. Examples of cancer types include pancreatic cancer, lung cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, gingival cancer, esophageal cancer, stomach cancer, bile duct cancer, breast cancer, kidney cancer, bladder cancer, uterine cancer, prostate cancer, malignant lymphoma and the like. It is.

[0134]

[Reference Example 1]

Preparation of anti-PTHrP (1-34) mouse monoclonal antibody-producing hybridoma Monoclonal antibody-producing hybridomas against human PTHrP (1-34) # 23-57-154 and # 23-57-137-
1 was made by K. Sato and others (Sato, K.et
al., J. Bone Miner. Res. 8, 849-860, 1993).

For use as an immunogen, PTHrP
(1-34) (manufactured by Peninsula) and thyroglobulin as a carrier protein were bound using carbodiimide (Dojinn). PTHrP bound to thyroglobulin
(1-34) was dialyzed, and the protein concentration was 2 μg / ml.
And then Freund's adjuvant (Di
fco) in a 1: 1 ratio to form an emulsion, and then subcutaneously or intraperitoneally into the back of 16 female BALB / C mice at 100 / animal.
μg was immunized 11 times. For the first immunization, Freund's complete adjuvant was used, and for the second and subsequent boosters, Freund's incomplete adjuvant was used.

The measurement of the antibody titer in the serum of the immunized mouse was performed by the following method. That is, blood was collected from the tail vein of the mouse, and after separating the serum, antiserum diluted with RIA buffer and ¹²⁵ I-labeled PTHrP (1-34) were mixed, and the binding activity was measured. PTHrP (1-34) having no carrier protein bound to the peritoneal cavity of the mouse with an increased antibody titer
Was final immunized with 50 μg per animal.

On the third day of the final immunization, the mice were sacrificed and the spleen was removed.
1 was subjected to cell fusion according to a conventional method using 50% polyethylene glycol 4000. 2 × 10 ⁴ cells
The cells were seeded into 85 96-well plates at a cell number per well.
Hybridomas were selected using a HAT medium.

Hybridoma screening was performed using HA
The culture supernatant in the well where growth was observed in T medium was
It was performed by measuring the presence or absence of a PTHrP-recognizing antibody by Method A and selecting it. Hybridomas were collected from the wells in which the ability to bind to the antibody was recognized, suspended in RPMI-1640 medium containing 15% FCS and supplemented with OPI-supplement (Sigma), and homogenized by limiting dilution. Was carried out. Clones # 23-57-154 and # 23-57-137-1 having strong binding ability to PTHrP (1-34) were obtained.

The hybridoma clone # 23-57-13
7-1 was designated as mouse-mouse hybridoma # 23-57-137-1 by the Institute of Biotechnology, Industrial Science and Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture) on August 15, 1996. , FERM
Deposited internationally under the Budapest Treaty as BP-5631.

Example 1 Human PTHrP (1-34)
Of the variable region of mouse monoclonal antibody # 23-57-137-1 against human PTHrP (1-34)
Was cloned as follows. (1) Preparation of mRNA Quick mRNA from hybridoma # 23-57-137-1
It was prepared using Prep mRNA Purification Kit (Pharmacia Biotech). The cells of hybridoma # 23-57-137-1 were completely homogenized with extraction buffer, and the oligo (dT) -cellulose span column (Ol
The mRNA was purified using igo (dT) -Cellulose Spun Column) and precipitated with ethanol. The mRNA precipitate was dissolved in the elution buffer.

(2) Preparation and Amplification of cDNA of Gene Encoding Mouse H Chain V Region (i) Cloning of # 23-57-137-1 Antibody H Chain V Region cDNA H of mouse monoclonal antibody against human PTHrP
Cloning of the DNA encoding the chain V region is performed by 5'-RAC
E method (Frohman, MA et al., Proc. Natl. Acad. Sci. U
SA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucl
eic Acids Res. 17, 2919-2932, 1989).
For 5'-RACE method, use 5'-Ampli FINDER RACE kit (CLONETECH
The operation was performed according to the prescription attached to the kit. As a primer used for cDNA synthesis, an MHC2 primer (SEQ ID NO: 1) that hybridizes with a mouse H chain constant region (C region) was used. MHC2 primer using about 2 μg of mRNA prepared as described above as a template
10 pmole was added, and the mixture was reacted with reverse transcriptase at 52 ° C. for 30 minutes to perform reverse transcription to cDNA.

Hydrolysis of RNA with 6N NaOH (65 ° C., 30
After that, cDNA was purified by ethanol precipitation. By reacting with T4 RNA ligase at 37 ° C. for 6 hours and at room temperature for 16 hours, Am was added to the 5 ′ end of the synthesized cDNA.
pli FINDER Anchor (SEQ ID NO: 42) was ligated. Using this as a template to amplify by PCR
Anchor primer (SEQ ID NO: 2) and MHC-G1 primer (SEQ ID NO: 3) (ST Jones, et al., Biotechno)
logy, 9, 88, 1991).

The PCR solution contained 10 mM Tris-
HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dCT
P, dTTP), 1.5 mM MgCl ₂ , 2.5 units TaKaRa Taq
(Takara Shuzo), 10pmole Anchor primer and MH
Contains 1 μl of reaction mixture of cDNA ligated with C-G1 primer and AmpliFINDER Anchor. In this solution
50 μl of mineral oil was overlaid. PCR is Thermal Cycler Mod
Using el 480J (Perkin Elmer) at 94 ° C for 45 seconds, 60 ° C
For 45 seconds and at a temperature of 72 ° C. for 2 minutes 30 times.

(Ii) # 23-57-137-1 cD of antibody L chain V region
Cloning of NA L of mouse monoclonal antibody against human PTHrP
Cloning of the DNA encoding the chain V region is performed by 5'-RAC
E method (Frohman, MA et al., Proc. Natl. Acad. Sci. U
SA 85, 8998-9002, 1988; Belyavsky, A. et al., Nucl
eic Acids Res. 17, 2919-2932, 1989). Five'
The 5'-Ampli Finder RACE Kit (Clonetech) was used for the -RACE method, and the operation was performed according to the attached prescription. The oligo-dT primer was used as a primer for cDNA synthesis. Using about 2 μg of the mRNA prepared as described above as a template, oligo-
Reverse transcription to cDNA was performed by adding dT primer and reacting with reverse transcriptase at 52 ° C. for 30 minutes. 6N NaOH
After hydrolyzing RNA (at 65 ° C. for 30 minutes), cDNA was purified by ethanol precipitation. The above-mentioned Ampli FINDER Anchor was ligated to the 5 ′ end of the synthesized cDNA by reacting with the above T4 RNA ligase at 37 ° C. for 6 hours and at room temperature for 16 hours.

From the conserved sequence of the mouse L chain λ chain constant region,
The CR primer MLC (SEQ ID NO: 4) was designed and 394 DN
It was synthesized using an A / RNA synthesizer (ABI). PC
The R solution contained 10 mM Tris-HCl (pH 8.
3), 50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dC
TP, dTTP), 1.5Mm MgCl ₂ , 2.5 units of AmpliTaq
(PERKIN ELMER), 50 pmole Anchor primer (SEQ ID NO: 2), MLC (SEQ ID NO: 4) and Ampli FI
Contains 1 μl of reaction mixture of cDNA linked to NDER Anchor. The solution was overlaid with 50 μl of mineral oil. PCR
Was performed 35 times using a Thermal Cycler Model 480J (Perkin Elmer) at a temperature cycle of 94 ° C. for 45 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 2 minutes.

(3) Purification and Fragmentation of PCR Product The DNA fragment amplified by the PCR method as described above was separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products). D of about 550 bp long as the H chain V region and 550 bp long as the L chain V region
Cut the agarose piece containing the NA fragment and use GENECLEAN
DNA using II Kit (BIO101)
The fragment was purified. After precipitating the purified DNA with ethanol, 10 mM Tris-HCl (pH 7.4), 20 mM 1 mM EDTA solution
l. 1 μl of the obtained DNA solution was mixed with restriction enzyme X
The digestion was carried out at 37 ° C. for 1 hour with maI (New England Biolabs) and then for 1 hour at 37 ° C. with the restriction enzyme EcoRI (Takara Shuzo). The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation. Thus, having an EcoRI recognition sequence at the 5′-end,
DNA encoding a mouse H chain V region having an XmaI recognition sequence at the 3'-end and D encoding an L chain V region
NA was obtained.

The DNA encoding the mouse H chain V region and the D encoding the L chain V region prepared as described above.
An EcoRI-XmaI DNA fragment containing NA and a pUC19 vector prepared by digestion with EcoRI and XmaI were ligated with DN.
A ligation kit ver.2 (Takara Shuzo) was used and reacted at 16 ° C. for 1 hour according to the attached prescription to ligate. Then 10μ
l of the above ligation mixture was added to 100 μl of E. coli JM109 competent cells (Nippon Gene), and the cells were allowed to stand on ice for 15 minutes, at 42 ° C. for 1 minute, and on ice for 1 minute. Then, 300 μl of SOC medium (Molecular Cloning:
A LabgoratoryManual, Sambrook, et al., Cold Spring
Harbor Laboratory Press, 1989)
100 μg / ml or 50 μg
Agar medium containing LB / ml ampicillin, 0.1 mM IPTG, 20 μg / ml X-gal or 2 × YT agar medium (Molecular Cloning: A Labgoratory Manual, Sambroo)
k, et al., Cold Spring Harbor Laboratory Press, 198
9) This Escherichia coli was sowed on the plate and incubated at 37 ° C overnight to obtain an Escherichia coli transformant.

This transformant was treated with 100 μg / ml or 50 μg / ml.
LB medium containing 2 g / ml ampicillin or 2 ×
After culturing overnight at 37 ° C. in 2 ml of YT medium, plasmid extractor PI-100Σ (Kurabo Industries) or QIAprep Spin
Plasmid DNA was prepared using Plasmid Kit (QIAGEN), and the nucleotide sequence was determined.

(4) DNA encoding mouse antibody V region
Determination of the nucleotide sequence of the cDNA coding region in the plasmid
Dye terminator cycle sequencing kit (D
ye Terminator Cycle Sequencing kit (Perkin-Elmer))
Using a DNA sequencer 373A (ABI Perkin-Elm
er). M13 as a sequencing primer
Primer M4 (Takara Shuzo) (SEQ ID NO: 5) and M13 Primer R
Using V (Takara Shuzo) (SEQ ID NO: 6), the sequence was determined by confirming the nucleotide sequence in both directions.

Hybridoma # 23-57- thus obtained
DNA encoding mouse H chain V region derived from 137-1
Was named MBC1H04, and the plasmid containing the DNA encoding the L chain V region was named MBC1L24. The nucleotide sequences (including the corresponding amino acid sequences) of the DNAs encoding the H chain V region and L chain V region of mouse # 23-57-137-1 antibody contained in plasmids MBC1H04 and MBC1L24 are shown in SEQ ID NOs: 57 and 65, respectively. Show. Both the polypeptide containing the H chain V region and the polypeptide containing the L chain V region start from the 58th position (encoding glutamine) of the nucleotide sequence represented by SEQ ID NOS: 57 and 65, respectively. These amino acid sequences are shown in SEQ ID NO: 46 for the polypeptide containing the H chain V region, and SEQ ID NO: 45 for the polypeptide containing the L chain V region.

The plasmids MBC1H04 and MBC1H04
Escherichia coli having L24 is Escherichia coli JM109 (MBC1
H04) and Escherichia coli JM109 (MBC1L24) to the Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture) on August 15, 1996.
FERM BP-562 for E. coli JM109 (MBC1H04)
8.FERM for Escherichia coli JM109 (MBC1L24)
Deposited internationally under the Budapest Treaty as BP-5627.

(5) Determination of CDR of mouse monoclonal antibody # 23-57-137-1 against human PTHrP The overall structures of the H chain V region and L chain V region are similar to each other, One framework part consists of three hypervariable regions, the complementarity determining regions (CD
R). The amino acid sequence of the framework is relatively well conserved, while the amino acid sequence of the CDR regions has extremely high variability (Kabat, EAet).
al., `` Sequence of Proteins of Immunological Inte
rest "US Dept. Health and Human Services, 1983).

Based on these facts, human PTHrP
The CDR region was determined as shown in Table 2 by applying the amino acid sequence of the variable region of the mouse monoclonal antibody to the database of the amino acid sequence of the antibody prepared by Kabat et al. The amino acid sequences of CDRs 1 to 3 in the L chain V region are shown in SEQ ID NOS: 59 to 61, respectively, and the amino acid sequences of CDRs 1 to 3 in the H chain V region are shown in SEQ ID NOs: 62 to 64, respectively.

[0154]

[Table 2]

Example 2 Construction of Chimeric Antibody (1) Construction of Chimeric Antibody H Chain (i) Construction of H Chain V Region Cloning to ligate to an expression vector containing genomic DNA of human H chain C region Cγ1 The DNA encoding the mouse H chain V region was modified by PCR. The backward primer MBC1-S1 (SEQ ID NO: 7) hybridizes to DNA encoding the 5'-side of the V region leader sequence and a Kozak consensus sequence (Kozak, M. et a).
l., J. Mol. Biol., 196, 947-950, 1987) and the restriction enzyme Hin.
It was designed to have a dIII recognition sequence. The forward primer MBC1-a (SEQ ID NO: 8) was designed to hybridize to the DNA sequence encoding the 3′-side of the J region and to have a splice donor sequence and a recognition sequence for the restriction enzyme BamHI. For PCR, TaKaRa Ex Taq (Takara Shuzo) was used as a template DNA in 50 μl of the reaction mixture as a template DNA.
07 μg of plasmid MBC1H04, MBC1-a as primer
And MBC1-S1 with 50pmole and 2.5U TaKaRa Ex
Under the conditions containing Taq and 0.25 mM dNTP, 50 μl of mineral oil was layered on the upper layer using the attached buffer solution.
30 minutes with a temperature cycle of 2 minutes at 72 ° C. P
DNA fragments amplified by the CR method were separated by agarose gel electrophoresis using 3% NuSieve GTG agarose (FMC Bio. Products).

A piece of agarose containing a DNA fragment of 437 bp was cut out, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. Purified D
After recovering NA by ethanol precipitation, 10 mM Tris-HCl (p
H7.4) and dissolved in 20 μl of a 1 mM EDTA solution. D obtained
Add 1 μl of NA solution to restriction enzymes BamHI and Hind III (Takara Shuzo)
For 1 hour at 37 ° C. The digestion mixture is extracted with phenol and chloroform, and ethanol
NA was recovered.

[0157] The Hind III-BamHI DNA fragment containing the DNA encoding the mouse H chain V region prepared as described above was digested with Hind III and BamHI to obtain p
It was subcloned into the UC19 vector. Die terminator cycle sequencing kit (Perkin-Elmer) using primers M13 Primer M4 and M13 Primer RV as primers to confirm the nucleotide sequence of this plasmid
Was used to determine the nucleotide sequence using a DNA sequencer 373A (Perkin-Elmer). Contains DNA encoding the mouse H chain V region derived from hybridoma # 23-57-137-1 having the correct nucleotide sequence, and recognizes a Hind III recognition sequence and a Kozak sequence on the 5'-side and BamHI recognition on the 3'-side. The plasmid having the sequence was named MBC1H / pUC19.

(Ii) Construction of H chain V region for preparing mouse-human chimeric H chain of cDNA type In order to ligate with cDNA of human H chain C region Cγ1, mouse H chain V constructed as described above The DNA encoding the region was modified by the PCR method. The backward primer MBC1HVS2 (SEQ ID NO: 9) for modifying the H chain V region converts the second asparagine in the sequence encoding the first of the V region leader sequence to glycine, and uses a Kozak consensus sequence (Kozak, M.et). al., J. Mol. Biol., 196, 947-950, 1987)
And were designed to have Hind III and EcoRI recognition sequences. Forward primer MB for modifying H chain V region
C1HVR2 (SEQ ID NO: 10) hybridizes to the DNA sequence encoding the 3'-side of the J region and binds to the 5'-
The flanking sequence was designed to have the ApaI and SmaI recognition sequences.

For PCR, TaKaRa Ex Taq (Takara Shuzo) was used. In 50 μl of the reaction mixture, 0.6 μg of plasmid MBC1H / pUC19 was used as a template DNA, MBC1HVS2 and MBC1HVR2 were used as primers at 50 pmole, and TaKaRa Ex Taq was added at 2.
Using conditions of 5 U and 0.25 mM dNTP, 50 μl of mineral oil was layered on the upper layer using the attached buffer solution, and the cycle was performed 30 times at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute 30 times. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 1% Sea Kem GTG agarose (FMC Bio. Products). An agarose piece containing a 456 bp DNA fragment was cut out, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. After ethanol precipitation of the purified DNA, 10 mM Tris-HC
l (pH 7.4) and dissolved in 20 μl of 1 mM EDTA solution.

[0160] 1 µl of the obtained DNA solution was subjected to restriction enzyme EcoR.
Digestion with I and SmaI (Takara Shuzo) for 1 hour at 37 ° C.
The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation. DNA encoding mouse H chain V region prepared as described above
Was subcloned into a pUC19 vector prepared by digestion with EcoRI and SmaI. To confirm the nucleotide sequence of this plasmid, the nucleotide sequence was determined using a DNA sequencer 373A (Perkin-Elmer) using a dye terminator cycle sequencing kit (Perkin-Elmer) with primers M13 Primer M4 and M13 Primer RV as primers. .
Hybridoma # 23-57-137-1 having the correct nucleotide sequence
DNA encoding the mouse H chain V region derived from E. coli, and EcoRI and Hind III recognition sequences and Koza
A plasmid having an ApaI and SmaI recognition sequence on the 3′-side of the k sequence was named MBC1Hv / pUC19.

(Iii) Construction of Expression Vector for Chimeric Antibody H Chain cDNA containing human antibody H chain C region Cγ1 was prepared as follows. That is, the genomic DN of the humanized PM1 antibody H chain V region and human antibody H chain C region IgG1
A (N. Takahashi, et al., Cell 29, 671-679 1982)
Expression vector DHFR- △ E-RVh-PM-1-f (WO92 /
19759) and the DN of the humanized PM1 antibody L chain V region.
MRNA was prepared from CHO cells transfected with A and an expression vector RV1-PM1a (see WO92 / 19759) encoding the genomic DNA of the L chain κ chain C region of a human antibody, and RT-PCR
The cDNA containing the humanized PM1 antibody H chain V region and the human antibody C region Cγ1 was cloned by the method and the pUC19 Hi
It was subcloned into ndIII and BamHI sites. After confirming the nucleotide sequence, insert the plasmid with the correct sequence into pRVh-PM1
It was named f-cDNA.

The HindIII site between the SV40 promoter and the DHFR gene on DHFR-ΔE-RVh-PM-1-f, and between the EF-1α promoter and the humanized PM1 antibody H chain V region. An expression vector lacking a certain EcoRI site was prepared and used to construct a cDNA expression vector containing the humanized PM1 antibody H chain V region and human antibody C region Cγ1.

The pRVh-PM1f-cDNA was digested with BamHI, blunted with a Klenow fragment, and further digested with HindIII to prepare a HindIII-BamHI blunted fragment. This Hi
The nd III-BamHI blunted fragment was ligated with the above Hind III site and
DHFR- △ E-RVh-PM1-f with EcoRI site deleted was replaced with Hind III
Then, the resultant was ligated to an expression vector prepared by digestion with SmaI to construct an expression vector RVh-PM1f-cDNA containing cDNA encoding the humanized PM1 antibody H chain V region and human antibody C region Cγ1.

After digesting the expression vector RVh-PM1f-cDNA containing cDNA encoding the humanized PM1 antibody H chain V region and human antibody C region Cγ1 with ApaI and BamHI, the DNA fragment containing the H chain C region is recovered. And ApaI and
It was introduced into MBC1Hv / pUC19 prepared by digestion with BamHI. The plasmid prepared in this manner was used for the MBC1HcDNA / pU
Named C19. This plasmid contains the H chain V of the mouse antibody.
Coding for region and human antibody C region Cγ1
And at the 5'-end EcoRI and HindIII recognition sequences,
It has a BamHI recognition sequence at the 3'-end.

The plasmid MBC1HcDNA / pUC19 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the base sequence encoding the H chain of the chimeric antibody was ligated with EcoRI and BamHI.
Expression vector pCOS1 prepared by digestion with
Was introduced. The expression plasmid of the chimeric antibody thus obtained was named MBC1HcDNA / pCOS1. The expression vector pCOS1 was prepared using HEF-PMh-gγ1 (WO92 / 1
9759), the antibody gene was deleted by EcoRI and SmaI digestion, and EcoRI-NotI-BamHI Adapter (Takara Shuzo) was ligated.

To prepare a plasmid for use in expression in CHO cells, plasmid MBC1HcDNA / pUC1
9 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the chimeric antibody H chain sequence was introduced into an expression plasmid pCHO1 prepared by digestion with EcoRI and BamHI. The expression plasmid for the chimeric antibody thus obtained was named MBC1HcDNA / pCHO1. The expression vector pCHO1 is obtained by deleting the antibody gene from DHFR-ΔE-rvH-PM1-f (see WO92 / 19759) by digestion with EcoRI and SmaI, and ligating EcoRI-NotI-BamHI Adapter (Takara Shuzo). It was constructed.

(2) Construction of human L chain constant region (i) Construction of cloning vector In order to construct a pUC19 vector containing a human L chain constant region, a Hind III site deleted pUC19 vector was constructed. pUC1
9 2 μg of vector was added to 20 mM Tris-HCl (pH 8.5), 10 mM M
The digestion was carried out at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing gCl ₂ , 1 mM DTT, 100 mM KCl, and 8 U of Hind III (Takara Shuzo). The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.

The recovered DNA was washed with 50 mM Tris-HCl (pH 7.
5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM NaCl, 0.5 mM dNTP,
50μ containing 6U Klenow fragment (GIBCO BRL)
The reaction was performed at room temperature for 20 minutes in 1 l of the reaction mixture to blunt the ends. The reaction mixture was extracted with phenol and chloroform, and the vector DNA was recovered by ethanol precipitation.

[0169] The recovered vector DNA was treated with 50 mM Tris-HCl.
(PH 7.6), 10 mM MgCl ₂ , 1 mM ATP, 1 mM DTT, 5% (v
/ v) polyethylene glycol-8000, 16 U in a 10 μl reaction mixture containing 0.5 U of T4 DNA ligase (GIBCO BRL).
The reaction was carried out at a temperature of 2 ° C. for 2 hours for self-ligation. Reaction mixture 5μ
l for E. coli JM109 competent cells (Nippon Gene)
Add 100 μl, leave on ice for 30 minutes,
And then left on ice for 1 minute. SOC medium 500 μ
After incubating at 37 ° C. for 1 hour,
2 × YT agar medium (50 μL) coated with X-gal and IPTG on the surface
g / ml ampicillin) (Molecular Cloning: A Lab
goratory Manual, Sambrook, et al., Cold Spring Harb
or Laboratory Press, 1989) and cultured at 37 ° C. overnight to obtain a transformant.

The transformant was cultured overnight at 37 ° C. in 20 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid DNA was purified from the cell fraction using a Plasmid Mini Kit (QIAGEN) according to the attached protocol. . The purified plasmid was digested with HindIII, and the plasmid confirmed to have deleted the HindIII site was named pUC19ΔHindIII.

(Ii) D encoding human L chain λ chain constant region
Construction of NA The human antibody light chain λ chain C region is Mcg + Ke + Oz−
, Mcg-Ke-Oz-, Mcg-Ke-Oz
+ And Mcg-Ke + Oz-, at least four isotypes are known (P. Dariavach, et al.,
Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987). # 23-5
7-137-1 A human antibody L chain λ chain C region having homology to the mouse L chain λ chain C region was searched for in the EMBL database, and as a result, the isotype was Mcg + Ke + Oz- (acce
ssion No. X57819) (P. Dariyavach, et al., Proc. Natl. A
cad. Sci. USA, 84, 9074-9078, 1987) shows the highest homology with the # 23-57-137-1 mouse L chain λ.
The homology with the chain C region was 64.4% in the amino acid sequence and 73.4% in the nucleotide sequence.

Therefore, construction of DNA encoding the human antibody L chain λ chain C region was carried out by PCR. The synthesis of each primer was performed using a 394 DNA / RNA synthesizer (ABI
Was carried out using HLAMB1 (SEQ ID NO: 11) and HLAMB
3 (SEQ ID NO: 13) has a sense DNA sequence and HLAMB2
(SEQ ID NO: 12) and HLAMB4 (SEQ ID NO: 14) have antisense DNA sequences, with complementary sequences of 20 to 23 bp at each end of each primer.

External primers HLAMBS (SEQ ID NO: 15), HL
AMBR (SEQ ID NO: 16) has sequences homologous to HLAMB1 and HLAMB4, respectively, and HLAMBBS is EcoRI, HindIII, Bl
HLAMBR contains an EcoRI recognition sequence, and HLAMBR contains an EcoRI recognition sequence. HLAMB1-HLAMB2 and HLAMB3-HLAMB4 in the first PCR
Was performed. After the reaction, they are mixed in equal amounts and the second P
Assembly was performed in CR. Further external primer HLAM
BS and HLAMBR were added, and the full-length DNA was
Was amplified.

PCR was performed using TaKaRa Ex Taq (Takara Shuzo) according to the attached prescription. In the first PCR, 5pm
ole HLAMB1 and 0.5pmole HLAMB2 and 5U TaKaRa
100 μl reaction mixture containing Ex Taq (Takara Shuzo), or 0.5 pmole HLAMB3 and 5 pmole HLAMB4
100μ containing 5U of TaKaRa Ex Taq (Takara Shuzo)
Using 1 l of the reaction mixture, 50 μl of mineral oil was applied to the upper layer and subjected to 5 times of temperature cycles at 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute. In the second PCR, the reaction solution was mixed in 50 μl portions, 50 μl of mineral oil was layered on the upper layer, and the mixture was heated at 94 ° C. for 1 minute at 60 ° C.
For 1 minute and a temperature cycle of 72 ° C. for 1 minute three times. In the third PCR, 50 pmole of each of the external primers HLAMBS and HLAMBR was added to the reaction solution, and the mixture was added at 94 ° C for 1 minute for 60 minutes.
The test was performed 30 times at a temperature cycle of 1 minute at 72 ° C and 1 minute at 72 ° C.

After the DNA fragment of the third PCR product was electrophoresed on a 3% low-melting point agarose gel (NuSieve GTG Agarose, FMC), the DNA fragment was recovered and purified from the gel using GENECLEANII Kit (BIO101) according to the attached instructions. The obtained DNA fragment was treated with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 1 mM
20 μl containing 00 mM NaCl, 8 U EcoRI (Takara Shuzo)
Was digested at 37 ° C. for 1 hour in the reaction mixture. The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation, and then 10 mM Tris-HCl (pH 7.4),
It was dissolved in 8 μl of a mM EDTA solution.

0.8 μg of the plasmid pUC19 ΔHind III was similarly digested with EcoRI, extracted with phenol and chloroform, and recovered by ethanol precipitation. The digested plasmid pUC19 ΔHind III was added to 50 mM Tris-HCl (pH 9.0),
The mixture was reacted at 37 ° C. for 30 minutes in 50 μl of a reaction mixture containing mM MgCl ₂ and alkaline phosphatase (E. coli C75, Takara Shuzo) for dephosphorylation (BAP treatment). The reaction solution was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation, and then 10 mM Tris-HCl (pH 7.4), 1 mM ED
It was dissolved in 10 μl of a TA solution.

The BAP-treated plasmid pUC19Δ
1 μl of Hind III and 4 μl of the PCR product were mixed with DNA Liga
Ligation Kit Ver.2 (Takara Shuzo) and E. coli JM10
Transformed into 9 competent cells. The obtained transformant was treated with 2 × YT medium 2 containing 50 μg / ml ampicillin.
After culturing overnight, the QIAprep Spin PlasmidKi
The plasmid was purified using t (QIAGEN).

With respect to the above plasmid, the nucleotide sequence of the cloned DNA was confirmed. The nucleotide sequence was determined using a 373A DNA sequencer (ABI), and M13 primer M4 and M13 primer RV (Takara Shuzo) were used as primers. As a result, the cloned DNA
Was found to have a 12 bp deletion inside. This DNA
Was named CλΔ / pUC19. Therefore, primer HCLMS (SEQ ID NO:
17), HCLMR (SEQ ID NO: 18) was newly synthesized, and the correct DNA was again constructed by PCR.

In the first PCR, plasmid CλΔ / pUC19 containing the deleted DNA was used as a template, and primers HLAMBS and HCLMR were used.
, HCLMS and HLAMB4. Each PCR product was purified and assembled in a second PCR. Add external primers HLAMBS and HLAMB4 and add 3rd PC
R amplified the full-length DNA.

In the first PCR, CλΔ / pUC1 was used as a template.
9 0.1μg, primer HLAMBS and HCLMR 50pmole each
Or 50pmole of HCLMS and HLAMB4 each, 5U of TaK
Using 100 μl of the reaction mixture containing aRa Ex Taq (Takara Shuzo), overlay 50 μl of mineral oil at 94 ° C. for 1 min.
The test was performed 30 times at a temperature cycle of 1 minute at 72 ° C and 1 minute at 72 ° C.

PCR products HLAMBS-HCLMR (236 bp), HCLMS-
HLAMB4 (147 bp) was electrophoresed on a 3% low melting point agarose gel, and then recovered and purified from the gel using GENECLEANII Kit (BIO101). In the second PCR, 20 ng each containing 40 ng of the purified DNA fragment and 1 U of TaKaRa Ex Taq (Takara Shuzo) were used.
Using 25 μl of mineral oil, add 94 μl of reaction mixture
Temperature cycles of 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute were performed five times.

In the third PCR, 2 μm of the second PCR reaction solution was used.
1, external primers HLAMBS, HLAMB4 50pmole each, 5U
Using 100 μl of the reaction mixture containing TaKaRa Ex Taq (Takara Shuzo), 50 μl of mineral oil was overlaid. PCR at 94 ° C
For 30 minutes at 60 ° C. for 1 minute and at 72 ° C. for 1 minute. The third PCR product, 357 bp D
After electrophoresis of the NA fragment on a 3% low melting point agarose gel, the gel was recovered from the gel using GENECLEANII Kit (BIO101).
Purified.

After digesting 0.1 μg of the obtained DNA fragment with EcoRI, BAP-treated plasmid pUC19ΔHindIII
Was subcloned. E. coli JM109 competent cells were transformed and cultured overnight in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin.
The plasmid was purified using p Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the purified plasmid was determined using M13 Primer M4 and M13 Primer RV (Takara Shuzo).
It was determined by A DNA sequencer (ABI). The plasmid confirmed to have the correct nucleotide sequence without deletion was designated as Cλ / pUC19.

(Iii) Construction of DNA encoding human L chain κ chain constant region L chain κ chain C from plasmid HEF-PM1k-gk (WO92 / 19759)
The DNA fragment encoding the region was cloned using the PCR method. 394 The forward primer HKAPS (SEQ ID NO: 19) synthesized using a DNA / RNA synthesizer (ABI)
EcoRI, HindIII and BlnI recognition sequences were added to the rear primer HK
APA (SEQ ID NO: 20) was designed to have an EcoRI recognition sequence.

Plasmid HEF-PM1k-gk as template 0.1 μm
g, primer HKAPS, HKAPA 50pmole each, 5U TaKa
Using 100 μl of the reaction mixture containing Ra Ex Taq (Takara Shuzo), 50 μl of mineral oil was overlaid. 60 minutes at 94 ° C for 1 minute
The reaction was carried out at 30 ° C. for 1 minute and at 72 ° C. for 1 minute for 30 cycles. After a 360 bp PCR product was electrophoresed on a 3% low melting point agarose gel, it was recovered and purified from the gel using a GENECLEANII Kit (BIO101).

The obtained DNA fragment was digested with EcoRI, and cloned into BAP-treated plasmid pUC19ΔHindIII. E. coli JM109 competent cells were transformed and 2 × YT containing 50 μg / ml ampicillin.
After culturing overnight in 2 ml of medium, QIAprep Spin Pla
The plasmid was purified using a smid Kit (QIAGEN). The nucleotide sequence of the purified plasmid was converted to M13 primers M4 and M13.
Using a primer RV (Takara Shuzo), determination was performed using a 373A DNA sequencer (ABI). The plasmid confirmed to have the correct nucleotide sequence was designated as Cκ / pUC19.

(3) Construction of Chimeric Antibody L Chain Expression Vector A chimeric # 23-57-137-1 antibody L chain expression vector was constructed. Plasmids Cλ / pUC19 and Cκ / pUC19 were ligated with HindIII and BlnI sites immediately before the human antibody constant region, and # 23-57-
The DNA encoding the 137-1 L chain V region was ligated to obtain the chimeric # 23-57-137-1 antibody L chain V region and D chain encoding the L chain λ chain or L chain κ chain constant region, respectively.
A pUC19 vector containing NA was prepared. The DNA encoding the chimeric antibody L chain was excised by EcoRI digestion and
Subcloning was performed into an EF expression vector.

Specifically, plasmids MBC1L24 to # 23-5
7-137-1 DNA encoding antibody L chain V region
Cloned using the method. The synthesis of each primer
This was performed using a 394 DNA / RNA synthesizer (ABI).
The rear primer MBCCHL1 (SEQ ID NO: 21) is composed of a Hind III recognition sequence and a Kozak sequence (Kozak, M. et al., J. Mol. Biol. 196,
947-950, 1987) was designed so that the forward primer MBCCHL3 (SEQ ID NO: 22) had BglII and EcoRI recognition sequences.

PCR was performed using 10 mM Tris-HCl (pH 8.3), 50 mM K
Cl, 1.5 mM MgCl ₂ , 0.2 mM dNTP, 0.1 μg MBC1L2
4.MBCCHL1 and MBCCHL3 as primers 50pm each
ole, 100 μl containing 1 μl AmpliTaq (PERKIN ELMER)
Using 50 μl of mineral oil, add 94 μl of reaction mixture
For 30 seconds at 45 ° C., 45 seconds at 60 ° C., and 2 minutes at 72 ° C.

After the 444 bp PCR product was electrophoresed on a 3% low-melting point agarose gel, the product was recovered from the gel using the GENECLEAN II kit (BIO101), purified, and then 10 mM Tris-HCl (pH 7.4).
And dissolved in 20 μl of a 1 mM EDTA solution. PCR product 1μ
1 with 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1
Digestion was performed at 37 ° C. for 1 hour in a 20 μl reaction mixture containing mM DTT, 50 mM NaCl, 8 U of Hind III (Takara Shuzo) and 8 U of EcoRI (Takara Shuzo). The digestion mixture was extracted with phenol and chloroform, the DNA was recovered by ethanol precipitation, and 10 mM Tris-HCl (pH 7.4), 8 mM
l.

Similarly, 1 μg of the plasmid pUC19 was added to HindIII.
And digested with EcoRI, extracted with phenol and chloroform, recovered by ethanol precipitation, and BAP-treated with alkaline phosphatase (E. coli C75, Takara Shuzo).
The reaction solution was extracted with phenol and chloroform, DNA
Was recovered by ethanol precipitation, and then 10 mM Tris-HCl (pH 7.
4) Dissolved in 10 μl of 1 mM EDTA solution.

1 μl of the BAP-treated plasmid pUC19 and 4 μl of the above PCR product were ligated using DNA Ligation Kit Ver. 2 (Takara Shuzo) and transformed into E. coli JM109 competent cells (Nippon Gene) in the same manner as described above. This was spread on a 2 × YT agar medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. overnight. The resulting transformant was transformed with 2 × YT containing 50 μg / ml ampicillin.
The cells were cultured overnight at 37 ° C in 2 ml of the medium. QIAprep from cell fraction
The plasmid was purified using Spin Plasmid Kit (QIAGEN). After determining the nucleotide sequence, the plasmid having the correct nucleotide sequence was designated as CHL / pUC19.

Each 1 μg of each of the plasmids Cλ / pUC19 and Cκ / pUC19 was added to 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ ,
20 μl of reaction mixture containing 1 mM DTT, 100 mM KCl, 8 U Hind III (Takara Shuzo) and 2 U BlnI (Takara Shuzo)
For 1 hour at 37 ° C. The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation, followed by BAP treatment at 37 ° C. for 30 minutes. The reaction solution was extracted with phenol and chloroform, the DNA was recovered by ethanol precipitation, and 10 mM Tris-HCl (pH 7.4), 1 mM
It was dissolved in 10 μl of a DTA solution.

# 23-57-137-1 D encoding L chain V region
8 μg of plasmid CHL / pUC19 containing NA
Digested with ndIII and BlnI. The resulting 409 bp DNA
After the fragments were electrophoresed on a 3% low melting point agarose gel,
It was recovered from the gel using the NECLEANII Kit (BIO101), purified, and dissolved in 10 mM Tris-HCl (pH 7.4) and 10 μl of 1 mM EDTA solution.

4 μl of this L chain V region DNA was treated with BAP-treated plasmid Cλ / pUC19 or Cκ / pUC19 for 1 each.
The cells were subcloned into μl and transformed into E. coli JM109 competent cells. The cells were cultured overnight in 3 ml of 2 × YT medium containing 50 μg / ml ampicillin.
The plasmid was purified using Aprep Spin Plasmid Kit (QIAGEN). These were respectively treated with plasmid MBC1L (λ) / pU
C19 and MBC1L (κ) / pUC19 were used. Plasmid MBC1L (λ) /
pUC19 and MBC1L (κ) / pUC19 were each digested with EcoRI, and electrophoresed on a 3% low melting point agarose gel.
The DNA fragment of p was recovered and purified from the gel using the GENECLEANII Kit (BIO101), and 10 mM Tris-HCl (pH 7.4), 1 mM EDT
A solution was dissolved in 10 μl.

Plasmid HEF-PM1k-g was used as an expression vector.
2.7 μg of k was digested with EcoRI, extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.
After BAP treatment of the recovered DNA fragment, it was electrophoresed on a 1% low melting point agarose gel, and the 6561 bp DNA fragment was
Collect and purify from the gel using CLEANII Kit (BIO101),
It was dissolved in 10 mM Tris-HCl (pH 7.4) and 10 μl of 1 mM EDTA solution.

2 μl of the BAP-treated HEF vector was ligated with 3 μl of each of the above-mentioned plasmid MBC1L (λ) or MBC1L (κ) EcoRI fragment and transformed into E. coli JM109 competent cells. 2 × containing 50 μg / ml ampicillin
After culturing in 2 ml of YT medium, QIAprep Spin Pla
The plasmid was purified using smid Kit (QIAGEN).

The purified plasmid was added to 20 mM Tris-HCl (p
H8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U Hi
Digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing ndIII (Takara Shuzo) and 2 U of PvuI (Takara Shuzo). If the fragment is inserted in the correct direction, 5104/2195 bp, if inserted in the reverse direction, a digested fragment of 4378/2926 bp will be generated, so that the plasmid inserted in the correct direction is MBC1L (λ) / neo, MBC1L (κ) / neo.

(4) Transfection of COS-7 cells In order to evaluate the antigen-binding activity and the neutralizing activity of the chimeric antibody, the expression plasmid was transiently expressed in COS-7 cells. That is, the transient expression of the chimeric antibody was determined by the plasmid MBC1HcDNA / pCOS1 and MBC1L.
(Λ) / neo, or MBC1H cDNA / pCOS
Gene P in combination with MBC1L (κ) / neo
COS-7 cells were co-transduced by electroporation using an ulser apparatus (Bio Rad). PBS
10 μl of each plasmid DNA was added to 0.8 ml of COS-7 cells suspended at a cell concentration of 1 × 10 ⁷ cells / ml in (−).
g, and a pulse was applied with a capacitance of 1,500 V and 25 μF. After a 10 minute recovery period at room temperature, the electroporated cells were suspended in DMEM medium (GIBCO) containing 2% Ultra Low IgG fetal calf serum (GIBCO) and CO2 was removed using a 10 cm culture dish. _The cells were cultured in _two incubators. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and used for ELISA samples. In addition, the purification of the chimeric antibody from the culture supernatant of the COS-7 cells was performed using an AffiGel Protein A MAPSII kit (BioRad) according to the instructions attached to the kit.

(5) ELISA (i) Measurement of Antibody Concentration An ELISA plate for antibody concentration measurement was prepared as follows. Fill each well of a 96-well ELISA plate (Maxisorp, NUNC) with a solid phase buffer (0.1 M NaHCO ₃ , 0.02% NaN).
₃ ) Immobilize with 100 µl of goat anti-human IgG antibody (TAGO) adjusted to a concentration of 1 µg / ml in 200 µl of dilution buffer (50 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05%
Tween20,0.02% NaN _3, 1% bovine serum albumin (B
After blocking at SA), pH 7.2), the culture supernatant of the COS cells expressing the chimeric antibody or the purified chimeric antibody was serially diluted and added to each well. After incubating for 1 hour at room temperature and washing with PBS-Tween20, 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubating for 1 hour at room temperature, washing with PBS-Tween20, 1 mg / ml substrate solution (Sigma104, p-nitrophenylphosphate, SIGMA) was added, and then the absorbance at 405 nm was measured using a microplate reader (Bio Rad ).
Hu IgG1λ Purified as a standard for concentration measurement
(The BindingSite) was used.

(Ii) Measurement of antigen-binding ability An ElISA plate for antigen-binding measurement was prepared as follows. Each well of the 96-well plate for ELISA was immobilized with 100 μl of human PTHrP (1-34) (Peptide Research Institute) adjusted to a concentration of 1 μg / ml with an immobilization buffer. After blocking with 200 μl of the dilution buffer, the culture supernatant of the COS cells expressing the chimeric antibody or the purified chimeric antibody was serially diluted and added to each well. After incubating at room temperature and washing with PBS-Tween20, alkaline phosphatase-conjugated goat anti-human IgG antibody (TAG
O) 100 μl was added. Incubate at room temperature in PBS
After washing with -Tween20, 1 mg / ml substrate solution (Sigma10
4, p-nitrophenyl phosphate, SIGMA), then add 4
Measure the absorbance at 05 nm using a microplate reader (Bio Ra
Measured in d).

As a result, the chimeric antibody was human PTHrP
(1-34), indicating that it has the correct structure of the cloned mouse antibody V region (FIG. 4). In the chimeric antibody, the L chain C region is λ
PTHrP of the antibody, whether it is a κ chain or a κ chain
Since the binding ability to (1-34) does not change, the L chain C region of the humanized antibody was constructed using the humanized antibody L chain λ chain.

(6) Establishment of Stable CHO Producing Cell Line In order to establish a stable chimeric antibody producing cell line, the above expression plasmid was introduced into CHO cells (DXB11). That is, the establishment of a cell line for stable production of chimeric antibodies was performed by using the expression plasmid MBC1HcDNA / pCHO1 for CHO cells and M
BC1L (λ) / neo or MBC1H cDNA /
CHO cells were co-transduced by electroporation using a combination of pCHO1 and MBC1L (κ) / neo using a Gene Pulser apparatus (Bio Rad). Each expression vector was cleaved with a restriction enzyme PvuI to obtain linear DNA, and extracted with phenol and chloroform.
DNA was recovered by ethanol precipitation and used for electroporation. 10 μg of each plasmid DNA was added to 0.8 ml of CHO cells suspended at a cell concentration of 1 × 10 ⁷ cells / ml in PBS (−), and pulsed at 1,500 V, 25 μF capacitance. After a 10 minute recovery period at room temperature, the electroporated cells were transformed into MEM-α medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO).
) And cultured in a CO ₂ incubator using three 96-well plates (Falcon). The day after the start of the culture, 10% fetal bovine serum (GIBCO) and 500 mg / ml GENE
TICIN (G418 Sulfate, GIBCO) was added, and the selection medium of MEM-α medium (GIBCO) free of ribonucleoside and deoxyribonucleoside was exchanged to select cells into which the antibody gene was introduced. After exchanging the selective medium, the cells were observed under a microscope about two weeks, and after successful cell growth was observed, the antibody production was measured by the antibody concentration measurement ELISA, and cells having a large antibody production were selected. .

The culture of the established antibody-producing stable cell line was expanded, and 2% Ultra Law IgG fetal bovine serum was added using a roller bottle, and a large-scale culture was performed using a MEM medium free of ribonucleoside and deoxyribonucleoside. Was. On the third or fourth day of the culture, the culture supernatant was collected, and cell debris was removed with a 0.2 μm filter (Millipore). CH
Purification of the chimeric antibody from the culture supernatant of the O cells was performed using PORO.
Using an S Protein A column (PerSeptive Biosystems), ConSep LC100 (Millipore) was used according to the attached prescription, and subjected to the measurement of neutralizing activity and the efficacy test in a model animal of hypercalcemia. The concentration and antigen-binding activity of the purified chimeric antibody obtained were determined by the ELISA
It was measured in the SA system.

[Example 3] Construction of humanized antibody (1) Construction of humanized antibody H chain (i) Construction of humanized H chain V region Humanized # 23-57-137-1 antibody H Strand
It was prepared by CDR-grafting according to the method. Human antibody S31679 (NBRF-PDB, Cuisinier AM et al., Eur.J.Immuno
l., 23, 110-118, 1993).
Six PCR primers were used for the production of the 3-57-137-1 antibody heavy chain (version "a"). CD
R-grafting primers MBC1HGP1 (SEQ ID NO: 23) and MBC1HGP3 (SEQ ID NO: 24) have a sense DNA sequence and CDR grafting primers MBC1HGP2 (SEQ ID NO: 25) and MBC1
1HGP4 (SEQ ID NO: 26) has an antisense DNA sequence and has 15 to 2
It has a 1 bp complementary sequence. External primer MBC1
HVS1 (SEQ ID NO: 27) and MBC1 HVR1 (SEQ ID NO: 28) are CDR grafting primers MBC1
It has homology with HGP1 and MBC1HGP4.

CDR-grafting primer MBC
1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 were separated using a urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Ma
nual, Sambrook et al., Cold Spring Harbor Laboratory Pr
ess, 1989), extraction from gels was carried out by the crush and soak method (Molec
ular Cloning: A Laboratory Manual, Sambrook et al., Col
d Spring Harbor Laboratory Press, 1989).

That is, each 1 nmole CDR
-The grafting primer is separated on a 6% denaturing polyacrylamide gel, the DNA fragment of the desired size is identified by irradiating it with ultraviolet light on a silica gel thin plate.
20 μl of 10 mM Tr collected from the gel by the d soak method
It was dissolved in is-HCl (pH 7.4) and 1 mM EDTA solution. PCR was performed using TaKaRa Ex Taq (Takara Shuzo) and adding 100 μl of the reaction mixture to the CD prepared as described above.
R-grafting primer MBC1HGP1, MB
C1HGP2, MBC1HGP3 and MBC1HGP
4 for 1 minute at 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. under the conditions containing 1 μl each of 0.25 mM dNTP and 2.5 U of TaKaRa Ex Taq, using the attached buffer.
5 times with a 1 minute temperature cycle. 50p more
mole primers MBC1HVS1 and MBC
1 HVR1 was added and the same temperature cycle was performed 30 times.
4% NuSieve GTG
Separation was performed by agarose gel electrophoresis using agarose (FMC Bio. Products).

A piece of agarose containing a 421 bp DNA fragment was cut out, and GENECLEAN II Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. After the purified DNA was precipitated with ethanol, 10 mM Tris-HCl (pH 7.4), 1 mM
It was dissolved in 20 μl of a mM EDTA solution. Obtained PC
The R reaction mixture was subcloned into pUC19 prepared by digestion with BamHI and HindIII, and the nucleotide sequence was determined. The plasmid having the correct sequence was named hMBCHv / pUC19.

(Ii) Construction of H chain V region for humanized H chain cDNA In order to ligate to the human H chain C region Cγ1 cDNA, the humanized H chain V region was constructed as described above. DNA was modified by the PCR method. Back primer MBC1HV
S2 hybridizes with a sequence encoding the 5'-side of the leader region of the V region, and has a Kozak consensus sequence (Kozak, M, et al., J. Mol. Biol. 196, 947-950, 1987), Hi.
Designed to have ndIII and EcoRI recognition sequences. The forward primer MBC1HVR2 for modifying the DNA of the H chain V region hybridizes to the DNA sequence encoding the 3′-side of the J region and 5 ′ of the C region.
The negative sequence was designed to encode the ApaI and SmaI recognition sequences.

PCR was performed using TaKaRa Ex Taq (Takara Shuzo) and 0.4 μg of hMBC as a template DNA.
Using Hv / pUC19, MBC1H was used as a primer.
50 pmol of VS2 and MBC1HVR2 respectively
e, 2.5 U TaKaRaEx Taq, 0.25 m
M dNTP, using attached buffer at 94 ° C
For 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute 30 times. D amplified by PCR
The NA fragment was ligated to 3% NuSieve GTG agarose (FMC
Separation by agarose gel electrophoresis using Bio. Products).

A piece of agarose containing a 456 bp DNA fragment was cut out, and GENECLEAN II Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. After the purified DNA was precipitated with ethanol, 10 mM Tris-HCl (pH 7.4), 1 mM
It was dissolved in 20 μl of a mM EDTA solution. Obtained PC
The R reaction mixture was subcloned into pUC19 prepared by digestion with EcoRI and SmaI, and the nucleotide sequence was determined. Hybridoma # 2 thus obtained
A plasmid containing a DNA encoding the mouse H chain V region derived from 3-57-137-1 and having an EcoRI and HindIII recognition sequence and a Kozak sequence on the 5'-side and an ApaI and SmaI recognition sequence on the 3'-side. Was designated as hMBC1Hv / pUC19.

(2) Construction of Expression Vector for Humanized Antibody H Chain Plasmid RV containing hPM1 antibody H chain cDNA sequence
h-PM1f-cDNA is digested with ApaI and BamHI, and DN containing a nucleotide sequence encoding the H chain C region.
The A fragment was recovered and introduced into hMBC1Hv / pUC19 prepared by digestion with ApaI and BamHI. The thus prepared plasmid was designated as hMBC1HcD.
Named NA / pUC19. This plasmid contains DNAs encoding the H chain V region and human H chain C region Cγ1 of the humanized # 23-57-137-1 antibody, and EcoRI and HindIII recognition sequences at the 5′-end and 3′-end. It has a BamHI recognition sequence. Plasmid hMBC1H
SEQ ID NO: 58 shows the nucleotide sequence and corresponding amino acid sequence of humanized H chain version "a" contained in cDNA / pUC19. The amino acid sequence of version a is shown in SEQ ID NO: 56.

The hMBC1H cDNA / pUC19 was replaced with Ec
The DNA fragment containing the H chain sequence obtained by digestion with oRI and BamHI was introduced into an expression plasmid pCOS1 prepared by digestion with EcoRI and BamHI. The expression plasmid for the humanized antibody thus obtained was named hMBC1HcDNA / pCOS1.

Further, hMBC1H cDNA / pU was used to prepare a plasmid for use in expression in CHO cells.
C19 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the heavy chain sequence was digested with EcoRI and BamHI.
Expression plasmid p prepared by digestion with mHI
CHO1. The expression plasmid for the humanized antibody thus obtained was named hMBC1HcDNA / pCHO1.

(3) Construction of L chain hybrid variable region (i) Preparation of FR1,2 / FR3,4 hybrid antibody DNA encoding L chain obtained by recombining FR regions of humanized antibody and mouse (chimeric) antibody was prepared. Each region was constructed and evaluated for humanization. By utilizing the restriction enzyme AflII cleavage site in CDR2, hybrid antibodies were prepared in which FR1 and FR2 were derived from human antibodies and FR3 and FR4 were derived from mouse antibodies.

10 μg of each of the plasmids MBC1L (λ) / neo and hMBC1L (λ) / neo was added to 10 mM T
ris-HCl (pH 7.5), 10 mM MgCl ₂ ,
1 mM DTT, 50 mM NaCl, 0.01% (w /
v) Digestion was performed at 37 ° C. for 1 hour in 100 μl of a reaction mixture containing 10 U of BSA and AflII (Takara Shuzo). The reaction solution was subjected to electrophoresis on a 2% low-melting point agarose gel, and a fragment (hereinafter referred to as c1) of 6282 bp and a fragment (hereinafter referred to as c2) of plasmid MBC1L (λ) / neo and plasmid hMBC1L (λ) / neo of 622 bp were obtained.
The 82 bp fragment (h1) and the 1022 bp fragment (h2) were combined with the GENECLEANII Kit.
It was recovered and purified from the gel using (BIO101). BAP treatment was performed on each 1 μg of the collected c1 and h1 fragments. The DNA was extracted with phenol and chloroform, collected by ethanol precipitation, and then 10 mM Tris-
HCl (pH 7.4), 10 μl of 1 mM EDTA solution
Was dissolved.

1 μl of the BAP-treated c1 and h1 fragments were ligated to 4 μl of the h2 and c2 fragments, respectively (4 ° C., overnight), and transformed into E. coli JM109 competent cells. The cells were cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and QIAprep Spin Plasmid was isolated from the cell fraction.
The plasmid was purified using Kit (QIAGEN).

The purified plasmid was added to 10 mM Tris
-HCl (pH 7.5), 10 mM MgCl ₂ and 1 m
MDTT and ApaLI (Takara Shuzo) 2U, BamHI
The digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing 8 U (Takara Shuzo) or 8 U HindIII (Takara Shuzo). If c1-h2 is correctly linked, ApaLI
5560/1246/498 bp, BamHI / Hi
The plasmid was confirmed by the generation of a digested fragment of 7134/269 bp by ndIII.

The expression vector encoding the human FR1,2 / mouse FR3,4 hybrid antibody L chain was expressed in h / mMB
C1L (λ) / neo. On the other hand, since no clone of h1-c2 was obtained, it was cloned into the HEF vector after recombination on the pUC vector. At this time, plasmid hMBC1Laλ / pUC1 containing DNA encoding a humanized antibody L chain V region without amino acid substitution
9, and a plasmid hMBC1Ldλ / pUC19 containing a DNA encoding a humanized antibody L chain V region in which tyrosine at position 91 (amino acid number 87 according to Kabat's definition) in FR3 was substituted with isoleucine was used as a template.

Plasmid MBC1L (λ) / pUC1
9, hMBC1Laλ / pUC19 and hMBC1Ld
10 μg of λ / pUC19 was added to 10 mM Tris-H
Cl (pH 7.5), 10 mM MgCl ₂ , 1 mM DT
T, 50 mM NaCl, 0.01% (w / v) BSA,
Digestion was performed at 37 ° C. for 1 hour in 30 μl of a reaction mixture containing 16 U of HindIII and 4 U of AflII. The reaction solution was electrophoresed on a 2% low melting point agarose gel, and the plasmid M
BC1L (λ) / pUC19 to 215 bp (c
2 ′), 3218 from plasmids hMBC1Laλ / pUC19 and hMBC1Ldλ / pUC19, respectively.
bp (ha1 ', hd1') DNA fragment
The gel was recovered and purified using EANII Kit (BIO101).

The ha1 ′ and hd1 ′ fragments were each ligated to the c2 ′ fragment and transformed into E. coli JM109 competent cells. 2 × containing 50 μg / ml ampicillin
The cells were cultured in 2 ml of YT medium, and QIAprep
Plasmids were purified using SpinPlasmidKit (QIAGEN). Plasmids containing the ha1 'and hd1' fragments were each cloned into plasmid m / hMBC1La
λ / pUC19 and m / hMBC1Ldλ / pUC19.

The resulting plasmid m / hMBC1Laλ
/ PUC19, m / hMBC1Ldλ / pUC19 to E
digested with coRI. Each 743 bp DNA fragment was electrophoresed on a 2% low melting point agarose gel, and then GEN
The gel was recovered and purified from the gel using ECLEANII Kit (BIO101), and 10 mM Tris-HCl (p
H7.4) was dissolved in 20 μl of a 1 mM EDTA solution.

4 μl of each DNA fragment was ligated to 1 μl of the above-mentioned BAP-treated HEF vector and transformed into E. coli JM109 competent cells. The cells were cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

[0224] Each purified plasmid was ligated with 20 mM Tri.
s-HCl (pH 8.5), 10 mM MgCl ₂ , 1 m
The digestion was carried out for 1 hour at 37 ° C. in a 20 μl reaction mixture containing MDTT, 100 mM KCl, 8 U of HindIII (Takara Shuzo) and 2 U of PvuI (Takara Shuzo). Plasmids were confirmed by generating digested fragments of 5104/2195 bp if the fragment was inserted in the correct direction and 4378/2926 bp if inserted in the opposite direction. These were used as expression vectors m / hM encoding mouse FR1,2 / human FR3,4 hybrid antibody L chains, respectively.
BC1Laλ / neo, m / hMBC1Ldλ / neo
And

(Ii) Preparation of FR1 / FR2 hybrid antibody A hybrid antibody of FR1 and FR2 was similarly prepared by utilizing the SnaBI cleavage site in CDR1. Plasmid MBC1L (λ) / neo and h /
10 μg of each of mMBC1L (λ) / neo was added to 10 mM
Tris-HCl (pH 7.9), 10 mM MgCl
₂ , 1 mM DTT, 50 mM NaCl, 0.01%
(W / v) Digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing 6 U of BSA and SnaBI (Takara Shuzo). Next, 20 mM Tris-HCl (pH 8.5), 1
0 mM MgCl ₂ , 1 mM DTT, 100 mM K
Digestion was performed for 1 hour at 37 ° C. in 50 μl of a reaction mixture containing 6 U of Cl, 0.01% (w / v) BSA, PvuI.

After electrophoresis of the reaction mixture on a 1.5% low-melting point agarose gel, plasmid MBC1L (λ) / neo
From 4955 bp (m1) and 2349 bp (m
2), 4955 bp (hm1) and 2349 bp (hm2) from plasmid h / mMBC1L (λ) / neo
Each DNA fragment of the GENECLEANII Kit
(BIO101) to recover and purify from the gel.
mM Tris-HCl (pH 7.4), 1 mM ED
Dissolved in 40 μl of TA solution.

1 µl of the m1 and hm1 fragments
2. Ligation was performed with 4 μl of the m2 fragment, and transformed into E. coli JM109 competent cells. The cells were cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and QIAprepSpinPlasmidKit (QIprep
(AGEN) to purify the plasmid. Each of the purified plasmids was added to 10 mM Tris-HCl (pH 7.
5), 10 mM MgCl ₂ , 1 mM DTT and ApaI
The digestion was carried out at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing 8 U (Takara Shuzo) or 2 U of ApaLI (Takara Shuzo).

If each fragment is correctly ligated, Apa
7304 bp for I and 5560/1246 / for ApaLI
498 bp (m1-hm2), 6538/7 with ApaI
66bp, 3535/2025/1246 with ApaLI
The plasmid was confirmed by the generation of a digested fragment of / 498 bp (hm1-m2). The expression vector encoding the human FR1 / mouse FR2,3,4 hybrid antibody L chain is hmmMBC1L (λ) / neo, and the expression vector encoding the mouse FR1 / human FR2 / mouse FR3,4 hybrid antibody L chain is mhmMBC1L.
(Λ) / neo.

(4) Construction of Humanized Antibody L Chain The humanized # 23-57-137-1 antibody L chain was subjected to PCR.
It was prepared by CDR-grafting according to the method. Human antibody HSU03868 (GEN-BANK, Deftos M et al., Scand. J.
Immunol., 39, 95-103, 1994) and human antibody S25755 (NBRF-PD
B) Humanized # 23-57-13 with FR4 derived
7-1 Preparation of Antibody L Chain (Version “a”)
PCR primers were used.

CDR-grafting primer MBC
1LGP1 (SEQ ID NO: 29) and MBC1LGP3 (SEQ ID NO: 30) have a sense DNA sequence and
The grafting primers MBC1LGP2 (SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 32) have an antisense DNA sequence, and each have a complementary sequence of 15 to 21 bp at each end of the primer. External primers MBC1LVS1 (SEQ ID NO: 33) and MBC
1LVR1 (SEQ ID NO: 34) has homology to CDR grafting primers MBC1LGP1 and MBC1LGP4.

The CDR-grafting primer MBC
1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 were separated using a urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manu).
al, Sambrook et al., Cold SpringHarbor Laboratory Press,
1989), extraction from gel was performed by the crush and soak method (Molecular
Cloning: A Laboratory Manual, Sambrook et al., Cold Spr
ing Harbor Laboratory Press, 1989).

That is, each 1 nmole CDR
-The grafting primer is separated on a 6% denaturing polyacrylamide gel, the DNA fragment of the desired size is identified by irradiating it with ultraviolet light on a silica gel thin plate.
The gel was recovered from the gel by the d soak method, and 20 μl of 10 mM T
It was dissolved in ris-HCl (pH 7.4), 1 mM EDTA solution.

PCR was performed using TaKaRa Ex Taq.
Using (Takara Shuzo), the CDR-grafting primer MBC1 prepared as described above was added to 100 μl of the reaction mixture.
1 μl each of LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4, 0.25 mM d
Under conditions containing NTP and 2.5 U of TaKaRa Ex Taq, at 94 ° C. for 1 minute using the attached buffer solution,
Performed 5 times at a temperature cycle of 55 ° C. for 1 minute and 72 ° C. for 1 minute, and added 50 pmole of external primers MBC1LVS1 and MBC1LVR1 to the reaction mixture.
The reaction was further performed 30 times in the same temperature cycle. The DNA fragment amplified by the PCR method is used for 3% Nu Sieve G
TG Agarose (FMC Bio. Products)
Separation was performed by agarose gel electrophoresis using.

A piece of agarose containing a 421 bp DNA fragment was cut out, and GENECLEAN II Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. The resulting PCR reaction mixture was purified using BamH
It was subcloned into pUC19 prepared by digestion with I and HindIII, and the nucleotide sequence was determined. The thus obtained plasmid was designated as hMBCL / pUC.
Named 19 However, position 104 of CDR4 (K
Since the amino acid at amino acid number 96 (defined by abat) was arginine, a modified primer MBC1LGP10R (SEQ ID NO: 35) for correcting this to tyrosine was designed and synthesized. PCR is TaKaRa
Using Taq (Takara Shuzo), 0.6 μg of plasmid hMBCL /
pUC19, MBC1LVS1 and M as primers
BC1LGP10R was 50 pmole and 2.5 pm, respectively.
U's TaKaRa Ex Taq (Takara Shuzo) 0.25m
M dNTPs and 50 mM using the attached buffer.
Add 1 μl of mineral oil at 94 ° C. for 1 minute,
For 30 minutes, a temperature cycle of 72 ° C. and 1 minute was performed 30 times. The DNA fragment amplified by the PCR method is 3% Nu S
ieve GTG agarose (FMC Bio. Pro)
(Ducts).

A piece of agarose containing a 421 bp long DNA fragment was cut out, and GENECLEAN II Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. The resulting PCR reaction mixture was purified with BamHI
And p prepared by digestion with HindIII
It was subcloned into UC19.

The nucleotide sequence was determined using the M13 Primer M4 primer and the M13 Primer RV primer. As a result, the correct sequence was obtained. This plasmid was digested with HindIII and BlnI, and the 416 bp fragment was digested with 1% agarose gel. Separated by electrophoresis. GENECLEANII Kit
(BIO101) and DN according to the prescription attached to the kit.
The A fragment was purified. The resulting PCR reaction mixture is
and introduced into plasmid Cλ / pUC19 prepared by digestion with ndIII and BlnI.
It was named MBC1Laλ / pUC19. This plasmid was digested with EcoRI to obtain a DNA encoding a humanized L chain.
A was introduced into plasmid pCOS1 such that the start codon of the humanized L chain was located downstream of the EF1α promoter. The thus obtained plasmid was designated as hMBC1La.
It was named λ / pCOS1. Humanized light chain version "
The nucleotide sequence of "a" (including the corresponding amino acid) is represented by SEQ ID NO: 6.
See Figure 6. The amino acid sequence of version a is shown in SEQ ID NO: 47.

[0237] Version "b" was prepared by introducing a mutation by PCR. 43rd in version "b" (Kab
It was designed so that glycine at amino acid number 43 defined by at was changed to proline and lysine at position 49 (amino acid number 49 defined by Kabat) was changed to aspartic acid. The mutagenic primer MBC1LGP5R (SEQ ID NO: 36) and the primer MBC1LVS1 were used to generate plasmid PC using plasmid hMBC1Laλ / pUC19 as a template.
R, and the obtained DNA fragment was ligated with BamHI and Hi.
digested with ndIII, BamHI, Hind of pUC19
It was subcloned into the dIII site. After determining the nucleotide sequence, hMB digested with restriction enzymes HindIII and AflII and digested with HindIII and AflII
It was ligated with C1Laλ / pUC19.

[0238] The thus obtained plasmid was designated as hMBC1.
Lbλ / pUC19, and this plasmid was EcoRI
The fragment containing DNA encoding the humanized L chain was introduced into plasmid pCOS1, so that the initiation codon of the humanized L chain was located downstream of the EF1α promoter. The thus obtained plasmid was designated as hMBC1Lbλ.
/ PCOS1.

[0239] Version "c" was prepared by using mutagenesis by the PCR method. 84th in version "c" (Kab
It was designed to change serine at amino acid number 80 (defined by at) to proline. Mutagenic primer MB
C1LGP6S (SEQ ID NO: 37) and primer M13
Plasmid hMBC1Laλ by Primer RV
/ PUC19 was used as a template for PCR and the resulting DN
The A fragment was digested with BamHI and HindIII and
It was subcloned into pUC19 prepared by digestion with amHI and HindIII. After determining the nucleotide sequence, hM digested with the restriction enzymes BstPI and Aor51HI, and digested with BstPI and Aor51HI
It was ligated with BC1Laλ / pUC19. The thus obtained plasmid was designated as hMBC1Lcλ / pUC19, and this plasmid was digested with the restriction enzyme EcoRI to obtain humanized L.
The sequence containing the sequence coding for the chain was converted into plasmid pCOS1.
Of the humanized L chain was located downstream of the EF1α promoter.
The thus obtained plasmid was designated as hMBC1Lcλ / pC
It was named OS1.

Versions "d", "e" and "f" are
It was prepared using mutagenesis by the R method. For each version, the “a”, “b”, and “c” versions are ranked 91st (Ka)
The tyrosine at amino acid number 87 according to the bat specification) was designed to be changed to isoleucine. Mutagen primer MBC1LGP11R (SEQ ID NO: 38) and primer M-S1 (SEQ ID NO: 44) were used to generate hMBC1
Laλ / pCOS1, hMBC1Lbλ / pCOS1,
PCR was performed using hMBC1Lcλ / pCOS1 as a template, and the obtained DNA fragment was analyzed using BamHI and HindI.
II and subcloned into pUC19 prepared by digestion with BamHI and HindIII. After determining the nucleotide sequence, HindIII and BlnI
And ligated to Cλ / pUC19 prepared by digestion with HindIII and BlnI.

[0241] The plasmids thus obtained were sequentially transformed into hMB.
C1Ldλ / pUC19, hMBC1Leλ / pUC1
9, hMBC1Lfλ / pUC19. These plasmids were digested with EcoRI, and the sequence containing the sequence encoding the humanized L chain was replaced with the EcoRI of plasmid pCOS1.
Site so that the start codon of the humanized light chain is located downstream of the EF1α promoter. The plasmids obtained in this manner were individually sequenced into hMBC1Ldλ / pC
OS1, hMBC1Leλ / pCOS1, hMBC1L
It was named fλ / pCOS1.

[0242] Versions "g" and "h" were prepared using mutagenesis by the PCR method. Each version in turn "
The histidine at position 36 (amino acid number 36 as defined by Kabat) of the "a" and "d" versions was designed to be changed to tyrosine.
P9R (SEQ ID NO: 39) and M13 PrimerR
HMBC1Laλ / pU using V as a primer
PCR was performed using C19 as a template, and further PCR was performed using the obtained PCR product and M13 Primer M4 as primers and using plasmid hMBC1Laλ / pUC19 as a template. The obtained DNA fragment is Hi
plasmid Cλ prepared by digestion with ndIII and BlnI and digestion with HindIII and BlnI.
/ PUC19. Using this plasmid as a template, a PC with primers MBC1LGP13R (SEQ ID NO: 40) and MBC1LVS1
R was performed. The obtained PCR fragment was digested with ApaI and Hi.
ndII was digested with I and introduced into plasmids hMBC1Laλ / pUC19 and hMBC1Ldλ / pUC19 digested with ApaI and HindIII. The nucleotide sequence was determined, and plasmids containing the correct sequence were sequentially identified as hMBC
1Lgλ / pUC19 and hMBC1Lhλ / pUC
These plasmids were digested with the restriction enzyme EcoRI, a sequence containing a sequence encoding the humanized L chain was introduced into the EcoRI site of the plasmid pCOS1, and EF1α
The start codon of the humanized light chain was located downstream of the promoter. The plasmids thus obtained were respectively sequenced into hMBC1Lgλ / pCOS1 and hMBC1
It was named Lhλ / pCOS1.

Versions "i", "j", "k", "l"
, "M", "n" and "o" were prepared using mutagenesis by PCR. Mutagenic primer MBC1LGP1
Plasmid hMBC1Laλ / pU by 4S (SEQ ID NO: 41) and primer VIRV (λ) (SEQ ID NO: 43)
PCR was performed using C19 as a template, and the obtained DNA fragment was digested with ApaI and BlnI,
Plasmid hMB prepared by digestion with InI
It was subcloned into C1Lgλ / pUC19. The nucleotide sequence was determined, and clones in which the mutation corresponding to each version was introduced were selected. The plasmid thus obtained was transformed into hMBC1Lxλ / pUC19 (x = i,
j, k, l, m, n, o).
oRI digested, a sequence containing a sequence encoding a humanized L chain was introduced into the EcoRI site of plasmid pCOS1,
The initiation codon of the humanized light chain was located downstream of the EF1α promoter. The plasmid thus obtained was transformed into hMBC1Lxλ / pCOS1 (x = i, j, k,
1, m, n, o). Version "j", "l"
, "M" and "o" (including the corresponding amino acids) are shown in SEQ ID NOs: 67, 68, 69 and 70, respectively. The amino acid sequences of these versions are shown in SEQ ID NOs: 48, 49, 50 and 51, respectively.

Versions "p", "q", "r", "s" and "t" are versions "i", "j", "m", "l".
Or, a version in which the tyrosine at position 87 in the amino acid sequence of "o" is replaced with isoleucine. Using the restriction enzyme Aor51MI cleavage site in FR3, version "h" is replaced with version "i", "j". , "M", "l" or "o". That is, the expression plasmid hMBC1Lxλ / pC
Aor51HI fragment 51 containing CDR3 and part of FR3 and FR4 in OS1 (x = i, j, m, l, o)
4 bp, except that the expression plasmid hMBC1Lhλ
/ PCOS1, CDR3 and part of FR3 and FR
No. 91 by connecting the 514 bp Aor51HI fragment containing No. 4 (amino acid 87 as defined by Kabat)
Tyrosine is isoleucine. The base sequence is determined and each version "i", "j", "m", "
Clones in which the tyrosine at position 91 (amino acid position 87 according to Kabat's definition) of l "and" o "were replaced with isoleucine were selected, and the corresponding versions were designated as" p "," q "," s ","", respectively. r "and" t ", and the obtained plasmid was designated as hMBC1Lxλ / pCOS1 (x =
p, q, s, r, t). Version "q", "
The nucleotide sequences of r "," s "and" t "(including the corresponding amino acids) are shown in SEQ ID NOs: 71, 72, 73 and 74. The amino acid sequences of these versions are shown in SEQ ID NOs: 52 and 53, respectively. , 54, 55.

Plasmid hMBC1Lqλ / pCOS1
Was digested with HindIII and EcoRI, and HindIII was digested with HindIII and EcoRI.
Plasmid pUC1 digested with III and EcoRI
9 and cloned into plasmid hMBC1Lqλ
/ PUC19. Table 3 shows the position of the substituted amino acid in each version of the humanized L chain.

[0246]

[Table 3]

In the table, Y represents tyrosine, P represents proline, K represents lysine, V represents valine, D represents aspartic acid, and I represents isoleucine.

The plasmid hMBC1HcDN
E. coli harboring A / pUC19 and hMBC1Lqλ / pUC19 were isolated from Escherichia c
oli JM109 (hMBC1HcDNA / pUC1
9) and Escherichia coli JM
Escher as 109 (hMBC1Lqλ / pUC19) at the Institute of Biotechnology and Industrial Technology (1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture) on August 15, 1996.
ichia coli JM109 (hMBC1HcD
NA / pUC19) for FERM BP-562
9, Escherichia coli JM109
(HMBC1Lqλ / pUC19) for FERM
It has been deposited internationally under the Budapest Treaty as BP-5630.

(5) Transfection into COS-7 cells Hybrid antibody and humanized # 23-57-137
The expression plasmid was transiently expressed in COS-7 cells to evaluate the antigen binding activity and neutralizing activity of the -1 antibody. That is, in the transient expression of the light chain hybrid antibody, plasmids hMBC1HcDNA / pCOS1 and h / mMBC1L (λ) / neo, hMBC1HcDN
A / pCOS1 and m / hMBC1Laλ / neo, hM
BC1H cDNA / pCOS1 and m / hMBC1Ldλ
/ Neo, hMBC1HcDNA / pCOS1 and hmm
MBC1L (λ) / neo or hMBC1HcDN
A / pCOS1 and mhmmBC1L (λ) / neo were combined using a GenePulser device (BioRa).
COS-7 by electroporation using d)
Cells were co-transduced. 10 μg of each plasmid DNA was added to 0.8 ml of COS-7 cells suspended at a cell concentration of 1 × 10 ⁷ cells / ml in PBS (−),
A pulse was applied with a capacitance of 1,500 V and 25 μF. After a recovery period of 10 minutes at room temperature, the electroporated cells were treated with 2% UltraLow Ig.
G bovine serum (GIBCO) and suspended in a DMEM culture medium (GIBCO) containing
The cells were cultured in an O ₂ incubator. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and used for an ELISA sample.

For transient expression of the humanized # 23-57-137-1 antibody, plasmid hMBC1HcDNA / p
COS1 and hMBC1Lxλ / pCOS1 (x = a to
Any combination of t) was transfected into COS-7 cells using a GenePulser device (BioRad) in the same manner as in the case of the hybrid antibody, and the obtained culture supernatant was subjected to ELISA.
Purification of a hybrid antibody or a humanized antibody from a culture supernatant of COS-7 cells was performed by AffiGel Pr
otein A MAPSII kit (BioRad)
Was performed according to the prescription attached to the kit.

(6) ELISA (i) Measurement of Antibody Concentration An ELISA plate for antibody concentration measurement was prepared as follows. 96-well plate for ELISA (Maxi
sorp, NUNC) into the solid phase buffer (0.
1 μg with 1M NaHCO ₃ , 0.02% NaN ₃ )
Goat anti-human IgG antibody (TAG
O) Immobilize with 100 μl, and add 200 μl of dilution buffer (50 mM Tris-HCl, 1 mM MgC
l ₂ , 0.1 M NaCl, 0.05% Tween2
After blocking with 0, 0.02% NaN ₃ , 1% bovine serum albumin (BSA), pH 7.2), COS-7 expressing hybrid antibody or humanized antibody
Cell culture supernatant or purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubating for 1 hour at room temperature and washing with PBS-Tween 20,
100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubating for 1 hour at room temperature and washing with PBS-Tween 20, 1 m
g / ml substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, then absorbance at 405 nm was measured using a microplate reader (BioRad).
Was measured. Hu I as a standard for concentration measurement
gG1λ Purified (TheBinding
Site).

(Ii) Measurement of antigen-binding ability An ELISA plate for antigen-binding measurement was prepared as follows. Each well of the 96-well ELISA plate was immobilized with 100 µl of human PTHrP (1-34) prepared at a concentration of 1 µg / ml with a solid phase buffer. 20
After blocking with 0 μl of dilution buffer, the culture supernatant of COS-7 cells expressing the hybrid antibody or humanized antibody or the purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubation at room temperature and washing with PBS-Tween 20, alkaline phosphatase-conjugated goat anti-human IgG antibody (TAG
O) 100 μl was added. Incubate at room temperature
After washing with BS-Tween 20, 1 mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate,
SIGMA) and then the absorbance at 405 nm was measured on a microplate reader (BioRad).

(7) Confirmation of Activity (i) Evaluation of Humanized H Chain The antibody obtained by combining the humanized H chain version “a” and the chimeric L chain had the same PTHrP binding ability as the chimeric antibody (FIG. 5). This result indicates that humanization of the H chain V region is sufficient with version "a". Hereinafter, the humanized H chain version "a" was used as the H chain of the humanized antibody.

(Ii) Activity of hybrid antibody (ii-a) FR1,2 / FR3,4 hybrid antibody When the L chain was h / mMBC1L (λ), no activity was observed, but m / hMBC1Laλ or m / hMBC1Laλ. / H
In the case of MBC1Ldλ, chimera # 23-57-
It showed a binding activity equivalent to that of the 137-1 antibody (FIG. 6). These results suggest that FR3,4 is not a problem as a humanized antibody, but that there are amino acid residues to be substituted in FR1,2.

(Ii-b) FR1 / FR2 Hybrid Antibody When the L chain was hmmMBC1L (λ), no activity was observed. It showed equivalent binding activity (FIG. 7). These results indicate that FR1 and FR2
1 indicates no problem as a humanized antibody, but suggests that an amino acid residue to be substituted exists in FR2.

(Iii) Activity of Humanized Antibody The antigen-binding activity of a humanized antibody using one of each of versions "a" to "t" as an L chain was measured. As a result, the light chain versions "j", "l", "m", "o"
, "Q", "r", "s", and "t" exhibited the same PTHrP binding ability as the chimeric antibody (FIG. 8).
~ 11).

(8) Establishment of a Stable CHO Producing Cell Line In order to establish a stable humanized antibody producing cell line, the above expression plasmid was introduced into CHO cells (DXB11).
That is, the establishment of a cell line for stably producing a humanized antibody is performed by CHO
Expression plasmid hMBC1HcDNA / pCHO for cells
1 and hMBC1Lmλ / pCOS1, or hMBC1
HcDNA / pCHO1 and hMBC1Lqλ / pCOS
1, or hMBC1HcDNA / pCHO1 and hM
Gene in combination with BC1Lrλ / pCOS1
CHO cells were co-transduced by electroporation using a Pulser device (BioRad). Each expression vector was cleaved with a restriction enzyme PvuI to obtain linear DNA, and extracted with phenol and chloroform.
DNA was recovered by ethanol precipitation and used for electroporation. 1 × 10 ⁷ cells / ml in PBS (−)
0.8 ml of CHO cells suspended at a cell concentration of
10 μg of each plasmid DNA was added, and 1,500 V, 2
A pulse was applied with a capacitance of 5 μF. After a recovery period of 10 minutes at room temperature, the electroporated cells were suspended in MEM-α medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) and used in a 96-well plate (Falcon). And cultured in a CO ₂ incubator. The day after the start of culture, 10% fetal bovine serum (G
IBCO) and 500 mg / ml GENETICI
N (G418 Sulfate, GIBCO) added, ribonucleoside and deoxyribonucleoside free ME
The cells were replaced with an M-α selection medium (GIBCO), and cells into which the antibody gene had been introduced were selected. The cells were observed under a microscope about two weeks after the selection medium was exchanged, and after successful cell growth was observed, the amount of antibody production was measured by the antibody concentration measurement ELISA, and cells having high antibody production ability were selected. .

The culture of the established antibody-producing stable cell line was expanded, and 2% UltraLow Ig was added using a roller bottle.
Mass culture was performed using a MEM-α selection medium without G bovine fetal serum, ribonucleoside and deoxyribonucleoside. On the third or fourth day of the culture, the culture supernatant was collected, and cell debris was removed using a 0.2 μm filter (Millipore). Purification of the humanized antibody from the culture supernatant of CHO cells was performed using a ConROS LC100 (Millipore) using a POROS protein A column (PerSeptive Biosystems).
In e), the measurement was performed according to the attached prescription, and the sample was subjected to measurement of neutralizing activity and a drug efficacy test in a hypercalcemia model animal.
The concentration and antigen-binding activity of the obtained purified humanized antibody were measured by the above-mentioned ELISA system.

Example 4 Measurement of Neutralizing Activity The neutralizing activity of a mouse antibody, a chimeric antibody and a humanized antibody was measured using a rat osteosarcoma cell line ROS17 / 2.8-5.
This was performed using cells. That is, ROS17 / 2.8-
5 cells were treated with Ha containing 10% fetal calf serum (GIBCO).
The cells were cultured in m'SF-12 medium (GIBCO) in a CO ₂ incubator. ROS17 / 2.8-5 cells were seeded at 10 ⁴ cells / 100 μl / well in a 96-well plate, cultured for 1 day, and added with 4 mM hydrocortisone and 10%
Ham'SF-12 medium (GIBCO) containing fetal calf serum
Replace with After further culturing for 3-4 days, 260
After washing with μl Ham'SF-12 medium (GIBCO), 1 mM isobutyl-1-methylxanthine (IBM
X, SIGMA) and 10% fetal calf serum and 10 mM
80 μl of Ham's F-12 containing HEPES was added and incubated at 37 ° C. for 30 minutes.

A mouse antibody, a chimeric antibody, or a humanized antibody whose neutralizing activity is to be measured was previously prepared at 10 μg / ml.
3.3 μg / ml, 1.1 μg / ml and 0.37 μ
g / ml group, 10 μg / ml, 2 μg / ml, 0.5
μg / ml and 0.01 μg / ml groups, or 10
μg / ml, 5 μg / ml, 1.25 μg / ml, 0.
PTHrP (1-34) serially diluted to a group of 63 μg / ml and 0.31 μg / ml and adjusted to 4 ng / ml
And 80 μl of a mixture of each antibody and PTHrP (1-34) was added to each well. The final concentration of each antibody is
One-fourth of the above antibody concentration, PTHrP (1-3
The concentration of 4) becomes 1 ng / ml. After treatment at room temperature for 10 minutes, the culture supernatant is discarded, and the cells are washed three times with PBS. Then, intracellular cAMP is extracted with 100 μl of 0.3% hydrochloric acid and 95% ethanol. Ethanol hydrochloride is evaporated with a water aspirator, and cAMP EIA ki
EIA included with t (CAYMANCHEMICAL'S)
After extracting cAMP by adding 120 μl of buffer, c
AMP EIA kit (CAYMANCHEMICA
L'S) cAMP was measured according to the attached prescription. As a result, a humanized antibody having versions “q”, “r”, “s”, and “t” in which the tyrosine at position 91 was replaced with isoleucine in the L chain version having the same antigen binding as the chimeric antibody was obtained. The neutralization ability was close to that of the chimeric antibody, and among them, version "q" showed the strongest neutralization ability (Figs. 12 to 14).

[Example 5] Drug efficacy test in a hypercalcemia model animal (1) A chimeric antibody against PTHrP and an L chain version "m" were used in a hypercalcemia model animal transplanted with a human tumor-nude mouse. , "R" and "q" were tested for their therapeutic effects on hypercalcemia.

As a model animal for hypercalcemia, human pancreatic cancer PAN-7 (purchased from Central Laboratory for Experimental Animals)
Transplanted nude mice were used. In nude mice transplanted with human pancreatic cancer PAN-7, blood calcium levels increase with an increase in tumors, and hypercalcemia such as weight loss and loss of exercise develops. Examination of the therapeutic effect on hypercalcemia was performed by evaluating that chimeric antibody and humanized antibody improve hypercalcemia caused by human pancreatic cancer PAN-7, using body weight and blood calcium concentration as indexes. did.

Passage of human pancreatic cancer PAN-7 was performed using BALB
/ C-nu / nu nude mice (Charles River Japan) were used in vivo. For efficacy evaluation,
Five week old male BALB / c-nu / nu nude mice (Charles River Japan) were purchased and, after one week acclimation, 6
Week-old animals were used. Preparation and grouping of hypercalcemia model animals were performed as follows. That is, human pancreatic cancer PAN-7 that has been passaged is excised and 3 m
The tumor mass finely chopped in the m-square block was implanted subcutaneously in the flank of each mouse, one per mouse. After confirming that the tumor volume has become sufficiently large two to three weeks after the transplantation of the tumor mass, the tumor volume, blood calcium concentration, and body weight are used as indices for grouping so that each index is averaged. The animals were modeled for calciumemia.

The examination of the therapeutic effect on hypercalcemia was performed as follows. That is, prepared above,
The grouped hypercalcemia model animals were administered a single dose of 10 μg or 30 μg of a chimeric antibody against PTHrP or a humanized antibody having L-chain versions m and r per mouse into the tail vein. L chain version "q"
The humanized antibody having a single dose of 20 μg or 60 μg per mouse was administered into the tail vein. On days 1, 4, 7, and 11 after administration of the chimeric antibody and the humanized antibody, the blood calcium concentration and body weight were measured, and the efficacy of each antibody was evaluated. The tumor volume was determined by measuring the major axis (amm) and minor axis (bmm) of the tumor and calculating Galant's formula ab ²
/ 2 was calculated as the tumor volume. Blood calcium concentration was determined by collecting blood from the orbit using a hematocrit tube and using a 643 automatic Ca ++ / pH analyzer (CIBA-CORNIN).
G) was used to measure the concentration of ionized calcium in whole blood.

As a result, by administering the chimeric antibody and the humanized antibody having the L chain versions “m”, “r” and “q”, rapid improvement and sustainability of body weight and blood calcium concentration were observed. Was done. This demonstrated the usefulness of the chimeric antibody and humanized antibody of the present invention as a therapeutic agent for hypercalcemia associated with malignant tumors (FIGS. 15 and 16).

[Example 6] Drug efficacy test in hypercalcemia model animal (2) Chimera antibody against PTHrP and light chain version "q" using hypercalcemia model animal transplanted with human tumor-nude mouse For a humanized antibody having
The therapeutic effect on hypercalcemia was examined. Examination of the therapeutic effect on hypercalcemia was performed as follows. That is, 10 μg or 3 μg / mouse was added to the hypercalcemia model animals prepared and grouped above.
A single dose of 0 μg of a chimeric antibody against PTHrP or a humanized antibody having the light chain version “q” was injected into the tail vein. 1 day after administration of chimeric antibody and humanized antibody, 3 days
On days 7, 7 and 10, blood calcium concentration and body weight were measured, and the efficacy of each antibody was evaluated. Blood calcium concentration was measured from the orbit using a hematocrit tube and 643 automatic Ca ++
/ Ionized calcium concentration in whole blood was measured using a pH / pH analyzer (CIBA-CORNING).

As a result, in a human pancreatic cancer PAN-7-transplanted hypercalcemia model, administration of a chimeric antibody and a humanized antibody having an L-chain version “q” resulted in a rapid increase in body weight and blood calcium concentration. Improvement and continuity were noted. This demonstrated the utility of the chimeric antibody and humanized antibody of the present invention as a therapeutic agent for hypercalcemia associated with malignant tumors (FIG. 17).

[Example 7] Drug efficacy test in a hypercalcemia model animal (3) Using a hypercalcemia model animal (human lung cancer LC-6 transplanted hypercalcemia model) in a human tumor-nude mouse transplantation system A chimeric antibody against PTHrP and
The therapeutic effect of humanized antibody having light chain version “q” on hypercalcemia was examined. As a model animal for hypercalcemia, nude mice transplanted with human lung cancer LC-6 (purchased from Central Laboratory for Experimental Animals) were used. In nude mice transplanted with human lung cancer LC-6, the blood calcium concentration increases with an increase in the tumor, and hypercalcemia such as weight loss and loss of exercise occurs.

The study of the therapeutic effect on hypercalcemia was conducted by examining that the chimeric antibody and the humanized antibody improve hypercalcemia caused by human lung cancer LC-6, using body weight and blood calcium concentration as indices. Was evaluated. Passage of human lung cancer LC-6 was performed in vivo using BALB / c-nu / nu nude mice (Charles River Japan).
For the efficacy evaluation, 5-week-old male BALB / c-nu / nu nude mice (Charles River Japan) were purchased, and after acclimation for 1 week, 6-week-old animals were used.

The preparation and grouping of hypercalcemia model animals were performed as follows. That is, human lung cancer LC-6 that had been subcultured was excised, and a tumor mass finely chopped into a 3 mm square block was implanted subcutaneously into the flank of a mouse one by one. After confirming that the tumor volume has become sufficiently large two to three weeks after the transplantation of the tumor mass, the tumor volume, blood calcium concentration, and body weight are used as indices for grouping so that each index is averaged. The animals were modeled for calciumemia.

[0271] The therapeutic effect on hypercalcemia was examined as follows. That is, prepared above,
The grouped hypercalcemia model animals received a single intravenous injection of 10 μg or 30 μg of a chimeric antibody against PTHrP or a humanized antibody having the light chain version “q” per mouse into the tail vein. On the 1st, 3rd, 6th, and 10th days after administration of the chimeric antibody and the humanized antibody, the blood calcium concentration and body weight were measured, and the efficacy of each antibody was evaluated. Blood calcium concentration was collected from the orbit using a hematocrit tube.
Whole blood ionized calcium concentration was measured using a 643 automatic Ca ++ / pH analyzer (CIBA-CORNING).

As a result, administration of a chimeric antibody and a humanized antibody having L-chain version “q” in a human lung cancer LC-6-transplanted hypercalcemia model was performed.
Immediate improvement and persistence of body weight and blood calcium levels were noted. This demonstrated the utility of the chimeric antibody and humanized antibody of the present invention as a therapeutic agent for hypercalcemia associated with malignant tumors (FIG. 18).

[Example 8] PTHrP and anti-P using BIACORE
Kinetic Analysis of THrP Antibody Interaction Kinetic analysis of antigen-antibody reaction was performed using BIACORE. Using PTHrP (1-34 + Cys) as an antigen, C-terminal site-specifically immobilized on a sensor chip, and purified antibodies prepared at various concentrations were used as analytes. From the obtained sensorgram, kinetic parameters (binding rate constant
kass and dissociation rate constant kdiss) were calculated. For the kinetic analysis, see the literature "Kinetic analysis of monoclonal ant
ibody-antigen interactions with a new biosensor ba
sed analytical system '' (Karlsson, R. et al., (1991)
J. Immunol. Methods 145, p229-240.).

(1) PTHrP (1-34 + C) on the sensor tip
Immobilization of PTHrP (1-34 + C) is immobilized on the sensor chip CM5 (Pharmacia). HBS (10 mM HEP as running buffer)
ES pH7.4, 0.15M NaCl, 3.4mM EDTA, 0.005% Surfacta
nt P20) and the flow rate was 5 μl / min. The carboxyl group of carboxymethyl dextran on the sensor chip CM5 was replaced with 100 μl of 0.05 M N-hydroxysuccinimide (N
HS) /0.2M HCl N-ethyl-N '-(3-dimethylaminopropyl) -carbodiimide (EDC) injection and 100 μl
l of 80 mM 2- (2-pyridinyldithio) ethanamine (P
Activated by injection of DEA) /0.1 M borate buffer pH 8.5. Subsequently, 10 μl of 5 μg / ml PTHrP (1-34 +
C) / 10 mM sodium acetate buffer (pH 5.0) was injected to specifically immobilize the C-terminal Cys residue of PTHrP (1-34 + C). In addition, 100 μl of 50 mM (L) -cysteine / 1 M NaCl /
Excess active groups were blocked by injecting 0.1 M sodium formate buffer pH 4.3. In addition, 10
μl 0.1 M glycine-HCl buffer pH 2.5 and 10 μl 1
Non-covalently bound substances were washed out by injecting 0 mM hydrochloric acid. At this time, the fixed amount of PTHrP (1-34 + C) was 226.4 RU (resonance units) (FIG. 1).
9).

(2) Immobilized PTHrP (1-34 + C) and mouse anti-PTHr
Interaction with P-purified antibody HBS was used as a running buffer, and the flow rate was 20 μl /
Minutes. For the antibody, hybridoma cells were ascitesed into Balb / c mice, and the collected ascites was purified using a protein A column. The purified # 23-57-137-1 antibody was designated as MBC, and the purified 3F5 antibody was designated as 3F5. These antibodies were prepared at concentrations of 1.25, 2.5, 5, 10, and 20 μg / ml using HBS.
In the analysis, the binding phase was set for 2 minutes in which 40 μl of the antibody solution was injected, and then switched to HBS, and the dissociation phase was set for 2 minutes. After completion of the dissociation phase, 10 μl of 10 mM hydrochloric acid was injected to regenerate the sensor chip. The binding, dissociation, and regeneration were regarded as one cycle of analysis, and various antibody solutions were injected to obtain sensorgrams.

(3) Immobilized PTHrP (1-34 + C) and humanized anti-PT
Interaction with HrP purified antibody HBS was used as a running buffer, and the flow rate was 20 μl /
Minutes. Antibodies are produced in CHO cells and protein A
Purification was performed using a column. Purify chimeric antibody with chMB
C, the purified humanized antibody version m was designated as hMBCm, and version q was designated as hMBCq. These antibodies were prepared at concentrations of 1.25, 2.5, 5, 10, and 20 μg / ml using HBS.
In the analysis, the binding phase was 2 minutes in which 40 μl of the antibody solution was injected, and then switched to HBS, and the dissociation phase was 2 minutes. After completion of the dissociation phase, the sensor chip was regenerated by injecting 10 μl of 10 mM HCl. This join
Dissociation and regeneration were regarded as one cycle of analysis, and various antibody solutions were injected to obtain sensorgrams.

(4) Kinetic analysis of interaction A target data file was read, and a target reaction region was compared with a reaction pattern by overwriting (FIG. 2).
0-24). Furthermore, the kinetic analysis of the interaction was performed using BIACORE's dedicated analysis software "BIAevaluation 2.1" (Pharmacia), which calculates kinetic parameters (association rate constant kass and dissociation rate constant kdiss) by curve fitting. (Tables 4 and 5).
20 to 24, each curve is 1.25, 2.5, 5, 10, 20 μg / ml from the top of the figure to the bottom.
Of the antibody concentration.

[0278]

[Table 4]

[0279]

[Table 5]

In determining the binding rate constant, an analysis model type 4 was used (BIAevaluation 2.1 Software
Handbook, A1 to A5).

[Example 9] Phosphorus excretion inhibitory action in a paraneoplastic hypercalcemia model Parathyroidism-associated hypercalcemia (HHM) is caused by PTHrP produced by a tumor, and PTHrP is It is known to enhance absorption and reabsorption of calcium in renal tubules, causing hypercalcemia. On the other hand, with respect to phosphorus, PTHrP suppresses reabsorption in renal tubules, thereby exhibiting an excretion promoting effect, and hypophosphatemia is often observed in clinical HHM patients. Therefore, using a rat model associated with paraneoplastic hypercalcemia, humanized anti-PTHr
The effect of P antibody on phosphorus excretion in the kidney was examined.

As a model animal, a human lung cancer cell line LC-6 (manufactured by
Nude rats transplanted with the Experimental Animal Central Laboratory) were used. In nude rats implanted with human lung cancer strain LC-6 subcutaneously, blood calcium levels increase with the increase in tumors,
It presents with hypercalcemic symptoms such as weight loss and decreased exercise. Using this model, the effect of humanized anti-PTHrP antibody on phosphorus excretion in the kidney was evaluated by the renal clearance method using the phosphorus excretion rate (described later) as an index. Passage of human lung cancer strain LC-6 was performed in vivo using BALB / c-nu / nu nude mice (CLEA Japan). For efficacy evaluation, 5-week-old male F344 / N
Jcl-rnu nude rats (CLEA Japan) were purchased, and after acclimation for one week, 6-week-old animals were used.

[0283] Preparation of a model for hypercalcemia associated with malignant tumor was performed as follows. That is, the passaged human lung cancer cell line LC-6 tumor was excised, and a tumor mass finely cut into a 3 mm square block was implanted subcutaneously into the rat's flank, one per rat. After confirming that the tumor volume had become sufficiently large (3000 mm ³ ) about 30 days after the transplantation of the tumor mass, the animal was used as a model animal for malignant tumor-associated hypercalcemia using blood calcium concentration and body weight as indices. Examination of phosphorus excretion by the renal clearance method was performed as follows.

(1) Renal clearance method A model animal associated with malignant tumor-associated hypercalcemia was anesthetized with pentobarbital (Nembutal, Dainippon Pharmaceutical Co., Ltd.), and was fixed in a dorsal position on a 37 ° C. heat insulation mat to collect urine. A bladder cannula (polyethylene tubing, PE50, Nippon Becton Dickinson) was inserted into the container. Next, a cannula (polyethylene tube, PE1
0, Nippon Becton Dickinson) and infusion solution (composition: 0.7% inulin, 5% mannitol, 0.2% pentobarbital, 0.9% sodium chloride) and infusion pump (Terfusion syringe pump, STC-525, Terumo) Infusion was performed at a flow rate of 2 ml / hr. After equilibration for 50 minutes, urine was collected from the bladder cannula five times (period-1 to period-5) at 20-minute intervals to obtain a urine sample. At the midpoint of each urine collection, about 0.25 ml of a blood sample was collected from the right jugular vein using a heparin-treated syringe.

(2) Administration of Antibody At the start of period-2 urine collection in the above clearance experiment, humanized anti-PTHrP antibody was intravenously administered at 1 mg / ml / kg. (3) Measurement of Inulin and Phosphorus Concentrations in Urine and Blood Urine samples obtained from period-1 to period-5 were measured for inulin and phosphorus concentrations after measuring urine volume. The blood sample obtained in the same manner was subjected to refrigerated centrifugation, and then inulin and phosphorus concentrations were measured as a plasma sample. Inulin concentration was determined by the anthrone-sulfuric acid method (Roe, JH et al., J Biol Ch.
em 178, 839-845, 1949). Phosphorus concentration was measured according to a manual for measurement using a reagent for measuring inorganic phosphorus, Autocera IP (Daiichi Pure Chemicals), using a Hitachi Automatic Analyzer Model 7170 (Fiske-Sabalow method).

(4) Calculation of inulin clearance, link clearance, and phosphorus excretion rate Inulin clearance (inulin clearance, Cin), link clearance (phosphate clearance, Cp), and phosphorus excretion rate (fractional excretion of phosphate, FEp)
Was calculated by the following equation.

Inulin clearance (inulin clearanc)
e, Cin) Calculation Cin = Uin V / Pin Cin represents inulin clearance (ml / kg / min). Uin
Represents urinary inulin concentration (mg / ml). V represents the amount of urine per unit time (ml / kg / min). Pin represents blood inulin concentration (mg / ml).

Calculation of Link Clearance (phosphate clearance, Cp) Cp = Up V / Pp Cp represents link clearance (ml / kg / min). Up represents the urinary phosphorus concentration (mg / ml). V represents the amount of urine per unit time (ml / kg / min). Pp is the blood phosphorus concentration (mg / ml)
Represents

The phosphorus excretion rate (fractional excretion of ph
Calculation of osphate, FEp) FEp = Cp / Cin FEp indicates the phosphorus excretion rate. Cin represents inulin clearance (ml / kg / min). Cp is the link clearance (ml / kg / m
in). The experiment was performed using four animals. The results are shown as the mean ± standard error.

The results of the phosphorus excretion rate and blood phosphorus concentration are shown.
25 and FIG. 26. FIG. 25 shows each period of the clearance (1 period is 20 minutes) and the phosphorus excretion rate from the kidney (=
It is a graph which shows the relationship with (link clearance / inulin clearance). In addition, humanized anti-PTHrP antibody, 1 mg /
kg (iv) was administered at the beginning of period-2.

FIG. 26 is a graph showing the relationship between each period of the clearance (1 period is 20 minutes) and the phosphorus concentration in plasma. Humanized anti-PTHrP antibody, 1 mg / kg (iv)
Was given at the beginning of period-2. From the above results, the phosphorus excretion rate (period-2 to period-5) after the antibody administration was clearly suppressed compared to the phosphorus excretion rate (period-1) before the antibody administration. That is, administration of a neutralizing antibody normalizes phosphorus reabsorption to a condition exhibiting a hypophosphatemic state due to enhanced phosphorus excretion (FEp> 0.2) (phosphorus reabsorption rate = 1-FEp> 0.8%). ) To recover to the vicinity,
Blood phosphorus levels tended to normalize. Thus, the usefulness of this antibody was shown as a therapeutic agent for hyperphosphatemia and hypophosphatemia caused by PTHrP.

Since PTHrP is a causative substance of hypercalcemia associated with malignant tumor, it is expected that PTHrP will increase phosphorus excretion and decrease the high-energy organic phosphate concentration in tissues. Therefore, in diseases associated with hypophosphatemia, for example, hypophosphatemic rickets, hypophosphatemic vitamin D-resistant rickets, etc., increased urinary excretion of phosphorus is the main etiology. Useful as

Example 10 Improvement of Clinical Symptoms of Hypercalcemia Associated with Malignant Tumors Hypercalcemia associated with malignant tumors was produced by PT
HrP is the causative substance, and PTHrP is known to enhance bone resorption and calcium reabsorption in renal tubules, causing hypercalcemia. In addition, hypercalcemia associated with malignant tumors has worsened performance status, impaired consciousness, general malaise, and worsened clinical symptoms such as dry mouth and nausea / vomiting (anorexia). The effects of anti-PTHrP antibodies on these clinical symptoms were examined using human tumor-nude mouse transplantation system and human tumor-nude rat transplantation system hypercalcemia model animals.

As a model animal for hypercalcemia, nude mice and nude rats transplanted with human lung cancer LC-6 (purchased from Central Laboratory for Experimental Animals) were used. Human lung cancer L
In nude mice and nude rats implanted with C-6, blood calcium concentration increases with an increase in tumor, and hypercalcemia symptoms such as a decrease in body temperature, weight loss, and a decrease in locomotor activity occur.

The improvement effect of mouse anti-PTHrP antibody on general clinical symptoms of paraneoplastic hypercalcemia was
Photographs were shown using a human lung cancer LC-6 nude mouse transplantation system. The effects of improving exercise, improving body temperature and reducing food intake were evaluated using a human lung cancer LC-6 nude rat transplantation system.

[0296] 1. Improvement of appearance clinical symptoms associated with hypercalcemia Passage of human lung cancer LC-6 was performed using BALB / c-nu / nu
In vivo was performed using nude mice (CLEA Japan). For the efficacy evaluation, 5-week-old male BALB / c-nu
/ Nu nude mice (CLEA Japan) were purchased, and after acclimation for one week, 6-week-old animals were used.

Preparation and grouping of hypercalcemia model animals were performed as follows. That is, a human lung cancer LC-6 that has been passaged is excised, and a tumor mass finely cut into a 3 mm square block is subcutaneously subcutaneously in the flank of a mouse at a rate of 1 per mouse.
Transplanted individually. After confirming that the tumor volume was sufficiently large on the 27th day after the tumor mass transplantation, the tumors were grouped so that each index was averaged using the tumor volume, blood calcium concentration and body weight as indexes, and high calcium It was used as a blood model animal.

[0298] Tumor volume tumors major axis (amm) and minor axis (b mm) were measured, Galant formula ab ^2/2
Was calculated as the tumor volume. Blood calcium concentration was determined by collecting blood from the orbit using a hematocrit tube.
a ++ / pH analyzer (CIBA-CORNIN
G) was used to measure the concentration of ionized calcium in whole blood.

Examination of the therapeutic effect on hypercalcemia was carried out as follows. That is, prepared above,
100 μg / mouse antibody to PTHrP / mouse was added to the grouped hypercalcemia model animals.
On days 27, 30, 34, and 37 after tumor implantation, administration was performed into the tail vein. For the control group, phosphate buffered saline was similarly administered into the tail vein. One typical animal was selected from each of the antibody administration group and the control group, and photographed together with normal animals on day 41 of tumor implantation.

As a result, in the model of hypercalcemia transplanted with human lung cancer LC-6, the animals to which the antibody was administered (center in FIG. 27 and center in FIG. 28) were the control animals (right in FIG. 27 and right in FIG. 28).
Despite having the same tumor mass as normal animals (Fig.
27 and the left of FIG. 28).
Improvement of apparent clinical symptoms was observed by administration of the P antibody (FIGS. 27 and 28).

[0301] 2. Improvement of locomotor activity associated with hypercalcemia Passage of human lung cancer strain LC-6 was performed using BALB / c-nu / n
In vivo using u nude mice (CLEA Japan)
I went in. To evaluate the efficacy, 5-week-old male F344 / NJ
cl-rnu nude rats (Clear Japan)
After a week of acclimation, 6 week old animals were used.

[0302] Preparation of the paracalcemia model associated with malignant tumor was performed as follows. That is, the human lung cancer cell line LC-6 which was subcultured was excised, and a tumor mass finely cut into a 3 mm square block was subcutaneously subcutaneously in the flank of a rat at a rate of 1 per mouse.
Transplanted individually. After confirming that the tumor volume was sufficiently large around day 30 after the transplantation of the tumor mass, the animal was used as a model animal for malignant tumor-associated hypercalcemia using blood calcium concentration and body weight as indices. Blood calcium concentration was determined by collecting blood from the orbit using a hematocrit tube and using 643 automatic Ca ++ / p.
The whole blood was measured as an ionized calcium concentration using an H analyzer (CIBA-CORNING).

(1) Spontaneous Momentum Measurement Method Spontaneous momentum is measured by a spontaneous momentum measuring device, ANIMEX activity meter type SE, FARAD Electronics,
Sweden), a poly cage (water supply, under feeding), which was individually bred for each individual, was placed at a predetermined position of the apparatus. This device measures the amount of movement of the rat and is recorded as a count per fixed time. 7 pm measurement
13 hours from time to the next day 8:00 am, the measurement result is 1
The number of counts per hour was used.

(2) Administration of Antibody As described above, a humanized anti-PTHrP antibody was used at 5 mg / 0.5 ml /
kg was administered via the tail vein. As a control, physiological saline was similarly administered into the tail vein. The measurement was performed alternately between the antibody-administered individual and the control individual. On the measurement day, the antibody administration was performed on the 0th day (the day before administration), 2, 4, 7, and 14 days for the antibody administration, and the control individual was performed on the 1, 3, 5, 8, 15 days. as a result,
The spontaneous locomotion of the control individual did not change or showed a decreasing tendency during the experiment period, whereas the spontaneous locomotor activity of the antibody-administered individual increased after the fourth day (FIG. 29).

[0305] 3. Improvement of hypothermia associated with hypercalcemia Passage of human lung cancer cell line LC-6 and preparation of a paraneoplastic hypercalcemia model were performed in the same manner as in the method described in the above 2. (1) Body temperature measurement method The body temperature was measured using a digital thermometer, the individual was anesthetized with pentobarbital (Nembutal, Dainippon Pharmaceutical Co., Ltd.), and a temperature sensor probe was inserted into the rectum.

(2) Administration of Antibody Humanized anti-PTHrP antibody was administered at 1 mg / ml / kg in the tail vein of a rat that had developed hypercalcemia as described above. As a control, physiological saline was similarly administered into the tail vein. Further, the body temperature of a normal rat (no administration) was also measured at the same time. The body temperature was measured on day 0, day 1, 2, and 3 of the administration of the antibody-administered individual, the control individual, and the normal rat.

As a result, the body temperature of a normal rat during the experiment
The temperature remained almost unchanged at 34.2 to 34.4 ° C. Compared to normal rats, paracalcemic rats with paraneoplastic hypercalcemia
A decrease in body temperature of about 2 ° C. was observed. It was confirmed that when a humanized anti-PTHrP antibody was administered to this model, it recovered to the body temperature of a normal rat 3 days after the administration. As described above, it was shown that the humanized anti-PTHrP antibody has an effect of improving body temperature decrease in a paraneoplastic hypercalcemia model (FIG. 30).

[0308] 4. Improvement of the decrease in food intake associated with hypercalcemia Passage of the human lung cancer strain LC-6 and preparation of a hypercalcemia model associated with malignant tumor were carried out in the same manner as in the method described in 2 above. The prepared models were grouped so that each index was averaged using blood calcium concentration and body weight as indexes, and used in the following experiments. (1) Food intake measurement method Rats were placed in metabolism cages for individual breeding during the experiment,
They were bred under water and feed. The food consumption was the 24-hour food consumption from 9:00 am on the day to 9:00 am the next day, the weight of the feeder was measured, and the difference from the previously measured weight (tare weight) was taken as the food consumption (g) of the individual. .

(2) Administration of Antibody As described above, rats (H
HM rat) and a humanized anti-PTHrP antibody
g / 0.5 ml / kg was administered via the tail vein. In the control group, physiological saline was similarly intravenously administered. further,
For normal rats, physiological saline was similarly administered into the tail vein. The food intake was measured for all groups including the antibody administration group, the control group and the normal rat group.
(From the day of administration to the next day), 3 (from the 3rd day of administration to the next day), 5
The test was performed on the day (from the 5th day of administration to the next day).

As a result, the amount of food consumed before administration was 8.11 g on average in hypercalcemic rats (individuals 5 to 9), and 12.06 g on normal rats. Thus, the food intake was clearly decreased in the hypercalcemic rats. When the humanized anti-PTHrP antibody was administered to this model, it was confirmed that the food intake in the control group did not change much, whereas the food intake in the antibody-administered group recovered to the normal rat intake from day 1 after administration. Thus, it was shown that the humanized anti-PTHrP antibody has an improving effect on the decrease in food intake in a model of hypercalcemia associated with malignancy (Table 6).

[0311]

[Table 6]

[0312] The above results indicate that the chimeric antibody and humanized antibody of the present invention are useful as agents for improving clinical symptoms of hypercalcemia associated with malignant tumors. 5. Improvement of Blood pH Associated with Hypercalcemia Passage of human lung cancer cell line LC-6 and preparation of a paraneoplastic hypercalcemia model were performed in the same manner as in the method described in the above 2. The prepared models were grouped so that each index was averaged using blood calcium concentration and body weight as indexes, and used in the following experiments. (1) Blood pH measurement method Blood pH was measured using a heparin-treated syringe, blood was collected by cardiac blood sampling, and blood pH was measured using a 643 automatic Ca ++ / pH analyzer (CIBA-CORNING). .

(2) Administration of Antibody As described above, rats (H
HM rat) and a humanized anti-PTHrP antibody
g / 0.5 ml / kg was administered via the tail vein (n = 3). In the control group, physiological saline was similarly intravenously administered.
(n = 2). Blood pH measurement was performed on day 0 (on the day of administration), day 1 and day 7 in both the antibody administration group and the control group.
The results were shown by the average value in each group.

As a result, the blood pH of the hypercalcemic rat before administration of the antibody was about 7.49 (the blood p
H was pH 7.40 ± 0.02), and this model clearly showed the pathology of metabolic alkalosis. When a humanized anti-PTHrP antibody was administered to this model, the blood pH was hardly changed in the control group, but improved to a value close to the blood pH of normal rats on day 7 in the antibody-administered group. It was confirmed that. Metabolic alkalosis based on inhibition of excretion of bicarbonate ion (HCO ₃ ⁻ ) in the kidney has been reported as one of the clinical symptoms in hypercalcemia associated with malignancy (HHM). Administration of the humanized anti-PTHrP antibody normalized the blood pH in this model.
M was shown to have the effect of improving the metabolic alkalosis seen in M (FIG. 31). The above results indicate that the chimeric antibody and the humanized antibody of the present invention are useful as ameliorating agents for improving clinical symptoms of hypercalcemia associated with malignant tumors.

[0315]

According to the present invention, an antibody, a chimeric antibody and a humanized antibody against PTHrP are provided. Since these antibodies have low antigenicity in humans, they are useful as therapeutic agents for hypercalcemia, hypophosphatemia, and the like.

[0316]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AAATAGCCCT TGACCAGGCA 20

SEQ ID NO: 2 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG 38

SEQ ID NO: 3 Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGATCCCGGG CCAGTGGATA GACAGATG 28

SEQ ID NO: 4 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGATCCCGGG TCAGRGGAAG GTGGRAACA 29

SEQ ID NO: 5 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTTTTCCCAG TCACGAC 17

SEQ ID NO: 6 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CAGGAAACAG CTATGAC 17

SEQ ID NO: 7 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGAA ACTTCGGGCT C 31

SEQ ID NO: 8 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGGATCC CTGCAGAGAC AGTGACCAGA 30

SEQ ID NO: 9 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTCCAC CATGGGGTTT GGGCTG 36

SEQ ID NO: 10 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TTTCCCGGGG CCTTGTGTGGA GGCTGAGGAG ACG
GTGACCA G41

SEQ ID NO: 11 Sequence length: 109 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCCCACGGTC ACCCTGTTCC 60 CGCCCTCCTC TGAGGAGCTC CAAGCCAACA AGGCCACACT AGTGTGTCT 109

SEQ ID NO: 12 Sequence length: 110 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGTTTGGTGG TCTCCACTCC CGCCTTGACG GGGCTGCCAT CTGCCTTCCA GGCCACTGTC 60 ACAGCTCCCG GGTAGAAGTC ACTGATCAGA CACACTAGTG TGGCCTTGTT 11
0

SEQ ID NO: 13 Sequence length: 98 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGAGTGGAGA CCACCAAAACC CTCCAAACAG AGC
AACAACA AGTACGCGGC CAGCAGCTAC 60 CTGAGCCTGA CGCCCGAGCA GTGGAAGTCC CACAGAAG 98

SEQ ID NO: 14 Sequence length: 106 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence: TGTTGAATTC TTACTATGAA CATTCTGTAG GGGCCACTGT CTTCTCCACG GTGCTCCCTT 60 CATGCGTGAC CTGGCAGCTG TAGCTTCTGT GGGACTTCCA CTGCTC 106

SEQ ID NO: 15 Sequence length: 43 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCC 43

SEQ ID NO: 16 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC TTACTATGAAA
20

SEQ ID NO: 17 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CAACAAGTAC GCGGCCAGCA GCTACCTGAG CCTGACGCC 39

SEQ ID NO: 18 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTAGCTGCTG GCCGCGTACT TGTTGTTGCT CTGTTTGGA 39

SEQ ID NO: 19 Sequence length: 46 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTAGTC CTAGGTCGAA CTGTGGCTGC ACCATC 46

SEQ ID NO: 20 Sequence length: 34 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC TTACTAACAC TCTCCCCTGT TGAA 34

SEQ ID NO: 21 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGGC CTGGACTCCT CTCTT 35

SEQ ID NO: 22 Sequence length: 48 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC AGATCTAACT ACTTACCTAG GACAGTGACC TTGGTCCC 48

SEQ ID NO: 23 Sequence length: 128 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGGG GTTTGGGCTG AGCTGGGTTT TCCTCGTTGC TCTTTTAAGA 60 GGTGTCCAGT GTCAGGTGCA GCTGGTGGAG TCTGGGGGAG GCGTGGTCCA GCCTGGGAGG 120 TCCCTGAG 128

SEQ ID NO: 24 Sequence length: 125 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence: ACCATTAGTA GTGGTGGTAG TTACACCTAC TATCCAGACA GTGTGAAGGG GCGATTCACC 60 ATCTCCAGAG ACAATTCCAA GAACACGCTG TATCTGCAAA TGAACAGCCT GAGAGCTGAG 120 GACAC 125

SEQ ID NO: 25 Sequence length: 132 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTACCACCAC TACTAATGGT TGCCACCCAC TCCAGCCCCT TGCCTGGAGC CTGGCGGACC60 CAAGACATGC CATAGCTACT GAAGGTGAAT CCAGAGGCTG CACAGGAGAG TCTCAGGGAC 120 CTCCCAGGCT GG 132

SEQ ID NO: 26 Sequence length: 110 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGGATCC CTGAGGAGAC GGTGACCAGG GTTCCCTGGC CCCAGTAAGC AAAGTAAGTC60 ATAGTAGTCT GTCTCGCACA GTAATACACA GCCGTGTCCT CAGCTCTCAG 110

SEQ ID NO: 27 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGGG GTTTGGGCTG 30

SEQ ID NO: 28 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGGATCC CTGAGGAGACGGTGGACCAGGG
30

SEQ ID NO: 29 Sequence length: 133 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACAAAGCTTC CACCATGGCC TGGACTCCTC TCTTCTTCTT CTTTGTTCTT CATTGCTCAG 60 GTTCTTTCTC CCAGCTTGTG CTGACTCAAT CGCCCTCTGC CTCTGCCTCC CTGGGAGCCT 120 CGGTCAAGCT CAC 133

SEQ ID NO: 30 Sequence length: 118 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AGCAAGATGG AAGCCACAGC ACAGGTGATG GGATTCCTGA TCGCTTCTCA GGCTCCAGCT 60 CTGGGGCTGA GCGCTACCTC ACCATCTCCA GCCTCCAGTC TGAGGATGAG GCTGACTA 118

SEQ ID NO: 31 Sequence length: 128 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTGTGGCTTC CATCTTGCTT AAGTTTCATC AAGTACCGAG GGCCCTTCTC TGGCTGCTGC 60 TGATGCCATT CAATGGTGTA CGTACTGTGC TGACTACTCA AGGTGCAGGT GAGCTTGACC 120 GAGGCTCC 128

SEQ ID NO: 32 Sequence length: 114 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CCCTCACAAA 60 TTGTTCCTTA ATTGTATCAC CCACACCACA GTAATAGTCA GCCTCATCCT CAGA 114

SEQ ID NO: 33 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACAAAGCTTC CACCATG 17

SEQ ID NO: 34 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCT 19

SEQ ID NO: 35 Sequence length: 75 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA 60 TTGTTCCTTA ATTGT 75

SEQ ID NO: 36 Sequence length: 43 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AAAGGATCCT TAAGATCCAT CAAGTACCGA GGGGGCTTCT CTG 43

SEQ ID NO: 37 Sequence length: 46 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACAAAGCTTA GCGCTACCTC ACCATCTCCA GCCTCCAGCC TGAGGA 46

SEQ ID NO: 38 Sequence length: 111 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA 60 TTGTTCCTTA ATTGTATCAC CCACACCACA GATATAGTCA GCCTCATCCT C 111

SEQ ID NO: 39 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTCTCTGGC TGCTGCTGAT ACCATTCAAT GGTGTACGTA CT 42

SEQ ID NO: 40 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CGAGGGCCCT TCTCTGGCTGCTGCTG
26

SEQ ID NO: 41 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GAGAAGGGCC CTARGTACST GATGRAWCTT AAGCA 35

SEQ ID NO: 42 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG 35

SEQ ID NO: 43 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGCTTGGAGC TCCTCAGA 18

SEQ ID NO: 44 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GACAGTGGTT CAAAGTTTTT 20

SEQ ID NO: 45 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Ser Ala Ser Phe Ser Leu Gly 1 5 10 15 Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg 65 70 75 Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Met Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 46 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu 35 40 45 Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 50 55 60 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp 80 85 90 Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe 95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 110 115

SEQ ID NO: 47 Sequence length: 116 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 110 115

SEQ ID NO: 48 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 49 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 50 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 51 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 52 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 53 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 54 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 55 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 56 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 50 55 60 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe 95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115

SEQ ID NO: 57 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG AAC TTC GGG CTC AGC TTG ATT TTC CTT GCC CTC ATT TTA AAA 45 Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys -15 -10 -5 GGT GTC CAG TGT GAG GTG CAA CTG GTG GAG TCT GGG GGA GAC TTA 90 Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu 1 5 10 GTG AAG CCT GGA GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA 135 Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly 15 20 25 TTC ACT TTC AGT AGC TAT GGC ATG TCT TGG ATT CGC CAG ACT CCA 180 Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Ile Arg Gln Thr Pro 30 35 40 GAC AAG AGG CTG GAG TGG GTC GCA ACC ATT AGT AGT GGT GGT AGT 225 Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser 45 50 55 TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 60 65 70 AGA GAC AAT GCC AAG AAC ACC CTA TAC CTG CAA ATG AGC AGT CTG 315 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Seruu 75 80 85 AAG TCT GAG GAC ACA GCC ATG TTT TAC TGT GCA AGA CAG ACT ACT 360 Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr 90 95 100 ATG ACT TAC TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC 405 Met Thr Tyr Pyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 105 110 115 TCT GCA 411 Ser Ala

SEQ ID NO: 58 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GGG TTT GGG CTG AGC TGG GTT TTC CTC GTT GCT CTT TTA AGA 45 Met Gly Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg -15 -10 -5 GGT GTC CAG TGT CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG 90 Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val 1 5 10 GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA 135 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 15 20 25 TTC ACC TTC AGT AGC TAT GGC ATG TCT TGG GTC CGC CAG GCT CCA 180 Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro 30 35 40 GGC AAG GGG CTG GAG TGG GTG GCA ACC ATT AGT AGT GGT GGT AGT 225 Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser 45 50 55 TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 60 65 70 AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG 315 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 75 80 85 AGA GCT GAG GAC ACG GCT GTG TAT TAC TGT GCG AGA CAG ACT ACT 360 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr 90 95 100 ATG ACT TAC TTT GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC 405 Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 105 110 115 TCC TCA 411 Ser Ser

SEQ ID NO: 59 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 60 Sequence length: 7 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 61 Sequence length: 9 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 62 Sequence length: 5 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 63 Sequence length: 16 Sequence type: amino acid Topology: Linear Sequence type: Peptide Sequence: Ser Ile Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly 1 5 10 15

SEQ ID NO: 64 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 65 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAA CTT GTG CTC ACT CAG TCA TCT TCA GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser 1 5 10 TTC TCC CTG GGA GCC TCA GCA AAA CTC ACG TGC ACC TTG AGT AGT 135 Phe Ser Leu Gly Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAA CAG CCA CTC 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu 30 35 40 AAG CCT CCT AAG TAT GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Pro Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCT GGA TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGT GCT GAT CGC TAC CTT AGC ATT TCC AAC ATC CAG CCA GAA GAT 315 Gly Ala Asp Arg Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp 75 80 85 GAA GCA ATG TAC ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Met Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAT GTT TTC GGC GGT GGG ACC AAG GTC ACT GTC CTA GGT 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 66 Sequence length: 405 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG CAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT CGG TAC TTG ATG AAA CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGT 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115

SEQ ID NO: 67 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 68 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 69 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 70 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 71 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 72 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 73 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 74 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 75 Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile 1 5 10 15 Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu 20 25 30 Ile His Thr Ala

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the antibody of the present invention.

FIG. 2 is a diagram showing an outline of CDR-grafting.

FIG. 3 is a diagram showing evaluation of FR and CDR in a V region.

FIG. 4 is a view showing the measurement results of antibody binding activity.

FIG. 5 is a view showing the measurement results of antibody binding activity.

FIG. 6 is a view showing the measurement results of antibody binding activity.

FIG. 7 shows the results of measuring antibody binding activity.

FIG. 8 is a view showing the measurement results of antibody binding activity.

FIG. 9 shows the results of measuring antibody binding activity.

FIG. 10 shows the results of measuring antibody binding activity.

FIG. 11 is a view showing the measurement results of antibody binding activity.

FIG. 12 shows the neutralizing activity of a humanized antibody.

FIG. 13 shows the neutralizing activity of a humanized antibody.

FIG. 14 shows the neutralizing activity of a humanized antibody.

FIG. 15 is a graph showing the effect of the antibody of the present invention on a hypercalcemia model animal.

FIG. 16 is a view showing the effect of the antibody of the present invention on a hypercalcemia model animal.

FIG. 17 is a view showing the effect of the antibody of the present invention on a hypercalcemia model animal.

FIG. 18 is a view showing the effect of the antibody of the present invention on a hypercalcemia model animal.

FIG. 19 is a diagram showing a sensorgram of PTHrP immobilization on a sensor chip.

FIG. 20 shows the results of kinetic analysis of the antibody of the present invention.

FIG. 21 shows the results of kinetic analysis of the antibody of the present invention.

FIG. 22 shows the results of kinetic analysis of the antibody of the present invention.

FIG. 23 shows the results of kinetic analysis of the antibody of the present invention.

FIG. 24 shows the results of kinetic analysis of the antibody of the present invention.

FIG. 25 is a view showing the results of testing the effect of the humanized antibody of the present invention on the phosphorus excretion rate.

FIG. 26 is a view showing the results of a test on the effect of the humanized antibody of the present invention on plasma phosphorus concentration.

FIG. 27 is a photograph showing the results of observing the apparent clinical symptoms after administration of an anti-PTHrP antibody to a hypercalcemic mouse (organism form).

FIG. 28 is a photograph showing the results of observing the apparent clinical symptoms after administration of an anti-PTHrP antibody to a hypercalcemic mouse (organism form).

FIG. 29. Anti-PTH using hypercalcemia model
FIG. 7 is a diagram comparing the daily change in locomotor activity after administration of an rP antibody with that of a control group (administration of physiological saline).

FIG. 30. Anti-PTH using hypercalcemia model
FIG. 7 is a diagram comparing daily changes in body temperature after administration of rP antibody with those of a control group (administration of physiological saline).

FIG. 31. Anti-PTH using hypercalcemia model
FIG. 7 is a diagram comparing daily changes in blood pH after administration of rP antibody with those of a control group (administration of physiological saline).

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 13, 1997

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] FIG. 27

[Correction method] Change

[Correction contents]

FIG. 27

[Procedure amendment 4]

[Document name to be amended] Drawing

[Correction target item name] FIG. 28

[Correction method] Change

[Correction contents]

FIG. 28

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 1/21 C12N 1/21 5/10 C12P 21/08 15/02 C12N 5/00 B 15/09 ZNA 15/00 C C12P 21/08 ZNAA // A61K 38/00 ADD A61K 37/02 ADD (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (84)

[Claims] 1. A chimeric L chain comprising an L chain C region of a human antibody and a V region of a mouse monoclonal antibody against a human parathyroid hormone-related peptide. 2. The chimeric L chain according to claim 1, wherein the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 45. 3. The chimeric L chain according to claim 1, wherein the C region is a Cλ region. 4. A chimeric H chain comprising the H chain C region of a human antibody and the H chain V region of a mouse monoclonal antibody against a human parathyroid hormone-related peptide. 5. The chimeric H chain according to claim 4, wherein the H chain V region comprises the amino acid sequence represented by SEQ ID NO: 46. 6. The chimeric H chain according to claim 4, wherein the C region is a Cγ1 region. 7. A chimera for a human parathyroid hormone-related peptide, comprising the chimeric L chain according to any one of claims 1 to 3 and the chimeric H chain according to any one of claims 4 to 6. Monoclonal antibody. 8. A humanized version comprising framework regions 1 to 4 of the L chain V region of a human antibody, and complementarity determining regions 1 to 3 of the L chain V region of a mouse monoclonal antibody to a human parathyroid hormone-related peptide. A polypeptide comprising the light chain V region of an antibody. 9. The polypeptide according to claim 8, wherein the complementarity determining regions 1 to 3 contain the amino acid sequences represented by SEQ ID NOS: 59 to 61, respectively. 10. The framework regions 1 to 3 are respectively derived from the framework regions 1 to 3 of the human antibody HSU03868, and the framework region 4 is a human antibody S257.
9. The polypeptide according to claim 8, which is derived from 55 framework 4. 11. The framework regions 1 to 3 are substantially the same as the framework regions 1 to 3 of the human antibody HSU03868, respectively, and the framework region 4 is substantially the same as the framework region 4 of the human antibody S25755. 9. The polypeptide according to claim 8, which is identical in nature. 12. The framework according to claim 8, wherein the amino acid at position 36 according to Kabat's definition is tyrosine and the amino acid at position 49 is aspartic acid.
The polypeptide of any one of the preceding claims. 13. The polypeptide according to claim 12, comprising any one of the amino acid sequences represented by SEQ ID NOs: 48 to 51. 14. The amino acid at position 45 according to the definition of Kabat in the framework region is lysine, and
The polypeptide according to claim 8, wherein the amino acid at position 87 is isoleucine. 15. The polypeptide according to claim 14, which comprises any one of the amino acid sequences represented by SEQ ID NOS: 52 to 55. 16. A humanized form comprising framework regions 1 to 4 of the H chain V region of a human antibody and complementarity determining regions 1 to 3 of the H chain V region of a mouse monoclonal antibody to a human parathyroid hormone-related peptide. A polypeptide comprising the H chain V region of an antibody. 17. The polypeptide according to claim 16, wherein the complementarity determining regions 1 to 3 include the amino acid sequences represented by SEQ ID NOs: 62 to 64, respectively. 18. The polypeptide of claim 16, wherein the framework regions 1-4 are derived from framework regions 1-4 of a human antibody of human subgroup III. 19. The polypeptide according to claim 16, wherein the framework regions 1 to 4 are derived from the framework regions 1 to 4 of the human antibody S31679, respectively. 20. The polypeptide of claim 16, wherein the framework regions 1-4 are substantially identical to framework regions 1-4 of human antibody S31679, respectively. 21. A polypeptide comprising the H chain V region of a humanized antibody, comprising the amino acid sequence represented by SEQ ID NO: 56. 22. A light chain of a humanized antibody against a human parathyroid hormone-related peptide, comprising a polypeptide comprising the C region of the L chain of a human antibody, and the polypeptide according to any one of claims 8 to 15. . 23. The C region is a Cλ region, the framework regions 1 to 3 are substantially the same as the framework regions 1 to 3 of the human antibody HSU03868, and the framework region 4 is the same as that of the human antibody S25755. Substantially the same as the framework region 4 and the complementarity determining region 1
23. The L chain of a humanized antibody according to claim 22, wherein the amino acid sequences of SEQ ID NOS: to 3 are represented by SEQ ID NOS: 59 to 61, respectively. 24. An H chain of a humanized antibody against a human parathyroid hormone-related peptide, comprising a polypeptide comprising the H chain C region of a human antibody, and the polypeptide according to any one of claims 16 to 21. . 25. The C region is a Cλ1 region, the framework regions 1 to 4 are respectively derived from the framework regions 1 to 4 of the human antibody HSGIII, and the complementarity determining regions 1 to 3 are each SEQ ID NO: 62. 25. The H chain of the humanized antibody according to claim 24, which comprises an amino acid sequence represented by any one of SEQ ID NOs: to 64. 26. A humanized antibody against a human parathyroid hormone-related peptide, comprising the light chain of the humanized antibody according to claim 22 or 23 and the heavy chain of the humanized antibody according to claim 24 or 25. 27. An antibody against a human parathyroid hormone-related peptide having a dissociation constant of 1.86 × 10 ⁻⁷ [M] or less. 28. An antibody against a human parathyroid hormone-related peptide having a dissociation rate constant of 1.22 × 10 ⁻¹ [1 / Sec] or less. 29. An antibody against a human parathyroid hormone-related peptide having a binding rate constant of 6.55 × 10 ⁴ [1 / M.Sec] or more. 30. An antibody against a human parathyroid hormone-related peptide having a dissociation rate constant of 1.22 × 10 ⁻¹ [1 / Sec] or less and a binding rate constant of 6.55 × 10 ⁴ [1 / M.Sec] or more. 31. The antibody according to claim 27, wherein the dissociation constant is measured by a surface plasmon resonance sensor. 32. The dissociation rate constant is measured by a surface plasmon resonance sensor.
The antibody according to 0. 33. The binding rate constant is measured by a surface plasmon resonance sensor.
The antibody according to 0. 34. A dissociation constant of 1.02 × 10 ^{-11 to} 1.86 × 10
28. The antibody according to claim 27, which is ^-7 [M]. 35. A dissociation constant of 1.02 × 10 ^{-10 to} 1.86 × 10
28. The antibody according to claim 27, which is ^-8 [M]. 36. A dissociation constant of 1.34 × 10 ^{-10 to} 3.58 × 10 ^-10
28. The antibody according to claim 27, which is [M]. 37. A dissociation rate constant of 7.38 × 10 ^{−6 to} 1.22 × 10
29. The antibody according to claim 28, which is ^-1 [1 / Sec]. 38. A dissociation rate constant of 7.38 × 10 ^{−5 to} 1.22 × 10
^29. The antibody according to claim 28, which is ^-2 [1 / Sec]. 39.A dissociation rate constant of 1.66 × 10 ^{−4 to} 3.16 × 10
^29. The antibody according to claim 28, which is ^-4 [1 / Sec]. 40. The antibody according to claim 28, wherein the dissociation rate constant is 2.32 × 10 ⁻⁴ [1 / Sec]. 41. A binding rate constant of 6.55 × 10 ^{4 to} 1.24 × 10 ^7.
30. The antibody according to claim 29, which is [1 / M.Sec]. 42. A binding rate constant of 6.55 × 10 ^{5 to} 1.24 × 10 ⁶
30. The antibody according to claim 29, which is [1 / M.Sec]. 43. A binding rate constant of 7.23 × 10 ^{5 to} 1.03 × 10 ⁶
30. The antibody according to claim 29, which is [1 / M.Sec]. 44. The antibody according to claim 29, wherein the binding rate constant is 1.03 × 10 ⁶ [1 / M.Sec]. 45. A human having a dissociation rate constant of 2.32 × 10 ^{−4 to} 3.16 × 10 ⁻⁴ [1 / Sec] and a binding rate constant of 0.883 × 10 ^{6 to} 1.03 × 10 ⁶ [1 / M.Sec]. Antibodies to parathyroid hormone-related peptides. 46. The antibody according to claim 27, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, or a primed antibody.
45. The antibody according to any one of the items 45. 47. A DNA comprising a base sequence encoding an L chain V region of a mouse monoclonal antibody against a human parathyroid hormone-related peptide. 48. The DNA according to claim 47, wherein the L chain V region comprises the amino acid sequence represented by SEQ ID NO: 45. 49. The DN according to claim 47, wherein the nucleotide sequence encoding the L chain V region is represented by SEQ ID NO: 65.
A. 50. A DNA comprising a nucleotide sequence encoding the V region of the H chain of a mouse monoclonal antibody against a human parathyroid hormone-related peptide. 51. The DNA according to claim 50, wherein the V region of the H chain comprises the amino acid sequence represented by SEQ ID NO: 46. 52. The DN according to claim 50, wherein the nucleotide sequence encoding the H chain V region is represented by SEQ ID NO: 57.
A. 53. A DNA encoding the chimeric L chain according to any one of claims 1 to 3. 54. The DNA encoding the chimeric L chain comprises the nucleotide sequence represented by SEQ ID NO: 65.
The described DNA. 55. A DNA encoding the chimeric H chain according to any one of claims 4 to 6. 56. The DNA encoding the chimeric H chain comprises the nucleotide sequence represented by SEQ ID NO: 57.
The described DNA. 57. A DNA comprising a base sequence encoding the polypeptide according to any one of claims 8 to 15. 58. The DNA according to claim 57, comprising any one of the nucleotide sequences represented by SEQ ID NOs: 66 to 74. 59. A DNA comprising a base sequence encoding the polypeptide according to any one of claims 16 to 21. 60. The DNA according to claim 59, comprising the base sequence represented by SEQ ID NO: 58. 61. A DNA encoding the L chain of the humanized antibody according to claim 22 or 23. 62. A humanized antibody L chain DNA comprising a base sequence encoding any one of the amino acid sequences represented by SEQ ID NOs: 47 to 55. 63. The DNA according to claim 62, wherein the L chain DNA of the humanized antibody comprises any one of the nucleotide sequences represented by SEQ ID NOs: 66 to 74. 64. A DNA encoding the H chain of the humanized antibody according to claim 24 or 25. 65. H chain DN of a humanized antibody, comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 56.
A. 66. The DNA according to claim 65, wherein the H chain DNA of the humanized antibody comprises the nucleotide sequence represented by SEQ ID NO: 58. 67. A recombinant vector comprising the DNA according to any one of claims 47 to 66. 68. A transformant transformed by the recombinant vector according to claim 67. 69. An expression vector comprising the DNA according to any one of claims 47 to 49 and 53 to 54, and an expression vector comprising the DNA according to any one of claims 50 to 52 and 55 to 56. A method for producing a chimeric antibody against a human parathyroid-related peptide, comprising culturing a transformant transformed with a vector and collecting a chimeric antibody against a human parathyroid-related peptide from the resulting culture. 70. An expression vector containing the DNA of any one of claims 57 to 58 and 61 to 63, and an expression vector containing the DNA of any one of claims 59 to 60 and 64 to 65. A method for producing a humanized antibody against a human parathyroid-related peptide, comprising culturing a transformant transformed with a vector and collecting a humanized antibody against the human parathyroid-related peptide from the obtained culture. 71. A pharmaceutical composition comprising a humanized antibody against a human parathyroid hormone-related peptide as an active ingredient. 72. A hypercalcemia inhibitor comprising a humanized antibody against a human parathyroid hormone-related peptide as an active ingredient. 73. An agent for suppressing hypercalcemia associated with a malignant tumor, comprising a humanized antibody against a human parathyroid hormone-related peptide as an active ingredient. 74. The malignant tumor is pancreatic cancer, lung cancer, pharyngeal cancer,
Laryngeal, tongue, gingival, esophageal, stomach, bile duct, breast,
74. The hypercalcemia inhibitor according to claim 73, which is at least one selected from the group consisting of kidney cancer, bladder cancer, uterine cancer, prostate cancer, and malignant lymphoma. 75. A pharmaceutical composition comprising the antibody according to any one of claims 27 to 46 as an active ingredient. A hypercalcemia inhibitor comprising the antibody according to any one of claims 27 to 46 as an active ingredient. 77. An agent for suppressing hypercalcemia associated with malignant tumor, comprising the antibody according to any one of claims 27 to 46 as an active ingredient. 78. The malignant tumor is pancreatic cancer, lung cancer, pharyngeal cancer,
Laryngeal, tongue, gingival, esophageal, stomach, bile duct, breast,
The hypercalcemia inhibitor according to claim 77, which is at least one selected from the group consisting of renal cancer, bladder cancer, uterine cancer, prostate cancer, and malignant lymphoma. 79. An agent for improving hypophosphatemia comprising, as an active ingredient, a humanized antibody against a human parathyroid hormone-related peptide. 80. The method according to claim 79, wherein the hypophosphatemia is hypophosphatemic rickets. 81. The method according to claim 79, wherein the hypophosphatemia is hypophosphatemic vitamin D-resistant rickets. 82. An agent for ameliorating hypophosphatemia comprising the antibody according to any one of claims 27 to 46 as an active ingredient. 83. The method according to claim 82, wherein the hypophosphatemia is hypophosphatemic rickets. 84. The method of claim 82, wherein the hypophosphatemia is hypophosphatemic vitamin D-resistant rickets.