JPWO2001064249A1 - Tissue decomposition inhibitor - Google Patents

Abstract

(57) [Abstract] A tissue degradation inhibitor comprising a substance that inhibits the binding of parathyroid hormone-related peptide to its receptor.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to parathyroid hormone-related peptides (PTH-related peptides).
The bond between the receptor and the PTHrP (protoplastin-related protein)
The present invention relates to a tissue degradation inhibitor containing a substance that inhibits the synthesis of parathyroid hormone-related peptide (PHR)
PTHrP is a steroid hormone-related peptide (PTHrP) that inhibits the production of calcium.
It is a protein produced by tumors that is the main cause of hypercalcemia, and is involved in bone resorption and calcification in renal tubules.
By promoting calcium resorption, it is possible to treat tumor-induced hypercalcemia (Humor
al hypercalcemia of malalignancy, hereinafter referred to as “HH
Currently, HHM is treated with anti-bone resorption drugs.
Calcitonin and bisphosphonates are used, but HHM progresses rapidly.
This significantly worsens the quality of life (QOL) of terminal cancer patients.
Therefore, there is a need to develop more effective treatments that address the underlying cause. Antibodies against parathyroid hormone-related peptide (hereinafter referred to as "anti-PTHrP antibodies")
) shows its effect on HHM immediately after administration, so it takes several days for the effect to appear.
Furthermore, it is superior to bisphosphonates, which require
It is also useful as a therapeutic agent for cachexia seen in
Cancer cachexia is a paraneoplastic syndrome characterized by rapid weight loss.
This weight loss is seen in many patients with advanced cancer.
The occurrence of cancer cachexia has a significant impact on survival time.
Not only does it have adverse effects on the patient's quality of life, but it also significantly impairs the quality of life of the patient.
Thus, controlling cachexia is an important part of cancer treatment.
It has important meaning. Incidentally, model rats transplanted with the human lung cancer line LC-6 cause HHM.
For this reason, existing HHM treatment drugs, bisphosphonates, are known to cause
However, in HHM model rats that show severe weight loss and other cachectic conditions,
Even with the use of bisphosphonates, weight recovery is minimal. Disclosure of the Invention The present invention is a treatment for rheumatoid arthritis using a substance that inhibits the binding of PTHrP to its receptor as an active ingredient.
The present inventors have conducted extensive research to solve the above-mentioned problems, and as a result have discovered that a parathyroid hormone
Substances that inhibit the binding of mon-related peptides to their receptors can suppress tissue degradation
This discovery led to the completion of the present invention. That is, the present invention provides a method for detecting the binding between parathyroid hormone-related peptide and its receptor.
The tissue decomposition inhibitor contains as an active ingredient a substance that inhibits the decomposition of tissue.
For example, antagonists of parathyroid hormone-related peptide receptors, antiparathyroid hormones,
glandular hormone-related peptide antibodies (e.g., humanized or chimeric monoclonal antibodies);
Examples of antibodies include human antibodies, fragments of such antibodies, and/or modified products thereof.
Examples of the humanized antibody include humanized #23-57-137-1 antibody.
Examples of tissues include muscle tissue and adipose tissue.
Tissue breakdown can occur due to cancer cachexia, sepsis, severe trauma, or muscular dystrophy.
The inhibitors include, for example, those that inhibit cytokines (e.g., IL-6
, G-CSF, IL-11, and LIF.
It is also effective in patients with higher than normal blood levels of IL-6,
Inflammatory cytokines such as IL-11 and LIF are said to be involved in tissue degradation.
Despite the fact that the present invention
Inhibitors of the above are effective. The present invention will be described in detail below. This specification is a copy of Japanese Patent Application No. 2000-52414, from which the present application claims priority.
The present invention encompasses the contents described in the specification and drawings of No. 2004/002262. The present invention relates to a parathyroid hormone-related peptide (PTH)
PTHrP) and its receptor (PT
It is a tissue decomposition inhibitor containing as an active ingredient a substance that inhibits binding to the HrP receptor.
The inventors have found that anti-PTHrP antibodies have the medicinal effect of correcting calcium levels as well as improving body weight.
Based on the findings that the antibody has a medicinal effect of restoring the body weight,
It is speculated that the weight loss caused by cachexia plays a role in the
It was thought that this was caused by tissue destruction.
The present invention was completed based on this discovery. In this specification, the term "PTHrP receptor" refers to a receptor that is a PTHrP receptor, as defined in, for example, JP-A-6-506598.
This refers to the receptors that bind to PTHrP, which is described in the literature, and is present on target organs (e.g., bone
It does not matter whether the PTHrP receptor is present in the blood or kidney. Furthermore, "a substance that inhibits the binding of PTHrP to the PTHrP receptor" means any of the following:
) Binding to PTHrP causes PTHrP to bind to the PTHrP receptor
(2) substances that inhibit PTHrP receptors (e.g., anti-PTHrP antibodies);
By binding to the body, it inhibits PTHrP from binding to the PTHrP receptor.
Substances that act as antagonists to PTHrP receptors (e.g., PTHrP antagonists)
agonist), specifically at least one amino acid of a PTHrP peptide
(referring to amino acid substitutions, deletions, or partial sequences of PTHrP peptides)
The anti-PTHrP antibody used in the present invention binds to PTHrP and inhibits tissue degradation.
If it has an inhibitory effect, its origin and type (monoclonal, polyclonal)
The anti-PTHrP antibody may be of any type (normal) and shape. Examples of the anti-PTHrP antibody include humanized antibodies and human antibodies (WO96/33
735), chimeric antibodies (Japanese Patent Laid-Open No. 4-228089), mouse antibodies (
For example, the antibody produced by hybridoma #23-57-137-1 (#
23-57-137-1 antibody).
Although a monoclonal antibody may be used, a monoclonal antibody is preferable. PTHrP antagonists include polypeptides and small molecules.
For example, the compounds that act antagonistically against PTHrP include, but are not limited to,
Substances that bind to THrP receptors include those disclosed in Japanese Patent Application Laid-Open No. 7-165790 and Japanese Patent Application Laid-Open No. 2005-200844.
5-509098 or Peptides (UNITED STATES)
1995, 16(6) 1031-1037, Biochemistry (UNI
TED STATES) April. 281992, 31 (16) 4026-403
3. A polypeptide having PTHrP antagonist activity is also included.
In addition, among the above-mentioned exemplary polypeptides, at least one amino acid is deleted or substituted.
and the like, which have been added or inserted into the polypeptide and have equivalent PTHrP antagonist activity.
The PTHrP antagonists of the present invention also include those having this property. In the present invention, "substances that inhibit the binding of PTHrP to the PTHrP receptor" are defined as
We will explain this using anti-PTHrP antibodies as an example. 1. Anti-PTHrP Antibodies The anti-PTHrP antibodies used in the present invention are polyclonal antibodies obtained using known methods.
Alternatively, it can be obtained as a monoclonal antibody.
As the HrP antibody, a monoclonal antibody derived from a mammal is particularly preferred.
Monoclonal antibodies derived from a substance include those produced by hybridomas and those derived from genetically modified organisms.
The antibody is produced in a host transformed with an expression vector containing an antibody gene by genetic engineering techniques.
This antibody binds to PTHrP, thereby inhibiting PTHrP from becoming P
Blocks PTHrP signal transduction by inhibiting binding to TH/PTHrP receptors
It is an antibody that blocks the biological activity of PTHrP. An example of such an antibody is hybridoma clone #23-57-13
Examples include the #23-57-137-1 antibody produced by mouse-m
ose hybridoma #23-57-137-1, Agency of Industrial Science and Technology
In August 1996, the Institute of Bioscience and Human-Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture)
On the 15th, it was deposited internationally under the Budapest Treaty as FERM BP-5631.
2. Antibody-producing hybridomas Monoclonal antibody-producing hybridomas can be produced as follows.
That is, PTHrP was used as a sensitizing antigen, and this was used as a standard immunization method.
The resulting immune cells are fused with known parent cells by a conventional cell fusion method.
Monoclonal antibody-producing cells are then screened using conventional screening methods.
First, human PTHrP, which is used as a sensitizing antigen for antibody production, is immunized with Suva, L
J. et al., Science (1987) 237, 893
It is obtained by expressing the PTHrP gene/amino acid sequence.
The gene sequence encoding HrP is inserted into a known expression vector system and expressed in a suitable host cell.
After transforming the cells, the target PTHrP is extracted from the host cells or the culture supernatant.
The protein is purified by known methods. The purified PTHrP protein is then used as a sensitizing antigen.
The 34 N-terminal peptides of THrP can also be produced by chemical synthesis.
This can also be used as a sensitizing antigen. Mammals that can be immunized with a sensitizing antigen are not particularly limited, but include:
It is preferable to select the host cell in consideration of its compatibility with the parent cell used for cell fusion.
Rodents (e.g., mice, rats, hamsters, etc.) or rabbits,
Monkeys and the like are used. Animals are immunized with the sensitizing antigen according to known methods. For example,
A common method is to inject a sensitizing antigen into a mammal intraperitoneally or subcutaneously.
Specifically, the sensitizing antigen is dissolved in PBS (Phosphate Buffer Solution)
Dilute and suspend in an appropriate amount of saline or physiological saline, etc., and use as desired.
A more conventional adjuvant, such as Freund's complete adjuvant, is mixed in an appropriate amount and the milk
After the vaccination, the vaccine is administered to the mammal several times every 4 to 21 days.
A carrier can also be used. After immunization in this way and confirming that the desired antibody level has increased in the serum,
Immune cells are collected from mammals and subjected to cell fusion.
Examples of the parent cells to be fused with the immune cells include spleen cells. Mammalian myeloma cells can be used as the other parent cell to be fused with the immune cells.
The myeloma cells used may be any of various known cell lines, such as P3 (P3x6
3Ag8.653) (J. Immnol. (1979) 123, 1548-15
50), P3x63Ag8U. 1 (Current Topics in Mic
robiology and immunology (1978) 81, 1-7
), NS-1 (Kohler.G. and Milstein, C.Eur.J
．． Immunol. (1976) 6, 511-519), MPC-11 (Mar
gulies. D. H. et al. , Cell (1976) 8, 405-41
5), SP2/0 (Shulman, M. et al., Nature (197
8) 276, 269-270), FO (de St. Groth, S.F. et
al. , J. Immunol. Methods (1980) 35, 1-21)
, S194 (Trowbridge, I. S. J. Exp. Med. (1978
) 148, 313-323), R210 (Galfre, G. et al., N
Cell fusion between the immune cells and myeloma cells can be carried out basically by known methods, for example,
For example, the method of Milstein et al. (Kohler, G. and Milstein,
C., Methods Enzymol. (1981) 73, 3-46)
More specifically, the cell fusion can be carried out by, for example, culturing the cells in the presence of a cell fusion promoter under normal nutrition.
The fusion is carried out in a culture medium. Examples of fusion promoters include polyethylene glycol.
(PEG), Sendai virus (HVJ), etc. are used, and if desired, a fusion agent may be used.
To increase the efficiency, an adjuvant such as dimethyl sulfoxide can be added.
The ratio of immune cells to myeloma cells can be set arbitrarily.
For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells.
The culture medium used for cell fusion may be, for example, a medium suitable for the growth of the myeloma cell line.
RPMI1640 culture medium, MEM culture medium, and other media used for this type of cell culture
In addition, serum supplements such as fetal calf serum (FCS) can be used.
Cell fusion can also be performed by fusion of predetermined amounts of the immune cells and myeloma cells in the culture medium.
The mixture was mixed with a PEG solution (for example, an average molecular weight of 1000-6000) preheated to about 37°C.
000) is usually added at a concentration of 30-60% (w/v) and mixed.
The desired fused cells (hybridomas) are formed by the above procedure.
The hybridization was carried out by repeating the steps of sequentially adding the above-mentioned components and centrifuging the mixture to remove the supernatant.
The cell fusion agent and other substances that are unfavorable to the growth of the hybridoma are removed. The hybridomas obtained in this way are cultured in a conventional selective culture medium, such as HAT
Culture in culture medium (containing hypoxanthine, aminopterin, and thymidine)
The culture in the HAT medium is carried out to select the desired hybrid.
Allow sufficient time for non-doma cells (unfused cells) to die (usually several days to several weeks).
Then, a conventional limiting dilution method is carried out to produce the desired antibody.
Hybridoma screening and single cloning are performed. In addition to obtaining the above hybridomas by immunizing non-human animals with antigens,
Lymphocytes were sensitized to PTHrP in vitro, and the sensitized lymphocytes were obtained from human
It is fused with myeloma cells having immortal division ability and has PTHrP binding activity.
It is also possible to obtain a desired human antibody (see Japanese Patent Publication No. 1-59878).
Furthermore, transgenic animals with a full repertoire of human antibody genes have been developed.
Anti-PTHrP antibody-producing cells are obtained by administering PTHrP as an antigen, and these are then used as anti-PTHrP antibodies.
Human antibodies against PTHrP may be obtained from the killed cells (International Publication No.
No. WO 94/25585, WO 93/12227, WO 92
(See WO 94/03918 and WO 94/02602.) The hybridomas producing the monoclonal antibodies thus produced are
It can be subcultured in normal culture medium and stored for a long period in liquid nitrogen.
To obtain a monoclonal antibody from the hybridoma,
A method of culturing a hamster according to a conventional method and obtaining the culture supernatant;
The hybridomas are administered to a compatible mammal and grown therein, and the ascites fluid is collected.
The former method is suitable for obtaining highly pure antibodies.
The latter method is suitable for mass production of antibodies. 3. Recombinant Antibodies In the present invention, antibody genes are cloned from hybridomas to produce monoclonal antibodies.
The cloned vector is then inserted into a suitable vector, which is then introduced into a host cell for genetic recombination.
Recombinant forms produced using recombinant techniques can be used (e.g., Va
ndamme, A. M. et al. , Eur. J. Biochem. (199
0) 192, 767-775, 1990). Specifically, anti-PTHrP was isolated from a hybridoma producing an anti-PTHrP antibody.
The mRNA encoding the variable (V) region of the antibody is isolated.
Known methods, such as the guanidine ultracentrifugation method (Chirgwin, J.M. et al.
l. , Biochemistry (1979) 18, 5294-5299), A
GPC method (Chomczynski, P. et al., Anal. Bioch
(1987) 162, 156-159) and other methods to prepare total RNA.
, mRNA Purification Kit (Pharmacia), etc.
The target mRNA is prepared using QuickPrep mRNA Preparation.
By using the Identification Kit (Pharmacia)
mRNA can be prepared directly. The obtained mRNA is then used to synthesize cDNA for the antibody V region using reverse transcriptase.
cDNA synthesis was carried out using AMV Reverse Transcriptase F.
irst-strand cDNA Synthesis Kit (Seikagaku Corporation)
For cDNA synthesis and amplification, a 5'-A primer is used.
mpli FINDER RACE Kit (Clontech) and PC
5'-RACE method using R (Frohman, M.A. et al., Proc.
c. Natl. Acad. Sci. USA (1988) 85, 8998-900
2, Belyavsky, A. et al. , Nucleic AcidsRe
(1989) 17, 2919-2932) or the like can be used. The desired DNA fragment is purified from the resulting PCR product and linked to vector DNA.
Furthermore, a recombinant vector is prepared from this and introduced into E. coli or the like to form colonies.
The desired recombinant vector is prepared by selecting the desired DNA.
The base sequence can be determined by known methods, for example, dideoxynucleotide chain termination.
After obtaining the DNA encoding the V region of the desired anti-PTHrP antibody,
into an expression vector containing DNA encoding the desired antibody constant region (C region)
To produce the anti-PTHrP antibody used in the present invention, the antibody gene is expressed and regulated.
The expression vector is designed to be expressed under the control of a region, e.g., an enhancer or promoter.
Next, host cells are transformed with this expression vector, and the
Expression of antibody genes involves the expression of DNA encoding the antibody heavy chain (H chain) or light chain (L chain).
A may be separately incorporated into expression vectors and co-transformed into host cells, or
Alternatively, DNA encoding the H chain and the L chain may be integrated into a single expression vector.
Host cells may also be transformed (see WO 94/11523). In addition to the above host cells, transgenic cells can also be used to produce recombinant antibodies.
For example, an animal can be used that expresses an antibody gene that is specifically produced in milk.
It is inserted into a gene encoding a protein (such as goat beta-casein) that is used to make a fusion gene.
The DNA fragment containing the fusion gene into which the antibody gene has been inserted is then transfected into a goat embryo.
The embryo is then injected into a female goat, and the resulting embryo is transferred to a female goat.
The desired antibody is obtained from the milk produced by the transgenic goat or its offspring.
To increase the amount of milk containing the desired antibody produced by transgenic goats,
For this purpose, appropriate hormones may be administered to transgenic goats (Ebert,
K. M. et al. , Bio/Technology (1994) 12, 69
9-702). 4. Modified Antibodies In addition to the antibodies described above, the present invention also provides modified antibodies that can reduce heterologous antigenicity to humans.
recombinant antibodies artificially modified for the purpose of
Humanized antibodies can be used. These modified antibodies can be prepared in the following manner:
Chimeric antibodies useful in the present invention can be produced using a method encoding the antibody V region obtained as described above.
The DNA is ligated to DNA encoding the C region of a human antibody, and the resulting DNA is incorporated into an expression vector.
Humanized antibodies can be obtained by incorporating the antibody into a host and introducing it into the host for production. Humanized antibodies are also called reshaped human antibodies, which are
Complementarity determining regions (CDRs) of non-human mammalian, e.g., mouse, antibodies
The human antibody
The general genetic recombination method is also known.
(European Patent Application Publication No. EP 125023, WO 96/025
76). Specifically, the CDR of a mouse antibody and the framework region of a human antibody (frame
A DNA sequence designed to link the fragment (F1) and the fragment (F2) (the working region; FR) was
The CDR and FR regions were designed to overlap each other at both ends.
The resulting oligonucleotides are used as primers to amplify the resulting DNA by PCR.
The resulting DNA was ligated to DNA encoding the C region of a human antibody, and then the resulting DNA was used as an expression vector.
The vector is then introduced into a host, where it is produced to produce a humanized antibody.
It can be obtained (EP 239400, WO 96/02576
(See references). The framework regions of a human antibody linked via CDRs are called complementarity-determining regions (CDRs).
If necessary, a reshaped human antibody may be used.
The complementarity determining regions of the antibody are then attached to the variable region of the antibody so that they form an appropriate antigen-binding site.
The amino acids in the framework regions may be substituted (Sato, K. et al.
I., Cancer Res. (1993) 53, 851-856). The C region of a chimeric antibody or a humanized antibody is derived from a human antibody, for example.
For example, Cγ1, Cγ2, Cγ3, and Cγ4 are used for the H chain, and Cκ and Cλ are used for the L chain.
Furthermore, in order to improve the stability of the antibody or its production, human antibodies can be used.
The C region may be modified. Chimeric antibodies are antibodies that combine the variable region of an antibody derived from a non-human mammal with the constant region of a human antibody.
On the other hand, humanized antibodies are composed of the complementary domains of antibodies derived from mammals other than humans.
It consists of a determining region and a framework region and C region derived from a human antibody.
The antigenicity of the modified antibody in the human body is reduced, and therefore the effectiveness of the inhibitor of the present invention is improved.
It is useful as an active ingredient. Humanized antibodies that can be used in the present invention include humanized #23-57-137-1
The humanized #23-57-137-1 antibody is a mouse-derived #
The complementarity determining regions of the 23-57-137-1 antibody were determined by the human antibody HS for the L chain.
U03868 (GEN-BANK, Deftos M et al., Scand. J. Im
munol., 39, 95-103, 1994)
, FR2 and FR3) and derived from human antibody S25755 (NBRF-PDB)
The H chain is linked to the FR fragment (FR4) of human antibody S316
79 (NBRF-PDB, Cuisinier AM et al., Eur. J. Immu
Nol., 23, 110-118, 1993),
Some amino acid residues in the framework region were replaced to retain antigen-binding activity.
The humanized #23-57-137-1 antibody L chain or H chain is encoded by
E. coli carrying a plasmid containing DNA was developed by the National Institute of Bioscience and Human-Related Technology, Agency of Industrial Science and Technology.
(1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture) on August 15, 1996.
Escherichia coli carrying a plasmid containing DNA encoding
E. coli JM109 (hMBC1HcDNA/pUC19)
M BP-5629 has a plasmid containing DNA encoding the L chain.
Escherichia coli JM109 (hMBC1Lq
λ/pUC19) is FERM BP-5630 under the Budapest Treaty.
Each of these has been internationally deposited under the Patent Application No. 2004-2006-10104. 5. Modified Antibodies The antibodies used in the present invention bind to PTHrP and inhibit its activity.
As long as the antibody fragment is a fragment of an antibody, the antibody fragment may be a fragment of an antibody or a modified antibody thereof.
Fab, F(ab')₂Fv, or Fv of the H chain or L chain,
Examples include single chain Fv (scFv) linked by a linker.
by treating antibodies with enzymes such as papain or pepsin to produce antibody fragments;
Alternatively, genes encoding these antibody fragments may be constructed and introduced into expression vectors.
After transfection, the vector is expressed in an appropriate host cell (see, for example, Co, M.S. et al.
, J. Immunol. (1994) 152, 2968-2976, Bette
r, M. & Horwitz, A. H. Methods in Enzymol
ogy (1989) 178, 476-496, Academic Press,
Inc. , Plueckthun, A. & Skerra, A. Methods
in Enzymology (1989) 178, 476-496, Acad
emic Press, Inc. , Lamoyi, E. ,Methods in
Enzymology (1989) 121, 652-663, Roussea
ux, J. et al. , Methods in Enzymology (19
89) 121, 663-669, Bird, R. E. et al. ,TIBTE
CH (1991) 9, 132-137). scFv is obtained by linking the heavy chain V region and light chain V region of an antibody.
In this scFv, the H chain V region and the L chain V region are linked by a linker, preferably a peptide.
linked via a peptide linker (Huston, J.S. et al., P
roc. Natl. Acad. Sci. U. S. A. (1988) 85,587
9-5883). The H chain V region and L chain V region of the scFv are
The antibody may be derived from any of the antibodies described above.
The peptide linker may be, for example, any single chain consisting of 12 to 19 amino acid residues.
A peptide is used. The DNA encoding the scFv encodes the H chain or H chain V region of the antibody.
DNA encoding the L chain or L chain V region, and DNA encoding the L chain or L chain V region.
The entire sequence or a DNA portion encoding the desired amino acid sequence is used as a template.
The primer pair is then amplified by PCR using a primer pair that defines both ends of the
DNA encoding the peptide linker portion, and its ends corresponding to the H chain and L chain, respectively
It is obtained by amplifying a primer pair that is designed to be linked together.
Once the DNA encoding the scFv is prepared, it is possible to produce a gene encoding the scFv.
The expression vector and the host transformed with the expression vector are obtained by conventional methods.
Furthermore, by using the host, scFv can be obtained according to a conventional method.
These antibody fragments can be obtained by obtaining and expressing the genes in the same manner as described above and injecting them into a host.
The "antibody" in the present invention includes fragments of these antibodies.
Also included are antibodies modified with various molecules such as polyethylene glycol (PEG).
The "antibody" in the present invention includes the following:
These antibody modifications are also included. Such antibody modifications are made by adding chemical modifications to the obtained antibody.
The antibody can be obtained by subjecting it to specific modification.
This has already been established in the field. 6. Expression and production of recombinant or modified antibodies The antibody genes constructed as described above can be expressed and obtained using known methods.
In the case of mammalian cells, useful promoters that are commonly used,
The gene is expressed by functionally linking a poly(A) signal to its 3' downstream.
For example, the promoter/enhancer can be human cytomegalovirus (HCV)
Viral early promoter/enhancer (human cytomegalovirus
irus immediately early promoter/enhance
r). Other promoters/encoders that can be used to express the antibodies used in the present invention include
As a marker, retroviruses, polyomaviruses, adenoviruses, and schizophrenia
Viral promoters/enhancers such as SV40 (Stevens-Van Virus 40)
or mammalian cell-derived factors such as human elongation factor 1α (HEF1α)
Examples of suitable promoters/enhancers include conventional promoters/enhancers. When using the SV40 promoter/enhancer, the method described in Mulligan et al.
(Nature (1979) 277, 108) and HEF1α
When a promoter/enhancer is used, the method of Mizushima et al.
(1990) 18, 5322)
Gene expression can be easily achieved in E. coli. In the case of E. coli, commonly used useful promoters and signal sequences for antibody secretion are
and functionally linking the sequence and the antibody gene to be expressed to express the gene.
Examples of promoters include lacz promoter and araB promoter.
When the lacz promoter is used, the War
(Nature (1098) 341, 544-546; FASEB
J. (1992) 6, 2422-2427) or by araB promoter
When using a ter, the method of Better et al. (Science (1988) 24
0, 1041-1043). As a signal sequence for antibody secretion,
If the pelB signal sequence (Lei, S.P. et al J. Bacter
(1987) 169, 4379).
After isolating the antibodies produced by the enzyme, the antibody structure is appropriately reassembled (ref
ld) are used. Replication origins include SV40, polyomavirus, adenovirus, and bovine
Those derived from human papillomavirus (BPV) and the like can be used.
For gene copy number amplification in primary cell lines, the expression vector is
aminoglycoside transferase (APH) gene, thymidine kinase (TK
) gene, Escherichia coli xanthine guanine phosphoribosyltransferase (Ec
ogpt gene, dihydrofolate reductase (dhfr) gene, etc.
Any expression system, such as eukaryotic cells or
Prokaryotic cell systems can be used. Eukaryotic cells include, for example, established mammalian cells.
Examples of such cells include mammalian cells, insect cells, filamentous fungi, and yeast cells.
Prokaryotic cells include bacterial cells such as Escherichia coli cells. Preferably, the antibodies used in the present invention are produced from mammalian cells, such as CHO and COS cells.
The gene is expressed in myeloma, BHK, Vero, and HeLa cells. The transformed host cells are then cultured in vitro or in vivo.
The host cells are cultured according to known methods to produce the desired antibody.
For example, DMEM, MEM, RPMI1640, and IMDM are used as culture media.
Serum supplements such as fetal calf serum (FCS) can also be used in combination.
7. Antibody isolation and purification The antibodies expressed and produced as described above are isolated from the cells and host animals and purified to homogeneity.
The antibodies used in the present invention can be separated and purified by affinity chromatography.
This can be done using a column. For example, a column using a protein A column
Hyper D, POROS, Sepharose F.F. (Pha
Other examples include those used for ordinary proteins.
Any separation and purification method may be used, and there is no limitation. For example,
Chromatography columns other than affinity columns, filters, and ultrafiltration
The antibody can be separated and purified by appropriately selecting and combining methods such as precipitation, salting out, and dialysis.
(Antibodies A Laboratory Manual
．． Ed Harlow, David Lane, Cold Spring Ha
Roberts Laboratory, 1988. 8. Confirmation of Antibody Activity The antigen-binding activity of the antibodies used in the present invention (Antibodies A Lab
oratory Manual. Ed Harlow, David Lane,
Cold Spring Harbor Laboratory, 1988)
Ligand-receptor binding inhibitory activity (Harada, A. et al., Inter
National Immunology (1993) 5, 681-690)
Known means can be used to measure the antigen binding activity of the anti-PTHrP antibody used in the present invention.
ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (
Radioimmunoassay or fluorescent antibody techniques can be used.
When using the measurement method, the following was added to a plate coated with PTHrP(1-34):
Samples containing anti-PTHrP antibodies, such as culture supernatants of anti-PTHrP antibody-producing cells,
Add purified antibodies. Add secondary antibodies labeled with enzymes such as alkaline phosphatase.
The plate was incubated and washed, and then p-nitrophenyl phosphate or the like was added.
The antigen-binding activity can be evaluated by adding the enzyme substrate and measuring the absorbance.
To confirm the activity of the antibodies used in the present invention, the neutralizing activity of the anti-PTHrP antibody
Measure the amount of PTHrP in the tissue breakdown inhibitor. 9. Administration Method and Formulation The tissue breakdown inhibitor containing the anti-PTHrP antibody of the present invention as an active ingredient is orally administered.
Although either oral or parenteral administration is possible, parenteral administration is preferred.
Pulmonary dosage forms (e.g., pulmonary administration using a device such as a nephrizer) and nasal administration
These include transdermal dosage forms (e.g., ointments, creams), and injection forms.
Examples of injections include intravenous injections such as drip infusions, intramuscular injections, intraperitoneal injections, and skin injections.
It can be administered systemically or locally by subcutaneous injection, etc. Also, depending on the patient's age and symptoms,
The administration method can be selected appropriately depending on the condition.
The dose is selected from the range of 0.001 mg to 1000 mg per kg.
The dosage can be selected from the range of 0.01 to 100,000 mg/body per subject.
However, the inhibitor containing the anti-PTHrP antibody of the present invention is not affected by these dosages.
The diseases to which the inhibitor of the present invention is administered include, but are not limited to, cancer cachexia, pneumonitis, and trauma (severe
These include, but are not limited to, muscular dystrophy, etc.
It also includes cases where multiple of the above diseases have developed or where the disease has been complicated with other diseases.
The inhibitor may be, for example, interleukin-6 (IL-6), granulocyte colony
Ronnie stimulating factor (G-CSF), IL-11, leukocyte inhibitory factor (LIF) and α
₁Acidic glycoprotein (α₁-AG) blood levels higher than normal
In the present invention, the "normal value" is defined as a value between 10 and 15.
The average blood concentration of each drug in 100 healthy individuals, or the commonly published
For example, the clinical test value for IL-6 is 0.2 to 4.
.6 pg/mL, and G-CSF was 7.7-38.9 pg/mL,
L-11 was less than 31.3 pg/mL, and α₁- 42-93 mg/AG
dl. The inhibitor of the present invention should be administered around the time when clinical symptoms of the above-mentioned diseases appear.
It may be administered at any time, or when weight loss is predicted. The inhibitor containing the anti-PTHrP antibody of the present invention as an active ingredient may be administered in a conventional manner.
(Remington's Pharmaceutics
cal Science,latest edition,Mark Publ
Ishiing Company, Easton, USA), a pharmaceutically acceptable carrier
It may contain both a carrier and an additive. Examples of such a carrier and additive include water, pharmaceutically acceptable organic solvents,
Agent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl
Nyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate
sodium alginate, water-soluble dextran, carboxymethyl starch
Sodium, pectin, methylcellulose, ethylcellulose, xanthan gum
, gum arabic, casein, agar, polyethylene glycol, diglycerin,
Glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol
Stearic acid, human serum albumin (HSA), mannitol, sorbitol
Examples of additives include ethanol, lactose, and surfactants acceptable as pharmaceutical additives. The actual additives may be selected from the above individually or in combination as appropriate depending on the dosage form of the inhibitor of the present invention.
The combinations of the above are not limited to, but include, for example, injectable formulations.
When used as a solvent, the purified anti-PTHrP antibody is dissolved in a solvent such as saline,
Dissolved in a buffer solution, glucose solution, etc., and then add an adsorption inhibitor, such as Tween 80,
Use a product containing Tween 20, gelatin, human serum albumin, etc.
Alternatively, it can be freeze-dried to prepare a dosage form that is reconstituted before use.
The excipient for freeze-drying may be, for example, mannitol,
Sugar alcohols such as glucose and other sugars can be used. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be explained in more detail below with reference to examples and reference examples. However, the present invention
The technical scope of the present invention is not limited to these examples. [Example 1] Tissue Degradation Inhibition Test (1) 1. Preparation of Humanized Anti-PTHrP Antibody In this example, an anti-PTHrP antibody against PTHrP(1-34) was used as an example of an anti-PTHrP antibody.
The humanized anti-PTHrP antibody was prepared by genetic recombination techniques as described in Reference Example 4.
The obtained humanized anti-PTHrP antibody (hereinafter referred to as "human") was prepared (L chain version q).
The antibody (hereinafter referred to as "antibody") was dissolved in 20 mmol/L citric acid solution and 1 mol/L Tris
Add the solution to adjust the pH to 6.6 (concentration 6.34 mg/mL), and then heat to -70°C or below.
The antibody used was confirmed to be the intended one by checking the SD of the molecular weight.
The binding was determined by S-PAGE and by an ELISA binding assay.
This was confirmed. After thawing the antibody solution, it was diluted under sterile conditions with Dulbecco's PBS (-) (Nippon Suido Co., Ltd.).
The solution was diluted to 1 mg/mL with PBS (Pharmaceutical Co., Ltd., code. 05913, hereinafter referred to as PBS).
2. Experimental animals The mouse colon cancer line Colon26 or the human oral cavity cancer line OCC-1-JCK was transplanted.
BALB/cAnNCrj mice are commonly used.
Mice (Colon 26 for transplantation) or BALB/cAJcl-nu mice (OC
Thirty male mice (C-1-JCK for transplantation) were used for each experiment.
The mice were selected from those aged 6 weeks at the time of tumor cell transplantation.
j is from Charles River Japan, and BALB/cAJcl-nu is from Claire Japan.
After acclimating the 30 purchased mice for about a week, seven were randomly selected and placed in the control
A group without tumor transplantation was prepared as a normal group. The remaining 23 mice were transplanted with tumors.
After 7 days from the transplantation, tumor engraftment was confirmed.
Twenty mice were selected. The mice were then averaged based on their body weight.
The mice were divided into two groups, 10 mice per group, administered with PBS and the humanized antibody of the present invention. The mice were kept in the following environment: Room temperature: 24±2°C Relative humidity: 55±10% Ventilation rate: 10-30 times/hour Lighting hours: 14 hours Rearing cage: M-4 cage (215 x 320 x 130 mm, polycarbonate)
(Product manufactured by CLEA Japan, Inc., with bedding) Breeding density: 5-7 mice/cage Feeding method: CE-2 (CLEA Japan, Inc.), ad libitum Drinking water method: Tap water, ad libitum water from a 200 mL water bottle 3. Experimental method 3-1. Creation of disease model Tumor lines used: Colon26 (mouse colon cancer line), OCC-1-JCK (
Human oral floor cancer cell line) Tumor cell line origin: Colon 26 (Japanese Cancer Research Association Chemotherapy Center), OCC-1-JC
K (Central Institute for Experimental Animals) Tumor line passage: Colon 26 was cultured in BALB/cAnNCrj.
OCC-1-JCK was subcultured in vivo every 2 weeks.
Colon 26, OCC.
The mouse model transplanted with α-1-JCK was prepared as follows:
On the day of transplantation, the mice used for in vivo passage were cervically dislocated, and the tumor was removed.
The tumor was then transplanted subcutaneously into mice.
Mice with tumors were used as established model mice. 3-2. Measurement of blood ionized calcium concentration (blood iCa concentration) Approximately 60 μL of blood was collected from the orbit and immediately analyzed for 643Ca++/pH analytes.
Measurements were made using a meter (Chiron). 3-3. Measurement of serum biochemical values (glucose, triglycerides, cholesterol)
Under anesthesia, approximately 1 mL of blood was withdrawn from the inferior vena cava into a Separator Pit tube (Sekisui Chemical Co., Ltd.).
After collecting the sample in a refrigerator (Kanagawa Prefecture, Ltd.), the sample was left to stand at room temperature for 30 minutes or more.
The serum was separated using a 20-minute syringe (HITACHI, 05PR-22) and then incubated at -20°C.
The samples were frozen and stored at the following temperature. Serum biochemical values (glucose, triglycerides, cholesterol) were measured by autoanalysis.
Measurements were performed using a Hitachi 7170 automatic analyzer. 3-4. Administration of humanized antibodies Humanized antibodies were administered intravenously (i.v.) at a dose of 0.1 mg/mouse.
In addition, Dulbecco's PBS (-
The PBS-administered group received 10 mg of 10 ...
The normal group, which did not receive tumor transplants, received no treatment. The mouse group composition and administration schedule are outlined in Table 1. The outline of the efficacy test is as follows: Test items: body weight, adipose tissue (around the testes) weight, muscle tissue (gastrocnemius) weight, serum
Glucose level, serum triglyceride level, serum cholesterol level, blood iCa concentration
, general condition In OCC-1-JCK-implanted mice, body weight was 7 and 10 days after tumor implantation.
The blood iCa levels were measured on the 7th and 13th days.
Other parameters were measured on day 13. In Colon 26-implanted mice, body weight was measured on days 7 and 10 after tumor implantation.
The blood iCa concentration was measured on the 7th and 14th days.
Other items were measured on day 14. Efficacy evaluation: Suppression of loss of body weight, fat tissue, and muscle tissue weight by administration of humanized antibody
The effects of the drug on blood iCa levels, serum glucose levels, and serum triglycerides were also investigated.
We also investigated the effect of restoring serum cholesterol levels to near-normal levels.
3-5. Examination of the Effects of Humanized Antibodies Comparison Control Groups: PBS-Administered Group and Humanized Antibody-Administered Group Hypothesis Tested: Compared to the PBS-Administered Group, the humanized antibody-administered group had lower body weight and adipose tissue.
Weight, muscle tissue weight, blood iCa concentration, serum glucose level, serum triglyceride
Changes in serum cholesterol levels. Statistical analysis methods used: body weight, adipose tissue weight, muscle tissue weight, blood iCa concentration
, serum glucose levels, serum triglyceride levels, serum cholesterol levels,
The normal group and the PBS-administered group were tested by t-test, and for those with significant differences, P
The significance between the BS-administered group and the humanized antibody-administered group was examined by t-test.
The significance level was set at 5% on both sides. SAS was used for these analyses. 4. Results 4-1. Effects in OCC-1-JCK-transplanted mice Effects on body weight, blood iCa concentration, and general condition (Figure 1) Body weight: The PBS-administered group showed a significant decrease in body weight compared to the control group (after tumor transplantation).
12 and 13 days, *: p<0.05 compared to the normal group, tested by t-test
The weight loss in the humanized antibody-administered group was significantly suppressed compared to the PBS-administered group.
(#: p<0.05 compared to the PBS-administered group, tested by t-test)
), and maintained a weight similar to that of the normal group (weight (g) on the 13th day after transplantation; normal
Normal group 24.64±0.61, PBS-administered group 19.37±0.59, humanized antibody
Treatment group: 24.11±0.47) (shown as mean±SE; same below). Blood iCa concentration: A significant increase was observed in the PBS-treated group compared to the normal group.
(13 days after tumor inoculation (*)). In the humanized antibody administration group, compared with the PBS administration group,
The increase in blood iCa concentration was significantly suppressed (#) (blood iCa concentration on the 13th day after transplantation)
(mmol/L); normal group 1.36±0.01, PBS-administered group 2.73±0.1
0, humanized antibody group 1.62±0.01). General condition: Macroscopically, the PBS-administered group showed a worsening of the general condition compared to the normal group.
Improvement in overall condition was observed in the humanized antibody-treated group. Effects on adipose tissue (around the testes) and muscle tissue (gastrocnemius) weight (Table 2) Adipose tissue weight: A significant decrease was observed in the PBS-administered group compared to the normal group (*
The decrease was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group.
(#). Muscle tissue weight: A significant decrease was observed in the PBS-administered group compared to the normal group (*
The decrease was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group.
(#). Effects on serum biochemical values (glucose, triglycerides, cholesterol) (
Table 2) The PBS-administered group showed significant increases in glucose, triglycerides, and cholesterol compared to the normal group.
A decrease in sterol concentration was observed (*). In the humanized antibody administration group, PBS administration
Although there was a tendency for these declines to be suppressed compared to the control group, there was a significant improvement effect.
This was only observed in triglycerides (#). 4-2. Effects in Colon 26-implanted mice Effects on body weight, blood iCa concentration, and general condition (Figure 2) Body weight: The PBS-administered group showed a significant decrease in body weight compared to the control group (10 days after tumor implantation).
From the day onwards, *: p<0.05 compared to the normal group, tested by t-test.
The humanized antibody-administered group showed significantly less weight loss than the PBS-administered group.
Controlled (#: p<0.05 compared to the PBS-administered group, tested by t-test)
The body weight (g) on the 14th day after transplantation was 26.78±0.38 in the normal group and 26.78±0.38 in the PBS group.
The mean values were 19.04±0.35 in the treatment group and 21.51±0.32 in the humanized antibody treatment group.
Blood iCa concentration: A significant increase was observed in the PBS-administered group compared to the normal group.
However, the humanized antibody administration group showed a significantly higher tumor density compared to the PBS administration group (14 days after tumor inoculation (*)).
The increase in blood iCa concentration (mmol) on the 14th day after transplantation was significantly suppressed (#).
l/L) was 1.34±0.01 in the normal group, 1.94±0.09 in the PBS-administered group, and
The mean age of the patients receiving the PBS-treated group was 1.54±0.03. General condition: Macroscopically, the general condition of the patients receiving the PBS-treated group was worse than that of the control group.
In the humanized antibody group, a slight improvement in the deterioration of the overall condition was observed.
Effects on adipose tissue (around the testes) and muscle tissue (gastrocnemius) weight (Table 3) Adipose tissue weight: A significant decrease was observed in the PBS-administered group compared to the normal group (
*) The decrease was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group.
(#). Muscle tissue weight: A significant decrease was observed in the PBS-administered group compared to the normal group (
*) The decrease was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group.
(#). Effects on serum biochemical values (glucose, triglycerides, cholesterol) (Table
3) Serum glucose concentrations were significantly lower in the PBS-administered group compared to the normal group.
However, (*) no improvement was observed with the administration of the humanized antibody.
The phosphate concentration was significantly lower in the PBS-administered group compared to the normal group, and
Although not significant, the decrease in serum cholesterol was suppressed.
No changes were observed in the BS-administered group or the normal group, nor in the humanized antibody-administered group.
4-3. Summary Cancer cachexia is a condition in which cancer patients experience significant weight loss due to loss of appetite and hypermetabolism.
It is a clinical syndrome characterized by low blood glucose and hyperlipidemia.
There are several tumors that induce cancer cachexia in tumor transplant models.
Representative tumor lines include SEKI melanoma (human melanoma), Col
on 26 (mouse colon cancer cell line), OCC-1-JCK (human oral floor cancer cell line)
When these tumors were transplanted into nude mice, significant weight loss was observed.
On the other hand, a significant weight loss similar to that seen in cancer cachexia was observed in the HHM model.
PTHrP has been identified as the causative agent of HHM (L.J. Suv
a, et al., Science 237, 893-896, 1987). Therefore, the present inventors investigated the human oral cavity cancer cell line OCC-1-JCK and mouse colon cancer cell line OCC-1-JCK.
We created a cancer cachexia model by transplanting two types of tumor lines, the cancer cell line Colon 26.
The effects of humanized antibodies were examined. OCC-1-JCK transplanted mice were treated with Colon 26 transplantation from 10 days after tumor transplantation.
The mice showed a significant weight loss (and adipose tissue weight and muscle tissue weight) from 7 days after tumor implantation.
The patient was in a state of low glucose and had a clinically significant decrease in cancer cachexia.
These models also showed high-quality characteristics.
This revealed that HHM occurred in conjunction with cancer cachexia. When humanized antibodies were administered to the two types of model mice mentioned above, body weight and blood iCa levels increased.
Significant improvements were observed not only in the concentration but also in the fat and muscle tissue weights.
Furthermore, OCC-1-JCK transplanted mice showed improvement in hypotriglycerides.
The effect was observed, and although not significant, a tendency for improvement in hypoglycemia was observed. In the present invention, the humanized anti-PTHrP antibody has the ability to inhibit the activity of PTHrP in adipose tissue and muscle tissue.
This indicates that the inhibitory effect on tissue degradation associated with various diseases such as cancer cachexia is
This compound is useful as a therapeutic or preventive agent for HHM. [Example 2] Tissue Degradation Inhibition Test (2) 1. Humanized Anti-PTHrP Antibody The humanized anti-PTHrP antibody prepared in Example 1 was used. 2. Experimental Animals Human tumor cell lines can be transplanted, allowing the creation of a stable HHM model.
As a rat that can be used, nude rat F344/N Jcl-rnu (Nippon Cre)
Eighty rats purchased at 5 weeks of age were acclimated for about a week.
Afterwards, 55 randomly selected mice were transplanted with tumors. Euthanasia was performed by ether inhalation.
The rats were kept in the following conditions: Room temperature: 24±2°C Relative humidity: 55±10% Ventilation rate: 10-30 times/hour Lighting hours: 5:00-19:00 Cages and breeding density: From the time of acclimation and tumor implantation until the start of the experiment, rats were kept in a stable cage.
Stainless steel wire mesh hanging cage (dimensions 660 x 310 x 200 mm) per cage
They were kept in groups of 8 to 11 and kept in polycarbonate cages (265 x 42 cm) during the experiment.
The mice were housed in cages measuring 5 x 160 mm, with one mouse per cage. Diet and feeding method: CE-2 (CLEA Japan, Inc.), available ad libitum. Drinking water and water supply method: Tap water, available ad libitum. Daily care: In accordance with the Laboratory Animal Care Standards (compiled by Chugai Pharmaceutical Laboratory Animal Care Committee).
3. Experimental Method 3-1. Creation of Disease Model Tumor Line Used: LC-6-JCK (Human Large Cell Lung Carcinoma Line) Origin of Tumor Line: Purchased from the Central Institute for Experimental Animals. Subculture of Tumor Line: Mice (BALB/cA Jcl-nu; CLEA Japan, Inc.)
Tumor blocks were subcutaneously transplanted into mice and passaged as in vivo tumor lines.
Subculture was performed every 3 to 4 weeks. Model preparation: Mice in which tumor lines were subcultured were cervically dislocated to remove the tumors.
The tumor was cut into 3 mm square blocks and implanted subcutaneously in rats.
The transplant was performed on 55 rats randomly selected from 80 rats. The remaining 25 rats
No treatment was performed. Model validity criteria: The transplanted tumor is growing macroscopically under the skin, and the blood i
The HHM model was established by showing higher Ca concentrations than the normal group.
Animals with a iCa concentration of 1.80 mmol/L or higher were used. Grouping method: Blood iCa concentration was measured 49 days after tumor transplantation to establish the HHM model.
The HHM model was stratified using the blood iCa concentration as an index, and
24 animals were randomly selected and administered saline to ensure there was no difference between the groups.
The mice were divided into three groups (8 mice/group): a group administered with the humanized antibody, and a group receiving tumor removal.
Eight rats were randomly selected from the 25 untreated rats to serve as the normal group. 3-2. Measurement of blood ionized calcium concentration (blood iCa concentration) Blood was collected from the tail vein and immediately analyzed using a calcium analyzer (automated pH/Ca
⁺⁺Measurements were made using an analyzer, Chiron 634 (Bayer Medical). 3-3. Serum collection and biochemical values (glucose, triglycerides, cholesterol)
rol, free fatty acids), cytokines (human IL-6, human G-C
SF, humanIL-11, humanLIF) and rat α₁-AG measurement
Serum was collected by dissolving blood in a separapit tube (Sekisui Chemical Co., Ltd.) from the descending aorta under anesthesia.
The samples were collected in a container (Corporation) and left to stand at room temperature for 30 minutes or more.
The serum was separated using a 20-minute incubation (Hitachi, 05PR-22).
The samples were frozen and stored at 0°C or below. Serum biochemical values (glucose, triglycerides, cholesterol, free fatty acids)
was measured using an autoanalyzer (Hitachi, 7170 model). Cytokines (human IL-6, human G-CSF, human IL
-11, human LIF) and rat α₁-AG was measured using commercially available E
Quantikine® human IL-6 (R&D) kit
systems), Quantikine® human G-CSF (
R&D systems), Quantikine registered trademark human IL
-11 (R&D systems), LIF/HILDA EASIA/K2 (
BIOSOURCE), Panatest Registered Trademark A Series Rat α₁-AG (
White blood cell count was measured using a Panapharm Laboratories. 3-4. White blood cell count 20 μL of blood was collected from the tail vein and immediately suspended in a Cell Pack (PL30L).
Then, Sysmex Microcell counter F-80
The white blood cell count was measured using a Shimazu 0. 3-5. Measurement of adipose tissue and muscle tissue weight Adipose tissue (around the testes) and muscle tissue (gastrocnemius) were removed and weighed.
Shimazu Libror EB-330H with EP-50 printer
The weight was measured. 3-6. Administration of Humanized Antibody The humanized antibody was administered intravenously (i.v.) at a dose of 3.0 mg/kg.
The negative control group was administered saline i.v.
(Saline administration group). The administration solution was administered at a volume of 0.1 mL per 100 g of body weight.
The normal group, which did not receive tumor transplants, received no administration. Tumor removal: A vinyl string (tie band) was wrapped around the skin at the base of the tumor.
The tumor was then tied with a band (used for bacterial bags) (tumor removal group). A few hours later and the next day, the band was tied again.
The animals were then re-tied. The next day, it was confirmed that the tumors had become necrotic. Test Schedule: On the 49th day after tumor transplantation, body weight, blood iCa concentration, and white blood cell count were measured.
Based on the results, the mice were divided into groups.
The groups were administered saline and humanized antibodies, respectively, and the tumor removal group
The tumor was removed. The body weight, blood iCa concentration, and
On the 10th day after administration, the weight of the adipose tissue and muscle tissue were measured.
Blood and serum samples were also collected and serum biochemical values and cytokines were measured.
3-7. Statistical Analysis Method The difference in the mean values for each measurement day between the normal group and the saline-administered group was analyzed unpaired.
The test was conducted using a t-test. First, the variance was tested using an F-test. If the F-value was 5% or more,
is equal variance, Student's t-test, if F value is 5% or less, it is unequal
Welch's t-test was used as a measure of statistical significance, with a two-sided significance level of 5%.
The saline administration group and the humanized antibody administration group or tumor removal group were compared.
The difference in the average values for each fixed day was tested using Dunnett's multiple comparison test, and the significance level was
The statistical analysis was performed using SAS Ver. 6.12.
4. Results 4-1. Effects on Body Weight and Blood iCa Concentration (Figure 3) Body weight (Figure 3, upper panel): In the saline-treated group (-■-), body weight was significantly lower on all measurement days.
The weight of the subjects was significantly decreased compared to the normal group (-▲-) (p < 0.05 compared to the normal group).
<0.05, tested by t-test.) Humanized antibody administration group (-●-)
In the group administered saline, the body weight increased significantly from the fourth day after administration.
A significant difference was observed up to the 10th day (#: p<0.05 compared to the saline-administered group).
.05, tested by Dunnett's multiple comparison. ) Tumor removal group (-◆-)
However, a similar level of weight recovery was observed, and the recovery rate was 4, 7, and 9 days after administration compared with the saline-treated group.
A significant difference was observed on the 10th day (#). Blood iCa concentration (Figure 3, lower panel): In the saline-administered group,
In the humanized antibody administration group, saline
Compared with the treatment group, the increase in blood iCa concentration was significant on days 1, 4, 7, and 10 after administration.
In the tumor-excised group, the tumor growth rate was suppressed at 1, 4, 7, and 8 days after administration compared to the saline-administered group.
The increase in blood iCa concentration was suppressed on day 10 (#). 4-2: Effects on adipose tissue (peri-testicular) and muscle tissue (gastrocnemius) weight (Table 4) Adipose tissue weight: A significant decrease was observed in the saline-treated group compared to the control group.
(*) In the humanized antibody administration group and the tumor removal group, compared with the saline administration group,
The decrease was significantly suppressed (#). Muscle tissue weight: A significant decrease was observed in the saline-administered group compared to the normal group.
(*) In the humanized antibody administration group and the tumor removal group, compared with the saline administration group,
The decrease was significantly suppressed by administering the supplement (#). 4-3. Serum biochemical values (glucose, triglycerides, cholesterol, free
Effects on glucose and free fatty acids (Table 4) The saline-treated group had significantly higher glucose and free fatty acid concentrations than the control group.
A decrease in blood cholesterol and an increase in cholesterol were observed in the humanized antibody group.
A significant decrease in cholesterol was observed in the group administered saline (
However, no significant improvement was observed in other parameters.
showed an improvement in glucose and cholesterol levels (#).4-4. Cytokines (hIL-6, hG-CSF, hIL-11, hLIF)
) and rα₁-Effects on AG (Table 5) The saline-treated group showed significant increases in hIL-6, hG-CSF, and
Increases in hIL-11 and hLIF were observed (*).
rα induced by kinases (hIL-6, hIL-11, LIF, etc.)₁-A
An increase in α1-acid glycoprotein (G) was also observed.
In the tumor removal group, no decrease in serum cholesterol was observed compared with the saline administration group.
The levels of hIL-6, hG-CSF, hIL-11 and hIL-11 were significantly higher in the IFN-γ-treated group than in the IFN-γ-treated group.
A decrease in inflammatory cytokines (hIL-6, hIL-1) was observed.
1, LIF, etc.)₁-A decrease in AG was also observed. Effects on white blood cells (Figure 4) The saline-treated group had significantly higher white blood cell counts than the normal group on all measurement days.
The results showed an increase (p<0.05 compared to the normal group, tested by t-test).
The white blood cell count in the treatment group was significantly reduced on the fourth day after administration compared to the saline treatment group.
(#, significance level: 5%, tested by Dunnett's multiple comparison), but other measurement dates
In the tumor removal group, there was no significant difference on days 1, 4, 7, and 10 after administration.
A significant decrease was observed (#). #: Significant difference compared to the saline-treated group. 6. Summary The present inventors conducted a randomized controlled trial of cancer cachexia in rats transplanted with the human large cell lung cancer line LC-6-JCK.
A model was created and the effects of humanized antibodies were compared with those observed in tumor-excised rats. After tumor transplantation, LC-6-JCK-implanted rats developed hypercalcemia accompanied by significant weight loss.
A model was established in which HHM developed along with cancer cachexia.
The reduction in fat and muscle tissue weight and the hypoglycemic state are clinically
Furthermore, tumor-derived cytokines and their induced proteins were also detected.
When the humanized antibody was administered to the above model rats, not only did the body weight and blood iCa concentration increase, but
Furthermore, significant improvements were observed in fat tissue weight and muscle tissue weight.
Furthermore, although not significant, a tendency for improvement in hypoglycemia was observed.
Inhibitory effect on the production of tumor-derived cytokines and their derived proteins, and suppressive effect on leukocytes
On the other hand, when the tumor was removed, the body weight, blood iCa concentration, and fat mass were at the same levels as those observed after antibody administration.
Significant improvements in tissue weight and muscle tissue weight were observed.
The effect of improving high cholesterol was also observed.
The humanized anti-PTHrP antibody of the present invention has been shown to have an inhibitory effect on the production of PTHrP and its derived proteins, as well as an inhibitory effect on the increase in white blood cells.
This indicates that the inhibitory effect on tissue degradation associated with various diseases such as cancer cachexia is
These results suggest that tumor-derived cytokines may be useful as a therapeutic agent for
Since the production of IL-6, G-CSF, IL-11, and LIF is not suppressed,
, α₁Even when the blood concentration of cytokines such as -AG is high, the PT of the present invention
Substances that inhibit the binding of HrP to its receptorofWhen administered, it can improve tissues associated with various diseases.
In particular, we found that the degradation of IL-6, IL-11, LIF, etc. can be suppressed.
Inflammatory cytokines are said to be involved in tissue degradation (e.g., Cl
(Initial Science, 89, 431-439, 1995)
Nevertheless, even in the presence of high concentrations of such cytokines, the PT
If we administer a substance that inhibits the binding of HrP to its receptor, we can prevent the formation of tissues associated with various diseases.
We found that this compound can inhibit tissue degradation. [Reference Example 1] Creation of a hybridoma producing an anti-PTHrP (1-34) mouse monoclonal antibody
Hybridoma producing a monoclonal antibody against human PTHrP (1-34)
#23-57-154 and #23-57-137-1 were prepared as follows:
(Sato, K. et al., J. Bone Miner. Res. 8, 84
9-860, 1993). The amino acid sequence of human PTHrP (1-34) is
The antibody is shown in SEQ ID NO: 75. For use as an immunogen, PTHrP(1-34) (Peninsula
The carrier protein thyroglobulin was then reacted with carbodiimide (Doji
PTHrP(1-34) bound to thyroglobulin
The mixture was dialyzed to a protein concentration of 2 μg/ml, and then diluted with furoin.
The mixture was mixed with an adjuvant (Difco) at a ratio of 1:1 to prepare an emulsion.
Female BALB/C mice were given 100 μg of the drug subcutaneously or intraperitoneally on the back of each animal.
The first immunization was with Freund's complete adjuvant, and the second and subsequent immunizations were with Freund's complete adjuvant.
Freund's incomplete adjuvant was used for immunization. The antibody titer in the serum of immunized mice was measured as follows:
Blood was collected from the tail vein of the mouse, and the serum was separated. After that, the antiserum was diluted with RIA buffer and then mixed with 12
5I-labeled PTHrP (1-34) was mixed and the binding activity was measured.
PTHrP (1-3) not conjugated to a carrier protein was injected into the abdominal cavity of the mice.
4) was administered to each animal as a final immunization at a dose of 50 μg. On the third day after the final immunization, the mice were sacrificed, and the spleens were removed. The spleen cells and mouse myelin were then extracted.
The human cell line P3x63Ag8U.1 was cultured in 50% polyethylene glycol 4000.
Cell fusion was carried out according to the standard method used in the past. The fused cells were collected at a concentration of 2 × 10⁴/Well's
The cells were plated onto 85 96-well plates. Hybridomas were selected using HAT
Hybridoma screening was performed using the culture medium. Hybridoma screening was performed by culturing the culture medium in wells where growth was observed in HAT medium.
The culture supernatant was analyzed for the presence or absence of PTHrP-recognizing antibodies using the solid-phase RIA method.
Hybridomas were collected from wells where antibody binding was confirmed, and
RPMI-1640 medium containing 100% FCS and OPI-supplement (Si
The hybridomas were then suspended in a medium containing α-gma and subjected to limiting dilution to isolate the hybridomas.
Clone #23-57-15, which has strong binding ability to PTHrP (1-34)
Hybridoma clone #23-57-137-1 was obtained from mouse-m
ose hybridoma #23-57-137-1, Agency of Industrial Science and Technology
In August 1996, the Institute of Bioscience and Human-Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture)
On the 15th, it was deposited internationally under the Budapest Treaty as FERM BP-5631.
Reference Example 2: Mouse monoclonal antibody against human PTHrP (1-34)
Cloning of DNA encoding the V region of the human PTHrP (1-34) mouse monoclonal antibody #23-57
The DNA encoding the variable region of -137-1 was cloned as follows.
(1) Preparation of mRNA mRNA from hybridoma #23-57-137-1 was purified using QuickPro
ep mRNA Purification Kit (Pharmacia B
Hybridoma #23-57-137-1 was prepared using a recombinant human IgG1 gene (manufactured by iotech).
The cells were thoroughly homogenized in the extraction buffer and extracted according to the instructions provided with the kit.
mRNA in ligo(dT)-Cellulose Spun Column
The mRNA was purified and precipitated with ethanol.
(2) Preparation and Amplification of cDNA for the Gene Encoding the Mouse H Chain V Region (i) Cloning of the #23-57-137-1 Antibody H Chain V Region cDNA The cDNA encoding the H chain V region of a mouse monoclonal antibody against human PTHrP
The gene was cloned using the 5'-RACE method (Frohman, M.A. et al.
al. , Proc. Natl. Acad. Sci. USA, 85, 8998-9
002, 1988; Belyavsky, A. et al. , Nucleic
Acids Res. 17, 2919-2932, 1989).
For the '-RACE method, the 5'-Ampli FINDER RACE kit (CL
The procedure was carried out according to the instructions attached to the kit.
The primers used for NA synthesis are hybrids with the mouse H chain constant region (C region).
The MHC2 primer (SEQ ID NO: 1) was used.
Approximately 2 μg of the mRNA was used as a template, and 10 pmoles of MHC2 primer was added.
Reverse transcription to cDNA was carried out by reacting with transcriptase at 52°C for 30 minutes.
After hydrolyzing the RNA with 6N NaOH (65°C, 30 minutes),
The cDNA was purified by precipitation with T4 RNA ligase at 37°C for 6 hours and at room temperature.
After 16 hours of reaction, Ampli FI was added to the 5' end of the synthesized cDNA.
NDER Anchor (SEQ ID NO: 42) was ligated. PCR was performed using this as a template.
Anchor primer (SEQ ID NO: 2) was used as a primer for amplification by
and MHC-G1 primer (SEQ ID NO: 3) (S.T. Jones, et al.
l., Biotechnology, 9, 88, 1991) was used. The PCR solution was 50 μl of 10 mM Tris-HCl (pH 8.3).
, 50mM KCl, 0.25mM dNTPs (dATP, dGTP, dCTP
, dTTP), 1.5mM MgCl₂, 2.5 units of TaKaRa Ta
q (Takara Shuzo), 10 pmoles of Anchor primer, and MHC-G1
Primer and Ampli FINDER Anchor linked cDNA
The reaction mixture contained 1 μl. This solution was overlaid with 50 μl of mineral oil. PCR was performed
Thermal Cycler Model 480J (Perkin Elm
er) at 94°C for 45 seconds, 60°C for 45 seconds, and 72°C for 2 minutes.
The temperature cycle was repeated 30 times. (ii) Cloning of the cDNA for the L chain V region of antibody #23-57-137-1 Encoding the L chain V region of a mouse monoclonal antibody against human PTHrP
The gene was cloned using the 5'-RACE method (Frohman, M.A. et al.
al. , Proc. Natl. Acad. Sci. USA 85,8998-9
002, 1988; Belyavsky, A. et al. , Nucleic
Acids Res. 17, 2919-2932, 1989).
For the '-RACE method, 5'-Ampli FinderRACE Kit (Clo
The procedure was carried out according to the attached instructions.
The primer used was an oligo-dT primer.
Approximately 2 μg of DNA was used as a template, and oligo-dT primer was added.
Reverse transcription to cDNA was carried out by reacting at 2°C for 30 minutes.
After hydrolysis of RNA with OH (65°C, 30 min), the RNA was precipitated by ethanol.
The DNA was purified. The Ampli FINDE was added to the 5' end of the synthesized cDNA.
R Anchor was reacted with T4 RNA ligase at 37°C for 6 hours and at room temperature for 16 hours.
The PCR primer MLC (SEQ ID NO: 4) was derived from the conserved sequence of the mouse L chain λ chain constant region.
) was designed and synthesized using a 394 DNA/RNA Synthesizer (ABI).
The PCR solution was prepared by adding 100 μl of 10 mM Tris-HCl.
l (pH 8.3), 50mM KCl, 0.25mM dNTPs (dATP,
dGTP, dCTP, dTTP), 1.5Mm MgCl₂, 2.5 units
AmpliTaq (PERKIN ELMER), 50 pmole of Anchor
r primer (SEQ ID NO: 2), and MLC (SEQ ID NO: 4) and Ampli
Contains 1 μl of FINDER Anchor-ligated cDNA reaction mixture
This solution was overlaid with 50 μl of mineral oil. PCR was performed using Thermal Cycle.
r Model 480J (Perkin Elmer) was used, and the temperature was 94°C for 45 minutes.
The temperature cycle was repeated 35 times, with the temperature at 60°C for 45 seconds and at 72°C for 2 minutes.
(3) Purification and fragmentation of PCR products The DNA fragments amplified by PCR as described above were purified using 3% NuSev
e Agarose using GTG agarose (FMC Bio. Products)
The H chain V region was approximately 550 bp long, and the L chain V region was approximately 550 bp long.
The agarose piece containing the DNA fragment of about 550 bp was excised and subjected to GENE
Using the CLEAN II Kit (BIO101), D was prepared according to the instructions attached to the kit.
The purified DNA was precipitated with ethanol and then diluted with 10 mM
The fragment was dissolved in 20 μl of Tris-HCl (pH 7.4) and 1 mM EDTA solution.
1 μl of the resulting DNA solution was digested with the restriction enzyme XmaI (New England Bio
labs) at 37°C for 1 hour, and then digested with the restriction enzyme EcoRI (Takara Shuzo)
The digestion mixture was then digested with phenol and chloroform at 37°C for 1 hour.
The DNA was extracted with PEG and recovered by ethanol precipitation. This resulted in a fragment containing an EcoRI recognition sequence at the 5'-end and an XmaI recognition sequence at the 3'-end.
D containing genes encoding mouse H chain V region and L chain V region having a recognition sequence
The NA fragment was obtained. The genes encoding the mouse H chain V region and L chain V region prepared as described above were
The EcoRI-XmaI DNA fragment containing the gene and the
The pUC19 vector prepared by the above procedure was ligated using a DNA ligation kit.
ver. 2 (Takara Shuzo) was used, and the reaction was carried out at 16°C for 1 hour according to the attached instructions.
Next, 10 μl of the ligation mixture was transferred to E. coli JM109 competent cells (Nicholson et al.
The cells were incubated on ice for 15 minutes and at 42°C for 1 minute.
Then, 300 μl of SOC medium (Molecule
lar Cloning: A Laboratory Manual, Sam
Brook, et al. , Cold Spring Harbor Labo
Cell Culture Press, 1989) and incubate at 37°C for 30 minutes.
After that, 100 μg/ml or 50 μg/ml ampicillin, 0.1 mM
LB agar medium or 2xYT agar containing IPTG and 20 μg/ml X-gal
Medium (Molecular Cloning: A Laboratory Medium
annual, Sambrook, et al. , Cold Spring Ha
The E. coli was then spread onto a plate (Borbor Laboratory Press, 1989).
The transformants were then incubated at 37°C overnight to obtain E. coli transformants. These transformants were then cultured in a broth containing 100 μg/ml or 50 μg/ml ampicillin.
The cells were cultured overnight at 37°C in 2 ml of LB medium or 2xYT medium.
Plasmid extractor PI-100Σ (Kurabo) or QIAprep Spin
Plasmid DNA was prepared using a Plasmid Kit (QIAGEN).
The base sequence was determined. (4) Determining the base sequence of the gene encoding the mouse antibody V region The base sequence of the cDNA coding region in the above plasmid was determined using Dye Terminator.
ator Cycle Sequencing kit (Perkin-Elm
er) and DNA Sequencer 373A (ABI Perkin
The sequencing primers were M13 Prime and M13 RI.
er M4 (Takara Shuzo) (SEQ ID NO: 5) and M13 Primer RV (Takara Shuzo)
) (SEQ ID NO: 6) was used to confirm the base sequence in both directions and determine the sequence.
Mice derived from the hybridoma #23-57-137-1 thus obtained
The plasmid containing the gene encoding the H chain V region was MBC1H04, and the plasmid containing the gene encoding the L chain V region was MBC1H05.
The plasmid containing the gene encoding the region was designated MBC1L24.
Mouse #23-57- contained in plasmids MBC1H04 and MBC1L24
The base sequences of the genes encoding the H chain V region and L chain V region of the 137-1 antibody (
The corresponding amino acid sequences are shown in SEQ ID NOs: 57 and 65, respectively.
The amino acid sequence of the H chain V region fragment is SEQ ID NO: 46, and the L chain V region fragment is SEQ ID NO: 47.
is shown in SEQ ID NO: 45. Note that E. coli carrying the plasmids MBC1H04 and MBC1L24
Escherichia coli JM109 (MBC1H04) and Es
Cherichia coli JM109 (MBC1L24)
In 1996, the Institute of Bioscience and Human Technology, Faculty of Medicine (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture)
On August 15, Escherichia coli JM109 (MBC1H0
4) FERM BP-5628, Escherichia coli
JM109 (MBC1L24) is branded as FERM BP-5627.
It has been internationally deposited under the Dapest Treaty. (5) Mouse monoclonal antibody #23-57-137 against human PTHrP
Determination of CDRs of H-1 The general structures of the H chain V region and L chain V region are similar to each other,
Each of the four framework regions is divided into three hypervariable regions, i.e., complementarity-determining regions.
The amino acid sequence of the framework is relatively well-defined.
The amino acid sequences of the CDR regions are highly conserved, whereas the amino acid sequences of the CDR regions are highly variable (
Kabat, E. A. et al. , “Sequence of Protei
ns of Immunological Interest” US Dept.
Health and Human Services, 1983). Based on these facts, a mouse monoclonal antibody against human PTHrP was developed.
The amino acid sequence of the variable region of the antibody prepared by Kabat et al.
The CDR regions were identified by examining the homology with the database shown in Table 6.
The amino acid sequences of CDR1 to 3 in the L chain V region were determined as follows:
The amino acid sequences of CDR1 to CDR3 of the H chain V region are shown in Table 5.
These are shown in SEQ ID NOs: 62 to 64. [Reference Example 3] Construction of Chimeric Antibody (1) Construction of Chimeric Antibody H Chain (i) Construction of H Chain V Region To ligate into an expression vector containing genomic DNA of the human H chain C region Cγ1,
The cloned mouse H chain V region was modified by PCR.
MBC1-S1 (SEQ ID NO: 7) encodes the 5'-side of the leader sequence of the V region
The DNA hybridizes to the Kozak consensus sequence (Kozak, M
．． et al. , J. Mol. Biol. , 196, 947-950, 1987
) and the recognition sequence for the restriction enzyme Hind III.
The DNA sequence encoding the 3'-side of the J region is the MBC1-a (SEQ ID NO: 8).
and recognizes the splice donor sequence and the restriction enzyme BamHI.
PCR was performed using TaKaRa Ex Taq (Takara Shuzo).
0.07 μg of plasmid DNA was added to a 50 μl reaction mixture.
The primers used were MBC1H04, MBC1-a and MBC1-S1, and
50 pmole, 2.5 U of TaKaRa Ex Taq, 0.25 mM
Using the attached buffer containing dNTP, 50 μl of mineral oil was layered on top, and the mixture was incubated at 94°C.
The temperature cycle was repeated 30 times at 1 minute, 55°C for 1 minute, and 72°C for 2 minutes.
The DNA fragments amplified by PCR were then transferred to 3% Nu Sieve GTG agarose gel.
(FMC Bio. Products) by agarose gel electrophoresis.
The agarose piece containing the 437 bp DNA fragment was excised and purified using GENECLE.
DNA fragmentation was performed using the AN II Kit (BIO101) according to the instructions attached to the kit.
The purified DNA was recovered by ethanol precipitation and then diluted with 10 mM Tr
The resulting mixture was dissolved in 20 μl of 1 mM EDTA solution containing 100 μl of 100 μl of 1 mM EDTA solution.
1 μl of the DNA solution was digested with restriction enzymes BamHI and Hind III (Takara Shuzo).
The digestion mixture was extracted with phenol and chloroform.
The DNA was then recovered by ethanol precipitation. The Hin containing the gene encoding the mouse H chain V region prepared as described above was used.
The dIII-BamHI DNA fragment was digested with HindIII and BamHI.
The fragment was subcloned into a pUC19 vector prepared by digesting the fragment.
Primer M13 and Primer M4 were used to confirm the base sequence of the plasmid.
and M13 Primer RV as primers, Dye Terminator
tor Cycle Sequencing kit (Perkin-Elme
r) and DNA Sequencer 373A (Perkin-Elme
The base sequence was determined by the nucleotide sequence determination method. Hybridoma #23 having the correct base sequence
-57-137-1, containing a gene encoding a mouse H chain V region derived from
Hind III recognition sequence and Kozak sequence on the 5'-side, BamH on the 3'-side
The plasmid containing the I recognition sequence was designated MBC1H/pUC19. (ii) H chain V region for the construction of a cDNA-type mouse-human chimeric H chain
Construction To link with the cDNA of human H chain C region Cγ1,
The mouse H chain V region was modified by PCR.
The primer MBC1HVS2 (SEQ ID NO: 9) encodes the beginning of the leader sequence of the V region.
The second asparagine in the sequence was changed to glycine, and the Kozak consensus
sequence (Kozak, M. et al., J. Mol. Biol., 196, 94
7-950, 1987) and has Hind III and EcoRI recognition sequences.
The forward primer for the H chain V region was MBC1HVR2 (sequence
Lane number 10) hybridizes to the DNA sequence encoding the 3'-side of the J region,
It also encodes the 5'-side sequence of the C region and has ApaI and SmaI recognition sequences.
PCR was performed using TaKaRa Ex Taq (Takara Shuzo) with a 50 μl reaction mixture.
The solution contained 0.6 μg of the plasmid MBC1H/pUC19 as a template DNA.
MBC1HVS2 and MBC1HVR2 were used as primers at 50 pmoles each.
, 2.5 U of TaKaRa Ex Taq, and 0.25 mM dNTP
50 μl of mineral oil was layered on top using the attached buffer, and the mixture was incubated at 94°C for 1 minute and at 55°C for 1 minute.
The temperature cycle was repeated 30 times, with 72°C for 1 minute and 72°C for 1 minute.
Fragment A was incubated with 1% Sea Kem GTG agarose (FMC Bio. Prod.
The DNA was separated by agarose gel electrophoresis using a 456 bp DNA fragment.
The agarose piece containing the A fragment was excised and purified using the GENECLEAN II Kit (
The DNA fragment was purified using a kit containing 1000 kJ/ml of ...
The DNA was ethanol precipitated and then diluted with 10 mM Tris-HCl (pH 7.4
), and dissolved in 20 μl of 1 mM EDTA solution. 1 μl of the resulting DNA solution was digested with restriction enzymes EcoRI and SmaI (Takara Shuzo).
The digestion mixture was diluted with phenol and chloroform for 1 hour at 37°C.
The DNA was extracted and then recovered by ethanol precipitation.
An EcoRI-SmaI DNA fragment containing the gene encoding the mouse H chain V region was
pUC19 vector prepared by digesting with EcoRI and SmaI
The nucleotide sequence of this plasmid was confirmed by using the primers
-M13 Primer M4 and M13 Primer RV were used as primers.
Then, Dye Terminator Cycle Sequencing
The DNA sequence was analyzed using a DNA Sequencer 373 (Perkin-Elmer).
The base sequence was determined by Perkin-Elmer A.
The mouse H chain V region derived from hybridoma #23-57-137-1 having
It contains a gene encoding the nucleotide sequence nucleotides ...
sequence and Kozak sequence, and has ApaI and SmaI recognition sequences on the 3'-side
The plasmid was named MBC1Hv/pUC19. (iii) Construction of an expression vector for a chimeric antibody H chain cDNA containing the C region Cγ1 of a human antibody H chain was prepared as follows.
That is, the humanized PM1 antibody H chain V region and the human antibody H chain C region IgG1
Nomu DNA (N. Takahashi, et al., Cell 29,671
The expression vector DHFR-ΔE-RVh-PM encodes the
-1-f (see WO92/19759), and humanized PM1 antibody L chain V region and
The expression vector RV1-P encodes the genomic DNA of the human antibody L chain κ chain C region.
mRNA was prepared from CHO cells transfected with M1a (see WO92/19759).
The humanized PM1 antibody H chain V region and human antibody C region C were isolated by RT-PCR.
The cDNA containing γ1 was cloned and inserted into pUC19 via the Hind III and Bam
After confirming the base sequence, a clone with the correct sequence was selected.
The plasmid was named pRVh-PM1f-cDNA. The SV40 promoter and DHFR on DHFR-ΔE-RVh-PM-1-f
The Hind III site between the EF-1α promoter and the gene
An expression vector lacking the EcoRI site between the H chain V region of the PM1 antibody
A humanized PM1 antibody H chain V region and a human antibody C region Cγ1 were prepared.
This was used to construct a cDNA expression vector. pRVh-PM1f-cDNA was digested with BamHI and then ligated with Klenow fragments.
The fragment was blunted with a Hind III fragment, further digested with Hind III, and the resulting Hind III-B fragment was
A HindIII-BamHI blunted fragment was prepared.
DHFR-Δ
E-RVh-PM1-f was digested with HindIII and SmaI to obtain
The humanized PM1 antibody H chain V region and human antibody
Expression vector RVh-PM1f-c containing cDNA encoding the C region Cγ1
DNA was constructed. A cDNA encoding the humanized PM1 antibody H chain V region and the human antibody C region Cγ1
The expression vector RVh-PM1f-cDNA containing NA was digested with ApaI and BamH.
After digestion with I, a DNA fragment containing the H chain C region was recovered and purified with ApaI and Bam
The vector was introduced into MBC1Hv/pUC19 prepared by digesting with HI.
The resulting plasmid was named MBC1HcDNA/pUC19.
The plasmid encodes the H chain V region of a mouse antibody and the C region Cγ1 of a human antibody.
DNA, with EcoRI and HindIII recognition sequences at the 5'-end and
-Contains a BamHI recognition sequence at the end. Plasmid MBC1HcDNA/pUC19 was digested with EcoRI and BamHI.
The resulting DNA fragment containing the base sequence encoding the H chain of the chimeric antibody is
The expression vector pCO was prepared by digesting with EcoRI and BamHI.
The expression plasmid for the chimeric antibody thus obtained was introduced into MBC1Hc
The expression vector pCOS1 was named DNA/pCOS1.
Mh-gγ1 (see WO92/19759) was digested with EcoRI and SmaI.
The antibody gene was deleted by transfection, and the fragment was inserted into the EcoRI-NotI-BamHI adapter (
It was constructed by ligating the nucleotides (Takara Shuzo) of the nucleotide sequence nucleotides (Takara Shuzo). Furthermore, to prepare a plasmid for expression in CHO cells,
MidMBC1H cDNA/pUC19 was digested with EcoRI and BamHI,
The resulting DNA fragment containing the chimeric antibody H chain sequence was digested with EcoRI and BamHI.
The resulting plasmid was introduced into the expression plasmid pCHO1 prepared by digestion with
The resulting expression plasmid for the chimeric antibody was named MBC1HcDNA/pCHO1.
The expression vector pCHO1 was DHFR-ΔE-rvH-PM1-f
(see WO92/19759) by digestion with EcoRI and SmaI.
The gene was deleted and the vector was cloned using EcoRI-NotI-BamHI Adaptor (Takara Shuzo).
) was ligated. (2) Construction of the human L chain constant region (i) Preparation of a cloning vector To construct a pUC19 vector containing the human L chain constant region, Hind I was used.
A pUC19 vector lacking the II site was prepared.
mM Tris-HCl (pH 8.5), 10mM MgCl₂, 1 mM DT
The reaction mixture contained 100 mM KCl, 8 U of Hind III (Takara Shuzo).
The digestion was carried out in 20 μl of the combined solution at 37° C. for 1 hour. The digestion mixture was diluted with phenol and chloramphenicol.
The DNA was extracted with chloroform and recovered by ethanol precipitation. The recovered DNA was dissolved in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl, and 100 mM MgCl.
gCl₂, 1mM DTT, 100mM NaCl, 0.5mM dNTP, 6
A 50 μl reaction mixture containing the Klenow fragment of U (GIBCO BRL) was
The reaction mixture was reacted at room temperature for 20 minutes to make the ends blunt.
The vector DNA was extracted with ethanol and chloroform, and then precipitated with ethanol.
The recovered vector DNA was diluted with 50 mM Tris-HCl (pH 7.6), 10
mM MgCl₂, 1 mM ATP, 1 mM DTT, 5% (v/v) polyethylene
PEG-8000, 0.5 U of T4 DNA ligase (GIBCO B
RL) at 16°C for 2 hours to induce self-ligation.
5 μl of the reaction mixture was transferred to Escherichia coli JM109 competent cells (Nippon Gene).
) and place on ice for 30 minutes, then heat at 42°C for 1 minute and then on ice for another 1 minute.
500 μl of SOC medium was added and incubated at 37°C for 1 hour.
After incubation, the cells were cultured on 2xYT agar medium (
50 μg/ml ampicillin) (Molecular Cloning:
A Laboratory Manual, Sambrook, et al.
, Cold Spring Harbor Laboratory Press
, 1989) and cultured overnight at 37°C to obtain transformants. The transformants were cultured in 20 ml of 2xYT medium containing 50 μg/ml ampicillin.
The cells were cultured at 37°C overnight, and the Plasmid Mini Kit (QIA) was used to isolate the plasmid from the bacterial cell fraction.
Plasmid DNA was purified using a PCR kit (Genesis Electronics, Inc.) according to the attached instructions.
The plasmid was digested with Hind III to remove the Hind III site.
The plasmid confirmed to be soluble was named pUC19 ΔHind III. (ii) Construction of genes encoding human L chain λ chain constant regions The human antibody L chain λ chain C region was constructed using the Mcg+Ke+Oz-, Mcg-Ke-Oz-, and
At least four isotypes of Mcg-Ke-Oz+, Mcg-Ke+Oz-
It is known that the
l. Acad. Sci. USA, 84, 9074-9078, 1987). #2
3-57-137-1 Human antibody L chain λ having homology to mouse L chain λ C region
As a result of searching the C region in the EMBL database, the isotype was Mcg+Ke+
Oz- (accession No. X57819) (P. Dariavach
, et al. , Proc. Natl. Acad. Sci. USA, 84, 90
The human antibody L chain λ chain of #2 (SEQ ID NO: 74-9078, 1987) showed the highest homology.
The amino acid sequence homology with the 3-57-137-1 mouse L chain λ chain C region is 64.
4% for the DNA sequence and 73.4% for the base sequence. Therefore, the gene encoding this human antibody L chain λ chain C region was constructed using PCR.
The synthesis of each primer was carried out using 394 DNA/RNA synthesis
The analysis was performed using a PCR sizer (ABI).
HLAMB3 (SEQ ID NO: 13) has the sense DNA sequence, and HLAMB2 (SEQ ID NO: 14) has the sense DNA sequence.
No. 12) and HLAMB4 (SEQ ID NO: 14) have antisense DNA sequences.
Each primer has a complementary sequence of 20 to 23 bp at both ends. External primers HLAMBS (SEQ ID NO: 15), HLAMBR (SEQ ID NO: 16)
) has sequences homologous to HLAMB1 and HLAMB4, respectively, and
AMBS has EcoRI, HindIII, and BlnI recognition sequences, and HLAMBR
Each of these contains an EcoRI recognition sequence.
After the reaction, equal amounts of HLAMB2 and HLAMB3-HLAMB4 were
The resulting mixture was mixed and assembled in a second PCR.
and HLAMBR were added, and full-length DNA was amplified by third PCR. PCR was performed using TaKaRa Ex Taq (Takara Shuzo) according to the attached instructions.
In the first PCR, 5 pmoles of HLAMB1 and 0.5 pmoles of
1 containing 100 μg of HLAMB2 and 5 U of TaKaRa Ex Taq (Takara Shuzo)
00 μl of reaction mixture, or 0.5 pmole of HLAMB3 and 5 pmole of
Contains 1000mg of HLAMB4 and 5U of TaKaRa Ex Taq (Takara Shuzo)
100 μl of the reaction mixture was layered with 50 μl of mineral oil and incubated at 94° C. for 1 minute.
The temperature cycle was repeated five times: 1 minute at 60°C, 1 minute at 60°C, and 1 minute at 72°C. The second PCR was performed by mixing 50 μl of the reaction mixture, overlaying 50 μl of mineral oil, and
The temperature cycle was repeated three times: 1 minute at 60°C, 1 minute at 60°C, and 1 minute at 72°C.
The third PCR was performed by adding external primers HLAMBS and HLAMBR to the reaction mixture.
50 pmoles each were added, and the mixture was incubated at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute.
The temperature cycle was repeated 30 times, each time for 1 minute. The DNA fragments of the third PCR product were separated by gel electrophoresis on a 3% low-melting-point agarose gel (NuSieve).
After electrophoresis on GTG Agarose (FMC), GENECLEAN II
The product was recovered and purified from the gel using a kit (BIO101) according to the attached instructions.
The resulting DNA fragment was diluted with 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl₂, 1 mM DTT, 100 mM NaCl, 8 U of EcoRI (Takara
The digestion was carried out for 1 hour at 37°C in a 20 µl reaction mixture containing 100 µL of PEG-400 (Sake Brewery).
The mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.
After that, 8 μL of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution was added.
1. 0.8 μg of plasmid pUC19 ΔHind III was similarly digested with EcoRI.
The DNA was digested with , extracted with phenol and chloroform, and recovered by ethanol precipitation.
The digested plasmid pUC19 ΔHind III was diluted with 50 mM Tris
-HCl (pH 9.0), 1mM MgCl₂, alkaline phosphatase (E.
The incubation was continued for 30 minutes at 37°C in 50 μl of a reaction mixture containing E. coli C75 (Takara Shuzo).
The reaction mixture was then dephosphorylated (BAP treatment).
After extraction with formaldehyde, the DNA was recovered by ethanol precipitation, and then diluted with 10 mM Tris
-HCl (pH 7.4), 10 μl of 1 mM EDTA solution. 1 μl of the above BAP-treated plasmid pUC19 ΔHind III
4 μl of the PCR product was ligated using DNA Ligation Kit Ver. 2 (Takarashu).
The resulting fragment was ligated using the DNA fragment (DNA fragment 102) and transformed into E. coli JM109 competent cells.
The transformants were cultured in 2 ml of 2xYT medium containing 50 μg/ml ampicillin.
After overnight culture, the bacterial cell fraction was purified using the QIAprep Spin Plasmid Kit.
The plasmid was purified using QIAGEN. The base sequence of the cloned DNA was confirmed for the above plasmid.
The base sequence was determined using a 373A DNA sequencer (ABI).
The primers used were M13 Primer M4 and M13 Primer
RV (Takara Shuzo) was used. As a result, 12b was found inside the cloned DNA.
The plasmid containing this DNA was designated CλΔ/pUC.
19. Therefore, the primer HCLMS (SEQ ID NO: 19) was used to complement this region.
No. 17), HCLMR (SEQ ID NO: 18) was newly synthesized and the correct DNA was again obtained by PCR.
NA was constructed. In the first PCR, the plasmid CλΔ/pUC19 containing the deleted DNA was used as a template.
Reactions were carried out with primer HLAMBS and HClMR, and with HClMS and HLAMB4.
The PCR products were purified and assembled in a second PCR.
Primers HLAMBS and HLAMB4 were added and a third PCR was performed to obtain the full-length DNA fragment.
NA was amplified. In the first PCR, 0.1 μg of CλΔ/pUC19 was used as a template, and primer H
LAMBS and HClMR, 50 pmole each, or HClMS and HL
AMB4 50 pmole each, 5U of TaKaRa Ex Taq (Takara Shuzo)
100 μl of the reaction mixture containing the above solution was overlaid with 50 μl of mineral oil and incubated at 94° C. for 1 hour.
The temperature cycle was repeated 30 times: 1 minute at 60°C, 1 minute at 60°C, and 1 minute at 72°C. PCR products: HLAMBS-HCLMR (236 bp), HCLMS-HLAM
B4 (147 bp) were electrophoresed on a 3% low-melting-point agarose gel, and then
The protein was recovered from the gel and purified using the ENECLEAN II Kit (BIO101).
In the second PCR, 40 ng of each purified DNA fragment and 1 U of TaKaRa Ex T
A 20 μl reaction mixture containing 20 μl of aq (Takara Shuzo) was used, and 25 μl of mineral oil was added as the top layer.
Then, a temperature cycle of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute was performed.
This was performed five times. The third PCR consisted of 2 μl of the second PCR reaction mixture, external primers HLAMBS and H
LAMB4 50 pmole each, 5U of TaKaRa Ex Taq (Takara Shuzo)
A 100 μl reaction mixture containing 50 μl of mineral oil was used as an overlay.
3 temperature cycles at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute
The third PCR product, a 357 bp DNA fragment, was diluted with 3% low-melting-point agarose.
After gel electrophoresis, the DNA was purified using GENECLEAN II Kit (BIO101).
The resulting DNA fragment (0.1 μg) was digested with EcoRI and then purified using a BAP-treated primer.
The plasmid pUC19ΔHind III was subcloned into E. coli JM1
Transformed into 0.09 competent cells and cultured in 2 medium containing 50 μg/ml ampicillin.
The cells were cultured overnight in 2 ml of 2×YT medium, and the bacterial cell fraction was purified using QIAprep Spin Pl.
The plasmid was purified using the asmid Kit (QIAGEN). The nucleotide sequence of the purified plasmid was determined using M13 Primer M4 and M13
Primer RV (Takara Shuzo) was used to generate 373A DNA sequence.
The DNA sequence was determined using a DNA fragment analysis kit (ABI).
The identified plasmid was designated Cλ/pUC19. (iii) Construction of a gene encoding the human L chain κ chain constant region The L chain κ chain C was constructed from the plasmid HEF-PM1k-gk (WO92/19759).
The DNA fragment encoding the region was cloned using PCR.
The forward primer was synthesized using an NA/RNA synthesizer (ABI).
The marker HKAPS (SEQ ID NO: 19) is EcoRI, Hind III, and BlnI.
The reverse primer HKAPA (SEQ ID NO: 20) is the EcoRI recognition sequence.
It was designed to have the following: Template plasmid HEF-PM1k-gk 0.1 μg, primer HK
APS, HKAPA, 50 pmole each, 5U of TaKaRaEx Taq (Takarashu)
100 μl of the reaction mixture containing the 90% EDTA (prepared by HPLC) was used and overlaid with 50 μl of mineral oil.
The reaction was carried out for 30 cycles at 4°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute.
The 360 bp PCR product was electrophoresed on a 3% low-melting-point agarose gel,
The protein was recovered from the gel and purified using the GENECLEAN II Kit (BIO101).
The resulting DNA fragment was digested with EcoRI and then transformed into the BAP-treated plasmid p
The vector was cloned into E. coli JM109 competent strain UC19 ΔHind III.
The cells were transformed with the 2×YT medium containing 50 μg/ml ampicillin.
The cells were cultured overnight in 100 ml of PBS, and the bacterial cell fraction was purified using QIAprep Spin Plasmid.
The plasmid was purified using a QIAGEN PCR Kit. The base sequence of the purified plasmid was confirmed using M13 Primer M4 and M13 Pr
The DNA sequence was sequenced using a 373A DNA sequencer (A
The plasmids that were confirmed to have the correct base sequence were
The plasmid was Cκ/pUC19. (3) Construction of a chimeric antibody L chain expression vector A chimeric #23-57-137-1 antibody L chain expression vector was constructed.
Hin immediately before the human antibody constant region of Cλ/pUC19 and Cκ/pUC19
dIII, BlnI site, encoding the #23-57-137-1 L chain V region
By linking the genes corresponding to the chimera #23-57-137-1,
pUC19 encoding antibody L chain V region and L chain λ or L chain κ constant region
The chimeric antibody L chain gene was excised by EcoRI digestion.
The L chain of antibody #23-57-137-1 was subcloned into the HEF expression vector.
The V region was cloned using PCR.
This was done using a DNA/RNA synthesizer (ABI).
Primer MBCCHL1 (SEQ ID NO: 21) has a Hind III recognition sequence and a Koz
ak sequence (Kozak, M. et al., J. Mol. Biol. 196, 9
47-950, 1987) with the forward primer MBCCHL3 (SEQ ID NO: 22)
was designed to have BgIII and EcoRI recognition sequences. PCR was performed in 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
1.5 mM MgCl₂, 0.2 mM dNTP, 0.1 μg MBC1L24
50 pmoles each of MBCCHL1 and MBCCHL3 as primers;
100 μl containing 1 μl of AmpliTaq (PERKIN ELMER)
The reaction mixture was overlaid with 50 μl of mineral oil and incubated at 94° C. for 45 seconds and at 60° C.
The PCR product was subjected to 30 temperature cycles of 45 seconds at 72°C and 2 minutes at 72°C. The 444 bp PCR product was electrophoresed on a 3% low-melting-point agarose gel and then
The protein was recovered from the gel and purified using the NECLEAN II kit (BIO101).
, 10mM Tris-HCl (pH 7.4), 20μl of 1mM EDTA solution
1 μl of each PCR product was dissolved in 10 mM Tris-HCl (pH 7
．． 5), 10mM MgCl₂, 1 mM DTT, 50 mM NaCl, 8 U of
Reaction containing Hind III (Takara Shuzo) and 8 U of EcoRI (Takara Shuzo)
The digestion was carried out in 20 μl of the mixture at 37° C. for 1 hour.
The DNA was extracted with chloroform, and then recovered by ethanol precipitation.
The plasmid pUC19 (1 μg) was similarly digested with Hind III and EcoRI.
digested, extracted with phenol and chloroform, and recovered by ethanol precipitation.
The BAP was treated with alkaline phosphatase (E. coli C75, Takara Shuzo).
The reaction mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.
After that, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution
1 μl of the BAP-treated plasmid pUC19 and 4 μl of the PCR product were dissolved in DNA.
The fragments were ligated using A Ligation Kit Ver. 2 (Takara Shuzo) and transformed into E. coli.
JM109 competent cells (Nippon Gene) were transformed in the same manner as above.
This was plated on 2xYT agar medium containing 50 μg/ml ampicillin and incubated at 37° C.
The resulting transformants were cultured overnight in 50 μg/ml ampicillin-containing medium.
The cells were cultured overnight at 37°C in 2 ml of 2xYT medium.
Plasmids were purified using the Spin Plasmid Kit (QIAGEN).
After determining the base sequence, the plasmid with the correct base sequence was cloned into CHL/pUC.
19. 1 μg each of plasmid Cλ/pUC19 and Cκ/pUC19 was diluted to 20 mM
Tris-HCl (pH 8.5), 10mM MgCl₂, 1 mM DTT,
100 mM KCl, 8 U of Hind III (Takara Shuzo) and 2 U of BlnI
The digestion was carried out for 1 hour at 37°C in 20 µl of a reaction mixture containing 100 µL of PEG-1000 (Takara Shuzo).
The mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.
After that, the reaction mixture was treated with BAP at 37°C for 30 minutes.
The DNA was extracted with chloroform, and the DNA was recovered by ethanol precipitation.
Dissolved in 10 μl of 1 mM EDTA solution (pH 7.4). #23-57-137-1 Plasmid CHL/pUC19 containing the L chain V region
8 μg of the resulting 40
The 9 bp DNA fragment was electrophoresed on a 3% low-melting-point agarose gel and then purified by GENEC
The resulting mixture was recovered from the gel using the LEAN II Kit (BIO101), purified, and then diluted to 10 ml.
Dissolved in 10 μl of 1 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.
4 μl of this L chain V region DNA was treated with BAP and transformed into the plasmid Cλ/pUC19
Alternatively, the plasmid was subcloned into 1 μl each of Cκ/pUC19 and transformed into E. coli JM109 colony.
The cells were transformed into 2xYT cells containing 50 μg/ml ampicillin.
The cells were cultured overnight in 3 ml of medium, and the bacterial cell fraction was purified using QIAprep Spin Plasma.
The plasmids were purified using the id Kit (QIAGEN).
The plasmids MBC1L(λ)/pUC19 and MBC1L(κ)/pUC19
Plasmids MBC1L(λ)/pUC19 and MBC1L(κ)/pUC1
9 were each digested with EcoRI and subjected to electrophoresis on a 3% low-melting-point agarose gel.
The 743 bp DNA fragment was purified using GENECLEAN II Kit (BIO101).
The resulting product was recovered and purified from the gel using 10 mM Tris-HCl (pH 7.4),
The expression vector was dissolved in 10 μl of 1 mM EDTA solution. 2.7 μg of the plasmid HEF-PM1k-gk was transfected with Eco
Digested with RI, extracted with phenol and chloroform, and DNA precipitated with ethanol
The recovered DNA fragment was treated with BAP and then soaked in 1% low-melting-point agarose gel.
The 6561 bp DNA fragment was isolated using the GENECLEAN II Kit.
(BIO101) and purified from the gel, and then diluted with 10 mM Tris-HCl.
(pH 7.4), dissolved in 10 μl of 1 mM EDTA solution. 2 μl of the BAP-treated HEF vector was added to the above-mentioned plasmid MBC1L(λ) or
The resulting mixture was ligated with 3 μl of each MBC1L(κ) EcoRI fragment and transformed into E. coli JM109 competent cells.
The transformants were grown in 2xYT medium containing 50 μg/ml ampicillin.
The culture was performed in 2 ml of the medium, and the bacterial cell fraction was purified using QIAprep Spin Plasmid.
The plasmid was purified using a PCR kit (QIAGEN). The purified plasmid was dissolved in 20 mM Tris-HCl (pH 8.5), 10 mM
MgCl₂, 1 mM DTT, 100 mM KCl, 8 U of HindIII
(Takara Shuzo) and 2 U of PvuI (Takara Shuzo) in 20 μl of reaction mixture
The digestion was carried out at 37°C for 1 hour. If the fragment was inserted in the correct direction,
If inserted in the reverse direction, a digestion fragment of 4378/2926 bp will be generated.
By doing so, the plasmids inserted in the correct direction were identified as MBC1L (
λ)/neo and MBC1L(κ)/neo. (4) Transfection of COS-7 cells To evaluate the antigen-binding and neutralizing activities of the chimeric antibodies, the expression plasmids were transfected with
The chimeric antibody was transiently expressed in COS-7 cells using the MBC1HcDNA/pCO plasmid.
S1 and MBC1L(λ)/neo or MBC1HcDNA/pCOS1 and MB
The combination of C1L(κ)/neo was used in the Gene Pulser device (Bio
Rad) into COS-7 cells by electroporation.
CO cells suspended in PBS(-) at a cell concentration of 1 x 107 cells/ml were
10 μg of each plasmid DNA was added to 0.8 ml of S-7 cells and incubated at 1,500 V.
After a 10-minute recovery period at room temperature,
The electroporated cells were incubated with 2% Ultra Low IgG.
The cells were suspended in DMEM medium (GIBCO) containing fetal bovine serum (GIBCO), and 1
0 cm culture dish and CO₂The cells were cultured in an incubator for 72 hours.
After that, the culture supernatant was collected, centrifuged to remove cell debris, and used as an ELISA sample.
In addition, chimeric antibodies were purified from the culture supernatant of COS-7 cells using AffiGel
Protein A MAPS II kit (BioRad) was used.
The procedure was carried out according to the attached recipe. (5) ELISA (i) Measurement of antibody concentration An ELISA plate for measuring antibody concentration was prepared as follows. ELISA
Each well of a 96-well plate for SA (Maxisorp, NUNC) was filled with solid-phase buffer.
-(0.1M NaHCO₃, 0.02% NaN₃) to a concentration of 1 μg/ml.
The solution was immobilized with 100 μl of the prepared goat anti-human IgG antibody (TAGO), and 200 μl of
Dilution buffer (50 mM Tris-HCl, 1 mM MgCl₂, 0.1M
NaCl, 0.05% Tween20, 0.02% NaN₃, 1% bovine blood
After blocking with serum albumin (BSA), pH 7.2, chimeric antibodies were expressed.
The culture supernatant of COS cells or purified chimeric antibody was serially diluted and added to each well.
After incubation at room temperature for 1 hour and washing with PBS-Tween 20,
100 μl of lysine phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added.
After incubation at room temperature for 1 hour and washing with PBS-Tween 20, 1 mg
/ml of substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA
) was added, and then the absorbance at 405 nm was measured using a microplate reader (BioRa
d) was measured. Hu IgG1λ Puri was used as a standard for concentration measurement.
Fied (The Binding Site) was used. (ii) Measurement of antigen binding ability ELISA plates for antigen binding measurement were prepared as follows.
Each well of a 96-well LISA plate was filled with immobilization buffer to a concentration of 1 μg/ml.
100 μl of human PTHrP (1-34) (Peptide Institute) prepared by
After blocking with 200 μl of dilution buffer, the chimeric antibody was expressed in C
The culture supernatant of OS cells or purified chimeric antibody was serially diluted and added to each well.
After incubation with PBS-Tween 20, the cells were washed with alkaline phosphatase.
100 μl of enzyme-conjugated goat anti-human IgG antibody (TAGO) was added.
After incubation and washing with PBS-Tween 20, 1 mg/ml of substrate solution (S
Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and then 405
The absorbance at 100 nm was measured using a microplate reader (Bio Rad). As a result, the chimeric antibody had the ability to bind to human PTHrP (1-34).
It has been shown to have the correct structure of the cloned mouse antibody V region.
In addition, in the chimeric antibody, the L chain C region is either a λ chain or a κ chain.
Since the binding ability of the antibody to PTHrP(1-34) remains unchanged,
The L chain C region of the chimeric antibody was constructed using the L chain λ chain of a humanized antibody. (6) Establishment of a CHO stable cell line To establish a cell line that stably produces the chimeric antibody, the expression plasmid was transfected into CHO cells.
The chimeric antibody was then introduced into CHO cells (DXB11). In other words, the establishment of a stable chimeric antibody producing cell line was achieved by using the CHO cell expression plasmid M
BC1HcDNA/pCHO1 and MBC1L(λ)/neO or MBC1Hc
The combination of DNA/pCHO1 and MBC1L(κ)/neo was used.
CH was generated by electroporation using a ulser device (Bio Rad).
The expression vectors were then co-transfected into 0 cells. Each expression vector was cleaved with the restriction enzyme Pvul.
The DNA was linearized by phenol and chloroform extraction, followed by ethanol precipitation.
The DNA was collected and used for electroporation.
Each plasmid was added to 0.8 ml of CHO cells suspended at a cell concentration of 7 cells/ml.
10 μg of DNA was added, and a pulse was applied at 1,500 V and a capacitance of 25 μF.
After a 10-minute recovery period at room temperature, the electroporated cells
MEM-α medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO)
and three 96-well plates (Falcon) were used to₂Incubation
The day after the start of the culture, 10% fetal bovine serum (GIBCO) and
500 mg/ml GENETICIN (G418 Sulfate, GIBCO
) supplemented, ribonucleoside and deoxyribonucleoside-free MEM-α medium (
The selective medium (GIBCO) was replaced with a new one to select cells into which the antibody gene had been introduced.
After changing the selective medium, the cells were observed under a microscope about two weeks later, and smooth cell growth was observed.
After the test, the amount of antibody produced was measured by the antibody concentration measurement ELISA.
The established stable antibody-producing cell line was cultured and expanded, and the cells with the highest concentration were cultured in roller bottles at 2% U.
Itra Low IgG with fetal bovine serum, ribonucleosides and deoxyribonucleic acid
Large-scale culture was carried out using MEM medium without ribonucleotides.
The culture supernatant was collected and filtered through a 0.2 μm filter (Millipore).
Cell debris was removed. The chimeric antibody was purified from the CHO cell culture supernatant using POROS protein A cap.
ConSept
The neutralizing activity was measured using LC100 (Millipore) according to the attached instructions.
The purified calcium phosphate was measured and used in a drug efficacy test in a hypercalcemia model animal.
The antibody concentration and antigen binding activity were measured using the ELISA system described above. [Reference Example 4] Construction of Humanized Antibody (1) Construction of Humanized Antibody H Chain (i) Construction of Humanized H Chain V Region The humanized #23-57-137-1 antibody H chain was constructed by PCR to obtain the CDR-Granule region.
Human antibody S31679 (NBRF-PDB, Cui
senior A. M. et al., Eur. J. Immunol. ,23,110-1
Humanized #23-57-137-1 antibody H having FRs derived from
Six PCR primers were used to generate the C strand (version "a").
DR-grafting primers MBC1HGP1 (SEQ ID NO: 23) and MBC
1HGP3 (SEQ ID NO: 24) has the sense DNA sequence and CDR grafts.
The binding primers MBC1HGP2 (SEQ ID NO: 25) and MBC1HGP4 (SEQ ID NO: 26) were used.
Column number 26) has the antisense DNA sequence, and both of the primers
The external primers MBC1HV (
MBC1HVR1 (SEQ ID NO: 27) and MBC1HVR1 (SEQ ID NO: 28) are CDR-grafted sequences.
It shares homology with primers MBC1HGP1 and MBC1HGP4. CDR-grafting primers MBC1HGP1, MBC1HGP2, M
BC1HGP3 and MBC1HGP4 were analyzed using urea-denatured polyacrylamide gel.
(Molecular Cloning: A Laboratory
Manual, Sambrook et al., Cold Spring Harbor
Laboratory Press, 1989), extraction from the gel was performed using crus
h and soak method (Molecular Cloning: A Labo
ratoryManual, Sambrook et al., Cold Spring H
The procedure was carried out using a 6% RT-PCR kit (Arbor Laboratory Press, 1989). That is, 1 nmole of CDR-grafting primer was used.
Separation on a denaturing polyacrylamide gel and identification of DNA fragments of the desired size were performed sequentially.
The crush and soak method was used to irradiate ultraviolet light onto a gel thin-layer plate.
The gel was recovered and resuspended in 20 μl of 10 mM Tris-HCl (pH 7.4), 1 mM
The PCR was performed using TaKaRa Ex Taq (Takara Shuzo).
Into 100 μl of the reaction mixture was added the CDR-grafting
Primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MB
1 μl of C1HGP4, 0.25 mM dNTP, 2.5 U of TaKa
The conditions were as follows: 1 minute at 94°C, 55°C, and 1 minute at 55°C using the attached buffer containing Ra Ex Taq.
5 times at 50 °C for 1 minute and 72 °C for 1 minute.
The external primers MBC1HVS1 and MBC1HVR1 were added to the 100-kDa DNA at the same temperature.
The PCR-amplified DNA fragment was then subjected to 30 cycles.
Agarose using eve GTG agarose (FMC Bio. Products)
The DNA fragment was separated by agarose gel electrophoresis. The agarose piece containing the 421 bp DNA fragment was excised and purified using GENECLE.
Using the ANII Kit (BIO101), DNA fragments were extracted according to the instructions attached to the kit.
The purified DNA was precipitated with ethanol and then diluted with 10 mM Tris
The resulting solution was dissolved in 20 μl of 1 mM EDTA solution.
Prepared by digesting the PCR reaction mixture with BamHI and HindIII
The fragment was subcloned into pUC19 and the nucleotide sequence was determined.
The resulting plasmid was designated hMBCHv/pUC19. (ii) Construction of the H chain V region for humanized H chain cDNA To link to the cDNA for the human H chain C region Cγ1, the plasmid constructed as described above was used.
The humanized H chain V region was modified by PCR.
S2 hybridizes to a sequence encoding the 5'-side of the leader sequence of the V region,
and the Kozak consensus sequence (Kozak, M, et al., J. Mol. Biol.
196, 947-950, 1987), HindIII and EcoRI recognition
The forward primer for the H chain V region was designed to have the sequence MBC1HV
R2 hybridizes to the DNA sequence encoding the 3'-side of the J region and
The 5'-terminal sequence of the nucleotide sequence encoding the nucleotide ...
PCR was performed using TaKaRa Ex Taq (Takara Shuzo) with 0.01% ribonucleotides as template DNA.
4 μg of hMBCHv/pUC19 was used, and MBC1HVS was used as a primer.
2 and MBC1HVR2 were used at 50 pmole and 2.5 U of TaKaRa
The attached buffer solution containing Ex Taq and 0.25 mM dNTP was used.
30 temperature cycles of 4°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute
The DNA fragments amplified by PCR were subjected to PCR with 3% Nu Sieve GTG
Agarose gel electrophoresis using agarose (FMC Bio. Products)
The agarose piece containing the 456 bp DNA fragment was excised and purified using GENECLE.
Using the ANII Kit (BIO101), DNA fragments were extracted according to the instructions attached to the kit.
The purified DNA was precipitated with ethanol and then diluted with 10 mM Tris
The resulting solution was dissolved in 20 μl of 1 mM EDTA solution.
pUC prepared by digesting the PCR reaction mixture with EcoRI and SmaI
The resulting hybrid was subcloned into 19 and the nucleotide sequence was determined.
A gene encoding the mouse H chain V region derived from mouse #23-57-137-1
and EcoRI and HindIII recognition sequences and Kozak
The plasmid containing the ApaI and SmaI recognition sequences on the 3'-side was used as hMBC.
The vector was named RVh-PM1f-cDNA. (2) Construction of an expression vector for the humanized antibody H chain Plasmid RVh-PM1f-cDNA containing the hPM1 antibody H chain cDNA sequence
A was digested with ApaI and BamHI to recover a DNA fragment containing the H chain C region.
hMBC1Hv/ was prepared by digesting the DNA with ApaI and BamHI.
The resulting plasmid was used as hMBC1H cDNA/
This plasmid was designated pUC19.
It contains the human H chain V region and the human H chain C region Cγ1, and has EcoRI and
The plasmid has a HindIII recognition sequence at the 3'-end and a BamHI recognition sequence at the 4'-end.
Humanized H chain version "a" contained in hMBC1H cDNA/pUC19
The base sequence and the corresponding amino acid sequence are shown in SEQ ID NO: 58.
The amino acid sequence of nucleotide a is shown in SEQ ID NO: 56. hMBC1H cDNA/pUC19 was digested with EcoRI and BamHI,
The resulting DNA fragment containing the H chain sequence was digested with EcoRI and BamHI.
The resulting human genomic DNA was introduced into the expression plasmid pCOS1 prepared by the above procedure.
The expression plasmid for the hMBC1H antibody was named hMBC1HcDNA/pCOS1. Furthermore, to prepare a plasmid for expression in CHO cells,
1HcDNA/pUC19 was digested with EcoRI and BamHI, and the resulting H
A DNA fragment containing the strand sequence was prepared by digesting with EcoRI and BamHI.
The resulting humanized antibody was introduced into the expression plasmid pCHO1.
The expression plasmid was named hMBC1HcDNA/pCHO1. (3) Construction of a hybrid light chain variable region (i) Preparation of a FR1,2/FR3,4 hybrid antibody An L chain gene was constructed by recombining the FR regions of a humanized antibody and a mouse (chimeric) antibody.
The restriction enzyme Af in CDR2 was used to construct the humanized fragment.
By utilizing the Ill cleavage site, FR1 and 2 are derived from a human antibody, FR3
and 4 were used to produce hybrid antibodies derived from mouse antibodies. 10 each of the plasmids MBC1L(λ)/neo and hMBC1L(λ)/neo
μg in 10mM Tris-HCl (pH 7.5), 10mM MgCl₂, 1
mM DTT, 50 mM NaCl, 0.01% (w/v) BSA, AflII
(Takara Shuzo) 10 U in 100 μl of reaction mixture at 37°C for 1 hour.
The reaction mixture was electrophoresed on a 2% low-melting-point agarose gel, and the plasmid MBC1L (
λ)/neo to a 6282 bp fragment (referred to as c1) and a 1022 bp fragment
(referred to as c2), a 6282 bp fragment from the plasmid hMBC1L(λ)/neo
The 1022 bp fragment (h1) and the 1022 bp fragment (h2) were purified by GENECLE.
The fragments were recovered from the gel and purified using the ANII Kit (BIO101). 1 μg each of the recovered c1 and h1 fragments was subjected to BAP treatment.
Extraction with ethanol and chloroform followed by ethanol precipitation was performed.
The fragments were dissolved in 10 μl of ris-HCl (pH 7.4), 1 mM EDTA solution. 1 μl of BAP-treated c1 and h1 fragments were connected to 4 μl of h2 and c2 fragments, respectively.
The mixture was lysed (4°C, overnight) and transformed into competent E. coli JM109 cells.
The cells were cultured in 2 ml of 2xYT medium containing μg/ml ampicillin, and the bacterial cell fraction was
Using QIAprep Spin Plasmid Kit (QIAGEN)
The plasmid was purified. The purified plasmid was diluted with 10 mM Tris-HCl (pH 7.5), 10 mM
MgCl₂, 1 mM DTT, ApaLI (Takara Shuzo) 2 U, or BamH
20 μL of reaction mixture containing 8 U of Hind I (Takara Shuzo) and 8 U of Hind III (Takara Shuzo)
The digestion was carried out in 1 ml at 37°C for 1 hour. If c1-h2 were correctly ligated,
5560/1246/498 bp with LI, 713 with BamHI/HindIII
The plasmid was confirmed by the generation of a 4/269 bp digestion fragment. This was used to identify the plasmid encoding the human FR1,2/mouse FR3,4 hybrid antibody L chain.
The expression vector was h/mMBC1L(λ)/neo.
Since no clone was obtained, the vector was recombined on a pUC vector and then transferred to the HEF vector.
At that time, the humanized antibody L chain V region without amino acid substitution was cloned into
Plasmid hMBC1Laλ/pUC19, containing the nucleotide sequence at position 91 in FR3 (Kaba
Human tyrosine (amino acid number 87 according to the t rule) is replaced with isoleucine
The plasmid hMBC1Ldλ/pUC19 containing the L chain V region of the antibody was used as a template.
The plasmids MBC1L(λ)/pUC19, hMBC1Laλ/pUC19, and
10 μg each of hMBC1Ldλ/pUC19 and hMBC1Ldλ/pUC19 was dissolved in 10 mM Tris-HCl (
pH 7.5), 10mM MgCl₂, 1mM DTT, 50mM NaCl,
Contains 0.01% (w/v) BSA, HindIII 16U, AflII 4U.
The reaction mixture was digested at 37°C for 1 hour in 30 µl of a reaction mixture containing 2% low-melting point ATP.
The plasmid MBC1L(λ)/pUC19 was electrophoresed on agarose gel and 21
5 bp (c2'), plasmids hMBC1Laλ/pUC19 and hMBC1
DNA of 3218 bp (ha1', hd1') from Ldλ/pUC19
The fragment was recovered from the gel using the GENECLEAN II Kit (BIO101).
The ha1' and hd1' fragments were ligated to the c2' fragment, and the resulting products were transformed into E. coli JM109.
The transformants were transformed into competent cells.
The cells were cultured in 2 ml of T medium, and the bacterial cell fraction was purified using QIAprep Spin Plasmi.
The plasmids were purified using the d Kit (QIAGEN).
Plasmids m/hMBC1Laλ/pUC19 and m/hMBC1Ldλ/pUC
19. The resulting plasmids m/hMBC1Laλ/pUC19 and m/hMBC1Ld
λ/pUC19 was digested with EcoRI. Two 743 bp DNA fragments were isolated.
After electrophoresis on a 100% low-melting-point agarose gel, the DNA fragments were purified using the GENECLEAN II Kit (
The resulting mixture was recovered from the gel using 10 mM Tris-HCl (
Each DNA fragment (4 μl) was ligated to 1 μl of the BAP-treated HEF vector.
The resulting product was transformed into E. coli JM109 competent cells.
The cells were cultured in 2 ml of 2xYT medium containing 100 mg of PEG-4000.
The plasmid was purified using the QIAGEN Plasmid Kit.
Each purified plasmid was diluted with 20 mM Tris-HCl (pH 8.5), 10
mM MgCl₂, 1mM DTT, 100mM KCl, HindIII (Takara)
In a 20 μl reaction mixture containing 8 U of PvuI (Takara Shuzo) and 2 U of PvuI (Takara Shuzo),
If the fragment was inserted in the correct direction, the digestion was
If it is inserted in the reverse direction, a digestion fragment of 4378/2926 bp will be generated.
These were confirmed as mouse FR1, FR2/human ...2/human FR1, FR2/human FR2/human FR1, FR2/human FR2/human FR2/human FR2/human FR2/human FR2/human FR2/human FR2/human FR2/human
The expression vector encoding the FR3,4 hybrid antibody L chain was designated m/hMBC1L
aλ/neo, m/hMBC1Ldλ/neo. (ii) Construction of FR1/FR2 hybrid antibodies By utilizing the SnaBI cleavage site within CDR1, we were able to construct FR1 hybrid antibodies.
A hybrid antibody was produced using the plasmids MBC1L(λ)/neo and h/mMBC1L(λ)/neo.
10μg of each was added to 10mM Tris-HCl (pH 7.9), 10mM MgCl
₂, 1mM DTT, 50mM NaCl, 0.01% (w/v) BSA, Sn
Digestion was carried out for 1 hour at 37°C in 20 μl of a reaction mixture containing 6 U of aBI (Takara Shuzo).
Next, the solution was diluted with 20 mM Tris-HCl (pH 8.5), 10 mM MgCl₂
, 1mM DTT, 100mM KCl, 0.01% (w/v) BSA, Pvu
The reaction mixture was digested at 37°C for 1 hour in 50 μl of a reaction mixture containing 6 U of I. The reaction mixture was electrophoresed on a 1.5% low-melting-point agarose gel, and the plasmid MBC was
1L(λ)/neo to 4955bp (ml) and 2349bp (m2),
Plasmid h/mMBC1L(λ)/neo to 4955 bp (hm1) and 2
Each 349 bp (hm2) DNA fragment was purified using the GENECLEAN II Kit (BI
The resulting product was recovered from the gel using 10 mM Tris-HCl (pH 8.0).
7.4), dissolved in 40 μl of 1 mM EDTA solution. 1 μl of m1 and hm1 fragments were ligated to 4 μl of hm2 and m2 fragments, respectively, and transformed into E. coli.
The resulting vector was transformed into JM109 competent cells containing 50 μg/ml ampicillin.
The cells were cultured in 2 ml of 2xYT medium containing 100% ethanol, and the cells were extracted with QIAprep Spin
Plasmids were purified using a Plasmid Kit (QIAGEN). Each purified plasmid was diluted with 10 mM Tris-HCl (pH 7.5), 10
mM MgCl₂, 1 mM DTT, ApaI (Takara Shuzo) 8U, or ApaL
The reaction mixture (20 μl) containing 2 U of I (Takara Shuzo) was digested at 37° C. for 1 hour.
If the fragments are ligated correctly, the fragment will be 7304 bp with ApaI and 500 bp with ApaLI.
560/1246/498 bp (m1-hm2), 6538/766 with ApaI
bp, 3535/2025/1246/498bp (hm1-m2
The plasmid was confirmed by the generation of digestion fragments of the following:
An expression vector encoding the human FR1/mouse FR2,3,4 hybrid antibody L chain was
The vectors were hmmMBC1L(λ)/neo, mouse FR1/human FR2/mouse
The expression vector encoding the FR3,4 hybrid antibody L chain was designated mhmMBC1L.
(λ)/neo. (4) Construction of humanized antibody L chain The humanized #23-57-137-1 antibody L chain was constructed by PCR using CDR-Gamma.
Human antibody HSU03868 (GEN-BANK, D
Eftos M et al., Scand. J. Immunol., 39, 95-103,
1994) and human antibody S25755 (
Humanized #23-57-137-1 antibody with FR4 from NBRF-PDB
Six PCR primers were used to generate the human light chain (version "a").
CDR-grafting primers MBC1LGP1 (SEQ ID NO: 29) and M
BC1LGP3 (SEQ ID NO: 30) has the sense DNA sequence and the CDR graph
Footing primers MBC1LGP2 (SEQ ID NO: 31) and MBC1LGP4
(SEQ ID NO: 32) has an antisense DNA sequence, and the primers
The external primers MBC1LV have complementary sequences of 15 to 21 bp on both ends.
S1 (SEQ ID NO: 33) and MBC1LVR1 (SEQ ID NO: 34) are CDR grafts.
It has homology with the primers MBC1LGP1 and MBC1LGP4.
. CDR-grafting primers MBC1LGP1, MBC1LGP2, M
BC1LGP3 and MBC1LGP4 were analyzed using urea-denatured polyacrylamide gel.
(Molecular Cloning: A Laboratory
Manual, Sambrook et al., Cold Spring Harbor
Laboratory Press, 1989), extraction from the gel was performed using crus
h and soak method (Molecular Cloning: A Labo
ratory Manual, Sambrook et al., Cold Spring
The procedure was carried out using a 6% RT-PCR kit (Harbor Laboratory Press, 1989). That is, 1 nmole of CDR-grafting primer was used.
Separation on a denaturing polyacrylamide gel and identification of DNA fragments of the desired size were performed sequentially.
The crush and soak method was used to irradiate ultraviolet light onto a gel thin-layer plate.
The gel was recovered and resuspended in 20 μl of 10 mM Tris-HCl (pH 7.4), 1 mM
The DNA was dissolved in EDTA solution. PCR was performed using TaKaRa Ex Taq (Takara Shuzo) in a 100 μl reaction.
The mixture was added with the CDR-grafting primer MBC1LGP prepared as above.
1, MBC1LGP2, MBC1LGP3 and MBC1LGP4, respectively.
μl, 0.25 mM dNTP, 2.5 U TaKaRa Ex Taq
Using the attached buffer, conditions were 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute.
The reaction mixture was subjected to five 1-minute temperature cycles, and 50 pmoles of external plug was added.
Add MBC1LVS1 and MBC1LVR1 to the buffer and repeat the same temperature cycle.
The DNA fragments amplified by PCR were incubated with 3% Nu Siev
e Agarose using GTG agarose (FMC Bio Products)
The agarose strip containing the 421 bp DNA fragment was excised and purified using GENECLE.
Using the ANII Kit (BIO101), DNA fragments were extracted according to the instructions attached to the kit.
The resulting PCR reaction mixture was digested with BamHI and HindIII.
The DNA was subcloned into pUC19 prepared by the above procedure, and the nucleotide sequence was determined.
The resulting plasmid was named hMBCL/pUC19.
However, the amino acid sequence at position 104 of CDR4 (amino acid number 96 according to Kabat)
Since the amino acid had become arginine, a correction process was performed to change this to tyrosine.
The primer MBC1LGP10R (SEQ ID NO: 35) was designed and synthesized.
Using TaKaRa Taq (Takara Shuzo), template DNA was added to 100 μl of the reaction mixture.
0.6 μg of plasmid hMBCL/pUC19 as a primer, MB
C1LVS1 and MBC1LGP10R were used at 50 pmole and 2.5 U, respectively.
TaKaRa Ex Taq (Takara Shuzo) was added under conditions containing 0.25 mM dNTP.
Using the buffer provided, 50 μl of mineral oil was overlaid and incubated at 94°C for 1 minute and at 55°C for 1 minute.
The PCR amplification was performed 30 times with a cycle of 1 minute at 72°C and 1 minute at 72°C.
The DNA fragment was then lysed in 3% Nu Sieve GTG agarose (FMC Bio.
The DNA fragment was separated by agarose gel electrophoresis using GENECLE® 1000. The agarose gel containing the 421 bp DNA fragment was excised and purified using GENECLE® 1000.
Using the ANII Kit (BIO101), DNA fragments were extracted according to the instructions attached to the kit.
The resulting PCR reaction mixture was digested with BamHI and HindIII.
The fragment was subcloned into pUC19 prepared by cloning the fragment. M13 Primer M4 primer and M13 Primer RV primer
As a result of determining the base sequence using the timer, the correct sequence was obtained.
This plasmid was digested with HindIII and BlnI to obtain a 416 bp fragment.
The fragments were separated by 1% agarose gel electrophoresis. GENECLEAN II Kit
The DNA fragment was purified using a kit containing 100% PEG/PPG-100 (BIO101) according to the instructions attached to the kit.
The PCR reaction mixture was digested with HindIII and BlnI.
The plasmid was introduced into the prepared plasmid Cλ/pUC19 to obtain the plasmid hMBC1Laλ/p
This plasmid was digested with EcoRI to encode the humanized L chain.
A sequence containing the EF1α promoter was introduced into the plasmid pCOS1.
The initiation codon of the humanized L chain was positioned downstream of the
The plasmid was named hMBC1Laλ/pCOS1. Humanized light chain version
The base sequence of "a" (including the corresponding amino acids) is shown in SEQ ID NO: 66.
The amino acid sequence of version a is shown in SEQ ID NO: 47. Version "b" was created using PCR mutagenesis.
In b", glycine at position 43 (amino acid number 43 according to Kabat) is protease.
Lysine at position 49 (amino acid number 49 according to Kabat) was added to loline.
Mutagenic primer MBC1LGP5R was designed to change the nucleotide sequence to a spartic acid.
(SEQ ID NO: 36) and primer MBC1LVS1 to clone plasmid hMBC1L
PCR was carried out using aλ/pUC19 as a template, and the resulting DNA fragment was digested with BamHI.
and HindIII to form the BamHI and HindIII sites of pUC19.
After determining the base sequence, the DNA was subcloned using the restriction enzymes HindIII and Af
hMBC1Laλ digested with HmcII and hMBC1Laλ digested with HindIII and AflII
/pUC19. The resulting plasmid was designated hMBC1Lbλ/pUC19.
The resulting mide was digested with EcoRI, and a fragment containing the DNA encoding the humanized L chain was isolated.
The humanized L chain was inserted into the plasmid pCOS1 and placed downstream of the EF1α promoter.
The initiation codon was positioned at the end of the plasmid. The resulting plasmid was designated hMBC1Lb
This was named λ/pCOS1. Version "c" was created using PCR-based mutagenesis.
In c", the serine at position 84 (amino acid number 80 according to Kabat) is protonated.
The mutagenic primer MBC1LGP6S (SEQ ID NO: 1) was designed to change the nucleotide sequence to phospho-.
No. 37) and primer M13Primer RV to obtain the plasmid hMBC1L.
PCR was carried out using aλ/pUC19 as a template, and the resulting DNA fragment was digested with BamHI.
and HindIII, and then digested with BamHI and HindIII.
After determining the base sequence, the fragment was digested with the restriction enzymes BstPI and Aor51HI and subcloned into pUC19.
The resulting plasmid was ligated with hMBC1Laλ/pUC19 digested with PI and Aor51HI.
The resulting plasmid was designated hMBC1Lcλ/pUC19.
The resulting mide was digested with the restriction enzyme EcoRI to obtain a sequence containing the sequence encoding the humanized L chain.
was introduced into the EcoRI site of the plasmid pCOS1 and placed under the EF1α promoter.
The initiation codon of the humanized L chain was positioned at the beginning of the sequence.
The resulting vector was named hMBC1Lcλ/pCOS1. Versions "d", "e", and "f" were created using PCR-based mutagenesis.
Each version was ranked 91st (Ka) for "a", "b" and "c".
Change tyrosine at amino acid position 87 (according to the bat rule) to isoleucine
The mutagenic primer MBC1LGP11R (SEQ ID NO: 38) and the primer
hMBC1Laλ/pCOS
1. hMBC1Lbλ/pCOS1 and hMBC1Lcλ/pCOS1 were used as templates.
PCR was performed using the above DNA fragments, and the resulting DNA fragments were digested with BamHI and HindIII.
pUC19 was prepared by digesting the
After determining the base sequence, the fragment was digested with HindIII and BlnI.
Cλ/pUC prepared by cloning and digesting with HindIII and BlnI
19. The resulting plasmids were named hMBC1Ldλ/pUC19, hMBC1Ldλ/pUC19, and hMBC1Ldλ/pUC19, respectively.
These plasmids were named hMBC1Lfλ/pUC19 and hMBC1Lfλ/pUC19.
The plasmid was digested with EcoRI, and a sequence containing the humanized L chain coding sequence was inserted into the plasmid.
The human EF1α promoter was inserted into the EcoRI site of pCOS1.
The plasmid thus obtained was then
These are hMBC1Ldλ/pCOS1, hMBC1Leλ/pCOS1, and hM
The vector was named BC1Lfλ/pCOS1. Versions "g" and "h" were created using PCR mutagenesis.
Both versions are ranked 36th in the order of "a" and "d" (according to Kabat's rules).
The histidine at amino acid position 36 was changed to tyrosine.
Heterogeneous primers MBC1LGP9R (SEQ ID NO: 39) and M13 Prime
rRV was used as a primer and hMBC1Laλ/pUC19 was used as a template.
PCR was performed using the obtained PCR product and M13 Primer M4 as primers.
The plasmid hMBC1Laλ/pUC19 was used as a template.
PCR was performed. The resulting DNA fragment was digested with HindIII and BlnI.
, HindIII and BlnI.
This plasmid was used as a template and subcloned into UC19 using primer M
PCR using BC1LGP13R (SEQ ID NO: 40) and MBC1LVS1 as primers
The resulting PCR fragment was digested with ApaI and HindII.
Plasmid hMBC1Laλ/pUC1 digested with paI and HindIII
The nucleotide sequence was determined and the correct
The plasmids containing the sequence were then separated into hMBC1Lgλ/pUC19 and hMBC1L
hλ/pUC19, and these plasmids were digested with the restriction enzyme EcoRI to
The sequence containing the sequence encoding the modified light chain was inserted into the EcoRI site of the plasmid pCOS1.
The initiation codon of the humanized L chain is located downstream of the EF1α promoter.
The plasmids thus obtained were each designated hMBC1Lgλ/p
They were named COS1 and hMBC1Lhλ/pCOS1. Versions "i", "j", "k", "l", "m", "n" and "o"
The mutagenic primer MBC1LGP was prepared using PCR mutagenesis.
14S (SEQ ID NO: 41) and primer VIRV(λ) (SEQ ID NO: 43).
PCR was performed using the plasmid hMBC1Laλ/pUC19 as a template, and the resulting
The NA fragment was digested with ApaI and BlnI, and
The plasmid was subcloned into hMBC1Lgλ/pUC19 prepared by
The base sequence was determined, and mutations corresponding to each version were introduced.
The resulting clones were selected.
UC19 (x = i, j, k, l, m, n, o), and this plasmid was
The plasmid pCOS1 was digested with .I. and the sequence containing the humanized L chain coding sequence was inserted into
The EcoRI site was inserted downstream of the EF1α promoter to insert the initiation gene of the humanized L chain.
The resulting plasmid was designated hMBC1Lxλ/
It was named pCOS1 (x = i, j, k, l, m, n, o). Version "j"
, "l", "m" and "o" base sequences (including the corresponding amino acids)
These are shown in SEQ ID NOs: 67, 68, 69, and 70.
The amino acid sequences 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 "o" amino acid sequence of tyrosine at position 87
This is a version where the amino acid sequence is substituted with isoleucine, and the restriction enzyme Aor51 in FR3
Using the MI cleavage site, version "h" was converted to each of versions "i", "j",
It is made by replacing it with "m", "l" or "o".
That is, the expression plasmid hMBC1Lxλ/pCOS1 (x=i, j, m, l,
o) Aor51HI fragment 51 including CDR3 and part of FR3 and FR4
Except for 4 bp, the CDR
3 and a 514 bp Aor51HI fragment containing part of FR3 and FR4.
As a result, the tyrosine at position 91 (amino acid number 87 according to Kabat)
The base sequence was determined and each version "i" and "
j”, m”, l” and o at position 91 (amino acids according to Kabat
A clone in which tyrosine at position 87 was replaced with isoleucine was selected, and the corresponding
Let the versions "p", "q", "s", "r" and "t" be the obtained versions respectively.
The resulting plasmid was hMBC1Lxλ/pCOS1 (x = p, q, s, r, t).
The base sequences of versions "q", "r", "s" and "t" (corresponding
The amino acids corresponding to the amino acids α, β ...
The amino acid sequences of these versions are SEQ ID NOs: 52, 53, 54, and 55, respectively.
55. Plasmid hMBC1Lqλ/pCOS1 was digested with HindIII and EcoRI.
and ligated into plasmid pUC19 digested with HindIII and EcoRI.
The plasmid was subcloned and designated hMBC1Lqλ/pUC19. The positions of the substituted amino acids in each version of the humanized L chain are shown in Table 7. In Table 7, Y is tyrosine, P is proline, K is lysine, V is valine, and D is aspartate.
I represents isoleucine, and I represents oleic acid. The above-mentioned plasmids hMBC1HcDNA/pUC19 and hMBC1L
The E. coli carrying qλ/pUC19 was Escherichia coli JM1
09 (hMBC1HcDNA/pUC19) and Escherichia
coli JM109 (hMBC1Lqλ/pUC19),
The Biotechnology and Life Sciences Research Institute (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture) was established on August 1, 1996.
On the 5th, Escherichia coli JM109 (hMBC1H cDNA
/pUC19) is FERM BP-5629, Escherichia
E. coli JM109 (hMBC1Lqλ/pUC19) was obtained from FERM.
It has been internationally deposited under the Budapest Treaty as BP-5630. (5) Transfection into COS-7 cells Antigen-binding activity of the hybrid antibody and humanized #23-57-137-1 antibody
To evaluate the potency and neutralizing activity, the expression plasmids were transiently transfected into COS-7 cells.
That is, for the transient expression of the L-chain hybrid antibody, the plasmid
hMBC1H cDNA/pCOS1 and h/mMBC1L(λ)/neo, hMB
C1H cDNA/pCOS1 and m/hMBC1Laλ/neo, hMBC1Hc
DNA/pCOS1 and m/hMBC1Ldλ/neo, hMBC1HcDNA/
pCOS1 and hmmMBC1L(λ)/neo or hMBC1HcDNA/
The combination of pCOS1 and mhmMBC1L(λ)/neo was
The cells were electroporated using a ulser device (BioRad) and CO
S-7 cells were co-transfected with 1 × 10⁷cells/ml of cells
10 μl of each plasmid DNA was added to 0.8 ml of COS-7 cells suspended at a concentration of
A pulse was applied at 1,500 V and a capacitance of 25 μF.
After a 0-minute recovery period, the electroporated cells were incubated with 2% Ult
DMEM culture medium containing ra Low IgG fetal bovine serum (GIBCO) (
The cells were suspended in CO2 solution (GIBCO) and incubated in a 10 cm culture dish.₂Cultivated in an incubator
After 72 hours of culture, the culture supernatant was collected and centrifuged to remove cell debris.
The humanized #23-57-137-1 antibody was transiently expressed using the hMB plasmid.
C1HcDNA/pCOS1 and hMBC1Lxλ/pCOS1 (x = a to t)
Either combination was analyzed using a Gene Pulser device (BioRad).
The hybrid antibody was transfected into COS-7 cells in the same manner as in the hybrid antibody.
The resulting culture supernatant was subjected to ELISA. Hybrid antibodies or humanized antibodies from the COS-7 cell culture supernatant were also tested.
Purification was performed using the AffiGel Protein A MAPS II kit (BioR
ad) according to the instructions provided with the kit. (6) ELISA (i) Measurement of antibody concentration An ELISA plate for measuring antibody concentration was prepared as follows. ELISA
Each well of a 96-well plate for SA (Maxisorp, NUNC) was filled with solid-phase buffer.
-(0.1M NaHCO₃, 0.02% NaN₃) to a concentration of 1 μg/ml
100 μl of the prepared goat anti-human IgG antibody (TAGO) was immobilized, and 200 μl
Dilution buffer (50 mM Tris-HCl, 1 mM MgCl₂, 0.1
M NaCl, 0.05% Tween20, 0.02% NaN₃, 1% cow
After blocking with bovine serum albumin (BSA), pH 7.2, hybrid antibodies were
The culture supernatant or purified high-density lipoprotein (HDL) from COS-7 cells expressing human or humanized antibodies
Hybridized or humanized antibodies were serially diluted and added to each well.
After incubation and washing with PBS-Tween 20, alkaline phosphatase
100 μl of conjugated goat anti-human IgG antibody (TAGO) was added.
After incubation and washing with PBS-Tween 20, 1 mg/ml of substrate solution (
Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and then 40
The absorbance at 5 nm was measured with a microplate reader (Bio Rad).
Hu IgGl λ Purified (Th
(e Binding Site) was used. (ii) Measurement of antigen binding ability ELISA plates for antigen binding measurement were prepared as follows.
Each well of a 96-well ISA plate is adjusted to a concentration of 1 μg/ml with immobilization buffer.
100 μl of diluted human PTHrP (1-34) was immobilized on the plate.
After blocking with buffer, hybrid or humanized antibodies are expressed.
COS-7 cell culture supernatant or purified hybrid antibody or humanized antibody
The diluted solution was added to each well.
After washing with 0, alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO)
100 μl of the mixture was added. Incubate at room temperature and wash with PBS-Tween 20.
Then, 1 mg/ml of substrate solution (Sigma 104, p-nitrophenyl phosphate
(Sigma) was added, and the absorbance at 405 nm was measured with a microplate reader.
(Bio Rad). (7) Activity Confirmation (i) Evaluation of Humanized H Chain The antibody combining humanized H chain version "a" with a chimeric L chain was used as a chimeric antibody.
This result indicates that humanization of the H chain V region is effective for
Hereinafter, humanized H chain version “a” will be referred to as humanized H chain version “a.”
The light chain was used as the heavy chain of the hybridized antibody. (ii) Hybrid antibody activity (ii-a) FR1,2/FR3,4 hybrid antibody When the light chain was h/mMBC1L(λ), no activity was observed, but when the light chain was m/
In the case of hMBC1Laλ or m/hMBC1Ldλ, both chimeras were #23.
These results indicate that the FR3, FR4, and FR5 antibodies showed binding activity equivalent to that of the FR5 antibody.
4 is a humanized antibody, but there are amino acid residues to be substituted in FR1 and FR2.
This suggests the presence of a hybrid antibody. (ii-b) FR1/FR2 hybrid antibody When the L chain was mhmMBC1L(λ), no activity was observed, but when the L chain was hm
In the case of mMBC1L(λ), binding was equivalent to that of chimeric #23-57-137-1 antibody.
These results show that FR1, out of FR1 and FR2, is a humanized antibody.
Although there is no problem, it suggests that there are amino acid residues that should be substituted within FR2. (iii) Activity of humanized antibodies Regarding humanized antibodies using each of versions "a" to "t" as the light chain,
The antigen binding activity was measured using the L chain versions "j", "l", and "
Humanized antibodies with m", o", q", r", s", and t are chimeric
The antibody showed PTHrP binding ability equivalent to that of the humanized antibody. (8) Establishment of a CHO stable-producing cell line To establish a stable-producing cell line for the humanized antibody, the expression plasmid was transfected into CHO cells.
The humanized antibody was then introduced into DXB11 cells. In other words, the establishment of a stable humanized antibody producing cell line was achieved by using an expression plasmid for CHO cells.
hMBC1HcDNA/pCHO1 and hMBC1Lmλ/pCOS1 or hM
BC1HcDNA/pCHO1 and hMBC1Lqλ/pCOS1 or hMB
The combination of C1HcDNA/pCHO1 and hMBC1Lrλ/pCOS1
Electroporation was performed using a Gene Pulser device (Bio Rad).
The expression vectors were co-transfected into CHO cells using the restriction enzyme P
The DNA was digested with vuI to form linear DNA, extracted with phenol and chloroform, and then
The DNA was recovered by ethanol precipitation and used for electroporation.
0.8 ml of CHO cells suspended in a solution at a cell concentration of 1 x 107 cells/ml
10 μg of each plasmid DNA was added, and the voltage was set to 1,500 V and a capacitance of 25 μF.
After a 10-minute recovery period at room temperature, electroporation
The treated cells were cultured in MEM-α medium (G
The cells were suspended in 1000 kJ/ml PBS (IBCO) and incubated in a 96-well plate (Falcon) with CO₂Incubus
The day after the start of the culture, 10% fetal bovine serum (GIBCO) and
and 500 mg/ml GENETICIN (G418 Sulfate, GI
MEM containing ribonucleosides and deoxyribonucleosides, supplemented with PEG-400 (BCO)
The medium was replaced with selective α medium (GIBCO) to select cells into which antibody genes had been introduced.
After changing the selective medium, the cells were observed under a microscope about two weeks later to confirm that the cells were growing well.
After the antibody concentration was confirmed, the amount of antibody produced was measured by the antibody concentration measurement ELISA.
Cells with high productivity were selected. The established stable antibody-producing cell line was expanded and cultured in roller bottles at 2% U.
ltra Low IgG with fetal bovine serum, ribonucleosides and deoxyribonucleic acid
Large-scale culture was carried out using ribonucleoside-free MEM-α medium.
On the fourth day, the culture supernatant was collected and filtered through a 0.2 μm filter (Millipore).
The humanized antibody was purified from the CHO cell culture supernatant.
, POROS Protein A column (PerSeptive Biosystem
s) and then treated with ConSep LC100 (Millipore) according to the attached instructions.
The neutralizing activity was measured and the efficacy was tested in a hypercalcemia model animal.
The concentration and antigen-binding activity of the purified humanized antibody obtained were measured using the same EL
Measurement was performed using the ISA system. [Reference Example 5] Measurement of Neutralizing Activity The neutralizing activity of mouse antibodies, chimeric antibodies, and humanized antibodies was measured using rat bone marrow smear.
The experiment was carried out using the tumor cell line ROS17/2.8-5 cells.
2.8-5 cells were cultured in Ham's F-1 medium containing 10% fetal bovine serum (GIBCO).
2 medium (GIBCO)₂The cells were cultured in an incubator.
/2.8-5 cells were seeded in a 96-well plate at 104 cells/100 μl/well and incubated for 1 day.
The cells were cultured for 1 h and then in H20 containing 4 mM Hydrocortisone and 10% fetal bovine serum.
The medium was replaced with am'S F-12 medium (GIBCO). Culture was continued for another 3 to 4 days.
After that, the cells were washed with 260 μl of Ham's F-12 medium (GIBCO) and
mM isobutyl-1-methyl xanthine (IBMX, SIGMA) and 1
80 μl of Ham's F-1 containing 0% fetal bovine serum and 10 mM HEPES
2 was added and incubated at 37°C for 30 minutes. The mouse antibody, chimeric antibody, or humanized antibody whose neutralizing activity was to be measured was prepared in advance.
10 μg/ml, 3.3 μg/ml, 1.1 μg/ml and 0.37 μg/ml
ml group, 10 μg/ml, 2 μg/ml, 0.5 μg/ml and 0.01 μg/ml
g/ml group, or 10 μg/ml, 5 μg/ml, 1.25 μg/ml, 0
Serial dilutions were made into groups of 0.63 μg/ml and 0.31 μg/ml, and 4 ng/ml
The prepared PTHrP(1-34) was mixed in equal amounts, and each antibody and PTHrP(1-34)
80 μl of the mixture of the above antibodies was added to each well. The final concentration of each antibody was 4 times the above antibody concentration.
The concentration of PTHrP(1-34) becomes 1 ng/ml.
After treatment at room temperature, the culture supernatant was discarded, and the cells were washed three times with PBS.
The cAMP in the cells was extracted with 1 ml of 0.3% hydrochloric acid and 95% ethanol.
The hydrochloric acid ethanol was evaporated using a evaporator, and the cAMP EIA kit (CAY
Add 120 μl of EIA buffer provided by MAN CHEMICALS.
After extracting AMP, the cAMP was analyzed using a cAMP EIA kit (CAYMAN CHEMICAL
'S) cAMP was measured according to the attached instructions. As a result, it was found that the cAMP level was equivalent to that of the chimeric antibody.
In the antigen-binding light chain version, the tyrosine at position 91 is replaced with an isoleucine.
Humanized antibodies with modified versions "q", "r", "s", and "t" are
Among them, version "q" shows the strongest neutralizing activity.
All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.
The present invention is incorporated herein by reference. Industrial Applicability The present invention provides a method for inhibiting the binding of parathyroid hormone-related peptide to its receptor.
The present invention provides a tissue decomposition inhibitor containing a substance as an active ingredient.
is useful as a pharmaceutical composition for suppressing weight loss associated with cancer cachexia, etc.
. Sequence Listing Free Text SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA SEQ ID NO: 8: Synthetic DNA SEQ ID NO: 9: Synthetic DNA SEQ ID NO: 10: Synthetic DNA SEQ ID NO: 11: Synthetic DNA SEQ ID NO: 12: Synthetic DNA SEQ ID NO: 13: Synthetic DNA SEQ ID NO: 14: Synthetic DNA SEQ ID NO: 15: Synthetic DNA SEQ ID NO: 16: Synthetic DNA SEQ ID NO: 17: Synthetic DNA SEQ ID NO: 18: Synthetic DNA SEQ ID NO: 19: Synthetic DNA SEQ ID NO: 20: Synthetic DNA SEQ ID NO: 21: Synthetic DNA SEQ ID NO: 22: Synthetic DNA SEQ ID NO: 23: Synthetic DNA SEQ ID NO: 24: Synthetic DNA SEQ ID NO: 25: Synthetic DNA SEQ ID NO: 26: Synthetic DNA SEQ ID NO: 27: Synthetic DNA SEQ ID NO: 28: Synthetic DNA SEQ ID NO: 29: Synthetic DNA SEQ ID NO: 30: Synthetic DNA SEQ ID NO: 31: Synthetic DNA SEQ ID NO: 32: Synthetic DNA SEQ ID NO: 33: Synthetic DNA SEQ ID NO: 34: Synthetic DNA SEQ ID NO: 35: Synthetic DNA SEQ ID NO: 36: Synthetic DNA SEQ ID NO: 37: Synthetic DNA SEQ ID NO: 38: Synthetic DNA SEQ ID NO: 39: Synthetic DNA SEQ ID NO: 40: Synthetic DNA SEQ ID NO: 41: Synthetic DNA SEQ ID NO: 42: Synthetic DNA SEQ ID NO: 43: Synthetic DNA SEQ ID NO: 44: Synthetic DNA

[Sequence List]

[Brief explanation of the drawings]

Figure 1 shows the effect of a humanized antibody on body weight and blood ionized calcium concentration in OCC-1-JCK transplanted mice. Figure 2 shows the effect of a humanized antibody on body weight and blood ionized calcium concentration in Colon 26 transplanted mice. Figure 3 shows the effect of a humanized antibody on body weight and blood ionized calcium concentration in LC-6-JCK transplanted rats. Figure 4 shows the time course of white blood cell count in LC-6-JCK transplanted rats.

⁷ 2-1-9 Kyobashi, Chuo-ku, Tokyo 105 43/00 105 111 111 (72) Inventor Isao Sato Chugai Pharmaceutical Co., Ltd. 2-1-9 Kyobashi, Chuo-ku, Tokyo (Note) This publication is based on the gazette published internationally by the International Bureau of Patents (WIPO). The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act) and is unrelated to this publication.

Claims (12)

[Claims] 1. A tissue decomposition inhibitor comprising a substance that inhibits the binding of parathyroid hormone-related peptide to its receptor. 2. The inhibitor according to claim 1, wherein the substance is an antagonist to the parathyroid hormone-related peptide receptor. 3. The inhibitor according to claim 1, wherein the substance is an anti-parathyroid hormone-related peptide antibody. 4. The inhibitor according to claim 1, wherein the substance is an anti-parathyroid hormone-related peptide antibody fragment and/or a modified product thereof. 5. The inhibitor according to claim 3 or 4, wherein the antibody is a monoclonal antibody. 6. The inhibitor according to claim 3 or 4, wherein the antibody is humanized or chimeric. 7. The inhibitor according to claim 6, wherein the humanized antibody is humanized #23-57-137-1 antibody. 8. The inhibitor according to any one of claims 1 to 7, wherein the tissue is muscle tissue or adipose tissue. 9. The inhibitor according to any one of claims 1 to 8, wherein the tissue degradation is caused by cancer cachexia, sepsis, severe trauma, or muscular dystrophy. 10. The inhibitor according to any one of claims 1 to 9, for administration to a patient whose blood concentration of cytokine is higher than normal. 11. The cytokines are IL-6, G-CSF, IL-11 and LIF.
The inhibitor according to claim 10, which is at least one selected from the group consisting of: 12. The inhibitor according to claim 10, wherein the cytokine is an inflammatory cytokine.