HK1096974B

HK1096974B - Antibody against human parathormone related peptides

Info

Publication number: HK1096974B
Application number: HK07101636.1A
Authority: HK
Inventors: 佐藤功; 若原裕二; 薮田尚弘
Original assignee: 中外制药株式会社
Priority date: 1996-09-26
Filing date: 2007-02-12
Publication date: 2011-10-21

Description

antibody against human parathyroid hormone related protein

The invention is a divisional application of international application PCT/JP97/03382 with application number 97199722.5, which enters the Chinese national stage at 14/5 of 1999.

Technical Field

The present invention relates to a human/mouse chimeric antibody comprising a variable region (V region) of a mouse monoclonal antibody against parathyroid hormone-related protein and a constant region (C region) of a human antibody, a humanized antibody in which complementarity determining regions of light (L chain) and heavy (H chain) chains of a V region of a mouse monoclonal antibody against parathyroid hormone-related protein (PTHrP) are grafted to a human antibody, the L chain and H chain of said antibody, and a polypeptide comprising a V region constituting the L chain or H chain of said antibody.

The present invention also relates to a DNA encoding the above antibody, particularly the base sequence of V region thereof, and a DNA encoding L or H chain of V region. The invention further relates to a recombinant vector comprising said DNA and a host transformed with said vector.

In addition, the present invention relates to a method for producing chimeric and humanized antibodies against PTHrP. And a pharmaceutical composition comprising the anti-PTHrP antibody as an active ingredient, which is a hypercalcemia-suppressing agent or a hypophosphatemia-promoting agent.

Background

Hypercalcemia associated with malignancy is a serious complication found in 5% to 20% of malignant patients, which is considered to be the ultimate symptom of malignancy, since it necessarily leads to death if the symptom is left untreated. Control of hypercalcemia greatly affects patient prognosis and QOL (quality of life); therefore, it will play an important role clinically.

Generally, hypercalcemia in patients with malignant tumors is roughly classified into HHM (malignant humoral hypercalcemia) based on tumor-producing humoral bone resorption factors, and LOH (local osteolytic hypercalcemia) based on local activity of tumors metastasizing or penetrating into bone. In HHM, it is considered that bone resorption or bone sclerosis is promoted to increase calcium flux and produce hypercalcemia accompanied by a decrease in renal calcium excretion ability (S.Wada and N.Nagata, International medicine, 69, 644-.

It is believed that serum calcium concentrations in excess of 12 mg/dl show symptoms of hypercalcemia; because of its symptoms: anorexia (loss of appetite), nausea and vomiting are non-specific symptoms in the early stages of malignant patients. When hypercalcemia worsens, reduction of water concentration capacity due to injury of the distal tubules leads to diuresis, anorexia nausea and dehydration due to lack of water intake.

Moseley, j.m. et al found that the parathyroid hormone-related protein (hereinafter referred to as "PTHrP") which is a substance like parathyroid hormone (PTH), is a humoral factor responsible for HHM in hypercalcemia associated with malignancies: annual proceedings of the national academy of sciences of the United states (1987)84, 5048-5052.

Thus, the gene encoding PTHrP was isolated (Suva, l.j. et al, science (1987)237, 893), and this analysis demonstrated that three human PTHrP with 139, 141 and 173 amino acids were produced due to the alternate splicing of the genes, since the restrictive degradation of the PTHrP (1-139) with its complete structure occurred in the blood: baba, h., clinical calcium (1995)5,229- 223. In PTHrP, 8 amino acids among 13 amino acids at the N-terminus are identical to the corresponding sites of PTH, and the amino acid sites at positions 14 to 34 thereof are presumed to have a steric structure similar to PTH; thus, PTHrP and PTH bind to the common PTH/PTHrP receptor at least in the N-terminal region: juepner, H. et al, science (1991)254, 1024-; Abou-Samra, A-B, et al, annual proceedings of the national academy of sciences of the United states (1992)89, 2732-.

A large number of tumor tissues have been reported to produce PTHrP, and it has been described that PTHrP is produced not only in tumors but also in various normal tissues of fetuses and adults, including skin, central nervous system, uterus, placenta, breast during lactation, thyroid, parathyroid, adrenal gland, liver, kidney, and bladder: burtis, w.j., clinical chemistry (1992)38, 2171-2183; stewart, A.F. and Broadus, A.E., J.Clin Endocrinology (1991)71, 1410-. In addition, PTHrP is thought to play an important role in the regulation of calcium metabolism during the fetal to neonatal period than in maintaining higher concentrations in the mother.

PTH/PTHrP receptors are now known to be present mainly in bone and kidney (C.Shigeno, clinical calcium (1995)5, 355-359), which activate most of the intercellular signaling systems by binding PTHrP to the receptor. One of them is adenylate cyclase, the other phospholipase C. Activation of adenylate cyclase increases the concentration of intercellular cAMP to activate protein kinase a. Phospholipase C cleaves 4, 5-diphosphoylinositol to produce inositol 1, 4, 5-triphosphate and diacylglycerol. G proteins are also involved in these signalling systems: coleman, d.t. et al, biochemical mechanism of parathyroid hormone activity, "parathyroid hormone" (Bilezikian, j.p. et al), Raven Press, new york (1994) page 239.

Through these signaling systems, PTHrP causes hypercalcemia, hypophosphatemia, a decrease in renal phosphate reabsorption ability, an increase in renal cAMP excretion, and the like, which are observed in HHM.

Therefore, it has been elucidated that PTHrP is closely associated with hypercalcemia associated with malignant tumors. In the treatment of hypercalcemia associated with malignant tumor, calcitonin, steroid drugs, indomethacin, inorganic phosphate, diphosphate, etc. have been used, and body fluid replacement has also been used. However, these drugs are found to have reduced effects, show severe side effects, or have slow expression of their pharmacological effects after continuous use; it is therefore highly desirable to use agents or drugs having higher therapeutic effects and lower side effects.

On the other hand, Kukreja, s.c. et al report a new method for treating hypercalcemia associated with malignancy: when a hypercalcemic-producing athymic mouse transplanted with human lung or laryngeal cancer cells was administered with a neutralizing antiserum against PTHrP, the blood calcium concentration and urinary cAMP levels decreased: journal of clinical research (1988)82, 1798-. Kanji Sato et al reported that hypercalcemia was alleviated and survival time of mice was significantly prolonged when an anti-PTHrP (1-34) antibody was administered to nude mice transplanted with PTHrP-producing human tumors: bone&MineRes. (1993)8, 849-. In addition, Japanese unexamined patent application publication No. 4-228089 discloses a mouse/human chimeric antibody against human PTHrP (1-34).

Murine monoclonal antibodies are highly immunogenic (sometimes also used as "antigenic") in humans, which limits the pharmaceutical therapeutic value of murine monoclonal antibodies in humans. For example, when a murine antibody is administered to a human, it may be metabolized as a foreign substance; therefore, the half-life of murine antibodies in humans is relatively short and their desired effects are not efficiently expressed. In addition, human anti-murine antibodies (HAMA) against administered murine antibodies may cause adverse and dangerous immune reactions to patients, such as serum sickness and other allergic reactions. Therefore, murine monoclonal antibodies cannot be frequently administered to humans.

To solve these problems, methods for reducing the immunogenicity of antibodies of non-human origin, for example, monoclonal antibodies derived from mice, have been developed. One of the methods is to prepare a chimeric antibody in which the variable region (V region) is derived from a murine monoclonal antibody and the constant region (C region) is derived from a suitable human antibody.

Since the resulting chimeric antibody has the entire variable region of the original murine antibody, it is considered that the chimeric antibody S has the same specific antigen binding as the original murine antibody. Further, such a chimeric antibody has a substantially reduced proportion of amino acid sequences derived from a non-human animal; thus, the monoclonal antibody binds to its antigen to an equivalent extent as the original murine antibody, with reduced immunogenicity, but may still produce some immune response to the murine variable region: LoBuglio, A.F. et al, annual proceedings of the national academy of sciences USA, 86, 4220-.

The second approach to reducing the immunogenicity of murine antibodies is still more complex, but is expected to further greatly reduce the potential immunogenicity of murine antibodies. In this method, only the Complementarity Determining Regions (CDRs) of the murine antibody variable region are grafted onto the human antibody variable region to produce an "engineered" human variable region. If necessary, a part of the amino acid sequence of the Framework Region (FR) supporting the CDR in the mouse antibody variable region may be grafted to the human antibody variable region to make the CDR structure of the altered human variable region closer to the similar structure of the original mouse antibody.

These humanized, engineered human antibody variable regions are then combined with human constant regions. In the final engineered humanized antibody, the portions derived from the non-human amino acid sequence are only the CDRs and a small portion of the FRs. CDRs consist of highly variable amino acid sequences and sequences that do not exhibit any species specificity. Thus, a humanized antibody comprising murine CDRs will no longer exhibit greater immunogenicity than a naturally occurring human antibody comprising human CDRs.

As for humanized antibodies, there are also the following references: riechmann, L, et al, Nature, 332, 323-; verhoeye, M. et al, science 239, 1534-1536, 1988; kettleborough, C.A., et al, protein engineering, 4, 773-; maeda, H, et al, human antibodies and hybridomas, 2, 124-; gorman, S.D., et al, annual newspaper of national academy of sciences USA, 88, 4181, 4185, 1991; tempest, P.R., et al, Bio/technology, 9, 266-271, 1991; co, M.S., et al, annual newspaper of national academy of sciences USA, 88, 2869-; carter, p. et al, annual newspaper of national academy of sciences USA, 89, 4285-; co, M.S. et al, J. immunology, 148, 1149-1154, 1992; sato, K. et al, cancer research, 53, 851-.

Although humanized antibodies are expected to be beneficial for the therapeutic purposes mentioned previously, the above references do not disclose humanized antibodies against PTHrP, nor do they suggest anything. In addition, there is generally no standard method applicable to any antibody in the preparation of humanized antibodies; there is a need to develop various means and methods for humanized antibodies that exhibit sufficient binding force and neutralizing activity to specific antigens: see, e.g., Sato, K. et al, cancer research, 53, 851-.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a human/mouse chimeric antibody comprising a variable region (V region) of a mouse anti-PTHrP monoclonal antibody and a constant region (C region) of a human antibody, a humanized antibody in which complementarity determining regions of the V region consisting of a light chain (L chain) and a heavy chain (H chain) of the mouse anti-PTHrP monoclonal antibody are grafted to a human antibody, the L chain and the H chain of said antibody, and a polypeptide comprising the V region consisting of the L chain or the H chain of said antibody.

Another object of the present invention is to provide a DNA comprising a base sequence encoding the above antibody, particularly the V region thereof, and a DNA encoding the L or H chain of a polypeptide comprising the V region. It is still another object of the present invention to provide a recombinant vector comprising said DNA and a host transformed with said vector. In addition, an object of the present invention is to provide a method for producing a chimeric humanized antibody against PTHrP. It is also an object of the present invention to provide an anti-PTHrP antibody having a high neutralizing activity. It is another object of the present invention to provide a pharmaceutical composition and a hypercalcemia-suppressing agent, a hypophosphatemia-ameliorating agent, or an alkalosis-ameliorating agent, each containing an antibody or a humanized antibody against PTHrP as an active ingredient.

As a result of intensive studies after combining the above objects, the present inventors have succeeded in obtaining an anti-PTHrP murine monoclonal antibody, the immunogenicity of which is reduced in humans; thus, the present invention has been achieved.

The present invention relates to a chimeric L chain comprising an L chain C region of a human antibody and an L chain V region of a murine monoclonal antibody against PTHrP. The L chain V region comprises a sequence shown as SEQ ID NO: 45 and the L chain C region comprises a C lambda region.

The present invention also relates to a chimeric H chain comprising a human antibody H chain C region and a murine monoclonal antibody H chain V region against PTHrP. The H chain V region comprises a nucleotide sequence shown as SEQ ID NO: 46, and the H chain C region includes a C.lamda.1 region.

In addition, the present invention relates to a chimeric monoclonal antibody against PTHrP comprising said chimeric L chain and said chimeric H chain.

Further, the present invention also includes a polypeptide of an L chain V region of a humanized antibody comprising framework regions 1 to 4 of an L chain V region of a human antibody and complementarity determining regions 1 to 3 of an L chain V region of a murine monoclonal antibody against PTHrP. Complementarity determining regions 1 to 3 are as set forth in SEQ ID NOs: 59-61; framework regions 1 to 3 are derived from framework regions 1 to 3 of human antibody HSU03868, respectively, and framework region 4 comprises framework region 4 derived from human antibody S25755; or the framework regions 1 to 3 comprise substantially the same sequences as the framework regions 1 to 3 of human antibody HSU03868, respectively, and the framework region 4 comprises substantially the same sequence as the framework region 4 of human antibody S25755.

The term "substantially the same" as used herein means that the framework region of a human antibody used in a humanized antibody has amino acid deletions, substitutions and/or additions necessary to form the complementarity determining regions of a murine monoclonal antibody so that the humanized antibody has an activity equivalent to that of the murine monoclonal antibody.

Accordingly, the present invention relates to a polypeptide comprising a humanized antibody L chain V region wherein amino acids 36 and 49 of the framework region are tyrosine and aspartic acid, respectively, according to Kabat's convention (Kabat, e.a., et al, U.S. department of health and human services, U.S. government printing office, 1991).

The present invention also relates to a polypeptide comprising an L chain V region of a humanized antibody comprising an amino acid sequence set forth in SEQ ID NO: 48-51.

The present invention further relates to a polypeptide comprising a humanized antibody L chain V region wherein amino acids at positions 45 and 87 of the framework region are lysine and isoleucine, respectively, according to Kabat's convention.

The present invention still further relates to a polypeptide comprising an L chain V region of a humanized antibody comprising an amino acid sequence as set forth in SEQ ID NO: 52-55.

The present invention further relates to a polypeptide comprising a H chain V region of a humanized antibody comprising framework regions 1 to 4 of a H chain V region of a human antibody and complementarity determining regions 1 to 3 of a H chain V region of a murine monoclonal antibody against human PTHrP. The complementarity determining regions 1 to 3 include the amino acid sequences comprising SEQ ID NOs: 62-64, the framework regions 1 to 4 comprising framework regions 1 to 4 of human antibodies derived from human subgroup III (hsg III), Kabat, e.a., et al, U.S. department of health and human services, U.S. government printing office, 1991), particularly those derived from framework regions 1 to 4, respectively, of human antibody S31679, or sequences substantially identical to framework regions 1 to 4, respectively, of human antibody S31679.

The invention also relates to a polypeptide comprising SEQ ID NO: 56 in the presence of a polypeptide in the H chain V region of a humanized antibody.

The present invention also relates to an L chain of a humanized antibody against human PTHrP comprising the humanized antibody L chain V region polypeptide and a human antibody L chain C region polypeptide. The C region includes C λ region, the framework regions 1 to 3 include sequences substantially identical to the framework regions 1 to 3 of human antibody HSU03868, respectively, the framework region 4 includes sequences substantially identical to the framework region 4 of human antibody S25755, and the amino acid sequences of the complementarity determining regions 1 to 3 include SEQ ID NOs: 59-61.

The present invention also relates to a H chain of a humanized antibody against human PTHrP, which comprises polypeptides of the H chain C region and the H chain V region of said human antibody. The C region includes a C γ 1 region, the framework regions 1 to 4 include framework regions 1 to 4 derived from a human antibody of HSG III, and the complementarity determining regions 1 to 3 are SEQ ID NOs: 62-64.

Still further, the present invention also relates to an anti-PTHrP antibody having weak antigenicity and high neutralizing activity. The PTHrP antibody includes human antibodies, humanized antibodies, chimeric antibodies and primatized antibodies which can be used in the treatment of human diseases. The antibody has a low dissociation constant. The antibody of the present invention has high neutralizing activity due to its low dissociation constant, and thus can treat human diseases.

The dissociation constant of the antibody of the present invention is 1.86X 10^-7[M]Or less, with a dissociation rate constant of 1.22X 10^-1[l/Sec]Or less, and has a binding rate constant of 6.55X 10⁴[l/M.Sec]Or higher. These constants were determined by Scatchard analysis using RI-labeled ligands or surface plasmid genome resonance sensors.

The present invention further relates to a DNA comprising a base sequence encoding the L chain V region or H chain V region of a mouse monoclonal antibody against human PTHrP. The L chain V region and the H chain V region respectively comprise SEQID NO: 45 to 46, and the DNA including a base sequence encoding an L chain V region includes, for example, the amino acid sequences shown in SEQ ID NOS: 65, and the DNA comprising a base sequence encoding an H chain V region comprises the nucleotide sequence shown in SEQ ID NO: 57, or a pharmaceutically acceptable salt thereof.

In addition, the present invention also relates to a DNA encoding the chimeric L or H chain. The DNA encoding the L chain includes, for example, SEQ ID NO: 65, and the DNA encoding the H chain comprises the nucleotide sequence shown in SEQ ID NO: 57, or a nucleotide sequence thereof.

Still further, the present invention also relates to a DNA comprising a base sequence encoding the L chain V region or the H chain V region of the humanized antibody. The DNA having a base sequence encoding the L chain V region includes SEQ ID NO: 66-74, and the DNA having a base sequence encoding the H chain V region includes the base sequence shown in SEQ ID NO: 58, or a sequence shown in fig.

The invention also relates to a polypeptide comprising a nucleotide sequence encoding SEQ ID NO: 47 to 55, or a nucleotide sequence of any one of the amino acid sequences shown in FIGS. The DNA comprises SEQ ID NO: 66-74.

Still further, the present invention relates to a polypeptide encoding SEQ ID NO: 56 in the presence of a DNA of the H chain V region of the humanized antibody. The DNA comprises SEQ ID NO: 58, or a nucleotide sequence represented by the formula (I).

The present invention further relates to a recombinant vector containing any one of the DNAs.

The present invention still further relates to a transformant transformed with the recombinant vector.

In addition, the present invention also relates to a method for producing a chimeric or humanized antibody against human parathyroid hormone related protein, comprising culturing the transformant and collecting the chimeric or humanized antibody against human parathyroid hormone related protein from the resulting culture.

Still further, the present invention also relates to a pharmaceutical composition comprising the antibody as an active ingredient, or a hypercalcemia-suppressing agent or a hypophosphatemia-promoting agent. Malignant tumors cause calcium blood, and hypophosphatemia symptoms are commonly observed in patients with hypercalcemia associated with malignant tumors. Thus, the antibodies of the invention may be used to treat malignancies or to ameliorate the symptoms of hypercalcemia or hypophosphatemia. Malignant tumors include, but are not limited to, at least one selected from the group consisting of: pancreas, lung, pharynx, larynx, tongue, gingiva, esophagus, stomach, bile duct, breast, kidney, bladder, uterus and prostate cancer, and malignant lymphoma. The hypercalcemia-suppressing agent of the present invention can be used for treating any malignant tumor that causes hypercalcemia.

The present invention will be described in detail below.

1. Preparation of mouse monoclonal antibody against human PTHrP

Hybridomas can be prepared by cell fusion between myeloma cells and antibody-producing cells derived from animals immunized with an antigen, and clones producing antibodies specifically inhibiting the activity of PTHrP are selected from the resulting hybridomas to obtain a murine monoclonal antibody against PTHrP.

(1) Preparation of antigens

The PTHrP used for immunization of animals includes polypeptides having the whole or partial amino acid sequence of PTHrP prepared using recombinant DNA technology or chemical synthesis, and PTHrP derived from the supernatant of hypercalcemic-producing cancer cells. For example, a polypeptide containing the 1 st to 34 th amino acids of known PTHrP (Kemp, B.E., et al, science (1987)238, 1568-1570) [ PTHrP (1-34) ] can be used as the above-mentioned antigen. Human PTHrP (1-34) has the amino acid sequence shown in SEQ ID NO: 75.

The resulting PTHrP is attached to a carrier protein such as thyroglobulin, followed by addition of an adjuvant. Can be mixed with any adjuvant, including Freund's complete and incomplete adjuvants.

(2) Immunization and Collection of antibody-producing cells

The antigen obtained above is administered to a mammal such as a mouse, rat, horse, monkey, rabbit, goat or sheep. Immunization can be carried out by known methods, including intravenous, subcutaneous, and intraperitoneal injection. The injection interval of the immunization is not particularly limited, and may be several days to several weeks, preferably 4 to 21 days.

Two or three days after the last immunization, antibody-producing cells were collected. Antibody producing cells including spleen, lymph nodes, and peripheral blood cells; spleen cells are commonly used. The single dose of antigen used for immunization was 100 μ g per mouse.

(3) Determination of antibody titres

In order to determine the level of immune response in the immunized animal and to select hybridomas of interest from the cells subjected to the cell fusion treatment, the antibody titer in the blood of the immunized animal or the antibody titer in the supernatant of the antibody-producing cells was measured.

Antibody assay methods are known, including EIA (enzyme immunoassay), RIA (radioimmunoassay), and ELISA (enzyme-linked immunosorbent assay).

(4) Cell fusion

Myeloma cells for fusion with antibody-producing cells include cell lines derived from various animals such as mice, rats, and humans, and such techniques are generally provided in the art. Suitable cell lines for use are those which are drug resistant, do not survive in the unfused state and survive in the fused state in a selective medium such as HAT medium. An 8-azaguanine-resistant cell line is commonly used, which lacks hypoxanthine-guanine-phosphoribosyl transferase and cannot grow in hypoxanthine-aminopterin-thymidine (HAT) medium.

Suitable myeloma cells that may be used include various known cell lines, such as P3(P3x63Ag8.653) (J.Immunol, (1979) 123: 1548-1550); P3x63Ag8U.1 (current topic of microbiology and immunology (1978) 81: 1-7); NS-1(Kohler, G and Milstein, C. European journal of immunology (1976) 6: 511-519); MPC-11(Margulies, D.H. et al, cells (1976) 8: 405-415); SP2/0(Shulman, M. et al, Nature (1978) 8: 276: 269-270); FO (de St.Groth, S.F., et al, J. Immunol methods (1980) 35: 1-21); s194(Trowbridge, I.S., J.Im.Med. (1978) 148: 313-; and R210(Galfre, G et al, Nature (1979) 277: 131-.

Antibody-producing cells can be obtained from spleen cells, lymph node cells, and the like. That is, spleen, lymph node, etc. are extracted or isolated from any of the above animals, and the tissue is crushed. The resulting crushed material is suspended in a medium or buffer such as PBS, DMEM or RPMI1640, filtered with a stainless steel sieve or the like and centrifuged to prepare the desired antibody-producing cells.

Then, the myeloma cells and the antibody-producing cells are subjected to cell fusion.

Cell fusion can be performed as follows: myeloma cells and antibody-producing cells are mixed at a ratio of 1: 1 to 1: 10 in a medium for culturing animal cells such as MEM, DMEM or RPME-1640, while adding a fusion promoter at 30 to 37 ℃ for 1 to 15 minutes. To accelerate cell fusion, some fusion promoters or viruses may be used, such as polyethylene glycol having an average molecular weight of 1000 to 6000, polyvinyl alcohol, or sendai virus. Antibody-producing cells and myeloma cells can also be fused in a commercial cell fusion instrument using electrical stimulation such as electroporation.

(5) Selection and cloning of hybridomas

After cell fusion, hybridomas of interest are selected from the cells, for example, by screening using methods that allow selective growth of the cells in selective media.

That is, the cell suspension is diluted with an appropriate medium and inoculated into a microplate for culture, and a selection medium such as HAT medium is added to each well for culture, and the selection medium is replaced with a fresh medium as appropriate for incubation.

Thus, the growing cells were selected as hybridomas.

These hybridomas are then screened using a restriction dilution, fluorescence activated cell sorter, or other method. Finally, hybridomas that produce monoclonal antibodies are obtained.

(6) Collection of monoclonal antibodies

Methods for collecting monoclonal antibodies from the obtained hybridomas include conventional cell culture methods and ascites formation methods.

In the cell culture method, the hybridoma is cultured in a medium for culturing animal cells, such as PRMI-1640 medium containing 10% to 20% fetal bovine serum, MEM medium or serum-free medium, under conventional conditions (e.g., 37 ℃ C., 5% CO)₂) After 2 to 14 days of culture, the antibody was collected from the supernatant.

In the ascites-forming method, the hybridoma is inoculated intraperitoneally to a mammal of the same origin as the myeloma cells to grow the hybridoma in large quantities. After 1 to 4 weeks, ascites or serum is collected.

If it is desired to purify the antibody in these methods, known methods such as ammonium sulfate precipitation, ion exchange chromatography and affinity chromatography may be arbitrarily selected or combined.

2. Construction of chimeric antibodies

(1) Cloning of DNA containing base sequence encoding V region of mouse monoclonal antibody against human PTHrP

(i) Preparation of mRNA

To clone DNA containing a base sequence encoding V region of a mouse monoclonal antibody against human PTHrP, the collected hybridoma is treated by a conventional method, for example, guanidine-ultracentrifugation (Chirgwin, J.M., et al, biochemistry (1979)18, 5294-. mRNA can also be prepared using a Rapid preparative mRNA purification kit (Pharmacia AB) without extraction of total RNA.

(ii) Preparation and amplification of cDNA

(ii) Using the mRNA obtained in the above (i), each cDNA in the V region of L and H chains was synthesized using reverse transcriptase. In the synthesis of cDNA, oligo-dT primers or other suitable primers hybridizing to L or H chain C regions, for example, a primer having the sequence of SEQ ID NO: 1, and an MHC2 primer having the base sequence shown in FIG. 1.

In the synthesis of cDNA, the mRNA is mixed with primers and the reaction is carried out in the presence of reverse transcriptase under the following conditions: for example, 52 ℃ for 30 minutes.

L-and H-chain cDNAs can be amplified by 5 '-RACE method-based PCR (polymerase chain reaction) using the 5' -AmpliFINDER RACE kit (CLONTECH Inc.) (Frohman, M.A., et al, annual proceedings of the national academy of sciences USA, 85, 8998-. Thus, the AmpliFINDER anchor (SEQ ID NO: 42) was ligated to the 5' -end of the above-synthesized cDNA, and DNA containing the base sequences encoding the L-and H-chain V regions was PCR-synthesized. (hereinafter, the DNA containing a base sequence encoding an L chain V region is sometimes simply referred to as "DNA of L chain V region" or "DNA encoding an L chain V region", and the same applies to H chain V region, C region, etc.)

Primers for amplifying L chain V region DNA that can be used include, for example, anchor primers (SEQ ID NO: 2) and primers designed from a conserved sequence of murine antibody L.lambda.chain constant region (C.lambda.region), such as those having the amino acid sequence shown in SEQ ID NO: 4 in the sequence of the nucleotide sequence shown in (4). Primers for amplifying H chain V region DNA that can be used include, for example, an anchor primer (SEQ ID NO: 2) and an MHC-G1 primer (SEQ ID NO: 3) (S.T. Jones, et al, Biotechnology, 9, 88, 1991).

(iii) Purification of DNA and determination of base sequence

The PCR product is subjected to agarose gel electrophoresis according to a conventional method to excise the DNA fragment of interest, and then recovered, purified and ligated to the vector DNA.

Purification of DNA can be carried out using commercial kits such as GENECLEAN II, BIO 101. Vector DNA for carrying the DNA fragment suitable for use herein is known, for example, pUC19 or Bluescript.

The DNA and the vector DNA were ligated by using a known ligation kit (Takara Shuzo) to prepare a recombinant vector. The resulting recombinant vector is introduced into, for example, Escherichia coli JM109, and ampicillin-resistant colonies are selected; vector DNA is prepared by known methods: sambrook, et al, "molecular cloning", Cold Spring Harbor Laboratory Press, 1989. After the vector DNA is digested with restriction enzymes, the base sequence of the desired DNA is determined by a known method such as the dideoxy method (J.Sambrook, et al, "molecular cloning", Cold Spring Harbor Laboratory Press, 1989). The present invention may employ an automatic base sequence determining apparatus (DNA sequencer 373A; ABI Inc.).

(iv) Complementarity determining region

The H and L chain V regions form antigen binding sites, and their overall structures bear some resemblance to each other. That is, four Framework Region (FR) portions are connected by three hypervariable regions or Complementarity Determining Regions (CDRs). The amino acid sequences in the FR regions are relatively highly conserved, while the variability of the amino acid sequences in the CDR regions is high: kabat, e.a., et al, "immune-related protein sequences," U.S. department of health and human services, 1983.

Many parts of the four framework regions have a β -sheet structure, and thus, three CDRs form a loop. CDRs also sometimes form part of the beta sheet structure. Thus, by binding three CDRs in the paired regions to form the FRs of the antigen binding site, the three CDRs are very close to each other in space.

In view of this fact, it was possible to compare the amino acid sequence of the variable region of a murine monoclonal antibody against human PTHrP with the amino acid sequence database of an antibody prepared by Kabat et al ("immune-related protein sequence" U.S. department of health and human services, 1983) to find CDR regions in order to investigate the homology between the two.

(2) Components of a chimeric antibody expression vector.

After cloning of DNA fragments encoding the L and H chain V regions of a murine monoclonal antibody (hereinafter sometimes the L or H chain of a murine antibody is referred to as "murine L chain" or the like, and the L or H chain of a human antibody is referred to as "human H chain" or the like), the DNA encoding the murine V region and the DNA encoding the constant region of the human antibody are ligated and expressed to produce a chimeric anti-human PTHrP antibody.

The standard method for making chimeric antibodies involves ligating the murine leader sequence and V region sequences present in the cloned cDNA to sequences encoding human antibody C regions already present in mammalian cell expression vectors. Alternatively, the murine leader sequence and V region sequences present in the cloned cDNA are ligated to sequences encoding human antibody C regions, followed by ligation to a mammalian cell expression vector.

The polypeptide comprising a human antibody C region can be any H or L chain C region of a human antibody, including, for example, C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4 of a human H chain, or C.lambda.or C.kappa.of an L chain.

To prepare the chimeric antibody, two expression vectors are first constructed; that is, an expression vector comprising DNAs encoding a murine L chain V region and a human L chain C region under the control of an expression control region such as an enhancer/promoter system and an expression vector comprising DNAs encoding a murine H chain V region and a human H chain C region under the control of an expression control region such as an enhancer/promoter system are constructed. These expression vectors are then used to co-transform host cells, such as mammalian cells, and the transformed cells are cultured in vitro or in vivo to produce chimeric antibodies: see, for example, WO 91/16928.

Alternatively, the murine leader sequence and the DNAs encoding the murine L chain V region and the human L chain C region and the murine leader sequence and the DNAs encoding the murine H chain V region and the human H chain C region present in the cloned cDNA are introduced into a single expression vector (see, for example, WO94/11523) which is used to transform a host cell; the transformed host is then cultured in vivo or in vitro to produce the desired chimeric antibody.

(i) Production of chimeric antibody H chain

A cDNA comprising a base sequence encoding a murine H chain V region (hereinafter also referred to as "cDNA for H chain V region") can be introduced into an appropriate expression vector containing a genomic DNA comprising a base sequence encoding an H chain C region of a human antibody (hereinafter also referred to as "genomic DNA for H chain C region") or a cDNA encoding the region (hereinafter also referred to as "cDNA for H chain C region"). The H chain C region includes, for example, a C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4 region.

(i-a) construction of chimeric H chain expression vector containing genomic DNA encoding H chain C region

Expression vectors having genomic DNA encoding H chain C region, particularly C.gamma.1 region, include, for example, HEF-PMh-g.gamma.1 (WO92/19759) and DHFR-. DELTA.E-RVh-PM 1-f (WO 92/19759).

When cDNA encoding mouse H chain V region is inserted into these expression vectors, an appropriate base sequence can be introduced into the cDNA by PCR. PCR can be performed using specifically designed PCR primers, for example, such that the cDNA has a5 'recognition sequence for a suitable restriction enzyme and a kozak consensus sequence immediately before the initiation codon to promote transcription efficiency, and such that the cDNA has a 3' recognition sequence for a suitable restriction enzyme and a splice donor site to correctly splice primary transcripts of genomic DNA to obtain mRNA, thereby introducing a suitable base sequence into the expression vector.

The constructed cDNA encoding the mouse H chain V region is treated with an appropriate restriction enzyme, and inserted into the expression vector to construct a chimeric H chain expression vector comprising genomic DNA encoding the H chain C region (C.gamma.1 region).

(i-b) construction of chimeric H chain expression vector containing cDNA comprising base sequence encoding H chain

An expression vector having a cDNA encoding the C region of H chain (e.g., C.gamma.1 region) can be constructed in the following manner: an expression vector DHFR-. DELTA.E-RVh-PM 1-f (see WO92/19759) comprising a DNA encoding a humanized PM1 antibody H chain V region and a genomic DNA of a human antibody H chain C region C γ 1(N.Takahashi et al, cell, 29, 671-679(1982)) and an expression vector RV1-PM1a (see WO92/19759) comprising a genomic DNA encoding a humanized PM1 antibody L chain V region and a genomic DNA encoding a human antibody L κ chain C region were introduced into CHO cells, mRNA was prepared from the above-mentioned CHO cells, and a cDNA encoding a humanized PM1 antibody H chain V region and a cDNA encoding a human antibody H chain C region (C γ 1) were cloned by RT-PCR method and ligated to an animal cell expression vector treated with an appropriate restriction enzyme to construct a desired expression vector.

When cDNA encoding a mouse H chain V region is directly ligated to cDNA encoding a human antibody H chain C region C.gamma.1, an appropriate base sequence can be introduced into a fragment containing cDNA encoding a H chain V region by PCR. For example, PCR may be performed using PCR primers specifically designed, for example, such that the cDNA has a5 'recognition sequence to recognize suitable restriction enzymes and a kozak consensus sequence immediately before codons to thereby promote transcription efficiency, and such that the cDNA has a 3' recognition sequence to recognize suitable restriction enzymes to thereby introduce these suitable base sequences into the cDNA.

Construction of cDNA encoding murine H chain V region the cDNA is treated with appropriate restriction enzymes, ligated to the cDNA encoding the H chain C region C.gamma.1, and inserted into an expression vector such as pCOS1 or pCHO1 to construct an expression vector comprising cDNA encoding chimeric antibody H chain.

(ii) Production of L chain of chimeric antibody

A vector for expressing the L chain of a chimeric antibody can be obtained by ligating cDNA encoding a mouse L chain V region with genomic DNA or cDNA encoding a human antibody L chain C region and introducing into an appropriate expression vector. The L chain C region includes, for example, a kappa chain and a lambda chain.

(ii-a) construction of an expression vector comprising cDNA encoding a chimeric L.lambda.chain

When an expression vector containing cDNA encoding a mouse L chain V region is constructed, an appropriate base sequence can be introduced into the expression vector by PCR. For example, PCR can be carried out using PCR primers specifically designed, for example, such that the cDNA has a recognition sequence for a suitable restriction enzyme at the 5 'end and a Kozak consensus sequence for promoting transcription efficiency immediately before the codon, and such that the cDNA has a recognition sequence for a suitable restriction enzyme at the 3' end, thereby introducing a suitable base sequence into the cDNA.

The entire base sequence of cDNA encoding the C region of the human L.lambda.chain can be synthesized using a DNA synthesizer and constructed by the PCR method. It is known that the human L.lambda.chain C region has at least four different isoforms, each of which can be used to construct an expression vector. For example, in searching for homology to the cloned murine monoclonal antibody L.lambda.chain C region, isoform Mcg + Ke + Oz of the human L.lambda.chain C region fragment (accession number X57819) (P.Dariavach et al, annual proceedings of the national academy of sciences USA, 84, 9074-. To construct a cDNA for a known human L.lambda.chain C region, e.g., Mcg + Ke + Oz-, for example, the following four sequences as shown in SEQ ID NO: 11-14, wherein: the primers MBC1HGP1(SEQ ID NO: 11) and MBC1HGP3(SEQ ID NO: 13) have sense DNA sequences, and the primers MBC1HGP2(SEQ ID NO: 12) and MBC1HGP4(SEQ ID NO: 14) have antisense DNA sequences, wherein each primer has a complementary sequence of 20 to 23 base pairs at each end.

MBC1HGPS (SEQ ID NO: 15) and MBC1HGPR (SEQ ID NO: 16) are called outer primers, have sequences homologous to MBC1HGP1 and MBC1HGP4, respectively, and have recognition sequences for suitable restriction enzymes. The four primers were assembled by the PCR method to synthesize a full-length cDNA, and the outer primers were added to amplify the cDNA.

Assembly by PCR means that MBC1HGP1 and MBC1HGP2 or MBC1HGP3 and MBC1HGP4 anneal through their complementary sequences to synthesize a MBC1HGP1-MBC1HGP2 fragment and a MBC1HGP3-MBC1HGP4 fragment, each of which fragments synthesizes a cDNA encoding the full-length human L.lambda.chain C region, again by annealing through complementary sequences.

The constructed cDNA encoding the human L λ chain C region and the above-described constructed cDNA encoding the murine L chain V region may be ligated between suitable restriction enzyme sites and inserted into an expression vector such as pCOS1 or pCHO1 to construct an expression vector including cDNA encoding the L λ chain of the chimeric antibody.

(ii-b) construction of an expression vector comprising cDNA encoding the chimeric L.kappa.chain

When an expression vector comprising cDNA encoding a mouse L chain V region is constructed, an appropriate base sequence can be introduced into the cDNA by PCR. For example, PCR may be performed using PCR primers specifically designed, for example, such that the cDNA has, at its 5 'end, a recognition sequence for a suitable restriction enzyme and a Kozak consensus sequence for promoting transcription efficiency, and such that the cDNA has a 3' end recognition sequence for a suitable restriction enzyme, thereby introducing a suitable base sequence into the cDNA.

The DNA encoding the human L.kappa.chain C region linked to the DNA encoding the murine L chain V region can be constructed, for example, from HEF-PM1k-gk containing genomic DNA (see WO 92/19759).

The recognition sequence of an appropriate restriction enzyme can be introduced into the 5 '-and 3' -ends of the DNA encoding L.kappa.chain C region by PCR, and the DNA encoding mouse L chain V region constructed as described above and the DNA encoding L.kappa.chain C region can be ligated to each other and inserted into an expression vector such as pCOS1 or pCHO1 to construct an expression vector including cDNA encoding chimeric antibody L.kappa.chain.

3. Preparation of humanized antibody

(1) Screening of human antibody homologs

To prepare a humanized antibody in which CDRs of a murine monoclonal antibody are grafted onto a human antibody, it is desirable that there be a high homology between FRs of the murine monoclonal antibody and FRs of the human antibody. Therefore, the V regions of H and L chains of the murine anti-human PTHrP monoclonal antibody were compared with those of all known antibodies whose structures were described using the protein database. They were also compared with the lengths, amino acid homologies, etc. of the FR antibodies of the human antibody subgroup classified by Kabat et al (HSG: human subgroup): kabat, e.a. et al, department of health and human services, U.S. government printing office, 1991.

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

When a mouse anti-human PTHrP monoclonal antibody is humanized, it is desirable to use a human H chain V region belonging to HSG III and having the highest degree of homology, or a human H chain V region having a corresponding canonical FR structure (Chothia C et al, J. mol. biol., 196, 901-917, 1987) as the human H chain V region, for constructing a humanized antibody. In addition, since the subgroup of human L λ chain V regions does not have a consensus sequence with the highest degree of homology, it is desirable to use human antibody L λ chain V regions with high homology that have been registered in the protein database when constructing a humanized antibody.

(2) Design of DNA encoding humanized antibody V region

The first step in designing DNA encoding humanized antibody V regions is to select human antibody V regions as the basis for the design.

In the present invention, the FR of the V region of a human antibody having a homology of more than 80% with the FR of the V region of a murine antibody can be used for a humanized antibody. As a framework of substantially identical FRs, the H chain V region FRs may include FRs derived from subgroup III, e.g., S31679: NBRF-PDB, Cuisinier A.M., et al, J.Eur. Immunity, 23, 110-. Further, as a framework of substantially the same FR, the FRs of the L chain V region may include, for example, FR1, FR2 and FR3 derived from the human antibody HSU03868(GEN-BANK, Deftos M. et al, Scand. immunojournal, 39, 95-103, 1994) and FR4 derived from the human antibody S25755 (NBRF-PDB).

Human antibody S31679 was cloned from a cDNA library of human fetal liver, while human antibody HSU03868 was cloned as a novel gene encoding a human L λ chain V region.

(3) Preparation of a polypeptide comprising a humanized antibody V region

In the humanized antibody of the present invention, the framework regions of the C region and the V region of the antibody are derived from human, and the Complementarity Determining Regions (CDRs) of the V region are derived from mouse (fig. 1). According to the present invention, a polypeptide comprising a humanized antibody V region can be obtained by a PCR method in a manner called CDR-grafting, as long as a DNA fragment of a human antibody can be used as a template. "CDR-grafting" refers to a method in which a DNA fragment encoding a mouse-derived CDR is prepared and used to replace the CDR of a human antibody as a template.

If a DNA fragment of a human antibody used as a template is not provided, a base sequence registered in a database can be synthesized by a DNA synthesizer, and a DNA encoding a humanized antibody V region can be prepared by a PCR method. In addition, when only one amino acid sequence is registered in the database, the entire base sequence can be deduced from the amino acid sequence based on the knowledge of the codon usage of antibodies reported by Kabat, E.A. et al (U.S. department of health and human services, U.S. government printed office, 1991). This base sequence is synthesized on a DNA synthesizer, and then DNA of the V region of the humanized antibody is prepared by a PCR method, and introduced into a suitable host, followed by expression therein to prepare the desired polypeptide.

When a human antibody DNA fragment as a template is present, a general procedure of CDR-grafting by the PCR method is described below.

(i) CDR grafting

It is assumed that the DNA encoding the V region includes DNAs encoding FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 linked to each other in this order as shown in FIG. 2.

First, DNA fragments corresponding to the respective CDRs derived from mouse were synthesized. CDRs 1 to 3 were synthesized on the basis of the base sequences of previously cloned murine H and L chain V regions. The graft primers B and E were synthesized so as to have sequences hybridizing to the murine CDR1 and the human antibody FR2 in the sense strand direction, and the graft primer E should have primers hybridizing to the CDR1 and the human antibody FR1 in the antisense strand direction (see FIG. 2 (1)). Similarly, the grafting primers C and F and the primers D and G were synthesized. In addition, suitable primers called "outer primers" which can hybridize to the upstream region of FR1 and the downstream region of FR4, and are denoted by A and H of FIG. 2(1), respectively, were also synthesized. The transplantation primers can be isolated and extracted according to known procedures: sambrook, et al, molecular cloning: a laboratory Manual, Cold Spring Harbor laboratory Press, 1989.

Then, first PCR was performed using the transfer primer E and the external primer A, the transfer primers B and F, the transfer primers C and G, and the transfer primer D and the external primer H to form fragments A-E, B-F, C-G and D-H, respectively (see FIG. 2 (2)).

Since the upstream region of the graft primer B and the downstream region of a part of the graft primer E are designed to overlap each other (the same applies to the graft primers C and F and D and G), these fragments can be annealed to complementary sequences respectively by reaction under appropriate temperature conditions, and then DNA of a length from A to H is combined by PCR. After obtaining a DNA fragment encoding a V region, external primers A and H may be added, and second PCR may be performed to produce a DNA encoding a V region of a humanized antibody FR1 to 4 derived from human and CDR1 to 3 derived from mouse. The DNA may then be introduced into a suitable host for expression to produce the desired polypeptide (FIG. 2 (3)).

(ii) Construction of DNA and expression vector encoding humanized H chain V region

In the present invention, the entire base sequence of the DNA encoding the H chain V region of a human antibody as a template for a humanized antibody can be synthesized by a DNA synthesizer and constructed by a PCR method even if the DNA cannot be obtained from a natural source.

The H chain V region of the murine anti-human PTHrP monoclonal antibody is highly homologous to S31679 belonging to human subgroup III. To construct a DNA encoding a humanized H chain V region using this human antibody as a template, a DNA encoding a humanized H chain V region such as SEQ ID NO: 23-26. The primers MBC1HGP1(SEQ ID NO: 23) and MBC1HGP3(SEQ ID NO: 24) have sense DNA sequences, and the primers MBC1HGP2(SEQ ID NO: 25) and MBC1HGP4(SEQ ID NO: 26) have antisense DNA sequences. Wherein each primer is designed to have a complementary sequence of 15 to 21 base pairs at each end.

The outer primers MBC1HVS1(SEQ ID NO: 27) and MBC1HVR1(SEQ ID NO: 28) have sequences homologous to MBC1HGP1 and MBC1HGP4, respectively, and have recognition sequences for suitable restriction enzymes, respectively. The four primers were assembled by the PCR method to synthesize full-length cDNA, and the outer primers were added to amplify the DNA. "Assembly by PCR" involves annealing MBC1HGP1 and MBC1HGP2 or MBC1HGP3 and MBC1HGP4 by their complementary sequences to synthesize a MBC1HGP1-MBC1HGP3 fragment and a MBC1HGP2-MBC1HGP4 fragment, each of which is annealed again by the complementary sequences to synthesize a full-length DNA encoding a humanized H chain V region.

The human antibody H chain C region may be any human H chain C region, for example, C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4 of human H chain.

The DNA encoding the humanized antibody H chain V region constructed as described above may be ligated to the DNA of any human antibody H chain C region, for example, human H chain C.gamma.1 region. As mentioned in the section "production of H chain of chimeric antibody", the DNA of H chain V region may be treated with an appropriate restriction enzyme and then ligated to the DNA encoding human H chain V region under the control of an expression regulatory region such as an enhancer/promoter system to prepare an expression vector comprising the DNA encoding humanized H chain V region and human H chain V region.

(iii) Construction of DNA and expression vector encoding humanized L chain V region

In the present invention, the entire base sequence of the DNA encoding the L chain V region of a human antibody used as a template can be synthesized by a DNA synthesizer and constructed by the PCR method even if the DNA encoding the L chain V region is not available as in the case of the DNA encoding the H chain V region.

In order to construct a DNA of a humanized L chain V region used as a template of human antibody SU03868 having the highest homology to the L chain V region of a mouse anti-human PTHrP monoclonal antibody, a DNA sequence shown in SEQ ID NO: 29-32. The primers MBC1LGP1(SEQ ID NO: 29) and MBC1LGP3(SEQ ID NO: 30) have sense DNA sequences and MBC1LGP2(SEQ ID NO: 31) and MBC1LGP4(SEQ ID NO: 32) have antisense DNA sequences. Wherein each end of each primer is designed with a complementary sequence of 15 to 21 base pairs.

The outer primers MBC1LVS1(SEQ ID NO: 33) and MBC1LVR1(SEQ ID NO: 34) have sequences homologous to MBC1LGP1 and MBC1LGP4, respectively, and have recognition sequences for the corresponding suitable restriction enzymes, respectively. The four primers were assembled by the PCR method to synthesize full-length DNA, and the outer primers were added to amplify the DNA. "Assembly by PCR" involves annealing MBC1LGP1 and MBC1LGP3 or MBC1LGP2 and MBC1LGP4 through their complementary sequences to synthesize a MBC1LGP1-MBC1LGP3 fragment and a MBC1LGP2-MBC1LGP4 fragment, each of which is annealed again through a complementary sequence to synthesize a full-length DNA encoding a humanized H chain V region.

The human antibody L chain C region may be any human L chain C region, for example, C.lamda.or C.kappa.of a human L chain.

The DNA encoding the humanized antibody L chain V region constructed as described above may be ligated to DNA of any human antibody L chain C region, for example, human L chain C.lamda.region. The DNA of the L chain V region may be treated with an appropriate restriction enzyme and then ligated to DNA encoding a human L.lambda.chain C region under the control of an expression regulatory region such as an enhancer/promoter system to prepare an expression vector comprising the humanized L chain V region and the human L.lambda.chain C region DNA.

If a polypeptide comprising a humanized antibody V region can be prepared as described above, it is not necessary to know whether the polypeptide has antibody activity, such as binding or neutralizing activity against its antigen. In particular, for the L chain, since the L chain V region of the murine anti-human PTHrP monoclonal antibody is derived from a very rare V.lambda.x gene, it is necessary to combine it with a humanized H chain and express it in animal cells such as COS7 to investigate the presence or absence of the activity.

Construction of the hybrid V region (Ohtomo, T. et al, molecular immunology, 32, 407-. In order to clarify that amino acids in the L chain V region of the humanized antibody of the present invention should be mutated to impart an activity thereto, a DNA in which a fragment of the FR region of the humanized antibody is recombined with a fragment of the FR region derived from mouse was constructed, and the humanization of each region was evaluated.

As shown in fig. 3, an antibody having a polypeptide comprising a recombinant V region derived from human antibodies FR1 and FR2 and from murine antibodies FR3 and FR4 (for example, an antibody having a recombinant fragment referred to as "hybrid antibody"), a hybrid antibody in which only FR1 is derived from a human, and a hybrid antibody in which only FR2 is derived from a human are prepared. Each DNA encoding these hybrid antibodies was introduced into an expression vector separately, and the humanized antibody was transiently expressed to investigate the presence or absence of antibody activity.

By this method, the present inventors investigated the antigen binding and neutralizing activity of a polypeptide comprising an L chain V region, and finally found that some amino acids present in FR2 and FR3 were substituted.

Having discovered the amino acids present in the FR2 and FR3 regions that contribute to activity, the present inventors further demonstrated that amino acids at positions 36, 45, 49 of the FR2 region and amino acid 87 of the FR3 region (numbering of antibody amino acids is determined by Kabat, e.a. et al (U.S. department of health and human services, U.S. government printed office, 1991)) contribute to activity.

Thus, the present invention produces polypeptides in which one or more amino acids of the V region are mutated (e.g., replaced).

First, a polypeptide comprising a V region having an introduced amino acid sequence or certain amino acid mutations is prepared as a base by the CDR-grafting method described above. This base polypeptide comprises SEQ id no: 47, and is referred to as "type a" (a in table 1).

Then, based on type a, various variant fragments in which one or some of the amino acids of the FR are mutated are prepared.

Mutations can be introduced by designing oligonucleotide primers (mutagenic primers) encoding amino acids that result in the desired mutation and performing PCR using the primers.

Thus, polypeptides in which specific amino acids of FR2 and FR3 of the V region (b to t type) were mutated (b to t in Table 1) can be prepared.

TABLE 1

FR1 CDR1

1 2 3

123456789012345678901234567890 12345

MBC H.PEP EVQLVESGGDLVKPGGSLKLSCAASGFTFS SYGMS

* ** * * *

S31679 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH

hMBC1-H.pep ------------------------------ SYGMS

FR2 CDR2

4 5 6

67890123456789 012A3456789012345

MBC H.PEP WIRQTPDKRLEWVA TISSGGSYTYYPDSVKG

* * * *

S31679 WVRQAPGKGLEWVA VISYDGSNKYYADSVKG

hMBC1-H.pep -------------- TISSGGSYTYYPDSVKG

FR3 CDR3 FR4

7 8 9 10 11

67890123456789012ABC345678901234 567890A12 34567890123

MBC H.PFP RFTISRDNAKNTLYLQMSSLKSEDTAMFYCAR QTTMTYFAY WGQGTLVTVSA

* * ** ** *

S31679 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ESRGDY WGQGTLVTVSS

hMBC1-H.pep -------------------------------- QTTMTYFAY -----------

Each type of DNA encoding the L chain V region of the humanized antibody constructed as described above may be ligated to DNA encoding the L chain C region of any human antibody, for example, the human L chain C.lamda.region. Thus, it is treated with an appropriate restriction enzyme and ligated to a DNA encoding a human L.lambda.chain C region under the control of an expression control sequence such as an enhancer/promoter system to construct an expression vector comprising each type of DNA encoding a humanized L chain V region and a DNA encoding a humanized L.lambda.chain C region.

The previously constructed DNAs encoding the humanized antibody H chain V region and human H chain C region and the DNAs encoding the humanized L chain V region and human L chain C region may also be introduced into a single expression vector such as that disclosed in WO94/11523, which may be used to transform a host cell, and the transformed host may be cultured in vivo or in vitro to produce the desired humanized antibody.

4. Preparation of chimeric and humanized antibodies

To prepare a chimeric or humanized antibody, two expression vectors as described above should be prepared. Thus, for the chimeric antibody, an expression vector comprising DNAs encoding a mouse H chain V region and a human H chain C region under the control of an expression regulatory region such as an enhancer/promoter system, and an expression vector comprising DNAs encoding a mouse L chain V region and a human L chain C region under the control of an expression regulatory region such as an enhancer/promoter system were constructed. For the humanized antibody, an expression vector comprising DNAs encoding a humanized H chain V region and a human H chain C region under the control of an expression regulatory region such as an enhancer/promoter system, and an expression vector comprising DNAs encoding a humanized L chain V region and a human L chain C region under the control of an expression regulatory region such as an enhancer/promoter system are constructed.

These expression vectors are then used to co-transform host cells such as mammalian cells, and the resulting transformed cells are cultured in vivo or in vitro to produce chimeric or humanized antibodies (see, e.g., WO 91/16928).

Alternatively, the DNA encoding the H chain V region and C region and the DNA encoding the L chain V region and C region may be ligated to a single vector and transformed into a suitable host cell to produce the antibody. Thus, in the expression of the chimeric antibody, DNAs encoding the murine leader sequence, the murine H chain V region and the human H chain C region present in the cloned cDNA, and DNAs encoding the murine leader sequence, the murine L chain V region and the human L chain C region are introduced into a single expression vector such as the one disclosed in WO 94/11523. In the expression of a humanized antibody, DNAs encoding a humanized H chain V region and a human H chain C region, and DNAs encoding a humanized L chain V region and a human L chain C region are introduced into a single expression vector such as that disclosed in WO 94/11523. Such a vector is used to transform a host cell, which is cultured in vivo or in vitro to produce the chimeric or humanized antibody of interest.

Thus, the chimeric or humanized antibody of interest prepared by culturing the transformant transformed with the DNA encoding the chimeric or humanized antibody can be isolated from the inside or outside of the cell and purified as a monomer.

The chimeric or humanized antibody of interest of the present invention can be isolated and purified using a protein a sepharose column, but a conventional protein isolation and purification method can also be used, and thus the procedure is not limited. For example, various methods of chromatography, ultrafiltration, salting out, and dialysis can be optionally selected or combined to isolate and purify the chimeric or humanized antibody.

Any expression system can be used to prepare the anti-human PTHrP chimeric or humanized antibody of the present invention. For example, eukaryotic cells include animal cells such as established mammalian cell lines, mold and fungal cells, and yeast cells; prokaryotic cells include bacterial cells such as Escherichia coli cells. The chimeric or humanized antibodies of the invention are preferably expressed in mammalian cells such as COS or CHO cells.

Any conventional promoter for mammalian cell expression may be used. For example, Human Cytomegalovirus (HCMV), an early promoter, is preferably used. Examples of expression vectors containing the HCMV promoter include HCMV-VH-HC γ 1 and HCMV-VL-HCK derived from pSV2neo (WO 92/19759).

In addition, the promoter for gene expression in mammalian cells which can be used in the present invention includes viral promoters such as retrovirus, polyoma virus, adenovirus and Simian Virus (SV)40, and promoters derived from mammalian cells such as human polypeptide chain elongation factor-1 α (HEF-1 α). For example, the SV40 promoter may be conveniently used according to the method of Mullingan et al (Nature, 277, 108, 1979); the promoter HEF-1. alpha. can be conveniently used according to the method of Mizushima et al (nucleic acids research, 18, 5322, 1990).

Origins of replication useful in the present invention include those derived from SV40, polyoma virus, adenovirus or Bovine Papilloma Virus (BPV). In addition, the expression vector may contain a phosphotransferase APH (3') II or I (neo) gene, a Thymidine Kinase (TK) gene, E.coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene or dihydrofolate reductase (DHFR) gene as a selectable marker for increasing the copy number of genes in the host cell system.

5. Evaluation of antigen binding and neutralizing Activity of chimeric antibody and humanized antibody

(1) Determination of antibody concentration

The concentration of the resulting purified antibody can be determined by ELISA.

According to the followingMode for preparation of ELISA plates for determination of antibody concentration: 100. mu.l of goat anti-human IgG antibody prepared at a concentration of, for example, 1. mu.g/ml is immobilized in each well of a 96-well plate for ELISA (e.g., Maxisorp, NUNC). After partitioning with 200. mu.l dilution buffer (e.g., 50mM Tris-HCl, 1mM MgCl)₂0.1M NaCl, 0.05% Tween20, 0.02% NaN₃1% Bovine Serum Albumin (BSA), ph7.2), gradually diluted COS-7 or CHO cell supernatants with expression of chimeric, hybrid or humanized antibody, or purified chimeric, hybrid or humanized antibody was added per well, 100 μ l of alkaline phosphatase conjugated goat anti-human IgG antibody was added, then 1 mg/ml base solution (Sigma 104, p-nitrophenyl phosphate, Sigma) was added, followed by measurement of absorbance at 405nm with a microplate reader (Bio Rad). Purified Hu IgG1 λ (binding site) can be used as a standard in concentration assays.

(2) Determination of antigen binding Capacity

ELISA plates for determination of antigen binding capacity were prepared as follows: 100. mu.l of human PTHrP (1-34) prepared at a concentration of 1. mu.g/ml was immobilized in each well of a 96-well plate for ELISA. After partitioning with 200. mu.l dilution buffer, gradually diluted COS-7 or CHO cell supernatants expressing chimeric, hybrid or humanized antibodies, or purified chimeric, hybrid or humanized antibodies were added to each well, 100. mu.l of alkaline phosphatase conjugated goat anti-human IgG antibody was added, then 1 mg/ml base solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and absorbance at 405nm was measured using a microplate reader (Bio Rad).

(3) Determination of neutralizing Activity

The neutralizing activity of murine, chimeric and humanized antibodies can be determined, for example, by the rat osteosarcoma cell line ROS17/2.8-5 cells (Sato, K et al, Endocrinology bulletin, 116, 113-120, 1987). Therefore, ROS17/2.8-5 cells were stimulated with 4mM hydrocortisone to induce PTH/PTHrP receptor. cAMP degrading enzyme was inhibited with 1mM isobutyl-1-methylxanthine (IBMX, SIGMA). The murine, chimeric or humanized antibody to be assayed for neutralizing activity was mixed with an equal amount of PTHrP (1-34), and the resulting mixture of each antibody and PTHrP (1-34) was added to each well of the plate. The neutralizing activity of the murine, chimeric or humanized antibody can be assessed by measuring the amount of ROS17/2.8-5 cells, a rat osteosarcoma cell line, that results in cAMP production due to stimulation with PTHrP.

(4) Kinetic analysis of the interaction between PTHrP and anti-PTHrP antibody

The present invention can analyze the kinetics of the interaction between PTHrP and anti-PTHrP using various methods and procedures. In particular, dissociation and association constants can be determined by Scatchard analysis and a surface plasmon resonance sensor (developed by Pharmacia Biotech) known as BIACORE. The surface plasmon resonance sensor assay, referred to as BIACORE, will be described hereinafter as an example.

The basic structure of BIACORE includes a light source, a prism, a detector, and a microchannel. In operation, the ligand is immobilized on top of the cartridge sensor and an analyte is injected into the sensor. When there is an affinity between them, the amount of binding can be determined optically.

The principle of detection is a phenomenon known as surface plasmon resonance. That is, incident light is irradiated to the surface between the metal films of the glass box so that total reflection occurs, and incident light at a certain angle is used to stimulate surface plasmon, followed by attenuation. The angle changes with the concentration of the solvent in contact with the metal film (sensor). BIACORE detects this change.

In BIACORE, this change is called a resonance signal (SPR signal), and the change of 0.1 degree is 1000RU (resonance unit). 1000RU corresponds to a change in binding of approximately 1 nanogram of protein to a gold thin layer sensor with a surface area of 1mm 2. For proteins, a change in 50RU (50pg) was fully detectable.

The detection signal is converted by the computer attached to the BIACORE into a binding curve, called sensorgram, which is displayed on-line by the computer: natsume, T. et al (1995), Experimental medicine, 13, 563-; karlsson, R. et al (1991) journal of immunological methodology 145, 229-240.

Kinetic parameters of the anti-PTHrP antibody of the present invention, i.e., dissociation constant (KD), dissociation rate constant (Kdis) and association rate constant (Kass), can be determined by the BIACORE method described above.

In view of neutralizing activity, the anti-PTHrP antibody of the present invention preferably has a dissociation constant (KD value) as small as possible. The anti-PTHrP antibody of the present invention preferably has a KD value of 1.86X 10^-7Or less, more preferably 1.8X 10^-8Or less, most preferably 3.58X 10^-10Or lower.

KD values are determined by two parameters, the dissociation rate constant (Kdiss) and the association rate constant (Kass) (KD ═ Kdiss/Kass). It can thus be seen that the KD values are smaller when the Kdiss value is smaller and the Kass value is larger.

In particular, the anti-PTHrP antibody of the present invention may have a Kdis value of 1.22X 10^-1[l/Sec]Or lower. The Kdis value is preferably 1.22X 10^-2[l/Sec]Or less, more preferably 3.16X 10^-4[l/Sec]Or less, most preferably 2.32X 10^-4[l/Sec]Or lower.

On the other hand, the Kass value may be 6.55X 10⁴[l/M.Sec]Or higher. The Kass value is preferably 6.55X 10⁵[l/M.Sec]Or higher, more preferably 0.883X 10⁶[l/M.Sec]Or higher, most preferably 1.03X 10⁶[l/M.Sec]Or higher.

And also preferably has a Kdis value of 1.22X 10^-1[l/Sec]And a Kass value of 6.55X 10⁴[l/M.Sec]Or higher anti-PTHrP antibody.

More specifically, the anti-PTHrP antibody of the present invention has a KD value ranging from 1.02X 10^-11To 1.86X 10^-7[M]Preferably 1.02X 10^-10To 1.86X 10^-8[M]More preferably 1.34X 10^-10To 3.58X 10^-10[M]Most preferably 2.25X 10^-10To 3.58X 10^-10[M]。

Kdis value at 7.38X 10^-6To 1.22X 10^-1[l/Sec]Preferably 7.38X 10^-5To 1.22X 10^-2[l/Sec]More preferably 1.66X 10^-4To 3.16X 10^-4[l/Sec]Most preferably 1.66X 10^-4To 2.32X 10^-4[l/Sec]In the meantime.

Kass value of 6.55X 10⁴To 1.24X 10⁷[l/M.Sec]Preferably 6.55X 10⁵To 1.24X 10⁶[l/M.Sec]More preferably 7.23X 10⁵To 1.03X 10⁶[l/M.Sec]Most preferably 0.883X 10⁶To 1.03X 10⁶[l/M.Sec]In the meantime.

These KD, Kdiss and Kass values can be determined by Scatchard analysis or surface plasmon resonance sensors such as BIACORE, and preferably BIACORE.

6. A pharmaceutical composition and a hypercalcemia-suppressing agent containing an anti-PTHrP antibody or a humanized antibody as an active ingredient.

The therapeutic effect of the humanized antibody against PTHrP can be determined by administering the anti-PTHrP antibody or the humanized antibody to an animal exhibiting hypercalcemia and measuring an index of hypercalcemia. Hypophosphatemia is often observed in animals exhibiting hypercalcemia or in hypercalcemic patients; the antibodies of the invention may also be used to ameliorate hypophosphatemia.

The antibody used in the present invention is an anti-PTHrP antibody, including an anti-PTHrP human antibody, a chimeric antibody and a primatized or humanized antibody having a dissociation constant, a dissociation rate constant and an association rate constant. The antibody neutralizes the activity of PTHrP by binding to PTHrP, and particularly preferably, the antibody is a humanized #23-57-137-1 antibody. The preparation of the humanized #23-57-137-1 antibody is described in examples 1 to 3.

The antibody used in the present invention can be obtained in a highly purified pure form by a combination of conventional purification methods such as salting out, gel filtration using HPLC or the like, and affinity chromatography using a protein A column or the like. The high accuracy of recognition of PTHrP by the purified antibody can thus be determined by conventional immunological methods such as Radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA) or immunofluorescence analysis.

Animals exhibiting hypercalcemia that can be used include model animals prepared by transplanting PTHrP-producing tumor cells into experimental animals having reduced or deficient immune function. The transplanted tumor cells are preferably derived from a human, including, for example, human pancreatic cancer PAN-7. Animals with poor or absent immune function implanted with tumor cells include nude mice and SCID mice.

Inhibition of hypercalcemia can be assessed by observing calcium concentration in blood, reduction in body weight, or decrease in the degree of exercise with the lapse of time, and determining the degree of improvement.

The pharmaceutical composition and hypercalcemia-suppressing agent comprising the anti-PTHrP antibody or anti-PTHrP humanized antibody of the present invention as an active ingredient may be administered parenterally or topically. For example, intravenous injection including instillation, intramuscular injection, intraperitoneal injection, or subcutaneous injection may be selected. The appropriate mode of administration may be selected according to the age and disease condition of the patient. An effective single dose may be selected from the range of 0.01 to 1000 mg per kg body weight. Alternatively, the dose administered is from 5 to 10000 mg/body weight, preferably from 50 to 1000 mg/volume.

The pharmaceutical composition and hypercalcemia-suppressing agent comprising the anti-PTHrP antibody or anti-PTHrP humanized antibody of the present invention as an active ingredient may further comprise pharmaceutically acceptable carriers and/or additives depending on the route of administration. Examples of such carriers and additives may include water, pharmaceutically acceptable organic solvents, collagen, polyallylic alcohol, polypropylenepolylidone, carboxypropylene polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, Human Serum Albumin (HSA), mannitol, sorbitol, lactose, and surfactants that may be used as pharmaceutical additives. The additives used may be selected from one or a combination of the above, but are not limited thereto.

The antibody of the present invention can be widely used for various cancers (malignant tumors) associated with hypercalcemia. These cancers are not particularly limited and include not only a single cancer but also a complication of many cancers. Cancers also include, for example, pancreatic, lung, pharyngeal, laryngeal, gingival, esophageal, gastric, biliary, breast, renal, bladder, uterine, and prostate cancers, as well as malignant lymphomas.

Brief description of the drawings

FIG. 1 is a schematic illustration of an antibody of the present invention;

FIG. 2 is a schematic illustration of CDR-grafting;

FIG. 3 is an illustration of the determination of the FR and V region CDRs;

FIG. 4 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 5 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 6 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 7 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 8 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 9 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 10 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 11 is a graph showing the measurement results of the antigen binding activity of the antibody;

FIG. 12 is a graph showing the neutralizing activity of the humanized antibody;

FIG. 13 is a graph showing the neutralizing activity of the humanized antibody;

FIG. 14 is a graph showing the neutralizing activity of the humanized antibody;

FIG. 15 is a graph illustrating the effect of an antibody of the present invention on the treatment of hypercalcemia model animals;

FIG. 16 is a graph illustrating the effect of an antibody of the present invention on the treatment of hypercalcemia model animals;

FIG. 17 is a graph illustrating the effect of an antibody of the present invention on the treatment of hypercalcemia model animals;

FIG. 18 is a graph illustrating the effect of an antibody of the present invention on the treatment of hypercalcemia model animals;

fig. 19 is a sensor diagram illustrating the fixing of PTHrP to the top of the sensor;

FIG. 20 is a graph showing the results of kinetic analysis of the antibody of the present invention;

FIG. 21 is a graph showing the results of kinetic analysis of the antibody of the present invention;

FIG. 22 is a graph showing the results of kinetic analysis of the antibody of the present invention;

FIG. 23 is a graph showing the results of kinetic analysis of the antibody of the present invention;

FIG. 24 is a graph showing the results of kinetic analysis of the antibody of the present invention;

FIG. 25 is a graph showing the results of measurement of the effect of the humanized antibody of the present invention on local phosphate secretion;

FIG. 26 is a graph showing the results of measurement of the effect of the humanized antibody of the present invention on plasma phosphorus concentration;

FIG. 27 is a photograph showing the clinical appearance of symptoms (morphology of living animals) in a hypercalcemia model mouse after administration of an anti-PTHrP antibody of the present invention;

FIG. 28 is a photograph showing the clinical appearance of symptoms (morphology of living animals) in a hypercalcemia model mouse after administration of an anti-PTHrP antibody of the present invention;

FIG. 29 is a graph showing the change in activity over time after administration of the anti-PTHrP antibody of the present invention to a hypercalcemia model animal, compared with a control model animal administered with physiological saline.

FIG. 30 is a graph showing the change in body temperature of hypercalcemia model animals over time after administration of the anti-PTHrP antibody of the present invention, compared with control model animals administered with physiological saline.

Fig. 31 is a graph showing the change in blood pH over time after administration of the anti-PTHrP antibody of the present invention to a hypercalcemia model animal, compared to a control model animal administered with physiological saline.

Examples

The present invention will be described in detail below with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Reference example 1

Preparation of hybridoma producing anti-PTHrP (1-34) murine monoclonal antibody

Hybridomas producing anti-human PTHrP (1-34), #23-57-154 and #23-57-137-1 monoclonal antibodies were prepared according to the method reported by Kanji Sato et al (Sato, K. et al, Bone Miner, J.Res., 8, 849-.

The immunogen used was PTHrP (1-34) (Peninsula), which was bonded to a carrier protein, thyroglobulin, using carbodiimide (Dojinn). Thyroglobulin-bound PTHrP (1-34) was dialyzed to obtain a solution having a protein concentration of 2. mu.g/ml. The resulting solution was mixed with Freund's adjuvant (Difco) in a ratio of 1: 1 to obtain an emulsion. The emulsion was injected subcutaneously or intraperitoneally to the backs of 16 female mice BALB/C at a dose of 100. mu.g/mouse, respectively, to immunize the mice. Injections were 11 times. As for the adjuvant, freund's complete adjuvant was used for the first immunization injection, and freund's incomplete adjuvant was used for the subsequent immunization injections.

Antibody titers in serum of each immunized mouse were determined in the following manner.

Blood was taken from each rat tail vein, and the blood sample was centrifuged to obtain serum. Serum was diluted with RIA buffer and reacted with¹²⁵I-labelled PTHrP (1-34) was mixed and then the binding activity was measured. It has been determined that mice with satisfactorily high antibody titers were given a final immunization by intraperitoneal injection of PTHrP (1-34) without carrier protein at a dose of 50. mu.g/mouse.

Three days after the final immunization, the mice were sacrificed and their spleens were excised. The spleen cells were then cell fused with the murine myeloma cell line P3x63Ag8U.1 using 50% polyethylene glycol 4000 according to a known conventional method. The thus-prepared fused cells were plated at 2X 10 in 96-well plates⁴The amount per well was cultured in 85 wells. Hybridomas of interest are screened by HAT culture as follows.

Hybridomas are screened by determining the presence of PTHrP-recognizing antibody in the culture supernatant of wells in HAT medium in which cell growth was observed by the solid-phase RIA method. Hybridomas were collected from wells that clearly had the ability to recognize the binding of PTHrP-antibody. The obtained hybridoma was suspended in RPMI-1640 medium containing 15% FCS and supplemented with OPI-supplement (Sigma), and then the hybridoma was homogenized by limiting dilution, thereby obtaining two hybridoma clone types, #23-57-154 and #23-57-137-1, both of which showed strong binding ability to PTHrP (1-34).

Hybridoma clone #23-57-137-1, designated "murine-murine hybridoma #23-57-137-1," has been deposited at the national institute of bioscience and human technology, agency of science and technology, in 1996, 8.15.d., under the terms of the Budapest treaty, Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan) under the accession number FERM BP-5631.

Example 1

Cloning of DNA encoding V region of mouse anti-human PTHrP (1-34) monoclonal antibody

The DNA encoding the V region of the above mouse anti-human PTHrP (1-34) monoclonal antibody obtained from #23-57-137-1 was cloned in the following manner.

(1) Preparation of mRNA

mRNA was prepared from hybridoma #23-57-137-1 using the Quick Prep mRNA purification kit (Pharmacia Biotech) as follows.

Hybridoma #23-57-137-1 cells obtained as described above were fully homogenized with extraction buffer, and mRNA was extracted therefrom using oligo (dT) -cellulose spin columns according to the procedure prescribed by the kit manufacturer. The extract solution was precipitated with ethanol to obtain mRNA as a precipitate. The mRNA pellet is dissolved in the elution buffer.

(2) Preparation and amplification of cDNA encoding murine H chain V region Gene

(i) Cloning of cDNA for H chain V region of antibody #23-57-137-1

The DNA encoding the H chain V region of the anti-human PTHrP murine monoclonal antibody was cloned by the 5' -RACE method (Frohman, M.A. et al, annual proceedings of the national academy of sciences USA, 85, 8998-. The procedure was performed using the 5 '-AmpliFINDER RACE kit (CLONECECH) following the manufacturer's protocol. In this method, the primer used for synthesizing cDNA is MHC2 primer (SEQ ID NO: 1), which hybridizes to murine H chain C region. Mu.g of the mRNA obtained above as a template for cDNA synthesis was mixed with 10pmoles of the MHC2 primer. The resulting mixture was reacted with reverse transcriptase at 52 ℃ for 30 minutes to prepare cDNA complementary to mRNA.

The product was mixed with 6N NaOH to hydrolyze mRNA therein (reaction at 65 ℃ C. for 30 minutes), and then ethanol-precipitated to isolate cDNA as a precipitate. The cDNA thus isolated was reacted with T4 RNA ligase at 37 ℃ for 6 hours and at room temperature for 16 hours to ligate the cDNA to the 5' end of the Ampli Finder Anchor (SEQ ID NO: 42). Anchor primers (SEQ ID NO: 2) and MHC-G1 primers (SEQ ID NO: 3) were used as primers for PCR amplification of cDNA (S.T. Jones et al, Biotechnology, 9, 88, 1991).

The PCR solution (50. mu.l) used in this method comprises 10mM Tris-HCl (pH8.3), 50mM KCl, 0.25mM dNTP (dATP, dGTP, dCTP, dTTP), 1.5mM MgCl₂2.5 units of TaKaRa Taq (Takara Shuzo), 10pmoles of Anchor primer, and 1. mu.l of the cDNA reaction mixture to which the MHC-G1 primer and the Ampli FINDER Anchor primer were ligated, and the mixture was sealed with 50. mu.l of mineral oil. By thermal cyclingPerforming PCR reaction for 30 cycles by using a cyclometer model 480J (Perkin Elmer), wherein the cycle temperature is 94 ℃ for 45 seconds; 45 seconds at 60 ℃; 72 ℃ for 2 minutes.

(ii) Cloning of cDNA for L chain V region of antibody #23-57-137-1

The DNA encoding the L chain V region of the mouse monoclonal antibody against human PTHrP was cloned by the 5' -RACE method (Frohman, M.A. et al, annual proceedings of the national academy of sciences USA, 85, 8998-. The method was performed using 5 '-AmpliFINDER RACE kit (Clonetech) following the manufacturer's protocol. In this method, oligo-dT primers are used as primers for synthesizing cDNA. Mu.g of the above mRNA (as template for cDNA synthesis) was mixed with oligo-dT primer. The resulting mixture was reacted with reverse transcriptase at 52 ℃ for 30 minutes to prepare cDNA. The product was mixed with 6N NaOH to hydrolyze RNA therein (reaction at 65 ℃ C. for 30 minutes), and the resulting mixture was precipitated with ethanol to isolate cDNA as a precipitate. The thus-synthesized cDNA was reacted with T4 RNA ligase at 37 ℃ for 6 hours and at room temperature for 16 hours to ligate the cDNA to the 5' -end of the Ampli FINDER Anchor.

PCR primers ML C (SEQ ID NO: 4) were designed based on the conserved sequence of the C region of the mouse L chain lambda strand and then synthesized using a 394DNA/RNA synthesizer (ABI). PCR solution (100. mu.l) for primer synthesis included 10mM Tris-HCl (pH8.3), 50mM KCl, 0.25mM dNTP (dATP, dGTP, dCTP, dTTP), 1.5mM MgCl₂2.5 units AmpliTaq (PerkinElmer), 50pmoles anchor primer (SEQ ID NO: 2), and 1. mu.l of the cDNA reaction mixture to which the ML C (SEQ ID NO: 4) primer and the Ampli FINDER anchor primer were ligated, overlaid with 50. mu.l of mineral oil. PCR was carried out for 35 cycles using a thermal cycler model 480J (Perkin Elmer) with a temperature cycle of 94 ℃ for 45 seconds; 45 seconds at 60 ℃; 72 ℃ for 2 minutes.

(3) Purification and cleavage of PCR products into fragments

Each DNA fragment amplified by the above PCR method was separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (product of FMC Bio. RTM.). Agarose gel fragments of each H chain V region and L chain V region of a DNA fragment of approximately 550 base pairs in length were cut out from the gel. Each gel fraction obtained was used to purify the DNA from the gel fraction using GENECLEAN II kit (BIO101) following the procedure provided by the manufacturer. The purified DNA was precipitated from the solution with ethanol and then dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA. Mu.l of the DNA solution thus prepared was digested with the restriction enzyme XmaI (New England Biolabs) at 37 ℃ for 1 hour, and additionally digested with the restriction enzyme EcoRI (Takara Shuzo) at 37 ℃ for 1 hour. The digestion mixture was extracted with phenol and chloroform, then precipitated with ethanol, and the DNA was collected.

In this manner, the DNA encoding the murine H chain V region and the DNA encoding the murine L chain V region were made to have both a5 '-terminal EcoRI recognition sequence and a 3' -terminal XmaI recognition sequence.

Each EcoRI-XmaI DNA fragment containing DNA encoding the mouse H chain V region and DNA encoding the mouse L chain V region was reacted with pUC19 vector previously digested with EcoRI and XmaI, respectively, using DNA ligation kit ver.2(Takara Shuzo) according to the procedure recommended by the manufacturer at 16 ℃ for 1 hour to ligate each other. The ligation mixture (10. mu.l) obtained was added to 100. mu.l of a solution containing competent cells of Escherichia coli JM109(Nippon Gene). The cell mixture was allowed to stand on ice for 15 minutes, at 42 ℃ for 1 minute, and then allowed to stand on ice for 1 minute. The product was mixed with 300. mu.l of SOC medium (molecular cloning: A laboratory Manual, Sambrook, et al, Cold spring harbor laboratory Press, 1989) and incubated at 37 ℃ for 30 minutes. The resulting cell solution was inoculated onto LB or 2XYT agar medium (molecular cloning: A laboratory Manual, Sambrook, et al, Cold Spring Harbor laboratory Press, 1989) supplemented with 100 or 50. mu.g/ml ampicillin, 0.1mM IPTG and 20. mu.g/ml X-gal, and then incubated at 37 ℃ overnight. In this manner, E.coli transformants were prepared.

The transformant was cultured overnight at 37 ℃ on2 ml of LB or 2XYT medium containing 100 or 50. mu.g/ml of ampicillin, and then plasmid DNA was prepared from the cell debris using a plasmid extractor PI-100 Sigma (Kurabou) or QIAprep Spin plasmid kit (QIAGEN). The sequence of the obtained plasmid DNA was determined.

(4) cDNA sequence coding for murine antibody V region

The coding region cDNA sequence carried on the plasmid was determined by DNA sequencer 373A (ABI; Perkin-Elmer) using dye-terminator cycle sequencing kit (Perkin-Elmer). This is a DNA sequencing method for determining the base sequence in both directions by using M13 primer M4(Takara Shuzo) (SEQ ID NO: 5) and M13 primer RV (Takara Shuzo) (SEQ ID NO: 6).

The obtained plasmids containing cDNA encoding murine H chain V region derived from hybridoma #23-57-137-1 and cDNA encoding murine L chain V region were designated "MBC 1H 04" and "MBC 1L 24", respectively. The DNA sequence encoding the H chain V region and the DNA sequence encoding the L chain V region of the murine #23-57-137-1 antibody (including the corresponding amino acid sequences) (carried on plasmid MBC1H04 and plasmid MBC1L24, respectively) are set forth in SEQ ID NO: 57 and 65. The polypeptides of the H chain V region fragment and the L chain V region fragment are all represented by SEQ ID NO: translation was initiated at the 58 th base (encoding glutamine) of the DNA sequences shown in 57 and 65. The amino acid sequences of the H chain V region and the L chain V region are respectively shown as SEQ ID NO: 46 and 45.

Escherichia coli having the plasmid MBC1H04 and Escherichia coli having the plasmid MBC1L24 were designated as "Escherichia coli JM109(MBC1H 04)" and "Escherichia coli JM109(MBC1L 24)", respectively. These E.coli strains have been deposited at the national institute of bioscience and human technology, agency of the scientific and technical institute, on 15.8.1996 under the terms of the Budapest treaty, Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan), with the accession number FERM BP-5628 for E.coli JM109(MBC1H04) and FERM BP-5627 for E.coli JM109(MBC1L 24).

(5) Determination of CDRs of anti-human PTHrP murine monoclonal antibody #23-57-137-1

The general structures of the H chain V region and the L chain V region are similar to each other. That is, both have a structure in which four framework regions are connected by three hypervariable regions, i.e., Complementarity Determining Regions (CDRs). The amino acid sequence of the framework regions is relatively conserved, while the amino acid sequence of the CDR regions exhibits a rather high mutagenicity (Kabat, E.A. et al, "protein sequences with immunological activity", U.S. department of health and human services, 1983).

Based on the above facts, CDRs can be determined by searching homology of the amino acid sequences of the V regions of the murine monoclonal antibody with reference to an antibody amino acid sequence database established by Kabat, e.a. etc.

The amino acid sequences of CDR1-3 in the L chain V region are respectively shown in SEQ ID NO: 59-61, the amino acid sequences of CDR1-3 in the H chain V region are shown in SEQ ID NO: shown at 62-64.

TABLE 2

Example 2

Construction of chimeric antibodies

(1) Construction of chimeric antibody H chain

(i) Construction of H chain V region

The cloned cDNA encoding the murine H chain V region was modified by PCR to ligate it into an expression vector carrying the human H chain V region C.gamma.1 genomic DNA. The downstream-side primer MBC1-S1(SEQ ID NO: 7) used was designed to hybridize with DNA encoding the 5' -terminal region of the V region leader sequence and had a Kozak consensus sequence (Kozak, M et al, J. mol. biol., 196, 947-950, 1987) and a HindIII-recognition sequence. The forward primer MBC1-a (SEQ ID NO: 8) used was designed to hybridize with DNA encoding the 3' -terminal region of the J region and has a junction donor sequence and a BamHI-recognition sequence. The PCR reaction was carried out using TaKaRa Ex Taq (Takara Shuzo) with addition of a buffer. The PCR solution (50. mu.l) used contained 0.07. mu.g of plasmid MBC1H04 as template DNA, 50pmole of MBC1-a and 50pmole of MBC1-S1 as primers, 2.5U of TaKaRaEx Taq and 0.25mM of dNTP in buffer, and the liquid surface was covered with 50. mu.l of mineral oil. The PCR reaction is carried out for 30 cycles, and the temperature cycle is 94 ℃ for 1 minute; 1 minute at 55 ℃; 72 ℃ for 2 minutes. The DNA fragments amplified by the PCR reaction were separated by agarose electrophoresis using 3% Nu Sieve GTG agarose (FMC bio. product).

Then, the agarose gel fragment containing the 437 base pair long DNA fragment was removed, and the DNA fragment was purified using GENECLEAN II kit (BIO101) according to the method described by the kit manufacturer. The purified DNA was collected by ethanol precipitation and then dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA. One microliter of the resulting DNA solution was digested with restriction enzymes BamHI and HindIII (Takara Shuzo) at 37 ℃ for 1 hour. The digestion mixture was extracted with phenol and chloroform, and then the DNA was collected by ethanol precipitation.

The obtained HindIII-BamHI DNA fragment containing DNA encoding the mouse H chain V region was subcloned into the pUCI9 vector digested with HindIII and BamHI. The resulting plasmids were sequenced by DNA sequencer 373A (Perkin-Elmer) using M13 primer M4 and M13 primer RV as primers and dye-terminator cycle sequencing kit (Perkin-Elmer). A plasmid containing DNA encoding the correct nucleotide sequence derived from the murine H chain V region of hybridoma #23-57-137-1 and having the HindIII recognition sequence and Kozak sequence of the 5 'terminal region and the BamHI recognition sequence of the 3' terminal region was designated "MBC 1H/pUC 19".

(ii) Construction of H chain V region for preparing mouse-human chimeric H chain cDNA type

The DNA encoding the mouse H chain V region constructed in the above-mentioned procedure was modified by PCR so as to be ligated to cDNA of human H chain C region C.gamma.1. The reverse primer MBC1HVS2(SEQ ID NO: 9) for modifying the H chain V region was designed so as to replace the second amino acid (i.e., aspartic acid) of the sequence encoding the front part of the V region leader sequence with glycine and has a Kozak consensus sequence (Kozak, M et al, J. Mol. biol., 196, 947-950, 1987) and HindIII-and EcoRI-recognition sequences. The forward primer MBC1HVR2(SEQ ID NO: 10) for modifying the H chain V region was also designed so as to be hybridizable to the DNA sequence coding for the 3 '-end region of the J region and the DNA coding for the 5' -end region of the C region, and had ApaI-and SmaI-recognition sequences.

The PCR reaction was carried out using TaKaRa Ex Taq (Takara Shuzo) and the attached buffer. The PCR solution (50. mu.l) used contained 0.6. mu.g of plasmid MBC1H/pUC19 as template DNA, 50pmole of MBC1HVS2 and 50pmole of MBC1HVR2 as primers, 2.5U of TaKaRa Ex Taq and 0.25mM dNTP in buffer, and the surface was covered with 50. mu.l of mineral oil. The PCR reaction is carried out for 30 cycles, and the temperature cycle is 94 ℃ for 1 minute; 1 minute at 55 ℃; 72 ℃ for 1 minute. The DNA fragments amplified by the PCR reaction were separated by agarose electrophoresis using 1% Sea KemGTG agarose (FMC Bio. product). Then, the agarose gel fragment containing the DNA fragment of 456 base pairs in length was excised, and the DNA fragment was purified using GENECLEAN II kit (BIO101) according to the method described by the kit manufacturer. The purified DNA was collected by ethanol precipitation and then dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

One microliter of the resulting DNA solution was digested with restriction enzymes EcoRI and SmaI (Takara Shuzo) at 37 ℃ for 1 hour. The digestion mixture was extracted with phenol and chloroform, and then the DNA was collected by ethanol precipitation. The obtained EcoRI-SmaI DNA fragment containing DNA encoding the mouse H chain V region was subcloned into the plasmid pUC19 vector digested with EcoRI and SmaI. The resulting plasmids were sequenced by DNA sequencer 373A (Perkin-Elmer) using M13 primer M4 and M13 primer RV as primers and dye-terminator cycle sequencing kit (Perkin-Elmer). A plasmid containing DNA encoding the correct nucleotide sequence derived from the murine H chain V region of hybridoma #23-57-137-1 and having a HindIII recognition sequence and a Kozak recognition sequence as 5 'end regions and ApaI and SmaI recognition sequences as 3' end regions was designated "MBC 1Hv/pUC 19".

(iii) Construction of chimeric antibody H chain expression vector

A cDNA comprising human antibody H chain C region C.gamma.1 was prepared as follows.

mRNA was prepared from CHO cells into which an expression vector DHFR-. DELTA.E-RVh-PM-1-f (see WO92/19759) encoding genomic DNA of the humanized PM1 antibody H chain V region and human antibody H chain C region IgG1 and an expression vector RV1-PM1a (see WO92/19759) encoding genomic DNA of the humanized PM1 antibody L chain V region and human antibody L chain kappa chain C region were introduced. From the obtained mRNA, cDNA comprising the humanized PM1 antibody H chain V region and human antibody C region C.gamma.1 was cloned by RT-PCR method, and then subcloned into the HindIII-BamHI site of plasmid pUC 19. The DNA sequence of the subcloned plasmid was determined and the plasmid with the correct nucleotide sequence was designated "pRVh-PM 1 f-cDNA".

An expression vector having a deletion of HindIII site between SV40 promoter and DHFR gene and a deletion of EcoRI site between EF-1. alpha. promoter and humanized PM1 antibody H chain V region was prepared, and used for constructing an expression vector of cDNA comprising humanized PM1 antibody H chain V region and human antibody C region C.gamma.1.

The obtained plasmid pRVh-PM1f-cDNA was digested with BamHI, blunt-ended with Klenow fragment, and further digested with HindIII to obtain a blunt-ended HindIII-BamHI fragment. This blunt-ended HindIII-BamHI fragment was ligated to the above-mentioned HindIII site and BamHI site-deleted expression vector DHFR-. DELTA.E-RVh-PM-1-f which had been digested with HindIII and BamHI, respectively, to construct an expression vector RVh-PM1f-cDNA comprising cDNA encoding the humanized PM1 antibody H chain V region and the human antibody C region C.gamma.1.

An expression vector RVh-PM1f-cDNA comprising cDNAs encoding the humanized PM1 antibody H chain V region and the human antibody C region Cgamma 1 was digested with ApaI and BamHI, from which a DNA fragment comprising the H chain V region was collected. The resulting DNA fragment was introduced into the above plasmid MBC1Hv/pUC19 digested with ApaI and BamHI. The plasmid thus prepared was designated "MBC 1HcDNA/pUC 19". This plasmid is a plasmid containing cDNAs encoding the H chain V region of a murine antibody and the C.gamma.1 region of a human antibody, respectively, and has EcoRI-and HindIII-recognition sequences at the 5 'ends and BamHI-recognition sequences at the 3' ends.

The plasmid MBC1HcDNA/pUC19 was digested with EcoRI and BamHI to obtain DNA encoding the H chain of the chimeric antibody. The resulting DNA fragment was introduced into the expression vector pCOS1 previously digested with EcoRI and BamHI. The expression vector encoding the chimeric antibody thus obtained was designated "MBC 1HcDNA/pCOS 1". Among them, HEF-PMh-g.gamma.1 (see W092/19759) antibody gene was deleted by digestion with EcoRI and SamI, and then it was ligated to EcoRI-NotI-BamHI conjugate (Takara Shuzo) to construct an expression vector pCOS 1.

To prepare a plasmid for expression in CHO cells, plasmid MBC1HcDNA/pUC19 was digested with EcoRI and BamHI to obtain a DNA encoding the H chain of the chimeric antibody, which was then introduced into an expression plasmid pCHO1 previously digested with EcoRI and BamHI. The expression plasmid encoding the chimeric antibody thus obtained was designated "MBC 1HcDNA/pCHO 1". Among them, DHFR-. DELTA.E-RVh-PM 1-f (see WO92/19759) antibody gene was deleted by digestion with EcoRI and SamI, and then it was ligated to EcoRI-NotI-BamHI conjugate (Takara Shuzo) to construct an expression vector pCHO 1.

(2) Construction of human L chain C region

(i) Preparation of cloning vectors

To construct pUC19 vector containing human L chain C region, pUC19 vector with HindIII site deleted was prepared. Mu.g of pUC19 vector contained 20mM Tris-HCl (pH8.5), 10mM MgCl in 20. mu.l₂1mM DTT, 100mM KCl, 8U HindIII (Takara Shuzo) reaction was digested at 37 ℃ for 1 hour. The resulting digestion mixture was extracted with phenol and chloroform, then precipitated with ethanol, and the DNA of interest was collected.

The collected DNA contained 50mM Tris-HCl (pH7.5), 10mM MgCl in 50. mu.l₂1mM DTT, 100mM NaCl, 0.5mM dNTP and 6U Klenow fragment (GIBCO BRL) were reacted at room temperature for 20 minutes to blunt the DNA ends. This reaction mixture was extracted with phenol and chloroform, and then precipitated with ethanol to collect the vector DNA.

The vector DNA thus collected contained 50mM Tris-HCl (pH7.6), 10mM MgCl in 10. mu.l₂1mM ATP, 1mM DTT, 5% (v/v) polyethylene glycol-8000, and 0.5U T4DNA ligase (GIBCO BRL) in a reaction solution at 16 ℃ for 2 hours to allow autonomous ligation of the vector DNA. Mu.l of the reaction solution was added to 100. mu.l of a solution containing competent Escherichia coli JM109(NipponGene), and the resulting solution was allowed to stand on ice for 30 minutes, reacted at 42 ℃ for 1 minute, and further placed on ice for 1 minute. 500 ml of SOC medium was added to the reaction solution, and the mixture was incubated at 37 ℃ for 1 hour. The resulting solution was plated on 2XYT agar medium (containing 50. mu.g/ml ampicillin) with X-gal and IPTG added to the surface (molecular cloning: A Laboratory Manual, Sambrook, et al, Cold Spri Nag Harbor Laboratory Press, 1989), and thenThe transformant was obtained by culturing at 37 ℃ overnight.

The transformants were cultured overnight at 37 ℃ in 2XYT agar medium containing 50. mu.g/ml ampicillin. Plasmid DNA was purified from the cell debris of the medium using the plasmid Mini kit (QIAGEN) according to the instructions on the kit. The purified plasmid was digested with HindIII. A plasmid with a deletion of the HindIII site was designated as "pUC 19. delta. HindIII".

(ii) Construction of DNA encoding human L chain lambda chain C region

It is known that the L chain lambda chain C region of a human antibody has at least four isotypes including Mcg⁺Ke⁺Oz^-，Mcg^-Ke^-Oz^-，Mcg^-Ke^-Oz⁺And Mcg^-Ke⁺Oz^-(P.Dariavach, et al, annual proceedings of the national academy of sciences USA, 84, 9074-. Human antibody L chain lambda chain C regions homologous to the #23-57-137-1 murine L chain lambda chain C region were retrieved based on the EMBL database. As a result, it was found that human antibody L chain lambda chain isoform Mcg⁺Ke⁺Oz^-(accession No. X57819) (P. Dariavach, et al, annual report of national academy of sciences USA, 84, 9074-.

Then, a DNA encoding the L chain lambda chain C region of the human antibody is constructed by PCR. Each of the primers used below was synthesized by a 394DNA/RNA synthesizer (ABI). Synthetic primers HLAMB1(SEQ ID NO: 11) and HLAMB3(SEQ ID NO: 13) each had a sense DNA sequence, primers HLAMB2(SEQ ID NO: 12) and HLAMB4(SEQ ID NO: 14) each had an antisense DNA sequence, and both ends of each primer contained a complementary sequence of 20-23 base pairs in length.

The outer primers HLAMBS (SEQ ID NO: 15) and HLAMBR (SEQ ID NO: 16) have sequences complementary to the primers HLAMB1 and HLAMB4, respectively, and comprise EcoRI-, HindIII-and BlnI-recognition sequences and EcoRI-recognition sequences, respectively. The first PCR reaction was performed with HLAMB1-HLAMB2 and HLAMB3-HLAMB4 reactions. After the reaction was completed, the two products were mixed in equal amounts and then subjected to a second PCR reaction. The external primers HLAMBS and HLAMBR are added into the reaction. The reaction mixture was subjected to a third PCR reaction for amplification of full length DNA.

The PCR reaction was carried out using TaKaRa Ex Taq (Takara Shuzo) according to the manufacturer's protocol. In the first PCR reaction, 100. mu.l of a reaction solution containing 5pmole HLAMB1, 0.5pmole HLAMB2 and 5U TaKaRa Ex Taq (Takara Shuzo) or a reaction solution containing 0.5pmole HLAMB3, 5pmole HLAMB4 and 5U TaKaRa Ex Taq (Takara Shuzo) was used, the liquid surface was covered with 50. mu.l of mineral oil, the PCR reaction was carried out 5 times, and the temperature cycle program was 94 ℃ for 1 minute; 1 minute at 60 ℃; 72 ℃ for 1 minute. In the second PCR reaction, 50. mu.l of each reaction solution was mixed, the liquid surface was covered with 50. mu.l of mineral oil, and 3 cycles of PCR reaction were carried out at 94 ℃ for 1 minute; 1 minute at 60 ℃; at 72 ℃ for 1 minute. In the third PCR reaction, 50pmole each of external primers HLAMBS and HLAMBR are added into the reaction liquid, 30 cycles of PCR reaction are carried out, and the temperature cycling program is 94 ℃ for 1 minute; 1 minute at 60 ℃; 72 ℃ for 1 minute.

The DNA fragment obtained from the third PCR reaction was electrophoresed on a 3% low melting point Agarose gel (NuSieve GTG Agarose, FMC), collected and purified from the gel using GENECLEAN II kit (BIO101) following the procedure described on the kit.

The DNA fragment thus obtained contained 50mM Tris-HCl (pH7.5), 10mM MgCl in 20. mu.l₂1mM DTT, 100mM NaCl, and 8U of EcoRI (Takara Shuzo) at 37 ℃ for 1 hour. The digested solution was extracted with phenol and chloroform, and then precipitated with ethanol to thereby obtain DNA. The DNA was collected and dissolved in 8. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

0.8. mu.g of plasmid pUC 19. delta. HindIII was digested with EcoRI in the same manner as described above. The digested liquid was extracted with phenol and chloroform, followed by precipitation with ethanol to obtain digested plasmid pUC 19. delta. HindIII. The plasmid thus digested contained 50mM Tris-HCl (pH9.0), 1mM MgCl in 50. mu.l₂And alkaline phosphatase (Escherichia coli C75; Takara Shuzo)The reaction was carried out at 37 ℃ for 30 minutes to dephosphorylate the plasmid (i.e., BAP-treatment). The reaction solution was extracted with phenol and chloroform, and the DNA was obtained by ethanol precipitation. The DNA thus obtained was dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

The thus prepared BAP-treated plasmid pUC 19. delta. HindIII 1. mu.l was mixed with 4. mu.l of the DNA obtained in the above PCR reaction, and the two were ligated by using DNA ligation kit Ver.2(Takara Shuzo). The obtained plasmid was introduced into competent Escherichia coli, and JM109 cells were used to form a transformant. The transformants were cultured overnight in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. The plasmid from the cells was purified using QIAprep Spin plasmid kit (QIAGEN).

For the above plasmids, the sequence of the cloned DNA was determined. For the determination of DNA sequence, 373A DNA sequencer (ABI) and primers "M13 primer M4" and "M13 primer RV" (Takara Shuzo) were used. As a result, it was found that the cloned DNA had a deletion portion of 12 base pairs in length. A plasmid containing the DNA was designated as "C.lamda.DELTA./pUC 19". Then, to compensate for this portion, primers, HCLMS (SEQ ID NO: 17) and HCLMR (SEQ ID NO: 18) were synthesized anew, and the correct DNA was reconstructed by the PCR method.

The first PCR reaction was performed using plasmid c.lamda.DELTA.pUC 19 containing the deleted DNA as a template and primers HLAMBS and HCLMS or primers HCLMS and HLAMB 4. Each PCR reaction product was purified separately. In the second PCR reaction, the PCR products were mixed with each other. The outer primers HLAMBS and HLAMB4 were added to the product, followed by a third PCR reaction to amplify full-length DNA.

In the first PCR reaction, 100. mu.l of a reaction solution containing 0.1. mu. g c. lambda. DELTA./pUC 19 as a template, 50pmole primers HLAMBS and HCLMR each or 50pmole primers HCLMS and HLAMB4 each, and 5U TaKaRa Ex Taq (Takara Shuzo) was used, and the liquid surface was covered with 50. mu.l of mineral oil, and the PCR reaction was carried out for 30 cycles using a temperature cycle program of 94 ℃ for 1 minute; 1 minute at 60 ℃; 72 ℃ for 1 minute.

The PCR products, HLAMBS-HCLMR (236 base pairs) and HCLMS-HLAMB4(147 base pairs), were electrophoresed on a 3% low melting agarose gel to separate DNA fragments. The DNA fragments were then collected and purified from the gel using GENECLEAN II kit (BIO 101). In the second PCR reaction, 20. mu.l of a reaction solution containing 40 ng of the purified DNA fragment and 1U of TaKaRa Ex Taq (Takara Shuzo) was used, and the liquid surface was covered with 25. mu.l of mineral oil, and the temperature cycle program was 94 ℃ for 1 minute; 1 minute at 60 ℃; 5 cycles of 1 minute at 72 ℃.

In the third PCR reaction, 100. mu.l of a reaction solution containing 2. mu.l of the reaction solution obtained in the second PCR reaction, 50pmole each of the external primers HLAMBS and HLAMB4, and 5UTaKaRa Ex Taq (Takara Shuzo) was used, the liquid surface was covered with 50. mu.l of mineral oil, and the PCR reaction was carried out for 30 cycles using a temperature cycle program of 94 ℃ for 1 minute; 1 minute at 60 ℃; this gave a DNA fragment of 357 base pairs in length (third PCR product) at 72 ℃ for 1 minute. The DNA fragments were separated by electrophoresis on a 3% low melting agarose gel. The resulting DNA fragment was collected and purified using GENECLEANII kit (BIO 101).

0.1. mu.g of the DNA fragment thus obtained was digested with EcoRI and then subcloned into the plasmid pUC 19. delta. HindIII previously treated with BAP. The resulting plasmid was introduced into competent Escherichia coli JM109 cells to form a transformant. The thus-prepared transformant was cultured overnight in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmids were purified from the cell debris using the QIAprep Spin plasmid kit (QIAGEN).

The DNA sequence of the thus obtained plasmid was determined in 373A DNA sequencer (ABI) using M13 primer M4 and M13 primer RV (Takara Shuzo). A plasmid having the correct DNA sequence without any deletion was specified as "C.lambda./pUC 19".

(iii) Construction of DNA encoding human L chain kappa chain C region

A DNA fragment encoding the C region of the L chain kappa chain was cloned by PCR from the plasmid HEF-PM1k-gk (WO 92/19759). The forward primers HKAPS (SEQ ID NO: 19) EcoRI-and HindIII-and BlnI-recognition sequences, and the reverse primer HKAPA (SEQ ID NO: 20) was designed to contain recognition sequences designed to contain EcoRI-both synthesized using a 394DNA/RNA synthesizer (ABI).

A PCR reaction was carried out using 100. mu.l of a reaction solution containing 0.1. mu.g of plasmid HEF-PM1k-gk as a template, 50pmole of each of primers HKAPS and HKAPA, and 5U of TaKaRa Ex Taq (Takara Shuzo), the surface of which was covered with 50. mu.l of mineral oil. The PCR reaction was carried out for 30 cycles using a temperature cycle of 94 ℃ for 1 minute; 1 minute at 60 ℃; this gave a DNA fragment of 360 base pairs in length at 72 ℃ for 1 minute. The DNA fragments were separated by 3% low melting point agarose gel electrophoresis, and then collected and purified using GENECLEAN II kit (BIO 101).

The DNA fragment thus obtained was digested with EcoRI and then cloned into plasmid pUC 19. delta. HindIII which had been treated with BAP. The obtained plasmid was introduced into competent Escherichia coli, and JM109 cells were used to form a transformant. The thus-prepared transformant was cultured overnight in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmids were purified from the cell debris using the QIAprep Spin plasmid kit (QIAGEN).

The purified plasmid DNA was sequenced with M13 primer M4 and M13 primer RV (Takara Shuzo) on 373A DNA sequencer (ABI). A plasmid having the correct nucleotide sequence was designated as "C.kappa./pUC 19".

(3) Construction of L chain expression vector for chimeric antibody

An L chain expression vector of the chimeric #23-57-137-1 antibody was constructed. The DNA encoding the L chain V region of the antibody #23-57-137-1 was ligated to HindIII and BlnI sites before the human antibody C region of each of the plasmids C.lambda./pUC 19 and C.kappa./pUC 19, thereby obtaining a pUC19 vector containing the DNA encoding the L chain V region and L chain. lambda. or kappa chain C region of the chimeric antibody # 23-57-137-1. Each of the resulting vectors was then digested with EcoRI to excise DNA encoding the L chain of the chimeric antibody, which was subcloned into the HEF expression vector.

The DNA encoding the L chain V region of the #23-57-137-1 antibody was cloned from the plasmid MBC1L24 by PCR. Primers were synthesized individually using a 394DNA/RNA synthesizer (ABI). The reverse primer MBCCHL1(SEQ ID NO: 21) used was designed to contain the Hind III-recognition sequence and the Kozak sequence (Kozak, M et al, J. mol. biol., 196, 947-950, 1987) and the forward primer MBCCHL3(SEQ ID NO: 22) was designed to contain the BglII-and RcoRI-recognition sequences.

With 100. mu.l of a solution containing 10mM Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl₂A PCR reaction was carried out using a reaction solution containing 0.2mM dNTP, 0.1. mu.g of MBC1L24, 50pmole of each of primers MBCCHL1 and MBCCHL3, and 1. mu.l of AmpliTaq (PERKIN ELMER), and the surface of the reaction solution was covered with 50. mu.l of mineral oil. The PCR reaction was carried out for 30 cycles, using a temperature cycle of 94 ℃ for 45 seconds; 45 seconds at 60 ℃; 72 ℃ for 2 minutes.

A DNA fragment 444 base pairs in length was separated by 3% low-melting agarose gel electrophoresis, and then collected and purified using GENECLEAN II kit (BIO 101). The purified DNA fragment was dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA. Mu.l of PCR product contained 10mM Tris-HCl (pH7.5), 10mM MgCl in 20. mu.l₂1mM DTT, 50mM NaCl, 8U of HindIII (Takara Shuzo) and 8U of EcoRI (Takara Shuzo) were digested at 37 ℃ for 1 hour. The digestion solution was extracted with phenol and chloroform, followed by ethanol precipitation to collect the precipitated DNA. The DNA thus obtained was dissolved in 8. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

Mu.g of plasmid pUC19 was digested with HindIII and EcoRI in the same manner as described above. Then phenol and chloroform extraction, followed by ethanol precipitation collection of digested plasmid. The product was treated with BAP (i.e., alkaline phosphate (Escherichia coli C75; Takara Shuzo)), followed by extraction with phenol and chloroform, and ethanol precipitation to obtain DNA. The DNA thus obtained was dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

Mu.l of BAP-treated plasmid pUC191 was mixed with 4. mu.l of the above PCR product, and the two were ligated by using DNA ligation kit Ver.2(Takara Shuzo). The resulting plasmid was introduced into competent Escherichia coli JM109 cells (Nippon Gene), and a transformant was formed in the same manner as described above. The transformants were cultured overnight at 37 ℃ on 2XYT agar medium containing 50. mu.g/ml ampicillin. The plasmid in the cell debris was purified using QIAprep Spin plasmid kit (QIAGEN). After DNA sequencing, the plasmid with the correct DNA sequence was designated "CHL/pUC 19".

Mu.g each of the plasmids Cλ/pUC19 and Cκ/pUC19 contained 20mM Tris-HCl (pH8.5), 10mM MgCl in 20 μ l₂1mM DTT, 100mM KCl, 8U HindIII (Takara Shuzo) and 2U BlnI (Takara Shuzo) were digested at 37 ℃ for 1 hour. The digested solution was extracted with phenol and chloroform, followed by precipitation with ethanol, thereby obtaining DNA. The DNA was treated with BAP at 37 ℃ for 30 minutes, then extracted with phenol and chloroform, followed by ethanol precipitation. The product was dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

Mu.g of plasmid CHL/pUC19 containing DNA encoding the #23-57-137-1L chain V region was digested with HindIII and BlnI in the same manner as described above. A DNA fragment of 409 base pairs in length was obtained by 3% low-melting agarose gel electrophoresis, and then collected and purified from the gel using GENECLEAN II kit (BIO 101). The DNA fragment was dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

Mu. l L chain V region DNA was subcloned into 1. mu.l of BAP-treated plasmid C.lambda./pUC 19 or C.kappa./pUC 19, respectively, and then introduced into competent E.coli, JM109 cells to form transformants. The transformants were cultured overnight in 3 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmids from the cell debris were isolated and purified using QIAprep Spin plasmid kit (QIAGEN). The plasmids thus prepared were designated as "MBC 1L (. lamda.)/pUC 19" and "MBC 1L (. kappa.)/pUC 19", respectively.

Plasmids MBC1L (. lamda.)/pUC 19 and MBC1L (. kappa.)/pUC 19 were digested with EcoRI, respectively, and then subjected to electrophoresis with 3% low-melting agarose gel. A DNA fragment 743 base pairs in length was isolated and purified from the gel using GENECLEAN II kit (BIO101), and dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

2.7. mu.g of the expression vector, plasmid HEF-PM1k-gk, was digested with EcoRI, followed by extraction with phenol and chloroform, followed by precipitation with ethanol, thereby obtaining a DNA fragment. The DNA fragments were treated with BAP and then electrophoresed on a 1% low melting agarose gel. DNA fragments of 6561 base pairs in length were isolated and purified from the gel using GENECLEAN II kit (BIO 101). The DNA fragment was dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

The HEF vector thus prepared was treated with BAP, and 2. mu.l of the BAP-treated HEF vector was mixed with 3. mu.l of EcoRI fragments of plasmids MBC1L (. lamda.)/pUC 19 and MBC1L (. kappa.)/pUC 19 to ligate each other. The ligation product was introduced into competent E.coli, JM109 cells, to form transformants. The transformants were cultured in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. The plasmid in the cell debris was purified using QIAprep Spin plasmid kit (QIAGEN).

The plasmid DNA thus purified contained 20mM Tris-HCl (pH8.5), 10mM MgCl in 20. mu.l₂1mM DTT, 100mM KCl, 8U of HindIII (Takara Shuzo) and 2UPvuI (Takara Shuzo) were digested at 37 ℃ for 1 hour. In this digestion reaction, it is presumed that a digested fragment of 5104/2195 base pairs is obtained when the above-mentioned DNA fragment is inserted into the vector in the forward direction, and a digested fragment of 4387/2926 base pairs is obtained when the above-mentioned DNA fragment is inserted into the vector in the reverse direction. Based on this presumption, plasmids of the forward insert were designated as "MBC 1L (λ)/neo" and "MBC 1L (κ)/neo", respectively.

(4) Transfection of COS-7 cells

The expression plasmids prepared above were transiently expressed using COS-7 cells, respectively, to evaluate the binding and neutralizing activity of the chimeric antibody to the antigen.

Plasmids MBC1HcDNA/pCOS1 and MBC1L (. lamda.)/neo or plasmids MBC1HcDNA/pCOS1 and MBC1L (. kappa.)/neo were bound by Gene Pulser (Bio Rad) electroporation for transient expression of chimeric antibodies to co-transfect these combinations of plasmid DNA into COS-7 cells. That is, cotransfected into 0.8 ml of a cell suspension in which COS-7 cells were suspended in PBS (-), at a concentration of 1X 10⁷Cells/ml, and 10. mu.l of each plasmid DNA was added. The resulting solution was electroporated using a pulse of 1500V and 2 μ F electrostatic capacity. After 10 min of room temperature recovery, cells were suspended in DMEM medium containing 2% Ultra Low IgG fetal bovine serum (GIBCO) and then plated on a10 cm petri dish in CO₂Culturing in an incubator. Cultured for 72 hoursAfter incubation, the culture supernatant was collected and centrifuged to remove cell debris, which was used as a sample for ELISA analysis.

In this procedure, purification of chimeric antibody from the supernatant of COS-7 cells was performed by AffiGelProtein A MAPSII kit (Bio Rad) following the procedure described above for the kit.

(5) ELISA assay

(i) Determination of antibody concentration

ELISA plates for determination of antibody concentration were prepared as follows. Each well of a 96-well plate for ELISA (Maxisorp, NUNC) was plated with 100. mu.l of a coating buffer (0.1M NaHCO)₃，0.02％NaN₃) The goat anti-human IgG antibody (TAGO) solution prepared in (1) μ g/ml was coated with 200 μ l of dilution buffer [50mM Tris-HCl, 1mM MgCl ]₂，0.1M NaCl，0.05％Tween20，0.02％NaN₃1% Bovine Serum Albumin (BSA); pH7.2]And (4) separating. COS-7 cell culture supernatant in which the chimeric antibody was expressed or purified chimeric antibody gradually diluted was added to each well. After incubation for 1 hour at room temperature and washing with PBS-Tween20, 100. mu.l of alkaline phosphatase conjugated goat anti-human IgG antibody (TAGO) was added to each well. After incubation for 1 hour at room temperature and washing with PBS-Tween20, 1 mg/ml of matrix solution ("Sigma 104", p-nitrophenyl phosphate, SIGMA) was added to each well. The absorbance of the solution was measured at 405nm using a microplate reader (Bio Rad). Purified Hu IgG1 λ (binding site) was used as a standard for this assay.

(ii) Determination of antigen binding Capacity

ELISA plates for determining antigen binding capacity were prepared as follows. Each well of a 96-well plate used for ELISA analysis was coated with 100. mu.l of a solution containing human PTHrP (1-34) (peptide research institute) prepared in the coating buffer at a concentration of 1. mu.g/ml, and then blocked with 200. mu.l of a dilution buffer. COS-7 cell culture supernatant in which the chimeric antibody was expressed or purified chimeric antibody gradually diluted was added to each well. After incubation at room temperature and washing with PBS-Tween20, 100. mu.l of alkaline phosphatase conjugated goat anti-human IgG antibody (TAGO) solution was added to each well. After incubation at room temperature and washing with PBS-Tween20, 1 mg/ml of matrix solution ("Sigma 104", p-nitrophenyl phosphate, SIGMA) was added to each well. The absorbance of the solution was measured at 405nm using a microplate reader (Bio Rad).

As a result, it was found that the chimeric antibody had binding ability to human PTHrP (1-34) and also had the correct structure of the cloned murine antibody V region (FIG. 4). It was also found that there was no difference in the binding ability to PTHrP (1-34) between the chimeric antibody having a. lamda. chain in the L chain C region and the chimeric antibody having a. kappa. chain in the L chain C region. Thus, a humanized antibody L chain C region was constructed by humanizing an antibody L chain lambda chain.

(6) Establishment of CHO stably transformed cell line

In order to establish stable transformants producing the chimeric antibody, the above expression plasmid was introduced into CHO cells (DXB 11).

The establishment of stable transformants of the chimeric antibody was achieved by the binding of the expression plasmid of CHO cells, plasmids MBC1HcDNA/pCHO1 and MBC1L (lambda)/neo or the expression plasmid of CHO cells, MBC1HcDNA/pCHO1 and MBC1L (kappa)/neo. These plasmid assemblies were separately co-transfected into CHO cells by Gene Pulser (Bio Rad) electroporation. Each expression vector was cleaved into linear DNA with the restriction enzyme PvuI. The resulting DNA was extracted with phenol and chloroform and collected by ethanol precipitation. The plasmid DNAs thus prepared were electroporated separately. Plasmid DNA 10. mu.g each was added to 0.8 ml of CHO-containing cells (concentration 1X 10)⁷Individual cells/ml) in PBS (-) cell suspension. The resulting mixture was subjected to electroporation using a pulse having an electrostatic capacity of 1500V and 2. mu.F. After 10 minutes of recovery at room temperature, the electroporated cells were suspended in MEM- α medium supplemented with 10% fetal bovine serum (GIBCO). The resulting suspension was plated in CO using three 96-well plates (Falcon)₂Culturing in an incubator. On the day of culture, the medium was supplemented with selective medium [ MEM-alpha medium supplemented with 10% fetal bovine serum (GIBCO) and 500 mg/ml GENETICIN (G418 Sulfate; GIBCO) ribonucleoside-free or deoxyribonucleoside]And (6) replacing. Cells into which the antibody gene has been introduced are selected from the culture medium. After replacement of the selective medium with fresh medium, before the start of the culture under microscopic observationAnd cells after 2 weeks of culture. When cell growth was observed, the amount of antibody produced was determined using the ELISA assay described above. Cells producing large amounts of antibody are selectively collected.

Antibody-stable transformants were established for large-scale culture in shake flasks in MEM medium supplemented with 2% Ultra Low IgG fetal bovine serum, ribonucleoside-free or deoxyribonucleoside. After 3 to 4 days of culture, the culture supernatant was collected and then filtered through a filter having a pore size of 0.2 μm (Millipore) to remove cell debris.

Subsequently, the resulting mixture was concentrated on a POROS Protein A column (PerSeptive Biosystems) at ConSep

Chimeric antibodies in CHO cell culture supernatants were purified on LC100(Millipore) according to the method described above. The purified chimeric antibody was used as a sample for determination of neutralizing activity and examination of efficacy against hypercalcemia model animals. The concentration and binding activity of the purified chimeric antibody antigen were determined using the same ELISA system.

Example 3

Construction of humanized antibody

(1) Construction of humanized antibody H chain

(i) Construction of humanized H chain V region

Humanized #23-57-137-1 antibody H chain was prepared by PCR using CDR-grafting technique. For the preparation of the humanized #23-57-137-1 antibody H chain ("type a") having FR derived from the human antibody S31679 (NMRF-PDB; Cuisinier, A.M., et al, J. Eur. Immunity, 23, 110-118, 1993), the following six types of PCR primers were used: CDR-grafting primers: MBC1HGP1(SEQ ID NO: 23) and MBC1HGP3(SEQ ID NO: 24) (both comprising sense DNA sequences) and MBCHGP2(SEQ ID NO: 25) and MBC1HGP4(SEQ ID NO: 26) (both comprising antisense DNA sequences) each comprising a complementary sequence 15-21 base pairs in length at each end; and an outer primer: MBC1HVS1(SEQ ID NO: 27) and MBC1HVR1(SEQ ID NO: 28) having homology to the CDR-grafting primers MBC1HGP1 and MBC1HGP4, respectively.

The CDR-grafting primers MBC1HGP1, MBCHGP2, MBC1HGP3 and MBC1HGP4 were isolated from the gel fragments by the crush-soak method (molecular cloning: A Laboratory Manual, Sambrook et al, Cold Spring Harbor Laboratory Press, 1989) using a urea-denatured polyacrylamide gel (molecular cloning: A Laboratory Manual, Sambrook et al, Cold Spring Harbor Laboratory Press, 1989) in the following manner.

Each CDR-grafting primer was separated at 1nmole using 6% denaturing polyacrylamide gel to obtain DNA fragments. DNA fragments of a desired length were identified from the product DNA fragments by UV irradiation of a silica gel thin-layer plate, and then collected by a crush-soak method. The product was dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA. The PCR reaction was carried out using TaKaRa Ex Taq (Takara Shuzo). The reaction solution (100. mu.l) used for the PCR reaction contained 1. mu.l each of the above CDR-grafted primers MBC1HGP1, MBCHGP2, MBC1HGP3 and MBC1HGP4, 0.25mM dNTP and 2.5U of TaKaRa Ex Taq buffer. The PCR reaction was performed for 5 cycles, using a temperature cycle of 94 ℃ for 1 minute; 1 minute at 55 ℃; 72 ℃ for 1 minute. 50pmole each of the outer primers MBC1HVS1 and MBC1HVR1 was added to the reaction mixture. The PCR reaction was performed using this reaction mixture for another 30 cycles using the same temperature cycle. The DNA fragment thus amplified was separated by gel electrophoresis using 4% Nu Sieve GTG agarose (FMC Bio.products).

The agarose fragment containing the DNA fragment 421 base pairs in length was excised, and the DNA fragment was purified using GENECLEAN II kit (BIO101) according to the method indicated on the kit. The purified DNA fragment was precipitated with ethanol and then dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA. The obtained PCR reaction mixture was used to subclone the DNA fragment onto plasmid pUC19 previously digested with BamHI and HindIII; subsequently, the nucleotide sequence of the resulting plasmid was determined. The plasmid with the correct sequence was designated "hMBCHv/pUC 19".

(ii) Construction of H chain V region for humanized H chain cDNA

The DNA of the humanized H chain V region constructed in the above-described procedure was modified by PCR for ligation to the cDNA of the humanized H chain C region C.gamma.1. In this method, the reverse primer MBC1HVS2 used was designed to hybridize with the sequence coding for the 5' -terminal region of the V region leader sequence and to carry a Kozak consensus sequence (Kozak et al, J. Mol. biol., 196, 947-950, 1987) and HindIII-and EcoRI-recognition sequences. The forward primer MBC1HVR2 for modifying the H chain V region DNA was designed so as to hybridize with the DNA sequence encoding the 3 '-terminal region of the J region and the DNA sequence encoding the 5' -terminal region of the C region and to carry ApaI-and SamI-recognition sequences.

The PCR reaction was carried out using TaKaRa Ex Taq (Takara Shuzo) to which a buffer was applied. The reaction solution used for the PCR reaction contained 0.4. mu.g of hMBCHv/pUC19 as a DNA template, 50pmole each of MBC1HVS2 and MBC1HVR2 as a primer, 2.5U of TaKaRaEx Taq and 0.25mM dNTP in a buffer. The PCR reaction was performed for 30 cycles, using a temperature cycle of 94 ℃ for 1 minute; 1 minute at 55 ℃; 72 ℃ for 1 minute. The DNA fragment thus amplified by PCR was separated by gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio.products).

The DNA fragment of 456 base pairs in length was excised and purified therefrom using GENECLEAN II kit (BIO101) according to the method indicated on the kit. The purified DNA fragment was precipitated with ethanol and then dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA. The obtained PCR reaction mixture was used for subcloning the DNA fragment onto the plasmid pUC19 previously digested with EcoRI and SamI; subsequently, the nucleotide sequence of the resulting plasmid was determined. The DNA thus prepared, which contained the mouse H chain V region derived from hybridoma #23-57-137-1 and plasmid DNA also containing the 5 '-terminal regions HindIH-and EcoRI-recognition sequences and Kozak sequences and the 3' -terminal regions ApaI-and SamI-recognition sequences, was designated "hMBC 1Hv/pUC 19".

(2) Construction of humanized antibody H chain expression vector

Plasmid RVh-PM1f-cDNA, which contains the cDNA sequence of the H chain of the hPM1 antibody, was digested with ApaI and BamHI to obtain a DNA fragment containing the base sequence of the H chain C region. This DNA fragment was introduced into plasmid hMBC1Hv/pUC19 previously digested with ApaI and BamHI. The plasmid thus prepared was designated "hMBC 1HcDNA/pUC 19". The plasmid contains DNA encoding the H chain V region of the humanized #23-57-137-1 antibody and DNA encoding the C.gamma.1 region of the human H chain and has recognition sequences for the 5 '-terminal regions HindIII-and EcoRI-and the 3' -terminal region BamHI-recognition sequences. The base sequence and the corresponding amino acid sequence of the humanized H chain "a" type contained in plasmid hMBC1HcDNA/pUC19 are respectively shown in SEQ ID NO: 58 and SEQ ID NO: as shown at 56.

The plasmid hMBC1HcDNA/pUC19 was digested with EcoRI and BamHI to obtain a DNA fragment containing a base sequence encoding H chain. This DNA fragment was introduced into an expression plasmid pCOS1 previously digested with EcoRI and BamHI. The expression plasmid of the humanized antibody obtained was designated as "hMBC 1HcDNA/pCOS 1".

To prepare a plasmid for expression in CHO cells, the plasmid hMBC1HcDNA/pUC19 was digested with EcoRI and BamHI to obtain a DNA fragment containing DNA encoding H chain. This DNA fragment was introduced into an expression vector pCHO1 previously digested with EcoRI and BamHI. The expression plasmid of the humanized antibody thus obtained was designated as "hMBC 1HcDNA/pCHO 1".

(3) Construction of L chain hybrid variable regions

(i) Preparation of FR1, 2/FR3, 4 hybrid antibody

A DNA encoding an L chain in which FR regions are recombined with humanized antibody and murine (chimeric) antibody FR regions was constructed, and suitability for the humanization of the regions was evaluated. In this step, antibodies comprising FR1 and FR2 derived from human antibodies and FR3 and FR4 derived from murine antibodies were prepared using the Afl II restriction site present in CDRZ.

10. mu.g of each of the plasmids MBC1L (. lamda.)/neo and hMBC1L (. lamda.)/neo contained 10mM Tris-HCl (pH7.5), 10mM MgCl and 100. mu.l each₂1mM DTT, 50mM NaCl, 0.01% (w/v) BSA and 10U Afl II (Takara Shuzo) were digested at 37 ℃ for 1 hour. The reaction solution was electrophoresed on a 2% low-melting agarose gel, and a DNA fragment (referred to as "c 1") having a length of 6282 base pairs and a DNA fragment (referred to as "h 1") having a length of 1022 base pairs were collected and purified from the gel using GENECLEANII kit (BIO 101).

Mu.g each of the obtained c1 and h1 fragments was treated with BAP. The DNA fragments were collected by phenol and chloroform extraction and ethanol precipitation, and then dissolved in 10. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

BAP-treated c1 and h1DNA fragments 1. mu.l each were mixed with 4. mu. l h2 and c2DNA fragments, respectively, to allow ligation to each other (overnight at 4 ℃). The ligation product was introduced into competent Escherichia coli JM109 cells to form transformants. The transformants were cultured in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmids were purified from the cell debris using the QIAprep Spin plasmid kit (QIAGEN).

The purified plasmid DNA contained 10mM Tris-HCl (pH7.5), 10mM MgCl in 20. mu.l₂1mM DTT, 2U ApaLI (Takara Shuzo), and 8U BamHI (Takara Shuzo) or HindIII (Takara Shuzo) were digested at 37 ℃ for 1 hour. In this step, plasmids were identified based on the assumption that if the c1-h2 fragment was ligated correctly to the plasmid, the ligation yielded either an ApaLI digested fragment of 5560/124/498 base pairs or a BamHI/HindIII digested fragment of 7134/269 base pairs.

The expression vector encoding the L chain of the human FR1, 2/murine FR3, 4 hybrid antibody is designated "h/mMBC 1L (lambda)/neo". On the other hand, no clone of h1-c1 was obtained. Thus, recombination was performed on a pUC vector, followed by cloning into a HEF vector. Among them, used as templates are a plasmid hMBC1La lambda/pUC 19 comprising a DNA encoding the L chain V region of a humanized antibody without amino acid substitution, and a plasmid hMBC1Ld lambda/pUC 19 comprising a DNA encoding the L chain V region of a humanized antibody in which tyrosine at position 91 (i.e., amino acid number 87 as defined in Kabat) is substituted with isoleucine in FR 3.

10 microliters each of plasmids MBC1L (. lamda.)/pUC 19, hMBC1 La. lamda./pUC 19 and hMBC1 Ld. lamda./pUC 19 contained 10mM Tris-HCl (pH7.5), 10mM MgCl in 30. mu.l₂1mM DTT, 50mM NaCl, 0.01% (w/v) BSA, 16U HindIII and 4U Afl II were digested at 37 ℃ for 1 hour. The reaction solution was electrophoresed on a 2% low-melting agarose gel, and then the following DNA fragments were collected and purified from the plasmid MBC1L (. lamda.)/pUC 19 using GENECLEANII kit (BIO 101): a DNA fragment of 215 base pairs in length (referred to as "c 2") orA DNA fragment 3218 base pairs in length (referred to as "hal" and "hdl", respectively) was collected and purified from plasmids hMBC1La λ/pUC19 and hMBC1Ld λ/pUC19, respectively.

Each of the hal ' and hdl ' fragments was ligated to the c2 ' fragment, respectively, and then introduced into competent E.coli JM109 cells to form transformants. The transformants were cultured in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmids were purified from the cell debris using the QIAprep Spin plasmid kit (QIAGEN). The resulting plasmid DNA containing the hal 'and hdl' fragments were designated "m/hMBC 1La λ/pUC 19" and "m/hMBC 1Ld λ/pUC 19", respectively.

Plasmids m/hMBC1La lambda/pUC 19 and m/hMBC1Ld lambda/pUC 19 were digested with EcoRI, respectively. A DNA fragment 743 bp in length was collected and purified from the gel by 2% low-melting agarose gel electrophoresis using GENECLEANII kit (BIO 101). The product was dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

Mu.l of the obtained DNA fragment was mixed with 1. mu.l of the above BAP-treated HEF vector to ligate each other. The ligation product was introduced into competent Escherichia coli JM109 cells to form transformants. The transformants were cultured in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmid DNA was purified from the cell debris using the QIAprep Spin plasmid kit (QIAGEN).

The purified plasmid DNA contained 20mM Tris-HCl (pH8.5), 10mM MgCl in 20. mu.l₂1mM DTT, 100mM KCl, 8U of HindIII (Takara Shuzo) and 2UPvuI (Takara Shuzo) were digested at 37 ℃ for 1 hour. In this step, plasmid DNA was identified based on the assumption that the ligation reaction resulted in a digested fragment of 5104/2195 base pairs if the DNA fragment was inserted forward into the plasmid, and 4378/2926 base pairs if the DNA fragment was inserted backward into the plasmid. The plasmids thus obtained were expression vectors encoding the L chains of the murine FR1, 2/human FR3, 4 hybrid antibody designated as expression vectors "m/hMBC 1 La. lambda./neo" and "m/hMBC 1 Ld. lambda./neo", respectively.

(ii) Preparation of FR1/FR2 hybrid antibody

The FR1/FR2 hybrid antibody was prepared in the same manner as described above using the SnaBI restriction sites present in CDR 1.

10. mu.g each of the plasmids MBC1L (. lamda.)/neo and mMBC1L (. lamda.)/neo contained 10mM Tris-HCl (pH7.9), 10mM MgCl and 20. mu.l each₂1mM DTT, 50mM NaCl, 0.01% (w/v) BSA, and 6U SnaBI (Takara Shuzo) was digested at 37 ℃ for 1 hour. The reaction solution obtained further contained 20mM Tris-HCl (pH8.5), 10mM MgCl in 50. mu.l₂1mM DTT, 100mM KCl, 0.01% (w/v) BSA, and 6U PvuI at 37 ℃ for 1 hour.

The resulting reaction solution was electrophoresed on a 1.5% low-melting agarose gel, and then a DNA fragment having a length of 4955 base pairs and 2349 base pairs was collected and purified from the gel using GENECLEANII kit (BIO 101). The DNA fragments obtained from plasmid MBC1L (λ)/neo were designated "m 1" (4955 base pairs) and "m 2" (2349 base pairs), and the DNA fragments obtained from plasmid h/mMBC1L (λ)/neo were designated "hm 1" (4955 base pairs) and "hm 2" (2349 base pairs). Each of the obtained DNA fragments was dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

mu.l each of the m1 and hm1 fragments were ligated to 4. mu.l each of the hm2 and m2 fragments, respectively, and then introduced into competent E.coli JM109 cells to form transformants. The obtained transformants were cultured in 2 ml of 2XYT medium containing 50. mu.g/ml ampicillin. Plasmid DNA was purified from the cell debris using the QIAprepspin plasmid kit (QIAGEN).

The purified plasmid DNA contained 10mM Tris-HCl (pH7.5), 10mM MgCl in 20. mu.l₂1mM DTT, and 8U ApaI (Takara Shuzo) or 2U ApalI (Takara Shuzo) at 37 ℃ for 1 hour.

Plasmids thus prepared (m1-hm2 and hm1-m2) were identified based on the assumption that if each DNA fragment was ligated to the plasmid in the correct orientation, the plasmid digested with ApaI and ApaLI (m1-hm2) would give a digested fragment of 7304 base pairs and a digested fragment of 5560/1246/498 base pairs, respectively, and the plasmid digested with ApaI and ApaLI (hm1-m2) would give a fragment of 6538/766 base pairs and a fragment of 3535/2025/1246/498 base pairs, respectively.

The expression vector encoding human FR 1/murine FR2, 3, 4 hybrid antibody L chain was designated "hmmbC 1L (λ)/neo", and the expression vector encoding murine FR 1/human FR 2/murine FR3, 4 hybrid antibody L chain was designated "mhmbC 1L (λ)/neo".

(4) Construction of humanized antibody L chain

The humanized #23-57-137-1 antibody L chain was prepared by PCR using CDR-grafting technique. That is, an L chain of a humanized #23-57-137-1 antibody ("type a") comprising FR1, FR2, FR3 derived from the human antibody HSU03868(GEN-BANK, Deftos M. et al, Scand. J. Immunol, 39, 95-103, 1994) and FR4 derived from the human antibody S25755(NBRF-PDB) FR4 was prepared using the following six types of PCR primers:

CDR-grafting primers with sense DNA sequence: MBC1LGP1(SEQ ID NO: 29) and MBC1LGP3(SEQ ID NO: 30), CDR-grafting primers with antisense DNA sequences: MBC1LGP2(SEQ ID NO: 31) and MBC1LGP4(SEQ ID NO: 32), all of which have 15-21 base pair complementary sequences at each end; and outer primers MBC1LVS1(SEQ ID NO: 33) and MBC1LVR1(SEQ ID NO: 34) homologous to the CDR-grafting primers MBC1LGP1 and MBC1LGP4, respectively.

The CDR-grafting primers MBC1LGP1, MBCLGP2, MBC1LGP3 and MBC1LGP4 were isolated from urea-denatured polyacrylamide gel (molecular cloning: A Laboratory Manual, Sambrook et al, Cold Spring Harbor Laboratory Press, 1989) and extracted from the gel fragments by crush-soak (molecular cloning: a Laboratory Manual, Sambrook et al, Cold Spring Harbor Laboratory Press, 1989).

1nmole of each CDR-grafting primer was separated on a 6% denaturing polyacrylamide gel. The DNA fragments of the desired length are identified on a thin silica gel plate irradiated with UV rays and then collected therefrom by a crush-soak method. The product was dissolved in 20. mu.l of a solution containing 10mM Tris-HCl (pH7.4) and 1mM EDTA.

The PCR reaction was carried out using TaKaRa Ex Taq (Takara Shuzo) buffer. The reaction solution (100. mu.l) used for the PCR reaction contained 1. mu.l each of the CDR-grafting primers MBC1LGP1, MBCLGP2, MBC1LGP3 and MBC1LGP4, 0.25mM dNTP, 2.5UTaKaRa Ex Taq in buffer. The PCR reaction was performed for 5 cycles using a temperature cycle of 94 ℃ for 1 minute; 1 minute at 55 ℃; 72 ℃ for 1 minute. To the reaction mixture were added 50pmole each of the outer primers MBC1LVS1 and MBC1LVR 1. The PCR reaction was performed using this reaction mixture for another 30 cycles using the same temperature cycle. The DNA fragment thus amplified was separated by gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio.products).

The agarose fragment containing the DNA fragment 421 base pairs in length was excised, and the DNA fragment was purified using GENECLEAN II kit (BIO101) according to the method indicated on the kit. The PCR reaction mixture thus prepared was used for subcloning of the DNA fragment onto the plasmid pUC19 previously digested with BamHI and HindIII. The DNA sequence of the resulting plasmid was determined. The plasmid thus prepared was designated "hMBCL/pUC 19". However, the 104 th amino acid (amino acid 96 according to Kabat determination) of the CDR4 of this plasmid was found to be substituted by arginine. To correct this amino acid to tyrosine, primer MBC1LGP10R (SEQ ID NO: 35) was designed and synthesized. Then, TaKaRa Ex Taq (Takara Shuzo) and a buffer were used to perform PCR reaction. The reaction solution (100. mu.l) used for the PCR reaction contained 0.6. mu.g of plasmid hMBCL/pUC19 as template DNA, 50pmole each of primers MBC1LVS1 and MBC1LVR1, 2.5U of TaKaRaEx Taq (Takara Shuzo), and 0.25mM dNTP in the buffer solution, and the liquid surface was covered with mineral oil. The PCR reaction was performed for 30 cycles, using a temperature cycle of 94 ℃ for 1 minute; 1 minute at 55 ℃; 72 ℃ for 1 minute. The DNA fragment thus amplified was separated by gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio.products).

A DNA fragment 421 base pairs in length was excised, and the DNA fragment was purified using GENECLEAN II kit (BIO101) according to the method indicated on the kit. The PCR reaction mixture thus prepared was used for subcloning the DNA fragment into plasmid pUC19 previously digested with BamHI and HindIII.

The DNA sequence of this plasmid was determined using M13 primer M4 and M13 primer RV. As a result, it was confirmed that the plasmid had the correct sequence. The plasmid was then digested with HindIII and BlnI, from which a 416 base pair DNA fragment was isolated by electrophoresis on a 1% agarose gel. The DNA fragment was purified using GENECLEAN II kit (BIO101) according to the method indicated on the kit. Then introduced into plasmid C lambda/pUC previously digested with HindIII and BlnI. The resulting plasmid was designated "hMBC 1 La. lambda./pUC 19". This plasmid was digested with EcoRI to give a DNA fragment encoding a humanized L chain. This DNA fragment was introduced into plasmid pCOS1 so that the start codon of the humanized L chain was positioned downstream of the EF 1. alpha. promoter. The plasmid thus obtained was designated "hMBC 1 La. lambda./pCOS 1". The DNA sequence (including the corresponding amino acid sequence) of the humanized L chain "a" type is shown in SEQ ID NO: as shown at 66. The amino acid sequence of the type "a" is shown in SEQ ID NO: shown at 47.

The "b" form was prepared by PCR using a mutagenesis technique. The "b" form was designed to replace glycine at position 43 (amino acid number 43 as defined by Kabat) with proline and lysine at position 49 (amino acid number 49 as defined by Kabat) with aspartic acid. A PCR reaction was carried out using plasmid hMBC1La lambda/pUC 19 as a template, with the mutated primer MBC1LGP5R (SEQ ID NO: 36) and the primer MBC1LVS 1. The obtained DNA fragment was digested with BamHI and HindIII, and the digested fragment was subcloned into BamHI-HindIII site of pUC 19. After sequencing, the plasmid DNA obtained was digested with HindIII and AflII and the resulting digested fragments were ligated to plasmid hMBC1 La. lambda./pUC 19 previously digested with HindIII and AflII.

The plasmid thus obtained was designated "hMBC 1 Lb. lambda./pUC 19". This plasmid DNA was digested with EcoRI to obtain a DNA fragment containing DNA encoding a humanized L chain. This DNA fragment was introduced into plasmid pCOS1 so that the start codon of the humanized L chain was located downstream of the EF 1. alpha. promoter. The plasmid thus obtained was designated "hMBC 1 Lb. lambda./pCOS 1".

"c" form was prepared by PCR using mutagenesis technique. The "c" form was designed to replace the serine at position 84 (amino acid number 80 as defined by Kabat) with proline. PCR was performed using plasmid hMBC1La lambda/pUC 19 as a template, and mutant primer MBC1LGP6S (SEQ ID NO: 37) and primer M13 primer RV. The obtained DNA fragment was digested with BamHI and HindIII, and then subcloned into pUC19 previously digested with BamHI and HindIII. After sequencing, the plasmid DNA obtained was digested with BstPI and Aor51HI, and the resulting DNA fragment was ligated to plasmid hMBC1 La. lambda./pUC 19 previously digested with BstPI and Aor51 HI. The plasmid thus obtained was designated "hMBC 1 Lc. lambda./pUC 19". This plasmid DNA was digested with EcoRI to obtain a DNA fragment containing a sequence encoding a humanized L chain. This sequence was introduced into the EcoRI site of plasmid pCOS1 so that the start codon of the humanized L chain was located downstream of the EF1 α promoter. The plasmids thus obtained, designated "hMBC 1Lc lambda/pCOS 1" "d", "e", "f" types, were also prepared by PCR using mutagenesis techniques. The "d", "e" and "f" forms were designed to replace tyrosine (amino acid 87 as defined by Kabat) with isoleucine of the "a", "b" and "c" forms, respectively, at position 91. The PCR reactions of the "d", "e" and "f" types were carried out using the plasmid hMBC1La λ/pCOS1 as the "d" type template, the plasmid hMBC1Lb λ/pUC19 as the "e" type template, the plasmid hMBC1Lc λ/pUC19 as the "f" type template, and the mutant primer MBC1LGP11R (SEQ ID NO: 38) and the primer M-S1(SEQ ID NO: 44), respectively. The obtained DNA fragment was digested with BamHI and HindIII, and then subcloned into pUC19 previously digested with BamHI and HindIII. After sequencing, the plasmid was digested with HindIII and BlnI, and the resulting digested fragment was ligated to plasmid C.lamda./pUC 19 previously digested with HindIII and BlnI.

The plasmids thus obtained were designated as "hMBC 1 Ld. lamda./pUC 19", "hMBC 1 Le. lamda./pUC 19" and "hMBC 1 Lf. lamda./pUC 19", respectively. Each plasmid was digested with EcoRI to obtain a DNA fragment containing DNA encoding a humanized L chain. This DNA fragment was introduced into the EcoRI site of plasmid pCOS1 so that the start codon of the humanized L chain was located downstream of the promoter of the plasmid EF1 alpha. The plasmids thus obtained were designated as "hMBC 1Ld lambda/pCOS 1", "hMBC 1Le lambda/pCOS 1" and "hMBC 1Lf lambda/pCOS 1", respectively.

The "g" and "h" forms were also prepared by PCR using mutagenesis techniques. The "g" and "h" forms were designed to replace the histidine at position 36 with tyrosine in the "a" and "d" forms, respectively (amino acid number 36 as defined by Kabat). PCR was performed using the mutation primer MBC1LGP9R (SEQ ID NO: 39) and the primer M13 primer RV, and using plasmid hMBC1 La. lambda./pUC 19 as a template. The PCR product was again subjected to another PCR reaction using M13 primer M4 as a primer and plasmid hMBC1 La. lambda./pUC 19 as a template. The obtained DNA fragment was digested with HindIII and BlnI, and then cloned by passage into plasmid C.lambda./pUC 19 previously digested with HindIII and BlnI. Using this plasmid as a template, PCR reaction was again carried out using the primers MBC1LGP13R (SEQ ID NO: 40) and MBC1LVS 1. The obtained PCR fragment was digested with ApaI and HindIII, and then introduced into plasmids hMBC1La lambda/pUC 19 and hMBC1Ld lambda/pUC 19 previously digested with ApaI and HindIII, respectively. The DNA sequence of the obtained plasmid was determined. Plasmids which clearly contain the correct sequence are designated "hMBC 1Lg λ/pUC 19" and "hMBC 1Lh λ/pUC 19", respectively. Each plasmid was digested with EcoRI to obtain a sequence comprising a sequence encoding a humanized L chain. This sequence was introduced into the EcoRI site of plasmid pCOS1 so that the start codon of the humanized L chain was located downstream of the EF1 α promoter. The plasmids thus obtained were designated "hMBC 1 Lg. lamda./pCOS 1" and "hMBC 1 Lh. lamda./pCOS 1", respectively. The "i", "j", "k", "l", "m", "n" and "o" forms were also prepared by PCR method using mutagenesis technique. PCR was performed using the mutation primer MBC1LGP14S (SEQ ID NO: 41) and the primer V1RV (lambda) (SEQ ID NO: 43), and using the plasmid hMBC1La lambda/pUC 19 as a template. The resulting DNA fragment was digested with ApaI and BlnI and then subcloned into the plasmid hMBC1 Lg. lambda./pUC 19 previously digested with ApaI and BlnI. The nucleotide sequence of the obtained plasmid was determined for each type of mutation-introduced clone. The plasmid thus obtained was designated "hMBC 1Lx λ/pUC19(x ═ i, j, k, l, m, n, or o)". This plasmid was digested with EcoRI to obtain a sequence containing a sequence encoding a humanized L chain. This sequence was introduced into the EcoRI site of plasmid pCOS1 so that the start codon of the humanized L chain was located downstream of the EF1 α promoter. The plasmid thus obtained was designated "hMBC 1Lx λ/pCOS1(x ═ i, j, k, l, m, n, or o)". The DNA sequences of the "j", "l", "m", and "o" types (including the corresponding amino acid sequences) are set forth in SEQ ID NOs: 67. 68, 69 and 70. These types of amino acid sequences are shown in SEQ ID NOs: 48. 49, 50 and 51. The "p", "q", "r", "s" and "t" forms are modified forms of the "i", "j", "m", "l" and "o" forms, respectively, in which tyrosine 87 is replaced by isoleucine. These types were prepared in the following manner using the Aor51MI restriction site in FR3, replacing form "i", "j", "m", "l" or "o" for form "h". The Aor51HI restriction fragment (514 base pairs) containing CDR3, part of FR3 and all of FR4 were deleted from the expression plasmid hMBC1Lx λ/pCOS1(x ═ i, j, m, l, or o). The Aor51HI restriction fragment (514 base pairs) containing CDR3, a portion of FR3, and the entire FR4 were ligated to the deleted portion of the expression plasmid, replacing the tyrosine at position 91 (amino acid 87 as defined by Kabat) with isoleucine. The DNA sequence of the resulting plasmid was determined and various clones of "i", "j", "m", "l" or "o" type were selected in which tyrosine 91 (amino acid 87 as defined by Kabat) was replaced by isoleucine. Types corresponding to "i", "j", "m", "l" or "o" types are designated as "p", "q", "r", "s" and "t" types, respectively, and plasmids of these types are designated as "hMBC 1Lx λ/pCOS1(x ═ p, q, s, r or t)". The DNA sequences of "q", "r", "s" and "t" types (including the corresponding amino acid sequences) are shown in SEQ ID NO: 71. 72, 73, 74. These types of amino acid sequences are shown in SEQ ID NOs: 52. 53, 54, 55.

The plasmid hMBC1Lq lambda/pCOS 1 was digested with HindIII and EcoRI and then subcloned into the plasmid pUC19 previously digested with HindIII and EcoRI. The resulting plasmid was designated "hMBC 1 Lq. lambda./pUC 19".

The positions of the substituted amino acids in each humanized L chain type are shown in table 3.

TABLE 3

Position of substituted amino acid in sequence Listing (amino acid numbering according to Kabat definition)

In table 3 above, capital letters represent the following amino acids: y: tyrosine; p: (ii) proline; k: lysine; v: valine; d: aspartic acid; i: isoleucine.

The E.coli strain containing the plasmid hMBC1HcDNA/pUC19 and the E.coli strain containing the plasmid hMBC1 Lq. lamda./pUC 19 were designated as "E.coli JM109(hMBC1HcDNA/pUC 19)" and "E.coli JM109(hMBC1 Lq. lamda./pUC 19)", respectively, which had been deposited at the national institute of bioscience and human technology, the agency of science and technology, on 8.15.1996 according to the terms of the Budapest treaty, and Japanese (1-3, Higashi 1-chome, Tsuuba-shi, Ibaragi-ken, Japan), the registration number of E.coli JM109(hMBC1HcDNA/pUC19) was JM 29 FERM BP-5629, and the registration number of E.coli JM109(hMBC1 Lq. lamda./pUC 19) was FERM BP-0.

(5) Transfection into COS-7 cells

To determine the antigen binding and neutralizing activities of the hybrid antibody and the humanized #23-57-137-1 antibody, the above expression plasmid was transiently expressed in COS-7 cells. For transient expression of hybrid antibody L chains, COS-7 cells were co-transfected by electroporation with a combination of the following plasmids using Gene Pulser (Bio Rad): hMBC1HcDNA/pCOS1 and h/mMBC1L (. lamda.)/neo; hMBC1HcDNA/pCOS1 and m/hMBC1La lambda/neo; hMBC1HcDNA/pCOS1 and m/hMBC1Ld lambda/neo; hMBC1HcDNA/pCOS1 and hmmMBC1L (lambda)/neo; hMBC1HcDNA/pCOS1 and mhMBC 1L (. lamda.)/neo. That is, 10. mu.g of each plasmid DNA was added to 0.8 ml of COS-7 cells suspended in PBS (-) at a concentration of 1X 10⁷Cell suspension of individual cells/ml. The resulting solution was pulsed with an electrostatic capacity of 1500V and 25. mu.F. The cells were recovered at room temperature for 10 minutes, and the electroporated cells were suspended in DMEM medium supplemented with 2% Ultra Low IgG fetal bovine serum (GIBCO), and then plated in a10 cm petri dish in CO₂Culturing in an incubator. After 72 hours of culture, the culture supernatant was collected and centrifuged to remove cell debris. The product was used as a sample for ELISA analysis.

To achieve transient expression of humanized #23-57-137-1 antibody, each plasmid combination hMBC1 hcna/pCOS 1 or hMBC1Lx λ/pCOS1(x ═ a-t) was transfected into COS-7 cells using Gene Pulser (Bio Rad) in the same manner as the hybrid antibody described above. The culture supernatants obtained were used as samples for ELISA analysis.

Wherein the hybrid or humanized antibody in COS-7 cell culture supernatant was purified using Affigel Protein A MAPSII kit (Bio Rad) according to the methods indicated on the kit.

(6) ELISA assay

(i) Determination of antibody concentration

ELISA plates for determination of antibody concentration were prepared as follows. Each well of a 96-well plate for ELISA (Maxisorp, NUNC) was coated with 100. mu.l of coating buffer (0.1M NaHCO) supplemented with 1. mu.g/ml goat anti-human IgG antibody (TAGO)₃，0.02％NaN₃) Coated and then diluted with 200. mu.l of dilution buffer [50mM Tris-HCl, 1mM MgCl ]₂，0.1M NaCl，0.05％Tween20，0.02％NaN₃1% Bovine Serum Albumin (BSA); pH7.2]And (4) separating. COS-7 cell culture supernatants expressing the hybrid or humanized antibodies or stepwise diluted solutions of purified hybrid or humanized antibodies were added to each well. After incubation for 1 hour at room temperature and washing with PBS-Tween20, 100. mu.l of alkaline phosphatase conjugated goat anti-human IgG antibody (TAGO) was added to each well. After incubation for 1 hour at room temperature and washing with PBS-Tween20, 1 mg/ml of matrix solution ("Sigma 104", p-nitrophenyl phosphate, SIGMA) was added to each well. The absorbance of the solution was measured at 405nm using a microplate reader (BioRad). Purified Hu IgG1 λ (binding site) was used as a standard for antibody concentration determination.

(ii) Determination of antigen binding Capacity

ELISA plates for determining antigen binding capacity were prepared as follows. Each well of a 96-well plate used for ELISA (Maxisorp, NUNC) was coated with 100. mu.l of a coating buffer to which 1. mu.g/ml of human PTHrP (1-34) was added, and then blocked with 200. mu.l of a dilution buffer. The hybrid antibody or humanized antibody is added to each well to obtain expressed COS-7 cell culture supernatant or stepwise diluted solution of purified hybrid antibody or humanized antibody. After incubation at room temperature and washing with PBS-Tween20, 100. mu.l of alkaline phosphatase conjugated goat anti-human IgG antibody (TAGO) was added to each well. After incubation at room temperature and washing with PBS-Tween20, 1 mg/ml of matrix solution ("Sigma 104", p-nitrophenyl phosphate, SIGMA) was added to each well. The absorbance of the solution was measured at 405nm using a microplate reader (Bio Rad).

(7) Determination of Activity

(i) Evaluation of humanized H chain

It was found that an antibody comprising the humanized H chain "a" type and a chimeric L chain exhibited the same level of binding activity to PTHrP as that of the chimeric antibody (see FIG. 5). This result indicates that the "a" form successfully achieves the humanization of the H chain V region. Thus, the humanized H chain "a" type was used as the humanized antibody H chain in the following experiment.

(ii) Activity of hybrid antibodies

(ii-a) FR1, 2/FR3, 4 hybrid antibody

When the L chain is h/mMBC1Ld (. lamda.), the antibody shows no antigen binding activity. However, when the L chain of the hybrid antibody was m/hMBC1 La. lamda. or m/hMBC1 Ld. lamda., the antibody exhibited the same level of antigen binding activity as the chimeric #23-57-137-1 antibody (FIG. 6). These results indicate that FR3 and FR4 are suitable for humanized antibodies, but that some amino acid residues required for the presence of FR1 and FR2 are substituted.

(ii-b) FR1/FR2 hybrid antibody

When the L chain of the hybrid antibody is mhmMBC1L (lambda), the antibody does not exhibit antigen binding activity. However, when the L chain of the hybrid antibody was hmmbC1L (. lamda.), the antibody exhibited the same level of antigen binding activity as the chimeric #23-57-137-1 antibody (FIG. 7). These results indicate that FR1 is suitable for a humanized antibody, but that some amino acid residues required for the presence of FR2 have been substituted.

(iii) Activity of humanized antibody

The antigen binding activity of each of the humanized antibodies for L chain of the "a" type to the "t" type was determined. As a result, it was found that the humanized antibodies having L chains of "j", "L", "m", "o", "q", "r", "s" and "t" types exhibited the same level of PTHrP-binding activity as that of the chimeric antibody (FIGS. 8 to 11).

(8) Establishment of CHO Stable production cell line

To establish stable transformants of the humanized antibody, the above expression plasmid was introduced into CHO cells (DXB 11).

Stable transformants of the humanized antibody were established using the following combination of plasmids as expression vectors for CHO cells: hMBC1HcDNA/pCHO1 and hMBC1Lm lambda/pCOS 1; hMBC1HcDNA/pCHO1 and hMBC1Lq lambda/pCOS 1; hMBC1HcDNA/pCHO1 and hMBC1Lr lambda/pCOS 1. The plasmids were co-transfected into CHO cells by electroporation using Gene Pulser (Bio Rad). Subsequently, each expression vector was cleaved with the restriction enzyme PvuI to obtain linear DNA. The resulting DNA was extracted with phenol and chloroform, and then precipitated with ethanol. The prepared DNAs were each subjected to electroporation as follows. That is, 0.8 ml of each plasmid DNA containing COS-7 cells suspended in PBS (-) at a concentration of 1X 10 was added at 10. mu.g per plasmid DNA⁷Cell suspension of individual cells/ml. The resulting mixture was pulsed with a static capacity of 1500V and 25. mu.F. The room temperature was returned for 10 minutes, and the electroporated cells were suspended in MEM-alpha medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), followed by CO-elution with 96-well plate (Falcon)₂Culturing in an incubator. On the day of culture, the original medium was replaced with MEM-alpha selection medium supplemented with 10% fetal bovine serum (GIBCO) and 500 mg/ml GENETICIN (G418 sulfate; GIBCO) but without ribonucleosides and deoxyribonucleosides. Cells into which the antibody gene has been introduced are selected from the culture medium. After replacing the original medium with fresh medium, cells were observed microscopically before and two weeks after the culture. When satisfactory cell growth was observed, the amount of antibody produced by the cells was determined using a conventional ELISA assay that determined the concentration of antibody as described above. Those cells producing the antibody in large quantities are selectively collected.

Stable transformants of the thus established antibodies were grown on a large scale in shake flasks in MEM-alpha medium supplemented with 2% Ultra Low IgG fetal bovine serum, ribonucleoside-free or deoxyribonucleoside. Culture medium supernatants were collected at 3 and 4 days after the culture and filtered with a 0.2 μm filter (Millipore) to remove cell debris therefrom. Humanized antibodies in CHO cell culture supernatants were purified on ConSepLC100(Millopore) using POROS Protein A columns (PerSeptive Biosystems) according to the instructions included therein. The humanized antibody was used as a sample for determination of neutralizing activity and examination of pharmacological efficacy for hypercalcemia model animals. The concentration and antigen binding activity of the purified humanized antibody were determined using the ELISA system described above.

Example 4

Determination of neutralizing Activity

The neutralizing activity of the murine, chimeric and humanized antibodies was determined by the rat myeloma cell line ROS17/2.8-5 cells. In CO₂In the incubator, ROS17/2.8-5 cells were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum (GIBCO) at a cell concentration of 10⁴Cells/100. mu.l/well, ROS17/2.8-5 cells were plated into each well in 96-well plates for 1 day of culture. The medium was replaced with Ham's F-12 medium (GIBCO) supplemented with 4mM hydrocortisone and 10% fetal bovine serum. After three to four days of culture, the cultured cells were washed with 260. mu.l of Ham's F-12 medium (GIBCO), to which was then added 80. mu.l of Ham's F-12 medium supplemented with 1mM isobutyl-1-methylxanthine (IBMX, SIGMA), 10% fetal bovine serum, and 10mM HEPES. The resulting mixture was incubated at 37 ℃ for 30 minutes.

The murine, chimeric and humanized antibodies used for the detection of neutralizing activity were diluted stepwise in advance according to the following groups: [ 10. mu.g/ml, 3.3. mu.g/ml, 1.1. mu.g/ml and 0.37. mu.g/ml ], [ 10. mu.g/ml, 2. mu.g/ml, 0.5. mu.g/ml and 0.01. mu.g/ml ] and [ 10. mu.g/ml, 5. mu.g/ml, 1.25. mu.g/ml, 0.63. mu.g/ml and 0.31. mu.g/ml ]. Each diluted antibody sample solution was mixed with an equal amount of 4 ng/ml PTHrP (1-34). 80. mu.l of the resulting mixed solution was added to each well. The final concentration of each antibody was one-fourth of the above antibody concentration, and thus the concentration of PTHrP (1-34) became 1 ng/ml. After ten minutes of treatment at room temperature, the culture supernatant was removed and the residue was washed three times with PBS. cAMP was extracted from the cells from the product with 10. mu.l of 0.3% HCl-95% ethanol, and then HCl-ethanol was removed by evaporation in a water vapor absorber. The residue was dissolved in 120. mu.l of EIA buffer adsorbed to cAMP EIA kit (CAYMAN CHEMICAL' S) to extract cAMP therefrom. The level of cAMP was determined using the cAMP EIA kit (CAYMANCHEMICAL' S) according to the method indicated therein. As a result, it was found that, among humanized antibodies of L chain type having the same level of antigen binding activity as that of the chimeric antibody, humanized antibodies of L chain type having "q", "r", "s" and "t" in which tyrosine at position 91 is substituted with isoleucine exhibited the most neutralizing activity as that of the chimeric antibody, and particularly, humanized antibodies having L chain of "q" exhibited the strongest neutralizing activity (FIGS. 12 to 14).

Example 5

Detection of pharmacological efficacy of hypercalcemia model animal (1)

The efficacy of the anti-PTHrP chimeric antibody and the humanized antibody having L chains of the "m", "r" and "q" types, respectively, for the treatment of hypercalcemia was examined using hypercalcemia model animals (human tumor-transplanted nude mice).

Nude mice transplanted with human pancreatic cancer PAN-7 (purchased from the center of laboratory animals) were used as hypercalcemia model animals. Nude mice transplanted with PAN-7, a human pancreatic cancer, are known to exhibit increased blood calcium concentrations with increased tumor volume and develop hypercalcemia associated with decreased body weight and spontaneous activity (for example). In this example, the efficacy of the chimeric and humanized antibodies of the invention for treating human pancreatic cancer PAN-7-induced hypercalcemia was examined by body weight and blood calcium concentration measurements in test animals.

BALB/c-nu/nu nude mice (Nippon Charles River) were used to transplant human pancreatic cancer PAN-7 in vivo. To evaluate pharmacological efficacy, 5-week-old male BALB/c-nu/nu nude mice (Nippon Charles River) were purchased and then acclimatized for one week, so that six-week-old mice were used in the above evaluation experiment. Hypercalcemia model mice were prepared and grouped as follows. Human pancreatic cancer PAN-7 was excised and finely cut into 3mm cubes. The resulting tumor mass was transplanted under the mouse flap, one for each mouse. Two or three weeks after the transplantation, when the tumor volume of each mouse was determined to be sufficiently large, the mice were divided into groups on the average according to the tumor volume, the blood calcium concentration, and the body weight, and thus the mice were used as hypercalcemia model animals.

The efficacy of the hypercalcemia treatment was examined as follows. The anti-PTHrP chimeric antibody or humanized antibody having L chains of the "m" or "r" type was administered to the tail vein of each of the above hypercalcemic model mice in a single dose of 10 or 30. mu.g/mouse, respectively. A single dose of the humanized antibody having L chain of "q" type was administered to the tail vein of each of the hypercalcemia model mice described above at a dose of 20 or 60. mu.g/mouse. The blood calcium concentration and body weight of each mouse were measured on day 1, day 4, day 7 and day 11 after the administration to evaluate the therapeutic efficacy of the antibody, respectively. Tumor volume was determined by measuring the maximum (a mm) and minimum (b mm) diameters of tumors and calculating the formula [ ab ] according to Galant' s²/2]And calculating two measurement values for determination. Blood calcium concentration blood was collected from the orbital of each mouse using a hematocrit tube and added to 643 AutoCa²⁺The whole blood calcium ion concentration was measured by pH analyzer (CIBA-CORNING) as the blood calcium concentration.

As a result, it was found that administration of the chimeric antibody and the humanized antibody having L chains of "m", "r" and "q" types to a subject results in rapid improvement in body weight and blood calcium concentration changes and an increase in retention for an extended period. This result indicates that the chimeric and humanized antibodies of the present invention can be used for the treatment of hypercalcemia-associated malignancies (see fig. 15 and 16).

Example 6

Detection of pharmacological efficacy of hypercalcemia model animals (2)

The efficacy of the anti-PTHrP chimeric antibody and humanized antibody having "q" -type L chain for the treatment of hypercalcemia was examined using hypercalcemia model animals (human tumor-transplanted nude mice) as follows.

The efficacy of treatment for hypercalcemia was examined as follows. A single dose of the anti-PTHrP chimeric antibody or humanized antibody having "q" -type L chain was administered to the tail vein of each of the above hypercalcemic model mice at a dose of 10 or 30. mu.g/mouse. Day 1, day 3, and day 7 after administrationAnd the blood calcium concentration and body weight of each mouse were measured on day 11, respectively, to evaluate the therapeutic efficacy of the antibody. Blood was collected from the orbit of each rat using a hematocrit tube and auto Ca was administered with 643²⁺The whole blood calcium ion concentration was measured by pH analyzer (CIBA-CORNING) as the blood calcium concentration.

As a result, it was found that administration of chimeric and humanized antibodies having "q" -type L chains to hypercalcemia model animals harboring human pancreatic cancer PAN-7 resulted in rapid improvement in body weight and blood calcium concentration of the recipient and an increase in retention for an extended period. This result indicates that the chimeric antibody and the humanized antibody of the present invention are effective drugs for treating hypercalcemia-associated malignant tumors (see FIG. 17).

Example 7

Detection of pharmacological efficacy of hypercalcemia model animal (3)

The efficacy of the anti-PTHrP chimeric antibody and humanized antibody having "q" -type L chain for the treatment of hypercalcemia was examined by hypercalcemia model animals (human lung cancer LC-6-transplanted nude mice).

In this experiment, nude mice transplanted with human lung cancer LC-6 (purchased from the center of experimental animals) were used as hypercalcemia model animals. Nude mice transplanted with LC-6 transplants of the known human lung carcinoma tend to show an increase in blood calcium concentration with an increase in tumor volume and develop hypercalcemia associated with body weight and spontaneous activity.

In this example, the therapeutic efficacy of the chimeric and humanized antibodies of the present invention for human lung cancer LC-6-induced hypercalcemia was examined by measuring the body weight and blood calcium concentration of the test animals.

Human lung cancer line LC-6 was transplanted in vivo in BALB/c-nu/nu nude mice (Nippon Charles River). Male BALB/c-nu/nu nude mice (Nippon Charles River) 5 weeks old were purchased for evaluation of pharmacological efficacy and then acclimatized for one week, so six weeks old mice were used for this experiment.

Hypercalcemia model mice were prepared and grouped as follows. Transplanted human lung carcinoma LC-6 was excised and finely cut into 3mm cubes. The resulting tumor mass was transplanted subcutaneously under the mouse flap, one for each mouse. Two or three weeks after the transplantation, when the tumor volume of each mouse was determined to be sufficiently large, the mice were divided into groups on the average according to the tumor volume, the blood calcium concentration, and the body weight, and the mice thus treated were used as hypercalcemia model animals.

The efficacy of the hypercalcemia treatment was examined as follows. A single dose of the chimeric antibody or humanized antibody having L chain of "q" type against PTHrP was administered at 10 or 30. mu.g/mouse to the tail vein of each of the above hypercalcemic model mice, respectively. The blood calcium concentration and body weight of each mouse were measured on day 1, day 3, day 6 and day 10 after the administration to evaluate the therapeutic efficacy of the antibody, respectively. Serum calcium concentration blood was obtained from the orbital of each mouse using a hematocrit tube and auto Ca was used with 643²⁺The whole blood calcium ion concentration was measured by pH analyzer (CIBA-CORNING) as the blood calcium concentration.

As a result, it was found that administration of the chimeric antibody and the humanized antibody having "q" -type L chain to a hypercalcemia model animal carrying human lung cancer LC-6 resulted in rapid improvement of body weight and blood calcium concentration of the recipient and increase of prolonged retention. This result indicates that the chimeric antibody and the humanized antibody of the present invention are effective drugs for treating hypercalcemia-associated malignant tumors (see FIG. 18).

Example 8

Analysis of PTHrP and anti-PTHrP Using BIACORE

Kinetics of interaction between antibodies

In this experiment, BIACORE was used to analyze the kinetics of antigen-antibody interactions. PTHrP (1-34+ Cys) was used as an antigen, and its C-terminus was specifically adsorbed to the tip of the sensor. Purified antibodies at various concentrations were used as analytes. Kinetic indices (association rate constant "kass" and dissociation rate constant "kdiss") were calculated from the sensorgrams obtained. Reference is made in kinetic analysis to the literature "kinetic analysis of monoclonal antibody-antigen interactions in novel biosensor-based assay systems", Karlsson, R.et al, (1991), J.Immunol.methods, 145, p.229-240.

(1) PTHrP (1-34+ C) was fixed to the sensor tip

PTHrP (1-34+ C) was adsorbed to the sensor tip CM5 (Pharmacia).

HBS (10mM HEPES, pH 7.4; 0.15M NaCl; 3.4mM EDTA; 0.005% surfactant P20) was used as a running buffer at a flow rate of 5. mu.l/min. The carbonyl group of carboxymethyldextran on the sensor tip CM5 was activated by injecting 100. mu.l of 0.05M N-hydroxysuccinimide (NHS)/0.2M N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 100. mu.l of 80mM 2- (2-pyridyldithio) acetamide (PDEA)/0.1M borate buffer (pH8.5), and 10. mu.l of 5. mu.g/ml PTHrP (1-34+ C)/10mM sodium acetate buffer (pH 5.0) was additionally injected so that the carbonyl group was specifically adsorbed to the cysteine residue at the C-terminus of PTHrP (1-34+ C). Subsequently, 100. mu.l of 50mM (L) -cysteine/1M NaCl/0.1M sodium formate buffer (pH 4.3) was injected to block excess activated groups. Subsequently, 10. mu.l of 0.1M glycine-HCl buffer (pH2.5) and 10. mu.l of 10mM HCl were injected to wash the matrix with non-covalent binding. The amount of fixed PTHrP (1-34+ C) was 226.4 RU (resonance unit) (FIG. 19).

(2) Interaction between immunized PTHrP (1-34+ C) and purified murine anti-PTHrP antibody

HBS was used as flow buffer at a flow rate of 20. mu.l/min. The antibody-producing hybridoma was injected into the abdominal cavity of Balb/c mice, and ascites was collected two weeks later and applied to a protein A column for antibody purification. The purified #23-57-137-1 antibody was designated "MBC" and the purified 3F5 antibody was designated "3F 5". These antibodies were diluted with HBS to a range of concentrations of 1.25, 2.5, 5, 10 and 20 μ g/ml.

In the assay, 40 μ l of antibody solution was injected for two minutes to obtain the binding phase, followed by two minutes of HBS injection to obtain the dissociation phase. After complete dissociation, the sensor tip was recovered by injection of 10 μ l 10mM HCl. Binding-dissociation-recovery as a cycle, various antibody solutions were injected to obtain sensorgrams for analysis.

(3) Interaction between immobilized PTHrP (1-34+ C) and purified humanized anti-PTHrP antibody

HBS was used as flow buffer at a flow rate of 20. mu.l/min. Antibodies were prepared from CHO cells and purified using a protein a column. The purified chimeric antibody was designated "chMBC" and the purified humanized antibodies of type m and q were designated "hMBCm" and "hMBCq", respectively, and these antibodies were diluted with HBS to a range of concentrations of 1.25, 2.5, 5, 10 and 20. mu.g/ml.

(4) Kinetic analysis of interactions

The data of interest were recorded and the reaction patterns were compared by overlaying the reaction zones of interest (FIGS. 20-24). In each of FIGS. 20-24, the top-to-bottom continuous lines represent data for antibody concentrations of 1.25, 2.5, 5, 10, and 20 μ g/ml. In addition, kinetic analysis of the interaction was performed by analysis software "BIAevaluation 2.1" (Pharmacia) specifically designed for BIACORE, which enables calculation of kinetic indices (association rate constant "kass" and dissociation rate constant "kdiss") by curve cutting (tables 4 and 5).

Table 4: kinetic indices of MBC and 3F5

Table 5: kinetic indices of chimeric and humanized antibodies

In this experiment, analytical model 4 (BIAevaluationionon 2.1 software handbook, A1-A5) was used to determine the binding rate constant.

Example 9

Inhibition of phosphorus secretion in a malignancy-associated hypercalcemia model

Hypercalcemia associated with malignancy (HHM) is a disease caused by the appearance of PTHrP, which is known to accelerate bone resorption and reabsorption of calcium by the kidney and ureter, resulting in hypercalcemia. On the other hand, in the case of phosphorus, PTHrP inhibits reabsorption of phosphorus by the kidney and ureter, resulting in excretion, and thus hypophosphatemia is frequently developed in clinical HHM patients. Wherein the effect of the humanized anti-PTHrP antibody on renal phosphorus excretion was examined using a hypercalcemia associated with malignant tumor model mouse.

Nude rats (purchased from experimental animal centers) transplanted with human lung cancer LC-6 were used as model animals. It is known that nude rats subcutaneously transplanted with human lung carcinoma LC-6 are prone to increased blood calcium concentration and increased tumor volume, and as a result, the rats develop hypercalcemia associated with, for example, decreased body weight and spontaneous activity. Using this animal model, the effect of the humanized anti-PTHrP antibody of the present invention on renal phosphorus excretion was examined based on the renal clearance method for partial excretion of phosphate as described below.

BALB/c-nu/nu nude rats (Nippon Kurea) were used for in vivo transplantation of human lung cancer LC-6. Five-week old male F344N/Jcl-rnu nude rats (Nippon Kurea) were purchased and acclimated for one week, and these six-week old rats were used to evaluate pharmacological efficacy.

The model animal of hypercalcemia associated with malignant tumor was prepared as follows. Transplanted human lung carcinoma LC-6 was excised and finely cut into 3mm cubes. The resulting tumor mass was transplanted subcutaneously under the mouse flap, one for each mouse. Approximately thirty days after transplantation, it was confirmed that the tumor volume of each mouse was sufficiently large (3000 mm)³) Rats that can be used as model animals for malignancy-associated hypercalcemia were selected based on blood calcium concentration and body weight.

Examination of phosphorus excretion by renal clearance method was performed as follows.

(1) Method for removing kidney

The malignant tumor-associated hypercalcemia model animals were anesthetized with pentobarbital (Nembutal, Dainippon Pharmaceutical co., Ltd), supine fixed on a 37 ℃ heat-insulating pad, and the bladder was cannulated to collect urine (polyethylene tubing, PE50, Nippon Beckton Dickinson). Subsequently, an infusion tube (polyethylene tube, PE50, Nippon Beckton Dickinson) was inserted into the femoral vein of the model animal, and then an infusion solution (0.7% inulin, 5% mannitol, 0.2% pentobarbital, 0.9% sodium chloride) was introduced into the model animal through the infusion tube at a flow rate of 2 ml/hr using an infusion pump (Terufusion syringe pump STC-525; TERUMO). After 50 minutes of equilibration, urine was collected 5 times (i.e., stages 1-5) through the cannula for 20 minutes to obtain a urine sample. At each intermediate time point of urine collection, approximately 0.25 ml of blood was collected from the right jugular vein of the model animal using a heparin-treated syringe.

(2) Administration of antibodies

In the above clearance test, the animals were intravenously injected with the humanized anti-PTHrP antibody at a dose of 1 mg/ml/kg at the time point immediately after the urine collection phase 2.

(3) Determination of inulin and phosphorus concentrations in urine and blood

The volumes of the urine samples obtained in stages 1 and 5 were measured and then their inulin and phosphorus concentrations were determined. The blood samples obtained above were centrifuged at low temperature to obtain plasma samples for the determination of inulin and phosphorus concentrations. The inulin concentration was determined by the sulfuric acid-anthrone method (Roe, L et al, J. biol. chem. 178, 839-845, 1949), and the phosphorus concentration was determined by an inorganic phosphorus determination reagent- -Autosera IP (Daiichi Pure Chemicals) by means of Hitachi autoanalyzer model 7170 according to the manual (Physke-Sabaroh method).

(4) Calculation of inulin clearance, phosphorus clearance and phosphorus moiety excretion

Inulin clearance (Cin), phosphorus clearance (Cp) and phosphorus relative excretion (FEp) were calculated according to the following formulas.

Calculation of inulin clearance (Cin):

Cin＝Uin V/Pin

where Cin represents inulin clearance (ml/kg/min); uin represents the concentration of inulin in urine (mg/ml); v represents the urine volume per unit time (ml/kg/min); pin represents the concentration of inulin in blood (mg/ml).

Calculation of phosphorus clearance (Cp):

Cp＝Up V/Pp

wherein Cp represents phosphorus clearance (ml/kg/min); up represents the concentration of phosphorus in urine (mg/ml); v represents the urine volume per unit time (ml/kg/min); pp represents the concentration of phosphorus in blood (mg/ml).

Calculation of relative phosphorus excretion (FEp):

FEp＝Cp/Cin

wherein FEp represents the relative excretion rate of phosphorus; cin stands for inulin clearance; cp represents phosphorus clearance. Four animals were used for the detection. Results are expressed as mean ± standard deviation.

The results of the phosphorus component excretion rate and the phosphorus concentration in blood are shown in fig. 25 and 26.

Fig. 25 illustrates the change in phosphorus relative to component excretion (phosphorus clearance/inulin clearance) as a function of clearance time (20 min phase 1). The humanized anti-PTHrP antibody (1 mg/kg) was administered at the beginning of phase 2 (intravenous injection).

Figure 26 illustrates plasma phosphorus concentration as a function of clearance time (20 min phase 1). The humanized anti-PTHrP antibody (1 mg/kg) was administered at the beginning of phase 2 (intravenous injection).

As a result, it was found that the excretion rates of phosphorus components obtained after the administration of the antibody (i.e., stages 2 and 5) were significantly suppressed as compared with the relative excretion rates of phosphorus obtained before the administration of the antibody (i.e., stage 1). In other words, it was found that administration of neutralizing antibodies to individuals who developed hypophosphatemia (resulting in accelerated phosphorus excretion, FEp > 0.2) restored approximately normal levels of receptor phosphorus reabsorption (phosphorus reabsorption fraction of 1-FEp > 0.8%), with the result that individual blood phosphorus concentrations were observed to be more normal. This result indicates that the antibody of the present invention can be an effective therapeutic agent for treating phosphorus accelerated excretion and hypophosphatemia caused by PTHrP.

Since PTHrP is a substance causing hypercalcemia associated with malignant tumors, it is predicted that PTHrP may promote phosphorus excretion and reduce high-energy organophosphorus in tissues. Accordingly, various diseases associated with hypophosphatemia, such as hypophosphatemic rickets and hypophosphatemic vitamin D-resistant rickets, are thought to be caused mainly by an increase in excretion of phosphorus through urine, and therefore the antibody of the present invention will also be effective for the treatment of such diseases.

Example 10

Amelioration of clinical symptoms of hypercalcemia associated with various malignancies

It is known that hypercalcemia associated with malignant tumor is caused by the generation of PTHrP by the tumor, and that PTHrP accelerates bone resorption and resorption of calcium by kidney and ureter (leading to hypercalcemia). In addition, clinical signs of exacerbation were observed in hypercalcemic patients, such as poor performance, low consciousness, systemic malaise, drinking habits, nausea and vomiting (anorexia). The effect of anti-PTHrP antibodies on these clinical symptoms was examined in hypercalcemia model animals using human tumor nude mice transplantation system and human tumor nude mice transplantation system.

Human lung carcinoma LC-6 transplanted nude mice and nude rats (purchased from the center of laboratory animals) were used as hyperemic model animals. Nude mice and nude rats transplanted with human lung carcinoma LC-6 tend to exhibit increased blood calcium concentrations and increased tumor volume, which results in hypercalcemia-associated reductions in body temperature and weight.

The improvement effect of the mouse anti-PTHrP antibody on the general clinical symptoms of hypercalcemia associated with malignant tumors was examined by using a human lung cancer LC-6 nude mice transplantation system, and the results were shown by photographs. The improvement of spontaneous activity and reduction of body temperature and anorexia by the antibody was examined by using the LC-6 nude mouse transplantation system for human lung cancer.

1. Amelioration of apparent clinical symptoms of hypercalcemia

The human lung cancer strain LC-6 was transplanted in vivo using BALB/c-nu/nu nude mice (Nippon Kurea). Five-week-old male BALB/c-nu/nu nude mice (Nippon Kurea) were purchased and acclimated for one week, and these six-week-old mice were used to evaluate pharmacological efficacy.

Hypercalcemia model mice were prepared and grouped as follows. Transplanted human lung carcinoma LC-6 was excised and finely cut into 3mm cubes. The resulting tumor mass was implanted subcutaneously, one for each mouse, under the mouse skin flap. Approximately twenty-seven days after transplantation, when it was confirmed that the tumor volume of each mouse had been sufficiently large, the mice were divided into groups on average in terms of tumor volume, blood calcium concentration and body weight of the mice, and then the mice were used as hypercalcemia model animals.

By measuring the maximum (a mm) and minimum (b mm) diameters of tumors and using these two measurements to calculate the formula [ ab ] as Galant' s²/2]The tumor volume was determined computationally.

Blood was collected from the orbit of each rat using a hematocrit tube and auto Ca was administered with 643²⁺The whole blood calcium ion concentration was measured by pH analyzer (CIBA-CORNING) as the blood calcium concentration.

The efficacy of the antibody for the treatment of hypercalcemia was examined in the following manner. The mouse anti-PTHrP antibody was administered to each of the hypercalcemic model animals at a dose of 100. mu.g/mouse by tail vein injection on days 27, 30, 34 and 37 after tumor transplantation. Phosphate buffered saline was administered in the same manner in place of the antibody for the preparation of the control. On day 41 after tumor transplantation, a typical mouse was selected from the group to which the antibody was administered and the control group, respectively, and photographed together with a normal mouse.

As a result, although the mice administered with the antibody (as shown in the middle panels of fig. 27 and 28) had the same level of tumor mass as the control mice (as shown in the right panels of fig. 27 and 28), they exhibited the same level of appearance as the normal mice (as shown in the graphs of fig. 27 and 28) in one hypercalcemia model animal transplanted with human lung cancer LC-6. The results indicate that administration of the anti-PTHrP antibody improved the apparent clinical symptoms (fig. 27 and 28).

2. Amelioration of hypercalcemia-related decreased spontaneous activity

Human lung carcinoma LC-6 was transplanted in vivo using BALB/c-nu/nu nude mice (Nippon Kurea). Five-week-old male F344/NJcl-run nude rats (Nippon Kurea) were purchased and acclimated for one week, and these six-week-old rats were used to evaluate pharmacological efficacy.

Hypercalcemia model animals were prepared in the following manner. Transplanted human lung carcinoma LC-6 was excised and finely cut into 3mm cubes. The resulting tumor mass was implanted subcutaneously, one for each mouse, under the mouse skin flap. Approximately thirty days after the transplantation, when it was confirmed that the tumor volume of each mouse had been sufficiently large, the mice were divided into groups on average in terms of tumor volume, blood calcium concentration and body weight of the mice, and then the mice were used as hypercalcemia model animals.

(1) Method for determining spontaneous activity

Spontaneous activity was determined using an ANIMEX Activity Meter model SE (FARAD, Electronics, Sweden) placed at the pre-determined position in polyethylene cages of model animals raised individually (water and food). The device was designed to measure the amount of exercise in each rat. With this device, the amount of movement is recorded as the number of times of a certain period of time. The measurement was taken 13 hours (from 7 o 'clock later on one day to 8 o' clock earlier on the next day) and the results were recorded as counts/hour.

(2) Administration of antibodies

Each of the hypercalcemic-forming rats prepared above was injected with a humanized anti-PTHrP antibody (dose of 5 mg/0.5 ml/kg) via the tail vein as a control. Another group of rats was injected with saline in the same manner. The antibody-dosed mice and the control mice were measured in turn.

The measurement was performed on 0 th (i.e., the day before the administration of the antibody), 2, 4, 7 and 14 days of the administration of the antibody mice, and the measurement was performed on 1 st, 3 rd, 5 th, 8 th and 15 th days of the administration of the antibody mice.

As a result, the control rats showed no change or decrease in spontaneous activity during the test period, while the mice using the antibody showed an increase in spontaneous activity after the 4 th day of administration (fig. 29).

3. Amelioration of hypercalcemia-related body temperature decline

Transplantation of human lung carcinoma LC-6 and preparation of a hypercalcemia model animal associated with malignant tumor were carried out in the same manner as in the above step 2.

(1) Body temperature measuring method

Animals were anesthetized with pentobarbital (Nembutal, Dainippon Pharmaceutical co., Ltd.), a temperature sensor was inserted rectally, and body temperature was measured by a digital thermometer.

(2) Administration of antibodies

The humanized anti-PTHrP antibody was injected into each of the hypercalcemic model rats at a dose of 1 mg/ml/kg via the caudal vein. The rats of other models were injected with saline via the tail vein as a control. In addition, the body temperature of normal rats not administered with the antibody was also measured. Body temperature was measured on day 0 of administration (i.e., day of administration), 1, 2 days and 3 days later for all the rats, control mice and normal mice using the antibody, respectively.

As a result, the normal mice showed no change in body temperature (34.2-34.4 ℃ C.) during the measurement, whereas the malignant tumor-associated hypercalcemia model rats had a temperature drop of about 2 ℃ from that of the normal rats. When a humanized anti-PTHrP antibody was administered to the model rats, the lowered body temperature of the malignant tumor-associated hypercalcemia model rats was significantly restored to the same level as that of normal rats three days after the administration. These results indicate that the humanized anti-PTHrP antibody of the present invention has a significant effect of ameliorating the hypothermia in the animal model of hypercalcemia associated with malignant tumor (FIG. 30).

4. Improvement of induced food intake reduction

Transplantation of human lung carcinoma LC-6 and preparation of hypercalcemic model animals were performed in the same manner as described in section 2 above. The prepared model animals were grouped on the average of blood calcium concentration and body weight, and such mice were used in the following experiments.

(1) Measurement of amount of food intake

During the test period, rats were placed individually in metabolic cages, fed water and food. For each rat, the intake was determined as the amount of food (g) in 24 hours (from 9 a day earlier to 9 a second day earlier). The total weight of the feed tank was measured at 9 am of the first day and 9 am of the second day, and the difference in weight was calculated to determine the amount of food intake.

(2) Administration of antibodies

Each of the hypercalcemia model rats (HHM mice) was injected with the humanized anti-PTHrP antibody at a dose of 5 mg/0.5 ml/kg via the tail vein. Each animal in the control group was injected with physiological saline via tail vein in the same manner. Normal rats were also injected with physiological saline via tail vein in the same manner. Food intake was measured for all of the antibody-used mice, control mice and normal rats on day 0 (i.e., the period from the day before administration to the day of administration), day 1 (i.e., the period from the day of administration to the day after administration), day 3 (i.e., the period from the third day to the fourth day after administration) and day 5 (i.e., the period from the day 5 to the day 6 after administration).

As a result, the intake amount of the hypercalcemic model rats (5 to 9 rats) was 8.11g on average and the intake amount of the normal rats was 12.06g on average before the administration of the antibody, indicating that the intake amount was significantly reduced in the hypercalcemic model rats. When the humanized anti-PTHrP antibody was used for the model rats, the intake of the mice administered with the antibody was restored to the level of normal mice on the day and after the administration of the antibody, although no change was observed in the intake of the control rats. These results indicate that the humanized anti-PTHrP antibody of the present invention has a significant improvement in the reduction in the uptake in the model animal of hypercalcemia associated with malignant tumor (Table 6).

Table 6: effects on food intake

^*Administration using saline: 0.5 ml/kg, via tail vein;

administration with antibodies: 5 mg/0.5 ml/kg via tail vein.

The above results demonstrate that the chimeric antibody and humanized antibody of the present invention can be effective as a drug for improving various clinical symptoms of hypercalcemia associated with malignant tumor.

5. Amelioration of blood pH reduction due to hypercalcemia

Transplantation of human lung carcinoma LC-6 and preparation of a hypercalcemia model animal associated with malignant tumor were carried out in the same manner as in the above step 2. Model animals were grouped according to blood calcium concentration and weight average.

(1) Determination of blood pH

Blood was collected from each test animal by a heparin-treated syringe by a heart blood collecting technique, and the collected blood was applied to 643Ca²⁺pH automatic analyzer (CIBA-CORNING) to determine pH of blood sample.

(2) Administration of antibodies

Each of the hypercalcemia model rats (HHM rats) described above was injected with the humanized anti-PTHrP antibody via tail vein at a dose of 5 mg/0.5 ml/kg (n ═ 3). Each animal in the control group (n-2) was injected with saline via tail vein in the same manner. Blood pH was measured on day 0 (i.e., day of administration), day 1 and day 7 for all rats and control mice using the antibody.

As a result, administration was carried out using an antibodyPreviously, hypercalcemic model rats received blood pH of about 7.49 (compared to normal rats of 7.40+0.02), which means that model rats developed significant metabolic alkalosis. When the humanized anti-PTHrP antibody of the present invention was administered to model rats, the pH of the blood of the mice administered with the antibody was restored to a value close to that of normal mice seven days after the administration of the antibody, although the pH of the blood of the control rats was almost unchanged. Metabolic alkalosis has been reported as one of the clinical symptoms of hypercalcemia associated with malignancy (HHM) due to renal bicarbonate ion (HCO)₃ ^-) Inhibition of excretion leads to this condition. Since the use of the humanized anti-PTHrP antibody of the present invention enabled blood pH to be normalized in the hypercalcemia model animal, it was suggested that the antibody could ameliorate metabolic alkalosis found in HHM (FIG. 31).

The above results demonstrate that the chimeric antibody and humanized antibody of the present invention can be effective as a drug for improving clinical symptoms of hypercalcemia associated with malignant tumor.

Industrial applicability

The present invention provides chimeric and humanized antibodies against PTHrP. These antibodies have low antigenicity to humans and are therefore useful as drugs for treating hypercalcemia, hypophosphatemia, and the like.

Sequence listing

(2) SEQ ID NO: 1, data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 1:

AAATAGCCCT TGACCAGGCA 20

(2) SEQ ID NO: 2, data:

(i) sequence characteristics:

(A) length: 38 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 2:

CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG 38

(2) SEQ ID NO: 3, data:

(i) sequence characteristics:

(A) length: 28 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 3:

CGATCCCGGG CCAGTGGATA GACAGATG 28

(2) SEQ ID NO: 4, data:

(i) sequence characteristics:

(A) length: 29 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 4:

GGATCCCGGG TCAGRGGAAG GTGGRAACA 29

(2) SEQ ID NO: 5, data:

(i) sequence characteristics:

(A) length: 17 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 5:

GTTTTCCCAG TCACGAC 17

(2) SEQ ID NO: data of 6:

(i) sequence characteristics:

(A) length: 17 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 6:

CAGGAAACAG CTATGAC 17

(2) SEQ ID NO: 7, data:

(i) sequence characteristics:

(A) length: 31 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 7:

GTCTAAGCTT CCACCATGAA ACTTCGGGCT C 31

(2) SEQ ID NO: data of 8:

(i) sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 8:

TGTTGGATCC CTGCAGAGAC AGTGACCAGA 30

(2) SEQ ID NO: 9, data:

(i) sequence characteristics:

(A) length: 36 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 9:

GTCTGAATTC AAGCTTCCAC CATGGGGTTT GGGCTG 36

(2) SEQ ID NO: data of 10:

(i) sequence characteristics:

(A) length: 41 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 10:

TTTCCCGGGG CCTTGGTGGA GGCTGAGGAG ACGGTGACCA G 41

(2) SEQ ID NO: data of 11:

(i) sequence characteristics:

(A) length: 109 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 11:

GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCCCACGGTC ACCCTGTTCC 60

CGCCCTCCTC TGAGGAGCTC CAAGCCAACA AGGCCACACT AGTGTGTCT 109

(2) SEQ ID NO: data of 12:

(i) sequence characteristics:

(A) length: 110 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 12:

GGTTTGGTGGTCTCCACTCC CGCCTTGACG GGCTGCCAT CTGCCTTCCA GGCCACTGTC 60

ACAGCTCCCG GGTAGAAGTC ACTGATCAGA CACACTAGTG TGGCCTTGTT 110

(2) SEQ ID NO: 13, data:

(i) sequence characteristics:

(A) length: 98 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 13:

GGAGTGGAGA CCACCAAACC CTCCAAACAG AGCAACAACA AGTACGCGGC CCAGCTAC 60

CTGAGCCTGA CGCCCGAGCA GTGGAAGTCC CACAGAAG

98

(2) SEQ ID NO: 14, data of:

(i) sequence characteristics:

(A) length: 106 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 14:

TGTTGAATTC TTACTATGAA CATTCTGTAG GGGCCACTGT CTTCTCCACG GTGCTCCCTT 60

CATGCGTGAC CTGGCAGCTG TAGCTTCTGT GGGACTTCCA CTGCTC 106

(2) SEQ ID NO: 15, data of:

(i) sequence characteristics:

(A) length: 43 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 15:

GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCC 43

(2) SEQ ID NO: data of 16:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 16:

TGTTGAATTC TTACTATGAA 20

(2) SEQ ID NO: 17 data:

(i) sequence characteristics:

(A) length: 39 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 17:

CAACAAGTAC GCGGCCAGCA GCTACCTGAG CCTGACGCC 39

(2) SEQ ID NO: data of 18:

(i) sequence characteristics:

(A) length: 39 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 18:

GTAGCTGCTG GCCGCGTACT TGTTGTTGCT CTGTTTGGA 39

(2) SEQ ID NO: data of 19:

(i) sequence characteristics:

(A) length: 46 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 19:

GTCTGAATTC AAGCTTAGTC CTAGGTCGAA CTGTGGCTGC ACCATC 46

(2) SEQ ID NO: 20, data:

(i) sequence characteristics:

(A) length: 34 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 20:

TGTTGAATTC TTACTAACAC TCTCCCCTGT TGAA 34

(2) SEQ ID NO: data of 21:

(i) sequence characteristics:

(A) length: 35 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 21:

GTCTAAGCTT CCACCATGGC CTGGACTCCT CTCTT 35

(2) SEQ ID NO: 22 data:

(i) sequence characteristics:

(A) length: 48 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 22:

TGTTGAATTC AGATCTAACT ACTTACCTAG GACAGTGACC TTGGTCCC 48

(2) SEQ ID NO: data of 23:

(i) sequence characteristics:

(A) length: 128 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 23:

and (3) labeling to SEQ ID NO: 23

(2) SEQ ID NO: data of 24:

(i) sequence characteristics:

(A) length: 125 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 24:

and (3) labeling to SEQ ID NO: 24

(2) SEQ ID NO: 25, data of:

(i) sequence characteristics:

(A) length: 132 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 25:

and (3) labeling to SEQ ID NO: 25

(2) SEQ ID NO: data of 26:

(i) sequence characteristics:

(A) length: 110 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 26:

and (3) labeling to SEQ ID NO: 26

(2) SEQ ID NO: data of 27:

(i) sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 27:

GTCTAAGCTT CCACCATGGG GTTTGGGCTG 30

(2) SEQ ID NO: data of 28:

(i) sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 28:

TGTTGGATCC CTGAGGAGAC GGTGACCAGG 30

(2) SEQ ID NO: 29 data:

(i) sequence characteristics:

(A) length: 133 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 29:

and (3) labeling to SEQ ID NO: 29

(2) SEQ ID NO: data of 30:

(i) sequence characteristics:

(A) length: 118 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 30:

and (3) labeling to SEQ ID NO: 30

(2) SEQ ID NO: 31 data:

(i) sequence characteristics:

(A) length: 128 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 31:

and (3) labeling to SEQ ID NO: 31

(2) SEQ ID NO: 32 data:

(i) sequence characteristics:

(A) length: 114 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 32:

and (3) labeling to SEQ ID NO: 32

(2) SEQ ID NO: 33 data of:

(i) sequence characteristics:

(A) length: 17 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 33:

ACAAAGCTTC CACCATG 17

(2) SEQ ID NO: 34, data of:

(i) sequence characteristics:

(A) length: 19 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 34:

CTTGGATCCG GGCTGACCT 19

(2) SEQ ID NO: data of 35:

(i) sequence characteristics:

(A) length: 75 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 35:

CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA 60

CGTACACAAA TTGTTCCTTA ATTGT 75

(2) SEQ ID NO: 36 of the data:

(i) sequence characteristics:

(A) length: 43 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 36:

AAAGGTCCT TAAGATCCAT CAAGTACCGA GGGGGCTTCT CTG 43

(2) SEQ ID NO: data of 37:

(i) sequence characteristics:

(A) length: 46 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 37:

ACAAAGCTTA GCGCTACCTC ACCATCTCCA GCCTCCAGCCTGAGGA 46

(2) SEQ ID NO: 38 of the data:

(i) sequence characteristics:

(A) length: 111 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 38:

and (3) labeling to SEQ ID NO: 38

(2) SEQ ID NO: 39 data:

(i) sequence characteristics:

(A) length: 42 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 39:

CTTCTCTGGC TGCTGCTGAT ACCATTCAAT GGTGTACGTA CT 42

(2) SEQ ID NO: data of 40:

(i) sequence characteristics:

(A) length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 40:

CGAGGGCCCT TCTCTGGCTG CTGCTG 26

(2) SEQ ID NO: data of 41:

(i) sequence characteristics:

(A) length: 35 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 41:

GAGAAGGGCC CTARGTACST GATGRAWCTT AAGCA 35

(2) SEQ ID NO: 42, data:

(i) sequence characteristics:

(A) length: 35 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 42:

CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG 35

(2) SEQ ID NO: 43 data:

(i) sequence characteristics:

(A) length: 18 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 43:

GGCTTGGAGC TCCTCAGA 18

(2) SEQ ID NO: data of 44:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: other nucleic acids

(A) Description of the drawings: "synthetic DNA" (/ desc) "

(xi) Description of the sequence: SEQ ID NO: 44:

GACAGTGGTT CAAAGTTTTT 20

(2) SEQ ID NO: 45, data:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 45:

Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser Leu Gly

1 5 10 15

Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys

35 40 45

Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg

65 70 75

Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Met Tyr

80 85 90

Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: 46 data:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 46:

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly

1 5 10 15

Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Gly Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu

35 40 45

Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr

50 55 60

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp

80 85 90

Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe

95 100 105

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

110 115

(2) SEQ ID NO: data of 47:

(i) sequence characteristics:

(A) length: 116 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 47:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg

35 40 45

Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

110 115

(2) SEQ ID NO: 48, data of:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 48:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys

35 40 45

Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: 49 data:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 49:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys

35 40 45

Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: data of 50:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 50:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg

35 40 45

Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: 51, data:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 51:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg

35 40 45

Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: data of 52:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 52:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys

35 40 45

Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: 53 data:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 53:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg

35 40 45

Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

2) SEQ ID NO: 54 data:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 54:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys

35 40 45

Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: data of 55:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 55:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly

1 5 10 15

Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr

20 25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg

35 40 45

Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp

50 55 60

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg

65 70 75

Tyr Leu Thr Ile Ser Ser LeuGln Ser Glu Asp Glu Ala Asp Tyr

80 85 90

Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val

95 100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

110 115

(2) SEQ ID NO: data of 56:

(i) sequence characteristics:

(A) length: 118 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 56:

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly

1 5 10 15

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr

50 55 60

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

65 70 75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe

95 100 105

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

110 115

(2) SEQ ID NO: data of 57:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 57:

ATG AAC TTC GGG CTC AGC TTG ATT TTC CTT GCC CTC ATT TTA AAA 45

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys

-15 -10 -5

GGT GTC CAG TGT GAG GTG CAA CTG GTG GAG TCT GGG GGA GAC TTA 90

Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu

1 5 10

GTG AAG CCT GGA GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA 135

Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly

15 20 25

TTC ACT TTC AGT AGC TAT GGC ATC TCT TGG ATT CGC CAG ACT CCA 180

Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Ile Arg Gln Thr Pro

30 35 40

GAC AAG AGG CTG GAG TGG GTC GCA ACC ATT AGT AGT GGT GGT AGT 225

Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser

45 50 55

TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270

Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

60 65 70

AGA GAC AAT GCC AAG AAC ACC CTA TAC CTG CAA ATG AGC AGT CTG 315

Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu

75 80 85

AAG TCT GAG GAC ACA GCC ATG TTT TAC TGT GCA AGA CAG ACT ACT 360

Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr

90 95 100

ATG ACT TAC TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC 405

Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

105 110 115

TCT GCA 411

Ser Ala

(2) SEQ ID NO: 58, data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 58:

ATG GGG TTT GGG CTG AGC TGG GTT TTC CTC GTT GCT CTT TTA AGA 45

Met Gly Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg

-15 -10 -5

GGT GTC CAG TGT CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG 90

Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

1 5 10

GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA 135

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

15 20 25

TTC ACC TTC AGT AGC TAT GGC ATG TCT TGG GTC CGC CAG GCT CCA 180

Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro

30 35 40

GGC AAG GGG CTG GAG TGG GTG GCA ACC ATT AGT AGT GGT GGT AGT 225

Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser

45 50 55

TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270

Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

60 65 70

AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG 315

Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

75 80 85

AGA GCT GAG GAC ACG GCT GTG TAT TAC TGT GCG AGA CAG ACT ACT 360

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr

90 95 100

ATG ACT TAC TTT GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC 405

Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

105 110 115

TCC TCA 411

Ser Ser

(2) SEQ ID NO: data of 59:

(i) sequence characteristics:

(A) length: 11 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 59:

Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

(2) SEQ ID NO: data of 60:

(i) sequence characteristics:

(A) length: 7 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 60:

Ser Ala Ser Asn Arg Tyr Thr

1 5

(2) SEQ ID NO: data of 61:

(i) sequence characteristics:

(A) length: 9 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 61:

Gln Gln His Tyr Ser Thr Pro Phe Thr

1 5

(2) SEQ ID NO: 62, data:

(i) sequence characteristics:

(A) length: 5 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 62:

Pro Tyr Trp Met Gln

1 5

(2) SEQ ID NO: 63, data:

(i) sequence characteristics:

(A) length: 16 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 63:

Ser Ile Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly

1 5 10 15

(2) SEQ ID NO: data of 64:

(i) sequence characteristics:

(A) length: 11 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 64:

Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

1 5 10

(2) SEQ ID NO: 65 data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 65:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAA CTT GTG CTC ACT CAG TCA TCT TCA GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser

1 5 10

TTC TCC CTG GGA GCC TCA GCA AAA CTC ACG TGC ACC TTG AGT AGT 135

Phe Ser Leu Gly Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAA CAG CCA CTC 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu

30 35 40

AAG CCT CCT AAG TAT GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Pro Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCT GGA TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGT GCT GAT CGC TAC CTT AGC ATT TCC AAC ATC CAG CCA GAA GAT 315

Gly Ala Asp Arg Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp

75 80 85

GAA GCA ATG TAC ATC TGT GGT GTG GGT GAT ACA ATT AAG CAA CAA 360

Glu Ala Met Tyr IIe Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAT GTT TTC GGC GGT GGG ACC AAG GTC ACT GTC CTA GGT 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 66 data:

(i) sequence characteristics:

(A) length: 405 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 66:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG CAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT CGG TAC TTG ATG AAA CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Arg Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGT 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu ThT Val Leu Gly

105 110 115

(2) SEQ ID NO: 67 data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 67:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AAG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAGGAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 68 data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 68:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AAG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 69 data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 69:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AGG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 70, data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 70:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AGG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 71 data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 71:

ATG GCC TGG ACT CGT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AAG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: data of 72:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 72:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AGG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 73, data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 73:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AAG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55

AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270

Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 74 data:

(i) sequence characteristics:

(A) length: 411 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(xi) Description of the sequence: SEQ ID NO: 74:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser

1 5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135

Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser

15 20 25

CAG CAC AGT ACG TAC ACCATT GAA TGG TAT CAG CAG CAG CCA GAG 180

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu

30 35 40

AAG GGC CCT AGG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225

Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His

45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

60 65 70

GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

75 80 85

GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360

Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln

90 95 100

TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405

PheVal Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro

(2) SEQ ID NO: 75, data:

(i) sequence characteristics:

(A) length: 34 amino acids

(B) Type (2): amino acids

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(xi) Description of the sequence: SEQ ID NO: 75:

Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile

1 5 10 15

Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu

20 25 30

Ile His Thr Ala

Claims

1. A humanized antibody of anti-human parathyroid hormone related protein comprises a humanized antibody containing amino acid sequences shown as SEQ ID NO: 59-61, and an L chain V region comprising a framework region having tyrosine and aspartic acid at amino acids 36 and 49 according to the Kabat definition.

2. The humanized antibody according to claim 1, wherein the amino acid at position 87 in the framework region of the L chain V region is isoleucine according to Kabat definition.

3. A humanized antibody according to claim 1, wherein the L chain V region comprises the amino acid sequence of SEQ ID NO: 48-51.

4. A humanized antibody according to claim 2, wherein the L chain V region comprises SEQ ID NO: 52-55.

5. The humanized antibody according to any one of claims 1 to 4, further comprising an amino acid sequence of a H chain V region of the humanized antibody comprising framework regions 1 to 4 of a H chain V region of a human antibody and complementarity determining regions 1 to 3 of a H chain V region of a mouse monoclonal antibody against human parathyroid hormone-related protein.

6. A humanized antibody according to claim 5, wherein the complementarity determining regions 1-3 comprise the amino acid sequences set forth as SEQ ID NOs: 62-64.

7. A humanized antibody according to claim 5, wherein the framework regions 1-4 are derived from the framework regions 1-4 of a human antibody of human subgroup III.

8. A humanized antibody according to claim 5, wherein the framework regions 1 to 4 are respectively derived from the framework regions 1 to 4 of the human antibody S31679.

9. The humanized antibody of claim 1, further comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 in the presence of a heavy chain antibody.

10. The humanized antibody according to claim 1, further comprising a polypeptide comprising an L chain C region of a human antibody.

11. A humanized antibody according to claim 10 wherein the C region is a C λ chain.

12. The humanized antibody according to claim 1, further comprising a polypeptide comprising a H chain C region of a human antibody.

13. A humanized antibody according to claim 12, wherein the C region is a C γ 1 chain.

A DNA comprising a base sequence of an amino acid sequence of an L chain V region of a humanized antibody encoding an anti-human parathyroid hormone-related protein, said amino acid sequence of the L chain V region comprising a sequence comprising amino acid sequences set forth as SEQ ID NOs: 59-61, and a framework region comprising tyrosine and aspartic acid at amino acids 36 and 49, respectively, according to the Kabat definition.

15. DNA according to claim 14, comprising SEQ ID NO: 66-74 in a nucleotide sequence set forth in any one of claims.

A DNA comprising a base sequence of an amino acid sequence of a H chain V region of a humanized antibody against human parathyroid hormone-related protein, the amino acid sequence of the H chain V region comprising framework regions 1 to 4 of a H chain V region of a human antibody, and amino acid sequences shown in SEQ ID NOs: complementarity determining regions 1-3 shown at 62-64.

17. DNA according to claim 16, comprising SEQ ID NO: 58, or a nucleotide sequence represented by the formula (I).

DNA encoding the L chain of a humanized antibody against human parathyroid hormone related protein, said L chain comprising the amino acid sequences set forth in SEQ ID NOs: 59-61, amino acids 36 and 49 according to the Kabat definition, respectively, contain framework regions of tyrosine and aspartic acid, and further include polypeptides comprising the L chain C region of a human antibody.

19. A DNA of a humanized antibody L chain comprising a sequence encoding SEQ ID NO: 47 to 55 in sequence.

20. DNA according to claim 19, wherein the DNA of the L chain of the humanized antibody comprises SEQ ID NO: 66-74 in a nucleotide sequence set forth in any one of claims.

DNA encoding the H chain of a humanized antibody against human parathyroid hormone related protein, said H chain comprising framework regions 1-4 of the H chain V region of a human antibody, as set forth in SEQ ID NOs: 62-64, and the H chain C region of a human antibody.

22. DNA of a humanized antibody H chain against human parathyroid hormone related protein comprising a sequence encoding SEQ ID NO: 56 in sequence listing.

23. The DNA according to claim 22, wherein the DNA of the H chain of the humanized antibody comprises SEQ ID NO: 58, or a nucleotide sequence represented by the formula (I).

24. A recombinant vector comprising the DNA of any one of claims 14-23.

25. A transformant transformed with the recombinant vector of claim 24.

26. A method for producing a humanized antibody against human parathyroid hormone related protein, comprising culturing a transformant transformed with an expression vector comprising the DNA of any one of claims 14, 15 and 18 to 20 and an expression vector comprising the DNA of any one of claims 16, 17, 21 and 22, and collecting a humanized antibody against human parathyroid hormone related protein from the resulting culture.

27. A pharmaceutical composition comprising as an active ingredient the humanized antibody against human parathyroid hormone related protein according to any one of claims 1 to 13.

28. A pharmaceutical agent for suppressing hypercalcemia comprising the humanized antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

29. A pharmaceutical agent for suppressing hypercalcemia associated with malignant tumor, which comprises the humanized antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

30. The agent for suppressing hypercalcemia according to claim 29, wherein the malignant tumor is at least one selected from the group consisting of: pancreatic, lung, pharyngeal, laryngeal, tongue, gingival, esophageal, gastric, biliary, breast, renal, bladder, uterine and prostate cancer, as well as malignant lymphomas.

31. A pharmaceutical agent for ameliorating hypophosphatemia, which comprises the humanized antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

32. The agent for improving hypophosphatemia according to claim 31, wherein the hypophosphatemia is rickets hypophosphatemia.

33. The agent for improving hypophosphatemia according to claim 31, wherein the hypophosphatemia is vitamin D-resistant rickets with hypophosphatemia.

34. A medicament for preventing or treating weight loss associated with malignant tumor, comprising the antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an effective ingredient.

35. An agent for preventing or ameliorating a decrease in food intake associated with malignant tumor, comprising the antibody against human parathyroid hormone related protein according to any one of claims 1 to 13 as an active ingredient.

36. An agent for preventing or ameliorating anorexia or nausea associated with malignant tumor, comprising the antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

37. A pharmaceutical agent for preventing or ameliorating metabolic alkalosis associated with malignant tumors, which comprises the antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

38. An agent for preventing or ameliorating blood pH abnormality associated with malignant tumor, comprising the antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

39. A medicament for controlling changes in body temperature associated with malignant tumors, comprising the antibody against human parathyroid hormone related protein according to any one of claims 1 to 13 as an active ingredient.

40. An agent for preventing or ameliorating a decrease in body temperature associated with malignant tumor, comprising the antibody against human parathyroid hormone related protein according to any one of claims 1 to 13 as an active ingredient.

41. An agent for preventing or ameliorating reduced spontaneous activity associated with malignant tumors, which comprises the antibody against human parathyroid hormone-related protein according to any one of claims 1 to 13 as an active ingredient.

42. An agent for preventing or improving a performance status associated with a malignant tumor, comprising an antibody against the human parathyroid hormone related protein according to any one of claims 1 to 13 as an active ingredient.

43. A pharmaceutical agent for preventing or ameliorating a decrease in the quality of life of a patient associated with a malignant tumor, comprising the antibody against human parathyroid hormone related protein according to any one of claims 1 to 13 as an active ingredient.